COMPOUND FOR USE IN RETINAL DISEASES

Abstract

Provided is an aldehyde binder, specifically, disclosed is a compound as represented by formula (II) or a pharmaceutically acceptable salt.

Claims

1. A compound represented by formula (II) or a pharmaceutically acceptable salt thereof, ##STR00063## wherein is selected from a single bond and a double bond; T.sub.1, T.sub.2, T.sub.3, and T.sub.4 are each independently selected from N, C, and CR.sub.1; T.sub.5 is selected from C, CR.sub.5, and C═O; T.sub.6 is selected from C, CR.sub.6, and N; T.sub.7 is selected from N and CR.sub.7; when T.sub.5 is selected from C═O and T.sub.6 is selected from N, then is selected from a single bond; L is selected from a single bond, —O—, —S—, —NR.sub.2— and —(CR.sub.3R.sub.4).sub.n—; each R.sub.1 is independently selected from H, F, Cl, Br, I, OH, and NH.sub.2; R.sub.2 is selected from H and C.sub.1-3 alkyl optionally substituted with 1, 2 or 3 R.sub.a; R.sub.3 and R.sub.4 are each independently selected from H, F, Cl, Br, I, OH, NH.sub.2, CN and C.sub.1-3 alkyl optionally substituted with 1, 2 or 3 R.sub.b; R.sub.5, R.sub.6 and R.sub.7 are each independently selected from H, F, Cl, Br and I; n is selected from 1, 2, and 3; R.sub.a and R.sub.b are each independently selected from H, F, Cl, Br, I, OH, NH.sub.2, CN, and CH.sub.3.

2. The compound or the pharmaceutically acceptable salt thereof as defined in claim 1, wherein R.sub.2 is selected from H, CH.sub.3 and CH.sub.2CH.sub.3; wherein the CH.sub.3 and the CH.sub.2CH.sub.3 are optionally substituted with 1, 2, or 3 R.sub.a.

3. The compound or the pharmaceutically acceptable salt thereof as defined in claim 2, wherein R.sub.2 is selected from H, CH.sub.3, and CH.sub.2CH.sub.3.

4. The compound or the pharmaceutically acceptable salt thereof as defined in claim 1, wherein R.sub.3 and R.sub.4 are each independently selected from H, F, Cl, Br, I, OH, NH.sub.2, CN, CH.sub.3 and CH.sub.2CH.sub.3, wherein the CH.sub.3 and the CH.sub.2CH.sub.3 are optionally substituted with 1, 2 or 3 R.sub.b.

5. The compound or the pharmaceutically acceptable salt thereof as defined in claim 4, wherein R.sub.3 and R.sub.4 are each independently selected from H, F, Cl, Br, I, OH, NH.sub.2, CN, CH.sub.3 and CH.sub.2CH.sub.3.

6. The compound or the pharmaceutically acceptable salt thereof as defined in claim 5, wherein L is selected from a single bond, —O—, —S—, —NH—, —(CH.sub.2).sub.2— and —CH.sub.2—.

7. The compound or the pharmaceutically acceptable salt thereof as defined in claim 1, wherein the compound is selected from ##STR00064## wherein T.sub.3 and T.sub.4 are each independently selected from N and CR.sub.1; R.sub.1 and L are as defined in claim 1.

8. The compound or the pharmaceutically acceptable salt thereof as defined in claim 7, wherein the compound is selected from ##STR00065## wherein R.sub.1 and L are as defined in claim 7.

9. A compound represented by the following formula or a pharmaceutically acceptable salt thereof, wherein the compound is selected from: ##STR00066## ##STR00067##

10. A pharmaceutical composition, comprising a therapeutically effective amount of the compound or the pharmaceutically acceptable salt thereof as defined in claim 1 as an active ingredient, and a pharmaceutically acceptable carrier thereof.

11-12. (canceled)

13. A method for binding the aldehydes in vivo in a subject in need thereof, comprising administering a therapeutically effective amount of the compound or the pharmaceutically acceptable salt thereof as defined in claim 1 to the subject.

14. A method for treating xerophthalmia in a subject in need thereof, comprising administering a therapeutically effective amount of the compound or the pharmaceutically acceptable salt thereof as defined in claim 1 to the subject.

15. The compound or the pharmaceutically acceptable salt thereof as defined in claim 2, wherein R.sub.3 and R.sub.4 are each independently selected from H, F, Cl, Br, I, OH, NH.sub.2, CN, CH.sub.3 and CH.sub.2CH.sub.3, wherein the CH.sub.3 and the CH.sub.2CH.sub.3 are optionally substituted with 1, 2 or 3 R.sub.b.

16. The compound or the pharmaceutically acceptable salt thereof as defined in claim 3, wherein R.sub.3 and R.sub.4 are each independently selected from H, F, Cl, Br, I, OH, NH.sub.2, CN, CH.sub.3 and CH.sub.2CH.sub.3, wherein the CH.sub.3 and the CH.sub.2CH.sub.3 are optionally substituted with 1, 2 or 3 R.sub.b.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0075] FIG. 1: Test results of in vitro aldehyde capture capacity;

[0076] FIG. 2: Tear secretion results of an efficacy test on xerophthalmia in mice;

[0077] FIG. 3: Fluorescence staining results of cornea in an efficacy test on xerophthalmia in mice.

TECHNICAL EFFECT

[0078] The compound of the present disclosure has excellent aldehyde complexing capacity, and is helpful to remit eye inflamed, thereby treating the xerophthalmia.

DETAILED DESCRIPTION OF THE EMBODIMENT

[0079] The following examples further illustrate the present disclosure, but the present disclosure is not adversely limited thereto. The present disclosure has been described in detail herein, wherein specific embodiments thereof are also disclosed, and it will be apparent to those skilled in the art to make various changes and improvements to specific embodiments of the present disclosure without departing from the spirit and scope of the present disclosure.

Example 1: Synthesis of Compound 1

[0080] ##STR00010##

Synthetic Route of the Compound 1

[0081] ##STR00011##

Step 1: Synthesis of Compound 1-2

[0082] ##STR00012##

[0083] A Compound 1-1 (1.5 g, 6.12 mmol, 1 eq), hexamethylditin (1.60 g, 4.90 mmol, 1.02 mL, 0.8 eq) and bis (tri-tert-butylphosphine) palladium(0) (938.39 mg, 1.84 mmol, 0.3 eq) were dissolved in toluene (20 mL), and stirred at 80° C. for 14 hours. TLC spot plate (dichloromethane:methanol=10:1) was used to test if the reaction was complete, and samples were concentrated and stirred directly. The samples were purified by a rapid silica gel column (mobile phase: 0-10% dichloromethane/methanol) to obtain Compound 1-2. [M+1].sup.+=330.9

Step 2: Synthesis of Compound 1

[0084] ##STR00013##

[0085] A substrate 1-2 (200 mg, 605 μmol, 1 eq) was dissolved in tetrahydrofuran (20 mL), methyl magnesium bromide (3 M, 4.04 mL, 20 eq) was slowly added dropwise at 0° C., and the mixture was reacted under stirring at 0° C. for 2 hours. A reaction solution was quenched by adding water (20 mL), then extracted with ethyl acetate (50 mL*3), and concentrated for organic phases. The mixture was dissolved in dimethylformamide (DMF) and purified by HPLC (neutral) to obtain Compound 1.

[0086] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.80 (d, J=8.3 Hz, 2H), 7.00 (d, J=8.3 Hz, 2H), 5.56 (s, 4H), 5.47 (s, 2H), 1.56 (s, 12H); LCMS: [M+H].sup.+=302.9

Example 2: Synthesis of Compound 2

[0087] ##STR00014##

Synthetic Route of Compound 2

[0088] ##STR00015##

Step 1: Synthesis of Compound 2-3

[0089] ##STR00016##

[0090] Substrates 2-1 (702.07 mg, 2.55 mmol, 1.5 eq) and 2-2 (310 mg, 1.70 mmol, 1 eq) were dissolved in acetonitrile (20 mL), cesium carbonate (1.11 g, 3.40 mmol, 2 eq) was added, and the mixture was reacted under stirring at 50° C. for 3 hours. After the reaction was over, a reaction solution was filtered directly, and filtrate was spin-dried to obtain crude. The crude was purified by a rapid silica gel column (petroleum ether:ethyl acetate=100:0 to 60:40) to obtain Compound 2-3.

[0091] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.42 (d, J=9.03 Hz, 1H), 7.24 (d, J=4.02 Hz, 1H), 7.15-7.20 (m, 1H), 7.07-7.13 (m, 1H), 5.82 (br s, 2H), 4.39 (dq, J=4.27, 7.11 Hz, 4H), 1.35 (td, J=7.15, 18.57 Hz, 6H).

[0092] Step 2: Synthesis of Compound 2-4

##STR00017##

[0093] The Compound 2-3 (360 mg, 956.63 μmol, 1 eq) and hydrochloric acid (12 M, 478.32 μL, 6 eq) were dissolved in ethanol (20 mL) and water (5 mL); reducing iron powders (534.23 mg, 9.57 mmol, 10 eq) were added, and stirred at 25° C. for 2 hours. After the reaction was over, a reaction solution was adjusted to pH=9 with a saturated sodium carbonate aqueous solution (30 mL), and extracted with ethyl acetate (30 mL*2). After organic phases were combined, the organic phases were dried over anhydrous sodium sulfate and spin-dried to obtain crude. The crude was purified by a rapid silica gel column (petroleum ether:ethyl acetate=100:0 to 40:60) to obtain Compound 2-4.

[0094] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.17-7.24 (m, 2H), 7.09-7.16 (m, 2H), 5.64 (br s, 4H), 4.37 (q, J=7.03 Hz, 4H), 1.37 (t, J=7.03 Hz, 6H).

Step 3: Synthesis of Compound 2

[0095] ##STR00018##

[0096] The Compound 2-4 (120 mg, 346.48 μmol, 1 eq) was dissolved in tetrahydrofuran (10 mL) and cooled to 0° C. Methyl magnesium bromide (3 M, 3.00 mL, 26 eq) was added dropwise in a 2-methyltetrahydrofuran solution at 0° C. The solution was reacted under stirring at 0° C. for 30 min, and then stirred at 25° C. for 2 hours. After the reaction was over, a reaction solution was quenched by adding a saturated amine chloride aqueous solution (40 mL), and then extracted with ethyl acetate (100 mL). The organic phases were dried over anhydrous sodium sulfate and spin-dried to obtain the crude. The crude was purified by HPLC separation (column model: Waters Xbridge 150*25 mm 5 μm; mobile phase: [water (10 mM amine bicarbonate solution)-acetonitrile]; % B (acetonitrile): 0%-50%, 10 min) to obtain Compound 2.

[0097] LCMS: [MS+H.sup.+]=318.9.

[0098] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.03 (d, J=8.28 Hz, 2H), 6.57 (d, J=8.53 Hz, 2H), 5.37 (s, 2H), 5.27 (s, 4H), 1.38 (s, 12H).

Example 3: Synthesis of Compound 3

[0099] ##STR00019##

Synthetic Route of Compound 3

[0100] ##STR00020##

Step 1: Synthesis of Compound 3-3

[0101] ##STR00021##

[0102] Compounds 3-1 (300 mg, 1.30 mmol, 1 eq) and 3-2 (379.73 mg, 1.69 mmol, 1.3 eq), potassium phosphate (551.24 mg, 2.60 mmol, 2 eq) and [1,1′-bis(diphenylphosphino) ferrocene] palladium (II) dichloromethane (530.18 mg, 649.22 μmol, 0.5 eq) were dissolved in dichloroethane (15 mL), and stirred at 80° C. for 15 hours. After the reaction was over, a reaction solution was filtered directly, and filtrate was spin-dried to obtain crude. The crude was purified by a rapid silica gel column (petroleum ether:ethyl acetate=100:0 to 50:50) to obtain Compound 3-3.

[0103] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.29 (s, 1H), 8.08 (s, 1H), 7.88 (s, 2H), 7.25 (s, 1H), 5.95 (br s, 2H), 4.03 (s, 3H), 3.96 (s, 3H).

Step 2: Synthesis of Compound 3-4

[0104] ##STR00022##

[0105] The Compound 3-3 (250 mg, 754.65 μmol, 1 eq) and hydrochloric acid (12 M, 1.26 mL, 20 eq) were dissolved in methanol (12 mL) and water (3 mL); reducing iron powders (421.43 mg, 7.55 mmol, 10 eq) were added, and stirred at 25° C. for 1 day and 15 hours. After the reaction was over, a reaction solution was adjusted to pH=9 with a saturated sodium carbonate aqueous solution (50 mL), and extracted with ethyl acetate (60 mL*2). After organic phases were combined, the organic phases were dried over anhydrous sodium sulfate and spin-dried to obtain crude. The crude was purified by a rapid silica gel column (petroleum ether:ethyl acetate=100:0 to 50:50) to obtain Compound 3-4.

[0106] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.09 (d, J=2.01 Hz, 1H), 7.82 (d, J=8.28 Hz, 1H), 7.43 (d, J=2.01 Hz, 1H), 7.08 (d, J=1.51 Hz, 1H), 6.71-6.86 (m, 5H), 3.83 (d, J=5.77 Hz, 6H).

Step 3: Synthesis of Compound 3

[0107] ##STR00023##

[0108] The Compound 3-4 (80 mg, 265.52 μmol, 1 eq) was dissolved in tetrahydrofuran (10 mL) and cooled to 0° C. Methyl magnesium bromide (33 M, 2 mL, 22.60 eq) was added dropwise in a 2-methyltetrahydrofuran solution at 0° C. The solution was reacted under stirring at 0° C. for 30 min, and then stirred at 25° C. for 15 hours. After the reaction was over, a reaction solution was quenched by adding a saturated amine chloride aqueous solution (20 mL), and then extracted in ethyl acetate (50 mL). The organic phases were dried over anhydrous sodium sulfate and spin-dried to obtain the crude. The crude was purified by thin-layer chromatographic silica gel plate separation (mobile phase: petroleum ether:ethyl acetate=2:5) to obtain Compound 3.

[0109] LCMS: [MS+H.sup.+]=301.9.

[0110] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.12 (d, J=1.76 Hz, 1H), 7.20 (d, J=8.03 Hz, 1H), 7.07 (d, J=2.01 Hz, 1H), 6.85 (dd, J=2.01, 8.03 Hz, 1H), 6.81 (d, J=2.01 Hz, 1H), 4.31-5.07 (m, 4H), 3.32 (br s, 2H), 1.71 (d, J=5.27 Hz, 12H).

Example 4: Synthesis of Compound 4

[0111] ##STR00024##

Synthetic Route of Compound 4

[0112] ##STR00025##

Step 1: Synthesis of Compound 4-2

[0113] ##STR00026##

[0114] The Compound 3-1 (3 g, 12.98 mmol, 1 eq) was dissolved in tetrahydrofuran (10 mL), a reaction temperature was reduced to −78° C., and lithium methyl (1 M, 64.92 mL, 5 eq) was added dropwise and stirred at −78° C. for 1 hour. TLC plate monitoring (petroleum ether:ethyl acetate=3:1) showed a new point generated in the reaction. A reaction solution was quenched by adding water (50 mL), and then extracted with ethyl acetate (50 mL*2). Organic phases were spin-dried. Products were purified by a rapid silica gel column (petroleum ether/ethyl acetate=3:1 to 1:1) to obtain Compound 4-2.

[0115] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.93 (d, J=2.0 Hz, 1H), 7.06 (d, J=2.0 Hz, 1H), 4.67 (br s, 2H), 1.64 (s, 6H)

Step 2: Synthesis of Compound 4

[0116] ##STR00027##

[0117] The Compound 4-2 (500 mg, 2.16 mmol), hexamethylditin (1.74 mmol, 360 mL), and bis (tri-tert-butylphosphine) palladium(0) (330 mg, 645.73 μmol) were added in toluene (15 mL), and stirred under nitrogen atmosphere for reaction at 80° C. for 12 hours. Thin layer chromatography (dichloromethane:methanol=10:1) showed formation of a new point with large polarity. After the reaction was over, methanol (10 mL) and the dichloromethane (100 mL) were added in the reaction; after solids were dissolved, the solids were filtered, and filtrate was concentrated under reduced pressure. The residue was subjected to column chromatography (dichloromethane:methanol=10:1) to obtain an impure product, and the impure product was separated by high performance liquid preparative chromatography (neutral, column: Waters Xbridge 150*25 mm 5 μm; mobile phase: [water (10 mM amine bicarbonate)-acetonitrile]; % B (acetonitrile): 10% -35%, 8.2 min) to obtain Compound 4.

[0118] LCMS (ESI): [M+H].sup.+=303.

[0119] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.91 (d, J=2.0 Hz, 2H), 7.12 (d, J=2.0 Hz, 2H), 5.68 (s, 4H), 5.48 (br s, 2H), 1.52 (m, 12H).

Example 5: Synthesis of Compound 5

[0120] ##STR00028##

Synthetic Route of Compound 5

[0121] ##STR00029## ##STR00030##

Step 1: Synthesis of Compound 5-3

[0122] ##STR00031##

[0123] Substrates 5-1 (3 g, 17.74 mmol, 1 eq) and 5-2 (4.03 g, 17.91 mmol, 1.01 eq) were dissolved in acetic acid (50 mL) and stirred at 25° C. for 2 hours. After the reaction was over, a reaction solution was spin-dried directly. Crude was dissolved in ethyl acetate (130 mL), and washed once with a saturated sodium carbonate aqueous solution (100 mL), sodium thiosulfate (100 mL, 1 M) and saturated saline (100 mL), respectively. The organic phases were dried over anhydrous sodium sulfate and spin-dried to obtain the crude. The crude was purified by a rapid silica gel column (petroleum ether:ethyl acetate=100:0 to 98:2) to obtain Compound 5-3.

[0124] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.59-7.67 (m, 2H), 6.22 (br s, 2H), 3.90 (s, 3H).

Step 2: Synthesis of Compound 5-5

[0125] ##STR00032##

[0126] Compounds 5-3 (1 g, 3.39 mmol, 1 eq) and 5-4 (860.66 mg, 3.39 mmol, 1 eq) and potassium acetate (332.62 mg, 3.39 mmol, 1 eq) were dissolved in toluene (15 mL), and stirred under nitrogen atmosphere at 25° C. for 10 min; [1,1′-bis(diphenylphosphino) ferrocene] palladium (II) dichloromethane [Pd(dppf)Cl.sub.2.CH.sub.2Cl.sub.2] (2.77 g, 3.39 mmol, 1 eq) was added. The solution was reacted under stirring for 15 hours after the temperature was increased to 100° C. After the reaction was over, a reaction solution was quenched by a saturated sodium carbonate aqueous solution (60 mL), and extracted with ethyl acetate (60 mL). Organic phases were washed with saturated saline (60 mL), dried over anhydrous sodium sulfate, and spin-dried to obtain crude. The crude was purified by a rapid silica gel column (petroleum ether:ethyl acetate=100:0 to 98.8:1.2) to obtain Compound 5-5.

[0127] LCMS: [MS+H.sup.+]=295.9.

Step 3: Synthesis of Compound 5-6

[0128] ##STR00033##

[0129] Compounds 2-1 (1.5 g, 5.45 mmol, 1 eq) and 5-5 (2.09 g, 7.09 mmol, 1.3 eq), potassium phosphate (3.47 g, 16.36 mmol, 3 eq) and Pd(dppf)Cl.sub.2.CH.sub.2Cl.sub.2 (1.34 g, 1.64 mmol, 0.3 eq) were dissolved in dimethoxyethane (60 mL), and stirred at 80° C. for 15 hours. After the reaction was over, a reaction solution was filtered directly, and filtrate was spin-dried to obtain crude. The crude was purified by a rapid silica gel column (petroleum ether:ethyl acetate=100:0 to 60:40) to obtain Compound 5-6.

[0130] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.46 (d, J=8.78 Hz, 1H), 8.12 (br s, 2H), 7.88 (d, J=8.78 Hz, 1H), 7.81 (dd, J=3.01, 9.03 Hz, 1H), 7.51 (dd, J=3.01, 9.03 Hz, 1H), 4.53 (q, J=7.03 Hz, 2H), 3.87-3.94 (m, 3H), 1.44 (t, J=7.15 Hz, 3H).

Step 4: Synthesis of Compound 5-7

[0131] ##STR00034##

[0132] The Compound 5-6 (890 mg, 2.45 mmol, 1 eq) was dissolved in ethyl acetate (100 mL), under nitrogen atmosphere, Pd/C (500 mg, 10% purity) was added, and a hydrogen balloon (15 psi) was replaced by gas for three times. The solution was stirred at 25° C. for 15 hours, and then stirred at 65° C. for 15 hours. After the reaction was over, a reaction solution was filtered by diatomaceous earth. Filtrate was spin-dried directly to obtain Compound 5-7.

[0133] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.54 (br s, 2H), 7.60-7.68 (m, 2H), 7.45 (dd, J=3.14, 9.66 Hz, 1H), 7.16 (d, J=8.78 Hz, 1H), 5.84 (br s, 2H), 4.42 (q, J=7.03 Hz, 2H), 3.90 (s, 3H), 1.45 (t, J=7.15 Hz, 3H).

Step 5: Synthesis of Compound 5-8

[0134] ##STR00035##

[0135] The Compound 5-7 (500 mg, 1.50 mmol, 1 eq) was dissolved in tetrahydrofuran (50 mL) and cooled to 0° C. Methyl magnesium bromide (3 M, 7.50 mL, 15 eq) was added dropwise in a 2-methyltetrahydrofuran solution at 0° C. The solution was reacted under stirring at 0° C. for 1.5 hours. After the reaction was over, a reaction solution was quenched by adding a saturated amine chloride aqueous solution (100 mL), and then extracted with ethyl acetate (100 mL*2). After organic phases were combined, the organic phases were dried over anhydrous sodium sulfate and spin-dried to obtain crude. The crude was purified by HPLC separation (column model: Boston Uni C18 40*150 mm*5 μm; mobile phase: [water (10 mM amine bicarbonate solution)-acetonitrile]; % B (acetonitrile): 28%-58%, 10 min) to obtain Compound 5-8.

[0136] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.30 (d, J=8.28 Hz, 1H), 6.99-7.10 (m, 2H), 6.89 (dd, J=2.89, 10.16 Hz, 1H), 1.71 (d, J=1.76 Hz, 12H).

Step 6: Synthesis of Compound 5

[0137] ##STR00036##

[0138] The Compound 5-8 (100 mg, 313.11 μmol, 1 eq) was dissolved in acetonitrile (5 mL), and then purified by separation with an HPLC machine (Column Model: Phenomenex Gemini-NX 150*30 mm*5 μm; mobile phase: [Water (0.04% amine aqueous solution+10 mM amine bicarbonate solution)-acetonitrile]; % B (acetonitrile): 39%-49%, 8 min) to obtain Compound 5.

[0139] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.36 (d, J=8.53 Hz, 1H), 7.05-7.13 (m, 2H), 6.89 (s, 2H), 6.84 (dd, J=3.01, 10.54 Hz, 1H), 5.72 (s, 2H), 5.55 (s, 1H), 5.47 (s, 1H), 1.53 (d, J=4.02 Hz, 12H).

Example 6: Synthesis of Compound 6

[0140] ##STR00037##

Synthetic Route of Compound 6

[0141] ##STR00038##

Step 1: Synthesis of Compound 6-2

[0142] ##STR00039##

[0143] The Compound 2-1 (10 g, 36.36 mmol, 1 eq) and benzyl alcohol (7.86 g, 72.71 mmol, 7.56 mL, 2 eq) were dissolved in acetonitrile (100 mL), and cesium carbonate (23.69 g, 72.71 mmol, 2 eq) was added at 20° C.; the mixed solution was stirred at 20° C. for 12 hours. After the reaction was over, a reaction solution was quenched in water (40 mL), and extracted with ethyl acetate (80 mL*3). After organic phases were combined, the organic phases were dried over anhydrous sodium sulfate, filtered, and spin-dried to obtain crude. The crude was purified by a rapid silica gel column (SiO.sub.2, petroleum ether/ethyl acetate=5:1 to 3:1) to obtain Compound 6-2.

[0144] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.39-8.33 (m, 1H), 7.48-7.38 (m, 5H), 6.99-6.93 (m, 1H), 5.52-5.48 (m, 2H), 4.57-4.48 (m, 2H), 1.48-1.41 (m, 3H).

Step 2: Synthesis of Compound 6-3

[0145] ##STR00040##

[0146] The Compound 6-2 (4.9 g, 16.21 mmol, 1 eq) was dissolved in ethanol (90 mL), under nitrogen atmosphere, palladium on carbon (Pd/C, 0.5 g, 16.21 mmol, 10% purity, 1 eq) was added, and a hydrogen balloon as replaced by gas for three times. The solution was stirred at 30° C. for 12 hours. After the reaction was over, a reaction solution was filtered, and filtrate was spin-dried to obtain crude. The crude was purified by a rapid silica gel column (SiO.sub.2, dichloromethane:methanol=10:0 to 10:1) to obtain Compound 6-3.

[0147] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 9.17 (br s, 1H), 7.10 (d, J=9.8 Hz, 1H), 6.73 (d, J=9.8 Hz, 1H), 5.41 (br s, 2H), 4.36 (q, J=7.0 Hz, 2H), 1.38 (t, J=7.2 Hz, 3H).

Step 3: Synthesis of Compound 6-5

[0148] ##STR00041##

[0149] The Compounds 6-4 (1 g, 5.34 mmol, 1 eq) and 6-3 (974.4 mg, 5.35 mmol, 1 eq) were dissolved in DMSO (20 mL), potassium phosphate (1.70 g, 8.00 mmol, 1.5 eq) was added, and the solution was stirred at 100° C. for 12 hours. After the reaction was over, a reaction solution was diluted in ethyl acetate (50 mL), and washed with water (30 mL*2) and saturated saline (30 mL) sequentially. Organic phases were dried over anhydrous sodium sulfate, filtered, and spin-dried to obtain crude. The crude was purified by a rapid silica gel column (SiO.sub.2, petroleum ether/ethyl acetate=5:1 to 2:1) to obtain Compound 6-5.

[0150] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.64 (d, J=11.8 Hz, 1H), 7.21-7.13 (m, 1H), 7.02-6.97 (m, 1H), 6.56 (d, J=6.8 Hz, 1H), 4.44-4.31 (m, 2H), 3.89 (s, 3H), 1.40 (t, J=7.2 Hz, 3H).

Step 4: Synthesis of Compound 6

[0151] ##STR00042##

[0152] The Compound 6-5 (200 mg, 572.55 μmol, 1 eq) was dissolved in tetrahydrofuran (40 mL) and cooled to 0° C. Methyl magnesium bromide (3 M, 5 mL, 26.20 eq) was added dropwise in a 2-methyltetrahydrofuran solution at 0° C. The solution was reacted under stirring at 0° C. for 1 hour, and then stirred at 15° C. for 15 hours. After the reaction was over, a reaction solution was quenched by adding a saturated amine chloride aqueous solution (50 mL), and then extracted in ethyl acetate (60 mL). The organic phases were dried over anhydrous sodium sulfate and spin-dried to obtain the crude. The crude was purified by HPLC separation (column model: Waters Xbridge 150*25 mm 5 μm; mobile phase: [water (10 mM amine bicarbonate solution)-acetonitrile]; % B (acetonitrile): 15%-45%, 10 min) to obtain Compound 2.

[0153] LCMS: [MS+H.sup.+]=336.0.

[0154] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.06 (d, J=8.28 Hz, 1H), 6.88 (d, J=13.05 Hz, 1H), 6.55 (d, J=8.28 Hz, 1H), 6.33 (d, J=7.53 Hz, 1H), 5.37 (s, 1H), 5.30 (br d, J=3.01 Hz, 4H), 5.26 (s, 1H), 1.48 (s, 6H), 1.38 (s, 6H).

Example 7: Synthesis of Compound 7

[0155] ##STR00043##

Synthetic Route of Compound 7

[0156] ##STR00044##

Step 1: Synthesis of Compound 7-2

[0157] ##STR00045##

[0158] The Compound 5-4 (3.65 g, 14.39 mmol, 2 eq), Pd(dppf)Cl.sub.2 (526.35 mg, 719.34 μmol, 0.1 eq), and potassium acetate (1.41 g, 14.39 mmol, 2 eq) were added in a toluene (30 mL) solution containing the Compound 7-1 (2 g, 7.19 mmol, 1 eq), and the mixed solution was reacted at 110° C. for 3 hours. After the reaction was over, a reaction solution was concentrated under reduced pressure to obtain crude 7-2.

Step 2: Synthesis of Compound 7-3

[0159] ##STR00046##

[0160] The Compound 3-1 (2.3 g, 7.07 mmol, 1 eq), cesium carbonate (4.61 g, 14.14 mmol, 2 eq), Pd(dppf)Cl.sub.2 (517.32 mg, 707.00 μmol, 0.1 eq) were added in a toluene (50 mL) and water (10 mL) solution containing the Compound 7-2 (1.63 g, 7.07 mmol, 1 eq), and the mixed solution was reacted at 110° C. under nitrogen atmosphere for 6 hours. After the reaction was over, a reaction solution was filtered with diatomaceous earth, and filtrate was concentrated under reduced pressure to obtain crude. The crude was separated by silica gel column chromatography (SiO.sub.2, petroleum ether/ethyl acetate=5:1 to 0:1) to obtain Compound 7-3.

[0161] LCMS: 350.1 [M+1].sup.+.

Step 3: Synthesis of Compound 7-4

[0162] ##STR00047##

[0163] Pd/C (1.7 g, 5% purity) was added in a methanol (50 mL) and ethyl acetate (50 mL) solution containing the Compound 7-3 (1.58 g, 4.52 mmol, 1 eq), and the mixed solution was reacted at 15 psi under hydrogen atmosphere at 20° C. for 3 hours. After the reaction was over, a reaction solution was filtered with diatomaceous earth, and filtrate was concentrated under reduced pressure to obtain Compound 7-4.

[0164] LCMS: 320 [M+1].sup.+.

[0165] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 7.96 (t, J=1.76 Hz, 1H), 7.54 (d, J=11.84 Hz, 1H), 7.39 (s, 1H), 6.95 (d, J=6.58 Hz, 1H), 6.82 (s, 2H), 6.66 (s, 2H), 3.83 (d, J=3.96 Hz, 6H).

Step 4: Synthesis of Compound 7

[0166] ##STR00048##

[0167] Methyl magnesium bromide (3M, 31.32 mL, 20 eq) was added in a tetrahydrofuran (150 mL) solution containing the Compound 7-4 (1.5 g, 4.70 mmol, 1 eq) at 0° C., and the mixed solution was reacted at 20° C. for 3 hours. After the reaction was over, a reaction solution was poured into a saturated amine chloride solution, 50 mL of ethyl acetate was added for liquid separation, and an aqueous phase was extracted with ethyl acetate (50 mL*3); organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain crude. The crude was separated by HPLC (column model: Xtimate C18 150*25 mm*5 μm; mobile phase: [water (10 mM amine bicarbonate solution)-acetonitrile]; % B (acetonitrile): 25%-45%, 10.5 min) to obtain Compound 7.

[0168] LCMS: 320.0 [M+1].sup.+;

[0169] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 7.77 (s, 1H), 7.05 (s, 1H), 6.91 (d, J=12.28 Hz, 1H), 6.67 (d, J=7.46 Hz, 1H), 5.63 (s, 2H), 5.27-5.48 (m, 4H), 1.51 (s, 12H)

Example 8: Synthesis of Compound 8

[0170] ##STR00049##

Synthetic Route of Compound 8

[0171] ##STR00050##

Step 1: Synthesis of Compound 8-1

[0172] ##STR00051##

[0173] The Compound 3-4 (1 g, 3.32 mmol, 1 eq) and NCS (509.67 mg, 3.82 mmol, 1.15 eq) were dissolved in glacial acetic acid (60 mL), and stirred at 25° C. for 15 hours. After the reaction was over, a reaction solution was adjusted to pH about 9 with a saturated sodium carbonate aqueous solution (200 mL), and extracted with ethyl acetate (150 mL). The organic phase was washed once with saturated saline (50 mL), then dried over anhydrous sodium sulfate, and spin-dried to obtain crude. The crude was purified by a rapid silica gel column (petroleum ether:ethyl acetate=100:0 to 70:30) to obtain Compound 8-1.

[0174] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.10 (d, J=1.76 Hz, 1H), 7.97 (s, 1H), 7.12 (d, J=1.76 Hz, 1H), 6.63 (s, 1H), 5.83 (br s, 4H), 4.01 (s, 3H), 3.91 (s, 3H).

Step 2: Synthesis of Compound 8

[0175] ##STR00052##

[0176] The Compound 8-1 (265 mg, 789.30 μmol, 1 eq) was dissolved in tetrahydrofuran (50 mL) and cooled to 0° C. Methyl magnesium bromide (3 M, 4 mL, 15.20 eq) was added dropwise in a 2-methyltetrahydrofuran solution at 0° C. The solution was reacted under stirring at 0° C. for 2 hours, and then stirred at 25° C. for 15 hours. After the reaction was over, a reaction solution was quenched by adding a saturated amine chloride aqueous solution (50 mL), and then extracted with ethyl acetate (60 mL*2). After organic phases were combined, the organic phases were dried over anhydrous sodium sulfate and spin-dried to obtain crude. The crude was purified by HPLC separation (column model: waters Xbridge 150*25 mm*5 μm; mobile phase: [water (10 mM amine bicarbonate solution)-acetonitrile]; % B (acetonitrile):25%-48%, 7.8 min) to obtain Compound 8.

[0177] LCMS: [MS+H+]=336.0;

[0178] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.66 (d, J=2.01 Hz, 1H), 7.07 (s, 1H), 6.96 (d, J=2.01 Hz, 1H), 6.61 (s, 1H), 5.61 (br d, J=17.32Hz, 4H), 5.47 (s, 1H), 5.34 (s, 1H), 1.51 (s, 12H).

Example 9: Synthesis of Compound 9

[0179] ##STR00053##

Synthetic Route of Compound 9

[0180] ##STR00054## ##STR00055##

Step 1: Synthesis of Compound 9-2

[0181] ##STR00056##

[0182] A Compound 9-1 (24.8 g, 120.37 mmol, 1 eq) and benzylamine (15.48 g, 144.45 mmol, 15.75 mL, 1.2 eq) were dissolved in DMF (200 mL), and triethylamine (36.54 g, 361.12 mmol, 50.26 mL, 3 eq) was added at 20° C. A reaction solution was stirred at 20° C. for 21 hours. After the reaction was complete, a reaction solution was quenched by adding water (250 mL), and then extracted with ethyl acetate (500 mL*2). Organic phases were spin-dried. Products were purified by a rapid silica gel column (SiO.sub.2, petroleum ether/ethyl acetate=5:1 to 3:1) to obtain Compound 9-2.

[0183] LCMS (ESI): [M+H].sup.+: 277.1.

[0184] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.71 (s, 1H), 8.55 (br s, 1H), 7.36-7.43 (m, 2H), 7.29-7.35 (m, 3H), 6.56 (s, 1H), 4.44 (d, J=5.52 Hz, 2H), 3.88-3.92 (m, 3H).

Step 2: Synthesis of Compound 9-3

[0185] ##STR00057##

[0186] The Compound 9-2 (5 g, 18.07 mmol, 1 eq) and sodium methoxide (9.76 g, 180.69 mmol, 10 eq) were dissolved in MeOH (50 mL), and stirred at 90° C. under nitrogen atmosphere for 15 hours. After the reaction was over, products were purified by a rapid silica gel column (SiO.sub.2, petroleum ether/ethyl acetate=5:1 to 3:1) to obtain Compound 9-3.

[0187] LCMS (ESI): [M+H]+: 273.

Step 3: Synthesis of Compound 9-4

[0188] ##STR00058##

[0189] The Compound 9-3 (3.26 g, 11.97 mmol, 1 eq), trimethylchlorosilane (5.20 g, 47.89 mmol, 6.08 mL, 4 eq) and NaI (7.18 g, 47.89 mmol, 4 eq) were dissolved in acetonitrile (50 mL), and then reacted under stirring at 80° C. under nitrogen atmosphere for 15 hours. After the reaction was over, a reaction solution was adjusted to PH=7 with sodium bicarbonate, then quenched with water (20 mL), and extracted twice with dichloromethane (100 mL); finally organic phases were combined, and spin-dried in a rotary evaporator. Crude was purified with a rapid silica gel column (SiO.sub.2, petroleum ether/ethyl acetate=5/1 to 3:1) to obtain Compound 9-4.

[0190] LCMS (ESI): [M+H].sup.+: 259.1

[0191] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.47 (br s, 1H), 6.47-6.58 (m, 5H), 4.65 (s, 1H), 3.55 (br d, J=5.27 Hz, 2H), 3.00-3.08 (m, 3H)

Step 4: Synthesis of Compound 9-6

[0192] ##STR00059##

[0193] The Compound 9-4 (2.29 g, 8.87 mmol, 1 eq), the Compound 9-5 (2.77 g, 10.64 mmol, 1.2 eq), cuprous iodide (337.73 mg, 1.77 mmol, 0.2 eq), potassium carbonate (2.45 g, 17.73 mmol, 2 eq) and (1S, 2S)-N1, N2-dimethylcyclohexane-1,2-diamine (252.24 mg, 1.77 mmol, 0.2 eq) were dissolved in toluene (30 mL), and reacted under stirring at 110° C. under nitrogen atmosphere for 13 hours. After the reaction was over, the solution was filtered and spin-dried in the rotary evaporator. The crude was purified by a rapid silica gel column (SiO.sub.2, petroleum ether/ethyl acetate=1:1 to 0:1) to obtain Compound 9-6.

[0194] LCMS (ESI): [M+H].sup.+: 438.1

Step 5: Synthesis of Compound 9-7

[0195] ##STR00060##

[0196] The Compound 9-6 (1 g, 2.29 mmol, 1 eq) was dissolved in tetrahydrofuran (30 mL), and palladium on carbon (1.5 g, 10% purity) was added in the mixed solution under nitrogen atmosphere. The mixed solution was stirred for 12 hours at 65° C. under hydrogen atmosphere. After the reaction was over, a reaction mixture was filtered and spin-dried. The crude was purified by a rapid silica gel column (SiO.sub.2, petroleum ether/ethyl acetate=1:1 to 0:1) to obtain Compound 9-7.

[0197] LCMS (ESI): [M+H].sup.+: 318.1

[0198] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.12 (s, 1H), 7.76 (d, J=8.53 Hz, 1H), 7.06 (br s, 2H), 6.84 (s, 2H), 6.77 (d, J=1.76 Hz, 1H), 6.51 (dd, J=2.01, 8.53 Hz, 1H), 5.42 (s, 1H), 3.81 (s, 3H), 3.74 (s, 3H)

Step 6: Synthesis of Compound 9

[0199] ##STR00061##

[0200] The Compound 9-7 (200 mg, 630.33 μmol, 1 eq) was dissolved in tetrahydrofuran (20 mL); under nitrogen atmosphere, a reaction solution was cooled to −78° C. in a dry ice-ethanol bath, and lithium methyl (1.6 M, 5.91 mL, 15 eq) was slowly added dropwise. The reaction solution was slowly heated to 0° C. thereafter and stirred for 30 min. After the reaction was over, a reaction solution was quenched in water (10 mL) at 0° C., diluted with water (5 mL) and extracted with THF (40 mL). After organic phases were combined, the organic phases were dried over anhydrous sodium sulfate, filtered, and spin-dried to obtain Compound 9.

[0201] LCMS (ESI): [M+H].sup.+: 318.

[0202] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 6.20 (d, J=8.28 Hz, 1H), 6.14 (s, 1H), 5.69 (d, J=2.26 Hz, 1H), 5.53 (dd, J=2.13, 8.16 Hz, 1H), 5.45 (s, 2H), 4.73 (s, 2H), 4.57 (s, 1H), 4.53 (s, 1H), 4.41 (s, 1H), 0.67 (s, 6H), 0.60 (s, 6H).

Experimental Example 1: In Vitro Aldehyde Capture Capacity Test

1. Experiment Objective and Process

[0203] Objective: Xerophthalmia is caused by inflammation in eyes, wherein the inflammation may produce aldehydes that will accelerate the inflammation symptoms and worsen the xerophthalmia if not eliminated in time. In the experiment, a better compound was selected according to the complexing capacity of the drug and aldehydes in vivo by simulating the in vivo environment.

[0204] Procedures: Sulfobutyl-B-cyclodextrin (310 mg) was dissolved in a phosphate buffer (1.25 mL) to prepare a solution.

[0205] Nonanal (5.0 mg, 32 μmol, 1.0 eq) and triolein (300 mg) were added to a reaction flask at room temperature, after addition of the prepared solution described above, linoleic acid (300 mg) was added, and finally a dimethyl sulfoxide (0.15 ml) solution containing the compound (32 μmol, 1.0 eq) of the present disclosure was added; the reaction solution was reacted at 20-23° C.

[0206] The reaction solution was reacted under stirring for 10 min, 100 min, 200 min, and 300 min respectively, and sampled for high performance liquid chromatography after the solution was stratified after standing for 2 min.

[0207] Sampling method: 25 μl of an upper emulsified layer and 50 μl of a lower aqueous phase were sampled by a pipette, and samples were diluted with 1 mL of methanol.

2. Test Results

[0208] The ultraviolet absorption of the nonanal was weak at 254 nm, so the nonanal had little overall effect on the content of complexed products. Therefore, the percentage contents of complexes at 254 nm under high performance liquid chromatography were compared to observe aldehyde capture and complexing capacities, as shown in FIG. 1 and Table 1:

##STR00062##

TABLE-US-00001 TABLE 1 Statistics of Test Results of Aldehyde Capture Capacity of the Compound Area under the Regression curve AUC Coefficient (min. Compound Linear Equation Slope R.sup.2 Conversion) Compound 1 y = 0.0157x + 1.718 0.0157 R.sup.2 = 0.7521 1257 Compound 2 y = 0.0103x + 2.7459 0.0103 R.sup.2 = 0.9712 1271 Compound 3 y = 0.0652x + 3.362 0.0652 R.sup.2 = 0.9891 3979 Compound 4 y = 0.0589x + 1.3064 0.0589 R.sup.2 = 0.9964 3061 Compound 5 y = 0.0271x + 0.1945 0.0271 R.sup.2 = 0.9326 1208 Compound 6 y = 0.1083x + 12.77 0.1083 R.sup.2 = 0.9027 8953 Compound 7 y = 0.1002x + 7.7455 0.1002 R.sup.2 = 0.9686 6944 Compound 8 y = 0.0519x + 4.9828 0.0519 R.sup.2 = 0.9535 3879 Compound 9 y = 0.1241x + 14.964 0.1241 R.sup.2 = 0.9299 10284

[0209] The HPLC method is as follows: 2.5 μm, 3.0*100 mm 5-95 CD_XBEH_12 min_0.8.1 cm or XBRIGE 2.5 μm, 3.0*100 mm 5-80 CD_XBEH_12 min_0.8.1 cm

[0210] Specific conditions: XBRIGE 2.5 μm, 3.0*100 mm 5-80 CD_XBEH_12 min_0.8.1 cm

TABLE-US-00002 Column XBridge BEHC18 3.0*100 mm, 2.5 μm Detection Wavelength 220, 254 nm Column Temperature 40° C. Flow Rate 0.8 mL/min Injection Volume 1 μL Mobile Phase A: 0.02% aqueous amine solution B: Acetonitrile Time (min) A % B % Gradient elution 0.01 95 5 6.00 40 60 9.00 20 80 12.00 20 80

XBRIGE 2.5 μm, 3.0*100 mm 5-95 CD_XBEH_12 min_0.8.1 cm

[0211]

TABLE-US-00003 Column XBridge BEHC18 3.0*100 mm, 2.5 μm Detection Wavelength 220, 254nm Column Temperature 40° C. Flow Rate 0.8 mL/min Injection Volume 1 μL Mobile Phase A: 0.02% aqueous amine solution B: Acetonitrile Time (min) A % B % Gradient elution 0.01 95 5 6.00 15 85 9.00 5 95 12.00 5 95

[0212] Taking The compound 7 as an example, after 300 min of reaction, the analytical method for HPLC was XBRIGE 2.5 μm, 3.0*100mm 5-80 CD_XBEH_12 min_0.8.1 cm;

[0213] Retention time 6.689 min, 6.787 min, 6.966 min, and 7.102 min were absorption peaks of products complexed with monomolecular aldehyde;

[0214] Retention time 8.905 min, 9.010 min, and 9.075 min were absorption peaks of products complexed with bimolecular aldehyde.

[0215] The percentage content of the specific complexed product at 254 nm was calculated as the sum of the percentage contents of the absorption peaks for the retention time described above, i.e. (1.839+1.715+14.993+13.029)%+(1.004+2.212+1.247)%=36.039%

[0216] Specific HPLC data of the percentage contents of complexed products of the compounds of the present disclosure are shown in Table 2 below:

TABLE-US-00004 Time (min) Compound 10 100 200 300 1 0.917 4.909 4.383 6.224 4 1.475 7.663 13.353 18.636 2 2.658 4.097 4.721 5.796 3 3.265 10.569 17.191 22.182 5 0.882 2.935 4.328 9.139 7 6.634 20.013 29.424 36.039 6 9.738 27.850 37.847 41.686 8 4.117 12.121 15.49 19.873 9 12.268 31.427 43.086 48.761

[0217] Conclusion: Experiment results showed that the compounds of the present disclosure had far significant aldehydes complexing capability and speed.

Experimental Example 2: In Vitro Evaluation

[0218] Objective: To study the inhibitory effect of compounds on human liver microsomal cytochrome P450.

[0219] Experimental procedures: In the study on inhibition of the compounds on the human liver microsomal cytochrome P450 (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4), fused human liver microsomes were selected as a CYP450 enzyme source. The compounds of different concentrations (10, 5, 1.5, 0.5, 0.15, 0.05, 0.015 mM) were incubated with probe substrates (where CYP3A4 employs two substrates) and cofactors (NADPH) of 5 CYP enzymes to determine the IC.sub.50 value of each compound for inhibition of each CYP enzyme. Test results are as follows:

TABLE-US-00005 TABLE 3 Inhibition Test Results of Compounds of the Present Disclosure on Human Liver Microsomal Cytochrome P450 Isoenzymes Compound No. CYP1A2/CYP2C9/CYP2C19/CYP2D6/CYP3A4(04) Compound 3 >50/>50/>50/>50/>50 Compound 7 >50/>50/>50/>50/>50 Compound 8 >50/>50/>50/>50/>50

[0220] Conclusion: The present disclosure has high safety and low possibility of drug interaction.

Experimental Example 3: Pharmacokinetic Evaluation of Compound

I

[0221] Objective: To study 5-Day toxicology of compounds in SD rats

[0222] Experimental materials: SD rats (male, 200-300 g, 7-9 weeks old, Shanghai Lingchang)

Experimental Procedures

[0223] Pharmacokinetic profiles of the compound were tested by intravenous injection in rodents according to the standard protocol. In the study, the candidate compound was prepared as a clear solution, and was administered to SD rats at 20 mg/kg by single intravenous injection for 5 consecutive days. A vehicle for intravenous injection was a 10% hydroxypropyl β-cyclodextrin aqueous solution. Whole blood samples were collected at 0.033, 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 hours postdose on Day 1 and Day 5 of dosing, centrifuged at 3000 r for 10 min, and separated from supernatant to obtain plasma samples. 15 μL of plasma samples were mixed with 300 μL of acetonitrile precipitated proteins containing an internal standard, and 2 μL of the supernatant was injected by centrifugation. Plasma concentrations were quantified by LC-MS/MS analysis, and pharmacokinetic parameters, such as clearance, half-life, and area under the concentration-time curve, were calculated.

Experiment Results

[0224]

TABLE-US-00006 TABLE 4 Pharmacokinetic Test Results Area under the concentration-time Clearance Half-time curve Test (mL/min/kg) T.sub.1/2 (h) AUC(nM .Math. hr) Sample Day 1 Day 5 Day 1 Day 5 Day 1 Day 5 Compound 3 7.11 9.04 2.45 2.72 155784 123277

[0225] Conclusion: The compound of the present disclosure has no risk of drug accumulation after injection for 5 consecutive days.

II

[0226] Objective: To study pharmacokinetics of the compound in Beagle dogs

[0227] Experimental material: Beagle dogs (male, 8-11 kg, greater than or equal to 6 months, Marshall)

Experimental Procedures

[0228] Pharmacokinetic profiles of the compound were tested by intravenous administration in rodents according to the standard protocol. In the study, the candidate compound was prepared as a clear solution, and was administered to Beagle dogs at 1 mg/kg by single intravenous injection. A vehicle for intravenous injection was a 10% hydroxypropyl β-cyclodextrin aqueous solution. Whole blood samples were collected at 0.033, 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours, respectively, centrifuged at 3000 r for 10 min, and separated from supernatant to obtain plasma samples. 20 μL of plasma samples were mixed with 400 μL of acetonitrile precipitated proteins containing an internal standard, and 2 μL of the supernatant was injected by centrifugation. Plasma concentrations were quantified by LC-MS/MS analysis, and pharmacokinetic parameters, such as clearance, half-life, and area under the concentration-time curve, etc. were calculated.

Experiment Results

[0229]

TABLE-US-00007 TABLE 5 Pharmacokinetic Test Results Area under the concentration-time Clearance Half-time curve Test Sample (mL/min/kg) T.sub.1/2 (h) AUC(nM .Math. hr) Compound 3 15 2.17 3709 Compound 7 11.3 2.94 4672

[0230] Conclusion: The present disclosure has high clearance, moderate half-life and good pharmacokinetic profile.

III

[0231] Objective: To study pharmacokinetics of the compound in cynomolgus monkeys

[0232] Experimental material: Cynomolgus monkeys (male, 2.5-4 kg, greater than or equal to 2 years, Jingang Biotech)

Experimental Procedures

[0233] Pharmacokinetic profiles of the compound were tested by intravenous administration in monkeys according to the standard protocol. In the study, the candidate compound was prepared as a clear solution, and was administered to cynomolgus monkeys at 1 mg/kg by single intravenous injection. A vehicle for intravenous injection was a 10% hydroxypropyl β-cyclodextrin aqueous solution. Whole blood samples were collected at 0.033, 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours, respectively, centrifuged at 3000 r for 10 min, and separated from supernatant to obtain plasma samples. 20 μL of plasma samples were mixed with 400 μL of acetonitrile precipitated proteins containing an internal standard, and 2 μL of the supernatant was injected by centrifugation. Plasma concentrations were quantified by LC-MS/MS analysis, and pharmacokinetic parameters, such as clearance, half-life, and area under the concentration-time curve, etc. were calculated.

Experiment Results

[0234]

TABLE-US-00008 TABLE 6 Pharmacokinetic Test Results Area under the concentration-time Clearance Half-time curve Test Sample (mL/min/kg) T.sub.1/2 (h) AUC(nM .Math. hr) Compound 3 6.2 3.81 9164 Compound 7 4.6 3.91 13016

[0235] Conclusion: The present disclosure has high clearance, moderate half-life and good pharmacokinetic profile.

Experimental Example 4: In Vivo Pharmacokinetic Study

[0236] Objective: To study the ratio of corneal to plasma drug concentration after eye drops in rats

[0237] Experimental materials: Male SD rats, 200-300 g, 7-9 weeks old, Shanghai Lingchang

[0238] Experimental procedures: The candidate compound was prepared as a clear solution, and administered to SD rats by eye drops. The vehicle for eye drops was 10% hydroxypropyl β-cyclodextrin aqueous solution, and the drug concentration in the eye drops was 5 mg/mL. Corneal and whole blood samples were collected at 1 and 4 hours, respectively. The corneal samples was homogenized in 15 mM phosphate buffer (PBS): MeoH (2:1, v:v) buffer, the whole blood samples were centrifuged at 3000 r for 10 min, and the supernatant plasma samples were separated. 20 μL of the plasma samples and homogenate samples were mixed with 400 μL of acetonitrile precipitated proteins containing an internal standard, respectively, and 2 μL of the supernatant was injected by centrifugation. The plasma concentrations were quantified by LC-MS/MS analysis; the drug concentrations in the cornea and plasma were measured respectively according to different time points, and the ratio of corneal/plasma concentrations was calculated.

Experiment Results

[0239]

TABLE-US-00009 TABLE 7 Pharmacokinetic Test Results Corneal Concentration/ Corneal Plasma Plasma Concentration Concentration Concentration Test Sample Time (nM) (nM) C/P ratio Compound 3 1 h 1796 162 14.3 4 h 581 43.8 16.6 Compound 7 1 h 2447 172 11.8 4 h 330 24.4 12.4 Compound 8 1 h 1663 148 12.0 4 h 167 36.4 3.72

[0240] Conclusion: As shown in the DMPK test, test compounds 3, 7 and 8 could enter the cornea by eye drops to show the efficacy. The ratio of cornea/plasma concentrations of the Compound 3 was relatively high (above 10). The drug took effect in the cornea, so the targeting property was good.

Experimental Example 5: In Vivo Efficacy Evaluation

Objective

[0241] Subcutaneous injection of scopolamine may induce the xerophthalmia in mice. Decreased tear secretion and inflammatory infiltration can be observed by tear tests and corneal fluorescence staining scores. It can predict whether a model can achieve the expected severity at the early stage of modeling.

Study Design

[0242] Twenty animals were selected from 25 female C57BL/6J mice and randomized into 4 groups by body weight via Provantis or Excel, 4 animals per group, and group assignment may also refer to the results of the tear test and the corneal fluorescence staining score of each animal prior to the experiment.

[0243] The mice were injected subcutaneously with scopolamine hydrobromide (3±0.5 h) for 4 times daily on Day 1 to Day 12 of the study to establish xerophthalmia models in the mice at 0.1 mL/animal/time.

[0244] On Day 1 to Day 13 of the study, animals were dosed by bilateral eye drops for 4 times daily (3±0.5 h) at 3 μL/eye (on Day 13, animals in Group 1 were not dosed). Each subcutaneous injection of the scopolamine hydrobromide was be given predose the eye drops (except examinations on Day 7 and Day 12).

[0245] Tear tests and corneal fluorescence staining scores were performed on all experimental animals on Day 7 and Day 12 prior to modeling.

[0246] Criteria for corneal fluorescence staining scores: The cornea of an animal was divided into 5 regions, i.e., superior, inferior, nasal, temporal and central regions, each with 0-3 scores. Each region was scored, and the monocular score was the sum of scores of the 5 regions. 0: no staining; 1: slight staining, punctate staining with less than 5 spots; 2: moderate staining, punctate staining but no plaque staining; 3: severe staining, marked fluorescent plaques.

[0247] The tear test was performed 30 min after the second dosing on Day 7 and Day 12 for all experimental animals, and the corneal fluorescence staining was scored for all experimental animals 30 min after the third dosing on Day 7 and Day 12.

TABLE-US-00010 TABLE 8 Efficacy Experiment Protocol of Scopolamine by Subcutaneous Injection in Mice Number of animals Therapy Method of Group (Female) Modeling Both eyes Administration Vehicles 4 Subcutaneous Vehicles Eye drops Compound 3 4 injection of 0.5% 3 μl/eye Compound 7 4 scopolamine for 4 0.5% 4 injections/day Compound 8 4 injections/day 0.3% (3 ± 0.5 h) for 12 days (5 mg/mL, 0.1 mL/injection, 3 ± 0.5 h)

[0248] Test results: See FIG. 2 and FIG. 3 (Note:* in the attached drawings indicates P≤0.5, ** indicates P≤0.01, and *** indicates P≤0.001).

[0249] Conclusion: Under the conditions of the experiment, it can be seen that all tested compounds can improve tear secretion and corneal inflammation, showing the efficacy of improving the xerophthalmia.