COMPOUND AS HIGHLY SELECTIVE ROS1 INHIBITOR AND USE THEREOF

Abstract

Disclosed are a class of compounds having a highly selective inhibition of ROS1, and the use thereof in the preparation of drugs for treating diseases related to abnormal ROS1 kinase expression. Specifically disclosed are compounds represented by formula (IV) and a pharmaceutically acceptable salt thereof.

##STR00001##

Claims

1. A compound represented by formula (IV) or a pharmaceutically acceptable salt thereof, ##STR00039## wherein, R.sub.1 is 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.a; R.sub.2 is selected from H and C.sub.1-3 alkyl optionally substituted with 1, 2 or 3 R.sub.b; L.sub.1 is selected from —C.sub.1-3 alkyl-, —C.sub.3-6 cycloalkyl- and —C.sub.3-6 cycloalkyl-C.sub.1-3 alkyl-, and the —C.sub.1-3 alkyl-, —C.sub.3-6 cycloalkyl- and —C.sub.3-6 cycloalkyl-C.sub.1-3 alkyl- are optionally substituted with 1, 2 or 3 R.sub.c; L.sub.2 is selected from —C.sub.1-3 alkyl- and —O—; R.sub.a, R.sub.b and R.sub.c are each independently selected from F, Cl, Br and CH.sub.3.

2. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein R.sub.1 is selected from H, F, Cl, Br, I, OH, NH.sub.2, CN and CH.sub.3, and the CH.sub.3 is optionally substituted with 1, 2 or 3 R.sub.a.

3. The compound or the pharmaceutically acceptable salt thereof according to claim 2, wherein R.sub.1 is selected from F.

4. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein R.sub.2 is selected from H and CH.sub.3.

5. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein L.sub.1 is selected from —CH.sub.2—, —CH(CH.sub.3)CH.sub.2—,-cyclopropyl-CH.sub.2— and -cyclobutyl-, and the —CH.sub.2—, —CH(CH.sub.3)CH.sub.2—, -cyclopropyl-CH.sub.2— and -cyclobutyl-are optionally substituted with 1, 2 or 3 R.sub.c.

6. The compound or the pharmaceutically acceptable salt thereof according to claim 5, wherein L.sub.1 is selected from —CH.sub.2—, —CH(CH.sub.3)CH.sub.2—, ##STR00040##

7. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein L.sub.2 is selected from —CH.sub.2— and —O—.

8. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the structural unit ##STR00041## is selected from and ##STR00042##

9. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the structural unit ##STR00043## is selected from ##STR00044##

10. The compound or the pharmaceutically acceptable salt thereof according to claim 1, selecting from: ##STR00045## wherein, R.sub.1 and L.sub.1 are defined as above.

11. A compound represented by the following formula or a pharmaceutically acceptable salt thereof, selecting from: ##STR00046##

12. The compound or the pharmaceutically acceptable salt thereof according to claim 11, selecting from: ##STR00047##

13. A compound represented by formula (V) and a compound represented by formula (VI), ##STR00048## wherein, R.sub.1 is 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.a; R.sub.2 is selected from H and C.sub.1-3 alkyl optionally substituted with 1, 2 or 3 R.sub.b; R.sub.3 is selected from OH and O—C.sub.1-3 alkyl; L.sub.1 is selected from —C.sub.1-3 alkyl-, —C.sub.3-6 cycloalkyl- and —C.sub.3-6 cycloalkyl-C.sub.1-3 alkyl-, and the —C.sub.1-3 alkyl-, —C.sub.3-6 cycloalkyl- and —C.sub.3-6 cycloalkyl-C.sub.1-3 alkyl- are optionally substituted with 1, 2 or 3 R.sub.c; R.sub.a, R.sub.b and R.sub.c are each independently selected from F, Cl, Br and CH.sub.3.

14. A preparation method of the compound according to claim 1, wherein the method comprises the following reaction routes: ##STR00049## or preparing the compound represented by formula (VI) from the compound represented by formula (V) under the condition of alkali and solvent A, and then preparing the compound represented by formula (IV) from the compound represented by formula (VI) under the condition of ligand and solvent B; ##STR00050## wherein, R.sub.1 is 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.a; R.sub.2 is selected from H and C.sub.1-3 alkyl optionally substituted with 1, 2 or 3 R.sub.b; R.sub.3 is selected from OH and O—C.sub.1-3 alkyl; L.sub.1 is selected from —C.sub.1-3 alkyl-, —C.sub.3-6 cycloalkyl- and —C.sub.3-6 cycloalkyl-C.sub.1-3 alkyl-, and the —C.sub.1-3 alkyl-, —C.sub.3-6 cycloalkyl- and —C.sub.3-6 cycloalkyl-C.sub.1-3 alkyl- are optionally substituted with 1, 2 or 3 R.sub.c; R.sub.a, R.sub.b and R.sub.c are each independently selected from F, Cl, Br and CH.sub.3; the alkali is selected from lithium hydroxide, sodium hydroxide, potassium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, calcium carbonate, magnesium carbonate, lithium bicarbonate, sodium bicarbonate, potassium bicarbonate, calcium bicarbonate, magnesium bicarbonate, sodium formate, potassium propionate, trimethylamine, triethylamine, pyridine, 4-dimethylaminopyridine or N-ethyldiisopropylamine; the solvent A is selected from N,N-dimethylformamide, acetonitrile, dichloromethane, dimethyl sulfoxide, N-methylpyrrolidone and N,N-dimethylacetamide; the ligand is selected from triphenylphosphine, trimethylphenylphosphine, tricyclohexylphosphine, tri-tert-butylphosphine, tributylphosphine and diethylphenylphosphine; the solvent B is selected from toluene, xylene, ethylene glycol dimethyl ether, dioxane, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide and N-methylpyrrolidone.

15. A method for inhibiting ROS1 in a subject in need thereof, comprising administering the compound or the pharmaceutically acceptable salt thereof according to claim 1 to the subject.

16. A method for treating cancer in a subject in need thereof, comprising administering the compound or the pharmaceutically acceptable salt thereof according to claim 1 to the subject.

17. A method for treating lung cancer in a subject in need thereof, comprising administering the compound or the pharmaceutically acceptable salt thereof according to claim 1 to the subject.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0129] FIG. 1: Ba/F3 CD74-ROS1-WT subcutaneous xenograft tumor model; and Vehicle represents solvent control group.

[0130] FIG. 2: Human-derived lung cancer LD1-0025-361019 PDX animal model; Vehicle control represents solvent control group.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0131] The present disclosure will be specifically described below by way of embodiments, but the scope of the present disclosure is not limited thereto. The present disclosure is described in detail herein, and specific embodiments thereof have also been disclosed. For those skilled in the art, it is obvious that various changes and improvements can be made to the specific embodiments of the present disclosure without departing from the spirit and scope of the present disclosure.

Reference Embodiment 2: Synthesis of Intermediate A-2

[0132] ##STR00034##

[0133] Step 1: Synthesis of Compound A-2-2

[0134] In a 5 L three-necked flask, under stirring, compound A-2-1 (170 g, 825.19 mmol, 1 eq) was added to a mixed solvent of N,N-dimethylformamide (850 mL) and water (340 mL), and then potassium carbonate (171.07 g, 1.24 mol, 1.5 eq), tetrabutylammonium acetate (497.61 g, 1.65 mol, 2 eq) and 3,3-dimethoxypropylene (252.83 g, 2.48 mol, 3 eq) were added sequentially. After nitrogen replacement for three times, palladium acetate (2.55 g, 11.36 mmol) was added, and the mixture was slowly heated to 95° C. in a nitrogen atmosphere (the heating time was 1.5 hours), stirred and reacted at this temperature for 16 hours. The reaction solution was cooled to 5° C., then the aqueous hydrochloric acid solution (4 M, 860 mL, 4.17 eq) was added dropwise, and an internal temperature was controlled at 10 to 20° C. (the dropwise addition time was 25 minutes); the mixture was stirred and reacted continuously at 20 to 25° C. for 30 minutes after the dropwise addition. Sodium bicarbonate was slowly added into the reaction flask, and the pH was adjusted to 6-7. After stirring for 20 minutes, the reaction solution was extracted with methyl tert-butyl ether for three times (the volume of the first and second extraction was 1360 mL, and the volume of the third extraction was 850 mL). All organic phases were combined, washed twice with saturated sodium chloride solution (850 mL each time), dried with anhydrous sodium sulfate (170 g), filtered under reduced pressure, and a filtrate was concentrated under vacuum at 45 to 50° C. to obtain a concentrate. The concentrate was slurried with a mixed solvent of n-heptane and methyl tert-butyl ether (510 mL; a volume ratio of the n-heptane to the methyl tert-butyl ether was 5.6:1) for 10 minutes, filtered, and the filter cake was dried under vacuum to obtain a crude product. (R)-diphenylprolinol trimethylsilyl ether (135.12 g, 415.09 mmol, 0.2 eq) was dissolved in dichloromethane (1500 mL), and the mixture was cooled to 0 to 5° C. The above crude product and benzoic acid (50.69 g, 415.09 mmol, 0.2 eq) were added under stirring, and the mixture was stirred at 0 to 5° C. for 0.5 hours. The temperature was then cooled to −5 to −7° C., and benzyl N-hydroxycarbamate (416.33 g, 2.49 mol, 1.2 eq) was added, and the mixture was stirred and reacted continuously at -5 to 10° C. for 4 hours. After the reaction was completed, the reaction solution was cooled to 0 to 5° C. and filtered. The filter cake was washed with dichloromethane (500 mL), and the filter cake was dried under vacuum at 45° C. for 16 hours to obtain compound A-2-2. .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 7.98 (d, J=2.8 Hz, 1H), 7.57 (dd, J=2.8, 8.2 Hz, 1H), 7.46-7.37 (m, 5H), 5.86 (d, J=4.6 Hz, 1H), 5.62 (t, J=7.8 Hz, 1H), 5.30 (q, J=12.3 Hz, 2H), 4.02 (s, 3H), 3.01 (dd, J=8.5, 12.9 Hz, 1H), 2.21-2.13 (m, 1H); LCMS m/z=349.2 [M+H].sup.+.

[0135] Step 2: Synthesis of Compound A-2-3

[0136] Compound A-2-2 (50 g, 143.54 mmol, 1 eq) was added to methanol (250 mL), and the mixture was stirred, cooled to 0° C.; and sodium borohydride (6.79 g, 179.43 mmol, 1.25 eq) was added in batches, then the internal temperature was controlled at 0 to 10° C.; the mixture was stirred and reacted at 20° C. for 60 minutes after the addition was completed. After the reaction was completed, 100 mL of saturated ammonium chloride solution was added to the reaction solution to quench the reaction, then 50 mL of water was added, and the reaction solution was extracted with methyl tert-butyl ether for three times, 200 mL each time. The organic phases were combined, washed once with 300 mL of saturated sodium chloride, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum at 45° C. to obtain compound A-2-3. .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 7.89 (d, J=2.8 Hz, 1H), 7.68 (br s, 1H), 7.66 (dd, J=2.8, 8.4 Hz, 1H), 7.42-7.27 (m, 5H), 5.52 (dd, J=4.8, 10.8 Hz, 1H), 5.27-5.07 (m, 2H), 4.01-3.84 (m, 1H), 3.88 (s, 3H), 3.82-3.70 (m, 1H), 2.29-2.07 (m, 2H); LCMS m/z=351.1[M+H].sup.+.

[0137] Step 3: Synthesis of Compound A-2-4

[0138] Compound A-2-3 (49 g, 139.86 mmol, 1 eq) was dissolved in THE (250 mL), and triphenylphosphine (44.02 g, 167.84 mmol, 1.2 eq) was added to the reaction solution, and diisopropyl azodicarboxylate (42.42 g, 209.80 mmol, 40.79 mL, 1.5 eq) was added dropwise at 0° C., controlling the reaction temperature to 0 to 10° C., and the mixture was stirred and reacted continuously at 0 to 10° C. for 0.5 hours. After the reaction was completed, the reaction solution was concentrated under vacuum at 45° C. The concentrate was stirred and slurried with a mixed solvent of methyl tert-butyl ether and n-heptane (100 mL; the volume ratio of methyl tert-butyl ether to n-heptane was 1:1) for 0.5 hours, filtered, and the filtrate was concentrated under vacuum to obtain a crude product. The crude product was separated and purified by silica gel column chromatography (gradient elution: n-heptane: ethyl acetate=100: 0 to 92:8) to obtain compound A-2-4. .sup.1H NMR (400 MHz, CDCl.sub.3) δ:7.90 (d, J=3.0 Hz, 1H), 7.50 (dd, J=2.4, 8.4 Hz, 1H), 7.42-7.32 (m, 5H), 5.44 (dd, J=5.2, 8.6 Hz, 1H), 5.31-5.18 (m, 2H), 4.20-4.09 (m, 1H), 3.96 (s, 3H), 3.95-3.83 (m, 1H), 2.97-2.80 (m, 1H), 2.23-2.09 (m, 1H); LCMS m/z=333.2[M+H].sup.+. SFC (column: ChiralpakAD-3, 3 μm, 0.46 cm id×5 cm L; mobile phase: A (CO.sub.2) and B (isopropanol, containing 0.05% diethylamine); gradient: B %=5-40%, 2 min, holding at 40%, 1.2 min, then 5%, 0.8 min; flow rate: 4 mL/min; wavelength: 220 nm; pressure: 1500 psi; retention time: 0.887 min, ee %=98.94%.

[0139] Step 4: Synthesis of Compound A-2

[0140] After compound A-2-4 (35 g, 105.32 mmol, 1 eq) was dissolved in dichloromethane (175 mL), then hydrobromic acid/acetic acid solution (68 mL, 413.24 mmol, mass fraction was 33%, 3.92 eq) was added thereto. After stirring and reacting at 25° C. for 5 days, an additional hydrobromic acid/acetic acid solution (51 mL, 3.0 eq) was added, and the reaction was continued for 1 day. Under the protection of nitrogen, the reaction solution was filtered under reduced pressure. After the filter cake was washed with 30 mL of methyl tert-butyl ether, the filter cake was dried under vacuum to obtain compound A-2 (identified by ion chromatography, the Br content was consistent with two salts). .sup.1H NMR (400 MHz, CD.sub.3OD) δ:7.93 (dd, J=2.4, 7.8 Hz, 1H), 7.69 (t, J=3.2 Hz, 1H), 5.13 (t, J=8.4 Hz, 1H), 4.59 (dt, J=3.6, 7.8 Hz, 1H), 4.41 (dt, J=6.0, 8.5 Hz, 1H), 3.00-2.89 (m, 1H), 2.87-2.73 (m, 1H); LCMS m/z=185.0[M+H].sup.+. SFC (column: Chiralpak AD-3, 3 μm, 0.46 cm id×5 cm L; mobile phase: A (CO.sub.2) and B (isopropanol, containing 0.05% diethylamine); gradient: B %=5-40%, 5 min, then 40-5%, 0.5 min, and finally holding at 5%, 1.5 min; flow rate: 2.5 mL/min; wavelength: 220 nm; pressure: 1500 psi; retention time: 3.624 min, ee %=98.32%.

Embodiment 1: Synthesis of Compound WX-001

[0141] ##STR00035## ##STR00036##

[0142] Step 1: Synthesis of Compound 1-2

[0143] Ethyl acetate (3000 mL) and potassium carbonate (13.36 g, 96.68 mmol, 0.05 eq) were added to compound 1-1 (300 g, 1.93 mol, 1 eq). Under the protection of nitrogen, the reaction solution was cooled to −5° C., and then N-bromosuccinimide (430.18 g, 2.42 mol, 1.25 eq) was added in batches. The reaction temperature was controlled at -5 to 5° C. with a feeding time of 2.5 hours, and then the mixture was stirred for 16 hours at room temperature (20° C.). Aqueous sodium sulfite solution (prepared by 400 g of anhydrous sodium sulfite and 2300 mL of water) was added to the reaction solution, and the mixture was continuously stirred for 10 minutes. The phases were separated, and the organic phase was washed once with 1000 mL of saturated brine. The organic phases and the aqueous phases obtained by the same treatment of this batch and another parallel batch reaction (same scale: 300 g of compound 1-1) were respectively combined for treatment. The aqueous phase (suspension) of the first separation was filtered, and the filtrate was extracted twice with ethyl acetate (1500 mL each time), and the combined organic phase was washed once with 2000 mL of saturated brine. All organic phases were combined, dried with anhydrous sodium sulfate, filtered, and the filter cake was washed with ethyl acetate (500 mL). The filtrate was concentrated under vacuum at 45° C. to obtain 880 g of solid crude product; the crude product was stirred and slurried with methanol (3960 mL) for 16 hours, filtered, and the filter cake was washed with methanol (300 mL), and the filtrate was concentrated under vacuum at 45° C. to obtain 780 g of crude product. The crude product was stirred and slurried with methyl tert-butyl ether (780 mL) at room temperature for 3 hours, filtered, and the filter cake was dried under vacuum to obtain compound 1-2. .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 5.33 (br s, 2H), 4.26 (q, J=7.0 Hz, 2H), 1.32 (t, J=7.2 Hz, 3H); LCMS m/z=233.8[M+1].sup.+.

[0144] Step 2: Synthesis of Compound 1-4

[0145] Compound 1-2 (294 g, 1.26 mol, 1 eq) and potassium phosphate (399.95 g, 1.88 mol, 1.5 eq) were sequentially added to N,N-dimethylformamide (2250 mL), stirred, and then ethyl 3-ethoxyacrylate (181.10 g, 1.26 mol, 181.46 mL, 1 eq) was added thereto, and the mixture was heated to 125° C. and reacted for 8 hours. After cooling to room temperature, 600 mL of cold water was added to the reaction solution, stirred for 5 minutes, then ice (2000 mL) was added to the reaction solution. Hydrochloric acid solution (3 M, 2000 mL) was slowly added thereto while stirring, and the pH was adjusted to 2; the mixture was stirred for 10 minutes, and then filtered. The filter cake was stirred and slurried with water (1000 mL) for 10 minutes, then filtered, and the obtained filter cake was stirred and slurried with methanol (1000 mL) for 20 minutes. After filtration, the filter cake was washed with methanol (200 mL) and dried under vacuum at 45° C. to obtain compound 1-4. .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 9.76 (br s, 1H), 7.97 (d, J=8.0 Hz, 1H), 6.09 (d, J=8.0 Hz, 1H), 4.34 (q, J=7.2 Hz, 2H), 1.35 (t, J=7.2 Hz, 3H).

[0146] Step 3: Synthesis of Compound 1-11

[0147] Compound 1-4 (50 g, 145.06 mmol, 1 eq) and cuprous cyanide (32.48 g, 362.66 mmol, 2.5 eq) were added to N,N-dimethylformamide (250 mL), and then the mixture was heated to 125° C. to react for 60 hours. After the reaction was completed, the reaction solution was cooled to room temperature, poured into water (500 mL) and stirred for 2 hours. The mixture solution was filtered under reduced pressure and the filter cake was collected, and the filter cake was dried under vacuum to obtain compound 1-11. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ: 12.28 (br s, 1H), 8.68 (br d, J=8.0 Hz, 1H), 6.41 (br d, J=8.0 Hz, 1H), 4.34 (q, J=6.8 Hz, 2H), 1.31 (t, J=7.0 Hz, 3H).

[0148] Step 4: Synthesis of Compound 1-12

[0149] Compound 1-11 (34 g, 146.43 mmol, 1 eq), triethylamine (44.45 g, 439.29 mmol, 3 eq) and 4-dimethylaminopyridine (3.58 g, 29.29 mmol, 0.2 eq) were sequentially added to dichloromethane (300 mL), and the system was replaced with nitrogen. Then, a dichloromethane (100 mL) solution of p-toluenesulfonyl chloride (69.79 g, 366.07 mmol, 2.5 eq) was added dropwise at 0° C. After the addition was completed, the reaction solution was slowly raised to room temperature of 20° C. to react for 6 hours. After the reaction was completed, the reaction solution was poured into water (1500 mL), and then dichloromethane (2500 mL) was added thereto, and the mixture was stirred for 1 hour, and then left to stand for liquid separation. After adding water (1500 mL) to the organic phase, the mixture was stirred for 1 hour, left to stand for liquid separation, and the aqueous phase was discarded. After adding dichloromethane (1500 mL) to the aqueous phase of the first liquid separation, the mixture was stirred for 1 hour and left to stand for liquid separation. The organic phases of the two liquid separations were combined, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain a crude product. The crude product was slurried and stirred with methanol (150 mL) at room temperature of 20° C. for 2 hours. After filtration, the filter cake was washed with a small amount of methanol (3 mL), and the filter cake was dried under vacuum to obtain compound 1-12. .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 8.68 (d, J=7.2 Hz, 1H), 8.36 (d, J=8.0 Hz, 2H), 7.43 (d, J=8.0 Hz, 2H), 6.87 (d, J=7.6 Hz, 1H), 4.54 (q, J=7.0 Hz, 2H), 2.48 (s, 3H), 1.52 (t, J=7.2 Hz, 3H); LCMS m/z=387.0[M+1]*.

[0150] Step 5: Synthesis of Compound 1-14

[0151] Compound A-2 (1.0 g, 2.89 mmol, 1 eq) was added to isopropanol (10 mL), then compound 1-12 (893.39 mg, 2.31 mmol, 0.80 eq) was added thereto, and finally N,N-diisopropylethylamine (1.12 g, 8.67 mmol, 1.51 mL, 3 eq) was added thereto, and the mixture was stirred at 40° C. for 3 hours. After the reaction was completed, the reaction system was cooled to room temperature, filtered to obtain a filter cake, and the filter cake was slurried with ethyl acetate (2 mL) at room temperature for 30 minutes, and then filtered to obtain compound 1-14. .sup.1HNMR (400 MHz, DMSO-d.sub.6) δ: 8.95 (d, J=7.6 Hz, 1H), 7.53 (s, 1H), 7.20 (s, 1H), 7.13 (d, J=8.0 Hz, 1H), 5.57-5.53 (m, 1H), 4.24-4.16 (m, 3H), 4.02-3.96 (m, 1H), 2.84-2.67 (m, 1H), 2.29-2.21 (m, 1H), 1.18 (t, J=7.2 Hz, 3H); LCMS m/z=399.1[M+1].sup.+.

[0152] Step 6: Synthesis of Compound 1-15

[0153] Compound 1-14 (8.7 g, 21.84 mmol, 1 eq) was dissolved in a mixed solution of water (22 mL) and tetrahydrofuran (65 mL), and then lithium hydroxide monohydrate (3.67 g, 87.36 mmol, 4 eq) was added, and the system was replaced with nitrogen. The reaction system changed from turbid to clear, and stirred at 25° C. for 4 hours. The reaction solution was adjusted to pH=6-7 with 1M hydrochloric acid solution (15 mL), and a solid was precipitated. The system was filtered with a funnel, and the filter cake was washed twice with 4 mL of water each time. The filter cake was dried under vacuum to obtain compound 1-15. LCMS m/z=371.0 [M+H].sup.+. SFC (column: ChiralpakAD-3, 3 μm, 0.46 cm id×5 cm L; mobile phase: A (CO.sub.2) and B (EtOH, containing 0.05% diethylamine); gradient: B %=5-50%, 3 min; flow rate: 3.4 mL/min; wavelength: 220 nm; pressure: 100 bar; retention time: 1.68 min, ee %=100%.

[0154] Step 7: Synthesis of Compound 1-16

[0155] Compound 1-15 (33.4 g, 90.20 mmol, 1 eq) and 1-aminocyclopropane methanol hydrochloride (12.26 g, 99.22 mmol, 1.1 eq, HCl) were dissolved in N,N-dimethylformamide (334 mL), and the system was replaced with nitrogen, and then HATU (37.73 g, 99.22 mmol, 1.1 eq) was added. The system was replaced with nitrogen again, and N,N-diisopropylethylamine (46.63 g, 360.79 mmol, 62.84 mL, 4 eq) was added. The reaction system changed from white turbidity to yellow clarification and then to yellow turbidity, and stirred at 25° C. for 3.5 hours. Saturated ammonium chloride solution (3.34, 0.1 V) was added to the reaction system, and then acetonitrile (133.6 mL, 4 V) was added thereto; the mixture was stirred for 30 minutes and filtered through a Buchner funnel, and the filter cake was washed with acetonitrile for three times, 10 mL each time. The filter cake was collected, and the filter cake was dried under vacuum to obtain compound 1-16. .sup.1HNMR (400 MHz, DMSO-d.sub.6) δ: 11.86 (s, 1H), 8.98 (d, J=7.6 Hz, 1H), 7.70 (s, 1H), 7.60 (s, 1H), 7.46-7.43 (m, 1H), 7.11 (d, J=8.0 Hz, 1H), 5.42 (t, J=6.8, 1H), 4.59-4.58 (m, 1H), 4.34-4.30 (m, 1H), 4.08-4.03 (m, 1H), 3.47-3.38 (m, 2H), 3.03-2.95 (m, 1H), 2.25-2.18 (m, 1H), 0.74-0.68 (m, 2H), 0.60-0.57 (m, 1H), 0.35-0.32 (m, 1H); LCMS m z=440.1 [M+H]f. SFC (column: (S,S)-WHELK-01, 3.5 μm, 0.46 cm id×5 cm L; mobile phase: A (CO.sub.2) and B (EtOH, containing 0.05% DEA); gradient: B %=5-50%, 3 min; flow rate: 3.4 mL/min; wavelength: 220 nm; column temperature: 35° C.; column pressure: 1800 psi, retention time: 1.65 min, ee %=100%.

[0156] Step 8: Synthesis of Compound WX-001

[0157] Compound 1-16 (45 g, 102.41 mmol, 1 eq) was dissolved in tetrahydrofuran (450 mL), and after the system was replaced with nitrogen, tri-n-butylphosphine (41.44 g, 204.83 mmol, 50.54 mL, 2 eq) was added thereto, then azodicarbonyl dipiperidine (51.68 g, 204.83 mmol, 2 eq) was added thereto; the mixture was reacted at 20° C. for 3 hours. After the reaction system was combined with the other two batches of reaction systems (4.45 g+5 g), methanol (220 mL, 4 V) was added to the system; and after stirring for 30 minutes, the filter cake solid was obtained by filtration, and then the filter cake solid was slurried with methanol (165 mL, 5V) for three times at room temperature and filtered after stirring for 30 minutes each time. The filter cake was collected, and the filter cake was dried under vacuum to obtain compound WX-001. .sup.1HNMR (400 MHz, DMSO-d.sub.6) δ: 9.26 (s, 1H), 8.98 (d, J=8.0 Hz, 1H), 8.16 (d, J=2.8 Hz, 1H), 7.89 (dd, J=8.4 Hz, 2.8 Hz, 1H), 7.04 (d, J=8.0 Hz, 1H), 5.97 (t, J=8.4, 1H), 4.76 (d, J=10.8 Hz, 1H), 4.59 (t, J=7.2 Hz, 1H), 4.02-3.99 (m, 1H), 3.91 (d, J=10.8 Hz, 1H), 3.07-3.01 (m, 1H), 2.63-2.57 (m, 1H), 2.13-2.08 (m, 1H), 1.28-1.24 (m, 1H), 0.98-0.89 (m, 2H); LCMS m/z=422.1 [M+H].sup.+. SFC (column: Chiralcel OD-3, 3 μm, 0.46 cm id×5 cm L; mobile phase: A (CO.sub.2) and B (MeOH, containing 0.05% isopropylamine); gradient: B %=10-40%, 3 min; flow rate: 4.0 mL/min; wavelength: 220 nm; pressure: 100 bar; retention time: 2.03 min, ee %=100%.

Embodiment 2: Synthesis of Compound WX-002A and WX-002B

[0158] ##STR00037##

[0159] Step 1: Synthesis of Compound 2-2

[0160] Compound 2-1 (0.2 g, 1.07 mmol, 1 eq) was dissolved in ethyl acetate (0.5 mL), then hydrochloric acid/ethyl acetate (2 mL, 4 M) was added thereto, and the mixture was reacted at room temperature of 20° C. for 10 minutes. The reaction solution was concentrated under reduced pressure at 45° C. to obtain compound 2-2 (crude product). 1.sup.1HNMR (400 MHz, DMSO-d.sub.6) δ: 8.34 (br s, 3H), 5.12 (br s, 1H), 4.47-4.40 (m, 1H), 3.68-3.60 (m, 1H), 2.35-2.27 (m, 2H), 2.15-2.09 (m, 2H).

[0161] Step 2: Synthesis of Compound 1-15A

[0162] Compound 1-14 (1.4 g, 3.51 mmol, 1 eq) was dissolved in acetonitrile (70 mL), then aluminum oxide (1.47 g, 14.41 mmol, 4.1 eq) and potassium trimethylsiliconate (901.76 mg, 7.03 mmol, 2 eq) were added thereto, and system was replaced with nitrogen, heated to 80° C. and stirred for 3 hours. After an additional potassium trimethylsiliconate (90.18 mg, 0.703 mmol, 0.2 eq) was added, stirring was continued at 80° C. for 1 hour. After cooling to room temperature, the reaction system was filtered, and the filter cake was washed twice with acetonitrile, 1.0 mL each time, and the obtained filter cake was the crude product. The crude product was slurried with a mixed solvent (17.5 mL of acetonitrile and 3.5 mL of methanol) at room temperature, filtered, and the filter cake was dried under vacuum to obtain compound 1-15A. LCMS m z=371.0 [M+H].sup.+. SFC (column: ChiralpakAD-3, 3 μm, 0.46 cm id×5 cm L; mobile phase: A (CO.sub.2) and B (EtOH, containing 0.05% diethylamine); gradient: B %=5-50%, 3 min; flow rate: 3.4 mL/min; wavelength: 220 nm; pressure: 100 bar; retention time: 1.68 min. ee %=79.56%, indicating that the product was partially racemic under this condition.)

[0163] Step 3: Synthesis of Compound 2-3

[0164] Compound 1-15A (570 mg, 769.66 μmol, 1 eq) was dissolved in N,N-dimethylformamide (12 mL), and then HATU (234.12 mg, 615.73 μmol, 0.8 eq), compound 2-2 (73.76 mg, 596.84 μmol, HCl) and N,N-diisopropylethylamine (298.41 mg, 2.31 mmol, 3 eq) were added, and the reaction solution was reacted at room temperature of 20° C. for 1 hour. After adding 2 drops of water to the reaction system to quench the reaction, the reaction solution was concentrated under vacuum at 45° C. to obtain a residue. The residue was separated and purified by silica gel column chromatography (eluent: dichloromethane: methanol=100:0-90:10) to obtain compound 2-3. LCMS m/z=440.1 [M+H].sup.+.

[0165] Step 4: Synthesis of Compounds WX-002A and WX-002B

[0166] Compound 2-3 (70 mg, 159.31 μmol, 1 eq) was dissolved in tetrahydrofuran (3 mL), and then triphenylphosphine (125.36 mg, 477.93 μmol, 3 eq) and diisopropyl azodicarboxylate (96.64 mg, 477.93 μmol, 3 eq) were added sequentially, and the mixture was stirred and reacted at room temperature of 20° C. for 1 hour after the addition was completed. After adding 2 drops of water to the reaction system to quench the reaction, the reaction solution was concentrated under vacuum to obtain the residue. The residue was purified by silica gel column chromatography (the eluent was dichloromethane: methanol=100: 0-90:10) to obtain a crude product. The above crude product was slurried and stirred with a mixed solution of 1 mL of dichloromethane and 1 mL of methanol for 10 minutes, and the mixture was filtered, and then the filter cake was dried under vacuum to obtain a product. The product was resolved by SFC (chromatographic column: REGIS (s,s) WHELK-O1 (250 mm*50 mm, 10 m); mobile phase: A: CO.sub.2; B: [MeOH with 0.1% ammonia]; gradient B %: 53%-53%). Compound WX-002A and compound WX-002B were obtained.

[0167] WX-002A: .sup.1HNMR (400 MHz, CDCl.sub.3) δ: 9.14 (d, J=10.4 Hz, 1H), 8.42 (d, J=8.0 Hz, 1H), 7.98 (d, J=2.8 Hz, 1H), 7.59-7.56 (m, 1H), 7.05 (d, J=7.2 Hz, 1H), 6.15 (t, J=8.0 Hz, 1H), 5.29-5.26 (m, 1H), 4.97-4.90 (m, 1H), 4.56 (t, J=8.0 Hz, 1H), 3.98-3.91 (m, 1H), 3.15-3.05 (m, 2H), 3.02-2.96 (m, 1H), 2.55-2.45 (m, 1H), 2.27-2.22 (m, 1H), 1.92-1.87 (m, 1H); LCMS m/z=422.1 [M+H].sup.+. SFC (column: REGIS (s,s) WHELK-O1 (50 mm*4.6 mm, 3 m); mobile phase: A: CO.sub.2; B: [MeOH with 0.05% diethylamine]; after the gradient B % was increased from 5% to 50% in 1.2 min, holding B %=50% for 1 min, and then decreased from 50% to 5% in 0.8 min); flow rate: 3.4 mL/min; temperature: 35° C.; pressure: 100 bar; retention time: 2.243 min, ee %=.sup.970.60%.

[0168] WX-002B: .sup.1HNMR (400 MHz, CDCl.sub.3) δ: 9.14 (d, J=10.4 Hz, 1H), 8.42 (d, J=8.0 Hz, 1H), 7.98 (d, J=3.2 Hz, 1H), 7.59-7.56 (m, 1H), 7.05 (d, J=7.2 Hz, 1H), 6.15 (t, J=8.0 Hz, 1H), 5.30-5.26 (m, 1H), 4.97-4.92 (m, 1H), 4.56 (t, J=7.6 Hz, 1H), 3.97-3.91 (m, 1H), 3.15-3.05 (m, 2H), 3.02-2.96 (m, 1H), 2.55-2.45 (m, 1H), 2.27-2.22 (m, 1H), 1.92-1.87 (m, 1H); LCMS m/z=422.2 [M+H].sup.+. SFC (column: REGIS (s,s) WHELK-O1 (50 mm*4.6 mm, 3 m); mobile phase: A: CO.sub.2; B: [MeOH with 0.05% diethylamine]; after the gradient B % was increased from 5% to 50% in 1.2 min, holding B %=50% for 1 min, and then decreased from 50% to 5% in 0.8 min); flow rate: 3.4 mL/min; temperature: 35° C.; pressure: 100 bar; retention time: 2.440 min, ee %=95.98%.

Embodiment 3: Synthesis of Compound WX-003

[0169] ##STR00038##

[0170] Step 1: Synthesis of Compound 3-1

[0171] Compound 1-15 (200.0 mg, 489.73 μmol, 1 eq) and (R)-(−)-2-amino-1-propanol (36.78 mg, 489.73 μmol, 1 eq) were dissolved in N,N-dimethylformamide (4 mL), and HATU (148.97 mg, 391.79 μmol, 0.8 eq) was added thereto. Then diisopropylethylamine (189.88 mg, 1.47 mmol, 3 eq) was added under the atmosphere of nitrogen. After the addition was completed, the reaction solution was stirred at 25° C. for 9 hours. The reaction solution was directly concentrated under vacuum to obtain a residue, and the residue was separated and purified by silica gel column chromatography (eluent: methanol: dichloromethane=0 to 1:1) to obtain compound 3-1. LCMS m/z=428.1 [M+H].sup.+.

[0172] Step 2: Synthesis of Compound WX-003

[0173] Compound 3-1 (67 mg, 156.77 μmol, 1 eq) was dissolved in dichloromethane (2 mL), then triphenylphosphine (123.35 mg, 470.30 μmol, 3 eq) was added thereto. After the system was replaced with nitrogen, diethyl azodicarboxylate (81.90 mg, 470.30 μmol, 3 eq) was added, and the mixture was stirred at 25° C. for 7 hours after the addition was completed. After 3 drops of water were dropped into the reaction solution to quench the reaction, the reaction solution was concentrated under vacuum, and the concentrated residue was separated and purified by preparative thin layer chromatography on silica gel plate (developing agent: ethyl acetate) to obtain compound WX-003. .sup.1HNMR (400 MHz, CDCl.sub.3) δ: 9.53 (d, J=7.2 Hz, 1H), 8.39 (d, J=7.6 Hz, 1H), 7.98 (d, J=2.8 Hz, 1H), 7.57-7.54 (m, 1H), 6.95 (d, J=7.6 Hz, 1H), 6.04-6.00 (m, 1H), 4.69 (dd, J=10.8, 4.0 Hz, 1H), 4.61-4.57 (m, 1H), 4.48-4.45 (m, 1H), 4.31-4.25 (m, 1H), 4.04-3.97 (m, 1H), 3.14-3.07 (m, 1H), 2.64-2.55 (m, 1H), 1.53 (d, J=6.4 Hz, 3H); LCMS m z=410.1 [M+H].sup.+.

[0174] Test Data

Experimental Embodiment 1: Kinase Inhibitory Activity of Compounds on TrkA and ROS1

[0175] The kinase inhibitory activity of compounds on TrkA and ROS1 was tested by Reaction Biology Corp. Company. A certain concentration of substrate, coenzyme factor, kinase and test compounds (10 doses, 3-fold serial dilution, 2% DMSO final concentration) were sequentially added to the reaction buffer (20 mM Hepes (pH 7.5), 10 mM MgCl.sub.2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mM Na.sub.3VO.sub.4, 2 mM DTT, 1% DMSO) and mixed well. The mixture was incubated at room temperature for 20 minutes. A certain concentration of .sup.33P-ATP was added to the reaction mixture to start the reaction, and then incubated at room temperature for 120 minutes. Finally, the radioactivity of reactants was detected by filter-binding method. The final kinase activity was expressed as the ratio of the remaining kinase activity in the test sample to the kinase activity of DMSO control group. The dose-effect curve was fitted by GraphPad software and IC.sub.50 was calculated. The results are shown in Table 1:

TABLE-US-00001 TABLE 1 Kinase half inhibitory concentration IC.sub.50 (nM) ROS1- Compound ROS1 G2032R TrkA ALK 1 TPX-0005 0.2 0.2 3.7 2.3 2 WX-001 0.8 0.3 231.0 103 4 WX-002B 0.2 0.1 597 25.1 5 WX-003 1.6 0.7 435 136

[0176] The results showed that the compound of the present disclosure showed high kinase inhibitory activity on a ROS1 kinase and a mutant ROS1-G2032R thereof, which was comparable to the activity of TPX-0005. However, the compound of the present disclosure had weak inhibitory activity on TrkA and ALK kinases, showing high selectivity, which was significantly better than that of TPX-0005.

Experimental Embodiment 2: Inhibitory Activity of Compounds on Cell Proliferation

[0177] Adenosine Tri-Phosphate (ATP) is an energy carrier shared by all kinds of life activities in nature, and it is the smallest unit of energy storage and transfer. The CellTiter-Glo™ living cell test reagent kit uses luciferase as the detector, and luciferase requires the participation of ATP in the process of luminescence. CellTiter-Glo™ reagent was added to a cell culture medium, and the luminescence value was measured. The light signal was directly proportional to the amount of ATP in the system, and ATP was positively correlated with the number of living cells. Therefore, cell proliferation can be detected by detecting ATP content with the CellTiter-Glo kit. In this test, the cell lines were Ba/F3 SLC34A2-ROS1-WT, Ba/F3 SLC34A2-ROS1-G2032R, Ba/F3 LMNA-NTRK1-WT stably transfected cell lines, with a number of 5000 cells/well.

[0178] IC.sub.50Determination Process:

[0179] 1 Cell Culture and Inoculation

[0180] a) Cells in logarithmic growth phase were harvested and counted using a platelet counter. Cell viability was detected by trypan blue exclusion method to ensure that the cell viability was above 90%.

[0181] b) Cell concentration was adjusted; 90 μL of cell suspension was added to a 96-well plate respectively.

[0182] c) Cells in the 96-well plate were cultured overnight at 37° C., 5% CO.sub.2, and 95% humidity.

[0183] 2 Drug Dilution and Dosing

[0184] a) A 10-fold drug solution was prepared with a maximum concentration of 10 μM, 9 concentrations and 3-fold dilution; 10 μL of drug solution was added to each well in the 96-well plate seeded with cells, and three duplicate wells were set for each drug concentration.

[0185] b) Cells in the dosed 96-well plate were incubated at 37° C., 5% CO.sub.2, and 95% humidity for a further 72 hours, and then CTG analysis was performed.

[0186] 3 End-Point Plate Reading

[0187] a) CTG reagent was melt and the cell plate was equilibrated to room temperature for 30 minutes.

[0188] b) An equal volume of CTG solution was added to each well.

[0189] c) Cells were lysed by shaking on an orbital shaker for 5 minutes.

[0190] d) The cell plate was placed at room temperature for 20 minutes to stabilize the luminescent signal.

[0191] e) The luminescence value was read.

[0192] 4 Data Processing

[0193] The data were analyzed using GraphPad Prism 5.0 software, and a dose-effect curve was obtained by fitting the data using a nonlinear S-curve regression, from which IC.sub.50 values were calculated. The data are shown in Table 2.

TABLE-US-00002 TABLE 2 Cell half inhibitory concentration IC.sub.50 (nM) Ba/F3 Ba/F3 Ba/F3 SLC34A2- SLC34A2- LMNA- Compound ROS1 ROS1-G2032R NTRK1-WT 1 TPX-0005 1.9 13.0 1.1 2 WX-001 4.6 5.6 37.0 3 WX-002B 24 20 157 4 WX-003 11 9.3 43

[0194] The results showed that the compounds of the present disclosure showed high cell proliferation inhibitory activity on the ROS1-fused cell line Ba/F3 SLC34A2-ROS1 and the mutant cell line Ba/F3 SLC34A2-ROS1-G2032R thereof. Meanwhile, the compounds of the present disclosure showed weak inhibitory activity on the Ba/F3 LMNA-NTRK1-WT cell line, and compared with Ba/F3 SLC34A2-ROS1 cell line and Ba/F3 SLC34A2-ROS1-G2032R cell line, especially Ba/F3 SLC34A2-ROS1-G2032R cell line, showing significant inhibitory selectivity.

[0195] Experimental embodiment 3: Cassette pharmacokinetics test of compounds in mice

[0196] Experimental objective: To study the pharmacokinetic behavior of the compounds of the present disclosure in mice, and evaluate its pharmacokinetic characteristics by taking 7-9 week-old male CD-1 mice as the test animals and using a LC/MS/MS method to determine the drug concentration of the compounds in plasma at different time after single intravenous injection (IV) and gavage (PO) administration.

[0197] Drug preparation: Compounds were formulated into clear solutions with 5% DMSO+10% solutol+85% water as the solvent for IV (intravenous) and PO (gavage) group administration. The dosing method was cassette dosing, and the dosage of each compound was IV 0.5 mg/kg; the dosage of PO was 2.5 mg/kg. The results of pharmacokinetic parameters are shown in Table 3:

TABLE-US-00003 TABLE 3 Results of cassette pharmacokinetic tests in mice Crizotinib Entrectinib Lorlatinib TPX-0005 WX-001 IV@ Starting 776 1280 905 936 994 0.5 concentration C.sub.0 mpk (nM) Half-life 2.54 2.52 2.39 4.38 1.66 T.sub.1/2 (h) Apparent volume 4.93 1.39 1.79 5.26 0.551 of distribution Vd (L/kg) Apparent 30.3 6.83 9.96 18.0 3.43 clearance rate Cl (mL/Kg/min) Area under curve 562 1912 1896 1282 5645 AUC.sub.0-last (nM .Math. hr) PO@ Peak 311 690 3225 3360 5380 2.5 concentration mpk C.sub.max (nM) Peak time 2.00 2.00 0.500 0.500 0.500 T.sub.max (h) Area under curve 1838 4363 13537 10602 25273 AUC.sub.0-last (nM .Math. hr) Bioavailability 65.4% 45.7% 143% 166% 89.8% F % Drug ND 12.7 622 42.2 486 concentration in brain at 0.5 hours Brain@0.5 h (nmol/kg) Drug 20.8 38.9 896 86.2 664 concentration in brain at 2 hours Brain@2 h (nmol/kg) Drug ND ND 105 5.46 26.9 concentration in cerebrospinal fluid at 0.5 hours CSF@0.5 h (nmol/kg) Drug ND ND 145 ND 36.4 concentration in cerebrospinal fluid at 2 hours CSF@2 h (nmol/kg) Drug 693 876 1344 1131 1770 concentration in lung at 0.5 hours Lung@0.5 h (nmol/kg) Drug 5088 5958 2016 1434 2862 concentration in lung at 2 hours Lung@2 h (nmol/kg) “ND”: Not detected.

[0198] The results showed that: at the same dosage, the total systemic exposure and peak concentration of compound WX-001 of the present disclosure after oral administration were significantly higher than those of Crizotinib, Entretinib, Lorlatinib and TPX-005, and its apparent clearance rate was significantly lower than those of Crizotinib, Entretinib, Lorlatinib and TPX-005, showing excellent pharmacokinetic characteristics. Compared with Entrectinib and Lorlatinib, the concentration of the compound of the present disclosure in brain and cerebrospinal fluid was significantly higher than that of Entrectinib and comparable to Lorlatinib after 0.5 hours and 2 hours of administration. Compared with TPX-0005, the concentrations of the compound of the present disclosure in lung, brain and cerebrospinal fluid were significantly higher at two time points after 0.5 hours and 2 hours of administration.

Experimental Embodiment 4: Pharmacokinetic Test of the Compounds in Mice

[0199] Experimental objective: To study the pharmacokinetic behavior of the compounds of the present disclosure in mice, and evaluate its pharmacokinetic characteristics by using 7-9 week-old male CD-1 mice as the test animals and using a LC/MS/MS method to determine the drug concentration of the compounds in plasma at different time after single intravenous injection (IV) and gavage (PO) administration.

[0200] Drug preparation: Compounds were formulated into clear solutions with 10% DMSO+10% solutol+80% water as the solvent for IV (intravenous) and PO (gavage) group administration. The dosage of each compound was: IV 1 mg/kg; the dosage of PO was 3 mg/kg. The results of pharmacokinetic parameters are shown in Table 4:

TABLE-US-00004 TABLE 4 Results of pharmacokinetic tests in mice WX-002B WX-003 IV Half-life T.sub.1/2 (h) 2.1 3.2 Apparent volume of 1.1 1.0 distribution Vd (L/kg) Apparent clearance rate 6.3 4.0 Cl (mL/Kg/min) Area under curve AUC.sub.0-last 5873 8665 (nM .Math. hr) PO Peak concentration C.sub.max 2170 3805 (nM) Peak time T.sub.max (h) 1.5 3.0 Area under curve AUC.sub.0-last 13350 23771 (nM .Math. hr) Bioavailability F % 76% 92%

[0201] The results showed that: the total systemic exposure, peak concentration and bioavailability of the compounds of the present disclosure after oral administration were all high, showing excellent pharmacokinetic characteristics.

Experimental Embodiment 5: Efficacy Test of the Compounds in Mice

[0202] Experimental objective: To evaluate the in vivo efficacy of WX-001 in Ba/F3 CD74-ROS1-WT subcutaneous xenograft tumor BALB/c nude mice model.

[0203] Drug preparation: Compounds were formulated into clear solutions with 10% DMSO+10% solutol+80% water as the solvent for PO (gavage) group administration.

[0204] Tumor measurement: Diameters of tumors were measured with vernier calipers twice a week. The calculation formula of tumor volume was: V=0.5×a×b.sup.2, wherein a and b represented

[0205] the long and short diameters of the tumor, respectively. The anti-tumor efficacy of the compounds was evaluated by TGI (%) or relative tumor proliferation rate T/C (%). TGI (%) reflects the tumor growth inhibition rate. Relative tumor proliferation rate T/C (%)=T.sub.RTV/C.sub.RTV×100% (T.sub.RTV: mean RTV of the treatment group; C.sub.RTV: mean RTV of the negative control group). The relative tumor volume (RTV) was calculated according to the results of tumor measurement, and the calculation formula was RTV=V.sub.t/V.sub.0, wherein V.sub.0 was the tumor volume measured at the time of group administration (that is, D.sub.0), V.sub.t was the tumor volume of the corresponding mouse at a certain measurement, and the data of T.sub.RTV and C.sub.RTV were taken on the same day. TGI (%)=[(1-(average tumor volume at the end of administration of a certain treatment group - average tumor volume at the beginning of administration of this treatment group))/(average tumor volume at the end of treatment in the solvent control group - average tumor volume at the beginning of treatment in the solvent control group)]×100%. The results are shown in FIG. 1.

[0206] Statistical analysis: Statistical analysis was based on the relative tumor volume and tumor weight at the end of the trial, which was analyzed by SPSS software. The comparison between multiple groups

[0207] was analyzed by using one-way ANOVA. If the variances were homogeneous (no significant difference in F-values), Tukey's method was used for analysis. If the variances were not homogeneous (significant difference in F-values), Games-Howell method was used for test. P<0.05 was considered as a significant difference.

[0208] Experimental results: In the Ba/F3 CD74-ROS1-WT subcutaneous xenograft tumor model, the TGIs of Crizotinib (30 mg/kg) and WX-001 (10 mg/kg) at 7 days of administration were 110.40% and 112.17% respectively, and the p values were all equal to 0.004, which showed significant anti-tumor effects.

[0209] Experimental conclusion: WX-001 has a significant inhibitory effect on the growth of Ba/F3 CD74-ROS1-WT nude mice transplanted tumor.

[0210] Experimental embodiment 6: Efficacy test of the compounds in mice

[0211] Experimental objective: To evaluate the anti-tumor effect of WX-001 in human-derived lung cancer LD1-0025-361019 PDX animal model.

[0212] Drug preparation: Compounds were formulated into clear solutions with 10% DMSO+10% solutol+80% water as the solvent for PO (gavage) group administration.

[0213] Tumor measurement: Diameters of tumors were measured with vernier calipers twice a week. The calculation formula of tumor volume was: V=0.5×a×b.sup.2, wherein a and b represented

[0214] the long and short diameters of the tumor, respectively. The anti-tumor efficacy of the compounds was evaluated by TGI (%). TGI (%) reflects the tumor growth inhibition rate. TGI (%)=[(1-(average tumor volume at the end of administration of a certain treatment group-average tumor volume at the beginning of administration of this treatment group))/(average tumor volume at the end of treatment in the solvent control group - average tumor volume at the beginning of treatment in the solvent control group)]×100%. The results are shown in FIG. 2.

[0215] Statistical analysis: All data were expressed by Mean±SEM. One-way ANOVA LSD test was used to compare whether there was a significant difference in tumor volume and tumor weight between the treatment group and the control group. All the data were analyzed by Graphpad. P<0.05 was considered as a significant difference.

[0216] Experimental results: At 21 days of administration on the human-derived lung cancer LD1-0025-361019 PDX animal model (CD74-ROS1 fusion & G2032R mutation), the tumor growth inhibition rate TGIs of Crizotinib (50 mg/kg) administration group, WX-001 high dose (20 mg/kg) administration group, WX-001 medium dose (15 mg/kg) administration group and WX-001 low dose (10 mg/kg) administration group were 35.43%, 82.38%, 70.03% and 60.83% respectively. Compared with the solvent control group, the WX-001 20 mg/kg administration group, the WX-001 15 mg/kg administration group and the WX-001 10 mg/kg administration group all showed significant inhibitory effects on tumor growth (P<0.01).

[0217] Experimental conclusion: In the human-derived lung cancer LD1-0025-361019 PDX animal model, WX-001 has a significant anti-tumor effect, and the anti-tumor effect has a dose-dependent trend (compared the high-dose group with the low-dose group p<0.05).