Diphenylaminopyrimidine and triazine compound, and pharmaceutical composition and use thereof

Abstract

A diphenylaminopyrimidine and triazine compound of Formula I, or a pharmaceutically acceptable salt, stereoisomer, hydrate or solvate thereof is disclosed ##STR00001##
In formula I, A is C or N; X and Y are independently selected from hydrogen, halo, cyano, trifluoromethyl, alkoxy, alkyl, aryl, alkenyl, alkynyl and nitro; or X and Y, together with the atoms to which they are attached, form a phenyl or an heteroaromatic ring; R.sub.1 is ##STR00002##
R.sub.2 is CD.sub.3 or CD.sub.2CD.sub.3; R.sub.3 is ##STR00003##
R.sub.4 is hydrogen, methyl, trifluoromethyl, cyano or halo; R.sub.5 is hydrogen, alkyl, substituted and unsubstituted phenyl, allyl or propargyl; R.sub.6 and R.sub.7 are independently selected from hydrogen, alkyl, substituted and unsubstituted phenyl, allyl and propargyl; or R.sub.6 and R.sub.7, together with the nitrogen atom to which they are attached, form a substituted or unsubstituted heterocycloalkyl group. The compound has pharmacodynamic and pharmacokinetic properties and ALK kinase inhibitory activity.

Claims

1. A diphenylaminopyrimidine and triazine compound of Formula I, or a pharmaceutically acceptable salt, stereoisomer, hydrate or solvate thereof: ##STR00226## wherein: A is C; X and Y are independently selected from the group consisting of hydrogen, halo, cyano, trifluoromethyl, alkoxy, alkyl, aryl, alkenyl, alkynyl, and nitro; or X and Y, together with the atoms to which they are attached, form a phenyl ring or an heteroaromatic ring, the heteroaromatic ring containing one or more of oxygen, sulfur and nitrogen heteroatoms; R.sub.1 is ##STR00227## R.sub.2 is CD.sub.3 or CD.sub.2CD.sub.3; R.sub.3 is selected from ##STR00228## R.sub.4 is selected from the group consisting of hydrogen, methyl, trifluoromethyl, cyano and halo; R.sub.5 is selected from the group consisting of hydrogen, alkyl, substituted and unsubstituted phenyl, allyl and propargyl; and R.sub.6 and R.sub.7 are independently selected from the group consisting of hydrogen, alkyl, substituted and unsubstituted phenyl, allyl and propargyl; or R.sub.6 and R.sub.7, together with the N atom to which they are attached, form a substituted or unsubstituted heterocycloalkyl group, the heterocycloalkyl group containing one or more of oxygen, sulfur, nitrogen, sulfoxide and sulfone groups.

2. The diphenylaminopyrimidine and triazine compound as claimed in claim 1, or a pharmaceutically acceptable salt, stereoisomer, hydrate or solvate thereof, wherein R.sub.1 is ##STR00229## R.sub.2 is CD.sub.3, X is halo, and Y is hydrogen.

3. The diphenylaminopyrimidine and triazine compound as claimed in claim 1, or a pharmaceutically acceptable salt, stereoisomer, hydrate or solvate thereof, wherein the diphenylaminopyrimidine and triazine compound is selected from the group consisting of: ##STR00230## ##STR00231## ##STR00232## ##STR00233## ##STR00234## ##STR00235## ##STR00236## ##STR00237## ##STR00238##

4. A pharmaceutical composition, comprising a diphenylaminopyrimidine and triazine compound, or a pharmaceutically acceptable salt, stereoisomer, hydrate or solvate thereof as claimed in claim 1, and a pharmaceutically acceptable carrier or excipient.

5. A method of treating a disease or disorder that benefits from inhibition of the ALK tyrosine kinase activity comprising: treating a subject in need thereof with an ALK tyrosine kinase inhibitor, wherein the ALK tyrosine kinase inhibitor is the diphenylaminopyrimidine and triazine compound of Formula I, or a pharmaceutically acceptable salt, stereoisomer, hydrate or solvate thereof as claimed in claim 1.

6. The method as claimed in claim 5, wherein the disease or disorder is selected from the group consisting of cancers, cell proliferative diseases, immune diseases or inflammation, infection, organ transplantation, viral diseases, cardiovascular diseases, and metabolic diseases.

7. The method as claimed in claim 6, wherein the cancers are selected from the group consisting of non-small cell lung cancer, neuroblastoma, melanoma leukemia and lymphoma.

8. The method as claimed in claim 6, wherein the immune diseases or inflammation are selected from the group consisting of rheumatoid arthritis, osteoarthritis, rheumatoid spondylitis, gout, asthma, bronchitis, rhinitis, chronic obstructive pulmonary disease and cystic fibrosis.

9. The method as claimed in claim 5, wherein the ALK tyrosine kinase inhibitor is administered alone or in combination with other therapeutic agents.

10. The method as claimed in claim 5, wherein the ALK tyrosine kinase inhibitor is administered orally, parenterally, transpulmonarily, intravenously or transdermally.

Description

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(1) The invention will be further illustrated in more detail with reference to embodiments. It is noted that, the following embodiments only are intended for purposes of illustration, but are not intended to limit the scope of the present invention.

I. Preparation of Compounds

1. Synthesis of Compound IX

(2) ##STR00027##

(3) Compound XI was subjected to a substitution reaction to obtain Compound IX. The result is shown in Table 1.

(4) TABLE-US-00001 TABLE 1 Structure of Compound IX No. R.sub.2 R.sub.4 LC-MS (M + 1) 1 CD.sub.3 H 191.0 2 CD.sub.3 CH.sub.3 205.0 3 CD.sub.3 F 209.0 4 CD.sub.3 Cl 225.0 5 CD.sub.3 CF.sub.3 259.0 6 CD.sub.3 CN 216.0 7 CD.sub.2CD.sub.3 H 207.1 8 CD.sub.2CD.sub.3 CH.sub.3 221.1 9 CD.sub.2CD.sub.3 Cl 241.0

Embodiment 1

Synthesis of Compound IX-1 (where R.SUB.2.H, and R.SUB.4.CD.SUB.3.)

(5) Under a nitrogen atmosphere, Compound (XI-1, where R.sub.4CD.sub.3)(2.3 g, 13.1 mmol) was added to DMSO (25 mL), and then cesium carbonate (20.8 g, 65.9 mmol) was added to the resulting solution. Deuterated methanol (2.0 mL) was added dropwise at room temperature, and the resulting mixture was heated to 50 C., and reacted for 1 hr, until the reaction was complete as monitored by TLC. The reaction solution was poured into iced water and extracted twice with ethyl acetate. Then the organic phase was dried. The organic solvent was removed by evaporation to obtain a solid. The solid was then recrystallized in methyl t-butyl ether, to obtain Compound (IX-1) (2.0 g, 80%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): =7.92 (d, 1H, J=6.8 Hz), 7.46 (s, 1H), 7.30 (d, 1H, J=6.8 Hz); LC-MS: m/z=191.0 (M+1).

Embodiment 2

Synthesis of Compound IX-2 (where R.SUB.2.=Me, and R.SUB.4.CD.SUB.3.)

(6) Under a nitrogen atmosphere, Compound (XI-2, where R.sub.4=Me) (2.5 g, 13.1 mmol) was added to DMSO (25 mL), and then cesium carbonate (20.8 g, 65.9 mmol) was added to the resulting solution. Deuterated methanol (2 mL) was added dropwise at room temperature, and the resulting mixture was heated to 50 C. and reacted for 1 hr, until the reaction was complete as monitored by TLC. The reaction solution was poured into iced water and extracted twice with ethyl acetate. Then the organic phase was dried. After filtering, the organic solvent was removed by evaporation to obtain a solid. The solid was then recrystallized in methyl t-butyl ether, to obtain Compound (IX-2) (2.2 g, 76.9%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): =7.90 (s, 1H), 7.52 (s, 1H), 2.30 (s, 3H); LC-MS: m/z=205.0 (M+1).

Embodiment 3

Synthesis of Compound IX-8 (where R.SUB.2.=Me, and R.SUB.4.CD.SUB.2.CD.SUB.3.)

(7) Under a nitrogen atmosphere, Compound (XI-8) (2.5 g, 13.1 mmol) was added to DMSO (25 mL), and then cesium carbonate (20.8 g, 65.9 mmol) was added to the resulting solution. Deuterated ethanol (2 mL) was added dropwise at room temperature, and the resulting mixture was heated to 50 C., and reacted for 1 hr, until the reaction was complete as monitored by TLC. The reaction solution was poured into iced water and extracted twice with ethyl acetate. Then the organic phase was dried. After filtering, the organic solvent was removed by evaporation to obtain a solid. The solid was then recrystallized in methyl t-butyl ether, to obtain Compound (IX-8) (2.4 g, 83.0%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): =7.92 (s, 1H), 7.50 (s, 1H), 2.30 (s, 3H); LC-MS: m/z=221.1 (M+1).

(8) Following the same procedure as shown in Embodiments 1-3, a series of Compounds (IX-1 to 9) were obtained, the structure of which was confirmed by LC-MS. The results are shown in Table 1.

2. Synthesis of Compound VIII

(9) Compound IX and Compound X were subjected to Suzuki coupling to obtain Compound VIII. The reaction route was as follows.

(10) ##STR00028##

(11) The results are shown in Table 2.

(12) TABLE-US-00002 TABLE 2 Structure of Compound VIII No. R.sub.2 R.sub.4 LC-MS (M + 1) 1 CD.sub.3 H 234.1 2 CD.sub.3 CH.sub.3 248.1 3 CD.sub.3 F 252.1 4 CD.sub.3 Cl 268.1 5 CD.sub.3 CF.sub.3 302.1 6 CD.sub.3 CN 259.1 7 CD.sub.2CD.sub.3 H 250.1 8 CD.sub.2CD.sub.3 CH.sub.3 264.1 9 CD.sub.2CD.sub.3 Cl 284.1

Embodiment 4

Synthesis of Compound VIII-2 (where R.SUB.2.=CD.SUB.3., and R.SUB.4.=Me)

(13) Under a nitrogen atmosphere, Compound (X) (1.47 g, 12 mmol) was added to a mixed solvent (100 mL/50 mL) of dioxane and water. Tris-(dibenzylideneacetone)dipalladium (1 g, 1.09 mmol), 2-dicyclohexylphosphino-2,6-dimethoxybiphenyl (1.12 g, 2.72 mmol), Compound (IX-2) (2.5 g, 10.9 mmol) and potassium phosphate (4.62 g, 21.8 mmol) were sequentially added to the mixture, and nitrogen was bubbled for 15 min. The resulting solution was heated to reflux and reacted for 6 hrs, until the reaction was complete as monitored by TLC. The reaction solution was diluted with ethyl acetate (200 mL), and washed with 1 N sodium hydroxide solution at room temperature. The organic solvent was removed by evaporation, and the residue was purified by column chromatography on silica gel to obtain Compound (VIII-2)(1.7 g, 68.8%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): =8.60 (d, 2H, J=7.2 Hz), 8.06 (d, 2H, J=7.0 Hz), 7.96 (s, 1H), 7.50 (s, 1H), 2.30 (s, 3H); LC-MS: m/z=248.1 (M+1).

Embodiment 5

Synthesis of Compound VIII-8 (where R.SUB.2.=CD.SUB.2.CD.SUB.3., and R.SUB.4.=Me)

(14) Under a nitrogen atmosphere, Compound (X) (1.47 g, 12 mmol) was added to a mixed solvent (100 mL/50 mL) of dioxane and water. Tris-(dibenzylideneacetone)dipalladium [Pd.sub.2dba.sub.3] (1 g, 1.09 mmol), 2-dicyclohexylphosphino-2,6-dimethoxybiphenyl (1.12 g, 2.72 mmol), Compound (IX-8) (2.5 g, 10.9 mmol) and potassium phosphate (4.62 g, 21.8 mmol) were sequentially added to the mixture, and nitrogen was bubbled for 15 min. The resulting solution was heated to reflux and reacted for 6 hrs, until the reaction was complete as monitored by TLC. The reaction solution was diluted with ethyl acetate (200 mL), and washed with 1 N sodium hydroxide solution at room temperature. The organic layer was separated, dried, evaporated to remove the organic solvent, and purified by column chromatography on silica gel to obtain Compound (VIII-8)(2.0 g, 62.8%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): =8.62 (d, 2H, J=7.0 Hz), 8.02 (d, 2H, J=7.0 Hz), 7.92 (s, 1H), 7.54 (s, 1H), 2.32 (s, 3H); LC-MS: m/z=264.1 (M+1).

(15) Using the same procedure as shown in Embodiments 4-5, a series of Compounds (VIII-1 to 10) were obtained, the structure of which was confirmed by LC-MS. The results are shown in Table 2.

(16) The coupling reaction of Compound (IX-1) with piperazine and other nitrogen-containing heterocyclic compounds was carried out as follows. The results are shown in Table 3.

(17) ##STR00029##

(18) TABLE-US-00003 TABLE 3 Structure of Compound VIII No. R.sub.4 0 LC-MS ( M + 1) 11 H 341.2 12 H 341.2 13 H 438.3 14 H 338.2 15 H 283.2 16 H 325.2 17 Me 355.2

Embodiment 6

Synthesis of Compound VIII-11

(19) The synthesis route of Compound VIII-11 was as follows.

(20) ##STR00038##

(21) Compound (IX-1) (3.8 g, 20 mmol) and Boc-piperazine (5.6 g, 30 mmol) were added to DMSO (50 mL) and then triethylamine (3 g, 30 mmol) was added. The resulting solution was heated to 70 C., and reacted for 30 hrs. The reaction solution was cooled to room temperature, stirred with water (100 mL) for 2 hrs to give a precipitate, which was filtered to obtain Compound (VIII-11) (4.80 g, 70%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): =7.80 (s, 1H, J=7.0 Hz), 6.62 (s, 1H, J=7.0 Hz), 6.50 (s, 1H), 3.32-3.42 (m, 8H), 1.34 (s, 9H); LC-MS: m/z=341.2 (M+1).

(22) Following the same procedure as shown in Embodiment 6, a series of Compounds (VIII-11 to 17) were obtained, the structure of which was confirmed by LC-MS. The results are shown in Table 3.

3. Synthesis of Compound VII

(23) The synthesis route of Compound VII was as follows.

(24) ##STR00039##

Embodiment 7

Synthesis of Compound VII-2 (where R.SUB.4.=Me, and R.SUB.2.=CD.SUB.3.)

(25) Compound (VIII-2) (4.38 g, 16.1 mmol), trifluoroacetic acid (2.4 ml, 32.2 mmol), and PtO.sub.2 (1.76 g, 40%) were added to acetic acid (200 mL), and the resulting solution was reacted under a hydrogen atmosphere for 36 hrs. After the reaction was complete as monitored by TLC, the solid was removed by filtration. The organic solvent in the mother liquid was removed by evaporation, and the solid was dissolved in ethyl acetate. Then, it was washed with a 1 N NaOH solution until pH value is 10, and the organic phase was dried, filtered, and concentrated to give Compound (VII-2) (2.5 g, 70%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): =9.50-8.04 (br, 1H), 6.53 (s, 1H), 6.44 (s, 1H), 4.44 (s, 2H), 3.03-2.96 (m, 2H), 2.87-2.84 (m, 2H), 2.83-2.80 (m, 1H), 2.12 (s, 3H), 1.78-1.72 (m, 4H); LC-MS: m/z=224.2 (M+1).

Embodiment 8

Synthesis of Compound VII-8 (where R.SUB.4.=Me, and R.SUB.2.=CD.SUB.2.CD.SUB.3.)

(26) Compound (VIII-8) (4.38 g, 16.1 mmol), trifluoroacetic acid (2.4 ml, 32.2 mmol), and PtO.sub.2 (1.76 g, 40%) were added to acetic acid (200 mL) and the resulting solution was reacted under a hydrogen atmosphere for 36 hrs. After the reaction was complete as monitored by TLC, the solid was removed by filtration. The solvent was removed by evaporation, and the solid was dissolved in ethyl acetate, and washed with a 1N NaOH solution until pH value is 10. The organic phase was dried, filtered, and concentrated to obtain Compound (VII-8) (2.65 g, 68.8%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): =9.52-8.00 (br, 1H), 6.53 (s, 1H), 6.48 (s, 1H), 4.40 (s, 2H), 3.00-2.92 (m, 2H), 2.84-2.86 (m, 2H), 2.80-2.82 (m, 1H), 2.14 (s, 3H), 1.78-1.70 (m, 4H); LC-MS: m/z=240.2 (M+1).

(27) Using the same procedure as shown in Embodiments 7-8, a series of Compounds (VII-1 to 9) were obtained, the structure of which was confirmed by LC-MS. The results are shown in Table 4.

(28) TABLE-US-00004 TABLE 4 Structure of Compound VII No. R.sub.2 R.sub.4 LC-MS (M + 1) 1 CD.sub.3 H 210.2 2 CD.sub.3 CH.sub.3 224.2 3 CD.sub.3 F 228.2 4 CD.sub.3 Cl 244.1 5 CD.sub.3 CF.sub.3 278.2 6 CD.sub.3 CN 235.2 7 CD.sub.2CD.sub.3 H 226.2 8 CD.sub.2CD.sub.3 CH.sub.3 240.2 9 CD.sub.2CD.sub.3 Cl 260.2

(29) The reduction of the nitrogen-containing heterocyclic Compound (VIII) was carried out according to a reaction route as follows.

(30) ##STR00040##

Embodiment 9

Synthesis of Compound VII-11

(31) The synthesis route of Compound VII-11 was as follows.

(32) ##STR00041##

(33) Compound (VIII-11) (6.8 g, 20 mmol), and 10% Pd/C (0.6 g) were added to methanol (100 mL), and reacted for 26 hrs under a hydrogen atmosphere. After the reaction was complete as monitored by TLC, the solid was removed by filtration. Methanol was concentrated to obtain Compound (VII-11) (5.7 g, 92%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): =6.48 (s, 1H, J=7.0 Hz), 6.26 (s, 1H, J=7.0 Hz), 6.00 (s, 1H), 3.30-3.40 (m, 8H), 1.34 (s, 9H); LC-MS: m/z=311.2 (M+1).

(34) Following the same procedure as shown in Embodiment 9, a series of Compounds (VII-1 to 9) were obtained, the structure of which was confirmed by LC-MS. The results are shown in Table 5.

(35) TABLE-US-00005 TABLE 5 Structure of nitrogen-containing heterocyclic Compound VII No. R.sub.4 LC-MS (M + 1) 11 H 311.2 12 H 311.2 13 H 408.3 14 H 308.3 15 H 253.2 16 H 295.2 17 CH.sub.3 325.2

4. Synthesis of Compound IV

(36) The synthesis route of Compound IV was as follows.

(37) ##STR00050##

Embodiment 10

Synthesis of Compound IV-2 (where R.SUB.4.=Me, and R.SUB.2.=CD.SUB.3.)

(38) Compound (VII-2) (2.0 g, 9.0 mmol), and triethyl amine (2.2 ml, 15.7 mmol) were dissolved in dichloromethane (100 mL), and cooled to 0 C. Then, di-tert-butyl dicarbonate (1.8 g, 9.0 mmol) was added in one portion, and reacted with stirring. After the reaction was complete as monitored by TLC, the organic solvent was removed by evaporation, and the residue was purified by column chromatography on silica gel to obtain Compound (IV-2) (2 g, 69.0%). .sup.1H NMR (400 MHz, CD.sub.3OD) =7.61 (s, 1H), 6.76 (s, 1H), 4.22 (d, J=13.2 Hz, 2H), 2.90 (m, 3H), 2.28 (s, 3H), 1.73 (d, J=12.2 Hz, 2H), 1.54 (d, J=11.4 Hz, 2H), 1.52 (s, 9H); LC-MS: m/z=324.2 (M+1).

Embodiment 11

Synthesis of Compound IV-8 (where R.SUB.4.=Me, and R.SUB.2.=CD.SUB.2.CD.SUB.3.)

(39) Compound (VII-8) (2.0 g, 9.0 mmol), and triethyl amine (2.2 mL, 15.7 mmol) were dissolved in dichloromethane (100 mL), and cooled to 0 C. Then, di-tert-butyl dicarbonate (1.8 g, 9.0 mmol) was added in one portion, and reacted with stirring. After the reaction was complete as monitored by TLC, the organic solvent was removed by evaporation, and the residue was purified by column chromatography on silica gel to obtain Compound (IV-8) (2.5 g, 81.8%). .sup.1H NMR (400 MHz, CD.sub.3OD) =7.64 (s, 1H), 6.72 (s, 1H), 4.20 (d, J=13.0 Hz, 2H), 2.92 (m, 3H), 2.24 (s, 3H), 1.70 (d, J=12.2 Hz, 2H), 1.52 (d, J=11.4 Hz, 2H), 1.50 (s, 9H); LC-MS: m/z=340.2 (M+1).

(40) Using the same procedure as shown in Embodiments 10-11, a series of Compounds (IV-1 to 9) were obtained, the structure of which was confirmed by LC-MS. The results are shown in Table 6.

(41) TABLE-US-00006 TABLE 6 Structure of Compound IV No. R.sub.2 R.sub.4 LC-MS (M + 1) 1 CD.sub.3 H 310.2 2 CD.sub.3 CH.sub.3 324.2 3 CD.sub.3 F 328.2 4 CD.sub.3 Cl 344.1 5 CD.sub.3 CF.sub.3 378.2 6 CD.sub.3 CN 335.2 7 CD.sub.2CD.sub.3 H 326.2 8 CD.sub.2CD.sub.3 CH.sub.3 340.2 9 CD.sub.2CD.sub.3 Cl 360.2

5. Synthesis of Compound III

(42) Compound V was coupled to the pyrimidine Compound VI, to obtain Compound III. The reaction route was as follows.

(43) ##STR00051##

Embodiment 12

Synthesis of Compound III-3

(44) At 0 C. and under a nitrogen atmosphere, NaH (1.51 g, 60%, 37.64 mmol) was added to DMF (50 mL) and DMSO (5 mL), and stirred for 5 min at 0 C. Then, a solution of Compound (V-3, where R.sub.1=i-PrSO.sub.2)(5 g, 25.09 mmol) in DMF/DMSO (18 mL/2 mL) was added dropwise to the mixed solution, and stirred for 45 min while the temperature was maintained at 0 C. A solution of 2, 4, 5-tricloropyrimidine (111-3) (9.20 g, 50.18 mmol) in DMF/DMSO (18 mL/2 mL) was added dropwise to the mixed solution. The resulting solution was stirred at 0 C. for 45 min and then at room temperature for 2 hrs. After the reaction was complete as monitored by TLC, the reaction solution was poured into iced water, and extracted twice with ethyl acetate. The organic solvent was removed by evaporation, and the residue was purified by column chromatography on silica gel to obtain Compound III-3 (5.2 g, 60.6%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): =9.81 (s, 1H), 8.57 (s, 1H), 8.32 (d, J=8.3 Hz, 1H), 7.96-7.82 (m, 2H), 7.56-7.42 (m, 1H), 3.61-3.46 (m, 1H), 1.16 (d, J=6.8 Hz, 6H); LC-MS: m/z=346.0 (M+1).

(45) Using the same procedure as shown in Embodiment 12, a series of Compounds (III-1 to 19) were obtained, the structure of which was confirmed by LC-MS. The results are shown in Table 7.

(46) TABLE-US-00007 TABLE 7 Structure of Compound III LC-MS No. R1: A X Y (M + 1) 1 i-PrSO.sub.2 C H H 312.1 2 i-PrSO.sub.2 C F H 330.0 3 i-PrSO.sub.2 C Cl H 346.0 4 i-PrSO.sub.2 C Br H 390.0 5 i-PrSO.sub.2 C CF.sub.3 H 380.0 6 i-PrSO.sub.2 C CN H 337.0 7 i-PrSO.sub.2 C OMe H 342.1 8 i-PrSO.sub.2 C Me H 326.1 9 i-PrSO.sub.2 C Et H 340.1 10 i-PrSO.sub.2 C NO.sub.2 H 357.0 11 i-PrSO.sub.2 C Ph H 388.1 12 i-PrSO.sub.2 C H 338.1 13 i-PrSO.sub.2 C H 336.1 14 i-PrSO.sub.2 C / 351.1 15 i-PrSO.sub.2 C / 351.1 16 i-PrSO.sub.2 C / 368.0 17 i-PrSO.sub.2 C / 368.0 18 i-PrSO.sub.2 C / 362.1 19 Me.sub.2P(O) C Cl H 316.0 20 i-PrSO.sub.2 N / H 313.0

6. Synthesis of Compound II

(47) In the presence of a palladium catalyst, Compound III was coupled to Compound VI, to obtain Compound II. The reaction route was as follows.

(48) ##STR00059##

Embodiment 13

Synthesis of Compound II-3

(49) Under a nitrogen atmosphere, Compound (IV-2) (1.7 g, 4.8 mmol), Compound (111-3) (1.69 g, 4.88 mmol), 4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene (Xantphos) (280 mg, 0.49 mmol), palladium acetate (55 mg, 0.25 mmol), and cesium carbonate (4.77 g, 14.6 mmol) were added to tetrahydrofuran, and heated to reflux and reacted for 36 hrs. After the reaction was complete as monitored by TLC, the organic solvent was removed by evaporation, and the residue was purified by column chromatography on silica gel to obtain Compound (II-3)(2.1 g, 69.3%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): =8.30 (s, 1H), 8.27 (d, J=7.2 Hz, 1H), 7.84 d, J=7.2 Hz, 1H), 7.65 (dd, J=7.2 Hz, 7.0 Hz, 1H), 7.44 (dd, J=7.2 Hz, 7.0 Hz, 1H), 7.40 (s, 1H), 6.80 (s, 1H), 3.49-3.32 (m, 3H), 3.10-2.91 (m, 3H), 2.09 (s, 3H), 1.89-1.77 (m, 4H), 1.36 (s, 9H), 1.13 (d, 6H); LC-MS: m/z=633.3 (M+1).

Embodiment 14

Synthesis of Compound II-16

(50) Under a nitrogen atmosphere, Compound (IV-12) (1.5 g, 4.8 mmol), Compound (111-3) (1.69 g, 4.88 mmol), 4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene (Xantphos) (280 mg, 0.49 mmol, 0.1 eq), palladium acetate (55 mg, 0.25 mmol), and cesium carbonate (4.77 g, 14.6 mmol) were added to tetrahydrofuran, and heated to reflux and reacted for 40 hrs. After the reaction was complete as monitored by TLC, the organic solvent was removed by evaporation, and the residue was purified by column chromatography on silica gel to obtain Compound (II-16)(1.8 g, 72%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): =8.32 (s, 1H), 8.24 (d, J=7.2 Hz, 1H), 7.82 (d, J=7.2 Hz, 1H), 7.65 (dd, J=7.2 Hz, 7.0 Hz, 1H), 7.40 (dd, J=7.2 Hz, 7.0 Hz, 1H), 7.32 (d, J=6.8 Hz, 1H), 6.80 (s, 1H), 3.20-3.32 (m, 9H), 1.36 (s, 9H), 1.13 (d, 6H); LC-MS: m/z=620.2 (M+1).

(51) Using the same procedure as shown in embodiments 13-14, a series of Compounds (11-1 to 43) were obtained, the structure of which was confirmed by LC-MS. The results are shown in Table 8.

(52) TABLE-US-00008 TABLE 8 Structure of Compound II LC-MS No. R.sub.1 A X Y R.sub.2 R.sub.4 R.sub.3-PG (M + 1) 1 i-PrSO.sub.2 C H H CD.sub.3 Me 0 599.3 2 i-PrSO.sub.2 C F H CD.sub.3 Me 617.3 3 i-PrSO.sub.2 C Cl H CD.sub.3 Me 633.3 4 i-PrSO.sub.2 C Br H CD.sub.3 Me 677.2 5 i-PrSO.sub.2 C CF.sub.3 H CD.sub.3 Me 667.3 6 i-PrSO.sub.2 C CN H CD.sub.3 Me 624.3 7 i-PrSO.sub.2 C OMe H CD.sub.3 Me 629.3 8 i-PrSO.sub.2 C Me H CD.sub.3 Me 613.3 9 i-PrSO.sub.2 C Et H CD.sub.3 Me 627.3 10 i-PrSO.sub.2 C NO.sub.2 H CD.sub.3 Me 644.3 11 i-PrSO.sub.2 C Ph H CD.sub.3 Me 0 675.3 12 i-PrSO.sub.2 C H CD.sub.3 Me 625.3 13 i-PrSO.sub.2 C H CD.sub.3 Me 623.3 14 i-PrSO.sub.2 C Cl H CD.sub.2CD.sub.3 Me 649.3 15 i-PrSO.sub.2 C Cl H CD.sub.3 H 619.3 16 i-PrSO.sub.2 C Cl H CD.sub.3 H 620.2 17 i-PrSO.sub.2 C Cl H CD.sub.3 Me 634.3 18 i-PrSO.sub.2 C Cl H CD.sub.3 Cl 653.2 19 i-PrSO.sub.2 C Cl H CD.sub.2CD.sub.3 Cl 0 669.2 20 i-PrSO.sub.2 C Cl H CD.sub.2CD.sub.3 H 635.3 21 i-PrSO.sub.2 C Cl H CD.sub.3 F 637.2 22 i-PrSO.sub.2 C Cl H CD.sub.3 CF.sub.3 687.2 23 i-PrSO.sub.2 C Cl H CD.sub.3 CN 644.2 24 i-PrSO.sub.2 C / CD.sub.3 H 622.3 25 i-PrSO.sub.2 C / CD.sub.3 H 622.3 26 i-PrSO.sub.2 C / CD.sub.3 H 0 584.2 27 i-PrSO.sub.2 C / CD.sub.3 H 584.2 28 i-PrSO.sub.2 C / CD.sub.3 H 620.3 29 i-PrSO.sub.2 C / CD.sub.3 Me 649.3 30 i-PrSO.sub.2 C / CD.sub.3 Me 638.3 31 i-PrSO.sub.2 C / CD.sub.3 Me 00 638.3 32 i-PrSO.sub.2 C 01 / CD.sub.3 Me 02 655.3 33 i-PrSO.sub.2 C Cl H CD.sub.3 H 03 620.2 34 i-PrSO.sub.2 C Cl H CD.sub.3 H 04 717.3 35 i-PrSO.sub.2 C Cl H CD.sub.3 H 05 604.3 36 i-PrSO.sub.2 C Cl H CD.sub.3 H 06 617.3 37 Me.sub.2P(O) C Cl H CD.sub.3 H 07 532.2 38 Me.sub.2P(O) C Cl H CD.sub.3 Me 08 603.3 39 i-PrSO.sub.2 N / H CD.sub.3 H 09 586.3 40 i-PrSO.sub.2 N / H CD.sub.3 Me 0 600.3 41 i-PrSO.sub.2 N / H CD.sub.3 H 587.3 42 i-PrSO.sub.2 N / H CD.sub.3 Me 601.3 43 i-PrSO.sub.2 N / H CD.sub.3 H 584.3

7. Synthesis of Compound I

(53) Compound II protected with a Boc group was deprotected, and then subjected to a salt formation reaction with a hydrogen chloride-ethanol solution to obtain a hydrochloride of the target Compound (I) of Formula (I). If no protecting group was present in Compound II, the salt formation reaction with the hydrogen chloride-ethanol solution was carried out directly without the deprotection reaction. The reaction route was as follows.

(54) ##STR00114##

Embodiment 15

Synthesis of Compound I-3

(55) Compound (11-3) (2.1 g, 3.3 mmol) was added to a solution of dichloromethane (10 mL) and trifluoroacetic acid (10 mL), and stirred at room temperature for 2 hrs. After the reaction was complete as monitored by TLC, the organic solvent was removed by evaporation, and the residue was dissolved in dichloromethane (100 mL), washed with a saturated sodium carbonate solution, dried, filtered and concentrated, to obtain a free amine. The free amine product was dissolved in dichloromethane (20 mL), a hydrogen chloride/ethanol solution (4 M, 10 mL) was added, and then the resulting solution was stirred at room temperature for 2 hrs. A white solid was precipitated, filtered under suction, and dried to obtain Compound (1-3) (1.8 g, 95.2%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) =8.32 (s, 1H), 8.27 (d, J=7.2 Hz, 1H), 7.88 (d, J=7.2 Hz, 1H), 7.67 (dd, J=7.2 Hz, 7.0 Hz, 1H), 7.45 (dd, J=7.2 Hz, 7.0 Hz, 1H), 7.42 (s, 1H), 6.79 (s, 1H), 3.49-3.32 (m, 3H), 3.10-2.91 (m, 3H), 2.09 (s, 3H), 1.89-1.77 (m, 4H), 1.13 (d, 6H); LC-MS: m/z=533.3 (M+1).

Embodiment 16

Synthesis of Compound 1-16

(56) Compound (11-16) (1.7 g, 3.3 mmol) was added to a solution of dichloromethane (10 mL) and trifluoroacetic acid (10 mL), and stirred at room temperature for 3 hrs. After the reaction was complete as monitored by TLC, the organic solvent was removed by evaporation, and the residue was then dissolved in dichloromethane (100 mL), washed with a saturated sodium carbonate solution, dried, filtered and concentrated, to obtain a free amine. The free amine product was dissolved in dichloromethane (20 mL), a hydrogen chloride/ethanol solution (4 M, 10 mL) was added, and then stirred at room temperature for 2 hrs. A white solid was precipitated, filtered under suction, and dried to obtain Compound (1-16) (1.4 g, 96%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): =8.30 (s, 1H), 8.20 (d, J=7.2 Hz, 1H), 7.82 (d, J=7.2 Hz, 1H), 7.62 (dd, J=7.2 Hz, 7.0 Hz, 1H), 7.42 (dd, J=7.2 Hz, 7.0 Hz, 1H), 7.36 (d, J=6.8 Hz, 1H), 6.80 (s, 1H), 3.20-3.32 (m, 9H), 1.13 (d, 6H); LC-MS: m/z=520.2 (M+1).

(57) Using the same procedure as shown in embodiments 15-16, a series of Compounds (I-1 to 43) were obtained, the structure of which was confirmed by LC-MS. The results are shown in Table 9.

(58) TABLE-US-00009 TABLE 9 Structure of Compound I LC-MS No. R1: A X Y R.sub.2 R.sub.4 R.sub.3 (M + 1) 1 i-PrSO.sub.2 C H H CD.sub.3 Me 499.3 2 i-PrSO.sub.2 C F H CD.sub.3 Me 517.3 3 i-PrSO.sub.2 C Cl H CD.sub.3 Me 533.3 4 i-PrSO.sub.2 C Br H CD.sub.3 Me 577.2 5 i-PrSO.sub.2 C CF.sub.3 H CD.sub.3 Me 567.3 6 i-PrSO.sub.2 C CN H CD.sub.3 Me 0 524.3 7 i-PrSO.sub.2 C OMe H CD.sub.3 Me 529.3 8 i-PrSO.sub.2 C Me H CD.sub.3 Me 513.3 9 i-PrSO.sub.2 C Et H CD.sub.3 Me 527.3 10 i-PrSO.sub.2 C NO.sub.2 H CD.sub.3 Me 544.3 11 i-PrSO.sub.2 C Ph H CD.sub.3 Me 575.3 12 i-PrSO.sub.2 C H CD.sub.3 Me 525.3 13 i-PrSO.sub.2 C H CD.sub.3 Me 523.3 14 i-PrSO.sub.2 C Cl H CD.sub.2CD.sub.3 Me 0 549.3 15 i-PrSO.sub.2 C Cl H CD.sub.3 H 519.3 16 i-PrSO.sub.2 C Cl H CD.sub.3 H 520.2 17 i-PrSO.sub.2 C Cl H CD.sub.3 Me 534.3 18 i-PrSO.sub.2 C Cl H CD.sub.3 Cl 553.2 19 i-PrSO.sub.2 C Cl H CD.sub.2CD.sub.3 Cl 569.2 20 i-PrSO.sub.2 C Cl H CD.sub.2CD.sub.3 H 535.2 21 i-PrSO.sub.2 C Cl H CD.sub.3 F 537.2 22 i-PrSO.sub.2 C Cl H CD.sub.3 CF.sub.3 587.2 23 i-PrSO.sub.2 C Cl H CD.sub.3 CN 544.3 24 i-PrSO.sub.2 C 0 / CD.sub.3 H 622.3 25 i-PrSO.sub.2 C / CD.sub.3 H 622.3 26 i-PrSO.sub.2 C / CD.sub.3 H 584.2 27 i-PrSO.sub.2 C / CD.sub.3 H 584.2 28 i-PrSO.sub.2 C / CD.sub.3 H 620.3 29 i-PrSO.sub.2 C 0 / CD.sub.3 Me 549.3 30 i-PrSO.sub.2 C / CD.sub.3 Me 538.3 31 i-PrSO.sub.2 C / CD.sub.3 Me 538.3 32 i-PrSO.sub.2 C / CD.sub.3 Me 555.3 33 i-PrSO.sub.2 C Cl H CD.sub.3 H 620.2 34 i-PrSO.sub.2 C Cl H CD.sub.3 H 617.3 35 i-PrSO.sub.2 C Cl H CD.sub.3 H 0 604.3 36 i-PrSO.sub.2 C Cl H CD.sub.3 H 617.3 37 Me.sub.2P(O) C Cl H CD.sub.3 H 532.2 38 Me.sub.2P(O) C Cl H CD.sub.3 Me 503.3 39 i-PrSO.sub.2 N / H CD.sub.3 H 486.3 40 i-PrSO.sub.2 N / H CD.sub.3 Me 500.3 41 i-PrSO.sub.2 N / H CD.sub.3 H 487.3 42 i-PrSO.sub.2 N / H CD.sub.3 Me 501.3 43 i-PrSO.sub.2 N / H CD.sub.3 H 484.3

II. Biological Activity Test

(59) Anaplastic Lymphoma Kinase (ALK) Inhibitory Activity

(60) The in-vitro kinase assay was performed using the HTRF kinEASE TK kit available from Cisbio. The operation steps are indicated in the instructions of the kit. This method was used to detect the inhibitory effect of the compound to be tested on the activity of ALK enzymes in vitro, including wild-type ALK (Cat.PV3867, Invitrogen), ALK L1196M (Cat. PV6168, Life technologies), and ALK F1174L (Cat. PV6160, Life technologies). The specific operation steps were as follows.

(61) (1) First, a 2.5% DMSO solution was prepared with 1 kinase buffer previously formulated (where a too high concentration of DMSO had influence on the reaction, and the final concentration of DMSO was controlled to 1%), and then the compound to be tested was diluted with the 2.5% DMSO solution corresponding to the enzyme. The compound had a screening concentration of 100 nM, 10 nM and 1 nM. In addition to the control wells, 4 L of the diluted compound solution to be tested was added to the reaction wells, and 4 L of the 2.5% DMSO solution corresponding to the ALK enzyme previously formulated was added to the control wells.

(62) (2) 2 L of a TK-biotin substrate solution previously formulated in a substrate concentration corresponding to the ALK enzyme was added to all reaction wells.

(63) (3) 2 L of a previously prepared enzyme solution of corresponding concentration was added to all the reaction wells except the negative wells, and 2 L of the 1 kinase buffer corresponding to the enzyme was added to the negative wells to make up the volume. The plate was sealed with a membrane, and incubated for 10 min at room temperature after mixing uniformly, such that the compounds were bond to the enzyme fully.

(64) (4) 2 L of an ATP solution in a concentration corresponding to the ALK enzyme was added to all the reaction wells to initiate the kinase reaction, where the enzyme reaction time by ALK was 60 minutes.

(65) (5) An ALK test solution was prepared 5 minutes before the end of the kinase reaction. Streptavidin-XL665 and TK antibody europium cryptate (1:100) solutions for assay having a concentration corresponding to the enzyme were prepared using the detection buffer in the kit.

(66) (6) After the completion of the kinase reaction, 5 l of diluted Streptavidin-XL665 was added to all the reaction wells respectively, and mixed uniformly, and then the diluted TK antibody europium cryptate solution for assay was added immediately.

(67) (7) The plate was sealed, mixed uniformly and allowed to react at room temperature for 1 h. The fluorescence signal (excitation at 320 nm, and emission at 665 nm and 615 nm) was detected using the ENVISION instrument (Perkinelmer). The inhibition rate for each well was calculated from the values of the fully active wells and the background signal wells. The values of replicated wells were averaged, and the half-maximal inhibitory activity (IC50) of each compound to be tested was fitted with a professional drawing analysis software PRISM 5.0.

(68) The inhibitory activity of each compound against ALK enzyme obtained by the above method is as shown in Table 10.

(69) TABLE-US-00010 TABLE 10 Inhibitory activity of each compound for ALK enzyme IC.sub.50 No. R1: A X Y R.sub.2 R.sub.4 R.sub.3 range 1 i-PrSO.sub.2 C H H CD.sub.3 Me +++ 2 i-PrSO.sub.2 C F H CD.sub.3 Me 0 +++ 3 i-PrSO.sub.2 C Cl H CD.sub.3 Me +++ 4 i-PrSO.sub.2 C Br H CD.sub.3 Me +++ 5 i-PrSO.sub.2 C CF.sub.3 H CD.sub.3 Me +++ 6 i-PrSO.sub.2 C CN H CD.sub.3 Me +++ 7 i-PrSO.sub.2 C OMe H CD.sub.3 Me +++ 8 i-PrSO.sub.2 C Me H CD.sub.3 Me +++ 9 i-PrSO.sub.2 C Et H CD.sub.3 Me +++ 10 i-PrSO.sub.2 C NO.sub.2 H CD.sub.3 Me +++ 11 i-PrSO.sub.2 C Ph H CD.sub.3 Me ++ 12 i-PrSO.sub.2 C 0 H CD.sub.3 Me +++ 13 i-PrSO.sub.2 C H CD.sub.3 Me +++ 14 i-PrSO.sub.2 C Cl H CD.sub.2CD.sub.3 Me +++ 15 i-PrSO.sub.2 C Cl H CD.sub.3 H +++ 16 i-PrSO.sub.2 C Cl H CD.sub.3 H +++ 17 i-PrSO.sub.2 C Cl H CD.sub.3 Me +++ 18 i-PrSO.sub.2 C Cl H CD.sub.3 Cl +++ 19 i-PrSO.sub.2 C Cl H CD.sub.2CD.sub.3 Cl +++ 20 i-PrSO.sub.2 C Cl H CD.sub.2CD.sub.3 H 0 +++ 21 i-PrSO.sub.2 C Cl H CD.sub.3 F +++ 22 i-PrSO.sub.2 C Cl H CD.sub.3 CF.sub.3 +++ 23 i-PrSO.sub.2 C Cl H CD.sub.3 CN +++ 24 i-PrSO.sub.2 C / CD.sub.3 H ++ 25 i-PrSO.sub.2 C / CD.sub.3 H ++ 26 i-PrSO.sub.2 C / CD.sub.3 H ++ 27 i-PrSO.sub.2 C 00 / CD.sub.3 H 01 ++ 28 i-PrSO.sub.2 C 02 / CD.sub.3 H 03 ++ 29 i-PrSO.sub.2 C 04 / CD.sub.3 Me 05 ++ 30 i-PrSO.sub.2 C 06 / CD.sub.3 Me 07 ++ 31 i-PrSO.sub.2 C 08 / CD.sub.3 Me 09 ++ 32 i-PrSO.sub.2 C 0 / CD.sub.3 Me ++ 33 i-PrSO.sub.2 C Cl H CD.sub.3 H +++ 34 i-PrSO.sub.2 C Cl H CD.sub.3 H +++ 35 i-PrSO.sub.2 C Cl H CD.sub.3 H +++ 36 i-PrSO.sub.2 C Cl H CD.sub.3 H +++ 37 Me.sub.2P(O) C Cl H CD.sub.3 H +++ 38 Me.sub.2P(O) C Cl H CD.sub.3 Me +++ 39 i-PrSO.sub.2 N / H CD.sub.3 H +++ 40 i-PrSO.sub.2 N / H CD.sub.3 Me +++ 41 i-PrSO.sub.2 N / H CD.sub.3 H 0 +++ 42 i-PrSO.sub.2 N / H CD.sub.3 Me +++ 43 i-PrSO.sub.2 N / H CD.sub.3 H +++ 44 i-PrSO.sub.2 C Cl H CD(CD.sub.3).sub.2 Me ++ 45 i-PrSO.sub.2 C Cl H Me Me ++ IC.sub.50 range: +++ represents 1-10 nM; ++ represents 10-100 nM; and + represents 100-1 uM.

(70) Growth Inhibition Test of the Series of Compounds on Various Tumor Cells (CCK8 Detection)

(71) 1. Cell lines:

(72) (1) Ba/F3 mouse IL-3 dependent pro-B lymphocyte cell line, RPMI 1640+10% FBS+10 ng/ml Interleukin-3+1% Sodium Pyruvate;

(73) (2) Karpas 299 human T lymphoma cell line, RPMI 1640+10% FBS+1% Sodium Pyruvate;

(74) (3) NCI-H2228 human non-small cell lung cancer cell line, RPMI 1640+10% FBS+1% Sodium Pyruvate;

(75) (4) NCI-H3122 human lung cancer cell line, RPMI 1640+10% FBS+1% Sodium Pyruvate;

(76) 2. Reagents and materials:

(77) CCK8 kit; anti-tumor compounds; DMSO

(78) 3. Test method:

(79) (1) Cell culture a) Cells in logarithmic growth phase were collected, counted, and re-suspended in a complete medium. b) The cell concentration was adjusted to an appropriate concentration, and inoculated into a 96-well plate in an amount of 100 l of cell suspension per well. c) The cells were incubated for 24 hrs in an incubator at 37 C. with 100% relative humidity and 5% CO.sub.2.

(80) (2) Test of relative inhibition rate 1) Cells in logarithmic growth phase were collected, counted, and re-suspended in a complete medium. The cell concentration was adjusted to an appropriate concentration (determined according to the optimal test result of cell density), and inoculated into a 96-well plate in an amount of 100 l of cell suspension per well. The cells were incubated for 24 hrs in an incubator at 37 C. with 100% relative humidity and 5% CO.sub.2. 2) The compound to be tested was diluted with the medium to the corresponding action concentration, and the cells were added in an amount of 25 l/well. The final concentration of the compound was from 10 M to 0 M, and a total of 10 concentration points were given by dilution over a 4-fold gradient. 3) The cells were incubated for 72 hrs in an incubator at 37 C. with 100% relative humidity and 5% CO.sub.2. 4) The medium was aspirated off, a complete medium containing 10% CCK-8 was added and the cells were incubated in an incubator at 37 C. for 2-4 hrs. 5) The absorbance at 450 nm was measured on the SpectraMax M5 Microplate Reader after gentle shaking, and the absorbance at 650 nm was used as a reference to calculate the inhibition rate.

(81) 4. Data processing and results

(82) The inhibition rate of the drug on tumor cell growth was calculated by a formula below: Tumor cell growth inhibition %=[(A.sub.cA.sub.s)/(A.sub.c-A.sub.b)]100%

(83) A.sub.s: OA of the sample (cells+CCK-8+compound to be tested)

(84) A.sub.c: OA of the negative control (cells+CCK-8+DMSO)

(85) A.sub.b: OA of the positive control (medium+CCK-8+DMSO)

(86) The IC.sub.50 curve was fitted using the software Graphpad Prism 5 and using the calculation formula log(inhibitor) vs response and the IC.sub.50 value was calculated. The inhibitory activities (IC.sub.50) of various compounds for the growth of various tumor cells are shown in Table 11.

(87) TABLE-US-00011 TABLE 11 Inhibitory activities (IC.sub.50) of various compounds against the growth of various tumor cells IC.sub.50 (nM) Ba/F3 Karpas 299 NCI-H2228 NCI-H3122 Compound cells cells cells cells I-3: 1242 7.643 41.3 4.031 I-14: 1993 5.23 45.5 6.09 2891 45.68 111.3 28.41 Ceritinib 2387 35.57 97.06 27.2

(88) In-Vivo Pharmacodynamic Evaluation

(89) SCID Beige mice, female, 5-6 weeks old, weighed 18+2 g, purchased from Beijing HFK Bioscience Co., Ltd., bred in an SPF level environment.

(90) NCI-H2228 cells were cultured in PRMI-1640 containing 10% fetal bovine serum (FBS) and 1% sodium pyruvate. The cells were cultured in an incubator at 37 C. with 5% CO.sub.2.

(91) Establishment of subcutaneous tumor transplantation model in nude mice by cell inoculation: Tumor cells in logarithmic growth phase were collected, counted and re-suspended in PRMI-1640 basal medium. Matrigel was added at a ratio of 1:1, and the concentration of the cell suspension was adjusted to 610.sup.7/ml. Tumor cells were subcutaneously inoculated to the right back of nude mice in an amount of 610.sup.6/0.1 mL/mouse using a 1 mL syringe (with a 4 gauge needle).

(92) When the tumor volume reached about 200 mm.sup.3, the animals were randomly grouped according to randomized block method, so that the difference in tumor volume of each group was within 10% of the mean. There were 5 groups in total, each having 8 animals. The day of grouping was recorded as Day 0, and the animals were administered immediately after grouping. Compound I-3 of the present invention and Ceritinib were orally administered once a day for consecutive 14 days, and then observed for 7 days. The weight and tumor size of the animals were measured twice a week during the experiment. The clinical symptoms were observed and recorded daily. After the animals were weighed for the last time on Day 21, they were sacrificed by euthanization with CO.sub.2. The tumors were taken, weighed and photographed, and the average tumor size was calculated, to investigate the growth inhibitory effect of the test substance Compound I-3 on human lung cancer NCI-H2228 nude mice xenografts under the experimental conditions.

(93) When the tumor in the tumor-bearing mice grew to a measurable size, the mice were randomly divided into 5 groups according to the principle of equal mean tumor volume using SPSS 17.0 software. Compound I-3 was administered by intragastric administration at a dosage of 30, 10, and 3 mg/kg per day. The positive control ceritinib was administered by intragastric administration at a dose of 10 mg/kg per day in a volume of 0.1 ml/10 g. These agents were administered once a day for consecutive 14 days. The animals in the negative control group were given an equal amount of solvent (1% DMSO in physiological saline). During the administration and recovery periods, the body weight and tumor size of the mice were measured 2-3 times per week. The tumor volume and relative tumor volume were calculated based on the measured data. The tumor volume (TV) was calculated by a formula below: TV=1/2ab.sup.2, where a and b represent the major and minor diameters of the tumor, respectively. According to the measurement results, the relative tumor volume (RTV) was calculated, by a formula below: RTV=V.sub.t/V.sub.0, where V.sub.0 is the tumor volume at the start of the experiment, and V.sub.t is the tumor volume measured each time. The evaluation indices for the anti-tumor activity were the relative tumor growth rate T/C (%), which was calculated by a formula below: T/C (%)=T.sub.RTV/C.sub.RTV100%, where T.sub.RTV is the RTV of the treatment group and C.sub.RTV is the RTV of the negative control group; and the relative tumor growth inhibition rate (tumor inhibition rate) (%)=(1T/C)100%. The results are shown in Table 12.

(94) TABLE-US-00012 TABLE 12 Therapeutic effect of the compound of the present invention on NCI-H2228 nude mice xenografts Dose Administration RTV IR (%) T/C (%) Group (mg/kg) mode d.sub.21 Blank 0 qd 3.75 0.27 group I-3: 3 qd 3.12 0.23 12.86 77.45 I-3: 10 qd 1.70 0.20*** 53.39 45.33 I-3: 30 qd 0.79 0.17*** 77.14 21.07 Ceritinib 10 qd 2.68 0.28* 26.07 71.47

(95) The above description is only preferred embodiments of the present invention and not intended to limit the present invention, it should be noted that those of ordinary skill in the art can further make various modifications and variations without departing from the technical principles of the present invention, and these modifications and variations also should be considered to be within the scope of protection of the present invention.