SALT AND CRYSTAL FORM OF MTORC1/2 DUAL KINASE ACTIVITY INHIBITOR AND PREPARATION METHOD THEREFOR

Abstract

Disclosed are a salt and a crystal form of an mTORC1/2 dual kinase inhibitor and a preparation method therefor. Also disclosed is use of the salt and crystal form in the preparation of a medicament related to the mTORC1/2 dual kinase inhibitor.

##STR00001##

Claims

1. A crystal form A of the compound of formula (I), wherein the X-ray powder diffraction pattern thereof has characteristic diffraction peaks at the following 2θ angles: 16.355±0.200°, 19.847±0.200°, and 21.319±0.200°, ##STR00017##

2. The crystal form A of the compound of formula (I) according to claim 1, wherein the X-ray powder diffraction pattern thereof has characteristic diffraction peaks at the following 2θ angles: 8.169±0.200°, 10.986±0.200°, 13.394±0.200°, 16.355±0.200°, 17.084±0.200°, 19.847±0.200°, 21.319±0.200°, and 22.154±0.200°, and/or, the differential scanning calorimetry curve thereof has an endothermic peak at 221.62±3.00° C.; and/or, the thermal gravimetric analysis curve thereof has a weight loss of 0.08225% at 122.98±3.00° C., and a further weight loss of 0.4156% at 262.62±3.00° C.

3. The crystal form A of the compound of formula (I) according to claim 2, wherein the X-ray powder diffraction pattern thereof has characteristic diffraction peaks at the following 2θ angles: 8.169±0.200°, 9.903±0.200°, 10.986±0.200°, 13.394±0.200°, 13.931±0.200°, 15.330±0.200°, 16.355±0.200°, 17.084±0.200°, 19.847±0.200°, 21.319±0.200°, 22.154±0.200°, and 23.475±0.200°.

4. The crystal form A of the compound of formula (I) according to claim 3, wherein the X-ray powder diffraction pattern thereof has characteristic diffraction peaks at the following 2θ angles: 8.169°, 9.119°, 9.551°, 9.903°, 10.986°, 12.525°, 13.394°, 13.931°, 15.330°, 15.589°, 15.923°, 16.355°, 16.743°, 17.084°, 17.815°, 18.268°, 19.149°, 19.414°, 19.847°, 20.273°, 21.319°, 22.154°, 23.475°, 23.953°, 24.710°, 25.134°, 25.805°, 26.054°, 26.232°, 26.772°, 28.048°, 29.254°, 29.469°, 29.958°, 31.617°, 31.838°, 33.074°, 33.924°, 34.281°, 36.061°, 36.520°, and 38.052°.

5. The crystal form A of the compound of formula (I) according to claim 1, wherein the XRPD pattern thereof is shown in FIG. 1. and/or, the DSC pattern thereof is shown in FIG. 2; and/or, the TGA graph thereof is shown in FIG. 3.

6. (canceled)

7. (canceled)

8. (canceled)

9. (canceled)

10. A crystal form B of the compound of formula (I), wherein the X-ray powder diffraction pattern thereof has characteristic diffraction peaks at the following 2θ angles: 6.295±0.200°, 7.851±0.200°, 11.324±0.200°, and 16.351±0.200°; ##STR00018##

11. The crystal form B of the compound of formula (I) according to claim 10, wherein the X-ray powder diffraction pattern thereof has characteristic diffraction peaks at the following 2θ angles: 6.295±0.200°, 7.851±0.200°, 8.621±0.200°, 11.324±0.200°, 14.184±0.200°, 15.287±0.200°, 16.351±0.200°, and 26.114±0.200°.

12. The crystal form B of the compound of formula (I) according to claim 11, wherein the X-ray powder diffraction pattern thereof has characteristic diffraction peaks at the following 2θ angles: 6.295±0.200°, 7.851±0.200°, 8.621±0.200°, 11.324±0.200°, 14.184±0.200°, 15.287±0.200°, 16.351±0.200°, 17.087±0.200°, 18.563±0.200°, 19.531±0.200°, 20.791±0.200°, and 26.114±0.200°.

13. The crystal form B of the compound of formula (I) according to claim 12, wherein the X-ray powder diffraction pattern thereof has characteristic diffraction peaks at the following 2θ angles: 6.295°, 7.851°, 8.166°, 8.621°, 9.109°, 9.528°, 9.897°, 10.985°, 11.324°, 11.973°, 12.586°, 12.876°, 13.391°, 13.928°, 14.184°, 15.287°, 15.879°, 16.351°, 16.590°, 17.087°, 17.276°, 18.563°, 18.933°, 19.531°, 19.827°, 20.791°, 21.285°, 22.136°, 22.585°, 23.489°, 23.650°, 24.151°, 24.970°, 25.328°, 25.900°, 26.114°, 26.731°, 28.109°, 28.659°, 29.551°, 29.908°, 31.539°, 36.554°, and 38.644°.

14. The crystal form B of the compound of formula (I) according to claim 13, wherein the XRPD pattern thereof is shown in FIG. 4.

15. Compound of formula (II), ##STR00019##

16. The crystal form C of the compound of formula (II) according to claim 15, wherein the X-ray powder diffraction pattern thereof has characteristic diffraction peaks at the following 2θ angles: 5.802±0.200°, 7.991±0.200°, 11.618±0.200°, and 16.567±0.200°.

17. The crystal form C of the compound of formula (II) according to claim 16, wherein the X-ray powder diffraction pattern thereof has characteristic diffraction peaks at the following 2θ angles: 5.802±0.200°, 7.991±0.200°, 11.618±0.200°, 13.138±0.200°, 15.487±0.200°, 15.959±0.200°, 16.567±0.200°, and 18.268±0.200°; and/or, the differential scanning calorimetry curve thereof has an endothermic peak at 229.91±3.00° C.; and/or, the thermal gravimetric analysis curve thereof has a weight loss of 0.1862% at 143.66±3.00° C.

18. The crystal form C of the compound of formula (II) according to claim 17, wherein the X-ray powder diffraction pattern thereof has characteristic diffraction peaks at the following 2θ angles: 5.802±0.200°, 7.991±0.200°, 11.618±0.200°, 13.138±0.200°, 15.487±0.200°, 15.959±0.200°, 16.567±0.200°, 18.268±0.200°, 18.802±0.200°, 19.505±0.200°, 22.309±0.200°, and 23.633±0.200°.

19. The crystal form C of the compound of formula (II) according to claim 18, wherein the X-ray powder diffraction pattern thereof has characteristic diffraction peaks at the following 2θ angles: 5.802°, 7.991°, 11.127°, 11.618°, 13.138°, 14.219°, 14.637°, 15.487°, 15.959°, 16.567°, 18.268°, 18.802°, 19.098°, 19.505°, 19.881°, 20.343°, 21.061°, 21.340°, 21.977°, 22.309°, 22.504°, 23.298°, 23.633°, 24.403°, 24.954°, 25.286°, 25.557°, 26.176°, 26.986°, 28.618°, 29.472°, 29.805°, 31.796°, 32.052°, 32.666°, 33.433°, 34.423°, 34.836°, 38.939°, and 39.312°.

20. The crystal form C of the compound of formula (II) according to claim 16, wherein the XRPD pattern thereof is as shown in FIG. 5; and/or, the DSC pattern thereof is shown in FIG. 6; and/or, the TGA pattern thereof is as shown in FIG. 7.

21. (canceled)

22. (canceled)

23. (canceled)

24. (canceled)

25. A method for preparing the crystal form A of compound of formula (I) according to claim 1, comprising: (a) adding the compound of formula (I) to a solvent; (b) stirring at 30-50° C. for 45-55 hours; (c) obtaining a solid by centrifugation as the crystal form A of compound of formula (I); wherein said solvent is water, acetone, acetonitrile, tetrahydrofuran, methyl t-butyl ether or ethyl acetate.

26. (canceled)

27. A method for inhibiting the mTORC1/2 dual kinase in a subject in need thereof, comprising administering a therapeutically effective amount of the crystal form A of compound of formula (I) according to claim 1 to the subject.

28. A method for inhibiting the mTORC1/2 dual kinase in a subject in need thereof, comprising administering a therapeutically effective amount of the crystal form B of compound of formula (I) according to claim 10 to the subject.

29. A method for inhibiting the mTORC1/2 dual kinase in a subject in need thereof, comprising administering a therapeutically effective amount of the compound of formula (II) according to claim 15 to the subject.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0105] FIG. 1 is XRPD pattern of the crystal form A of the compound of formula (I) measured by Cu-Kα radiation;

[0106] FIG. 2 is DSC pattern of the crystal form A of the compound of formula (I);

[0107] FIG. 3 is TGA graph of the crystal form A of the compound of formula (I);

[0108] FIG. 4 is XRPD pattern of the crystal form B of the compound of formula (I) measured by Cu-Kα radiation;

[0109] FIG. 5 is XRPD pattern of the crystal form C of the compound of formula (II) measured by Cu-Kα radiation;

[0110] FIG. 6 is DSC pattern of the crystal form C of the compound of formula (II);

[0111] FIG. 7 is TGA pattern of the crystal form C of the compound of formula (II);

[0112] FIG. 8 is XRPD pattern of the crystal form D of the compound of formula (III) measured by Cu-Kα radiation;

[0113] FIG. 9 is XRPD pattern of the crystal form E of the compound of formula (IV) measured by Cu-Kα radiation;

[0114] FIG. 10 is XRPD pattern of the crystal form F of the compound of formula (V) measured by Cu-Kα radiation;

[0115] FIG. 11 is XRPD pattern of the crystal form G of the compound of formula (VI) measured by Cu-Kα radiation;

[0116] FIG. 12 is XRPD pattern of the crystal form H of the compound of formula (VII) measured by Cu-Kα radiation;

[0117] FIG. 13 is XRPD pattern of the crystal form I of the compound of formula (VIII) measured by Cu-Kα radiation;

[0118] FIG. 14 is XRPD pattern of the crystal form J of the compound of formula (IX) measured by Cu-Kα radiation;

[0119] FIG. 15 is DVS pattern of the crystal form A of the compound of formula (I).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0120] In order to better understand the content of the invention, the following will be further described in combination with specific examples, but the specific embodiments do not limit the content of the invention.

EXAMPLE 1: MANUFACTURE OF THE COMPOUND OF FORMULA (I)

[0121] ##STR00013## ##STR00014## ##STR00015##

[0122] First Step

[0123] Compound 1a (20.0 g, 104 mmol, 1.00 eq) and concentrated ammonia (200 ml, 1.45 mol, 14.0 eq) were sealed in an autoclave and stirred at 130° C. for 24 hours under a pressure of about 0.9 MPa. The reaction solution was concentrated to obtain compound 1b.

[0124] MS-ESI calculated [M+H].sup.+ 173 and 175, founded 173 and 175. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ: 8.03 (d, J=8.0 Hz, 1H), 7.56 (br s, 2H), 6.61 (d, J=8.0 Hz, 1H).

[0125] Second Step

[0126] Compound 1b (17.0 g, 98.5 mmol, 1.00 eq), ammonium chloride (10.5 g, 197 mmol, 2.00 eq), 1-hydroxybenzotriazole (13.3 g, 98.5 mmol, 1.00 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (18.9 g, 98.5 mmol, 1.00 eq) and diisopropyl ethylamine (38.2 g, 296 mmol, 3.00 eq) were dissolved in N, N-dimethylformamide (200.0 mL). The mixture was stirred at 20° C. for 16 hours. After the completion of the reaction, the solvent was subjected to a rotary drying under reduced pressure, water (200 mL) was added, and the resultant was extracted with ethyl acetate (200 mL×3). The combined organic phases were dried on anhydrous sodium sulfate, filtered, and then subjected to column chromatography (1/1 petroleum ether/ethyl acetate, Rf=0.4) to obtain the compound. The ethyl acetate (50 mL) was beaten for ten minutes to obtain compound 1c.

[0127] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ: 7.96 (d, J=8.0 Hz, 2H), 7.62 (br s, 2H), 7.40 (br s, 1H), 6.61 (d, J=8.0 Hz, 1H).

[0128] Third Step

[0129] Compound 1c (8.00 g, 46.6 mmol, 1.00 eq) and oxalyl chloride (7.1 g, 56.0 mmol, 4.9 ml, 1.00 eq) were successively added to toluene (200 mL). The mixture was stirred at 110° C. for 15 hours, cooled to room temperature, filtered and dried to obtain compound 1d.

[0130] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ: 8.24 (d, J=8.0 Hz, 1H), 7.30 (d, J=8.0 Hz, 1H).

[0131] Fourth Step

[0132] Compound 1d (6.00 g, 30.4 mmol, 1.00 eq) and diisopropyl ethylamine (11.8 g, 91.1 mmol, 15.9 ml, 3.00 eq) were successively added to toluene (100 mL), and the resulting mixture was stirred at 70° C. for half an hour, cooled to room temperature and added dropwise with phosphorus oxychloride (14.0 g, 91.1 mmol, 8.5 ml, 3.00 eq). The mixture was stirred at 100° C. for 2 hours, cooled to room temperature, concentrate and subjected to column chromatography (3/1 petroleum ether/ethyl acetate, Rf=0.4) to obtain compound 1e.

[0133] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ: 8.45 (d, J=8.0 Hz, 1H), 7.63 (d, J=8.3 Hz, 1H).

[0134] Fifth Step

[0135] Compound 1e (1.90 g, 8.10 mmol, 1.00 eq), (S)-2-methylmorphorphine (819 mg, 8.10 mmol, 1.00 eq) and diisopropyl ethylamine (2.09 g, 16.2 mmol, 2.83 ml, 2.00 eq) were dissolved in dichloromethane (50 mL). The resulting solution was reacted at 25° C. for 2 hours. After the completion of the reaction, the resultant was concentrated and subjected to column chromatography (3/1 petroleum ether/ethyl acetate) to obtain compound 1f.

[0136] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ: 8.47 (d, J=8.8 Hz, 1H), 7.55 (d, J=8.8 Hz, 1H), 4.71-4.72 (m, 1H), 4.12-4.09 (m, 1H), 3.92-3.91 (m, 1H), 3.84-3.74 (m, 1H), 3.73-3.64 (m, 2H), 3.54-3.53 (m, 1H), 1.46 (d, J=6.8 Hz, 3H).

[0137] Sixth Step

[0138] Compounds 1f (2 g, 6.0 mmol, 1 eq), 35a (993 mg, 6.0 mmol, 1 eq), sodium carbonate (1.9 g, 18.1 mmol, 3 eq) and dichloro(triphenylphosphine) palladium (211 mg, 301 μmol, 0.05 eq) were dissolved in anhydrous dioxane (35 mL) and water (7.0 mL). The above solution was reacted at 70° C. for 19 hours in nitrogen environment. After the completion of the reaction, the reaction solution was concentrated under reduced pressure, then added with water (15 mL) and extracted with ethyl acetate (20 mL×3) The organic phase was treated with anhydrous sodium sulfate, collected under reduced pressure and separated by column chromatography (methanol/ethyl acetate; Rf=0.28) to obtain 35b.

[0139] MS-ESI calculated [M+H].sup.+ 384 and 386, founded 384 and 386.

[0140] Seventh Step

[0141] Compound 35b (100 mg, 261 μmol, 1.00 eq), 1i (46.8 mg, 313 μMol, 1.20 eq) and DIPEA (101 mg, 782 μmol, 136 μL, 3.00 eq) were dissolved in DMSO (2.00 mL), and the mixed solution was reacted at 80° C. for 18 hours. After the completion of the reaction, the reaction solution was purified through high performance liquid chromatography to obtain the compound of formula (I).

[0142] MS-ESI calculated [M+H].sup.+ 461, founded 461. .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 8.60 (s, 1H), 8.18 (br d, J=8.0 Hz, 1H), 7.98 (d, J=8.4 Hz, 1H), 7.92 (br d, J=8.0 Hz, 1H), 7.51 (t, J=8.0 Hz, 1H), 7.43 (d, J=8.4 Hz, 1H), 6.46 (br s, 1H), 5.62 (br s, 1H), 4.30 (br s, 1H), 4.05-3.59 (m, 12H), 1.40 (d, J=6.8 Hz, 3H), 0.80-0.73 (m, 2H), 0.61 (br s, 2H).

EXAMPLE 2: PREPARATION OF THE CRYSTAL FORM A OF COMPOUND OF FORMULA (I)

[0143] About 50.74 mg of compound of formula (I) was weighed and charged into a 2.0 mL glass vial, and thereto 200 μL of water was added to form a suspension. After adding magnetons, the above sample was placed on a magnetic oscillator (40° C., 700 rpm) and stirred for 53 hours (away from light), then the solid was separated through centrifugation and dried overnight in a vacuum drying oven to obtain the crystal form of compound A of formula (I).

EXAMPLE 3: PREPARATION OF THE CRYSTAL FORM B OF COMPOUND OF FORMULA (I)

[0144] About 50.63 mg of compound of formula (I) was weighed and charged into a 2.0 mL glass vial, and thereto 200 μL of methanol was added to form a suspension or solution. After adding magnetons, the above sample was placed on a magnetic oscillator (40° C., 700 rpm) and stirred for 53 hours (away from light), then the solid was separated through centrifugation and dried overnight in a vacuum drying oven to obtain the crystal form of compound B of formula (I).

EXAMPLE 4: PREPARATION OF THE CRYSTAL FORM C OF COMPOUND OF FORMULA (II)

[0145] ##STR00016##

[0146] About 92 mg (0.2 mmol) of the compound was weighed and charged into a 40 mL vial, and thereto 10 mL of THF was added. The sample added with stirrer was placed on a magnetic stirrer (50° C., 700 rpm) and stirred to obtain the suspension. The sample solution was added with tetrahydrofuran solution (553.41 μL, 0.057 mg/mL) of tartaric acid such that the molar ratio of its addition amount to the compound is 1:1.05. The acid sample was placed on the magnetic stirrer (50° C., 700 rpm) and stirred overnight. The mixed solution became transparent after acid addition and then was stirred overnight to form a yellow suspension. Then, the solid was separated through centrifugation and dried overnight in a vacuum drying oven to obtain the crystal form C of compound of formula (II).

EXAMPLE 5

[0147] About 92 mg (0.2 mmol) of the compound was weighed and charged into a 40 mL vial. 7 parts of the same amount of compounds were weighed and labelled as hydrochloric acid, sulfuric acid, phosphoric acid, methanesulfonic acid, maleic acid, citric acid and fumaric acid respectively. 14 mL of THF was added to the each of samples, and then the sample with stirrer was placed on a magnetic stirrer (50° C., 700 rpm) and stirred to dissolve. The sample solution was added with an acid sample such that the molar ratio of its addition amount to the compound is 1:1.05, and the acid sample was shown as in Table 11. The resulting sample was placed on the magnetic stirrer (50° C., 700 rpm) and stirred overnight, during which the phenomenon was shown in Table 11. Then, the solid was separated through centrifugation and dried overnight in a vacuum drying oven to obtain the corresponding crystal form.

TABLE-US-00012 TABLE 11 Acid mass volume percentage (mg/ ml) Actual weighed mass of solid acid THF (mg) actual Acid mass Actual volume of addition volume amount of liquid acid volume percentage acid added Crystal Acid (μL) (μL) (mg/ml) V (μL) Phenomenon form hydrochloric 100.00 900.00 0.120 172.44 Yellow suspension Crystal acid form D sulfuric acid 100.00 900.00 0.184 114.09 Milky white suspension Crystal form E phosphoric acid 100.00 900.00 0.169 143.09 After adding acid, it Crystal started to be yellow form F colloid, which became yellow suspension after stirring overnight Methanesulfonic 100.00 900.00 0.148 136.09 Milky white suspension Crystal acid form G Maleic acid 129.50 1000.00 0.130 188.01 After adding acid and Crystal stirring overnight, the form H solution was clear and transparent, and precipitate was formed after volatilizing in an open vessel overnight (50° C., 400 rpm) Fumaric acid 110.60 2000.00 0.055 440.26 After adding acid and Crystal stirring overnight, the form I solution was clear and transparent followed by volatilization in an open vessel (50° C., 400 rpm) until it was 5 mL, and when 20 mL of anti solvent n-heptane was added, precipitate was formed Citric acid 105.60 1000.00 0.106 419.51 After adding acid and Crystal stirring overnight, the form J solution was clear and transparent followed by volatilization in an open vessel (50° C., 400 rpm) until it was 5 mL, and when 20 mL of anti solvent MTBE was added, precipitate was formed

EXAMPLE 6: STUDY ON HYGROSCOPICITY OF THE CRYSTAL FORM A OF THE COMPOUND FORMULA (I)

[0148] Experimental materials:

[0149] DVS Advantage dynamic vapour adsorbent device

[0150] Experimental method:

[0151] Taking 27.36 mg of the crystal form A of the compound of formula (I) and putting it into a DVS sample disk for test.

[0152] Experimental results:

[0153] DVF graph of the crystal form A of the compound of formula (I) was shown as in FIG. 15, ΔW %<0.2%.

[0154] Experimental conclusion:

[0155] The crystal form A of formula (I) had a hygroscopic weight gain<0.2% at 25.6° C. and 80% RH, without hygroscopicity.

EXAMPLE 7: SOLID STABILITY TEST OF THE CRYSTAL FORM OF A OF THE COMPOUND OF FORMULA (I)

[0156] According to the guiding principles for stability test of bulk medicament and preparations (general rule 9001 of Part IV of Chinese Pharmacopoeia 2015 Edition), the crystal form A of the compound of formula (I) was investigated for its stability under long-term (25° C./60% RH) and accelerated (30° C./65% RH; 40° C./75% RH) conditions.

[0157] 51 g of the crystal form A of the compound of formula (I) was weighed and divide it into 34 parts, about 1.5 g each. Each sample was put into a double-layer LDPE Bag with each layer buckled and sealed respectively, and then the LDPE Bag was put into an aluminum foil bag and heat sealed. The samples were investigated for XRPD under the conditions of 25° C./60% RH (14 bags), 30° C./65% RH (10 bags) and 40° C./75% RH (10 bags). The test results were compared with the initial test results of 0 days. The test results were shown in Table 12 below:

[0158] Table 12 Solid stability test results of the crystal form A of the compound of formula (I)

TABLE-US-00013 Test conditions Time point Crystal form — Day 0 Crystal form A 25° C./60% RH 3 months Crystal form A 6 months Crystal form A 40° C./75% RH 3 months Crystal form A 6 months Crystal form A Note: If the crystal form was stable under the condition of 40° C./75% RH, the test will not be carried out under the condition of 30° C./65% RH.

[0159] Conclusion: the crystal form A of the compound of formula (I) had good stability under the conditions of high temperature, high humidity and strong light.

EXPERIMENTAL EXAMPLE 1: EVALUATION OF mTOR KINASE INHIBITORY ACTIVITY IN VITRO

[0160] Experimental materials:

[0161] This experiment was tested on discoverx, and all materials and methods were from discoverx.

[0162] Experiment operation:

[0163] Kinase activity analysis:

[0164] 1. The labeled mTOR kinase was stably expressed in HEK-293 cells.

[0165] 2. Streptavidin magnetic beads were treated with biotinylated small molecule ligands for 30 minutes at room temperature to produce an affinity resin for kinase analysis;

[0166] 3. The ligand beads were blocked with excess biotin and washed with buffer (1% bovine serum albumin, 20 mL of 0.05% Tween, 1 mL of dithiothreitol) to wash off unbound ligands and nonspecifically bound ligands;

[0167] 4. The binding reactions of kinase ligand affinity beads, assembly and test compounds were realized in buffer (20% blocking buffer, 0.17× phosphate buffer, 0.05% Tween 20, 6 mL of dithiothreitol);

[0168] 5. The test compound was dissolved in dimethyl sulfoxide;

[0169] 6. All compounds used for measurement were dissolved in DMSO and then diluted directly to a concentration of 0.9%;

[0170] 7. The solution was placed on 384 well polypropylene plates with a volume of 0.02 mL of each well;

[0171] 8. The plate was shaked at room temperature for 1 hour;

[0172] 9. The plate was washed with buffer (1× PBS, 0.05% Tween 20);

[0173] 10. Affinity beads were resuspended in buffer (1× PBS, 0.05% Tween 20, 0.5 μM nonbiotin-affinity ligands) and incubated at room temperature for 30 minutes;

[0174] 11. The kinase in eluent was measured for its concentration by qPCR.

[0175] The experimental results were shown in table 13.

TABLE-US-00014 TABLE 13 Activity test results of mTORC1 and mTORC2 kinase complexes inhibitory activity of mTORC1 and mTORC2 kinase Test sample complexes Kd (nM) Compound of 0.3 formula (I)

[0176] Conclusion: the compound of formula (I) had significant or even unexpected mTOR kinase inhibitory activity.

EXPERIMENTAL EXAMPLE 2: EVALUATION OF CELL PROLIFERATION INHIBITORY ACTIVITY

[0177] Objective: the inhibitory activity of the compound to be tested was tested on cell proliferation.

[0178] Experimental principle: Luciferase in Cell-Titer-Glo reagent uses luciferin, oxygen and ATP as reaction substrates to produce oxidized luciferin and release energy in the form of light. Since ATP is required for luciferase reaction, the total amount of light produced by the reaction is directly proportional to the total amount of ATP in response to cell viability.

[0179] Experimental materials:

[0180] Cell lines: MCF-7 cell line (ATCC-CRL-22), HT-29 cell line (ATCC-HTB-38), OE21 (ECACC-96062201), NCI-N87 cell line (ATCC-CRL-5822)

[0181] Cell culture medium: (RPMI 1640 medium (Invitrogen #1868546; 10% serum Invitrogen #1804958; L-glutamine 1×), Invitrogen #1830863; double antibody Hyclone #J170012))

[0182] Cell Titer-Glo® Luminescent cell viability test kit (Promega #G7573)

[0183] 384-well cell culture plate (Greiner #E15103MA)

[0184] Compound plate (LABCYTE #0006346665)

[0185] CO.sub.2 incubator (Thermo #371)

[0186] Vi-cell Cell counter (Beckman Coulter)

[0187] Transferpettor (Eppendorf)

[0188] Pipette (Greiner)

[0189] Transfer liquid gun (Eppendorf)

[0190] Multifunctional enzyme labeling instrument (Envision Reader)

[0191] ECHO Liquid-handling workstation (Labcyte-ECHO555)

[0192] Experimental steps and methods:

[0193] 2.1 Day 1:

[0194] According to the schematic diagram of cell seed plate, 384 or 96 well plate was seeded based on 1000 cells per well with a density of 25 μL per well, and the edge well was supplemented with 25 μL PBS, instead of cells.

[0195] 2.2 Day 0:

[0196] (1) The mother liquor of the compound was 10 mM, and the compound was diluted with DMSO to obtain its initial concentration of 4 mM. The compound was added into the mother liquor plate with 9 μL per well.

[0197] (2) The compound was diluted with ECHO liquid workstation and the compound was added into the cell plate with 125 nL of the compound per well, whereinto each well in columns 2 and 23 was added with 125 nL of DMSO, and PBS well in columns 1 and 24 was added with 125 nL of DMSO.

[0198] (3) Each well in the cell plate was supplemented with 25 μL of the medium to obtain a final 50 μL per well. The compound had a concentration of 1 μM, which was diluted 3 times to obtain 10 concentrations. Duplicated wells were set left and right, and thus DMSO had a final concentration of 0.25%.

[0199] 2.3 After the compound is added, the cell plate was centrifuged at 1000 rpm for 1 min, and then placed in an incubator under 37° C. and 5% CO.sub.2 for 3 days.

[0200] 2.4 Day 3:

[0201] The cell plate was taken from the incubator and balanced at room temperature for 30 minutes. 25 μL of Cell-Titer-Glo reagent was added to each well, and the plate was shaken for one minute to mix the reagent evenly followed by centrifugation at 1000 rpm for 1 minute. After 10 minutes, the plate reading was performed on PerkinElmer envision with setting the fluorescence reading time of 0.2 seconds. Test results: the test results were shown in table 14.

TABLE-US-00015 TABLE 14 Screening test results of in vitro cell proliferation inhibitory activity of the compound of formula (I) MCF-7 cells N87 cells Test sample IC.sub.50 (nM) IC.sub.50 (nM) Compound of 208 59 formula (I) “ND” means not detected.

[0202] Conclusion: the compound of formula (I) according to the present invention had obvious inhibitory activity on the proliferation of MCF-7 and N87 cells.

EXPERIMENTAL EXAMPLE 3: PHARMACOKINETIC EVALUATION

[0203] Experimental method

[0204] The tested compound was mixed with 5% DMSO/95% 10% polyoxyethylene castor oil (Cremophor EL) followed by vortex and ultrasonic treatment to prepare 1 mg/mL approximate clear solution, which was filtered by microporous membrane before used. BALB/C female mice of 18 to 20 g were injected with the compound solution intravenously at a dose of 1 or 2 mg/kg. The tested compound was mixed with 1% Tween 80, 90% polyethylene glycol 400, 90% aqueous solution followed by vortex and ultrasonic treatment to prepare 1 mg/mL approximate clear solution, which was filtered by microporous membrane before used. BALB/C female mice of 18 to 20 g were given the candidate compound solution orally at a dose of 2 or 10 mg/kg. Whole blood was collected for a certain time to prepare plasma. The medicament was analyzed for its concentration by LC-MS/MS, and the pharmacokinetic parameters were calculated by Phoenix winnonlin software (pharsight company).

[0205] Test results:

[0206] Test results were shown in Table 15 to Table 16.

TABLE-US-00016 TABLE 15 Pharmacokinetic (PK) parameters in plasma of reference compound (AZD2014) PK parameters in plasma of reference compound (AZD2014) PK IV IV PO PO parameters (1 mg/kg) (2 mg/kg) (2 mg/kg) (10 mg/kg) C.sub.0 (nM) 4786 8067 — — C.sub.max (nM) — — 4370 16967 T.sub.max (h) — — 0.500 0.25 T.sub.1/2 (h) 0.967 1.05 1.14 3.46 V.sub.dss (L/kg) 0.677 0.599 — — Cl (mL/min/kg) 8.41 8.08 — — T.sub.last (h) 8.00 8.00 12.0 24.0 AUC.sub.0-last (nM .Math. h) 4270 8880 8237 41443 AUC.sub.0-inf (nM .Math. h) 4281 8914 7241 41508 MRT.sub.0-last (h) 0.268 1.20 1.95 2.45 MRT.sub.0-inf (h) 1.88 1.24 1.95 2.50 AUC.sub.Extra (%) — 0.384 0.0496 0.156 AUMC.sub.Extra (%) — 2.95 0.347 1.82 F (%) — — 96.2 93.1

TABLE-US-00017 TABLE 16 PK parameters in plasma of the compound of formula (I) PK IV PO parameters (2 mg/kg) (10 mg/kg) C.sub.0 (nM) 8237 — C.sub.max (nM) — 12850 T.sub.max (h) — 0.25 T.sub.1/2 (h) 1.10 1.22 V.sub.dss (L/kg) 0.739 — Cl (mL/min/kg) 7.72 — T.sub.last (h) 12.0 12.0 AUC.sub.0-last (nM .Math. h) 9730 41869 AUC.sub.0-inf (nM .Math. h) 9374 41963 MRT.sub.0-last (h) 1.59 3.02 MRT.sub.0-inf (h) 1.60 3.04 AUC.sub.Extra (%) 0.0469 0.225 AUMC.sub.Extra (%) 0.400 1.02 F (%) — 89.5 Note: “—” means that no test or data was obtained

[0207] Test conclusion: the compound of formula (I) showed the same or even better pharmacokinetic properties as the reference compound; the compound of formula (I) according to the present invention had a bioavailability close to 100%, which was a good exploitable molecule for oral administration.

EXPERIMENT 4: IN VIVO PHARMACODYNAMIC STUDY OF SUBCUTANEOUS XENOGRAFTS OF HUMAN BREAST CANCER MCF-7 CELL IN NUDE BALB/c MICE MODEL

[0208] Experimental objective: to study the in vivo efficacy of the compound to be tested on subcutaneous xenografts of human breast cancer MCF-7 in nude BALB/c mice model.

[0209] Experimental animals: female BALB/C nude mice, 6-8 weeks old, weighing 18-22 g; Supplier: Shanghai Xipur Bikai Experimental Animal Co., Ltd.

[0210] Experimental methods and steps:

[0211] 4.1 cell culture

[0212] Human breast cancer MCF-7 cells (ECACC, item number: 86012803) were cultured in monolayer in vitro. The culture conditions were EMEM (EBSS)+2 mM glutamic acid+1% Non Essential Amino Acids (NEAA) medium plus 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin, incubated in 5% CO.sub.2 incubator at 37° C. Cells were passaged through routine digestion using Trypsin EDTA twice a week. When the cell saturation was 80%-90% and the number reached the requirements, cells were collected, counted and inoculated.

[0213] 4.2 Tumor cell inoculation (tumor inoculation)

[0214] Estrogen tablets (0.18 mg) were inoculated on the left back of each mouse 3 days before cell inoculation. 0.2 mL (1×10.sup.7) of MCF-7 cells (with matrix glue and volume ratio of 1:1) was subcutaneously inoculated on the right back of each mouse. The medicament was administered in groups when the average tumor volume reached 142 mm.sup.3.

[0215] 4.3 Formulation of the test compound

[0216] The test compound was formulated into clear solutions of 0.75 mg/mL, 1.5 mg/mL and 3 mg/mL respectively, and the solvent was 5% DMSO+30% polyethylene glycol 300+65% water.

[0217] 4.4 Tumor measurement and experimental indicators

[0218] The experimental index is used for investigating whether tumor growth is inhibited, delayed or cured. The tumor diameter was measured with a vernier caliper twice a week. The calculation formula of tumor volume is: V=0.5a×b.sup.2, wherein a and b represent the long diameter and short diameter of the tumor, respectively.

[0219] The antitumor effect of the compound was evaluated by TGI (%) or relative tumor proliferation rate T/C(%). TGI (%), reflecting tumor growth inhibition rate. TGI (%) was calculated: TGI (%)=[1−(average tumor volume at the end of administration in a certain treatment group−average tumor volume at the beginning of administration in the 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)]×control group.

[0220] Relative tumor proliferation rate T/C (%): the calculation formula is as follows: T/C %=TRTV/CRTV (TRTV:RTV in treatment group; CRTV:RTV in negative control group). The relative tumor volume (RTV) is calculated according to the results of tumor measurement. The calculation formula is RTV=V.sub.t/V.sub.0, where V.sub.0 is the average tumor volume measured in group administration (i.e. d.sub.0), V.sub.t is the average tumor volume measured in a certain measurement, and data on the same day were taken for TRTV and CRTV.

[0221] After the experiment, the tumor weight will be detected and the T/C weight percentage will be calculated. T weight and C weight represent the tumor weight of the administration group and the solvent control group respectively.

[0222] 4.5 Statistical analysis

[0223] Statistical analysis included the mean and standard error (SEM) of tumor volume at each time point of each group. The treatment group showed the best treatment effect on the 21st day after administration at the end of the trial. Therefore, statistical analysis was conducted based on these data to evaluate the difference between the groups. The comparison between the two groups was analyzed by T-test, and the comparison between the three or more groups was analyzed by one-way ANOVA. If there was a significant difference in F value, the Games-Howell method was used for test. If there was no significant difference in F value, Dunnet (2-sided) method was used for analysis. All data were analyzed with SPSS 17.0. If P<0.05, the difference was considered significant.

[0224] 4.6 The experimental results were shown in Table 17.

TABLE-US-00018 TABLE 17 Anti-tumor effect of the compound on subcutaneous xenografts of human breast cancer MCF-7 cell model Tumor Tumor volume volume (mm.sup.3).sup.a (mm.sup.3).sup.a RTV T/C.sup.b TGI.sup.b Group (day 0) (day 27) (day 27) (%) (%) Solvent 142 ± 7  588 ± 75  4.17 ± 0.52 — — AZD2014 142 ± 9  315 ± 65  2.25 ± 0.47 53.95 61.10 (7.5 mg/kg) AZD2014 141 ± 8  121 ± 41  0.85 ± 0.26 20.36 104.37   (15 mg/kg) Compound of 142 ± 11  323 ± 25  2.41 ± 0.36 57.84 59.27 formula (I)  (15 mg/kg) Note: “—” does not need to be calculated .sup.aMean ± SEM. .sup.bTumor growth inhibition was calculated by T/C and TGI (TGI (%) = [1-(T.sub.21-T.sub.0)/(V.sub.21-V.sub.0)+ × 100).

[0225] Experimental conclusion: in the MCF-7 Xenograft tumor model, the efficacy of the compound of formula (I) was equivalent to that of AZD2014.