DIARYL MACROCYCLIC COMPOUND AND PHARMACEUTICAL COMPOSITION, AND USE THEREOF

Abstract

The present invention provides a compound as represented by formula (1) or a pharmaceutically acceptable salt, a solvate, an active metabolite, a polymorph, an isotope label, an isomer or a prodrug thereof. The present invention also provides a pharmaceutical composition containing the same and the use of the compound and the pharmaceutical composition in preparation of drugs for treating tyrosine kinase-mediated diseases. The compound and the pharmaceutical composition comprising same provided by the present disclosure have significant tyrosine kinase inhibitory activity, can overcome tumor drug resistance, and break through through blood-brain barrier, also have excellent pharmacokinetic properties and excellent oral bioavailability, and can be administered in a small dosage, thereby reducing treatment costs of and possible side effects to a patient. Thus, the application potential is very great.

##STR00001##

Claims

1. A compound represented by formula (1) or a pharmaceutically acceptable salt, a solvate, an active metabolite, a polymorph, an isotope label, an isomer or a prodrug thereof, ##STR00074## wherein, X is selected from —O—, —S— or —CR.sub.aR.sub.b—; R.sub.a and R.sub.b are each independently selected from the following substituted or unsubstituted groups: hydrogen, halogen, a C.sub.1˜8 alkyl, a C.sub.1˜8 alkoxy, a C.sub.1˜8 haloalkyl, a C.sub.3˜8 cycloalkyl, a C.sub.3˜8 heterocyclyl, a C.sub.6˜20 aryl, a C.sub.5˜20 heteroaryl, hydroxyl, mercapto, carboxy, ester group, acyl, amino, amide, sulfonyl, cyano, or CR.sub.aR.sub.b together forms a 3-10 membered cycloalkyl group or a 3-10 membered heterocyclic group containing at least one heteroatom; L.sub.1 is selected from —O—, —S—, —S(═O)—, —S(═O).sub.2—, —NR.sub.6— or a single bond; L.sub.2 is selected from —O—, —S—, —S(═O)—, —S(═O).sub.2— or —NR.sub.6—; R.sub.1, R.sub.2, and R.sub.5 are each independently selected from the following substituted or unsubstituted groups: hydrogen, halogen, a C.sub.1˜8 alkyl, a C.sub.1˜8 alkoxy, a C.sub.1˜8 haloalkyl, a C.sub.3˜8 cycloalkyl, a C.sub.3˜8 heterocyclic group, a C.sub.6-20 aryl group, a C.sub.5-20 heteroaryl group, hydroxyl, mercapto, carboxyl, ester group, acyl, amino, amide, sulfonyl or cyano; the substituents R.sub.3 and R.sub.4 on each C atom are each independently selected from the following substituted or unsubstituted groups: hydrogen, halogen, a C.sub.1˜8 alkyl, a C.sub.1˜8 alkoxy, a C.sub.1˜8 haloalkyl, a C.sub.3˜8 cycloalkyl, a C.sub.3˜8 heterocyclyl, a C.sub.6˜20 aryl, a C.sub.5˜20 heteroaryl, hydroxy, mercapto, carboxy, ester group, acyl, amino, amido, sulfonyl, cyano, or the substituents R.sub.3 and R.sub.4 together with X group form a 3-10 membered cycloalkyl group, a 3-10 membered heterocyclic group containing at least one heteroatom, or a 5-10 membered heteroaryl group containing at least one heteroatom; or R.sub.3 and R.sub.4 are each independently a single bond connecting the C atom and the adjacent macrocyclic ring atom; R.sub.3′ and R.sub.4′ are each independently selected from the following substituted or unsubstituted groups: hydrogen, halogen, a C.sub.1˜8 alkyl, a C.sub.1˜8 alkoxy, a C.sub.1˜8 haloalkyl, a C.sub.3˜8 cycloalkyl, a C.sub.3˜8 heterocyclic group, a C.sub.6-20 aryl group, a C.sub.5-20 heteroaryl group, hydroxyl group, mercapto group, carboxyl group, ester group, acyl group, amino group, amide group, sulfonyl group, cyano group, or R.sub.3′ and R.sub.4′ together with the connected C and L.sub.2 form a 3-10 membered heterocyclic group containing at least one heteroatom or a 5-10 membered heteroaryl group containing at least one heteroatom; R.sub.6 is selected from the following substituted or unsubstituted groups: hydrogen, halogen, a C.sub.1˜8 alkyl, a C.sub.1˜8 alkoxy, a C.sub.1˜8 haloalkyl, a C.sub.3˜8 cycloalkyl, a C.sub.3˜8 heterocyclyl, a C.sub.6˜20 aryl, a C.sub.5˜20 heteroaryl, hydroxyl, mercapto, carboxy, ester, acyl, amino, amido, sulfonyl or cyano; Z represents C or heteroatom as a ring atom; n represents an integer from 1 to 10; the substituents of the above-mentioned groups may be selected from halogen, a C.sub.1˜8 alkyl, a C.sub.1˜8 haloalkyl, a C.sub.1˜8 alkoxy, a C.sub.3˜8 cycloalkyl, a C.sub.3˜8 heterocyclyl, a C.sub.6˜20 aryl, a C.sub.5-20 heteroaryl, hydroxyl, mercapto, carboxy, ester group, acyl, amino, amido, sulfonyl or cyano; the above-mentioned heteroatoms are selected from N, O or S.

2. The compound or a pharmaceutically acceptable salt, a solvate, an active metabolite, a polymorph, an isotope label, an isomer or a prodrug thereof according to claim 1, wherein the compound has the structure of formula (2), ##STR00075## wherein R.sub.1 is fluorine or bromine; and/or R.sub.2 is hydrogen, fluorine or bromine.

3. The compound or a pharmaceutically acceptable salt, a solvate, an active metabolite, a polymorph, an isotope label, an isomer or a prodrug thereof according to claim 1 or 2, wherein in Formula (1) or Formula (2), L.sub.1 is selected form —O—, —S— or a single bond; and/or L.sub.2 is —NR.sub.6—.

4. The compound or a pharmaceutically acceptable salt, a solvate, an active metabolite, a polymorph, an isotope label, an isomer or a prodrug thereof according to any of claims 1 to 3, wherein in Formula (1) or Formula (2), n represents an integer of 2, 3, 4, 5 or 6.

5. The compound or a pharmaceutically acceptable salt, a solvate, an active metabolite, a polymorph, an isotope label, an isomer or a prodrug thereof according to any of claims 1 to 4, wherein in Formula (1) or Formula (2), the substituents R.sub.3 and R.sub.4 on each C atom are each independently selected from the following substituted or unsubstituted groups: hydrogen, halogen, a C.sub.1˜5 alkyl, a C.sub.1˜5 alkoxy, a C.sub.1˜5 haloalkyl, a C.sub.3˜6 cycloalkyl, or R.sub.3 and R.sub.4 are each independently a single bond connecting the C atom and adjacent macrocyclic ring atoms; and/or R.sub.3′ and R.sub.4′ are each independently selected from the following substituted or unsubstituted groups: hydrogen, halogen, a C.sub.1˜5 alkyl, a C.sub.1˜5 alkoxy, a C.sub.1˜5 haloalkyl, a C.sub.3˜6 cycloalkyl, or R.sub.3′ and R.sub.4′ together with the connected C and L.sub.2 form a 4-8 membered heterocyclic group containing at least one heteroatom; and/or R.sub.5 is selected from the following substituted or unsubstituted groups: hydrogen, halogen, a C.sub.1˜5 alkyl, a C.sub.1˜5 alkoxy, a C.sub.1˜5 haloalkyl, a C.sub.3˜6 cycloalkyl, hydroxyl, mercapto, carboxyl, amino or cyano; and/or R.sub.6 is selected from the following substituted or unsubstituted groups: hydrogen, halogen, a C.sub.1˜5 alkyl, a C.sub.1˜5 alkoxy, a C.sub.1˜5 haloalkyl or a C.sub.3˜6 cycloalkyl; and/or the above substituents may be selected from fluorine, bromine, —CN, —OH, —CF.sub.3, —NH.sub.2, —NH(C.sub.1˜4 alkyl), —N(C.sub.1˜4 alkyl).sub.2, —CO.sub.2C.sub.1˜4 alkyl, —CO.sub.2H, —NHC(O)C.sub.1˜4 alkyl, —SO.sub.2C.sub.1˜4 alkyl, —C(O)NH.sub.2, —C(O)NH(C.sub.1˜4 alkyl), —C(O)N(C.sub.1˜4 alkyl).sub.2, a C.sub.1˜5 alkyl, a C.sub.3˜6 cycloalkyl, a C.sub.3˜6 heterocyclyl, a C.sub.6˜10 aryl or a C.sub.5˜10 heteroaryl.

6. The compound or a pharmaceutically acceptable salt, a solvate, an active metabolite, a polymorph, an isotope label, an isomer or a prodrug thereof according to any of claims 1 to 5, wherein the compound is selected from the following structures: ##STR00076## ##STR00077## ##STR00078## ##STR00079## ##STR00080##

7. A pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt, a solvate, an active metabolite, a polymorph, an isotope label, an isomer or a prodrug thereof according to any of claims 1 to 6, and a pharmaceutically acceptable carrier.

8. A use of the compound or a pharmaceutically acceptable salt, a solvate, an active metabolite, a polymorph, an isotope label, an isomer or a prodrug thereof according to any of claims 1 to 6 and the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating tyrosine kinase-mediated diseases.

9. The use according to claim 8, wherein the tyrosine kinase is selected from one or more of the following: ALK, ROS1, TRKA, TRKB, TRKC, JAK2, SRC, FYN, LYN, YES, FGR, FAK, AXL, ARK5.

10. The use according to claim 8, wherein the tyrosine kinase-mediated diseases include cancer, pain, neurological diseases, autoimmune diseases and inflammation.

11. A method for treating tyrosine kinase-mediated diseases, comprising administering to a patient an effective amount of the compound or a pharmaceutically acceptable salt, solvate, or active metabolite, polymorph, isotope label, isomer or prodrug thereof according to any of claims 1 to 6, or the pharmaceutical composition of claim 7.

12. The method according to claim 11, wherein the tyrosine kinase is selected from one or more of the following: ALK, ROS1, TRKA, TRKB, TRKC, JAK2, SRC, FYN, LYN, YES, FGR, FAK, AXL, ARK5.

13. The method according to claim 11, wherein the tyrosine kinase-mediated diseases include cancer, pain, neurological diseases, autoimmune diseases, and inflammation.

Description

DESCRIPTION OF THE DRAWINGS

[0102] FIG. 1 is a schematic diagram of the synthetic route of Example 1.

DETAILED DESCRIPTION

[0103] To make the objectives, technical solutions, and advantages of the present disclosure clearer, the technical solutions of the exemplary embodiments of the present disclosure will be further described below.

[0104] In the present disclosure, the compounds described in the present disclosure may be prepared by the following methods. The following methods and examples are to illustrate these methods. These procedures and examples should not be construed as limiting the present disclosure in any way. The compounds described herein may also be synthesized using standard synthesis techniques known to those skilled in the art, or methods known in the art and methods described herein may be used in combination.

[0105] In the present disclosure, the compounds described in the present disclosure may be prepared by the following methods. The following methods and examples are to illustrate these methods. These procedures and examples should not be construed as limiting the present disclosure in any way. The compounds described herein may also be synthesized using standard synthesis techniques known to those skilled in the art, or methods known in the art and methods described herein may be used in combination.

[0106] The chemical reactions in the embodiments of the present disclosure are completed in a suitable solvent, and the solvent must be suitable for the chemical changes of the present disclosure and the reagents and materials required. In order to obtain the compounds of the present disclosure, it is sometimes necessary for those skilled in the art to modify or select the synthesis steps or reaction schemes based on the existing embodiments.

[0107] An important consideration in the planning of any synthetic route in the art is to select an appropriate protecting group for the reactive functional group (such as the amino group in the present disclosure). For trained practitioners, Greene and Wuts (Protective Groups In Organic Synthesis, Wiley and Sons, 1991) is the authority in this regard. All references cited in the present disclosure are incorporated into the present disclosure in their entirety.

[0108] The reactions described herein may be monitored according to any suitable method known in the art. For example, product formation may be monitored by a broad spectrum method such as nuclear magnetic resonance spectroscopy (such as .sup.1H or .sup.13C), infrared spectroscopy, spectrophotometry (such as UV-visible light), mass spectrometry, etc., or chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.

Example 1: (1.SUP.3.E,1.SUP.4.E,3R,6S)-4.SUP.5.-fluoro-3,6-dimethyl-5,8-dioxa-2-aza-1(5,3)-pyrazolo[1,5-a]pyrimidina-4(1,2)-benzenacyclononaphan-9-one

[0109] ##STR00012##

[0110] Synthetic route was shown in FIG. 1.

Step A: (E)-N-(5-fluoro-2-hydroxybenzylidene)-2-methylpropane-2-sulfinamide

[0111] ##STR00013##

[0112] 5.8 g (41.4 mmol, 1.0 eq) 5-fluoro-2-hydroxybenzaldehyde and 5.0 g (41.4 mmol, 1 eq) (R)-2-methylpropane-2-sulfinamide was added to 100 mL methylene chloride (DCM), then 21.5 g (66.3 mmol, 1.6 eq) Cs.sub.2CO.sub.3 was added under magnetic stirring. The resultant solution was stirred at rt for overnight. The mixture was filtered, and the filter cake was rinsed with dichloromethane. The filtrate was concentrated to give the crude product, which was used in next step without further purification (8.5 g, 85% yield).

Step B: N—((R)-1-(5-fluoro-2-hydroxyphenyl)ethyl)-2-methylpropane-2-sulfinamide

[0113] ##STR00014##

[0114] 24 g (100 mmol, 1.0 eq) (E)-N-(5-fluoro-2-hydroxybenzylidene)-2-methylpropane-2-sulfinamide was added to 300 mL THE and the mixture was cooled to to −65° C., 100 mL (3N, 3.0 eq) methyl magnesium bromide was slowly added under N.sub.2, and the temperature was maintained below −50° C. After adding, the mixture was slowly raised to room temperature for overnight. TLC analysis (CH.sub.2Cl.sub.2:hexane=3:1) showed that the reaction was complete. The reaction mixture was quenched with water (500 mL). 500 mL ethyl acetate (EA) was added for extraction. The combined organic layers were washed twice with water and saturated brine in turn, and dried over anhydrous Na.sub.2SO.sub.4. The solvent was removed by concentration to afford the crude product, which was purified by silica gel column chromatography (8.5 g, 33% yield).

[0115] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.02 (s, 1H), 6.79 (d, J=4.0 Hz, 1H), 6.65-6.51 (m, 1H), 6.50-6.41 (m, 1H), 5.04 (d, J=8.0 Hz, 1H), 4.45-4.30 (m, 1H), 1.53 (d, J=8.0 Hz, 3H), 1.29 (s, 9H).

Step C: (R)-2-(1-aminoethyl)-4-fluorophenol Hydrochloride

[0116] ##STR00015##

[0117] 8.5 g (32.8 mmol, 1.0 eq) N—((R)-1-(5-fluoro-2-hydroxyphenyl)ethyl)-2-methylpropane-2-sulfinamide was dissolved in 100 mL dioxane (4N) solution. The reaction was carried out at room temperature for 3 hours, and TLC analysis showed that the reaction was complete. The solvent was removed by concentration. The crude product was dissolved in 100 mL EA, filtered and rinsed with EA. The solid was collected and dried to obtain the target product (5.4 g, 87% yield).

Step D: ethyl (R)-5-((1-(5-fluoro-2-hydroxyphenyl)ethyl)amino)pyrazolo[1,5-a] pyrimidine-3-carboxylate

[0118] ##STR00016##

[0119] 5.3 g (27.9 mmol, 1.0 eq) (R)-2-(1-aminoethyl)-4-fluorophenol hydrochloride, 6.29 g (27.9 mmol, 1.0 eq) ethyl 5-chloropyrazolo[1,5-a]pyrimidine-3-carboxylate and diisopropylethylamine (DIEA) (12 mL, 167 mmol, 6.0 eq) were dissolved in 60 ml N,N-dimethylformamide (DMF). The mixture was heated to 120° C. for 5 hours. TLC analysis showed that the reaction was complete, and the solvent was removed by concentration. Water (100 mL) and ethyl acetate (200 mL) were added for extraction. The combined organic layers were washed twice with water and saturated brine in turn, and dried over anhydrous Na.sub.2SO.sub.4. The solvent was removed by concentration to afford the crude product, which was purified by silica gel column chromatography (2.9 g, 31% yield).

[0120] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.17 (brs, 1H), 8.24 (s, 1H), 8.14 (d, J=8.0 Hz, 1H), 6.93-6.90 (m, 2H), 6.83 (d, J=8.0 Hz, 1H), 6.13 (d, J=8.0 Hz, 1H), 5.81 (d, J=8.0 Hz, 1H), 5.64 (t, J=8.0 Hz, 1H), 4.42 (q, J=8.0 Hz, 2H), 1.61 (d, J=8.0 Hz, 3H), 1.41 (t, 3H).

Step E: (R)-2-hydroxypropyl Pivalate

[0121] ##STR00017##

[0122] To a solution of (R)-propane-1,2-diol (7.6 g, 100 mmol, 1.0 eq), and pyridine (16 mL, 200 mmol, 2.0 eq) in 80 mL dichloromethane (DCM) was slowly added pivaloyl chloride (12.6 g, 100 mmol, 1.0 eq) at ice bath. After adding, the mixture was raised to room temperature for overnight. TLC analysis showed that the reaction was complete, and the solvent was removed by concentration. Water (100 mL) and ethyl acetate (250 mL) were added for extraction. The combined organic layers were washed twice with water and saturated brine in turn, and dried over anhydrous Na.sub.2SO.sub.4. The solvent was removed by concentration to afford the crude product, which was purified by silica gel column chromatography (10.5 g, 66% yield).

Step F: Compound 11

[0123] ##STR00018##

[0124] To a solution of (R)-ethyl (R)-5-((1-(5-fluoro-2-hydroxyphenyl)ethyl)amino)pyrazolo [1,5-a]pyrimidine-3-carboxylate (2.0 g, 5.8 mmol, 1.0 eq), (R)-2-hydroxypropyl pivalate (1.39 g, 8.7 mmol, 1.5 eq) and PPh.sub.3 (3.04 g, 11.6 mmol, 2.0 eq) in DCM (35 mL) was slowly added diisopropyl azodicarboxylate (DIAD) (2.02 g, 11.6 mmol, 2.0 eq) at ice bath. After adding, the mixture was raised to room temperature for overnight. TLC analysis showed that the reaction was complete, and the solvent was removed by concentration. Water (100 mL) and ethyl acetate (250 mL) were added for extraction. The combined organic layers were washed twice with water and saturated brine in turn, and dried over anhydrous Na.sub.2SO.sub.4. The solvent was removed by concentration to afford the crude product, which was purified by silica gel column chromatography (2.1 g, 75% yield).

[0125] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.25 (s, 1H), 8.16 (d, J=8.0 Hz, 1H), 7.10 (d, J=8.0 Hz, 1H), 6.90 (d, J=8.0 Hz, 2H), 6.08 (d, J=8.0 Hz, 1H), 5.24 (s, 1H), 4.74-4.65 (m, 1H), 4.38 (q, J=8.0 Hz, 2H), 4.28-4.13 (m, 3H), 1.56 (d, J=8.0 Hz, 3H), 1.40 (t, J=8.0 Hz, 3H), 1.25 (d, J=8.0 Hz, 3H), 1.20 (t, 9H).

Step G: Compound 12

[0126] ##STR00019##

[0127] To a solution of compound 11 (1.5 g, 3.1 mmol, 1.0 eq) in MeOH (10 mL) was added NaOH solution (3.1 mL, 10N, 10.0 eq) at ice bath. After adding, the mixture was heated to room temperature for 2.0 hours. TLC analysis showed that the reaction was complete, and the solution was removed by concentration. 1N HCl solution was added until pH 7. The resulting suspension was collected by filtration and washed with 200 mL water. The crude product was dried to obtain the target compound 12 (0.72 g, 63% yield).

[0128] .sup.1H NMR (400 MHz, DMSO) δ 11.50 (brs, 1H), 8.55 (d, J=9.2 Hz, 1H), 8.28 (d, J=9.2 Hz, 1H), 8.10 (s, 1H), 7.15-6.94 (m, 3H), 6.45 (d, J=9.2 Hz, 1H), 5.61 (d, J=8.0 Hz, 1H), 4.52-4.35 (m, 1H), 3.68-3.42 (m, 2H), 1.43 (d, J=8.0 Hz, 3H), 1.24 (d, J=8.0 Hz, 3H).

Step H: (1.SUP.3.E,1.SUP.4.E,3R,6S)-4.SUP.5.-fluoro-3,6-dimethyl-5,8-dioxa-2-aza-1(5,3)-pyrazolo[1,5-a]pyrimidina-4(1,2)-benzenacyclononaphan-9-one

[0129] ##STR00020##

[0130] To a solution of compound 12 (150 mg, 0.40 mmol, 1.0 eq), 2,4,6-trichlorobenzoyl chloride (489 mg, 2.0 mmol, 5.0 eq) in 10 mL THF was added Et.sub.3N (280 mg, 2.4 mmol, 6.0 eq), the reaction solution was stirred at ambient temperature for 30 min. Then the reaction solution was slowly added to a solution of 4-dimethylaminopyridine (488 mg, 4.0 mmol, 10.0 eq) in 500 mL toluene. The resultant mixture was heated to 100° C. under stirring for 2 hours, then the reaction was stirred at ambient temperature for overnight. TLC analysis showed that the reaction was complete, and the solvent was removed by concentration, and water (30 mL) and EA (50 mL) was added for extraction. The organic layers were washed twice with water and saturated brine. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4. The solvent was removed by concentration to afford the crude product, which was purified by silica gel column chromatography (23 mg, 16% yield).

[0131] LC-MS: m/z=357 [M+H].sup.+.

[0132] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.26-8.20 (m, 2H), 7.01 (d, J=8.0 Hz, 1H), 6.87-6.69 (m, 2H), 6.19 (d, J=4.0 Hz, 1H), 6.01-5.85 (m, 1H), 5.55 (s, 1H), 4.89 (dd, J=8.0, 4.0 Hz, 1H), 4.62 (s, 1H), 4.13 (t, J=8.0 Hz, 1H), 1.63-1.52 (m, 6H).

Example 2: (1.SUP.3.E,1.SUP.4.E,3S,6S)-4.SUP.5.-fluoro-3,6-dimethyl-5,8-dioxa-2-aza-1(5,3)-pyrazolo [1,5-a] pyrimidina-4(1,2)-benzenacyclononaphan-9-one

[0133] ##STR00021##

[0134] The compound (20 mg, 15%) was prepared according to the steps described in example 1. LC-MS: m/z=357 [M+H].sup.+.

Example 3: (1.SUP.3.E,1.SUP.4.E,3S,6R)-4.SUP.5.-fluoro-3,6-dimethyl-5,8-dioxa-2-aza-1(5,3)-pyrazolo [1,5-a]pyrimidina-4(1,2)-benzenacyclononaphan-9-one

[0135] ##STR00022##

[0136] The compound (31 mg, 22%) was prepared according to the steps described in example 1. LC-MS: m/z=357 [M+H].sup.+.

Example 4: (1.SUP.3.E,1.SUP.4.E,3R,6R)-4.SUP.5.-fluoro-3,6-dimethyl-5,8-dioxa-2-aza-1(5,3)-pyrazolo [1,5-a]pyrimidina-4(1,2)-benzenacyclononaphan-9-one

[0137] ##STR00023##

[0138] The compound (35 mg, 24%) was prepared according to the steps described in example 1. LC-MS: m/z=357 [M+H].sup.+.

[0139] The following compounds were prepared by a similar method according to example 1:

TABLE-US-00001 Example 5: (1.sup.3E,1.sup.4E,3R,6S)-4.sup.5-fluoro-3,6-dimethyl-5,9-dioxa-2- aza-1(5,3)-pyrazolo[1,5-a]pyrimidina-4(1,2)- benzenacyclodecaphan-10-one [00024] Example 7: (1.sup.3E,1.sup.4E,3R,8R)-4.sup.5-fluoro-3,8-dimethyl-5,9-dioxa-2- aza-1(5,3)-pyrazolo[1,5-a]pyrimidina-4(1,2)- benzenacyclodecaphan-10-one [00025] Example 8: (1.sup.3E,1.sup.4E,3R,6S)-4.sup.5-fluoro-2,3,6-trimethyl-5,8-dioxa- 2-aza-1(5,3)-pyrazolo[1,5-a]pyrimidina-4(1,2)- benzenacyclononaphan-9-one [00026] Example 10: (1.sup.3E,1.sup.4E,3R,6S)-4.sup.5-fluoro-2,3,6-trimethyl-8-oxa-5- thia-2-aza-1(5,3)-pyrazolo[1,5-a]pyrimidina-4(1,2)- benzenacyclononaphan-9-one [00027] Example 11: (1.sup.3E,1.sup.4E,2.sup.2R,5S)-3.sup.5-fluoro-5-methyl-4,7-dioxa- 1(5,3)-pyrazolo[1,5-a]pyrimidina-2(1,2)-pyrrolidina- 3(1,2)-benzenacyclooctaphan-8-one [00028] Example 12: (1.sup.3E,1.sup.4E,2.sup.2R,5S)-3.sup.5-fluoro-5-methyl-4,8-dioxa- 1(5,3)-pyrazolo[1,5-a]pyrimidina-2(1,2)-pyrrolidina- 3(1,2)-benzenacyclononaphan-9-one [00029] Example 14: (1.sup.3E,1.sup.4E,2.sup.2R,5S)-3.sup.5-fluoro-5-methyl-4,8-dioxa- 1(5,3)-pyrazolo[1,5-a]pyrimidina-2(1,2)-pyrrolidina- 3(1,2)-benzenacyclononaphan-9-one [00030] Example 15: (1.sup.3E,1.sup.4E,2.sup.2R,2.sup.4S,5S)-2.sup.4,3.sup.5-difluoro-5-methyl-4,7- dioxa-1(5,3)-pyrazolo[1,5-a]pyrimidina-2(1,2)- pyrrolidina-3(1,2)-benzenacyclooctaphan-8-one [00031] Example 16: (R,1.sup.3E,1.sup.4E)-4.sup.5-fluoro-3-methyl-5-oxa-8-thia-2-aza- 1(5,3)-pyrazolo[1,5-a]pyrimidina-4(1,2)- benzenacyclononaphan-9-one [00032] Example 17: (R,1.sup.3E,1.sup.4E)-4.sup.5-fluoro-3-methyl-5-oxa-2-aza-1(5,3)- pyrazolo[1,5-a]pyrimidina-4(1,2)- benzenacyclononaphan-9-one [00033] Example 18: (1.sup.3E,1.sup.4E,3R,6S)-1.sup.2-amino-4.sup.5-fluoro-3,6-dimethyl-5,8- dioxa-2-aza-1(5,3)-pyrazolo[1,5-a]pyrimidina-4(1,2)- benzenacyclononaphan-9-one [00034] Example 19: (1.sup.3Z,1.sup.4E,3R,6S)-1.sup.2-bromo-4.sup.5-fluoro-3,6-dimethyl- 5,8-dioxa-2-aza-1(5,3)-pyrazolo[1,5-a]pyrimidina- 4(1,2)-benzenacyclononaphan-9-one [00035] Example 20: (1.sup.3E,1.sup.4E,3R,6S)-4.sup.5-fluoro-3,6-dimethyl-9-oxo-5,8- dioxa-2-aza-1(5,3)-pyrazolo[1,5-a]pyrimidina-4(1,2)- benzenacyclononaphane-12-carbonitrile [00036] Example 21: (1.sup.3E,1.sup.4E,2.sup.2R,6R)-3.sup.5-fluoro-6-methyl-7-oxa-1(5,3)- pyrazolo[1,5-a]pyrimidina-3(3,2)-pyridina-2(1,2)- pyrrolidinacyclooctaphan-8-one [00037] Example 22: (R,1.sup.3E,1.sup.4E,7E)-4.sup.5-fluoro-3-methyl-5-oxa-2-aza- 1(5,3)-pyrazolo[1,5-a]pyrimidina-4(1,2)- benzenacyclononaphan-7-en-9-one [00038]

Example 6: (1.SUP.3.E,1.SUP.4.E,3R,7R)-4.SUP.5.-fluoro-3,7-dimethyl-5,8-dioxa-2-aza-1(5,3)-pyrazolo [1,5-a]pyrimidina-4(1,2)-benzenacyclononaphan-9-one

[0140] ##STR00039##

Step A: (R)-2-hydroxypropyl 4-methylbenzenesulfonate

[0141] ##STR00040##

[0142] To a solution of (R)-propane-1,2-diol (2.08 g, 27.3 mmol, 1.0 eq) and Et.sub.3N (8.3 g, 81.9 mmol, 3.0 eq) in 40 mL CH.sub.2Cl.sub.2, 4-methylbenzenesulfonyl chloride (5.2 g, 27.34 mmol, 1.0 eq) was slowly added at room temperature. Catalytic quantity of DMAP was added. The reaction was stirred overnight at room temperature. After adding water, the product was extracted three times with CH.sub.2Cl.sub.2, and washed once with saturated brine. The organic phases were dried over anhydrous Na.sub.2SO.sub.4 and evaporated in vacuum. The residure was purified by column chromatography on silica to yield the pure product (2.1 g, 33% yield).

[0143] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.85 (d, J=8.2 Hz, 2H), 7.40 (d, J=8.0 Hz, 2H), 4.16-3.98 (m, 2H), 3.92-3.87 (m, 1H), 2.50 (s, 3H), 1.20 (d, J=6.4 Hz, 3H).

Step B: ethyl 5-(((R)-1-(5-fluoro-2-((R)-2-hydroxypropoxy)phenyl)ethyl)amino) pyrazolo[1,5-a]pyrimidine-3-carboxylate

[0144] ##STR00041##

[0145] To a solution of ethyl (R)-5-((1-(5-fluoro-2-hydroxyphenyl)ethyl)amino) pyrazolo[1,5-a]pyrimidine-3-carboxylate (680 mg, 1.97 mmol, 1.0 eq) in DMF, K.sub.2CO.sub.3 (1.36 g, 9.85 mmol, 5.0 eq) and (R)-2-hydroxypropyl 4-methylbenzenesulfonate (500 mg, 2.17 mmol, 1.1 eq) was added in turn. After adding, the reaction mixture was heated to 80° C. at oil bath for 4 hours. After cooling to room temperature, the reaction was quenched with water, the product was extracted three times with EA. The combined organic phases were washed three times with water, dried over anhydrous Na.sub.2SO.sub.4 and evaporated in vacuum. The residure was purified by column chromatography on silica to yield the pure product (278 mg, 35% yield). LC-MS: m/z=403 [M+H].sup.+.

[0146] LC-MS: m/z=403 [M+H].sup.+.

Step C: 5-(((R)-1-(5-fluoro-2-((R)-2-hydroxypropoxy)phenyl)ethyl)amino) pyrazolo[1,5-a]pyrimidine-3-carboxylic Acid

[0147] ##STR00042##

[0148] Procedure referred to step C of example 13.

[0149] LC-MS: m/z=375 [M+H].sup.+.

Step D: (1.SUP.3.E,1.SUP.4.E,3R,7R)-4.SUP.5.-fluoro-3,7-dimethyl-5,8-dioxa-2-aza-1(5,3)-pyrazolo[1,5-a]pyrimidina-4(1,2)-benzenacyclononaphan-9-one

[0150] ##STR00043##

[0151] Procedure referred to step D of example 13.

[0152] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.24 (s, 1H), 8.18 (d, J=7.2 Hz, 1H), 7.01 (d, J=9.0 Hz, 1H), 6.88-6.86 (m, 1H), 6.79 (s, 1H), 6.17-6.15 (m, 2H), 5.62 (s, 1H), 5.52 (s, 1H), 4.37 (d, J=10.4 Hz, 1H), 4.08 (d, J=10.0 Hz, 1H), 1.71 (d, J=6.4 Hz, 3H), 1.57 (d, J=7.0 Hz, 3H). LC-MS: m/z=357 [M+H].sup.+.

Example 9: (1.SUP.3.E,1.SUP.4.E,3R,6S)-4.SUP.5.-fluoro-3,6-dimethyl-8-oxa-5-thia-2-aza-1(5,3)-pyrazolo[1,5-a]pyrimidina-4(1,2)-benzenacyclononaphan-9-one

[0153] ##STR00044##

Step A: ethyl (R)-5-((1-(5-fluoro-2-(((trifluoromethyl)sulfonyl)oxy) phenyl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylate

[0154] ##STR00045##

[0155] To a solution of ethyl (R)-5-((1-(5-fluoro-2-hydroxyphenyl)ethyl)amino)pyrazolo [1,5-a]pyrimidine-3-carboxylate in CH.sub.2Cl.sub.2 under an ice bath, pyridine (489 mg, 3.18 mmol, 3.0 eq) and trifluoromethanesulfonic anhydride (872 mg, 3.09 mmol, 1.5 eq) were slowly added in turn. The reaction was stirred for 2 hours at room temperature. After adding water, the product was extracted three times with CH.sub.2Cl.sub.2. The combined organic phases were washed once with saturated brine, dried over anhydrous Na.sub.2SO.sub.4 and evaporated in vacuum. The residure was purified by column chromatography on silica to yield the pure product (785 mg, 80% yield).

[0156] LC-MS: m/z=477 [M+H].sup.+.

Step B: (R)-1-((tert-butyldimethylsilyl)oxy)propan-2-yl 4-methylbenzenesulfonate

[0157] ##STR00046##

[0158] Procedure referred to step A of example 6.

Step C: (S)—S-(1-((tert-butyldimethylsilyl)oxy)propan-2-yl) ethanethioate

[0159] ##STR00047##

[0160] To a solution of (S)—S-(1-((tert-butyldimethylsilyl)oxy)propan-2-yl) ethanethioate (2.0 g, 5.8 mmol, 1.0 eq) in DMF, potassium ethanethioate (796 mg, 6.96 mmol, 1.2 eq) was slowly added. The reaction mixture was heated to 60° C. at oil bath for 4 hours. After cooling to RT, water was added. The product was extracted three times with EA. The combined organic phases were washed three times with water, dried over anhydrous Na.sub.2SO.sub.4 and evaporated in vacuum. The residure was purified by column chromatography on silica to yield the pure product (1.0 g, 69% yield).

Step D: (S)-1-((tert-butyldimethylsilyl)oxy)propane-2-thiol

[0161] ##STR00048##

[0162] To a solution of (S)—S-(1-((tert-butyldimethylsilyl)oxy)propan-2-yl)ethanethioate (1.0 g, 4.02 mmol, 1.0 eq) in MeOH/H.sub.2O (5:1), NaOH (241 mg, 6.03 mmol, 1.5 eq) was slowly added under an ice bath. The reaction was stirred for further 0.5 hours. MeOH was removed via vacuum distillation, and the product was extracted three times with CH.sub.2Cl.sub.2. The combined organic phases were washed once with water, dried over anhydrous Na.sub.2SO.sub.4 and evaporated in vacuum. The residure was purified by column chromatography on silica to yield the pure product (700 mg, 84% yield).

Step E: ethyl 5-(((R)-1-(2-(((S)-1-((tert-butyldimethylsilyl)oxy)propan-2-yl)thio)-5-fluorophenyl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylate

[0163] ##STR00049##

[0164] To a solution of ethyl (R)-5-((1-(5-fluoro-2-(((trifluoromethyl) sulfonyl)oxy)phenyl) ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (646 mg, 1.35 mmol, 1.0 eq) in 1,4-dioxane, (S)-1-((tert-butyldimethylsilyl)oxy)propane-2-thiol (700 mg, 3.39 mmol, 2.5 eq), Xantphos (156 mg, 0.27 mmol, 0.2 eq), DIEA (1.3 mL, 8.1 mmol, 6.0 eq) and Pd.sub.2(dba).sub.3 (124 mg, 0.135 mmol, 0.1 eq) were added. It was replaced 3 times with N.sub.2, and heated to 120° C. overnight via oil bath. After cooling to RT, water was added. The product was extracted 3 times with EA. The combined organic phases were washed once with saturated water, dried over anhydrous Na.sub.2SO.sub.4 and evaporated in vacuum. The residure was purified by column chromatography on silica to yield the pure product (390 g, 54% yield).

[0165] LC-MS: m/z=533 [M+H].sup.+.

Step F: ethyl 5-(((R)-1-(5-fluoro-2-(((S)-1-hydroxypropan-2-yl)thio)phenyl) ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylate

[0166] ##STR00050##

[0167] Procedure referred to step B of example 13.

[0168] LC-MS: m/z=419 [M+H].sup.+.

Step G: 5-(((R)-1-(5-fluoro-2-(((S)-1-hydroxypropan-2-yl)thio)phenyl)ethyl)amino) pyrazolo[1,5-a]pyrimidine-3-carboxylic Acid

[0169] ##STR00051##

[0170] Procedure referred to step C of example 13.

[0171] LC-MS: m/z=391 [M+H].sup.+.

Step H: (1.SUP.3.E,1.SUP.4.E,3R,6S)-4.SUP.5.-fluoro-3,6-dimethyl-8-oxa-5-thia-2-aza-1 (5,3)-pyrazolo[1,5-a]pyrimidina-4(1,2)-benzenacyclononaphan-9-one

[0172] ##STR00052##

[0173] Procedure referred to step D of example 13.

[0174] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.36-8.24 (m, 2H), 7.49-7.47 (m, 1H), 7.13-6.94 (m, 2H), 6.28 (d, J=7.6 Hz, 1H), 6.12-6.01 (m, 1H), 5.66 (s, 1H), 5.03 (dd, J=11.6, 3.8 Hz, 1H), 3.87 (t, J=11.2 Hz, 1H), 3.50-3.49 (m, 1H), 1.74 (d, J=7.2 Hz, 3H), 1.50 (d, J=7.0 Hz, 3H).

[0175] LC-MS: m/z=373 [M+H].sup.+.

Example 13: (R,1.SUP.3.E,1.SUP.4.E)-4.SUP.5.-fluoro-3-methyl-5,8-dioxa-2-aza-1(5,3)-pyrazolo[1,5-a]pyrimidina-4(1,2)-benzenacyclononaphan-9-one

[0176] ##STR00053##

Step A: ethyl (R)-5-((1-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-5-fluorophenyl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylate

[0177] ##STR00054##

[0178] To a solution of ethyl (R)-5-((1-(5-fluoro-2-hydroxyphenyl)ethyl)amino) pyrazolo[1,5-a]pyrimidine-3-carboxylate (600 mg, 1.74 mmol, 1.0 eq), (R)-2-hydroxypropyl pivalate (614 mg, 2.48 mmol, 2.0 eq), PPh.sub.3 (911 mg, 3.48 mmol, 2.0 eq) in 35 mL CH.sub.2Cl.sub.2, DIAD (703 mg, 3.48 mmol, 2.0 eq) was slowly added under an ice bath. After heating to temperature, the reaction mixture was stirred overnight. TLC analysis showed that the reaction was complete, and the content was evaporated in vacuum to obtain the residure, which was purified by column chromatography on silica to yield the pure product (719 g, 82% yield).

[0179] LC-MS: m/z=503 [M+H].sup.+

Step B: ethyl (R)-5-((1-(5-fluoro-2-(2-hydroxyethoxy)phenyl)ethyl)amino) pyrazolo[1,5-a]pyrimidine-3-carboxylate

[0180] ##STR00055##

[0181] To a solution of ethyl (R)-5-((1-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-5-fluorophenyl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (719 mg, 1.43 mmol, 1.0 eq) in THF, tetrabutylammonium fluoride trihydrate (900 mg, 2.86 mmol, 2.0 eq) was added under an ice bath. After adding and heating to room temperature, the reaction was stirred for further 1.5 hours at room temperature. After adding water, the product was extracted 3 times with EA. The combined organic phases were washed once with water, dried over anhydrous Na.sub.2SO.sub.4 and concentrated via vacuum distillation. The residure was purified by column chromatography on silica to yield the pure product (519 mg, 93% yield).

[0182] LC-MS: m/z=389 [M+H].sup.+.

Step C: (R)-5-((1-(5-fluoro-2-(2-hydroxyethoxy)phenyl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylic Acid

[0183] ##STR00056##

[0184] To a solution of ethyl (R)-5-((1-(5-fluoro-2-(2-hydroxyethoxy)phenyl)ethyl) amino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (519 mg, 1.34 mmol, 1.0 eq) in MeOH/H.sub.2O (5:1) was added NaOH (1.6 g, 40.2 mmol, 40.0 eq) and heated to 80° C. for 5.0 hour under oil bath. The solution was cooled to room temperature, partially concentrated via vacuum distillation, and acidified with 1N HCl solution until pH 5. The resulting suspension was extracted twice with EtOAc. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4 and evaporated in vacuum to obtain the product. (482 mg, 100% yield).

[0185] LC-MS: m/z=361 [M+H].sup.+.

Step D: (R,1.SUP.3.E,1.SUP.4.E)-4.SUP.5.-fluoro-3-methyl-5,8-dioxa-2-aza-1(5,3)-pyrazolo[1,5-a] pyrimidina-4(1,2)-benzenacyclononaphan-9-one

[0186] ##STR00057##

[0187] To a solution of (R)-5-((1-(5-fluoro-2-(2-hydroxyethoxy)phenyl)ethyl) amino)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (150 mg, 0.42 mmol, 1.0 eq) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (161 mg, 0.84 mmol, 2.0 eq) in DCM was added 4-dimethylaminopyridine (DMAP) (5 mg, 0.1 eq). The mixture was heated via oil bath to reflex for 5.0 hour. After cooling to RT, water was added. The resulting suspension was extracted 3 times with DCM. The combined extracts were washed once with saturated brine, dried with anhydrous Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residure was purified by column chromatography on silica to yield the pure product (60 mg, 42% yield).

[0188] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.26 (s, 1H), 8.20 (d, J=7.4 Hz, 1H), 7.04 (dd, J=9.0, 2.6 Hz, 1H), 6.95-6.78 (m, 2H), 6.21 (d, J=7.4 Hz, 1H), 6.14-6.01 (m, 1H), 5.85 (d, J=5.9 Hz, 1H), 5.06 (d, J=11.8 Hz, 1H), 4.62-4.59 (m, 1H), 4.49-4.45 (m, 1H), 4.39-4.19 (m, 2H), 1.56 (d, J=7.0 Hz, 3H). LC-MS: m/z=343 [M+H].sup.+.

Example 23: (R,1.SUP.3.E,1.SUP.4.E)-4.SUP.5.-fluoro-2,3-dimethyl-5,8-dioxa-2-aza-1(5,3)-pyrazolo[1,5-a]pyrimidina-4(1,2)-benzenacyclononaphan-9-one

[0189] ##STR00058##

Step A: tert-butyl (R)-(1-(5-fluoro-2-hydroxyphenyl)ethyl)carbamate

[0190] ##STR00059##

[0191] To a solution of (R)-2-(1-aminoethyl)-4-fluorophenol hydrochloride (3.7 g, 19.3 mmol, 1.0 eq) and trimethylamine (TEA) (5.4 mL, 38.6 mmol, 2.0 eq) in DCM was added di-tert-butyl dicarbonate (Boc.sub.2O) (4.6 g, 21.2 mmol, 1.1 eq). After adding, the mixture was stirred at rt for 2 hours. The reaction mixture was concentrated under reduced pressure. The residure was purified by column chromatography on silica to yield the product (4.9 g, 100% yield).

[0192] LC-MS: m/z=256 [M+H].sup.+.

Step B: tert-butyl (R)-(1-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-5-fluorophenyl) ethyl)carbamate

[0193] ##STR00060##

[0194] To a solution of (600 mg, 2.35 mmol, 1.0 eq) and K.sub.2CO.sub.3 (1.3 g, 9.4 mmol, 4.0 eq) in DMF was added (2-bromoethoxy)(tert-butyl)dimethylsilane(1.1 g, 4.7 mmol, 2.0 eq) and heated to 80° C. under oil bath for 4.0 hour. The mixture was cooled to RT. After adding water, the mixture was extracted 3 times with ethyl acetate. The combined organic layers were washed 3 times with water, and dried with anhydrous Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residure was purified by column chromatography on silica to yield the product (670 mg, 69% yield).

[0195] LC-MS: m/z=414 [M+H].sup.+.

Step C: tert-butyl (R)-(1-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-5-fluorophenyl) ethyl)(methyl)carbamate

[0196] ##STR00061##

[0197] To a solution of (670 mg, 1.62 mmol, 1.0 eq) in DMF was added NaH (194 mg, 4.86 mmol, 3.0 eq) under ice bath. After adding, the mixture was stirred at the temperature for 15 min and was added CH.sub.3I (460 mg, 3.24 mmol, 2.0 eq). The reaction was performed for further 2 h. After adding water, the mixture was extracted 3 times with ethyl acetate. The combined organic layers were washed 3 times with water, and dried with anhydrous Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residure was purified by column chromatography on silica to yield the product (692 mg, 100% yield).

[0198] LC-MS: m/z=428 [M+H].sup.+.

Step D: tert-butyl (R)-(1-(5-fluoro-2-(2-hydroxyethoxy)phenyl)ethyl)(methyl)carbamate

[0199] ##STR00062##

[0200] Procedure referred to step B of example 13.

[0201] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.12-6.94 (m, 2H), 6.84-6.80 (m Hz, 1H), 5.92 (bRs, 1H), 4.63 (bRs, 1H), 4.15-4.13 (m, 1H), 3.92-3.88 (m, 3H), 2.55 (s, 3H), 1.53 (s, 9H), 1.46 (d, J=7.0 Hz, 3H). LC-MS: m/z=314 [M+H].sup.+.

Step E: (R)-2-(4-fluoro-2-(1-(methylamino)ethyl)phenoxy)ethan-1-ol

[0202] ##STR00063##

[0203] Procedure referred to step C of example 24.

[0204] LC-MS: m/z=214 [M+H].sup.+.

Step F: ethyl (R)-5-((1-(5-fluoro-2-(2-hydroxyethoxy)phenyl)ethyl)(methyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylate

[0205] Procedure referred to step D of example 1.

[0206] LC-MS: m/z=403 [M+H].sup.+.

Step G: (R)-5-((1-(5-fluoro-2-(2-hydroxyethoxy)phenyl)ethyl)(methyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylic Acid

[0207] ##STR00064##

[0208] Procedure referred to step C of example 13.

[0209] LC-MS: m/z=375 [M+H].sup.+.

Step H: (R,1.SUP.3.E,1.SUP.4.E)-4.SUP.5.-fluoro-2,3-dimethyl-5,8-dioxa-2-aza-1(5,3)-pyrazolo[1,5-a] pyrimidina-4(1,2)-benzenacyclononaphan-9-one

[0210] ##STR00065##

[0211] Procedure referred to step D of example 13.

[0212] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.39-8.25 (m, 2H), 7.01-6.69 (m, 4H), 6.42 (d, J=7.8 Hz, 1H), 5.0-5.07 (m, 1H), 4.58-5.56 (m, 1H), 4.44-4.42 (m, 1H), 4.17-4.15 (m, 1H), 3.43 (s, 3H), 1.62 (d, J=7.4 Hz, 3H). LC-MS: m/z=357 [M+H].sup.+.

Example 24: (R,1.SUP.3.E,1.SUP.4.E)-4.SUP.5.,6,6-trifluoro-3-methyl-5,8-dioxa-2-aza-1(5,3)-pyrazolo [1,5-a]pyrimidina-4(1,2)-benzenacyclononaphan-9-one

[0213] ##STR00066##

Step A: ethyl (R)-2-(2-(1-((tert-butoxycarbonyl)amino)ethyl)-4-fluorophenoxy)-2,2-difluoroacetate

[0214] ##STR00067##

[0215] To a solution of (700 mg, 2.75 mmol, 1.0 eq) and ethyl 2-bromo-2,2-difluoroacetate (1.4 g, 6.86 mmol, 2.5 eq) in DMF was added DBU (1.06 g, 6.86 mmol, 2.5 eq) and heated to 70° C. under oil bath for 4.0 hour. The mixture was cooled to RT. After adding water, the mixture was extracted 3 times with ethyl acetate. The combined organic layers were washed 3 times with saturated brine, and dried with anhydrous Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residure was purified by column chromatography on silica to yield the product (720 mg, 69% yield).

[0216] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.33-7.23 (m, 1H), 7.10-7.07 (m, 1H), 6.97-5.93 (m, 1H), 5.19 (s, 1H), 4.98 (s, 1H), 4.45 (q, J=7.0 Hz, 2H), 1.49-1.39 (m, 15H).

Step B: tert-butyl (R)-(1-(2-(1,1-difluoro-2-hydroxyethoxy)-5-fluorophenyl)ethyl) carbamate

[0217] ##STR00068##

[0218] To a solution of ethyl (R)-2-(2-(1-((tert-butoxycarbonyl)amino)ethyl)-4-fluoro phenoxy)-2,2-difluoroacetate (720 mg, 1.9 mmol, 1.0 eq) in THF was added lithium aluminum hydride (160 mg, 4.2 mmol, 2.2 eq) under ice bath. The mixture was stirred at 0° C. for 2 hours. The reaction was quenched by adding water. The mixture was extracted 3 times with ethyl acetate. The combined organic layers were washed 3 times with saturated brine, and dried with anhydrous Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residure was purified by column chromatography on silica to yield the product (450 mg, 71% yield).

[0219] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.36-7.33 (m, 1H), 7.14-6.94 (m, 2H), 5.27 (s, 1H), 4.87 (bRs, 2H), 4.05-3.97 (m, 2H), 1.51-1.41 (m, 12H).

Step C: (R)-2-(2-(1-aminoethyl)-4-fluorophenoxy)-2,2-difluoroethan-1-ol

[0220] ##STR00069##

[0221] To a solution of (450 mg, 1.34 mmol, 1.0 eq) in DCM was added 4 mL trifluoroacetic acid (TFA). The mixture was stirred for 3 hours. Saturated sodium bicarbonate solution was added until it was alkaline. The mixture was extracted 3 times with ethyl acetate. The combined extracts were dried with anhydrous Na.sub.2SO.sub.4 and concentrated under reduced pressure to yield the product (315 mg, 100% yield).

[0222] LC-MS: m/z=236 [M+H].sup.+.

Step D: ethyl (R)-5-((1-(2-(1,1-difluoro-2-hydroxyethoxy)-5-fluorophenyl) ethyl) amino)pyrazolo[1,5-a]pyrimidine-3-carboxylate

[0223] ##STR00070##

[0224] Procedure referred to step D of example 1.

[0225] LC-MS: m/z=425 [M+H].sup.+.

Step E: (R)-5-((1-(2-(1,1-difluoro-2-hydroxyethoxy)-5-fluorophenyl)ethyl)amino) pyrazolo[1,5-a]pyrimidine-3-carboxylic Acid

[0226] ##STR00071##

[0227] Procedure referred to step C of example 13.

[0228] LC-MS: m/z=397 [M+H].sup.+.

Step F: (R,1.SUP.3.E,1.SUP.4.E)-4.SUP.5.,6,6-trifluoro-3-methyl-5,8-dioxa-2-aza-1(5,3)-pyrazolo[1,5-a]pyrimidina-4(1,2)-benzenacyclononaphan-9-one

[0229] ##STR00072##

[0230] Procedure referred to step D of example 13.

[0231] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.27-8.25 (m, 2H), 7.23 (s, 1H), 7.08 (dd, J=8.8, 3.0 Hz, 1H), 6.94-6.90 (m, 1H), 6.26 (d, J=7.5 Hz, 1H), 5.85 (bRs, 1H), 5.57 (bRs, 1H), 5.01-4.93 (m, 1H), 4.82-4.77 (m, 1H), 1.58 (d, J=7.0 Hz, 3H). LC-MS: m/z=379 [M+H].sup.+.

Example 25: (R,1.SUP.3.E,1.SUP.4.E)-4.SUP.5.-fluoro-3-methyl-8-oxa-5-thia-2-aza-1(5,3)-pyrazolo[1,5-a]pyrimidina-4(1,2)-benzenacyclononaphan-9-one

[0232] ##STR00073##

[0233] The compound was synthesised according to example 9.

[0234] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.33-8.20 (m, 2H), 7.44-7.42 (m, 1H), 7.06-7.04 (m, 1H), 6.96-6.94 (m, 1H), 6.25 (d, J=7.4 Hz, 1H), 6.17-5.98 (m, 1H), 5.64 (s, 1H), 5.02-5.00 (m, 1H), 4.21-4.06 (m, 1H), 3.78-3.76 (m, 1H), 3.33-3.18 (m, 1H), 1.49 (d, J=7.0 Hz, 3H). LC-MS: m/z=359 [M+H].sup.+.

Compound Evaluation

1. Inhibitory Activity of Compounds on TRKA, TRKB, TRKC and ROS1 (IC.SUB.50.)

[0235] The inhibitory activity of compounds on TRKA, TRKB, TRKC and ROS1 kinase was analyzed with Mobility shift assay. The screening platform is MSA based microfluidic chip technology, which applies the basic concept of capillary electrophoresis to microfluidic environment. The substrate used in the experiment is a poly-peptide labeled with fluorescent. Under the catalysis of enzyme in the reaction system, the substrate is transformed into a product, with the charge changed accordingly. MSA technology could detect the substrate and the product with different charge separately.

[0236] The operation is described as follows:

[0237] The compound powder was dissolved in 100% DMSO (Sigma, Cat. D8418-1l) to prepare a 10 mM storage solution. The compounds had an initial test concentration of 100 nM, were 3-fold serially diluted to obtain 10 samples for multiple hole inspection. For TRKA, TRKB and TRKC kinase targets, Loxo-101 (Selleckchem, Cat. S7960) was used as positive reference compound; for ROS1, Staurosporine (Selleckchem, Cat. S1421) was used as positive reference compound. The gradient diluted compounds were mixed with TRKA/TRKB/TRKC/ROS1 kinase (Carna, Cat. 08-186/08-187/08-197/08-163) with final concentration of 2.5 nM/2.55 nM/2.5 nM/0.3 nM in a Optiplate-384F plate (PerkinElmer, Cat. 6007270), and incubated at room temperature for 10 minutes. After that, ATP was added with final concentration of 47.8 μM/71.2 μM/44.4 μM/26.7 μM, 3 μM Kinase Substrate 22 (GL Biochem, Cat. 112393) was added. The reaction was carried out at room temperature for 30 min/40 min/20 min/20 min respectively. After the reaction was terminated, the conversion rate was read by Caliper EZ Reader II.

[0238] Data analysis:

[00001]$\%\mathrm{Inhibition}=\frac{\mathrm{Conversion}\%\mathrm{\_max}-\mathrm{Conversion}\%\mathrm{\_sample}}{\mathrm{Conversion}\%\mathrm{\_max}-\mathrm{Conversion}\%\mathrm{\_min}}\times 100$

[0239] Conversion %_Sample: conversion rate of sample;

[0240] Conversion %_Min: mean value of negative control, representing the conversion rate without enzyme activity;

[0241] Conversion %_Max: mean value of positive control, which represents the conversion rate reading without compound inhibition.

[0242] Taking the log value of concentration as X axis and the percentage inhibition rate as Y axis, the dose response curve was fitted by the analysis software GraphPad Prism 5, thus the IC.sub.50 value of each compound on enzyme activity inhibition was obtained.

2. Inhibitory Activity of Compounds to ROS1-G2032R (IC.SUB.50.)

[0243] Based on LanceUltra (Perkin Elmer, CR97-100) principle, ROS1-G2032R kinase activity detection platform was established to determine the inhibitory activity of compounds.

[0244] The compound powder was dissolved in 100% DMSO (Sigma, Cat. D8418-1l) to prepare a 10 mM storage solution. The compounds had an initial test concentration of 1000 nM, were 3-fold serially diluted to obtain 11 samples for multiple hole inspection. TPX-0005 (WuXi AppTec. supplied) was used as positive reference compound. The gradient diluted compounds were mixed with 0.016 nM ROS1-G2032R kinase (Abcam, Cat. ab206012), 50 nM LANCE Ultra ULight-poly GT peptide (PerkinElmer, Cat. TRF0100-M) and 2.6 μM ATP (Sigma, Cat. A7699) in the Optiplate-384F plate (PerkinElmer, Cat. 6007299) and incubated at room temperature for 60 mins. 5 μl 40 mM EDTA was used to stop the reaction. Then 2 nM Europium-anti-phosphotyrosine (PT66) (PerkinElmer, Cat. AD0069) was added and incubated at room temperature for 60 mins. The LANCE signal was obtained by EnVision™ (PerkinElmer, 2014) (Excitation light, 320 nm; Emission light, 665 nm). The IC.sub.50 values of the compounds were calculated using XLFIT5 (IDBS) software.

3. Inhibitory Activity of Compounds on TRKA and ALK-L1196M (IC.SUB.50.)

[0245] Based on HTRF of Cisbio (Cisbio, Cat. 08-52) principle, TRKA and ALK-L1196M kinase activity detection platform were established to determine the inhibitory activity of compounds. The compound powder was dissolved in 100% DMSO (Sigma, Cat. D8418-1l) to prepare a 10 mM storage solution. The compounds had an initial test concentration of 1000 nM and 10,000 nM respectively, were 3-fold serially diluted to obtain 11 samples for multiple hole inspection. RXDX-101 (WuXi AppTec. supplied) or Crizotinib (WuXi AppTec. Supplied) was used as positive reference compound.

[0246] The gradient diluted compounds were mixed with 0.5 nM TRKA (Carna, Cat. 08-186)/ALK-L1196M (Carna, Cat. 08-529), 0.3 μM/1 μM TK Substrate-biotin and 90 μM/30 μM ATP (Sigma, Cat. A7699) in a Optiplate-384F plate (PerkinElmer, Cat. 6007299) and incubated at room temperature for 90 mins/120 mins. Then 0.67 nM Eu-TK-Antibody and 50 nM Streptavidin-XL-665 were added, mixed and incubated at room temperature for 60 min. The fluorescence value was obtained by Envision (PerkinElmer, #2014) (Excitation light, 320 nm; Emission light, 665 nm). The IC.sub.50 values of the compounds were calculated using XLFIT5 (IDBS) software.

4. Inhibitory Effect of Compounds on the Proliferation of TRK Fusion and Mutant Stable Cell Lines (IC.SUB.50.)

[0247] Inhibitory effects of compounds on six cell lines were tested, while LOXO-101 (Selleckchem, Cat. S7960) and TPX-0005 (Selleckchem, Cat. S8583) were used as control compound. Six cell lines including Ba/F3 LMNA-NTRK1-WT, Ba/F3 LMNA-NTRK1-G595R, Ba/F3 ETV6-NTRK2-WT, Ba/F3 ETV6-NTRK2-G639R, Ba/F3 ETV6-NTRK2-G639R and Ba/F3 ETV6-NTRK3-G623R were used in this experiment. For the six cell lines, the maximum test concentration of the compound was 1 μM, 1 μM, 10 μM, 100 μM, 1 μM and 10 μM, were 3.16-fold gradient diluted to obtain 9 samples.

[0248] The operation is described briefly as follows:

[0249] The cells in logarithmic growth phase were harvested to ensure that the cell viability was above 90%. 3000 cell/well were seeded in 96-well plate (Corning, Cat #3603). The cells were incubated overnight with 5% CO2, 95% humidity at 37° C. Then each well containing the cells of the 96-well plate was added the compound solutions, 3 wells for each compound solution, and continued to incubate for another 72 hrs. Then CellTiter-Glo® kit (Promega, Cat #G7572) was used to do the detection. First, the CTG reagent was melted and the cell plate was balanced to room temperature. The same volume of CTG solution was added into each well and vibrated on the shaker for 5 minutes to lyse the cells. The cell plate was kept at room temperature for 20 minutes to stabilize the luminescent signal. The luminescent value was collected by SpectraMax microplate detector (MD, 2104-0010A). The dose response curve was fitted by the analysis software GraphPad Prism 5, thus the IC.sub.50 value of each compound on cell proliferation inhibition was obtained.

Cell survival rate (%)=(lum.sub.drug−lum.sub.medium)/(lum.sub.cell−lum.sub.medium)×100%

TABLE-US-00002 TABLE 1 Inhibitory activity on TRKA and ALK-L1196M kinases TRKA ALK L1196M Compound IC.sub.50 (nM) IC.sub.50 (nM) Crizotinib ND 106 Ceritinib 182 ND Alectinib 60.4 ND Lorlatinib 6.2 9 TPX-0005 0.067 39 Example 1 0.13 50

TABLE-US-00003 TABLE 2 Inhibitory activity on TRKA, TRKB, TRKC, ROS1 and ROS1-G2032R kinases TRKA TRKC ROS1- IC.sub.50 TRKB IC.sub.50 ROS1 G2032R Compound (nM) IC.sub.50 (nM) (nM) IC.sub.50 (nM) IC.sub.50 (nM) LOXO-101 1.1 1.9 2.0 — — TPX-0005 0.32 0.44 0.55 0.17 1.01 Example 1 0.42 0.50 0.61 0.33 0.36 Example 13 0.62 0.90 0.73 1.9 — Example 6 0.75 1.20 1.20 0.78 — Example 23 0.39 0.48 0.63 4.8 — Example 24 0.56 0.60 0.75 0.29 — Example 9 0.59 0.86 0.76 0.54 — Example 25 1.10 1.60 1.10 1.20 —

TABLE-US-00004 TABLE 3 Inhibitory activity on proliferation of TRK fusion and mutant stable cell lines IC.sub.50 (nM) TRKC Cell line LOXO-101 TPX-0005 Example 1 Ba/F3 LMNA-NTRK1-WT 47.4* 0.7.sup.# 5.4*/1.04 Ba/F3 LMNA-NTRK1-G595R 2174.3* 2.0.sup.# 7.8*/1.34 Ba/F3 ETV6-NTRK2-WT 75.3* 2.5.sup.# 7.8*/4.24 Ba/F3 ETV6-NTRK2-G639R 8183.5* 86.6.sup.# 100.5*/64.64  Ba/F3 ETV6-NTRK3-WT 6.8* 0.8.sup.# 0.2*/1.04 Ba/F3 LMNA-NTRK3-G623R 1095.7* 9.0.sup.# 8.4*/8.34 Note: *the same batch of test; .sup.#the same batch of test.

5. Analysis of Synergistic Effect

[0250] H1975 (L858R and T790M double mutation) cells were cultured in RPMI-1640 medium containing 10% FBS and 1% P/S (promycin/streptomycin). In the testing of the compounds, H1975 cells were seeded in 96-well plate (Corning, Cat #3917) at 3000 cell/well and the compounds were added at 195 μL solution/well. The initial test concentration of the compound was 10 μM, 3-fold gradient diluted to obtain 11 samples. 4 μL of each sample was added to 96 μL RPMI-1640 medium to dilute the 25× Compound. 5 μL was added to 195 μL cell medium (the final concentration of DMSO is 0.1%, v/v). After 72 hr incubation, 35 μL of CellTiter-Blue® (Promega, Cat #G8082) was added to the cells. The fluorescence signal was measured with FlexStation 3 (Molecular Devices) according to the instructions, and the IC.sub.50 value of the compound on cell proliferation inhibition was calculated by Graphpad Grism 5.0 software. The Chou-Talalay index method was used to analyze the effect of the combination. 0.9≤Combination Index (CI)≤1.1 means additive effect, 0.8≤CI<0.9 means low synergistic effect, 0.6≤CI<0.8 means medium synergistic effect, 0.4≤CI<0.6 means high synergistic effect, 0.2≤CI<0.4 means strong synergistic effect.

[0251] The data showed that the combination of example 1 and AZD9291 showed medium to high synergistic effect on the EGFR double mutant H1975 (L858R and T790M double mutation) cells (Example 1, CI=0.53-0.67), indicating that the combination of the test compound and the EGFR inhibitor may overcome EGFR resistance.

6. Pharmacokinetic Analysis

[0252] Male SD rats were grouped, each group having 3 rats. Each group was given the compound of Example 1 (5 mg/kg) or TPX-0005 (5 mg/kg) by single oral gavage, or the compound of Example 1 (1 mg/kg) by intravenous injection. Animals were fasted overnight from 10 hours before administration to 4 hours after administration. Blood samples were collected at 0.25, 0.5, 1, 2, 4, 8 and 24 hours after administration in the oral group and at 0.083, 0.25, 0.5, 1, 2, 4, 8 and 24 hours after injection in the intravenous group. After isoflurane anesthesia, 0.3 ml whole blood collected through the fundus vein plexus was put into the heparin anticoagulant tube. The sample was centrifuged at 4° C. at 4000 rpm for 5 min. The plasma was transferred into the centrifuge tube and stored at −80° C. until analysis. The extract from the plasma extracted by protein precipitation was analyzed by LC/MS/MS. The PK results were shown in Table 4 and table 5.

TABLE-US-00005 TABLE 4 Rat PK analysis (PO, 5 mg/kg) Example 1 TPX-0005 T.sub.1/2 (hr) 1.84 3.55 Tmax (hr) 0.667 1.33 Cmax (ng/mL) 952 503 AUC.sub.0-inf (hr * ng/mL) 3548 2948 F (%) 114 91.5

TABLE-US-00006 TABLE 5 Rat PK analysis (IV, 1 mg/kg) Example 1 T.sub.1/2 (hr) 1.70 C.sub.0 (ng/mL) 217 Vdss (L/kg) 4.26 C1 (mL/min/kg) 27.2 AUC.sub.0-inf (hr * ng/mL) 621

[0253] The data showed that the compound of Example 1 had the better oral pharmacokinetic performance than that of TPX-0005, and the oral bioavailability of Example 1 reached 100%. According to the same method, all the other compounds tested also showed better PK properties profile than TPX-0005.

7. Blood-To-Brain Distribution

[0254] Male SD rats were grouped, each group having 12 rats. Each group was given the compound of Examples (10 mg/kg) by single oral gavage. The animals were fasted overnight from 10 hours before administration to 4 hours after administration. The rats were killed at 0.5, 1, 4 and 12 hours after administration, and blood and brain tissues were collected. The samples were centrifuged at 4000 rpm at 4° C. for 5 min and plasma was transferred into the centrifuge tube and stored at −80° C. until analysis. The extract from the plasma extracted by protein precipitation was analyzed by LC/MS/MS.

TABLE-US-00007 TABLE 6 Blood-to-brain distribution TPX-0005 Example 1 PK parameter Plasma Brain Plasma Brain AUC.sub.0-inf (nM .Math. h) 13947 2351 18163 20766 Ratio (Brain/Plasma) 0.17 1.14

[0255] Although the preferred embodiments of the present disclosure have been disclosed in order to illustrate the present disclosure, those skilled in the art should understand that various modifications, addition and replacement may be made to the present disclosure without departing from the concept and scope of the present disclosure defined by the claims.