Tropomyosin receptor kinase inhibitor, preparation method therefor and use thereof

Abstract

Disclosed in the present invention are a pyrazolo [1,5-a]pyrimidine derivative having a structure of formula (I), a pharmaceutical composition comprising the compound of formula (I), and use of the compound in the preparation of a medicament for preventing or treating diseases associated with tropomyosin receptor kinases, in particular for preventing or treating cancers associated with tropomyosin receptor kinases. Each substituent in formula (I) has the same definition as that in the description. ##STR00001##

Claims

1. A compound represented by formula (I), stereoisomer thereof, or pharmaceutically acceptable salt thereof: ##STR00056## wherein R.sup.1 is selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8 alkynyl, —COR.sup.5, —SO.sub.2R.sup.5, and —SOR.sup.5, and optionally further substituted with one or more substituents selected from deuterium, halogen, hydroxyl, cyano, nitro, —NR.sup.6R.sup.8, —NR.sup.6COR.sup.7, —COR.sup.7, —SO.sub.2R.sup.7, —SOR.sup.7, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.8 alkoxy, and a 4-10 membered heterocyclic group; R.sup.2 and R.sup.4 are each independently selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.2-C.sub.8 alkenyl, and C.sub.2-C.sub.8 alkynyl, and optionally further substituted with one or more substituents selected from deuterium, halogen, hydroxyl, cyano, nitro, —NR.sup.6R.sup.8, —NR.sup.6COR.sup.5, —COR.sup.5, —SO.sub.2R.sup.5, —SOR.sup.5, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.8 alkoxy, and a 4-10 membered heterocyclic group; R.sup.5 and R.sup.7 are each independently selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, and —NR.sup.8; R.sup.6 and R.sup.8 are each independently selected from H, C.sub.1-C.sub.8 alkyl, and C.sub.3-C.sub.8 cycloalkyl; Y is —(CH.sub.2).sub.n— or —O(CH.sub.2).sub.n—; n is 0, 1, 2 or 3; alternatively, any two of R.sup.1, R.sup.2, and R.sup.4 independently form a C.sub.3-C.sub.8 cycloalkyl group or a 4-10 membered heterocyclic group; R.sup.3 is selected from H, deuterium, halogen, cyano, nitro, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, and C.sub.1-C.sub.8 alkoxy; and X is CH or N.

2. The compound of general formula (I), stereoisomer thereof, or pharmaceutically accepted salt thereof according to claim 1, which is a compound of general formula (II), stereoisomer thereof, or pharmaceutically acceptable salt thereof: ##STR00057## wherein R.sup.1 is selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8 alkynyl, —COR.sup.5, —SO.sub.2R.sup.5, and —SOR.sup.5, and optionally further substituted with one or more substituents selected from deuterium, halogen, hydroxyl, cyano, nitro, —NR.sup.6R.sup.8, —NR.sup.6COR.sup.7, —COR.sup.7, —SO.sub.2R.sup.7, —SOR.sup.7, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.8 alkoxy, and a 4-10 membered heterocyclic group; R.sup.2 and R.sup.4 are each independently selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.2-C.sub.8 alkenyl, and C.sub.2-C.sub.8 alkynyl, and optionally further substituted with one or more substituents selected from deuterium, halogen, hydroxyl, cyano, nitro, —NR.sup.6R.sup.8, —NR.sup.6COR.sup.5, —COR.sup.5, —SO.sub.2R.sup.5, —SOR.sup.5, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.8 alkoxy, and a 4-10 membered heterocyclic group; R.sup.5 and R.sup.7 are each independently selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, and —NR.sup.8; R.sup.6 and R.sup.8 are each independently selected from H, C.sub.1-C.sub.8 alkyl, and C.sub.3-C.sub.8 cycloalkyl; alternatively, any two of R.sup.1, R.sup.2, and R.sup.4 independently form a C.sub.3-C.sub.8 cycloalkyl group or a 4-10 membered heterocyclic group; R.sup.3 is selected from H, deuterium, halogen, cyano, nitro, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, and C.sub.1-C.sub.8 alkoxy; and X is CH or N.

3. The compound according to claim 2, wherein: R.sup.1 is selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.2-C.sub.8 alkenyl, and C.sub.2-C.sub.8 alkynyl, and optionally further substituted with one or more substituents selected from deuterium, halogen, hydroxyl, cyano, —NR.sup.6R.sup.8, —NR.sup.6COR.sup.7, —COR.sup.7, —SO.sub.2R.sup.7, —SOR.sup.7, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.8 alkoxy, and a 4-10 membered heterocyclic group; R.sup.2 and R.sup.4 are each independently selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.2-C.sub.8 alkenyl, and C.sub.2-C.sub.8 alkynyl, and optionally further substituted with one or more substituents selected from deuterium, halogen, hydroxyl, cyano, —NR.sup.6R.sup.8, —NR.sup.6COR.sup.5, —COR.sup.5, —SO.sub.2R.sup.5, —SOR.sup.5, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.8 alkoxy, and a 4-10 membered heterocyclic group; R.sup.5 and R.sup.7 are each independently selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, and —NR.sup.8; R.sup.6 and R.sup.8 are each independently selected from H, C.sub.1-C.sub.8 alkyl, and C.sub.3-C.sub.8 cycloalkyl; alternatively, any two of R.sup.1, R.sup.2, and R.sup.4 independently form a C.sub.3-C.sub.8 cycloalkyl group, and a 4-10 membered heterocyclic group; R.sup.3 is halogen; and X is CH or N.

4. The compound according to claim 3, wherein: R.sup.1 is selected from H, C.sub.1-C.sub.8 alkyl, and C.sub.3-C.sub.8 cycloalkyl, and optionally further substituted with one or more selected from deuterium, halogen, hydroxyl, cyano, —NR.sup.6R.sup.8, —NR.sup.6COR.sup.7, —COR.sup.7, —SO.sub.2R.sup.7, —SOR.sup.7, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.8 alkoxy, and a 4-10 membered heterocyclic group; R.sup.2 and R.sup.4 are each independently selected from H, C.sub.1-C.sub.8 alkyl, and C.sub.3-C.sub.8 cycloalkyl, and optionally further substituted with one or more substituents selected from deuterium, halogen, hydroxyl, cyano, —NR.sup.6R.sup.8, —NR.sup.6COR.sup.5, —COR.sup.5, —SO.sub.2R.sup.5, —SOR.sup.5, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.8 alkoxy, and a 4-10 membered heterocyclic group; R.sup.5 and R.sup.7 are each independently selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, or —NR.sup.8; R.sup.6 and R.sup.8 are each independently selected from H, C.sub.1-C.sub.8 alkyl, or C.sub.3-C.sub.8 cycloalkyl; Alternatively, any two of R.sup.1, R.sup.2, and R.sup.4 independently form a C.sub.3-C.sub.8 cycloalkyl group or a 4-10 membered heterocyclic group; R.sup.3 is halogen; and X is CH or N.

5. The compound according to claim 4, wherein: R.sup.1 is selected from H, C.sub.1-C.sub.8 alkyl, and C.sub.3-C.sub.8 cycloalkyl, and optionally further substituted with one or more substituents selected from deuterium, halogen, hydroxyl, cyano, —SO.sub.2R.sup.7, C.sub.1-C.sub.4 alkyl, C.sub.3-C.sub.6 cycloalkyl, and C.sub.1-C.sub.4 alkoxy; R.sup.7 is selected from H, C.sub.1-C.sub.8 alkyl, and C.sub.3-C.sub.8 cycloalkyl; R.sup.2 and R.sup.4 are each independently selected from H, C.sub.1-C.sub.8 alkyl, and C.sub.3-C.sub.8 cycloalkyl, and optionally further substituted with one or more substituents selected from deuterium, halogen, hydroxyl, cyano, C.sub.1-C.sub.4 alkyl, C.sub.3-C.sub.6 cycloalkyl, and C.sub.1-C.sub.4 alkoxy; alternatively, any two of R.sup.1, R.sup.2, and R.sup.4 independently form a C.sub.3-C.sub.8 cycloalkyl group or a 4-10 membered heterocyclic group; R.sup.3 is halogen; and X is selected from CH or N.

6. The compound according to claim 5, wherein: R.sup.1 is selected from H, C.sub.1-C.sub.8 alkyl, and C.sub.3-C.sub.8 cycloalkyl, and optionally further substituted with one or more substituents selected from deuterium, hydroxyl, halogen, —SO.sub.2R.sup.7, and C.sub.1-C.sub.4 alkoxy; R.sup.7 is H or C.sub.1-C.sub.8 alkyl; R.sup.2 and R.sup.4 are each independently selected from H, C.sub.1-C.sub.8 alkyl, and C.sub.3-C.sub.8 cycloalkyl; alternatively, any two of R.sup.1, R.sup.2, and R.sup.4 independently form a 4-10 membered heterocyclic group; R.sup.3 is fluorine or chlorine; and X is CH or N.

7. The compound according to claim 6, wherein: R.sup.1 is selected from H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl, and optionally further substituted with one or more substituents selected from deuterium, hydroxyl, F, Cl, —SO.sub.2CH.sub.3, and methoxy; R.sup.2 and R.sup.4 are each independently selected from H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl; alternatively, any two of R.sup.1, R.sup.2, and R.sup.4 independently form a morpholinyl; R.sup.3 is fluorine or chlorine; and X is CH or N.

8. A compound, stereoisomer thereof or pharmaceutically acceptable salt thereof, wherein the compound has a structural formula selected from the following: ##STR00058## ##STR00059## ##STR00060## ##STR00061## ##STR00062##

9. A compound, stereoisomer thereof or pharmaceutically acceptable salt thereof, wherein the compound has a structural formula selected from the following: ##STR00063## ##STR00064##

10. A pharmaceutical composition comprising the compound, stereoisomer thereof, or pharmaceutically acceptable salt thereof according to claim 1, and a pharmaceutically acceptable carrier.

11. The pharmaceutical composition according to claim 10, wherein the pharmaceutical composition is a form of capsule, powder, tablet, granule, pill, injection, syrup, oral liquid, inhalant, ointment, suppository, or patch.

12. A method for inhibiting tropomyosin receptor kinase activity diseases, comprising administering to a subject in need thereof an effective amount of the compound, stereoisomer thereof, or pharmaceutically acceptable salt thereof according to claim 1.

13. The method according to claim 12, wherein the tropomyosin receptor kinase is TrkA, TrkB, or TrkC.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is the 1H NMR spectrum of a compound of Example 1;

(2) FIG. 2 is the 1H NMR spectrum of a compound of Example 2;

(3) FIG. 3 is the 1H NMR spectrum of a compound of Example 3a;

(4) FIG. 4 is the 1H NMR spectrum of a compound of Example 3b;

(5) FIG. 5 is the 1H NMR spectrum of a compound of Example 4;

(6) FIG. 6 is the 1H NMR spectrum of a compound of Example 5;

(7) FIG. 7 is the 1H NMR spectrum of a compound of Example 6;

(8) FIG. 8 is the 1H NMR spectrum of a compound of Example 7a;

(9) FIG. 9 is the 1H NMR spectrum of a compound of Example 7b;

(10) FIG. 10 is the 1H NMR spectrum of a compound of Example 8a;

(11) FIG. 11 is the 1H NMR spectrum of a compound of Example 8b;

(12) FIG. 12 is the 1H NMR spectrum of a compound of Example 9; and

(13) FIG. 13 is the 1H NMR spectrum of a compound of Example 10.

DETAILED DESCRIPTION OF THE INVENTION

(14) The present invention will be described below with reference to specific examples. Those skilled in the art can understand that these examples are only used for illustrating the present invention and are not intended to limit the scope of the present invention in any way.

(15) The experimental methods in the examples below are conventional methods, unless otherwise specified. Medicinal raw materials and reagent materials used in the examples below are commercially available products, unless specifically stated.

Example 1

(R,I.SUP.3.E,I.SUP.4.E)-6-cyclopropyl-3.SUP.5.-fluoro-6,7-diazepine-1(5,3)-pyrazolo[1,5-a]pyrimidine-3(3,2)-pyridine-2(1,2)-pyrrolidine cyclooctane-8-one

(16) ##STR00011##

Step 1: Synthesis of (R)-tert-butyl 2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidine-1-carboxylate

(17) ##STR00012##

(18) At room temperature, tert-butyl pyrrolidine-1-carboxylate (2.86 mL, 16.03 mmol), (−)-Sparteine (4.50 g, 19.20 mmol) and MTBE (35.0 mL) were added into a 250 mL three-necked flask. The atmosphere in the flask was replaced with nitrogen for three times, and the temperature was lowered slowly to −78° C. Sec-BuLi (17.01 mL, 21.70 mmol, 1.3N in cyclohexane) was added dropwise after the temperature was stabilized, and stirred at −75° C. for 3 hours. ZnCl.sub.2 (14.68 mL, 14.36 mmol, 1 N in THF) was slowly added dropwise at −65° C., and stirred for 20 minutes after the temperature was lowered to around −78° C. The temperature was increased to room temperature, and under the protection of nitrogen, 3-bromo-5-fluoro-2-methoxypyridine (3.44 g, 16.70 mmol), Pd(OAc).sub.2 (0.18 g, 0.84 mmol), and t-Bu.sub.3P—HBF.sub.4 (0.28 g, 1.00 mmol) were sequentially added. After the addition was completed, the reaction was carried out at room temperature for 16 hours. 1.50 mL ammonia water was added dropwise to the reaction system and stirred at room temperature for 1 hour. The reaction solution was filtered through celite, washed with MTBE (20 mL×3), and separated. The organic phase was concentrated and subjected to column chromatography (Hexane:EA=20:1). After concentration under reduced pressure, 2.60 g of a yellow oil was obtained, with a yield of 52.53%. .sup.1H NMR (400 MHz, Chloroform-d) δ 7.84 (d, J=7.2 Hz, 1H), 7.09 (dd, J=29.5, 6.1 Hz, 1H), 4.99 (dd, J=52.8, 8.0 Hz, 1H), 3.93 (s, 3H), 3.53 (m, 2H), 2.28 (m, 1H), 1.82 (m, 3H), 1.46 (s, 9H).

Step 2: Synthesis of (R)-5-fluoro-2-methoxy-3-(pyrrolidin-2-yl)pyridine

(19) ##STR00013##

(20) At room temperature, (R)-tert-butyl 2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidine-1-carboxylate (2.60 g, 8.77 mmol), trifluoroacetic acid (7.80 mL) and dichloromethane (7.80 mL) were sequentially added into a 100 mL single-necked flask, and stirred at room temperature for 1.5 hours. After the raw materials were completely reacted, the reaction was stopped. The reaction solution was evaporated to dryness under reduced pressure to obtain a yellow oil of 3.94 g, which was directly used in the next reaction step.

Step 3: Synthesis of (R)-ethyl 5-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate

(21) ##STR00014##

(22) At room temperature, (R)-5-fluoro-2-methoxy-3-(pyrrolidin-2-yl)pyridine (1.72 g, 8.78 mmol), ethyl 5-chloropyrazolo[1,5-a]pyrimidine-3-carboxylate (2.38 g, 10.54 mmol), DIEA (5.25 g, 40.65 mmol), and DMF (19.30 mL) were added into a 30 mL microwave sealed tube, slowly warmed to 90° C., and reacted with stirring for 15.5 h. After the reaction was cooled, it was dissolved and dispersed by adding EA (100 mL), washed by adding water (100 mL), and separated. The organic phase was washed sequentially with saturated brine (100 mL) and a saturated aqueous solution of sodium bicarbonate (100 mL), dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. Column chromatography (PE:EA=1:1) was performed to obtain a yellow solid of 1.83 g. MS (ESI) m/z: 386[M+H].sup.+. .sup.1H NMR (400 MHz, Chloroform-d) δ 8.28 (s, 1H), 8.15 (d, J=5.2 Hz, 1H), 7.91 (s, 1H), 7.04 (d, J=7.2 Hz, 1H), 5.84 (d, J=4.5 Hz, 1H), 5.09 (m, 1H), 4.37 (m, 2H), 4.18-4.11 (m, 1H), 4.02 (s, 3H), 3.96 (m, 1H), 2.47 (m, 1H), 2.04 (m, 2H), 1.95 (m, 1H), 1.42 (m, 3H).

Step 4: Synthesis of (R)-ethyl 5-(2-(5-fluoro-2-hydroxypyridin-3-yl)pyrrolidin-1-yl)pyrazolo [1,5-a]pyrimidine-3-carboxylate

(23) ##STR00015##

(24) At room temperature, (R)-ethyl 5-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (1.83 g, 4.75 mmol), HBr (18.30 mL, 33% in AcOH) were added into a 100 mL single-necked flask, slowly warmed to 90° C., and reacted with stirring for 4.5 hours. The reaction was dissolved and dispersed by adding EA (100 mL), washed by adding water (100 mL), and separated. The organic phase was washed sequentially with a saturated NaHCO.sub.3 solution (100 mL) and saturated brine (100 mL), dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. Column chromatography (DCM:EtOH=20:1) was performed to obtain a yellow solid of 1.34 g. MS (ESI) m/z: 372[M+H].sup.+. .sup.1H NMR (400 MHz, Chloroform-d) δ 12.92 (br, 1H), 8.29 (s, 1H), 8.21 (d, J=7.5 Hz, 1H), 7.30 (s, 1H), 7.17 (s, 1H), 6.93 (d, J=4.7 Hz, 1H), 5.13 (m, 1H), 4.37 (q, J=8.7 Hz, 2H), 4.16 (m, 1H), 3.95 (m, 1H), 2.49 (m, 1H), 2.13-2.07 (m, 2H), 1.95 (m, 1H), 1.42 (t, J=8.8 Hz, 3H).

Step 5: Synthesis of (R)-ethyl 5-(2-(5-fluoro-2-(trifluoromethyl-sulfonyloxy)pyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate

(25) ##STR00016##

(26) At room temperature, (R)-ethyl 5-(2-(5-fluoro-2-hydroxypyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (1.34 g, 3.61 mmol) and DMF (13.0 mL) were added into a 100 mL single-necked flask, and ultrasonically dispersed to be completely dissolved. 1,1,1-trifluoro-N-phenyl-N-((trifluoromethyl)sulfonyl)methylsulfonamide (1.42 g, 3.97 mmol) and TEA (0.44 g, 4.33 mmol) were sequentially added to the reaction system and reacted with stirring at room temperature for 24 hours. EA (110 mL) was added to the reaction system. The reaction system was washed sequentially with a saturated NaHCO.sub.3 solution (100 mL), water (100 mL) and saturated brine (100 mL), dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. Column chromatography (PE:EA=1:1) was performed to obtain a foamy white solid of 1.60 g. MS (ESI) m/z: 504.0[M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.78 (d, J=7.8 Hz, 1H), 8.37 (s, 1H), 8.17 (s, 1H), 7.93 (d, J=8.8 Hz, 1H), 6.69 (d, J=7.8 Hz, 1H), 5.37 (m, 1H), 4.08 (m, 1H), 4.04 (q, J=7.2 Hz, 2H), 3.65 (m, 1H), 2.08 (m, 3H), 1.91 (m, 1H), 1.11 (t, J=7.2 Hz, 3H).

Step 6: Synthesis of ethyl (S)-5-(2-(5-fluoro-2-vinylpyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate

(27) ##STR00017##

(28) At room temperature, (R)-ethyl 5-(2-(5-fluoro-2-(trifluoromethyl-sulfonyloxy)pyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (1.75 g, 3.47 mmol), tributyl(vinyl)tin (1.73 g, 5.21 mmol), lithium chloride (0.74 g, 17.38 mmol), (Ph.sub.3P).sub.2PdCl.sub.2 (0.24 g, 0.34 mmol), and DMF (35.0 mL) were sequentially added into a 100 mL single-necked flask, replaced with nitrogen for three times, slowly warmed to 80° C., and reacted with stirring for for 4 hours. The reaction solution was cooled to room temperature, sequentially washed with a saturated NaHCO.sub.3 solution (100 mL), water (100 mL) and saturated brine (100 mL), dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. Column chromatography (PE:EA=1:1) was performed to obtain a white solid of 1.23 g. MS (ESI) m/z: 382.0[M+H].sup.+. .sup.1H NMR (400 MHz, Chloroform-d) δ 8.36 (s, 1H), 8.29 (s, 1H), 8.16 (br, 1H), 7.05 (br, 2H), 6.42 (d, J=16.8 Hz, 1H), 5.81-5.53 (m, 2H), 5.20 (m, 1H), 4.36 (m, 2H), 4.11 (q, J=7.2 Hz, 2H), 2.58 (m, 1H), 2.07 (m, 3H), 1.26 (t, J=7.2 Hz, 3H).

Step 7: Synthesis of ethyl (S)-5-(2-(2-(2-(cyclopropylamino)ethyl)-5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate

(29) ##STR00018##

(30) At room temperature, ethyl(S)-5-(2-(5-fluoro-2-vinylpyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (1.23 g, 3.22 mmol), cyclopropylamine (5.52 g, 96.75 mmol), glacial acetic acid (5.81 g, 96.75 mmol), and ethanol (12.30 mL) were added into a 100 mL single-necked flask. Under the protection of nitrogen, the temperature was slowly increased to 75° C., and the reaction was carried out under reflux for 20.5 hours. The reaction solution was cooled to room temperature, adjusted to a neutral pH with a saturated aqueous solution of sodium carbonate, and extracted by adding EA (150 mL). The organic phase was dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. Column chromatography (DCM:EtOH=25:1) was performed to obtain a yellow solid of 378.2 mg, with 605.4 mg of the raw materials recovered. The above operations were carried out again on the recovered raw materials, and the products were combined to obtain a yellow solid of 507.2 mg. MS (ESI) m/z: 439.0[M+H].sup.+.

Step 8: Synthesis of ethyl (S)-5-(2-(2-(2-(2-(tert-butylcarbonyl)-1-cyclopropylhydrazino)ethyl)-5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate

(31) ##STR00019##

(32) At room temperature, ethyl (S)-5-(2-(2-(2-(cyclopropylamino)ethyl)-5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (0.20 g, 0.45 mmol) and tetrahydrofuran (4.0 mL) were added into a 10 mL single-necked flask, ultrasonically dissolved, and cooled to 0° C. N-Boc-O-p-toluenesulfonyl-hydroxylamine (131.0 mg, 0.45 mmol) and N-methylmorpholine (22.5 mg, 0.23 mmol) were added. The reaction solution was returned to room temperature and allowed to react with stirring overnight. The reaction solution was diluted with dichloromethane (40 mL), washed with a saturated aqueous solution of sodium bicarbonate (300 mL), and extracted with di chloromethane (40 mL×3). The organic phase was dried over anhydrous Na.sub.2SO.sub.4, and evaporated to dryness under reduced pressure. Column chromatography (PE:EA=1:1) was performed to obtain a yellow solid of 100.0 mg. MS (ESI) m/z: 554.0[M+H].sup.+.

Step 9: Synthesis of(S)-5-(2-(2-(2-(2-(tert-butylcarbonyl)-1-cyclopropylhydrazino)ethyl)-5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid

(33) ##STR00020##

(34) At room temperature, ethyl (S)-5-(2-(2-(2-(2-(tert-butylcarbonyl)-1-cyclopropylhydrazino)ethyl)-5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (85.0 mg, 0.15 mmol) and ethanol (3.0 mL) were added into a 10 mL single-necked flask, and stirred. After complete dissolution, lithium hydroxide monohydrate (38.6 mg, 0.92 mmol, dissolved in 0.5 mL water) was added, the temperature was slowly increased to 70° C., and the reaction was carried out with stirring for 15 hours. The reaction solvent was evaporated to dryness under reduced pressure at 40° C. to obtain a crude product as a yellow solid. MS(ESI) m/z: 526.0[M+H].sup.+.

Step 10: Synthesis of (R,1.SUP.3.E,1.SUP.4.E)-6-cyclopropyl-3.SUP.5.-fluoro-6,7-diazepine-1(5,3)-pyrazolo[1,5-a]pyrimidine-3(3, 2)-pyridine-2(1,2)-pyrrolidine cyclooctane-8-one

(35) ##STR00021##

(36) At room temperature, the crude product from the above step was added into a 10 mL single-necked flask, followed by addition of methylene chloride (1.0 mL) and trifluoroacetic acid (1.0 mL), ultrasonically dispersed, and reacted with stirring for 2 hours. The reaction solvent was evaporated to dryness under reduced pressure at 40° C. to obtain a yellow oil, which was further suctioned by an oil pump at room temperature for 4 hours to obtain a pale yellow crude product 1. MS(ESI) m/z: 426.0[M+H].sup.+.

(37) At room temperature, the above crude product 1 was placed into a 10 mL single-necked flask, followed by addition of HOBt (103.7 mg, 0.77 mmol), EDCI (310.7 mg, 0.77 mmol) and DMF (4.0 mL), and ultrasonically dispersed. TEA (310.7 mg, 3.07 mmol) was added. Under the protection of nitrogen, the reaction was carried out overnight at room temperature. The reaction solution was evaporated to dryness under reduced pressure and the product was purified by reverse column chromatography (0.1% formic acid in water/acetonitrile, 95%˜0) to obtain a white solid of 11.0 mg. MS (ESI) m/z: 408.0[M+H].sup.+. .sup.1H NMR (300 MHz, Chloroform-d) δ 9.73 (br, 1H), 8.33 (d, J=7.7 Hz, 1H), 8.31 (d, J=2.5 Hz, 1H), 8.29 (s, 1H), 7.08 (dd, J=9.4, 2.8 Hz, 1H), 6.33 (d, J=7.7 Hz, 1H), 5.58 (t, J=7.4 Hz, 1H), 4.01-3.65 (m, 6H), 2.94 (dd, J=16.4, 8.6 Hz, 1H), 2.62 (dt, J=13.3, 6.7 Hz, 1H), 2.43 (dt, J=12.2, 6.0 Hz, 1H), 2.24 (dt, J=13.9, 7.5 Hz, 1H), 1.96 (dt, J=13.7, 6.7 Hz, 1H), 1.17 (dd, J=10.3, 5.6 Hz, 1H), 0.87 (dd, J=10.3, 4.0 Hz, 1H), 0.51 (d, J=6.3 Hz, 2H).

Example 2

(1.SUP.3.E,1.SUP.4.E,2.SUP.2.R)-6-cyclopropylamino-3.SUP.5.-fluoro-5-methyl-6,7-diazepine-1(5,3)-pyrazolo[1,5-a]pyrimidine-3(3,2)-azabenzene-2(1,2)-pyrrolidine cyclooctane-8-one

(38) ##STR00022##

(39) The preparation method of (1.sup.3E,1.sup.4E,2.sup.2R)-6-cyclopropylamino-3.sup.5-fluoro-5-methyl-6,7-diazepine-1(5,3)-pyrazolo[1,5-a]pyrimidine-3(3,2)-azabenzene-2(1,2)-pyrrolidine cyclooctane-8-one was similar to that of Example 1.

(40) .sup.1H NMR (300 MHz, Chloroform-d) δ 8.33 (d, J=9.0 Hz, 1H), 8.31 (d, J=6.0 Hz 1H), 8.24 (s, 1H), 7.21 (dd, J=9.0, 3.0 Hz, 0.5H), 7.11 (dd, J J=9.0, 3.0 Hz, 0.5H), 6.33 (d, J=7.6 Hz, 1H), 5.55 (t, J=6.0 Hz, 0.5H), 5.48 (t, J=6.0 Hz, 0.5H), 4.17 (m, 1H), 3.97 (m, 1H), 3.78 (m, 1H), 3.64 (m, 1H), 3.41 (m, 1H), 2.57 (ddq, J=19.1, 9.9, 8.6 Hz, 1H), 2.40 (dd, J=13.1, 6.7 Hz, 1H), 2.23 (m, 1H), 2.01 (m, 1H), 1.88 (m, 6.3 Hz, 1H), 1.49 (d, J=6.9 Hz, 1.5H), 1.16 (m, 1.5H), 1.00 (m, 1H), 0.88 (m, 1H), 0.70 (m, 1H), 0.43 (m, 1H). MS (ESI) m/z: 422.2[M+H].sup.+.

Example 3

(1.SUP.3.E,1.SUP.4.E,2.SUP.2.R,5R)-6-cyclopropyl-3.SUP.5.-fluoro-5-methyl-6,7-diazepine-1(5,3)-pyrazolo[1,5-a]pyrimidine-3(3,2)-pyridine-2(1,2)-pyrrolidine cyclooctane-8-one & (1.SUP.3.E,1.SUP.4.E,2.SUP.2.R,5S)-6-cyclopropyl-3.SUP.5.-fluoro-5-methyl-6,7-diazepine-1(5,3)-pyrazolo[1,5-a]pyrimidine-3(3,2)-pyridine-2(1,2)-pyrrolidine cyclooctane-8-one

(41) ##STR00023##

Step 1: Synthesis of (1.SUP.3.E,1.SUP.4.E,2.SUP.2.R)-6-cyclopropylamino-3.SUP.5.-fluoro-5-methyl-6,7-diazepine-1 (5,3)-pyrazolo[1,5-a]pyrimidine-3(3,2)-azabenzene-2(1,2)-pyrrolidine cyclooctane-8-one following the method of Example 2

Step 2: Preparation of (1.SUP.3.E,1.SUP.4.E,2.SUP.2.R,5R)-6-cyclopropyl-3.SUP.5.-fluoro-5-methyl-6,7-diazepine-1(5,3)-pyrazolo[1,5-a]pyrimidine-3(3,2)-pyridine-2(1,2)-pyrrolidine cyclooctane-8-one & (1.SUP.3.E,1.SUP.4.E,2.SUP.2.R,5S)-6-cyclopropyl-3.SUP.5.-fluor-5-methyl-6,7-diazepine-1(5,3)-pyrazolo[1,5-a]pyrimidine-3(3,2)-pyridine-2(1,2)-pyrrolidine cyclooctane-8-one

(42) ##STR00024##

(43) (1.sup.3E,1.sup.4E,2.sup.2R)-6-cyclopropylamino-3.sup.5-fluoro-5-methyl-6,7-diazepine-1(5,3)-pyrazolo[1,5-a]pyrimidine-3(3,2)-azabenzene-2(1,2)-pyrrolidine cyclooctane-8-one (200 mg, 0.47 mmol) was subjected to chiral separation on a preparative column to obtain Example 3a: (1.sup.3E,1.sup.4E,2.sup.2R,5R or S)-6-cyclopropyl-3.sup.5-fluoro-5-methyl-6,7-diazepine-1(5,3)-pyrazolo[1,54]pyrimidine-3(3,2)-pyridine-2(1,2)-pyrrolidine cyclooctane-8-one (28.3 mg, yield 28.3%) and Example 3b: (1.sup.3E,1.sup.4E,2.sup.2R,5R or S)-6-cyclopropyl-3.sup.5-fluoro-5-methyl-6,7-diazepine-1(5,3)-pyrazolo(1,5-a]pyrimidine-3(3,2)-pyridine-2(1,2)-pyrrolidine cyclooctane-8-one (35.6 mg, yield 35.6%).

(44) Separation and Preparation Method:

(45) Instrument: waters SFC200

(46) Chromatographic column: ChiralPak OD, 250×30 mm I.D., 5 μm

(47) Mobile phase: A: CO.sub.2; B: MEOH (0.1% NH.sub.3H.sub.2O)

(48) Wavelength: 254 nm

(49) Column temperature: 38° C.

(50) Flow rate: 60 g/min

(51) Single injection volume: 5 mL

(52) Single injection concentration: 10 mg/mL

(53) Injection times: 4

(54) Solvent: MeOH

(55) Back pressure: 100 bar

(56) Gradient condition: B 40%

(57) Cycle time: 8 min

(58) Collection condition: concentration under reduced pressure at 40° C.

(59) Separation and Detection Method:

(60) Instrument: waters UPCC

(61) Chromatographic column: ChiralPak OD, 2.1×150 mm I.D., 3 μm

(62) Mobile phase: A: CO.sub.2; B: MEOH (0.1% DEA)

(63) Wavelength: 254 nm

(64) Column temperature: 40° C.

(65) Flow rate: 1 mL/min

(66) Injection volume: 2 μL

(67) Injection concentration: 2 mg/mL

(68) Solvent: MeOH

(69) Back pressure: 1500 psi

(70) Gradient condition: B 5%-40%

(71) Runtime: 10 min

(72) Retention time: 4.471 min, 4.788 min

(73) Example 3a: (1.sup.3E,1.sup.4E,2.sup.2R,5R or S)-6-cyclopropyl-3.sup.3-fluoro-5-methyl-6,7-diazepine-1(5,3)-pyrazolo(1,5-a]pyrimidine-3(3,2)-pyridine-2(1,2)-pyrrolidine cyclooctane-8-one, retention time 4.471 min. MS (ESI) m/z: 422.2 [M+H].sup.+. .sup.1H NMR (400 MHz, CDCl3) δ 8.63 (br, 1H), 8.31 (dd, J=23.1, 15.6 Hz, 3H), 7.16-7.07 (m, 1H), 6.33 (d, J=0.6 Hz, 1H), 5.49 (t, J=17.1 Hz, 1H), 4.16 (t, J=8.0 Hz, 1H), 4.01-3.92 (m, 1H), 3.81 (m, 1H), 3.46 (dd, J=33.0, 21.6 Hz, 2H), 3.18 (m, 1H), 2.60 (m, 1H), 2.39 (m, 1H), 2.24 (m, 1H), 1.89 (d, J=11.6 Hz, 1H), 1.29-1.21 (d, J=7.6 Hz, 3H), 1.07-0.93 (m, 2H), 0.74-0.66 (m, 1H), 0.51-0.42 (m, 1H).

(74) Example 3b: (1.sup.3E,1.sup.4E,2.sup.2R,5R or S)-6-cyclopropyl-3.sup.5-fluoro-5-methyl-6,7-diazepine-1(5,3)-pyrazolo(1,5-a]pyrimidine-3(3,2)-pyridine-2(1,2)-pyrrolidine cyclooctane-8-one, retention time 4.788 min. MS(ESI) m/z: 422.2 [M+H].sup.+. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.11 (br, 1H), 8.42-8.22 (m, 3H), 7.20 (d, J=9.3 Hz, 1H), 6.39-6.25 (d, J=7.6 Hz, 1H), 5.56 (t, J=12 Hz, 1H), 4.18 (t, J=8.0 Hz, 1H), 3.98 (s, 1H), 3.86 (d, J=24.8 Hz, 2H), 2.80 (d, J=16.3 Hz, 1H), 2.53 (m, 1H), 2.42 (m, 1H), 2.30 (m, 1H), 2.22 (m, 1H), 1.88 (d, J=11.6 Hz, 1H), 1.57-1.42 (d, J=7.6 Hz, 3H), 1.02 (m, 1H), 0.93-0.85 (m, 1H), 0.44 (m, 2H).

Example 4

Synthesis of (R,1.SUP.3.E,1.SUP.4.E)-3.SUP.5.-fluoro-6-methyl-6,7-diazepine-1(5,3)-pyrazolo[1,5-a]pyrimidine-3(3,2)-azabenzene-2(1,2)-pyrrolidine cyclooctane-8-one

(75) ##STR00025##

Step 1: Synthesis of ethyl (S)-5-(2-(5-fluoro-2-(2-(methylamino)ethyl)pyridin-3-yl)pyrrolidi n-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate

(76) ##STR00026##

(77) At room temperature, ethyl (S,E)-5-(2-(5-fluoro-2-(prop-1-en-1-yl)pyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a] pyrimidine-3-carboxylate (1.00 g, 2.62 mmol), 30% methylamine in ethanol (30 mL), and glacial acetic acid (10 mL) were added into a 100 mL single-necked flask, evacuated, replaced with nitrogen, slowly warmed to 75° C., and reacted under reflux for 19 hours. The reaction was stopped. The reaction solution was cooled to room temperature, and EA was added. The reaction solution was washed with a saturated aqueous solution of sodium carbonate. The aqueous phase was back extracted with EA. The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. Column chromatography (DCM/EtOH system) was performed to obtain a yellow solid of 544.3 g, with a yield of 45.74%. MS (ESI) m/z:413.3[M+H].sup.+.

Step 2: Synthesis of ethyl (S)-5-(2-(2-(2-(2-(tert-butylcarbonyl)-1-methylhydrazino)ethyl)-5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate

(78) ##STR00027##

(79) At room temperature, ethyl (S)-5-(2-(5-fluoro-2-(2-(methylamino)ethyl)pyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (439.0 mg, 1.04 mmol), tetrahydrofuran (9.00 mL) and N-methylmorpholine (158.0 mg, 1.56 mmol) were added into a 50 mL single-necked flask. The temperature was reduced to 0° C., and N-Boc-O p-toluenesulfonyl-hydroxylamine (898.3 mg, 3.12 mmol) was added in portions. After the addition was completed and the temperature was returned to room temperature, the reaction was carried out for 19 hours. The reaction was stopped, and EA was added. The reaction solution was washed with a saturated aqueous solution of sodium carbonate. The aqueous phase was extracted with EA. The organic phases were combined, dried over anhydrous Na.sub.2SO.sub.4, and evaporated to dryness under reduced pressure. Column chromatography (PE/EA system) was performed to obtain a product of 68.2 mg. MS (ESI) m/z: 528.3 [M+H].sup.+.

Step 3: Synthesis of (S)-5-(2-(2-(2-(2-(tert-butylcarbonyl)-1-methylhydrazino)ethyl)-5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid

(80) ##STR00028##

(81) At room temperature, ethyl (S)-5-(2-(2-(2-(2-(tert-butylcarbonyl)-1-methylhydrazino)ethyl)-5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (62.8 mg, 0.13 mmol) and ethanol (2.00 mL) were added into a 25 mL single-necked flask. After complete dissolution, lithium hydroxide monohydrate (32.5 mg, 0.77 mmol) in water (0.5 mL) was added, the temperature was slowly increased to 70° C., and the reaction was carried out with stirring for 6 hours, at which the reaction was complete. The reaction solution was cooled to room temperature, and evaporated to dryness under reduced pressure to obtain a crude product 1 as a yellow solid. MS (ESI) m/z: 500.1[M+H].sup.+.

Step 4: Synthesis of (R,1.SUP.3.E,1.SUP.4.E)-3.SUP.5.-fluoro-6-methyl-6,7-diazepine-1(5,3)-pyrazolo[1,5-a]pyrimidine-3(3,2)-azabenzene-2(1,2)-pyrrolidine cyclooctane-8-one

(82) ##STR00029##

(83) At room temperature, the above crude product 1 was added into a 25 mL single-necked flask, followed by addition of methylene chloride (2.00 mL) and trifluoroacetic acid (2.00 mL), ultrasonically dissolved, and stirred for 2 hours at room temperature, at which the reaction was complete. The reaction was stopped. The reaction system was evaporated to dryness under reduced pressure to obtain a pale yellow crude product 2. MS (ESI) m/z: 400.5[M+H].sup.+.

(84) At room temperature, the above crude product 2 was placed into a 25 single-necked flask, followed by addition of HOBt (87.3 mg, 0.64 mmol), EDCI (123.9 mg, 0.64 mmol), DMF (3.00 mL), and TEA (261.6 mg, 2.58 mmol), evacuated, replaced with nitrogen, and reacted at room temperature overnight, at which the reaction was complete. The reaction system was evaporated to dryness under reduced pressure, and subjected to column chromatography (0.1% formic acid in water/acetonitrile system) to obtain a product of 16.5 mg. MS (ESI) m/z: 382.4[M+H].sup.+. .sup.1H NMR (300 MHz, Chloroform-d) δ 8.35 (d, J=9.4, 1H), 8.34 (d, 2.8, 1H), 8.31 (s, 1H), 7.10 (dd, J=9.4, 2.8 Hz, 1H), 6.34 (d, J=7.6 Hz, 1H), 5.54 (t, J=7.5 Hz, 1H), 4.12 (dd, J=15.0, 6.2 Hz, 1H), 3.99 (dd, J=7.9, 7.2 Hz, 1H), 3.86 (dd, J=8.7, 7.8 Hz, 1H), 3.72-3.69 (m, 1H), 3.67-3.65 (m, 1H), 2.87-2.79 (m, 1H), 2.77 (s, 3H), 2.66-2.56 (m, 1H), 2.47-2.41 (m, 1H), 2.30-2.23 (m, 1H), 1.97-1.90 (m, 1H).

Example 5

(R,1.SUP.3.E,1.SUP.4.E)-6-ethyl-3.SUP.3.-fluoro-6,7-diazepine-1(5,3)-pyrazolo[1,5-a]pyrimidine-3(3,2)-azabenzene-2(1,2)-pyrrolidine cyclooctane-8-one

(85) ##STR00030##

Step 1: Synthesis of ethyl (S)-5-(2-(2-(2-(ethylamino)ethyl)-5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate

(86) ##STR00031##

(87) At room temperature, ethyl (S)-5-(2-(5-fluoro-2-vinylpyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (1.52 g, 3.98 mmol), 30% ethylamine in ethanol (40.80 mL), and glacial acetic acid (13.60 mL) were added into a 100 mL single-necked flask, evacuated, replaced with nitrogen, slowly warmed to 75° C., and reacted under reflux. After stirring for 23.5 hours, the reaction was stopped. The reaction solution was cooled to room temperature, and EA was added. The reaction solution was washed with a saturated aqueous solution of sodium carbonate. The aqueous phase was back extracted with EA. The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. Column chromatography (DC M/EtOH system) was performed to obtain a yellow solid of 610 mg, with a yield of 33.66%. MS (ESI) m/z: 427.5[M+H].sup.+.

Step 2: Synthesis of ethyl (S)-5-(2-(2-(2-(2-(tert-butylcarbonyl)-1-ethylhydrazino)ethyl)-5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate

(88) ##STR00032##

(89) At room temperature, ethyl (S)-5-(2-(2-(2-(ethylamino)ethyl)-5-fluropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (753.7 mg, 1.77 mmol), tetrahydrofuran (15 mL) and N-methylmorpholine (178.8 mg, 1.77 mmol) were added into a 50 mL single-necked flask. The temperature was reduced to 0° C., and N-Boc-O p-toluenesulfonyl-hydroxylamine (2.54 g, 8.84 mmol) was added in portions. After the addition was completed and the temperature was returned to room temperature, the reaction was carried out for 17 hours. The reaction was stopped, and EA was added. The reaction solution was washed with a saturated aqueous solution of sodium carbonate. The aqueous phase was extracted with EA. The organic phases were combined, dried over an hydrous Na.sub.2SO.sub.4, and evaporated to dryness under reduced pressure. Column chromatography (PE/EA system) was performed to obtain a product of 101.6 mg. MS (ESI) m/z: 542.6[M+H].sup.+.

Step 3: Synthesis of (S)-5-(2-(2-(2-(2-(tert-butylcarbonyl)-1-ethylhydrazino)ethyl)-5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid

(90) ##STR00033##

(91) At room temperature, ethyl (S)-5-(2-(2-(2-(2-(tert-butylcarbonyl)-1-ethylhydrazino)ethyl)-5-fluoropyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (101.6 mg, 0.19 mmol) and ethanol (4.00 mL) were added into a 25 mL single-necked flask. After complete dissolution, lithium hydroxide monohydrate (47.2 mg, 1.13 mmol) in water (0.8 mL) was added, and the temperature was slowly increased to 70° C. After stirring for 5 hours, the reaction was complete. The re action system was cooled to room temperature, and evaporated to dryness under reduced pressure to obtain a crude product 1 as a yellow solid. MS (ESI) m/z: 514.5[M+H].sup.+.

Step 4: Synthesis of (R,1.SUP.3.E,1.SUP.4.E)-3.SUP.5.-fluoro-6-ethyl-6,7-diazepine-1(5,3)-pyrazolo[1,5-a]pyrimidine-3(3,2)-azabenzene-2(1,2)-pyrrolidine cyclooctane-8-one

(92) ##STR00034##

(93) At room temperature, the above crude product 1 was added into a 25 mL single-necked flask, followed by addition of methylene chloride (3.00 mL) and trifluoroacetic acid (3.00 mL), ultrasonically dissolved, and stirred for 1.5 hours at room temperature, at which the reaction was complete. The reaction was stopped. The reaction system was evaporated to dryness under reduced pressure to obtain a pale yellow crude product 2. MS (ESI) m/z: 414.4[M+H].sup.+.

(94) At room temperature, the above crude product 2 was placed into a 25 mL single-necked flask, followed by addition of HOBt (126.7 mg, 0.94 mmol), EDCI (180.0 mg, 0.94 mmol), DMF (6.00 mL), and TEA (380.0 mg, 3.75 mmol), evacuated, replaced with nitrogen, and reacted at room temperature overnight, at which the reaction was complete. The reaction system was evaporated to dryness under reduced pressure, and subjected to column chromatography (0.1% formic acid in water/acetonitrile system) to obtain a product of 14.4 mg. MS (ESI) m/z: 396.4 [M+H]. HPLC: 97.87%. .sup.1H NMR (300 MHz, Chloroform-d) δ 9.42 (br, 1H), 8.41-8.31 (m, 3H), 7.10 (dd, J=8.4, 2.8 Hz, 1H), 6.36 (d, J=7.9 Hz, 1H), 5.57 (t, J=7.5 Hz, 1H), 4.04-3.95 (m, 2H), 3.92-3.83 (m, 2H), 3.78-3.70 (m, 1H), 3.63 (d, J=17.8 Hz, 1H), 2.98 (q, J=8.4 Hz, 2H), 2.66-2.57 (m, 1H), 2.29-2.18 (m, 2H), 1.26-1.20 (t, J=6.0 Hz, 3H).

Example 6

Synthesis of (R,1.SUP.3.E,1.SUP.4.E)-3.SUP.5.-fluoro-6-isopropyl-6,7-diazepine-1(5,3)-pyrazolo[1,5-a]pyrimidine-3(3,2)-pyridine-2(1,2)-pyrrolidine cyclooctane-8-one

(95) ##STR00035##

(96) The preparation method of (R,1.sup.3E,1.sup.4E)-3.sup.5-fluoro-6-isopropyl-6,7-diazepine-1(5,3)-pyrazolo [1,5-a]pyrimidine-3(3,2)-pyridine-2(1,2)-pyrrolidine cyclooctane-8-one was similar to that of Example 1.

(97) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.41 (br, 1H), 8.33 (d, J=8.0 Hz, 1H), 8.32 (s, 2H), 7.09 (dd, J=9.4, 2.3 Hz, 1H), 6.34 (d, J=7.9 Hz, 1H), 5.56 (t, J=7.3 Hz, 1H), 4.20-4.14 (m, 1H), 4.00 (q, J=8.0 Hz, 1H), 3.88-3.82 (m, 1H), 3.65 (dd, J=15.4, 4.0 Hz, 1H), 3.56 (d, J=16.2 Hz, 1H), 3.09 (p, J=6.2 Hz, 1H), 2.86 (dd, J=15.1, 10.1 Hz, 1H), 2.61 (dd, J=13.4, 6.8 Hz, 1H), 2.44 (dt, J=12.7, 6.3 Hz, 1H), 2.25 (dt, J=13.7, 7.3 Hz, 1H), 1.94 (dd, J=13.5, 6.8 Hz, 1H), 1.29 (d, J=6.3 Hz, 3H), 1.22 (d, J=6.3 Hz, 3H). MS (ESI) m/z: 410.2[M+H].sup.+.

Example 7

(1.SUP.3.E,1.SUP.4.E,2.SUP.2.R,5R)-3.SUP.5.-fluoro-5,6-dimethyl-6,7-diazepine-1(5,3)-pyrazolo[1,5-a]pyrimidine-3 (3,2)-pyridine-2(1,2)-pyrrolidine cyclooctane-8-one & (1.SUP.3.E,1.SUP.4.E,2.SUP.2.R,5S)-3.SUP.5.-fluoro-5,6-dimethyl-6,7-diazepine-1(5,3)-pyrazolo[1,5-a]pyrimidine-3(3,2)-pyridine-2(1,2)-pyrrolidine cyclooctane-8-one

(98) ##STR00036##

Step 1: Synthesis of (1.SUP.3.E,1.SUP.4.E,2.SUP.2.R)-5,6-dimethyl-3.SUP.5.-fluoro-6,7-diazepine-1(5,3)-pyrazolo[1,5-a]pyrimidine-3(3,2)-azabenzene-2(1,2)-pyrrolidine cyclooctane-8-one following the method of Example 2

Step 2: Preparation of (1.SUP.3.E,1.SUP.4.E,2.SUP.2.R,5R)-3.SUP.5.-fluoro-5,6-dimethyl-6,7-diazepine-1(5,3)-pyrazol o[1,5-a]pyrimidine-3(3,2)-pyridine-2(1,2)-pyrrolidine cyclooctane-8-one & (1.SUP.3.E,1.SUP.4.E,2.SUP.2.R,5S)-3.SUP.5.-fluoro-5,6-dimethyl-6,7-diazepine-1(5,3)-pyrazolo[1,5-a]pyrimidine-3(3,2)-pyridine-2(1,2)-pyrrolidine cyclooctane-8-one

(99) ##STR00037##

(100) (1.sup.3E,1.sup.4E,2.sup.2R)-5,6-dimethyl-3.sup.5-fluoro-6,7-diazepine-1(5,3)-pyrazolo[1,5-a]pyrimidine-3(3,2)-azabenzene-2(1,2)-pyrrolidine cyclooctane-8-one (260 mg, 0.65 mmol) was subjected to separation on a preparative column to obtain Example 7a: (1.sup.3E,1.sup.4E,2.sup.2R,5R or S)-3.sup.5-fluoro-5,6-dimethyl-6,7-diazepine-1(5,3)-pyrazolo[1,5-a]pyrimidine-3(3,2)-pyridine-2(1,2)-pyrrolidine cyclooctane-8-one (23 mg, yield 17.7%) and Example 7b: (1.sup.3E,1.sup.4E,2.sup.2R,5R or S)-3.sup.5-fluoro-5,6-dimethyl-6,7-diazepine-1(5,3)-pyrazolo[1,5-a]pyrimidine-3(3,2)-pyridine-2(1,2)-pyrrolidine cyclooctane-8-one (56 mg, yield 43.1%).

(101) Separation and Preparation Method:

(102) Instrument: Qingbohua LC2000 High Performance Liquid Chromatograph

(103) Chromatographic column: YMC Triart C18 250*20 mm 10 um

(104) Mobile phase: A: acetonitrile; mobile phase B: 0.1% trifluoroacetic acid in water

(105) Wavelength: 230 nm

(106) Column temperature: 35° C.

(107) Flow rate: 10 ml/min

(108) Solvent: acetonitrile

(109) Preparation conditions: 0 min A25B75 25 min A25B75 30 min A26B74 35 min A28B72

(110) Collection condition: concentration under reduced pressure at 40° C.

(111) Separation and Detection Method:

(112) Instrument: Agilent 1260 Infinity II

(113) Chromatographic column: Xtimate C18 4.6*50 mm 3 um

(114) Mobile phase: A: acetonitrile; B: 0.1% trifluoroacetic acid in water

(115) Wavelength: 230 nm

(116) Column temperature: 35° C.

(117) Flow rate: 1 mL/min

(118) Injection volume: 20 μL

(119) Retention time: 2.371 min, 2.844 min

(120) Solvent: acetonitrile

(121) Detection condition: B 10%-76%

(122) Example 7a: (1.sup.3E,1.sup.4E,2.sup.2R,5R or S)-3.sup.5-fluoro-5,6-dimethyl-6,7-diazepine-1(5,3)-pyrazolo[1,5-a]pyrimidine-3(3,2)-pyridine-2(1,2)-pyrrolidine cyclooctane-8-one, retention time 2.371 min. MS (ESI) m/z: 396.2 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d6) δ 9.55 (br, 1H), 8.82 (d, J=7.7 Hz, 1H), 8.37 (m, 1H), 8.11 (s, 1H), 7.54 (d, J=9.8 Hz, 1H), 6.72 (d, J=7.8 Hz, 1H), 5.40 (t, J=6.8 Hz, 1H), 4.20 (m, 1H), 4.08 (q, J=7.5 Hz, 1H), 3.84 (m, 1H), 3.53-3.42 (m, 1H), 3.23 (dd, J=14.5, 10.6 Hz, 1H), 3.04 (s, 3H), 2.62 (dt, J=12.6, 6.5 Hz, 1H), 2.24 (dt, J=11.7, 5.9 Hz, 1H), 2.08 (d q, J=12.9, 7.4 Hz, 1H), 1.83-1.72 (m, 1H), 1.31 (d, J=6.9 Hz, 3H).

(123) Example 7b: (1.sup.3E,1.sup.4E,2.sup.2R,5R or S)-3.sup.5-fluoro-5,6-dimethyl-6,7-diazepine-1(5,3)-pyrazolo[1,5-a]pyrimidine-3(3,2)-pyridine-2(1,2)-pyrrolidine cyclooctane-8-one, retention time 2.844 min. MS (EST) m/z: 396.2 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d6) δ 9.64 (br, 1H), 8.80 (d, J=7.7 Hz, 1H), 8.42 (d, J=2.6 Hz, 1H), 8.13 (s, 1H), 7.59 (d, J=12.6 Hz, 1H), 6.69 (d, J=7.8 Hz, 1H), 5.61 (t, J=7.1 Hz, 1H), 4.14 (d, J=7.2 Hz, 1H), 3.96 (s, 1H), 3.91 (d, J=5.0 Hz, 1H), 3.88-3.75 (m, 2H), 2.65 (s, 3H), 2.59 (s, 1H), 2.31 (dq, J=11.7, 5.7, 5.1 Hz, 1H), 2.14-1.99 (m, 1H), 1.80 (dq, J=13.5, 7.1 Hz, 1H), 1.46 (d, J=6.9 Hz, 3H).

Example 8

(124) (1.sup.3E,1′E,2.sup.2R,5R)-3.sup.5-fluoro-6-ethyl-5-methyl-6,7-diazepine-1(5,3)-pyrazolo[1,5-a]pyrimidin e-3(3,2)-pyridine-2(1,2)-pyrrolidine cyclooctan-8-one & (1.sup.3E,1.sup.4E,2.sup.2R,5S)-3.sup.5-fluoro-6-ethyl-5-methyl-6,7-diazepine-1(5,3)-pyrazolo[1,5-a]pyrimidine-3(3,2)-pyridine-2(1,2)-pyrrolidine cyclooctan-8-one

(125) ##STR00038##

Step 1: Synthesis of (1.SUP.3.E,1.SUP.4.E,2.SUP.2.R)-3.SUP.5.-fluoro-6-ethyl-5-methyl-6,7-diazepine-1(5,3)-pyrazolo [1,5-a]pyrimidine-3(3,2)-pyridine-2(1,2)-pyrrolidine cyclooctan-8-one following the method of Example 2

Step 2: Preparation of (1.SUP.3.E,1.SUP.4.E,2.SUP.2.R,5R)-3.SUP.5.-fluoro-6-ethyl-5-methyl-6,7-diazepine-1(5,3)-pyrazolo[1,5-a]pyrimidine-3(3,2)-pyridine-2(1,2)-pyrrolidine cyclooctan-8-one & (1.SUP.3.E,1.SUP.4.E,2.SUP.2.R,5S)-3.SUP.5.-fluoro-6-ethyl-5-methyl-6,7-diazepine-1(5,3)-pyrazolo[1,5-a]pyrimidine-3(3,2)-pyridine-2(1,2)-pyrrolidine cyclooctan-8-one

(126) ##STR00039##

(127) (1.sup.3E,1.sup.4E,2.sup.2R)-3.sup.5-fluoro-6-ethyl-5-methyl-6,7-diazepine-1(5,3)-pyrazolo[1,5-a]pyrimidine-3 (3,2)-pyridine-2(1,2)-pyrrolidine cyclooctane-8-one (320 mg, 0.78 mmol) was subjected to separation on a preparative column to obtain Example 8a: (1.sup.3E,1.sup.4E,2.sup.2R,5R or S)-3.sup.5-fluoro-6-ethyl-5-methyl-6,7-diazepine-1(5,3)-pyrazolo[1,5-a]pyrimidine-3(3,2)-pyridine-2(1,2)-pyrrolidine cyclooctane-8-one (26 mg, yield 16.2%) and Example 8b: (1.sup.3E,1.sup.4E,2.sup.2R,5R or S)-3.sup.5-fluoro-6-ethyl-5-methyl-6,7-diazepine-1(5,3)-pyrazolo[1,5-a]pyrimidine-3(3,2)-pyridine-2(1,2)-pyrrolidine cyclooctane-8-one (53 mg, yield 33.1%).

(128) Separation and Preparation Method:

(129) Instrument: Qingbohua LC2000 High Performance Liquid Chromatograph

(130) Chromatographic column: YMC Triart C18 250*20 mm 10 um

(131) Mobile phase: A: acetonitrile; mobile phase B: 0.05% trifluoroacetic acid in water

(132) Wavelength: 230 nm

(133) Column temperature: 35° C.

(134) Flow rate: 10 ml/min

(135) Solvent: acetonitrile

(136) Preparation conditions: 0 min A33B67 25 min A33B67 30 min A35B65

(137) Collection condition: concentration under reduced pressure at 40° C.

(138) Separation and Detection Method:

(139) Instrument: Agilent 1260 Infinity II

(140) Chromatographic column: Xtimate C18 4.6*50 mm 3 um

(141) Mobile phase: A: acetonitrile; B: 0.1% trifluoroacetic acid in water

(142) Wavelength: 230 nm

(143) Column temperature: 35° C.

(144) Flow rate: 1 mL/min

(145) Injection volume: 20 μL

(146) Retention time: 1.898 min, 2.369 min

(147) Solvent: acetonitrile

(148) Detection condition: B10%-72%

(149) Example 8a: (1.sup.3E,1.sup.4E,2.sup.2R,5R or S)-3.sup.5-fluoro-6-ethyl-5-methyl-6,7-diazepine-1(5,3)-pyrazolo[1,5-a]pyrimidine-3(3,2)-pyridine-2(1,2)-pyrrolidine cyclooctane-8-one, retention time 1.898 min. MS (ESI) m/z: 410.1 [M+H].sup.+. .sup.1NMR (400 MHz, DMSO-d6) δ9.55 (br, 1H), 8.82 (d, J=7.7 Hz, 1H), 8.37 (m, 1H), 8.11 (s, 1H), 7.54 (d, J=9.8 Hz, 1H), 6.72 (d, J=7.8 Hz, 1H), 5.40 (t, J=6.8 Hz, 1H), 4.20 (m, 1H), 4.08 (q, J=7.5 Hz, 1H), 3.84 (m, 1H), 3.53-3.42 (m, 1H), 3.23 (dd, J=14.5, 10.6 Hz, 1H), 3.04 (s, 3H), 2.62 (dt, J=12.6, 6.5 Hz, 1H), 2.24 (dt, J=11.7, 5.9 Hz, 1H), 2.08 (d q, J=12.9, 7.4 Hz, 1H), 1.83-1.72 (m, 1H), 1.04 (m, 6H).

(150) Example 8b: (1.sup.3E,1.sup.4E,2.sup.2R,5R or S)-3.sup.5-fluoro-6-ethyl-5-methyl-6,7-diazepine-1(5,3)-pyrazolo[1,5-a]pyrimidine-3(3,2)-pyridine-2(1,2)-pyrrolidine cyclooctane-8-one, retention time 2.369 min. MS (ESI) m/z: 410.1 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d6) δ 9.02 (br, 1H), 8.76 (d, J=7.7 Hz, 1H), 8.39 (d, J=2.5 Hz, 1H), 8.06 (s, 1H), 7.56 (dd, J=10.1, 2.4 Hz, 1H), 6.66 (d, J=7.8 Hz, 1H), 5.59 (s, 1H), 4.22-3.94 (m, 1H), 3.90-3.73 (m, 2H), 3.68-3.59 (m, 1H), 2.90-2.74 (m, 1H), 2.67 (p, J=8.3, 7.5 Hz, 1H), 2.53 (m, 1H), 2.48-2.40 (m, 1H), 2.30 (dq, J=12.0, 6.6 Hz, 1H), 2.07 (dt, J=13.2, 7.2 Hz, 1H), 1.77 (td, J=12.9, 6.3 Hz, 1H), 1.44 (d, J=6.9 Hz, 3H), 0.98 (t, J=6.9 Hz, 3H).

Example 9

(R,1.SUP.3.E,1.SUP.4.E)-3.SUP.5.-fluoro-6-isobutyl-6,7-diazepine-1(5,3)-pyrazolo[1,5-a]pyrimidine-3(3,2)-pyridine-2(1,2)-pyrrolidine cyclooctane-8-one

(151) ##STR00040##

(152) The preparation method of (R,1.sup.3E,1.sup.4E)-3.sup.5-fluoro-6-isobutyl-6,7-diazepine-1(5,3)-pyrazolo[1, 5-a]pyrimidine-3(3,2)-pyridine-2(1,2)-pyrrolidine cyclooctane-8-one was similar to that of Example 1.

(153) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.29 (br, 1H), 8.30 (dd, J=9.5, 7.2 Hz, 3H), 7.07 (dd, J=9.4, 2.8 Hz, 1H), 6.32 (d, J=7.7 Hz, 1H), 5.57-5.53 (t, J=7.3 Hz, 1H), 3.99-3.91 (m, 2H), 3.87-3.80 (m, 2H), 3.63 (dt, J=16.0, 3.5 Hz, 1H), 2.86-2.76 (m, 2H), 2.65 (dt, J=13.3, 6.8 Hz, 1H), 2.54 (dd, J=11.8, 8.4 Hz, 1H), 2.43 (dt, J=12.6, 6.2 Hz, 1H), 2.25 (dt, J=13.5, 7.5 Hz, 1H), 1.97-1.88 (dt, J=14.2, 7.2 Hz, 1H), 1.82 (dt, J=13.6, 6.6 Hz, 1H), 1.12 (d, J=6.5 Hz, 3H), 0.96 (d, J=6.7 Hz, 3H). MS (ESI) m/z: 424.2[M+H].sup.+.

Example 10

Synthesis of (R,1.SUP.3.E,1.SUP.4.E)-6-cyclopropyl-3.SUP.5.-fluoro-6,7-diaza-1(5,3)-pyrazoline[1,5-a]pyrimidine-3(3,2)-pyridine-2(1,2)-pyrrolidine cyclotridecane-8-one

(154) ##STR00041##

Step 1: 2-(2-bromo-4-fluorophenyl)ethanol

(155) ##STR00042##

(156) 2-(2-bromo-4-fluorophenyl)acetic acid (46.63 g, 200.00 mmol) was dissolved in 500 mL of THF, and borane in tetrahydrofuran (1.0 M, 300 mL) was added at 0° C. The reaction was carried out for 16 h at 18° C. and stopped. The reaction solution was quenched with 1 N diluted hydrochloric acid, and extracted with EA. The organic phase was washed with saturated brine, dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated to obtain a yellow oil of 42.57 g, with a yield of 97%.

Step 2: 2-bromo-4-fluoro-1-(2-(4-methoxybenzyloxy)ethyl)benzene

(157) ##STR00043##

(158) At room temperature, NaH (4.10 g, 102.00 mmol) and 200 mL of THF were added into a four-necked flask. 2-(2-bromo-4-fluorophenyl)ethanol (14.90 g, 102.00 mmol) was added at 0° C. The reaction was carried out at 0° C. for 0.5 h and tetrabutylammonium iodide (36.28 g, 96.00 mmol) and p-methoxybenzyl chloride (12.78 g, 82.00 mmol) were added. The reaction was carried out for 4 h at 50° C. and stopped. The reaction solution was quenched with 1 N diluted hydrochloric acid, and extracted with EA. The organic phase was washed with saturated brine, dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated to obtain a colorless oil of 15.37 g, with a yield of 67%.

Step 3: 5-fluoro-2-(2-(4-methoxybenzyloxy)ethyl) benzaldehyde

(159) ##STR00044##

(160) At room temperature, 2-bromo-4-fluoro-1-(2-(4-methoxybenzyloxy)ethyl)benzene (15.37 g, 45.50 mmol) was dissolved in THF (500 mL). Under the protection of N2, sec-butyllithium (70 mL) was added at −78° C. and stirred at −78° C. for 1 h. Then, DMF (18.17 g, 230.00 mmol) was added. The reaction was carried out for 0.5 h while controlling the temperature at −78° C., and stopped. The reaction solution was quenched with a saturated ammonium chloride solution. It was extracted with EA. The organic phase was washed with water, dried over anhydrous MgSO.sub.4, filtered, concentrated, and purified by column chromatography (EA/PE system), to obtain a yellow oil of 8.01 g, with a yield of 62%.

Step 4: (R,E)-N-(5-fluoro-2-(4-methoxybenzyloxy)ethyl)benzylidene)-2-tert-butyl-2-sulfonimide

(161) ##STR00045##

(162) At room temperature, 5-fluoro-2-(2-(4-methoxybenzyloxy)ethyl)benzaldehyde (8.00 g, 28.00 mmol), R-tert-butylsulfinamide (3.56 g, 30.00 mmol), Cs.sub.2CO.sub.3 (6.33 g, 19.00 mmol), and 100 mL of DCM were sequentially added into a single-necked flask. The reaction was carried out under the protection of N.sub.2 at 25° C. for 16 h, and stopped. The reaction solution was poured into water, and separated. The organic phase was washed with saturated brine, dried over anhydrous MgSO.sub.4, filtered, and concentrated to obtain a yellow oil of 10.88 g, with a yield of 100%.

Step 5: N—((R)-3-((1,3-dioxan-2-yl)-1-(5-fluoro-2-(2-(4-methoxybenzyloxy)ethyl)phenyl)propyl)-2-tert-butyl-2-sulfonimide

(163) ##STR00046##

(164) At room temperature, Mg powder (0.87 g, 36.00 mmol), 1-bromo-3,3-dimethoxypropane (7.10 g, 36.00 mmol) and THF (100 mL) were sequentially added into a four-necked flask, evacuated, and replaced with N.sub.2. Under the protection of N.sub.2, the reaction was initiated by heating, and the reaction was carried out at 25° C. for 2 h. (R,E)-N-(5-fluoro-2-(4-methoxybenzyloxy)ethyl)methylene)-2-tert-butyl-2-sulfonimide (10.88 g, 28.00 mmol) in THF (40 mL) was added dropwise in an ice bath. After the addition, the temperature was increased to 25° C., and the reaction was carried out for 2 h and stopped. A saturated NH.sub.4Cl solution was added in an ice bath to quench the reaction. Layers were separated. The aqueous phase was extracted with EA. The organic phases were combined, dried over anhydrous MgSO.sub.4, filtered and concentrated to obtain a white solid of 14.20 g, with a yield of 100%.

Step 6: (R)-2-(2-(3,4-dihydro-2H-pyrrol-2-yl)-4-fluoro)phenethyl alcohol

(165) ##STR00047##

(166) At room temperature, (S)—N—((R)-3-((1,3-dioxan-2-yl)-1-(5-fluoro-2-(2-(4-methoxybenzyloxy)ethyl)phenyl)propyl)-2-tert-butyl-2-sulfonimide (14.20 g, 28.00 mmol) and H.sub.2O (25 mL) were sequentially added into a single-necked flask. TFA (100 mL) was added dropwise in an ice bath. After the addition, the reaction was carried out with stirring for 20 min. The temperature was raised to 25° C., and the reaction was carried out for 22 h and stopped. The reaction solution was directly used in the next step.

Step 7: (R)-2-(4-fluoro-2-(pyrrolidin-2-yl)phenethyl alcohol

(167) ##STR00048##

(168) At room temperature, (R)-2-(2-(3,4-dihydro-2H-pyrrol-2-yl)-4-fluoro)phenethyl alcohol (the reaction solution from the previous step) was added into a single-necked flask. Triethylsilane (9.77 g, 84.00 mmol) was added in portions in an ice salt bath. After the addition, the temperature was raised to 17° C. and the reaction was carried out for 1 h. A 2N HCl aqueous solution was added to quench the reaction and adjust the system to a pH of 2-3. The system was washed with EA, adjusted to a pH of 9-10 with a IN NaOH aqueous solution, and extracted with DCM. The organic phase was dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated to obtain a pale yellow oil of 3.80 g, with a total yield over two steps of 65%.

Step 8: ethyl (R)-5-(2-(5-fluoro-2-(2-hydroxyethyl)phenyl)pyrrol-1-yl)pyrazoline[1,5-a]pyrimidine-3-carboxylate

(169) ##STR00049##

(170) At room temperature, (R)-2-(4-fluoro-2-(pyrrolidin-2-yl)phenethyl alcohol (3.80 g, 18.00 mmol), ethyl 5-chloropyrazolo[1,5-A]pyrimidine-3-carboxylate (4.10 g, 18.00 mmol), TEA (3.64 g, 36.00 mmol), and EtOH (50 mL) were sequentially added into a single-necked flask, evacuated, and replaced with N.sub.2. Under the protection of N.sub.2, the reaction was carried out for 16 h at 24° C. and stopped. Ethanol was removed by rotary evaporation, and DCM and water were added. The reaction solution was stirred and separated. The aqueous phase was removed. The organic phase was washed with saturated brine and a saturated aqueous NaHCO.sub.3 solution, dried over anhydrous Na.sub.2SO.sub.4, filtered, concentrated, and purified by column chromatography (EA/PE system) to obtain a yellow foamy solid of 3.56 g, with a yield of 50%.

Step 9: ethyl(R)-5-(2-(5-fluoro-2-(2-methylsulfonyl)ethyl)phenyl)pyrrol-1-yl)pyrazoline[1,5-a]pyrimidine-3-carboxylate

(171) ##STR00050##

(172) At room temperature, ethyl (R)-5-(2-(5-fluoro-2-(2-hydroxyethyl)phenyl)pyrrol-1-yl)pyrazoline[1,5-a]pyrimidine-3-carboxylate (2.00 g, 5.02 mmol), TEA (1.52 g, 15.06 mmol) and 50 mL of DCM were sequentially added into a single-necked flask, and methanesulfonyl chloride (1.16 g, 10.04 mmol) was added in an ice bath. The temperature was naturally increased to 26° C., and the reaction was carried out for 2 h and stopped. The reaction solution was poured into water, stirred, extracted, and separated. The organic phase was dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated to obtain a yellow liquid. The crude product was directly used in the next step, with a yield of 100%.

Step 10: ethyl (R)-5-(2-(2-(2(cyclopropylamino)ethyl)-5-fluorophenyl)pyrrol-1-yl)pyrazoline[1,5-a]pyrimidine-3-carboxylate

(173) ##STR00051##

(174) At room temperature, ethyl (R)-5-(2-(5-fluoro-2-(2-methylsulfonyl)ethyl)phenyl)pyrrol-1-yl) pyrazoline[1,5-a]pyrimidine-3-carboxylate (the crude product from the previous step, 5.02 mmol), cyclopropylamine (0.86 g, 15.06 mmol) and DMF (30 mL) were sequentially added into a single-necked flask. Under the protection of N.sub.2, the reaction was carried out at 80° C. for 3 h and stopped. The reaction solution was poured into water, and EA was added for extraction. The EA phase was washed with IN diluted hydrochloric acid. The aqueous phase was added with sodium carbonate to adjust to PH-12. EA was added for extraction. The organic phase was washed with saturated brine, dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated to obtain a pale yellow liquid of 1.69 g, with a yield of 77%.

Step 11: Synthesis of ethyl (R)-5-(2-(2-(2-(2-(tert-butylcarbonyl)-1-cyclopropylhydrazino)ethyl)-5-fluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate

(175) ##STR00052##

(176) At room temperature, ethyl (R)-5-(2-(2-(2(cyclopropylamino)ethyl)-5-fluorophenyl)pyrrol-1-yl)pyrazoline[1,5-a]pyrimidine-3-carboxylate (1.67 g, 3.82 mmol), N-tert-butoxycarbonyl-3-(4-cyanophenyl) oxaziridine (1.25 g, 4.97 mmol) and DMF (25 mL) were sequentially added into a single-necked flask. The reaction was carried out for 16 h at 21° C. and stopped. EA was added, and the reaction solution was washed with water. The aqueous phase was back extracted with EA. The organic phases were combined, dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated. Column chromatography (PE/EA system) was performed to obtain a yellow oily solid of 1.02 g, with a yield of 68%.

Step 12: Synthesis of (R)-5-(2-(2-(2-(2-(tert-butylcarbonyl)-1-cyclopropylhydrazino)ethyl)-5-fluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid

(177) ##STR00053##

(178) At room temperature, ethyl (R)-5-(2-(2-(2-(2-(tert-butylcarbonyl)-1-cyclopropylhydrazino)ethyl)-5-fluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (0.70 g, 1.34 mmol) and ethanol (10 mL) were sequentially added into a single-necked flask and stirred to be completely dissolved. Then, a solution of sodium hydroxide (0.32 g, 8.04 mmol) in water (5 mL) was add ed. The temperature was raised to 70° C., and the reaction was carried out for 16 h and stopped. It was cooled to room temperature, concentrated to remove most of ethanol, and adjusted to a pH of 3-4 by adding DCM, H.sub.2O and 1 N HCl, and layers were separated after stirring. The aqueous phase was again extracted with DCM. The organic phases were combined, dried over anhydrous Mg.sub.2 SO.sub.4, filtered and concentrated to obtain a white solid of 0.63 g, with a yield of 95%.

Step 13: (R)-5-(2-(2-(2-(1-cyclopropylhydrazino)ethyl)-5-fluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid

(179) ##STR00054##

(180) At room temperature, (R)-5-(2-(2-(2-(2-(tert-butylcarbonyl)-1-cyclopropylhydrazino)ethyl)-5-fluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (0.63 g, 1.20 mmol) and a solution of HCl in ethanol (10 mL) were added to a single-necked flask, and stirred to be completely dissolved. The reaction was carried out at 32° C. for 1 h and stopped. The reaction system was concentrated to dryness under reduced pressure to obtain a pale yellow solid of 0.60 g, with a yield of 100%.

Step 14: (R,.SUP.13.E,.SUP.14.E)-6-cyclopropyl-3.SUP.5.-fluoro-6,7-diaza-1(5,3)-pyrazoline[1,5-a]pyrimidine-3 (3,2)-phenyl-2(1,2)-pyrrolidine cyclotridecane-8-one

(181) At room temperature, TBTU (0.68 g, 2.12 mmol), DMAP (0.034 g, 0.30 mmol), DMF (6 mL), and DCM (30 mL) were sequentially added to a four-necked flask, evacuated, and replaced with N.sub.2. Under the protection of N.sub.2, DIEA (1.09 g, 8.46 mmol) was added. (R)-5-(2-(2-(2-(1-cyclopropylhydrazino)ethyl)-5-fluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (0.60 g, 1.41 mmol) was dissolved with a mixed solution of DMF (6 mL) and DCM (6 mL) to form a clear solution. The clear solution was divided into five equal parts. One aliquot of the above-mentioned solution was added at 32° C. every 1 h. After the completion of the addition, the reaction was carried out at 32° C. for 1 h and stopped. The reaction system was concentrated to dryness under reduced pressure, and purified by column chromatography (DCM/CH.sub.3OH system) to obtain an off-white solid of 0.22 g, with a yield of 42%. .sup.1H NMR (400 MHz, Chloroform-d) 9.85 (br, 1H), 8.30 (d, J=9.3 Hz, 211), 7.20 (m, 1H), 6.90 (t, J=8.0 Hz, 1H), 6.78 (d, J=10.2 Hz, 1H), 6.33 (d, J=7.7 Hz, 11H), 5.67 (t, J=6.6 Hz, 1H), 3.99 (dt, J=14.0, 7.9 Hz, 2H), 3.90-3.72 (m, 1H), 3.65-3.50 (m, 2H), 2.94-2.88 (m, 111), 2.67-2.50 (m, 2H), 2.50-2.33 (m, 1H), 2.21 (dt, J=13.3, 7.0 Hz, 1H), 1.93 (dt, J=12.7, 6.5 Hz, 1H), 1.14 (m, 1H), 0.90 (d, J=10.2 Hz, 1H), 0.54 (m, 2H). MS (ESI) m/z: 407 [M+H].sup.+.

Example 11

(182) Inhibitory Activity Test for Trk Kinases

(183) In this experiment, the γ-.sup.33P-ATP isotope test was used to test the inhibition effect of a compound on kinases TrkA, TrkB and TrkC, and the half maximal inhibitory concentration IC.sub.50 of the inhibitory activity of the compound on the enzymes was obtained. The Trk inhibitor LOXO-101 reported in the literature was used as a positive control, and LOXO-101 was purchased from Shanghai Sinochemtech Co., Ltd., under lot number: SCT0142170801.

(184) 1. Basic Reaction Buffer

(185) 20 mM Hepes (pH 7.5), 10 mM MgCl.sub.2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na.sub.3VO.sub.4, 2 mM DTT, 1% DMSO.

(186) 2. Formulation of Compounds

(187) A compound was dissolved in 100% DMSO to a particular concentration, and then the resulting solution was gradient-diluted by an automatic loading device to different concentrations of the test samples (DMSO dissolving solutions).

(188) 3. Reaction Procedure

(189) 3.1. The reaction substrate was diluted using the base reaction buffer;

(190) 3.2. The kinase was added into the substrate solution and mixed gently and uniformly;

(191) 3.3. The compounds of different concentrations diluted in 100% DMSO were added into the kinase solution using an automatic loading system, and incubated for 20 min at room temperature;

(192) 3.4. .sup.33P-ATP (10 μM, 10 μCi/μl) was added at room temperature to initiate the kinase reaction, and the reaction was carried out for 2 h.

(193) 4. Detection

(194) The reaction solution was subjected to an ion exchange filtration system to remove unreacted ATP and the generated ADP ions during the reaction. Then, the radiometric quantity of .sup.33P isotope in the substrate was detected.

(195) 5. Data Processing

(196) The kinase activity in the system with the addition of the inhibitors of different concentrations was calculated from the radiometric quantity, to obtain the inhibitory effect of the compounds with different concentrations on the kinase activity. Fitting was performed using graphpad prism to obtain IC.sub.50 values of the compounds for inhibition.

(197) The biochemical activity of the compounds of the present invention was determined by the above experiment, and the measured IC.sub.50 values are shown in table 1:

(198) TABLE-US-00001 TABLE 1 Test results of inhibitory activity for Trk kinases Kinase IC.sub.50 (nM) Compound TrkA TrkB TrkC Example 1 4.23 0.57 0.25 Example 2 3.81 — — Example 3a 2.27 1.58 0.46 Example 3b 2.16 1.13 0.38 Example 4 12.6  — — Example 5 7.92 — — Example 6 2.09 0.83 0.29 Example 7a 3.20 4.04 0.58 Example 7b 2.62 0.71 0.19 Example 8a 4.01 6.19 0.76 Example 8b 1.85 1.35 0.34 Example 9 2.36 2.16 0.32 LOXO-101 5.58 1.10 0.67 Note: “—” in the table indicates not tested.

(199) Conclusion: The compounds of the present invention have better inhibitory activity for Trk kinases than the positive control.

Example 12

(200) Growth Inhibition Test for Tel-NTRK1-BaF3 and BaF3-LMNA-NTRK1 Cells

(201) In this experiment, the CellTiter-Glo cell proliferation fluorescence assay was used to test the growth inhibitory effect of a compound on tel-NTRK1-BaF3 and BaF3-LMNA-NTRK1 transgenic cells, and the half maximal growth inhibitory concentration GI.sub.50 of the compound on the cells was obtained. Trk inhibitor LOXO-101 reported in the literature was used as a positive control, and LOXO-101 was purchased from HaoyuanChemexpress Co., Ltd.

(202) 1. Experimental Instruments and Consumables

(203) I) PreceDo target equivalent gene stable cell line library;

(204) II) CellTiter-Glo cell proliferation fluorescence assay reagent (Promega, USA);

(205) III) Special 96-well plate for drug screening (Corning, Rochester, N.Y.);

(206) IV) Compounds for testing.

(207) 2. Preparation of Compound Plates

(208) A compound to be tested was dissolved in DMSO to formulate 10 mM of a mother solution. The mother solution was 3× diluted to 10 mM, 3.333 mM, 1.111 mM, 0.370 mM, 0.123 mM, 0.041 mM, 0.014 mM, 0.005 mM, and 0.002 mM. The prepared solutions were each stored in a 0.5 ml sterilized dorft tube (Corning, USA). In addition, an equal volume of DMSO solvent was used as a blank control. With 10 concentration gradients, the plate was stored in vacuum at −20° C.

(209) 3. Cell Culture Conditions

(210) The tel-NTRK1-BaF3 and BaF3-LMNA-NTRK1 cell lines were cultured with RPMI 1640 (Corning, N.Y., USA)+10% fetal bovine serum (Gibico, Invitrogen, USA). After the cells were thawed, they were cultured for two generations to be tested.

(211) 4. Testing and Data Processing

(212) Logarithmic growth phase cells (2000-2500 cells/well) were inoculated in a 12×8 96-well white opaque cell culture plate (Corning 3570, NY, USA) in a volume of 100 μL per well, and a drug was added to the cell plate (0.1 μL/well) at the final concentration of the compound of 10 μM, 3.3 μM, 1.1 μM, 0.37 μM, 0.12 IM, 0.04 μM, 0.014 IM, 0.005 μM, and 0.002 μM (0.4 uL of the drug solution was added to 400 ul of the cell homogenate, and then mixed uniformly, and the resulting mixture was added at 100 ul per well). The plate was incubated at 37° C. in a 5% CO.sub.2 incubator for 72 hours, and then 20 μL CellTiter-Glo cell proliferation fluorescence assay reagent was added. The plate was allowed to stand for 10 min, and read in the Envision Plate-Reader.

(213) 5. Experimental Verification

(214) A vehicle group (only DMSO added) was used as a negative control in the plates.

(215) 6. Results

(216) A corresponding fluorescence value RLU of each well was obtained by reading in the Envision Plate-reader. Raw data RLU.sub.Drug for the compounds to be tested were normalized to the RLU.sub.Drug for the DMSO control group:
Cell Viability %=(RLU.sub.Drug/RLU.sub.DMSO)*100%

(217) Nonlinear regression curve fitting was performed for the cell inhibition value of a single concentration of the compound to be tested by using Graph Pad Prism version 6.0, to obtain the GI.sub.50 values.

(218) 7. Basic Reaction Buffer

(219) 20 mM Hepes (pH 7.5), 10 mM MgCl.sub.2, 1 mM EGTA, 0.02% mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO.

(220) The biochemical activity of the compounds of the present invention was determined by the above experiment, and the measured GI.sub.50 values are shown in table 2:

(221) TABLE-US-00002 TABLE 2 Test results of inhibitory activity for TRK fusion cell lines Cell GI.sub.50 (nM) Compound BaF3-tel-NTRK1 BaF3-LMNA-NTRK1 Example 1 12.0 15.1 Example 2 18.0 — LOXO-101 24.6 35.7 Note: “—” in the table indicates not tested.

(222) Conclusion: The compounds of the present invention have better growth inhibitory activity for TRK fusion cell lines than the positive control.

Example 13

(223) Testing on Metabolic Stability of Compounds in Human Liver Microsomes

(224) The total volume of an incubation system was 250 μL, and 50 mmol/L PBS buffer (pH=7.4) was used to prepare incubation solutions of liver microsomes from various species with a protein concentration of 0.5 mg/mL. Before the start of the incubation, 2.5 μL of a 100 μmol/L compound to be tested was mixed with 197.5 μL of the above incubation solution, pre-incubated in a 37° C. water bath for 5 min, and then added with a 50 μL reducing coenzyme II solution (5 mmol/L) that was likewise pre-incubated for 5 min to start the reaction (in the reaction system, the protein content of liver microsomes from various species was 0.5 g/L, and the final concentration of the compound to be tested was 1 μmol/L), incubated in a 37° C. water bath with shaking, and taken out at 0, 5, 15, 30, and and 60 min. 600 μL of a methanol solution of mixed positive and negative internal standards with internal standards Terfenadine (positive ion internal standard, 25 ng/mL) and Tolbutamide (negative ion internal standard, 50 ng/mL) was immediately added to terminate the reaction. The incubation solution after termination was shaken for 2 min and centrifuged (4° C., 16000 r/min) for 10 min, and the supernatant was taken for LC-MS/MS detection to quantitatively analyze the remaining amount of the parent drug. (DMSO<0.1%).

(225) The concentration of the compound at 0 min incubation was regarded as 100%, and the concentrations at other incubation time points were converted into the remaining percentages. The natural logarithm of the remaining percentage at each time point was linearly regressed against the incubation time, and the slope k was calculated. According to the formula T.sub.1/2=−0.693/k, the in vitro half-life was calculated. Clearance in liver microsomes (CLint (μL/min/mg protein)=Ln (2)*1000/T.sub.1/2 (min)/Protein Conc(mg/ml)).

(226) Test data of metabolic stability of the compounds of the present invention in human liver microsomes are listed in detail in table 3:

(227) TABLE-US-00003 TABLE 3 Test results of metabolic stability in human liver microsomes Results of metabolic stability of tested substances in human liver microsomes Negative Clint control Tested Remaining % T.sub.1/2 (μL/min/ Remaining % substance No. (60 min) (min) mg protein) (60 min) Example 1 52.26 66.6 20.8 98.1 Example 4 88.39 217.0 6.40 108.0 LOXO-101 47.78 59.2 23.4 95.9 LOXO-195 6.70 15.3 90.6 102 Note: Trk inhibitor, LOXO-195 was prepared with reference to the method of patent WO2011146336.

(228) Conclusion: Compared with the control compound, the compounds of the present invention have better metabolic stability in human liver microsomes and better druggability than the positive control drug.