Di(hetero)aryl macrocyclic compound for inhibiting protein kinase activity

Abstract

A di(hetero)aryl macrocyclic compound having an inhibitory effect on protein kinase activity, preparation and the use thereof. Specifically, disclosed are a di(hetero)aryl macrocyclic compound represented by formula (I), or a pharmaceutically acceptable salt, an enantiomer, a diastereomer, a racemate, a solvate, a hydrate, a polymorph, a prodrug or an active metabolite thereof, a pharmaceutical composition comprising said compound and the derivative thereof, and methods of using the same, including methods of treating cancers, pain, neurological diseases, autoimmune diseases and inflammation. ##STR00001##

Claims

1. A compound of formula (I): ##STR00057## wherein, A.sub.1 is CR.sub.1; A.sub.2 is CR.sub.2; A.sub.3 is CR.sub.3; A.sub.4 is CR.sub.4; wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are independently selected from H, D, halogen, —CN, and —NO.sub.2; L.sub.1 is C(R.sub.1a)(R.sub.2a); L.sub.2 is C(R.sub.1b)(R.sub.2b); X is selected from O, S, and N(R.sub.1c); Y is selected from O, S, N(R.sub.1d) and C(R.sub.1d)(R.sub.2d); W is selected from O, S, and N(R.sub.1e); R is selected from H, D, C.sub.1-6 alkyl, and C.sub.1-6 haloalkyl; wherein the above groups are optionally substituted by one or more D atoms until completely deuterated; m is selected from 1, 2, 3, 4 and 5; n is selected from 1, 2 and 3; wherein, R.sub.1a and R.sub.2a are each independently selected from H, D, halogen, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, and C.sub.2-6 alkynyl; or R.sub.1a, R.sub.2a together with the atom to which they are attached form a C.sub.3-7 cycloalkyl; wherein the above groups are optionally substituted by one or more D atoms until completely deuterated; R.sub.1b and R.sub.2b are each independently selected from H, D, halogen, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, and C.sub.2-6 alkynyl; wherein the above groups are optionally substituted by one or more D atoms until completely deuterated; R.sub.1c is selected from H, D, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, and C.sub.2-6 alkynyl; wherein the above groups are optionally substituted by one or more D atoms until completely deuterated; R.sub.1d and R.sub.2d are each independently selected from H, D, halogen, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, and C.sub.2-6 alkynyl; or R.sub.1d, R.sub.2d together with the atom to which they are attached form a C.sub.3-7 cycloalkyl; wherein the above groups are optionally substituted by one or more D atoms until completely deuterated; R.sub.1e is selected from H, D, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, and C.sub.2-6 alkynyl; wherein the above groups are optionally substituted by one or more D atoms until completely deuterated; and substituents on different atoms in -(L.sub.2).sub.n-W— can be connected to form a-3- to 10-membered heterocyclyl; wherein the above groups are optionally substituted by one or more D atoms until completely deuterated; or a pharmaceutically acceptable salt, enantiomer, diastereomer, or racemate thereof; wherein, “3- to 7-membered heterocyclyl” is a radical of a 3- to 7-membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms; “5- to 10-membered heteroaryl” refers to a radical of a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur.

2. The compound according to claim 1, which is a compound of formula (III-1) or (III-2): ##STR00058## wherein, A.sub.1 is CR.sub.1; A.sub.4 is CR.sub.4; wherein R.sub.1, R.sub.3 and R.sub.4 are independently selected from H, D, halogen, —CN, and —NO.sub.2; X is O; Y is selected from N(R.sub.1d) and C(R.sub.1d)(R.sub.2d); W is selected from O and NH; m is selected from 1, 2 and 3; R.sub.1a and R.sub.2a are each independently selected from H, D, halogen, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, and C.sub.2-6 alkynyl; or R.sub.1a, R.sub.2a together with the atom to which they are attached form a C.sub.3-7 cycloalkyl; wherein the above groups are optionally substituted by one or more D atoms until completely deuterated; R.sub.1b is selected from H, D, halogen, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, and C.sub.2-6 alkynyl; wherein the above groups are optionally substituted by one or more D atoms until completely deuterated; and wherein, R.sub.1d and R.sub.2d are each independently selected from H, D, halogen, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, and C.sub.2-6 alkynyl; or R.sub.1d, R.sub.2d together with the atom to which they are attached form a C.sub.3-7 cycloalkyl; wherein the above groups are optionally substituted by one or more D atoms until completely deuterated; or a pharmaceutically acceptable salt, enantiomer, diastereomer, or racemate thereof.

3. The compound according to claim 1, which is a compound of formula (IV-1) or (IV-2): ##STR00059## wherein, R.sub.3 is selected from H, D, halogen, —CN, and —NO.sub.2; X is O; Y is selected from NH, CH.sub.2 and C(Me)(Me); wherein the above groups are optionally substituted by one or more D atoms until completely deuterated; m is selected from 1, 2 and 3; R.sub.1a and R.sub.2a are each independently selected from H, D, halogen, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, and C.sub.2-6 alkynyl; or R.sub.1a, R.sub.2a together with the atom to which they are attached form a C.sub.3-7 cycloalkyl; wherein the above groups are optionally substituted by one or more D atoms until completely deuterated; and R.sub.1b is selected from H, D, halogen, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, and C.sub.2-6 alkynyl; wherein the above groups are optionally substituted by one or more D atoms until completely deuterated; or a pharmaceutically acceptable salt, enantiomer, diastereomer, or racemate thereof.

4. The compound according to claim 1, which is a compound of formula (VI-1) or (VI-2): ##STR00060## wherein, R.sub.3 is selected from H, D, and halogen; R.sub.1a and R.sub.2a are each independently selected from H, D, C.sub.1-6 alkyl and C.sub.1-6 haloalkyl; wherein the above groups are optionally substituted by one or more D atoms until completely deuterated; and R.sub.1b is selected from H, D, C.sub.1-6 alkyl and C.sub.1-6 haloalkyl; wherein the above groups are optionally substituted by one or more D atoms until completely deuterated; or a pharmaceutically acceptable salt, enantiomer, diastereomer, or racemate thereof.

5. The compound according to claim 1, which is a compound of formula (I′): ##STR00061## A.sub.1 is CR.sub.1; A.sub.2 is CR.sub.2; A.sub.3 is CR.sub.3; A.sub.4 is CR.sub.4; wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are independently selected from H, D, halogen, —CN, and —NO.sub.2; L.sub.1 is C(R.sub.1a)(R.sub.2a); X is selected from O, S, and N(R.sub.1c); Y is selected from O, S, N(R.sub.1d) and C(R.sub.1d)(R.sub.2d); L.sub.3 is C(R.sub.1f)(R.sub.2f); L.sub.4 is C(R.sub.1g)(R.sub.2g); L.sub.5 is C(R.sub.1h)(R.sub.2h); R is selected from H, D, C.sub.1-6 alkyl, and C.sub.1-6 haloalkyl; wherein the above groups are optionally substituted by one or more D atoms until completely deuterated; m is selected from 1, 2, 3, 4 and 5; n is selected from 1, 2 and 3; wherein, R.sub.1a and R.sub.2a are each independently selected from H, D, halogen, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, and C.sub.2-6 alkynyl; or R.sub.1a, R.sub.2a together with the atom to which they are attached form a C.sub.3-7 cycloalkyl; wherein the above groups are optionally substituted by one or more D atoms until completely deuterated; R.sub.1d and R.sub.2d are each independently selected from H, D, halogen, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, and C.sub.2-6 alkynyl; or R.sub.1d, R.sub.2d together with the atom to which they are attached form a C.sub.3-7 cycloalkyl; wherein the above groups are optionally substituted by one or more D atoms until completely deuterated; R.sub.1f and R.sub.2f are each independently selected from H, D, halogen, C.sub.1-6 alkyl, and C.sub.1-6 haloalkyl; wherein the above groups are optionally substituted by one or more D atoms until completely deuterated; R.sub.1g and R.sub.2g are each independently selected from H, D, halogen, C.sub.1-6 alkyl, and C.sub.1-6 haloalkyl; wherein the above groups are optionally substituted by one or more D atoms until completely deuterated; and R.sub.1b and R.sub.2h are each independently selected from H, D, halogen, C.sub.1-6 alkyl, and C.sub.1-6 haloalkyl; wherein the above groups are optionally substituted by one or more D atoms until completely deuterated; or a pharmaceutically acceptable salt, enantiomer, diastereomer, or racemate thereof.

6. The compound according to claim 5, which is a compound of formula (III′-1) or (III′-2): ##STR00062## wherein, A.sub.1 is CR.sub.1; wherein R.sub.1 and R.sub.3 are each independently selected from H, D, halogen, —CN, and —NO.sub.2; X is O; Y is selected from CH.sub.2, and CH(R.sub.1d); wherein the above groups are optionally substituted by one or more D atoms until completely deuterated; R.sub.1a and R.sub.2a are each independently selected from H, D, halogen, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, and C.sub.2-6 alkynyl; or R.sub.1a, R.sub.2a together with the atom to which they are attached form a C.sub.3-7 cycloalkyl; wherein the above groups are optionally substituted by one or more D atoms until completely deuterated; R.sub.1d is selected from halogen, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, and C.sub.2-6 alkynyl; wherein the above groups are optionally substituted by one or more D atoms until completely deuterated; R.sub.1f and R.sub.2f are each independently selected from H, D, halogen, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, and C.sub.2-6 alkynyl; wherein the above groups are optionally substituted by one or more D atoms until completely deuterated; m is selected from 1, 2 and 3; or a pharmaceutically acceptable salt, enantiomer, diastereomer, or racemate thereof.

7. The compound according to claim 5, which is a compound of formula (V′-1) or (V′-2): ##STR00063## wherein, R.sub.3 is selected from H, D, halogen, —CN and —NO.sub.2; R.sub.1a and R.sub.2a are each independently selected from H, D, C.sub.1-6 alkyl and C.sub.1-6 haloalkyl; wherein the above groups are optionally substituted by one or more D atoms until completely deuterated; or a pharmaceutically acceptable salt, enantiomers, diastereomer, or racemate thereof.

8. The compound according to claim 1, which is selected from the group consisting of: ##STR00064## ##STR00065## ##STR00066## ##STR00067## ##STR00068## or a pharmaceutically acceptable salt, enantiomer, diastereomer, or racemates thereof.

9. A pharmaceutical composition, comprising a compound according to claim 1, or a pharmaceutically acceptable salt, enantiomer, diastereomer, or racemate thereof, and pharmaceutically acceptable excipient(s).

10. The compound according to claim 3, wherein, R.sub.3 is selected from H, D, halogen, —CN and —NO.sub.2; X is O; Y is selected from NH, CH.sub.2 and C(Me)(Me); wherein the above groups are optionally substituted by one or more D atoms until completely deuterated; m is selected from 1, 2 and 3; R.sub.1a and R.sub.2a are each independently selected from H, D, C.sub.1-6 alkyl and C.sub.1-6 haloalkyl; wherein the above groups are optionally substituted by one or more D atoms until completely deuterated; R.sub.1b is selected from C.sub.1-6 alkyl and C.sub.1-6 haloalkyl; wherein the above groups are optionally substituted by one or more D atoms until completely deuterated; or a pharmaceutically acceptable salt, enantiomer, diastereomer, or racemate thereof.

11. The compound according to claim 3, wherein, R.sub.3 is selected from H, D, and halogen; X is O; Y is selected from CH.sub.2 and C(Me)(Me); wherein the above groups are optionally substituted by one or more D atoms until completely deuterated; m is selected from 1, 2 and 3; R.sub.1a and R.sub.2a are each independently selected from H, D, C.sub.1-6 alkyl and C.sub.1-6 haloalkyl; wherein the above groups are optionally substituted by one or more D atoms until completely deuterated; R.sub.1b is selected from C.sub.1-6 alkyl and C.sub.1-6 haloalkyl; wherein the above groups are optionally substituted by one or more D atoms until completely deuterated; or a pharmaceutically acceptable salt, enantiomer, diastereomer, or racemate thereof.

12. The compound according to claim 7, wherein, R.sub.3 is selected from H, D, and halogen; R.sub.1a and R.sub.2a are each independently selected from H, D, C.sub.1-6 alkyl and C.sub.1-6 haloalkyl; and R.sub.1a and R.sub.2a are not H or D at the same time; wherein the above groups are optionally substituted by one or more D atoms until completely deuterated; or a pharmaceutically acceptable salt, enantiomer, diastereomer, or racemate thereof.

Description

EXAMPLES

(1) The present disclosure will be further described below in combination with specific embodiments. It should be understood that these examples are only used to illustrate the present disclosure and not to limit the scope of the present disclosure. The experimental methods without specific conditions in the following examples generally follow the conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, parts and percentages are parts by weight and weight percent.

(2) Generally, in the preparation process, each reaction is carried out in an inert solvent at a temperature from room temperature to reflux temperature (such as 0° C. to 100° C., alternatively 0° C. to 80° C.). The reaction time is usually 0.1-60 hours, alternatively 0.5-24 hours.

Example 1: Preparation of (13R)-11-fluoro-13-methyl-6,7,13,14-tetrahydro-1,15-ethenopyrazolo[4,3-f][1,4,8,10]benzoxatriazacyclotridecin-5(6H)-one (Compound T-1)

(3) ##STR00022##

(4) The following route was used for the synthesis:

(5) ##STR00023## ##STR00024##

(6) Step 1 Synthesis of Compound 1

(7) 5-fluoro-2-hydroxybenzaldehyde (1.4 g, 10.0 mmol), and R-tert-butylsulfinamide (1.21 g, 10.0 mmol) were added to a reaction flask, which were dissolved in 20 ml dichloromethane, cesium carbonate was added (5.21 g, 16.0 mmol), and the reaction was stirred at room temperature for 18 hours under nitrogen protection. The reaction was quenched by adding an excess amount of water, and extracted 3-4 times with dichloromethane. The organic phases were combined, washed with saturated brine, concentrated, purified by column chromatography, and dried in vacuum to afford 2.11 g of the product. Yield: 86.8%.

(8) Step 2 Synthesis of Compounds 2 and 3

(9) Compound 1 (1.86 g, 7.67 mmol) was added to a reaction flask, which was dissolved by adding 25 ml of anhydrous terahydrofuran, and a solution of 3M methylmagnesium bromide in anhydrous tetrahydrofuran (12.8 ml, 38.4 mmol) was slowly added at −65° C. After the addition, the reaction was warmed to room temperature and stirred overnight. TLC was used to monitor the completion of the reaction. 20 ml of water was added dropwise to quench the reaction in an ice bath, the organic phase was separated, and the aqueous phase was extracted with ethyl acetate for 3-4 times. The organic phases were combined, washed with saturated brine, concentrated and then purified by silica gel column chromatography (petroleum ether:ethyl acetate=2:1) to afford 0.98 g of compound 2 and 1.1 g of compound 3. Yield: 100%.

(10) Step 3 Synthesis of Compound 4

(11) Compound 3 (1.14 g, 4.4 mmol) was added to a reaction flask, to which a solution of 4 N hydrogen chloride in dioxane (13.7 ml, 54.77 mmol) was added, and the reaction was stirred at room temperature for 4 hours. A white solid was precipitated out, which was filtered to afford 798 mg of the product. The product was directly used in the next step without purification. Yield: 95%.

(12) Step 4 Synthesis of Compound 5

(13) Compound 4 (382 mg, 2.0 mmol), 5-chloro-3-nitropyrazolo[1,5-a]pyrimidine (396 mg, 2.0 mmol) and DIPEA (N,N-diisopropylethylamine, 1.29 g, 10 mmol) were added to a reaction flask, and 8 ml anhydrous ethanol was added. The reaction was heated to 80° C. for 1 hour. TLC was used to monitor the completion of the reaction. The reaction was concentrated to remove the solvent, and purified by silica gel column chromatography to afford 538 mg of the product. Yield: 85%. LC-MS (APCI): m/z=318.3 (M+1).sup.+.

(14) Step 5 Synthesis of Compound 6

(15) Compound 5 (100 mg, 0.315 mmol), ethyl 3-hydroxypropionate (55.8 mg, 0.473 mmol) and triphenylphosphine (124.1 mg, 0.473 mmol) were added to a reaction flask, to which 5 ml anhydrous tetrahydrofuran was added under nitrogen protection, and DIAD (diisopropyl azodicarboxylate, 95.6 mg, 0.473 mmol) was added dropwise at 0° C. After the addition, the reaction was warmed to room temperature and stirred for 15 hours. The reaction was concentrated to remove the solvent, and purified by silica gel column chromatography to afford 51.6 mg of the product. Yield: 38%. LC-MS (APCI): m/z=418.1 (M+1).sup.+.

(16) Step 6 Synthesis of Compound 7

(17) Compound 6 (192 mg, 0.46 mmol) was added to a reaction flask, which was dissolved in 8 ml methanol, and a solution of lithium hydroxide monohydrate (96.6 mg, 2.3 mmol) in 4 ml water was added. The solution was heated to 50° C. and reacted for 4-5 hours. TLC was used to monitor the completion of the reaction. The reaction was cooled to 0° C., pH thereof was adjusted to weak acidic with 1N dilute hydrochloric acid, and the reaction was extracted with ethyl acetate for 3-4 times. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to afford 174 mg of product. Yield: 97%. LC-MS (APCI): m/z=390.5 (M+1).sup.+.

(18) Step 7 Synthesis of Compound 8

(19) Compound 7 (174 mg, 0.45 mmol) was added to a reaction flask, which was dissolved in 5 ml methanol, and a catalytic amount of Pd/C was added. The reaction was filled with hydrogen gas, stirred and reacted at room temperature for 5-7 hours. TLC was used to monitor the completion of the reaction. After completion, the catalyst was removed by filtration, and the filtrate was concentrated to afford crude product of compound 8. The crude product was directly used in the next step without purification. LC-MS (APCI): m/z=360.4 (M+1).sup.+.

(20) Step 8 Synthesis of Compound T-1

(21) Compound 8 (161 mg, 0.45 mmol) was added to a reaction flask, which was dissolved in 15 ml anhydrous DMF, and FDPP (pentafluorophenyl diphenylphosphinate, 207.5 mg, 0.54 mmol) and DIPEA (290.8 mg, 2.25 mmol) were added. The reaction was stirred at room temperature overnight under nitrogen protection. TLC was used to monitor the completion of the reaction. After completion, an excess amount of water was added to the reaction, which was extracted with ethyl acetate for 3-4 times. The organic phases were combined, washed to with saturated brine, concentrated and then purified by silica gel column chromatography to afford 106 mg of the title product. Yield: 69%. LC-MS (APCI): m/z=342.5 (M+1).sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 10.11 (s, 1H), 8.56 (s, 1H), 8.12 (d, J=2.3 Hz, 1H), 7.35 (t, J=4.4 Hz, 1H), 6.98 (m, 2H), 6.52 (d, J=2.3 Hz, 1H), 4.25 (t, J=3.3 Hz, 2H), 4.01 (m, 1H), 2.66 (t, J=3.3 Hz, 2H), 1.21 (d, J=5.5 Hz, 3H).

Example 2-1: Preparation of (7S,13R)-11-fluoro-7,13-dimethyl-6,7,13,14-tetrahydro-1,15-ethenopyrazolo[4,3-f][1,4,8,10]benzoxatriazacyclotiidecin-5(6H)-one (Compound T-2-A)

(22) ##STR00025##

(23) The following route was used or the synthesis:

(24) ##STR00026##

(25) Step 1 Synthesis of Compound 9

(26) Compound 5 (200 mg, 0.63 mmol), ethyl (R)-3-hydroxybutyrate (125 mg, 0.946 mmol) and triphenylphosphine (248.2 mg, 0.946 mmol) were added to a reaction flask, 10 ml anhydrous tetrahydrofuran was added under nitrogen protection, and DIAD (191.2 mg, 0.946 mmol) was added dropwise at 0° C. After the addition, the reaction was warmed to room temperature and stirred for 15 hours. The reaction was concentrated to remove the solvent, and purified by silica gel column chromatography to afford 78.8 mg of the product. Yield: 29%. LC-MS (APCI): m/z=432.1 (M+1).sup.+.

(27) Step 2 Synthesis of Compound 10

(28) Compound 9 (200 mg, 0.46 mmol) was added to a reaction flask, which was dissolved in 8 ml methanol, and a solution of lithium hydroxide monohydrate (96.6 mg, 2.3 mmol) in 4 ml water was added. The solution was heated to 50° C. and reacted for 4-5 hours. TLC was used to monitor the completion of the reaction. The reaction was cooled to 0° C., pH thereof was adjusted to weak acidic with 1N dilute hydrochloric acid, and the reaction was extracted with ethyl acetate for 3-4 times. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to afford 182 mg of product. Yield: 98%. LC-MS (APCI): m/z=404.5 (M+1).sup.+.

(29) Step 3 Synthesis of Compound 11

(30) Compound 10 (209 mg, 0.52 mmol) was added to a reaction flask, which was dissolved in 8 ml methanol, and a catalytic amount of Pd/C was added. The reaction was filled with hydrogen gas, stirred and reacted at room temperature for 5-7 hours. TLC was used to monitor the completion of the reaction. After completion, the catalyst was removed by filtration, and the filtrate was concentrated to afford crude product of compound 11. The crude product was directly used in the next step without purification. LC-MS (APCI): m/z=374.6 (M+1).sup.+.

(31) Step 4 Synthesis of Compound T-2-A

(32) Compound 11 (168 mg, 0.45 mmol) was added to a reaction flask, which was dissolved in 15 ml anhydrous DMF, and FDPP (207.5 mg, 0.54 mmol) and DIPEA (290.8 mg, 2.25 mmol) were added. The reaction was stirred at room temperature overnight under nitrogen protection. TLC was used to monitor the completion of the reaction. After completion, an excess amount of water was added to the reaction, which was extracted with ethyl acetate for 3-4 times. The organic phases were combined, washed with saturated brine, concentrated and then purified by silica gel column chromatography to afford 81 mg of the title product. Yield: 51%. LC-MS (APCI): m/z=356.3 (M+1).sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 10.13 (s, 1H), 8.53 (s, 1H), 8.11 (d, J=2.7 Hz, 1H), 7.35 (t, J=4.4 Hz, 1H), 6.99 (m, 2H), 6.50 (d, J=2.7 Hz, 1H), 4.06 (m, 1H), 3.99 (m, 1H), 2.66 (t, J=3.3 Hz, 2H), 1.26-1.21 (m, 6H).

Example 2-2: Preparation of (7S,13S)-11-fluoro-7,13-dimethyl-6,7,13,14-tetrahydro-1,15-ethenopyrazolo[4,3-f][1,4,8,10]benzoxatriazacyclotidecin-5(6H)-one (Compound T-2-B)

(33) ##STR00027##

(34) The following route was used for the synthesis:

(35) ##STR00028##

(36) Step 1 Synthesis of Compound 12

(37) Compound 2 (1.14 g, 4.4 mmol) was added in a reaction flask, to which a solution of 4 N hydrogen chloride in dioxane (13.7 ml, 54.77 mmol) was added, and the reaction was stirred at room temperature for 4 hours. A white solid was precipitated out, which was filtered to afford 798 mg of product. The product was directly used in the next step without purification. Yield: 95%.

(38) Step 2 Synthesis of Compound 13

(39) Compound 12 (382 mg, 2.0 mmol), 5-chloro-3-nitropyrazolo[1,5-a]pyrimidine (396 mg, 2.0 mmol) and DIPEA (N,N-diisopropylethylamine, 1.29 g, 10 mmol) were added to a reaction flask, and 8 ml absolute ethanol was added. The reaction was heated to 80° C. for 1 hour. TLC was used to monitor the completion of the reaction. The reaction was concentrated to remove the solvent, and purified by silica gel column chromatography to afford 538 mg of the product. Yield: 85%. LC-MS (APCI): m/z=318.3 (M+1).sup.+.

(40) Step 3 Synthesis of Compound 14

(41) Compound 13 (200 mg, 0.63 mmol), ethyl (R)-3-hydroxybutyrate (125 mg, 0.946 mmol) and triphenylphosphine (248.2 mg, 0.946 mmol) were added to a reaction flask, and 10 ml anhydrous tetrahydrofuran was added under nitrogen protection, and DIAD (191.2 mg, 0.946 mmol) was added dropwise at 0° C. After the addition, the reaction was warmed to room temperature and stirred for 15 hours. The reaction was concentrated to remove the solvent, and purified by silica gel column chromatography to afford 78.8 mg of the product. Yield: 29%. LC-MS (APCI): m/z=432.1 (M+1).sup.+.

(42) Step 4 Synthesis of Compound 15

(43) Compound 14 (200 mg, 0.46 mmol) was added to a reaction flask, which was dissolved in 8 ml methanol, and a solution of lithium hydroxide monohydrate (96.6 mg, 2.3 mmol) in 4 ml water was added. The solution was heated to 50° C. and reacted for 4-5 hours. TLC was used to monitor the completion of the reaction. The reaction was cooled to 0° C., pH thereof was adjusted to weak acidic with 1N dilute hydrochloric acid, and the reaction was extracted with ethyl acetate for 3-4 times. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to afford 182 mg of product. Yield: 98%. LC-MS (APCI): m/z=404.5 (M+1).sup.+.

(44) Step 5 Synthesis of Compound 16

(45) Compound 15 (209 mg, 0.52 mmol) was added to a reaction flask, which was dissolved in 8 ml methanol, and a catalytic amount of Pd/C was added. The reaction was filled with hydrogen gas, stirred and reacted at room temperature for 5-7 hours. TLC was used to monitor the completion of the reaction. After completion, the catalyst was removed by filtration, and the filtrate was concentrated to afford crude product of compound 16. The crude product was directly used in the next step without purification. LC-MS (APCI): m/z=374.6 (M+1).sup.+.

(46) Step 6 Synthesis of Compound T-2-B

(47) Compound 16 (168 mg, 0.45 mmol) was added to a reaction flask, which was dissolved in 15 ml anhydrous DMF, and FDPP (207.5 mg, 0.54 mmol) and DIPEA (290.8 mg, 2.25 mmol) were added. The reaction was stirred at room temperature overnight under nitrogen protection. TLC was used to monitor the completion of the reaction. After completion, an excess amount of water was added to the reaction, which was extracted with ethyl acetate for 3-4 times. The organic phases were combined, washed with saturated brine, concentrated and then purified by silica gel column chromatography to afford 81 mg of the title product. Yield: 51%. LC-MS (APCI): m/z=356.3 (M+1).sup.+. .sup.1H NMR (400 MHz. DMSO-4) δ 10.13 (s, 1H), 8.53 (s, 1H), 8.11 (d, J=2.7 Hz, 1H), 7.35 (t, J=4.4 Hz, 1H), 6.99 (m, 2H), 6.50 (d, J=2.7 Hz, 1H), 4.06 (m, 1H), 3.99 (m, 1H), 2.66 (t, J=3.3 Hz, 2H), 1.26-1.21 (m, 6H).

Example 3: Preparation of ((13R)-11-fluoro-6,6,13-trimethyl-6,7,13,14-tetrahydro-1,15-ethenopyrazolo[4,3-f][1,4,8,10]benzoxatriazacyclotridecin-5(6H)-one (Compound T-3)

(48) ##STR00029##

(49) The following route was used for the synthesis:

(50) ##STR00030##

(51) Step 1 Synthesis of Compound 17

(52) Ethyl isobutyrate (2.5 g, 21.5 mmol) was added to a reaction flask, and 40 ml anhydrous tetrahydrofuran was added under nitrogen protection. The mixture was cooled to −40° C., and LDA (lithium diisopropylamide, 11.8 ml, 23.6 mmol) was slowly added dropwise. After the addition, the reaction was gradually warmed to room temperature and stirred to react for half an hour. After cooled to −40° C., a solution of diiodomethane (5.76 g, 21.5 mmol) in 10 ml anhydrous tetrahydrofuran was slowly added. After the addition, the temperature of the reaction was warmed to room temperature and the reaction was reacted overnight. Water was added to quench the reaction, the organic phase was separated, and the aqueous phase was extracted with ethyl acetate for 3-4 times. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to afford 4.62 g of the product. Yield: 84%.

(53) Step 2 Synthesis of Compound 18

(54) Compound 5 (300 mg, 0.95 mmol), compound 17 (291 mg, 1.14 mmol) and potassium carbonate (525.2 mg, 3.8 mmol) were added into a reaction flask, and DMF was added. The reaction was heated to 80° C. and stirred to react overnight. TLC was used to monitor the completion of the reaction. After completion, the reaction was cooled to room temperature, added with an excess amount of water, and extracted with ethyl acetate for 3-4 times. The organic phases were combined, washed with saturated brine, concentrated and then purified by silica gel column chromatography to afford 321.3 mg of the title product. Yield: 76%, LC-MS (APCI): m/z=446.1 (M+1).sup.+.

(55) Step 3 Synthesis of Compound 19

(56) Compound 18 (321 mg, 0.72 mmol) was added to a reaction flask, which was dissolved in 10 ml methanol, a solution of lithium hydroxide monohydrate (151.5 mg, 3.5 mmol) in 3 ml water was added, and the reaction was heated to 50° C. and reacted for 4-5 hours. TLC was used to monitor the completion of the reaction. The reaction was cooled to 0° C., pH thereof was adjusted to weak acidic with 1N dilute hydrochloric acid, and the reaction was extracted with ethyl acetate for 3-4 times. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to afford 279 mg of the product. Yield: 93%. LC-MS (APCI): m/z=418.3 (M+1).sup.+.

(57) Step 4 Synthesis of Compound 20

(58) Compound 19 (279 mg, 0.67 mmol) was added to a reaction flask, which was dissolved in 10 ml methanol, and a catalytic amount of Pd/C was added. The reaction was filled with hydrogen gas, stirred and reacted at room temperature for 5-7 hours. TLC was used to monitor the completion of the reaction. After completion, the catalyst was removed by filtration, and the filtrate was concentrated to afford crude product of compound 20, which was directly used in the next reaction without purification. LC-MS (APCI): m/z=387.9 (M+1).sup.+.

(59) Step 5 Synthesis of Compound T-3

(60) Compound 20 (259 mg, 0.67 mmol) was added to a reaction flask, which was dissolved in 20 ml anhydrous DMF, and FDPP (309 mg, 0.80 mmol) and DIPEA (433 mg, 3.35 mmol) were added. The reaction was stirred to react at room temperature overnight under nitrogen protection. TLC was used to monitor the completion of the reaction. After completion, the reaction was diluted with an excess amount of water, and extracted with ethyl acetate for 3-4 times. The organic phases were combined, washed with saturated brine, concentrated and then purified by silica gel column chromatography to afford 136 mg of the title product. Yield: 55%. LC-MS (APCI): m/z=370.3 (M+1).sup.+. .sup.1H NMR (400 MHz. DMSO-d.sub.6) δ 10.15 (s, 1H), 8.56 (s, 1H), 8.12 (d, J=2.3 Hz, 1H), 7.37 (t, J=4.4 Hz, 1H), 6.99 (m, 2H), 6.52 (d, J=2.3 Hz, 1H), 4.23 (s, 2H), 4.00 (m, 1H), 1.21 (d, J=5.5 Hz, 3H).

Example 4: Preparation of (14R)-12-fluoro-14-methyl-5,6,7,8,14,15-hexahydro-1,16-ethenopyrazolo[4,3-g][1,4,9,11]benzoxatetraazacyclotetradecin-5(6H)-one (Compound T-4)

(61) ##STR00031##

(62) The following route was used or the synthesis:

(63) ##STR00032##

(64) Step 1 Synthesis of Compound 21

(65) Compound 5 (300 mg, 0.95 mmol), N-tert-butoxycarbonyl-2-chloroethylamine (203.3 mg, 1.13 mmol) and potassium carbonate (525.2 mg, 3.8 mmol) were added to a reaction flask, and dissolved in 20 ml anhydrous DMF. The reaction was heated to 80° C. and reacted overnight. TLC was used to monitor the completion of the reaction. After completion, the reaction was diluted with an excess amount of water, and extracted with ethyl acetate for 3-4 times. The organic phases were combined, washed with saturated brine, concentrated and then purified by silica gel column chromatography to afford 345 mg of the product. Yield: 79%. LC-MS (APCI): m/z=461.2 (M+1).sup.+.

(66) Step 2 Synthesis of Compound 22

(67) Compound 21 (346 mg, 0.75 mmol) was added to a reaction flask, which was dissolved in 12 ml anhydrous methanol, and a catalytic amount of Pd/C was added. The reaction was filled with hydrogen gas, and reacted at room temperature for 1-2 hours. TLC was used to monitor the completion of the reaction. The reaction was filtered after completion, the filtrate was concentrated to afford 346 mg of the product, which was directly used in the next reaction without purification. LC-MS (APCI): m/z=431.3 (M+1).sup.+.

(68) Step 3 Synthesis of Compound 23

(69) Compound 22 (346 mg, 0.75 mmol) was added to a reaction flask, and 4M hydrogen chloride in dioxane (10 ml, 40 mmol) was added. The reaction was stirred at room temperature for 2-3 hours. After monitoring the completion of the reaction with MS, the reaction was concentrated to remove the solvent, which is then directly used in the next reaction. LC-MS (APCI): m/z=331.6 (M+1).sup.+.

(70) Step 4 Synthesis of Compound T4

(71) Compound 23 (200 mg, 0.61 mmol) was added to a reaction flask, to which 15 ml of dichloromethane solvent was added, and CDI (N,N′-carbonyldiimidazole, 196 mg, 1.21 mmol) was then added. The reaction was stirred at room temperature for 6 hours. TLC was used to monitor the completion of the reaction. The reaction was concentrated to remove the solvent, and purified by silica gel column chromatography to afford 134 mg of the product. Yield: 62%. LC-MS (APCI): m/z=357.5 (M+1).sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 10.11 (s, 1H), 9.85 (s, 1H), 8.56 (s, 1H), 8.12 (d, J=2.3 Hz, 1H), 7.35 (t, J=4.4 Hz 1H), 6.98 (m, 2H), 6.51 (d, J=2.3 Hz, 1H), 4.35 (t, J=3.5 Hz, 2H), 4.01 (m, 1H), 3.85 (t, J=3.5 Hz, 2H), 1.24 (d, J=5.5 Hz, 3H).

Example 5: Preparation of (14R)-12-fluoro-14-methyl-5,6,7,8,14,15-hexahydro-1,16-ethenopyrazolo[4,3-g][1,4,9,11]benzodioadiazatridecin-5(6H)-one (Compound T-5)

(72) ##STR00033##

(73) The following route was used for the synthesis:

(74) ##STR00034##

(75) Step 1 Synthesis of Compound 24

(76) 5-fluoro-2-iodoacetophenone (3.5 g, 13.26 mmol), (R)-tert-butylsulfinamide (2.41 g, 19.88 mmol) and tetraethyl titanate (7.54 g, 26.52 mmol) were added to a reaction flask, 50 ml anhydrous tetrahydrofuran was added, and the reaction was heated to reflux and stirred for 12 hours under nitrogen protection. TLC was used to monitor the completion of the reaction. The reaction was diluted with water and extracted 3-4 times with ethyl acetate. The organic phases were combined, washed with saturated brine, concentrated and then purified by silica gel column chromatography to afford 3.94 g of the product. Yield: 81%. LC-MS (APCI): m/z=368.4 (M+1).sup.+.

(77) Step 2 Synthesis of Compound 26

(78) Compound 24 (3.5 g, 9.54 mmol) was added to a reaction flask, which was dissolved in 25 ml tetrahydrofuran and 0.5 ml water, and sodium borohydride (0.54 g, 14.3 mmol) was added in batches at 0° C. After the addition, the reaction was warmed to room temperature and stirred to react for 12 hours. TLC was used to monitor the completion of the reaction. After completion, the reaction was diluted with water and extracted 3-4 times with ethyl acetate. The organic phases were combined, washed with saturated brine, concentrated and then purified by silica gel column chromatography to afford 3.27 g of the product. Yield: 93%. LC-MS (APCI): m/z=370.6 (M+1).sup.+.

(79) Step 3 Synthesis of Compound 27

(80) Compound 26 (3.27 g, 8.87 mmol) was added to a reaction flask, to which 4 M hydrogen chloride in dioxane (20 ml, 80 mmol) was then added, and the reaction was stirred at room temperature for 4-5 hours. A white solid was precipitated out, the resultant product was filtered and directly used in the next reaction after it was dried in vacuum. LC-MS (APCI): m/z=266.4 (M+1).sup.+.

(81) Step 4 Synthesis of Compound 28

(82) Compound 27 (300 mg, 1.13 mmol), 5-chloro-3-nitropyrazolo[1,5-a]pyrimidine (224 mg, 1.13 mmol) were added to a reaction flask, which was dissolved by adding 10 ml ethanol, and DIPEA (584 mg, 4.52 mmol) was added. The reaction was heated to 80° C. and stirred for 1 hour. TLC was used to monitor the completion of the reaction. The reaction was cooled to room temperature, concentrated and then purified by silica gel column chromatography to afford 371 mg of the product. Yield: 77%. LC-MS (APCI): m/z=428.1 (M+1).sup.+.

(83) Step 5 Synthesis of Compound 29

(84) Compound 28 (371 mg, 0.87 mmol), 3-phthalimido-1-propyne (241 mg, 1.3 mmol), bis(triphenylphosphine)palladium dichloride (30.5 mg, 0.043 mmol) and copper iodide (16.4 mg, 0.086 mmol) were added to a reaction flask, which was dissolved in 10 ml anhydrous tetrahydrofuran, and DIPEA (337.3 mg, 2.61 mmol) was added. The reaction was stirred and reacted overnight at room temperature under nitrogen protection. TLC was used to monitor the completion of the reaction. The reaction solution was concentrated and purified by silica gel column chromatography to afford 274 mg of the product. Yield: 65%. LC-MS (APCI): m/z=485.5 (M+1).sup.+.

(85) Step 6 Synthesis of Compound 30

(86) Compound 29 (274 mg, 0.57 mmol) was added to a reaction flask, which was dissolved in 5 ml methanol, and a catalytic amount of Pd/C was then added. The reaction was filled with hydrogen gas, stirred and reacted at room temperature overnight. TLC was used to monitor the completion of the reaction, and the reaction solution was filtered to afford 258 mg of the crude product, which was directly used in the next reaction. LC-MS (APCI): m/z=459.4 (M+1).sup.+.

(87) Step 7 Synthesis of Compound 31

(88) Compound 30 (258 mg, 0.56 mmol) was added to a reaction flask, which was dissolved in 10 ml methanol, then an excess amount of hydrazine hydrate was added. The reaction was heated to reflux and reacted for 4-6 hours. TLC was used to monitor the completion of the reaction. The reaction was concentrated to remove the solvent, and diluted with water, extracted with chloroform/isopropanol (3:1) for 4-5 times. The organic phases 0.5 were combined, washed with saturated brine, concentrated, and then purified by column chromatography to afford 71.6 mg of the product. Yield: 39%, LC-MS (APCI): m/z=329.6 (M+1).sup.+.

(89) Step 8 Synthesis of Compound T-5

(90) Compound 31 (71 mg, 0.22 mmol) was added to a reaction flask, and 10 ml dichloromethane solvent was added, then CDI (54 mg, 0.33 mmol) was added. The reaction was stirred and reacted for 6 hours at room temperature. TLC was used to monitor the completion of the reaction. The reaction was concentrated to remove the solvent, and purified by silica gel column chromatography to afford 51 mg of the product. Yield: 66%. LC-MS (APCI): m/z=355.1 (M+1).sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 10.11 (s, 1H), 8.56 (s, 1H), 8.12 (d, J=2.3 Hz, 1H), 7.35 (t, J=4.4 Hz, 1H), 6.98 (m, 2H), 6.52 (d, J=2.3 Hz, 1H), 4.01 (m, 1H), 2.66 (t, J=3.8 Hz, 2H), 2.45 (t, J=5.2 Hz, 2H), 1.75 (m, 2H), 1.21 (d, J=5.5 Hz, 3H).

Example 6: Preparation of (13R)-11-fluoro-13-methyl-6,7,13,14-tetrahydr-1,15-ethenopyrazolo[4,3-f][1,4,8,10]benzodioadiazatridecin-5(6H)-one (Compound T-6)

(91) ##STR00035##

(92) The following route was used for the synthesis:

(93) ##STR00036##

(94) Step 1 Synthesis of Compound 32

(95) 5-fluoro-2-hydroxyacetophenone (4.5 g, 29.2 mmol) was added to a reaction flask, which was dissolved in 40 ml anhydrous THF, and R—CBS (1.46 ml, 1.46 mmol) was added dropwise at 0° C. under nitrogen protection. After the addition, the reaction was stirred for 20 minutes, borane in tetrahydrofuran (29.2 ml, 29.2 mmol) was then slowly added dropwise. After the addition, the reaction was stirred and reacted for half an hour at low temperature. TLC was used to monitor the completion of the reaction. After completion, a small amount of methanol was added to quench the reaction. The reaction was concentrated to remove the solvent, ethyl acetate and water were added, the organic phase was separated, and aqueous phase was extracted with ethyl acetate for 3-4 times. The organic phases were combined, washed with saturated brine, concentrated and purified by column chromatography to afford 4.33 g of the product. Yield: 95%.

(96) Step 2 Synthesis of Compound 33

(97) Compound 32 (1.0 g, 6.41 mmol), 5-chloro-3-nitropyrazolo[1,5-α]pyrimidine (1.27 g, 6.41 mmol) were added to a reaction flask, which was dissolved in 20 ml ethanol, and NaH (2.48 g, 19.23 mmol) was added. The reaction was heated to 80° C., stirred and reacted for 1 hour. TLC was used to monitor the completion of the reaction. The reaction was cooled to room temperature, concentrated and purified by column chromatography to afford 1.79 g of the product. Yield: 88%. LC-MS (APCI): m/z=319.2 (M+1).sup.+.

(98) Step 3 Synthesis of Compound 34

(99) Compound 33 (1.2 g, 3.77 mmol), ethyl 3-hydroxypropionate (0.67 g, 5.66 mmol) and triphenylphosphine (1.48 g, 5.66 mmol) were added to a reaction flask, to which 5 ml anhydrous tetrahydrofuran was added under nitrogen protection, and DIAD (1.14 g, 5.66 mmol) was added dropwise under 0° C. After the addition, the reaction was warmed to room temperature and stirred for 15 hours. The reaction was concentrated to remove the solvent, and purified by silica gel column chromatography to afford 488.5 mg of the product. Yield: 31%. LC-MS (APCI): m/z=419.7 (M+1).

(100) Step 4 Synthesis of Compound 35

(101) Compound 34 (488 mg, 1.17 mmol) was added to a reaction flask, which was dissolved in 10 ml methanol, and a solution of lithium hydroxide monohydrate (245 mg, 5.84 mmol) in 4 ml water was added. The reaction was heated to 50° C. and reacted for 4-5 hours. TLC was used to monitor the completion of the reaction. The reaction was cooled to 0° C., pH thereof was adjusted to weak acidic with 1N dilute hydrochloric acid, and the reaction was extracted with ethyl acetate for 3-4 times. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to afford 433.4 mg of the product. Yield: 95%. LC-MS (APCI): m/z=391.5 (M+1).sup.+.

(102) Step 5 Synthesis of Compound 36

(103) Compound 35 (433 mg, 1.11 mmol) was added to a reaction flask, which was dissolved in 10 ml methanol, and a catalytic amount of Pd/C was added. The reaction was filled with hydrogen gas, stirred and reacted at room temperature for 5-7 hours. TLC was used to monitor the completion of the reaction. After completion, the catalyst was removed by filtration, and the filtrate was concentrated to afford crude product of compound 36, which was directly used in the next reaction without purification. LC-MS (APCI): m/z=361.4 (M+1).sup.+.

(104) Step 6 Synthesis of Compound T-6

(105) Compound 36 (399 mg, 1.11 mmol) was added to a reaction flask, which was dissolved in 25 ml anhydrous DMF, and FDPP (511.8 mg, 1.33 mmol) and DIPEA (717.3 mg, 5.55 mmol) were added. The reaction was stirred at room temperature overnight under nitrogen protection. TLC was used to monitor the completion of the reaction. After completion, the reaction was diluted with an excess amount of water, and extracted with ethyl acetate for 3-4 times. The organic phases were combined, washed with saturated brine, concentrated and then purified by silica gel column chromatography to afford 221 mg of the title product. Yield: 58%. LC-MS (APCI): m/z=343.5 (M+1).sup.+. LC-MS (APCI): m/z=342.5 (M+1).sup.+. .sup.1H NMR (400 MHz. DMSO-d.sub.6) δ 10.13 (s, 1H), 8.59 (s, 1H), 8.10 (d, J=2.3 Hz, 1H), 7.35 (t, J=4.4 Hz, 1H), 6.98 (m, 2H), 6.52 (d, J=2.3 Hz, 1H), 4.25 (t, J=3.3 Hz, 2H), 4.12 (m, 1H), 2.68 (t, J=3.3 Hz, 2H), 1.23 (d, J=5.5 Hz, 3H).

Example 7: Preparation of 9-fluoro-13-oxa-2,17,20,21,24-pentaazapentacyclo[16.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.21,25.]pentacosane-1(24),7,9,11,18(25),19,22-heptaene-16-one (Compound 9a), (6R)-9-fluoro-13-oxa-2,7,20,21,24-pentaazapentacyclo[16.5.2.0.SUP.26..0.SUP.7,12..0.SUP.21,25.]pentacosane-1(24),7,9,11,18(25),19,22-heptaene-16-one (Compound L-1-A), and (6S)-9-fluoro-13-oxa-2,17,20,21,24-pentaazapentacyclo[16.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.21,25.]pentacosane-1(24),7,9,11,18(25),19,22-heptaene-16-one (Compound L1-1-B)

(106) ##STR00037##

(107) The following route was used for the synthesis:

(108) ##STR00038##

(109) Step 1 Synthesis of Compound 1a

(110) 2-bromo-4-fluoro-1-methoxybenzene (18.95 g, 93 mmol) was dissolved in anhydrous THF (100 mL), and isopropyl magnesium chloride solution (43.4 mL, 86.8 mmol) was slowly added dropwise at −40° C. After the addition, the mixture was naturally warmed to 0° C. and stirred for 1 h, and a solution of N-tert-butoxycarbonyl-2-pyrrolidone (11.46 g, 62.0 mmol) in anhydrous tetrahydrofuran (30 mL) was then slowly added dropwise at −40° C. After the addition, the mixture was stirred at room temperature for 30 min. The reaction solution was poured into 100 mL of saturated ammonium chloride solution and stirred for 10 min, then the reaction solution was separated by standing. The aqueous phase was extracted three times with 40 mL ethyl acetate. The organic phases were combined and washed with saturated brine, and dried over anhydrous sodium sulfate. The residue was filtered and concentrated, purified by column chromatography to afford 17.68 g of compound 1a as light yellow liquid. Yield: 61.1%. LC-MS (APCI): m/z=312.1 (M+1).sup.+.

(111) Step 2 Synthesis of Compound 2a

(112) Compound 1a (1.2 g, 3.85 mmol) was dissolved in toluene (10 mL), to which 0.7 mL concentrated hydrochloric acid was added. The reaction was heated to 65° C., stirred and reacted overnight. The reaction was cooled to room temperature, pH thereof was adjusted to 14 with 2 M sodium hydroxide, and stirred for 1 h. TLC was used to monitor the completion of the reaction. The organic phase was separated, and the aqueous phase was extracted 3 times with ethyl acetate. The organic phases were combined, washed with saturated brine, concentrated, and purified by column chromatography to afford 557 mg of compound 2a as yellow liquid. Yield: 75%. LC-MS (APCI): m/z=194.3 (M+1).sup.+.

(113) Step 3 Synthesis of Compound 3a

(114) Compound 2a (557 mg, 2.89 mmol) was dissolved in anhydrous methanol (10 mL), Pd/C (50 mg) was added, and the reaction was hydrogenated at room temperature overnight. The reaction was filtered, the filter residue was washed with 20 mL ethyl acetate, and the filtrate was concentrated to afford 556 mg of compound 3a as colorless oily liquid which was directly used in the next step. Yield: 98.5%. LC-MS (APCI): m/z=196.3 (M+1).sup.+.

(115) Step 4 Synthesis of Compound 4a

(116) Compound 3a (722 mg, 3.7 mmol) and 5-chloro-3-nitropyrazolo[1,5-a]pyrimidine (733 mg, 3.7 mmol) were dissolved in anhydrous ethanol (10 mL), DIPEA (N,N-diisopropylethylamine, 1.91 g, 14.8 mmol) was added at room temperature, and heated to reflux for 30 min. The reaction solution was concentrated, purified by column chromatography (PE/EA, 30%-50%) to afford 1.15 g of compound 4a as light yellow solid powder. Yield: 81%. LC-MS (APCI): m/z=385.5 (M+1).sup.+.

(117) Step 5 Synthesis of Compound 5a

(118) Compound 4a (1.57 g, 4.1 mmol) was dissolved in 10 ml anhydrous dichloromethane, 1 M boron tribromide solution (20.5 ml, 20.5 mmol) was added dropwise at 0° C. After the addition, the reaction was warmed to room temperature and reacted for 1-2 hours. TLC was used to monitor the completion of the reaction. After completion, a small amount of water was added dropwise to quench the reaction under ice bath, the organic phase was separated, and the aqueous phase was extracted with dichloromethane for 3-4 times. The organic phases were combined, washed with saturated brine, concentrated and then purified by silica gel column chromatography to afford 880 mg of the title product. Yield: 58%. LC-MS (APCI): m/z=371.6 (M+1).sup.+.

(119) Step 6 Synthesis of Compound 6a

(120) Compound 5a (116 mg, 0.315 mmol), ethyl 3-hydroxypropionate (55.8 mg, 0.473 mmol) and triphenylphosphine (124.1 mg, 0.473 mmol) were added to a reaction flask, to which 5 ml anhydrous tetrahydrofuran was added under nitrogen protection, and DIAD (diisopropyl azodicarboxylate, 95.6 mg, 0.473 mmol) was added dropwise at 0° C. After the addition, the reaction was warmed to room temperature and stirred to react for 15 hours. The reaction was concentrated to remove the solvent, and purified by silica gel column chromatography to afford 53 mg of the product. Yield: 38%. LC-MS (APCI): m/z=444.1 (M+1).sup.+.

(121) Step 7 Synthesis of Compound 7a

(122) Compound 6a (204 mg, 0.46 mmol) was added to a reaction flask, which was dissolved in 8 ml methanol, and a solution of lithium hydroxide monohydrate (96.6 mg, 2.3 mmol) in 4 ml water was added. The reaction was heated to 50° C. and reacted for 4-5 hours. TLC was used to monitor the completion of the reaction. The reaction was cooled to 0° C., pH thereof was adjusted to weak acidic with 1N dilute hydrochloric acid, and the reaction was extracted with ethyl acetate for 3-4 times. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to afford 181 mg of the product. Yield: 95%. LC-MS (APCI): m/z=416.5 (M+1).sup.+.

(123) Step 8 Synthesis of Compound 8a

(124) Compound 7a (186 mg, 0.45 mmol) was added to a reaction flask, which was dissolved in 5 ml methanol and a catalytic amount of Pd/C was added. The reaction was filled with hydrogen gas, stirred and reacted at room temperature for 5-7 hours. TLC was used to monitor the completion of the reaction. After completion, the catalyst was removed by filtration, and the filtrate was concentrated to afford crude product of compound 8a, which was directly used in the next reaction without purification. LC-MS (APCI): m/z=386.4 (M+1).sup.+.

(125) Step 9 Synthesis of Compound 9a

(126) The product obtained in the previous step was dissolved in 20 ml of anhydrous DMF, FDPP (pentafluorophenyl diphenylphosphinate, 240 mg, 0.62 mmol) and DIPEA (336 mg, 2.6 mmol) were added at room temperature, and the reaction was stirred and reacted overnight under nitrogen protection. TLC was used to monitor the completion of the reaction. After completion, the reaction was diluted by water, and extracted with ethyl acetate for 3-4 times. The organic phases were combined, washed with saturated brine, concentrated and then purified by silica gel column chromatography to afford 71 mg of compound 9a as off-white solid. Yield: 43%. LC-MS (APCI): m/z=368.3 (M+1).sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 10.11 (s, 1H), 8.87 (s, 1H), 8.04 (d, J=2.3 Hz, 1H), 7.52 (s, 1H), 7.44 (d, J=3.3 Hz, 1H), 7.28 (d, J=3.3 Hz, 1H), 6.74 (d, J=2.3 Hz, 1H), 4.38 (t, J=4.4 Hz, 2H), 4.12 (m 1H), 3.61 (dd, J=17.0, 9.3 Hz, 2H), 2.36 (t, J=4.4 Hz, 2H), 2.01-1.65 (m, 2H), 1.22 (d, J=4.5 Hz, 2H).

(127) Step 10 Synthesis of Compounds L-1-A and L-1-B

(128) Preparative chiral column: CHIRALPAK IC (brand name), 4.6 mm×250 mm (internal diameter×length), 5 μm (particle size of the filler)

(129) Column temperature: 30° C.

(130) Flow rate: 1.0 mL/min

(131) UV detection wavelength: 254 nm

(132) Mobile phase: methyl tert-butyl ether:methanol=70:30

(133) Under the above preparative chiral column and chiral resolution conditions, racemic compound 9a was separated to afford target compound L-1-A (retention time: 20.33 min, relative amount: 43.4%) and L-1-B (retention time: 6.31 min, relative amount: 44.0%).

Example 8: Preparation of (14S)-9-fluoro-4-methyl-3-oxa-2,17,20,21,24-pentaazapentacyclo[16.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.21,25.]pentacosane-1(24),7,9,11,18(25),19,22-heptaene-16-one (Compound 13a), (6R,14S)-9-fluoro-14-methyl-13-oxa-2,17,20,21,24-pentaazapentacyclo[16.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.21,25.]pentacosane-1(24),7,9,11,18(25),19,22-heptaene-16-one (Compound L-2-A), and (6S,14S)-9-fluoro-14-methyl-13-oxa-2,17,20,21,24-pentaazapentacyclo[16.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.21,25.]pentacosane-1(24),7,9,11,18(25),19,22-heptaene-16-one (Compound L-2-B)

(134) ##STR00039##

(135) The following route was used for the synthesis:

(136) ##STR00040##

(137) Step Synthesis of Compound 10a

(138) Compound 5a (216 mg, 0.63 mmol), ethyl (R)-3-hydroxybutyrate (125 mg, 0.946 mmol) and triphenylphosphine (248.2 mg, 0.946 mmol) was added to a reaction flask, to which 10 ml anhydrous tetrahydrofuran was added under nitrogen protection, and DIAD (191.2 mg 0.946 mmol) was added dropwise at 0° C. After the addition, the reaction was warmed to room temperature and stirred for 15 hours. The reaction was concentrated to remove the solvent, purified by silica gel column chromatography to afford 77.7 mg of the product. Yield 27%. LC-MS (APCI): m/z=458.1 (M+1).sup.+.

(139) Step 2 Synthesis of Compound 11a

(140) Compound 10a (210 mg, 0.46 mmol) was added to a reaction flask, which was dissolved in 8 ml methanol, and a solution of lithium hydroxide monohydrate (96.6 mg, 2.3 mmol) in 4 ml water was added. The reaction was heated to 50° C. and reacted for 4-5 hours. TLC was used to monitor the completion of the reaction. The reaction was cooled to 0° C., pH thereof was adjusted to weak acidic with 1N dilute hydrochloric acid, and the reaction was extracted with ethyl acetate for 3-4 times. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to afford 193 mg of the product. Yield: 98%. LC-MS (APCI): m/z=430.5 (M+1).sup.+.

(141) Step 3 Synthesis of Compound 12a

(142) Compound 1a (223 mg, 0.52 mmol) was added to a reaction flask, which was dissolved in 8 ml methanol and a catalytic amount of Pd/C was added. The reaction was filled with hydrogen gas, stirred and reacted at room temperature for 5-7 hours, TLC was used to monitor the completion of the reaction. After completion, the catalyst was removed by filtration, and the filtrate was concentrated to afford crude product of compound 12a, which was directly used in the next reaction without purification. LC-MS (APCI): m/z=400.6 (M+1).sup.+.

(143) Step 4 Synthesis of Compound 13a

(144) Compound 12a (180 mg, 0.45 mmol) was added to a reaction flask, which was dissolved in 15 ml anhydrous DMF, and FDPP (207.5 mg, 0.54 mmol) and DIPEA (290.8 mg, 2.25 mmol) was added. The reaction was stirred at room temperature overnight under nitrogen protection. TLC was used to monitor the completion of the reaction. After completion, the reaction was diluted with an excess amount of water, and extracted with ethyl acetate for 3-4 times. The organic phases were combined, washed with saturated brine, concentrated and then purified by silica gel column chromatography to afford 103 mg of the title product. Yield: 60%. LC-MS (APCI): m/z=382.3 (M+1).sup.+. .sup.1H NMR (400 MHz. DMSO-d.sub.6) δ 10.08 (s, 1H), 8.83 (s, 1H), 8.04 (d, J=2.3 Hz, 1H), 7.52 (s, 1H), 7.42 (d, J=3.3 Hz, 1H), 7.28 (d, J=3.3 Hz, 1H), 6.74 (d, J=2.3 Hz, 1H), 4.31 (m, 1H), 4.12 (m, 1H), 3.55 (dd, J=17.0, 9.3 Hz, 2H), 2.32 (t, J=4.4 Hz, 2H), 1.95-1.61 (m, 2H), 1.22 (d, J=4.5 Hz, 2H), 1.15 (d, J=4.4 Hz, 3H).

(145) Step 5 Synthesis of Compounds L-2-A and L-2-B

(146) Preparative chiral column: CHIRALPAK IC (brand name), 4.6 mm×250 mm (internal diameter×length), 5 μm (particle size of the filler)

(147) Column temperature: 30° C.

(148) Flow rate: 1.0 mL/min

(149) UV detection wavelength: 254 nm

(150) Mobile phase: methyl tert-butyl ether:methanol=70:30

(151) Under the above preparative chiral column and chiral resolution conditions, racemic compound 13a was separated to afford target compound L-2-A (retention time: 27.06 min, relative amount: 27.7%) and L-2-B (retention time: 7.02 min, relative amount: 67.8%).

Example 9: Preparation of 9-fluoro-15,15-dimethyl-13-oxa-2,17,20,21,24-pentaazapentacyclo[16.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.21,25.]pentacosane-1(24),7,9,11,18(25),19,22-heptaene-16-one (Compound 18a) (6R)-9-fluoro-15,15-dimethyl-13-oxa-2,17,20,21,24-pentaazapentacyclo[16.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.21,25.]pentacosane-1(24),7,9,11,18(25),19,22-heptaene-16-one (Compound L-3-A), and (6S)-9-fluoro-15,15-dimethyl-13-oxa-2,17,20,21,24-pentaazapentacyclo[16.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.21,25.]pentacosane-1(24),7,9,11,18(25),19,22-heptaene-16-one (Compound L-3-B)

(152) ##STR00041##

(153) The following route was used for the synthesis:

(154) ##STR00042##

(155) Step 1 Synthesis of Compound 14a

(156) Ethyl isobutyrate (2.5 g, 21.5 mmol) was added to a reaction flask, and 40 ml anhydrous tetrahydrofuran was added under nitrogen protection. The mixture was cooled to −40° C., and LDA (11.8 ml, 23.6 mmol) was slowly added dropwise. After the addition, the reaction was gradually warmed to room temperature and stirred for half an hour. Then it was cooled to −40° C., and a solution of diiodomethane (5.76 g, 21.5 mmol) in 10 ml anhydrous tetrahydrofuran was slowly added dropwise. After the addition, the reaction was warmed to room temperature and reacted overnight. Water was added to quench the reaction, the organic phase was separated, and the aqueous phase was extracted with ethyl acetate for 3-4 times. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to afford 4.62 g of the product. Yield: 84%.

(157) Step 2 Synthesis of Compound 15a

(158) Compound 5a (326 mg, 0.95 mmol), compound 14a (291 mg, 1.14 mmol) and potassium carbonate (525.2 mg, 3.8 mmol) were added to a reaction flask, and DMF was added. The reaction was heated to 80° C. stirred and reacted overnight. TLC was used to monitor the completion of the reaction. After completion, the reaction was cooled to room temperature, added with an excess amount of water, and extracted with ethyl acetate for 3-4 times. The organic phases were combined, washed with saturated brine, concentrated and then purified by silica gel column chromatography to afford 300 mg of the title product. Yield: 67%, LC-MS (APCI): m/z=472.1 (M+1).sup.+.

(159) Step 3 Synthesis of Compound 16a

(160) Compound 15a (339 mg, 0.72 mmol) was added to a reaction flask, which was dissolved in 10 ml methanol, and a solution of lithium hydroxide monohydrate (151.5 mg, 3.5 mmol) in 3 ml water was added. The reaction was heated to 50° C. and reacted for 4-5 hours. TLC was used to monitor the completion of the reaction. The reaction was cooled to 0° C. pH thereof was adjusted to weak acidic with 1N dilute hydrochloric acid, and the reaction was extracted with ethyl acetate for 3-4 times. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to afford 296 mg of the product. Yield: 93%. LC-MS (APCI): m/z=444.3 (M+1).sup.+.

(161) Step 4 Synthesis of Compound 17a

(162) Compound 16a (297 mg, 0.67 mmol) was added to a reaction flask, which was dissolved in 10 ml methanol, and a catalytic amount of Pd/C was added. The reaction was filled with hydrogen gas, stirred and reacted at room temperature for 5-7 hours. TLC was used to monitor the completion of the reaction. After completion, the catalyst was removed by filtration, and the filtrate was concentrated to afford crude product of compound 17a, which was directly used in the next reaction without purification. LC-MS (APCI): m/z=414.9 (M+1).sup.+.

(163) Step 5 Synthesis of Compound 18a

(164) Compound 17a (277 mg, 0.67 mmol) was added to a reaction flask, which was dissolved in 20 ml anhydrous DMF, and FDPP (309 mg, 0.80 mmol) and DIPEA (433 mg, 3.35 mmol) were added. The reaction was stirred at room temperature overnight under nitrogen protection. TLC was used to monitor the completion of the reaction. After completion, the reaction was diluted with an excess amount of water, and extracted with ethyl acetate for 3-4 times. The organic phases were combined, washed with saturated brine, concentrated and then purified by silica gel column chromatography to afford 111 mg of the title product. Yield: 42%. LC-MS (APCI): m/z=396.3 (M+1).sup.+. .sup.1H NMR (400 MHz, DMSO-d) δ 10.10 (s, 1H), 8.85 (s, 1H), 8.04 (d, J=2.3 Hz, 1H), 7.52 (s, 1H), 7.42 (d, J=3.3 Hz, 1H), 7.28 (d. J=3.3 Hz, 1H), 6.74 (d, J=2.3 Hz, 1H), 4.32 (t, J=4.4 Hz, 2H), 4.12 (m, 1H), 3.61 (dd, J=17.0, 9.3 Hz, 2H), 2.01-1.65 (m, 2H), 1.22 (d, J=4.5 Hz, 2H), 1.05 (s, 6H).

(165) Step 6 Synthesis of Compounds L-3-A and L-3-B

(166) Preparative chiral column: CHIRALPAK IC (brand name), 4.6 mm×250 mm (internal diameter×length), 5 μm (particle size of the filler)

(167) Column temperature: 30° C.

(168) Flow rate: 1.0 mL/min

(169) UV detection wavelength: 254 nm

(170) Mobile phase: methyl tert-butyl ether:methanol=70:30

(171) Under the above preparative chiral column and chiral resolution conditions, racemic compound 18a was separated to afford target compound L-3-A (retention time: 24.2 min, relative amount: 33.1%) and L-3-B (retention time: 8.34 min, relative amount: 61.0%).

Example 10: Preparation of 9-fluoro-15-methyl-13-oxa-2,17,20,21,24-pentaazapentacyclo[16.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.21,25.]pentacosane-1(24),7,9,11,18(25),19,22-heptaene-16-one (Compound 23a), (6R,1SR)-9-fluoro-15-methyl-13-oxa-2,17,20,21,24-pentaazapentacyclo[16.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.21,25.]pentacosane-1(24),7,9,11,18(25),19,22-heptaene-16-one (Compound L-4-A), (6R,1SS)-9-fluoro-15-methyl-13-oxa-2,17,20,21,24-pentaazapentacyclo[16.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.21,25.]pentacosane-1(24),7,9,11,18(25),19,22-heptaene-16-one (Compound L-4-B), (6S,15R)-9-fluoro-15-methyl-13-oxa-2,17,20,21,24-pentaazapentacyclo[16.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.21,25.]pentacosane-1(24),7,9,11,18(25),19,22-heptaene-16-one (Compound L-4-C), and (6S,15S)-9-fluoro-15-methyl-13-oxa-2,17,20,21,24-pentaazapentacyclo[16.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.21,25.]pentacosane-1(24),7,9,11,18(25),19,22-heptaene-16-one (Compound L-4-D)

(172) ##STR00043##

(173) The following route was used for the synthesis:

(174) ##STR00044##

(175) Step 1 Synthesis of Compound 19a

(176) 2-methyl-1,3-propanediol (2.0 g, 22.2 mmol) was dissolved in dichloromethane (30 ml), triethylamine (3.37 g, 33.3 mmol) was added under ice bath, and p-toluenesulfonyl chloride (TsCl, 4.23 g, 22.2 mmol) was added in batches while maintaining the temperature no higher than 5° C. After the addition, the reaction was stirred and reacted at the low temperature for 2-3 hours. TLC was used to monitor the completion of the reaction. After completion, 20 ml water was added, and the organic phase was separated, washed with saturated brine, concentrated and then purified by column chromatography to afford 3.52 g of compound 19a as colorless oily liquid. Yield: 65%. LC-MS (APCI): m/z=245.6 (M+1).sup.+.

(177) Step 2 Synthesis of Compound 20a

(178) Compound 5a (800 mg, 2.33 mmol) was dissolved in DMF (20 ml), and potassium carbonate (805 mg, 5.82 mmol) and compound 19a (854 mg, 3.49 mmol) were added. The reaction was heated to 80°, and stirred and reacted for 4-6 hours. TLC was used to monitor the completion of the reaction. After completion, the reaction was cooled to room temperature, added with an excess amount of water, and extracted with ethyl acetate for 3-4 times. The organic phases were combined, washed with saturated brine, concentrated and then purified by column chromatography to afford 691 mg of the light yellow solid. Yield: 71.4%. LC-MS (APCI): m/z=416.1 (M+1).sup.+.

(179) Step 3 Synthesis of Compound 21a

(180) Compound 20a (606 mg, 1.46 mmol) was dissolved in dichloromethane (20 ml), Dess-Martin oxidant (928 mg, 2.19 mmol) was added in batches under ice bath, and the reaction was gradually warmed to room temperature and stirred for 1 hour. TLC was used to monitor the completion of the reaction. After completion, the reaction was diluted with dichloromethane, washed 2-3 times with saturated sodium bicarbonate solution, and the organic phase was concentrated and directly used in the next reaction.

(181) The intermediate obtained in the previous step was dissolved in acetonitrile (20 ml), sodium dihydrogen phosphate (876 mg, 7.3 mmol) was added, and a solution of sodium chlorite (264 mg, 2.92 mmol) in 5 ml water was added under ice bath, the reaction was gradually warmed to room temperature and stirred for 1 hour. TLC was used to monitor the completion of the reaction. After completion, the reaction was added with an excess amount of water, and extracted with ethyl acetate for 3-4 times. The organic phases were combined, washed with saturated brine, concentrated and then purified by column chromatography to afford 552 mg of compound 21a. Yield: 88%. LC-MS (APCI): m/z=430.4 (M+1).sup.+.

(182) Step 4 Synthesis of Compound 22a

(183) Compound 21a (540 mg, 1.26 mmol) was dissolved in methanol (15 ml), a catalytic amount of Pd/C was added, and the reaction was put under a balloon filled with hydrogen gas. The reaction was stirred and reacted at room temperature overnight. TLC was used to monitor the completion of the reaction. After completion, the catalyst was removed by filtration, and the filtrate was concentrated to afford 478 mg crude product of compound 22a, which was directly used in the next reaction. Yield: 95%. LC-MS (APCI): m/z=400.8 (M+1).sup.+.

(184) Step 5 Synthesis of Compound 23a

(185) Compound 22a (447 mg, 1.12 mmol) was dissolved in anhydrous DMF (20 ml), and DIPEA (580 mg, 4.48 mmol) and FDPP (516.4 mg, 1.34 mmol) were added. The reaction was stirred at room temperature overnight under nitrogen protection. TLC was used to monitor the completion of the reaction. After completion, the reaction was diluted with an excess amount of water, and extracted with ethyl acetate for 3-4 times. The organic phases were combined, washed with saturated brine, concentrated and then purified by column chromatography to afford 184 mg of compound 23a as light yellow solid. Yield: 43%. LC-MS (APCI): m/z=382.3 (M+1).sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.58 (s, 1H), 8.54 (d, J=7.6 Hz, 1H), 7.76 (s, 1H), 7.09 (d, J=7.2 Hz, 1H), 7.01-6.87 (m, 2H), 6.44 (d, J=7.6 Hz, 1H), 5.54 (s, 1H), 4.35-4.25 (m, 1H), 4.18 (t, J=10.6 Hz, 1H), 3.90 (d, J=9.2 Hz, 1H), 3.61 (s, 1H), 2.96 (s, 1H), 2.31 (s, 2H), 1.97 (d, J=7.0 Hz, 1H), 1.83 (d, J=5.0 Hz, 1H), 1.23 (d, J=7.1 Hz, 3H).

(186) Step 6 Synthesis of Compounds L-4-A, L-4-B. L-4-C and L-4-D

(187) Preparative chiral column: CHIRALPAK IC (brand name), 4.6 mm×250 mm (internal diameter×length), 5 μm (particle size of the filler)

(188) Column temperature: 30° C.

(189) Flow rate: 1.0 mL/min

(190) UV detection wavelength: 254 nm

(191) Mobile phase: methyl tert-butyl ether:methanol=70:30

(192) Under the above preparative chiral column and chiral resolution conditions, racemic compound 23a was separated to afford target compound L.sub.4-A (retention time: 6.28 min, relative amount: 22.3%), L-4-B (retention time: 18.46 min, relative amount: 22.4%), L-4-C (retention time: 30.45 min, relative amount: 22.2%) and L-4-D (retention time: 37.26 min, relative amount: 22.4%).

Example 11: Preparation of 9-fluoro-2,17,20,21,24-pentaazapentacyclo[16.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.21,25.]pentacosane-1(24),7,9,11,18(25),19,22-heptaene-16-one (Compound 28a), (6R)-9-fluoro-2,17,20,21,24-pentaazapentacyclo[16.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.21,25.]pentacosane-1(24),7,9,11,18(25),19,22-heptaene-16-one (Compound L-5-A), and (6S)-9-fluoro-2,17,20,21,24-pentaazapentacyclo[16.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.21,25.]pentacosane-1(24),7,9,11,18(25),19,22-heptaene-16-one (Compound L-5-B)

(193) ##STR00045##

(194) The following route was used for the synthesis:

(195) ##STR00046##

(196) Step 1 Synthesis of Compound 24a

(197) Compound 5a (1.0 g, 2.91 mmol) was dissolved in dichloromethane (30 ml), triethylamine (589.5 mg, 5.82 mmol) was added, and N,N-bis(trifluoromethylsulfonyl)aniline (1.25 g, 3.49 mmol). The reaction was stirred at room temperature overnight. TLC was used to monitor the completion of the reaction. After completion, the reaction was concentrated to remove the solvent, purified by silica gel column chromatography to afford 1.02 g of compound 24a as light yellow solid. Yield: 74%. LC-MS (APCI): m/z=476.4 (M+1).sup.+.

(198) Step 2 Synthesis of Compound 25a

(199) Compound 24a (1.42 g, 3 mmol) and 3-butyn-1-ol (315 mg, 4.5 mmol) were dissolved in dioxane (30 ml), bis(triphenylphosphine)palladium dichloride (21 mg, 0.03 mmol) and copper iodide (57 mg, 0.3 mmol) were added under nitrogen protection. The reaction was heated to 100° C. and stirred for 5 hours. TLC was used to monitor the completion of the reaction. After completion, the reaction was cooled to room temperature, diluted with water, and extracted with ethyl acetate for 3-4 times. The organic phases were combined, washed with saturated brine, concentrated and then purified by column chromatography to afford 854 mg of compound 25a as light yellow solid. Yield: 72%. LC-MS (APCI): m/z=396.8 (M+1).sup.+.

(200) Step 3 Synthesis of Compound 26a

(201) Compound 25a (577 mg, 1.46 mmol) was dissolved in dichloromethane (20 ml), Dess-Martin oxidant (928 mg, 2.19 mmol) was added in batches under ice bath, and the reaction was gradually warmed to room temperature and stirred for 1 hour. TLC was used to monitor the completion of the reaction. After completion, the reaction was diluted with dichloromethane, the resultant solution was washed 2-3 times with saturated sodium bicarbonate solution. After concentration, the organic phase was directly used in the next reaction.

(202) The intermediate obtained in the previous step was dissolved in acetonitrile (20 ml), sodium dihydrogen phosphate (876 mg, 7.3 mmol) was added, and a solution of sodium chlorite (264 mg, 2.92 mmol) in 5 ml water was added under ice bath, and the reaction was gradually warmed to room temperature and stirred for 1 hour. TLC was used to monitor the completion of the reaction. After completion, the reaction was added with an excess amount of water, and extracted with ethyl acetate for 3-4 times. The organic phases were combined, washed with saturated brine, concentrated and then purified by column chromatography to afford 466 mg of compound 26a. Yield: 78%. LC-MS (APCI): m/z=410.2 (M+1).sup.+.

(203) Step 4 Synthesis of Compound 27a

(204) Compound 26a (515 mg, 1.26 mmol) was dissolved in methanol (15 ml), a catalytic amount of Pd/C was added, and the reaction was put under a balloon filled with hydrogen gas. The reaction was stirred and reacted at room temperature overnight. TLC was used to monitor the completion of the reaction. After completion, the catalyst was removed by filtration, and the filtrate was concentrated to afford 468.6 mg crude product of compound 27a, which was directly used in the next reaction. Yield: 97%. LC-MS (APCI): m/z=384.6 (M+1).sup.+.

(205) Step 5 Synthesis of Compound 28a

(206) Compound 27a (429 mg, 1.12 mmol) was dissolved in anhydrous DMF (20 ml), and DIPEA (580 mg, 4.48 mmol) and FDPP (516.4 mg, 1.34 mmol) were added. The reaction was stirred at room temperature overnight under nitrogen protection. TLC was used to monitor the completion of the reaction. After completion, the reaction was diluted with an excess amount of water, and extracted with ethyl acetate for 3-4 times. The organic phases were combined, washed with saturated brine, concentrated and then purified by column chromatography to afford 160 mg of compound 28a as light yellow solid. Yield: 39%. LC-MS (APCI): m/z=366.1 (M+1).sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.76 (d, J=7.7 Hz, 1H), 8.42 (d, J=2.4 Hz, 1H), 7.62 (d, J=6.6 Hz, 1H), 7.59 (d, J=6.4 Hz, 1H), 7.57 (dd, J=9.9, 2.4 Hz, 1H), 7.22 (d, J=6.4 Hz, 1H), 6.60 (d, J=7.7 Hz, 1H), 5.36 (t, J=6.5 Hz, 1H), 4.05 (dd, J=16.9, 7.0 Hz, 2H), 3.75 (dd, J=14.0, 8.6 Hz, 1H), 3.67-3.51 (m, 1H), 2.95 (d, J=16.9 Hz, 1H), 2.55 (dd, J=14.0, 10.9 Hz, 1H), 2.42 (dd, J=13.4, 6.5 Hz, 2H), 2.23 (dd, J=12.0, 6.2 Hz, 2H), 2.12-1.96 (m, 1H), 1.79-1.66 (m 1H).

(207) Step 6 Synthesis of Compounds L-5-A and L-5-B

(208) Preparative chiral column: CHIRALPAK IC (brand name), 4.6 mm×250 mm (internal diameter×length), 5 μm (particle size of the filler)

(209) Column temperature: 30° C.

(210) Flow rate: 1.0 mL/min

(211) UV detection wavelength: 254 nm

(212) Mobile phase: methyl tert-butyl ether:methanol=70:30

(213) Under the above preparative chiral column and chiral resolution conditions, racemic compound 28a was separated to afford target compound L-5-A (retention time: 6.28 min, relative amount: 44.6%) and L-5-B (retention time: 18.46 min, relative amount: 44.8%).

Example 12: Preparation of 9-fluoro-15-methyl-2,17,20,21,24-pentaazapentacyclo[16.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.21,25.]pentacosane-1(24),7,9,11,18(25),19,22-heptaene-16-one (Compound 32a), (6R,15R)-9-fluoro-15-methyl-2,17,20,21,24-pentaazapentacyclo[16.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.21,25.]pentacosane-1(24),7,9,11,18(25),19,22-heptaene-16-one (Compound L-6-A), (6R,15S)-9-fluoro-15-methyl-2,17,20,21,24-pentaazapentacyclo[16.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.21,25.]pentacosane-1(24),7,9,11,18(25),19,22-heptaene-16-one (Compound L-6-B), (6S,15R)-9-fluoro-15-methyl-2,17,20,21,24-pentaazapentacyclo[16.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.21,25.]pentacosane-1(24),7,9,11,18(25),19,22-heptaene-16-one (Compound L-6-C), and (6S,15S)-9-fluoro-15-methyl-2,17,20,21,24-pentaazapentacyclo[16.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.21,25.]pentacosane-1(24),7,9,11,18(25),19,22-heptaene-16-one (Compound L-6-D)

(214) ##STR00047##

(215) The following route was used for the synthesis:

(216) ##STR00048##

(217) Step 1 Synthesis of Compound 29a

(218) Compound 24a (1.42 g, 3 mmol) and 2-methyl-3-butyn-1-ol (364 mg, 4.5 mmol) were dissolved in dioxane (30 ml), bis(triphenylphosphine)palladium dichloride (21 mg, 0.03 mmol) and copper iodide (57 mg, 0.3 mmol) were added under nitrogen protection. The reaction was heated to 100° C. and stirred for 5 hours. TLC was used to monitor the completion of the reaction. After completion, the reaction was cooled to room temperature, diluted with water, and extracted with ethyl acetate for 3-4 times. The organic phases were combined, washed with saturated brine, concentrated and then purified by column chromatography to afford 773 mg of compound 29a as light yellow solid. Yield: 63%. LC-MS (APCI): m/z=410.1 (M+1).sup.+.

(219) Step 2 Synthesis of Compound 30a

(220) Compound 29a (597 mg, 1.46 mmol) was dissolved in dichloromethane (20 ml), Dess-Martin oxidant (928 mg, 2.19 mmol) was added in batches under ice bath, and the reaction was gradually warmed to room temperature and stirred for 1 hour. TLC was used to monitor the completion of the reaction. After completion, the reaction was diluted with dichloromethane, and washed 2-3 times with saturated sodium bicarbonate solution. After concentration, the organic phase was directly used in the next reaction.

(221) The intermediate obtained in the previous step was dissolved in acetonitrile (20 ml), sodium dihydrogen phosphate (876 mg, 7.3 mmol) was added, and a solution of sodium chlorite (264 mg, 2.92 mmol) in 5 ml water was added under ice bath, and the reaction was gradually warmed to room temperature and stirred for 1 hour. TLC was used to monitor the completion of the reaction. After completion, the reaction was added with an excess amount of water, and extracted with ethyl acetate for 3-4 times. The organic phases were combined, washed with saturated brine, concentrated and then purified by column chromatography to afford 550 mg of compound 30a. Yield: 89%. LC-MS (APCI): m/z=424.2 (M+1).sup.+.

(222) Step 3 Synthesis of Compound 31a

(223) Compound 30a (533 mg, 1.26 mmol) was dissolved in methanol (15 ml), a catalytic amount of Pd/C was added, and the reaction was put under a balloon filled with hydrogen gas. The reaction was stirred and reacted at room temperature overnight. TLC was used to monitor the completion of the reaction. After completion, the catalyst was removed by filtration, and the filtrate was concentrated to afford 485.7 mg crude product of compound 31a, which was directly used in the next reaction. Yield: 97%. LC-MS (APCI): m/z=398.9 (M+1).sup.+.

(224) Step 4 Synthesis of Compound 32a

(225) Compound 31a (445 mg, 1.12 mmol) was dissolved in anhydrous DMF (20 ml), and DIPEA (580 mg, 4.48 mmol) and FDPP (516.4 mg, 1.34 mmol) were added. The reaction was stirred at room temperature overnight under nitrogen protection. TLC was used to monitor the completion of the reaction. After completion, the reaction was diluted with an excess amount of water, and extracted with ethyl acetate for 3-4 times. The organic phases were combined, washed with saturated brine, concentrated and then purified by column chromatography to afford 187 mg of compound 32a as light yellow solid. Yield: 44%. LC-MS (APCI): m/z=380.1 (M+1).sup.+. .sup.1H NMR (500 MHz, DMSO-d) δ 8.76 (d, J=7.7 Hz, 1H), 8.42 (d, J=2.4 Hz, 1H), 7.62 (d, J=6.6 Hz, 1H), 7.59 (d, J=6.4 Hz, 1H), 7.57 (dd, J=9.9, 2.4 Hz, 1H), 7.22 (d, J=6.4 Hz, 1H), 6.60 (d, J=7.7 Hz, 1H), 5.36 (t, J=6.5 Hz, 1H), 4.05 (dd, J=16.9, 7.0 Hz, 2H), 3.75 (dd, J=14.0, 8.6 Hz, 1H), 3.67-3.51 (m, 1H), 2.95 (d, J=16.9 Hz, 1H), 2.42 (dd, J=13.4, 6.5 Hz, 2H), 2.23 (dd, J=12.0.6.2 Hz, 2H),2.12-1.96 (m, 1H), 1.88 (d, J=6.9 Hz, 3H), 1.79-1.66 (m, 1H).

(226) Step 5 Synthesis of Compounds L-6-A, L-6-B, L-6-C and L.sub.4-D

(227) Preparative chiral column: CHIRALPAK IC (brand name), 4.6 mm×250 mm (internal diameter×length), 5 μm (particle size of the filler)

(228) Column temperature: 30° C.

(229) Flow rate: 1.0 mL/min

(230) UV detection wavelength: 254 nm

(231) Mobile phase: methyl tert-butyl ether:methanol=70:30

(232) Under the above preparative chiral column and chiral resolution conditions, racemic compound 32a was separated to afford target compound L-6-A (retention time: 7.14 min, relative amount: 21.6%), L-6-B (retention time: 15.69 min, relative amount: 22.8%). L-6-C (retention time: 29.54 min, relative amount: 17.6%) and L-6-D (retention time: 34.26 min. relative amount: 16.9%).

Example 13: Preparation of 9-fluoro-15-methyl-2,11,17,20,21,24-hexaazapentacyclo[16.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.21,25.]pentacosane-1(24),7,9,11,18(25),19,22-heptaene-16-one (Compound 41a), (6R,15R)-9-fluoro-15-methyl-2,11,17,20,21,24-hexaazapentacyclo[16.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.21,25.]pentacosane-1(24),7,9,11,18(25),19,22-heptaene-16-one (Compound L-7-A), (6R,15S)-9-fluoro-15-methyl-2,11,17,20,21,24-hexaazapentacyclo[16.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.21,25.]pentacosane-1(24),7,9,11,18(25),19,22-heptaene-16-one (Compound L-7-B), (6S,15R)-9-fluoro-15-methyl-2,11,17,20,21,24-hexaazapentacyclo[16.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.21,25.]pentacosane-1(24),7,9,11,18(25),19,22-heptaene-16-one (Compound L-7-C), and (6S,15S)-9-fluoro-15-methyl-2,11,17,20,21,24-hexaazapentacyclo[16.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.21,25.]pentacosane-1(24),7,9,11,18(25),19,22-heptaene-16-one (Compound L-7-D)

(233) ##STR00049##

(234) The following route was used for the synthesis:

(235) ##STR00050##

(236) Step 1 Synthesis of Compound 33a

(237) 2-chloro-3-bromo-5-fluoropyridine (19.53 g, 93 mmol) was dissolved in anhydrous THF (100 mL), and isopropyl magnesium chloride solution (43.4 mL, 86.8 mmol) was slowly added dropwise at −40° C. After the addition, the mixture was naturally warmed to 0° C. and stirred for 1 h and a solution of N-tert-butoxycarbonyl-2-pyrrolidone (11.46 g, 62.0 mmol) in anhydrous tetrahydrofuran (30 mL) was then slowly added dropwise at −40° C. After the addition, the mixture was stirred at room temperature for 30 min. The reaction solution was poured into 100 mL of saturated ammonium chloride solution and stirred for 10 min, then the reaction solution was separated by standing. The aqueous phase was extracted three times with 40 mL ethyl acetate. The organic phases were combined and washed with saturated brine, dried over anhydrous sodium sulfate. The residue was filtered and concentrated, purified by column chromatography to afford 19.5 g of compound 33a as light yellow liquid. Yield: 66.3%. LC-MS (APCI): m/z=317.2 (M+1).sup.+.

(238) Step 2 Synthesis of Compound 34a

(239) Compound 33a (1.22 g, 3.85 mmol) was dissolved in dichloromethane (10 mL), 2 mL trifluoroacetic acid was added, and the mixture was stirred at room temperature for 1 h. TLC was used to monitor the completion of the reaction. The reaction solution was washed with saturated sodium bicarbonate aqueous solution, the organic phase was separated, and the aqueous phase was extracted 3 times with dichloromethane. The organic phases were combined, washed with saturated brine, concentrated to afford crude product of compound 34a, which was directly used in the next reaction. LC-MS (APCI): m/z=199.3 (M+1).sup.+.

(240) Step 3 Synthesis of Compound 35a

(241) Compound 34a (764 mg, 3.85 mmol) was dissolved in anhydrous methanol (10 mL), Pd/C (50 mg) was added, and hydrogenated at room temperature overnight. The reaction was filtered, the filter residue was washed with 20 mL ethyl acetate, and the filtrate was concentrated to afford 726 mg of compound 35a as colorless oily liquid which was directly used in the next step. Yield: 95%. LC-MS (APCI): m/z=201.3 (M+1).sup.+.

(242) Step 4 Synthesis of Compound 36a

(243) Compound 35a (742 mg, 3.7 mmol) and 5-chloro-3-nitropyrazolo[1,5-a]pyrimidine (733 mg, 3.7 mmol) were dissolved in absolute ethanol (10 mL), DIPEA (1.91 g, 14.8 mmol) was added at room temperature, and the reaction was heated to reflux for 30 min. The reaction solution was concentrated, purified by column chromatography (PE/EA, 30%-50%) to afford 1.09 g of compound 36a as light yellow solid powder. Yield: 81%. LC-MS (APCI): m/z=363.5 (M+1).sup.+.

(244) Step 5 Synthesis of Compound 37a

(245) Compound 36a (1.09 g, 3 mmol) and 4-((tert-butyldimethylsilyl)oxo)-3-methylbutyne (893 mg, 4.5 mmol) were dissolved in dioxane (30 ml), bis(triphenylphosphine)palladium dichloride (21 mg, 0.03 mmol) and copper iodide (57 mg, 0.3 mmol) were added under nitrogen protection. The reaction was heated to 100° C. and stirred for 5 hours. TLC was used to monitor the completion of the reaction. After completion, the reaction was cooled to room temperature, diluted with water, and extracted with ethyl acetate for 3-4 times. The organic phases were combined, washed with saturated brine, concentrated and then purified by column chromatography to afford 936 mg of compound 37a as light yellow solid. Yield: 59.5%. LC-MS (APCI): m/z=525.8 (M+1).sup.+.

(246) Step 6 Synthesis of Compound 38a

(247) Compound 37a (936 mg, 1.78 mmol) was dissolved in anhydrous tetrahydrofuran (20 ml), a solution of 1M tetrabutylammonium fluoride in tetrahydrofuran (3.6 ml, 3.6 mmol) was slowly added dropwise. After the addition, the reaction was stirred and reacted for half an hour. TLC was used to monitor the completion of the reaction. After completion, the reaction was concentrated to remove the solvent, purified by silica gel column chromatography to afford 599 mg of compound 38a. Yield: 82%. LC-MS (APCI): m/z=411.1 (M+1).sup.+.

(248) Step 7 Synthesis of Compound 39a

(249) Compound 38a (599 mg, 1.46 mmol) was dissolved in dichloromethane (20 ml), Dess-Martin oxidant (928 mg, 2.19 mmol) was added in batches under ice bath, the reaction was gradually warmed to room temperature and stirred for 1 hour. TLC was used to monitor the completion of the reaction. After completion, the reaction was diluted with dichloromethane, and washed 2-3 times with saturated sodium bicarbonate solution. After concentration, the organic phase was directly used in the next reaction.

(250) The intermediate obtained in the previous step was dissolved in acetonitrile (20 ml), sodium dihydrogen phosphate (876 mg, 7.3 mmol) was added, and a solution of sodium chlorite (264 mg, 2.92 mmol) in 5 ml water was added under ice bath, and the reaction was gradually warmed to room temperature and stirred for 1 hour. TLC was used to monitor the completion of the reaction. After completion, the reaction was added with an excess amount of water, and extracted with ethyl acetate for 3-4 times. The organic phases were combined, washed with saturated brine, concentrated and then purified by column chromatography to afford 533 mg of compound 39a. Yield: 86.1%. LC-MS (APCI): m/z=425.7 (M+1).sup.+.

(251) Step 8 Synthesis of Compound 40a

(252) Compound 39a (533 mg, 1.26 mmol) was dissolved in methanol (15 ml), a catalytic amount of Pd/C was added, and the reaction was put under a balloon filled with hydrogen gas. The reaction was stirred and reacted at room temperature overnight. TLC was used to monitor the completion of the reaction. After completion, the catalyst was removed by filtration, and the filtrate was concentrated to afford 446.7 mg crude product of compound 40a, which was directly used in the next reaction. Yield: 89%. LC-MS (APCI): m/z=399.7 (M+1).sup.+.

(253) Step 9 Synthesis of Compound 41a

(254) Compound 40a (446.7 mg, 1.12 mmol) was dissolved in anhydrous DMF (20 ml), and DIPEA (580 mg, 4.48 mmol) and FDPP (516.4 mg, 1.34 mmol) were added. The reaction was stirred at room temperature overnight under nitrogen protection. TLC was used to monitor the completion of the reaction. After completion, the reaction was diluted with an excess amount of water, and extracted with ethyl acetate for 3-4 times. The organic phases were combined, washed with saturated brine, concentrated and then purified by column chromatography to afford 200.2 mg of compound 41a as light yellow solid. Yield: 47%. LC-MS (APCI): m/z=381.4 (M+1).sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.70 (d, J=7.7 Hz, 1H), 8.39 (d, J=2.4 Hz, 1H), 8.01 (s, 1H), 7.66 (d. J=6.6 Hz, 1H), 7.57 (dd, J=9.9, 2.4 Hz, 1H), 6.60 (d, J=7.7 Hz, 1H), 5.36 (t, J=6.5 Hz, 1H), 4.03 (dd, J=16.9, 7.0 Hz, 2H), 3.75 (dd, J=14.0, 8.6 Hz, 1H), 3.67-3.51 (m, 1H), 2.91 (d, J=16.9 Hz, 1H), 2.54 (dd, J=14.0, 10.9 Hz, 1H), 2.44 (dd, J=13.4, 6.5 Hz, 1H), 2.23 (dd, J=12.0, 6.2 Hz, 2H), 2.12-1.96 (m, 1H), 1.79-1.66 (m, 1H), 1.13 (d, J=6.6 Hz, 3H).

(255) Step 10 Synthesis of Compound L-7-A, L-7-B, L-7-C and L-7-D

(256) Preparative chiral column: CHIRALPAK IC (brand name), 4.6 mm×250 mm (internal diameter×length), 5 μm (particle size of the filler)

(257) Column temperature: 30° C.

(258) Flow rate: 1.0 mL/min

(259) UV detection wavelength: 254 nm

(260) Mobile phase: methyl tert-butyl ether:methanol=70:30

(261) Under the above preparative chiral column and chiral resolution conditions, racemic compound 41a was separated to afford target compound L-7-A (retention time: 4.77 min, relative amount: 30.21%), L-7-B (retention time: 15.68 min, relative amount: 22.5%). L-7-C (retention time: 26.31 min, relative amount: 10.66%) and L-7-D (retention time: 29.67 min, relative amount: 23.4%).

Example 14: Preparation of 9-fluoro-2,11,17,20,21,24-hexaazapentacyclo[16.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.21,25.]pentacosane-1(24),7,9,11,18(25),19,22-heptaene-16-one (Compound 45a), (6R)-9-fluoro-2,11,17,20,21,24-hexaazapentacyclo[16.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.21,25.]pentacosane-1(24),7,9,11,18(25),19,22-heptaene-16-one (Compound L-8-A), and (6S′)-9-fluoro-2,11,17,20,21,24-hexaazapentacyclo[16.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.21,25.]pentacosane-1(24),7,9,11,18(25),19,22-heptaene-16-one (Compound L-8-B)

(262) ##STR00051##

(263) The following route was used for the synthesis:

(264) ##STR00052##

(265) Step 1 Synthesis of Compound 42a

(266) Compound 36a (1.09 g, 3 mmol) and 3-butyn-1-ol (315 mg, 4.5 mmol) were dissolved in dioxane (30 ml), bis(triphenylphosphine)palladium dichloride (21 mg, 0.03 mmol) and copper iodide (57 mg, 0.3 mmol) were added under nitrogen protection. The reaction was heated to 100° C. and stirred for 5 hours. TLC was used to monitor the completion of the reaction. After completion, the reaction was cooled to room temperature, diluted with water, and extracted with ethyl acetate for 3-4 times. The organic phases were combined, washed with saturated brine, concentrated and then purified by column chromatography to afford 845 mg of compound 42a as light yellow solid. Yield: 66%. LC-MS (APCI): m/z=397.1 (M+1).sup.+.

(267) Step 2 Synthesis of Compound 43a

(268) Compound 42a (578 mg, 1.46 mmol) was dissolved in dichloromethane (20 ml), Dess-Martin oxidant (928 mg, 2.19 mmol) was added in batches under ice bath, and the reaction was gradually warmed to room temperature and stirred for 1 hour. TLC was used to monitor the completion of the reaction. After completion, the reaction was diluted with dichloromethane, the resultant solution was washed 2-3 times with saturated sodium bicarbonate solution. After concentration, the organic phase was directly used in the next reaction.

(269) The intermediate obtained in the previous step was dissolved in acetonitrile (20 ml), sodium dihydrogen phosphate (876 mg, 7.3 mmol) was added, and a solution of sodium chlorite (264 mg, 2.92 mmol) in 5 ml water was added under ice bath, and the reaction was gradually warmed to room temperature and stirred for 1 hour. TLC was used to monitor the completion of the reaction. After completion, the reaction was added with an excess amount of water, and extracted with ethyl acetate for 3-4 times. The organic phases were combined, washed with saturated brine, concentrated and then purified by column chromatography to afford 485 mg of compound 43a. Yield: 81%. LC-MS (APCI): m/z=411.4 (M+1).sup.+.

(270) Step 3 Synthesis of Compound 44a

(271) Compound 43a (517 mg, 1.26 mmol) was dissolved in methanol (15 ml), a catalytic amount of Pd/C was added, and the reaction was put under a balloon filled with hydrogen gas. The reaction was stirred and reacted at room temperature overnight. TLC was used to monitor the completion of the reaction. After completion, the catalyst was removed by filtration, and the filtrate was concentrated to afford 450.4 mg crude product of compound 44a, which was directly used in the next reaction. Yield: 93%. LC-MS (APCI): m/z=385.7 (M+1).sup.+.

(272) Step 4 Synthesis of Compound 45a

(273) Compound 44a (430.5 mg, 1.12 mmol) was dissolved in anhydrous DMF (20 ml), and DIPEA (580 mg, 4.48 mmol) and FDPP (516.4 mg, 1.34 mmol) were added. The reaction was stirred at room temperature overnight under nitrogen protection. TLC was used to monitor the completion of the reaction. After completion, the reaction was diluted with an excess amount of water, and extracted with ethyl acetate for 3-4 times. The organic phases were combined, washed with saturated brine, concentrated and then purified by column chromatography to afford 232.7 mg of compound 45a as light yellow solid. Yield: 56.7%. LC-MS (APCI): m/z=367.6 (M+1).sup.+. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.76 (d. J=7.7 Hz, 1H), 8.42 (d, J=2.4 Hz, 1H), 8.01 (s, 1H), 7.62 (d. J=6.6 Hz, 1H), 7.57 (dd, J=9.9, 2.4 Hz, 1H), 6.60 (d, J=7.7 Hz, 1H), 5.36 (t, J=6.5 Hz, 1H), 4.05 (dd, J=16.9, 7.0 Hz, 2H), 3.75 (dd, J=14.0, 8.6 Hz, 1H), 3.67-3.51 (m, 1H), 2.91 (d, J=16.9 Hz, 1H), 2.54 (dd, J=14.0, 10.9 Hz, 1H), 2.44 (dd, J=13.4, 6.5 Hz, 2H), 2.23 (dd, J=12.0, 6.2 Hz, 2H), 2.12-1.96 (m 1H), 1.79-1.66 (m, 1H).

(274) Step 5 Synthesis of Compounds L-8-A and L-8-B

(275) Preparative chiral column: CHIRALPAK IC (brand name), 4.6 mm×250 mm (internal diameter×length), 5 μm (particle size of the filler)

(276) Column temperature: 30° C.

(277) Flow rate: 1.0 mL/min

(278) UV detection wavelength: 254 nm

(279) Mobile phase: methyl tert-butyl ether:methanol=70:30

(280) Under the above preparative chiral column and chiral resolution conditions, racemic compound 45a was separated to afford target compound L-8-A (retention time: 20.15 min, relative amount: 43.7%) and L-8-B (retention time: 8.25 min, relative amount: 44.0%).

Example 15: Preparation of (4R)-9-fluoro-4-hydroxy-2,11,17,20,21,24-hexaazapentacyclo[16.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.21,25.]pentacosane-1(24),7,9,11,18(25),19,22-heptaene-16-one (Compound 56a), (4R,6R)-9-fluoro-4-hydroxy-2,11,17,20,21,24-hexaazapentacyclo[16.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.21,25.]pentacosane-1(24),7,9,11,18(25),19,22-heptaene-16-one (Compound L-9-A), and (4R,6S)-9-fluoro-4-hydroxy-2,11,17,20,21,24-hexaazapentacyclo[16.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.21,25.]pentacosane-1(24),7,9,11,18(25),19,22-heptaene-16-one (Compound L-9-B)

(281) ##STR00053##

(282) The following route was used for the synthesis:

(283) ##STR00054##

(284) Step 1 Synthesis of Compound 46a

(285) (R)-4-hydroxypyrrolidin-2-one (5.0 g, 49.4 mmol) was dissolved in dichloromethane (60 ml), imidazole (4.04 g, 59.3 mmol) was added, and triisopropylchlorosilane (TISCl, 10.5 g, 54.3 mmol) was added under ice bath. After the addition, the reaction was stirred and reacted at room temperature for 1-2 hours. TLC was used to monitor the completion of the reaction. After completion, the reaction was diluted with dichloromethane, and washed 3 times with water. After concentration, the organic phase was purified by column chromatography to afford 10.3 g of compound 46a as colorless oily liquid. Yield: 81%. LC-MS (APCI): m/z=258.3 (M+1).sup.+.

(286) Step 2 Synthesis of Compound 47a

(287) Compound 46a (10.3 g, 40 mmol) was dissolved in dichloromethane (100 ml), triethylamine (6.07 g, 60 mmol) was added, and di-tert-butyl dicarbonate (10.5 g, 48 mmol) was added under ice bath. After the addition, the reaction was heated to room temperature and reacted for 3-4 hours. TLC was used to monitor the completion of the reaction. After completion, the reaction was concentrated to remove the solvent, purified by column chromatography to afford 12.73 g of compound 47a as colorless oily liquid. Yield: 89%. LC-MS (APCI): m/z=358.3 (M+1).sup.+.

(288) Step 3 Synthesis of Compound 48a

(289) 2-chloro-3-bromo-5-fluoropyridine (19.53 g, 93 mmol) was dissolved in anhydrous THF (100 mL), isopropyl magnesium chloride solution (43.4 mL, 86.8 mmol) was slowly added dropwise at −40° C., and after the addition, the mixture was naturally warmed to 0° C. and stirred for 1 h. Then a solution of compound 47a (22.1 g, 62.0 mmol) in anhydrous tetrahydrofuran (30 mL) was slowly added dropwise at −40° C., and the mixture was stirred at room temperature for 30 min after the addition. The reaction solution was poured into 100 mL of saturated ammonium chloride solution and stirred for 10 min, then the reaction solution was separated by standing. The aqueous phase was extracted three times with 40 mL ethyl acetate. The organic phases were combined and washed with saturated brine, dried over anhydrous sodium sulfate. The residue was filtered and concentrated, purified by column chromatography to afford 18.5 g of compound 48a as light yellow liquid. Yield: 61.1%. LC-MS (APCI): m/z=490.2 (M+1).sup.+.

(290) Step 4 Synthesis of Compound 49a

(291) Compound 48a (1.88 g, 3.85 mmol) was dissolved in dichloromethane (10 mL), 2 mL trifluoroacetic acid was added, and the mixture was stirred at room temperature for 1 h. TLC was used to monitor the completion of the reaction. The reaction solution was washed with saturated sodium bicarbonate aqueous solution, the organic phase was separated, and the aqueous phase was extracted 3 times with dichloromethane. The organic phases were combined, washed with saturated brine, concentrated to afford crude product of compound 49a, which was directly used in the next reaction. LC-MS (APCI): m/z=372.3 (M+1).sup.+.

(292) Step 5 Synthesis of Compound 50a

(293) Compound 49a (1.43 g, 3.85 mmol) was dissolved in anhydrous methanol (20 mL), Pd/C (100 mg) was added, and hydrogenated at room temperature overnight. The reaction was filtered, the filter residue was washed with 20 mL ethyl acetate, and the filtrate was concentrated to afford 1.36 g of compound 50a as colorless oily liquid which was directly used in the next step. Yield: 95%. LC-MS (APCI): m/z=374.3 (M+1).sup.+.

(294) Step 6 Synthesis of Compound 51a

(295) Compound 50a (1.38 g, 3.7 mmol) and 5-chloro-3-nitropyrazolo[1,5-a]pyrimidine (733 mg, 3.7 mmol) were dissolved in absolute ethanol (10 mL), DIPEA (1.91 g, 14.8 mmol) was added at room temperature, and the mixture was heated to reflux for 30 min. The reaction solution was concentrated, purified by column chromatography (PE/EA, 30%-50%) to afford 1.74 g of compound 51a as light yellow solid powder. Yield: 88%. LC-MS (APCI): m/z=535.5 (M+1).sup.+.

(296) Step 7 Synthesis of Compound 52a

(297) Compound 51a (1.6 g, 3 mmol) and 3-hydroxybutyn-1-ol (893 mg, 4.5 mmol) were dissolved in dioxane (30 ml), bis(triphenylphosphine)palladium dichloride (21 mg, 0.03 mmol) and copper iodide (57 mg, 0.3 mmol) were added under nitrogen protection. The reaction was heated to 100° C. and stirred for 5 hours. TLC was used to monitor the completion of the reaction. After completion, the reaction was cooled to room temperature, diluted with water, and extracted with ethyl acetate for 3-4 times. The organic phases were combined, washed with saturated brine, concentrated and then purified by column chromatography to afford 938 mg of compound 52a as light yellow solid. Yield: 55%. LC-MS (APCI): m/z=569.8 (M+1).sup.+.

(298) Step 8 Synthesis of Compound 53a

(299) Compound 52a (1.01 g, 1.78 mmol) was dissolved in anhydrous tetrahydrofuran (20 ml), a solution of 1M tetrabutylammonium fluoride in tetrahydrofuran (3.6 ml, 3.6 mmol) was slowly added dropwise. After the addition, the reaction was stirred at room temperature for half an hour. TLC was used to monitor the completion of the reaction. After completion, the reaction was concentrated to remove the solvent, purified by silica gel column chromatography to afford 565 mg of compound 53a. Yield: 77%. LC-MS (APCI): m/z=4131 (M+1).sup.+.

(300) Step 9 Synthesis of Compound 54a

(301) Compound 53a (601 mg, 1.46 mmol) was dissolved in dichloromethane (20 ml), Dess-Martin oxidant (928 mg, 2.19 mmol) was added in batches under ice bath, the reaction was gradually warmed to room temperature and stirred for 1 hour. TLC was used to monitor the completion of the reaction. After completion, the reaction was diluted with dichloromethane, and washed 2-3 times with saturated sodium bicarbonate solution. After 0.5 concentration, the organic phase was directly used in the next reaction.

(302) The intermediate obtained in the previous step was dissolved in acetonitrile (20 ml), sodium dihydrogen phosphate (876 mg, 7.3 mmol) was added, and a solution of sodium chlorite (264 mg, 2.92 mmol) in 5 ml water was added under ice bath, and the reaction was gradually warmed to room temperature and stirred for 1 hour. TLC was used to monitor the completion of the reaction. After completion, the reaction was added with an excess amount of water, and extracted with ethyl acetate for 3-4 times. The organic phases were combined, washed with saturated brine, concentrated and then purified by column chromatography to afford 553 mg of compound 54a. Yield: 89%. LC-MS (APCI): m/z=427.7 (M+1).sup.+.

(303) Step 10 Synthesis of Compound 55a

(304) Compound 54a (536 mg, 1.26 mmol) was dissolved in methanol (15 ml), a catalytic amount of Pd/C was added, and the reaction was put under a balloon filled with hydrogen gas. The reaction was stirred and reacted at room temperature overnight. TLC was used to monitor the completion of the reaction. After completion, the catalyst was removed by filtration, and the filtrate was concentrated to afford 449 mg crude product of compound 55a, which was directly used in the next reaction. Yield: 89%. LC-MS (APCI): m/z=401.7 (M+1).sup.+.

(305) Step 11 Synthesis of Compound 56a

(306) Compound 55a (448 mg, 1.12 mmol) was dissolved in anhydrous DMF (20 ml), and DIPEA (580 mg, 4.48 mmol) and FDPP (516.4 mg, 1.34 mmol) were added. The reaction was stirred at room temperature overnight under nitrogen protection, TLC was used to monitor the completion of the reaction. After completion, the reaction was diluted with an excess amount of water, and extracted with ethyl acetate for 3-4 times. The organic phases were combined, washed with saturated brine, concentrated and then purified by column chromatography to afford 248 mg of compound 56a as light yellow solid. Yield: 58%. LC-MS (APCI): m/z=383.4 (M+1).sup.+. .sup.1H NMR (500 MHz. DMSO-d.sub.6) δ 8.73 (d. J=7.7 Hz, 1H), 8.41 (d, J=2.4 Hz, 1H), 8.00 (s, 1H), 7.62 (d, J=6.6 Hz, 1H), 7.57 (dd, J=9.9, 2.4 Hz, 1H), 6.62 (d, J=7.7 Hz, 1H), 5.36 (t, J=6.5 Hz, 1H), 4.16 (m, 1H), 4.05 (dd, J=16.9, 7.0 Hz, 2H), 3.75 (dd, J=14.0, 8.6 Hz, 1H), 3.64-3.51 (m, 1H), 2.91 (d, J=16.9 Hz, 1H), 2.54 (dd, J=14.0, 10.9 Hz, 1H), 2.44 (dd, J=13.4, 6.5 Hz, 2H), 2.23 (dd, J=12.0, 6.2 Hz, 1H), 2.12-1.96 (m, 11H), 1.79-1.66 (m, 1H).

(307) Step 12 Synthesis of Compounds L-9-A and L-9-B

(308) Preparative chiral column: CHIRALPAK IC (brand name), 4.6 mm×250 mm (internal diameter×length), 5 μm (particle size of the filler)

(309) Column temperature: 30° C.

(310) Flow rate: 1.0 L/min

(311) UV detection wavelength: 254 nm

(312) Mobile phase: methyl tert-butyl ether:methanol=70:30

(313) Under the above preparative chiral column and chiral resolution conditions, racemic compound 56a was separated to afford target compound L-9-A (retention time: 14.26 min, relative amount: 45.0%) and L-9-B (retention time: 5.38 min, relative amount: 42.9%).

Example 16: Preparation of 9-fluoro-13-oxa-2,11,18,21,22,25-hexaazapentacyclo[17.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.22,26.]hexadecane-1(25),7,9,11,19(26),20,23-heptaene-17-one (Compound 64a), (6R)-9-fluoro-13-oxa-2,11,18,21,22,25-hexaazapentacyclo[17.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.22,26.]hexadecane-1(2),7,9,11,19(26),20,23-heptaene-17-one (Compound L-10-A), and (6S)-9-fluoro-13-oxa-2,11,18,21,22,25-hexaazapentacyclo[17.5.2.0.SUP.2,6..0.SUP.7,12..0.SUP.22,26.]hexadecane-1(25),7,9,11,19(26),20,23-heptaene-17-one (Compound L-10-B)

(314) ##STR00055##

(315) The following route was used for the synthesis:

(316) ##STR00056##

(317) Step 1 Synthesis of Compound 57a

(318) 2-methoxy-3-bromo-5-fluoropyridine (19.16 g, 93 mmol) was dissolved in anhydrous THF (100 mL), and isopropyl magnesium chloride solution (43.4 mL, 86.8 mmol) was slowly added dropwise at −40° C. After the addition, the mixture was naturally warmed to 0° C. and stirred for 1 h. A solution of N-tert-butoxycarbonyl-2-pyrrolidone (11.46 g, 62.0 mmol) in anhydrous tetrahydrofuran (30 mL) was then slowly added dropwise at −40° C. After the addition, the mixture was stirred at room temperature for 30 min. The reaction solution was poured into 100 mL of saturated ammonium chloride solution and stirred for 10 min, then the reaction solution was separated by standing. The aqueous phase was extracted three times with 40 mL ethyl acetate. The organic phases were combined and washed with saturated brine, dried over anhydrous sodium sulfate. The residue was filtered and concentrated, purified by column chromatography to afford 16.2 g of compound 57a as light yellow liquid. Yield: 56%. LC-MS (APCI): m/z=313.2 (M+1).sup.+.

(319) Step 2 Synthesis of Compound 58a

(320) Compound 57a (1.2 g, 3.85 mmol) was dissolved in dichloromethane (10 mL), 2 mL trifluoroacetic acid was added, and the mixture was stirred at room temperature for 1 h. TLC was used to monitor the completion of the reaction. The reaction solution was washed with saturated sodium bicarbonate aqueous solution, the organic phase was separated, and the aqueous phase was extracted 3 times with dichloromethane. The organic phases were combined, washed with saturated brine, concentrated to afford crude product of compound 58a, which was directly used in the next reaction. LC-MS (APCI): m/z=195.2 (M+1).sup.+.

(321) Step 3 Synthesis of Compound 59a

(322) Compound 58a (747 mg, 3.85 mmol) was dissolved in anhydrous methanol (10 mL), Pd/C (50 mg) was added, and hydrogenated at room temperature overnight. The reaction was filtered, the filter residue was washed with 20 mL ethyl acetate, and the filtrate was concentrated to afford 731 mg of compound 59a as colorless oily liquid which was directly used in the next step. Yield: 97%. LC-MS (APCI): m/z=197.3 (M+1).sup.+.

(323) Step 4 Synthesis of Compound 60a

(324) Compound 59a (725 mg, 3.7 mmol) and 5-chloro-3-nitropyrazolo[1,5-a]pyrimidine (733 mg, 3.7 mmol) were dissolved in absolute ethanol (10 mL), DIPEA (1.91 g, 14.8 mmol) was added at room temperature, and the mixture was heated to reflux for 30 min. The reaction solution was concentrated, purified by column chromatography (PE/EA, 30%-50%) to afford 1.01 g of compound 60a as light yellow solid powder. Yield: 76%. LC-MS (APCI): m/z=359.5 (M+1).sup.+.

(325) Step 5 Synthesis of Compound 61a

(326) Compound 60a (1.01 g, 2.80 mmol) was dissolved in methanol (20 ml), a catalytic amount of Pd/C was added, and the reaction was put under a balloon filled with hydrogen gas. The reaction was stirred and reacted at room temperature for 4-5 hours. TLC was used to monitor the completion of the reaction. After completion, the catalyst was removed by filtration, and the filtrate was concentrated to afford 920 mg crude product of the title product, which was directly used in the next reaction. LC-MS (APCI): m/z=329.6 (M+1).sup.+.

(327) Step 6 Synthesis of Compound 62a

(328) Compound 61a (920 mg, 2.8 mmol) was dissolved in anhydrous DMF (15 ml), 4-chlorobutyric acid (360 mg, 2.94 mmol) and HATU (1.28 g, 3.36 mmol) were added, and then DIPEA (1.08 g, 8.4 mmol) was added, and the reaction was stirred at room temperature and reacted for 5 hours. TLC was used to monitor the completion of the reaction. After completion, the reaction was diluted with an excess amount of water, and extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, concentrated and then purified by column chromatography to afford 679 mg of compound 62a as light yellow solid. Yield: 56%. LC-MS (APCI): m/z=433.6 (M+1).sup.+.

(329) Step 7 Synthesis of Compound 63a

(330) Compound 62a (679 mg, 1.57 mmol) was added in a solution of 4N hydrogen chloride in dioxane (10 ml). The reaction was heated to reflux and stirred overnight. TLC was used to monitor the completion of the reaction. After completion, the reaction was concentrated to remove the solvent, which was directly used in the next reaction. LC-MS (APCI): m/z=−419.2 (M+1).sup.+.

(331) Step 8 Synthesis of Compound 64a

(332) Compound 63a (655 mg, 1.57 mmol) was dissolved in anhydrous DMF (20 ml), and potassium carbonate (434 mg, 3.14 mmol) was added. The reaction was heated to 80° C. and stirred to react overnight. TLC was used to monitor the completion of the reaction. After completion, the reaction was added with an excess amount of water, and extracted with ethyl acetate for 3-4 times. The organic phases were combined, washed with saturated saline, and then concentrated to remove the solvent, purified by silica gel column chromatography to afford 252 mg of compound 64a as white solid. Yield: 41%. LC-MS (APCI): m/z=383.3 (M+1).sup.+. .sup.1H NMR (500 MHz, DMSO-d) δ 8.76 (d, J=7.7 Hz, 1H), 8.42 (d, J=2.4 Hz, 1H), 8.01 (s, 11H), 7.62 (d, J=6.6 Hz, 11H), 7.57 (dd, J=9.9, 2.4 Hz, 1H), 6.60 (d, J=7.7 Hz, 1H), 5.36 (t, J=6.5 Hz, 1H), 4.05 (dd, J=16.9, 7.0 Hz, 2H), 3.75 (dd, J=14.0, 8.6 Hz, 1H), 3.67-3.51 (m, 1H), 2.91 (d, J=16.9 Hz, 1H), 2.54 (dd, J=14.0, 10.9 Hz, 1H), 2.44 (dd, J=13.4, 6.5 Hz, 2H), 2.23 (dd, J=12.0, 6.2 Hz, 2H), 2.12-1.96 (m, 1H), 1.79-1.66 (m, 1H).

(333) Step 9 Synthesis of Compounds L-10-A and L-10-B

(334) Preparative chiral column: CHIRALPAK IC (brand name), 4.6 mm×250 mm (internal diameter×length), 5 μm (particle size of the filler)

(335) Column temperature: 30° C.

(336) Flow rate: 1.0 mL/min

(337) UV detection wavelength: 254 nm

(338) Mobile phase: methyl tert-butyl ether:methanol=70:30

(339) Under the above preparative chiral column and chiral resolution conditions, racemic compound 64a was separated to afford target compound L-10-A (retention time: 27.54 min, relative amount: 49.4%) and L-10-B (retention time: 38.22 min, relative amount: 49.3%).

Biological Activity Assay

Biological Example 1: Biochemical Kinase Analysis

(340) Inhibition of TRKA, TRKB, TRKC kinases can be measured by HTRF (High Fluorescence Resonance Energy Transfer) method. The reaction was performed in a 384-well plate at 23° C. with a volume of 20 μL. TRKA TRKB or TRKC kinase were mixed with pre-formulated and diluted compounds of different concentrations for ten minutes in duplicate for each concentration, wherein 11 concentrations made with a 3-fold gradient dilution from the starting concentration of 300 nM were made and the final concentration of DMSO was 2%. The corresponding substrate and ATP were added thereto, and reacted at room temperature for 20 minutes (both a negative and a positive control were set: negative control is blank control, positive control is Entrectinib). After the reaction, detection reagent was added thereto (reagent in HTRF Kinase TK kit), and after incubation at room temperature for 30 minutes, detection was carried out by PerkinElmer Envision microplate reader for determining enzyme activity in the presence of the compounds of the present disclosure at various concentrations, and the inhibitory activity of different concentrations of compounds against enzyme activity was calculated. After that, in accordance with four-factors function and Graphpad 5.0 software, inhibitory activity on enzyme activity under different concentrations of compounds was fitted and IC.sub.50 values were calculated. The data of the compounds tested in this analysis is shown in Table 1.

Biological Example 2: KM12 Cell Proliferation Analysis

(341) The CGT method was used to detect the in vitro anti-proliferative activity of the compounds of the present disclosure on tumor cells cultured in vitro. The KM12 cell line was maintained in RPMI-1640 medium containing 10% fetal bovine serum and antibiotics, the cells in the logarithmic growth phase were harvested and planted in a 96-well plate. The plate was incubated in an incubator at 37° C., containing 5% carbon dioxide gas overnight. After the test compounds were dissolved in DMSO, a 3-fold concentration gradient dilution with 9 compound concentrations was made. Pre-prepared compounds of different concentrations were transferred to the cell plate in triplicate for each concentration, and continue culturing for 72 h. The final concentration of DMSO in the system was 0.1%, and the initial concentration of the test compound was 300 nM.

(342) CellTiter-Glo reagent was added to the cell plate, and incubated at room temperature for 30 minutes to stabilize the optical signal. Detection was carried out by PerkinElmer Envision microplate reader for determining the inhibitory activity of the compound of the present disclosure at various concentrations on cell proliferation. The inhibitory activity of compounds at different concentrations on cell proliferation was fitted according to Graphpad 5.0 software, and IC.sub.50 values were calculated. The data of the compounds tested in this analysis is shown in Table 1.

(343) TABLE-US-00001 TABLE 1 Kinase IC.sub.50 (nM) Cell IC.sub.50 (nM) TRK A TRK B TRK C KM12 TPX-005 0.11 0.09 0.12 1.97 LOXO-195 0.19 0.09 0.09 2.73 T-1 <0.17 <0.17 <0.17 5.13 T-2-A 0.19 0.10 0.06 1.60 T-2-B 0.36 0.09 0.09 4.79 T-3 0.34 0.57 0.52 T-4 4.91 5.31 5.41 T-5 93.88 225.32 257.93 L-1-A 0.18 0.10 0.14 0.55 L-1-B 21.07 15.54 14.20 161.51 Compound 9a 0.17 0.10 0.10 1.78 L-2-A 0.17 0.10 0.10 0.25 L-2-B 0.41 0.13 0.18 0.65 L-3-B >100 70.75 >100

Biological Example 3: Ba/F3 Cell Proliferation Analysis

(344) The CGT method was used to detect the in vitro anti-proliferative activity of the compounds of the present disclosure on three cell lines cultured in vitro.

(345) The Ba/F3 parent cells, Ba/F3 LMNA-NTRK1 and Ba/F3 LMNA-NTRK1-G595R cells were maintained in RPMI-1640 medium containing 10% fetal bovine serum and antibiotics, respectively, and the cells in the logarithmic growth phase were harvested and planted in a 96-well plate. The plate was incubated in a incubator at 37° C., containing 5% carbon dioxide gas overnight, wherein 8 ng/ml IL-3 was added to the Ba/F3 parent cells. After dissolving the test compounds in DMSO, 9 compound concentrations were made with a 3.16-fold gradient dilution. The pre-prepared compounds of different concentrations were transferred to the cell plate in triplicate for each concentration, and continued for culturing for 72 h. The final concentration of DMSO in the system was 0.1%, and the initial concentration of the test compounds in the Ba/F3 parent cell was 10 M, and the initial concentration of the test compounds in Ba/F3 LMNA-NTRK1 and Ba/F3 LMNA-NTRK1-G595R cells was 1 μM. CellTiter-Glo reagent was added to the cell plate, and incubated at room temperature for 30 minutes to stabilize the optical signal. Detection was carried out by PerkinElmer Envision microplate reader for determining the inhibitory activity of the compound of the present disclosure at various concentrations on cell proliferation. The inhibitory activity of compounds at different concentrations on cell proliferation was fitted according to Graphpad 5.0 software, and IC.sub.50 values were calculated. The results showed that the compound of the present disclosure has almost no inhibitory effect on Ba/F3 parent cells but has an inhibitory effect on Ba/F3 LMNA-NTRK1 and Ba/F3 LMNA-NTRK1-G595R cells.

Biological Example 4: Pharmacokinetic Experiment in Rats

(346) 6 male Sprague-Dawley rats (7-8 weeks old, and weighing approximately 210 g) were divided into 2 groups with 3 rats in each group. The rats were intravenously or orally administered a single dose of compounds (3 mg/kg intravenously, 10 mg/kg orally) to compare pharmacokinetic differences.

(347) The rats were feeded on standard food and water. Fasting was started 16 hours before the test. The drug was dissolved in PEG400 and dimethyl sulfoxide. The blood samples were collected from eyelids at the time points of 0.083 hour, 0.25 hour, 0.5 hour, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, and 24 hours after administration.

(348) Rats were briefly anesthetized after inhalation of diethyl ether and 300 μL of blood sample was collected from the eyelids into test tubes. There was 30 μL of 1% heparin salt solution in the test tube. Tubes were dried at 60° C. overnight before use. After the blood sample was collected at the last time point, the rats were sacrificed after ether anesthesia.

(349) Immediately after the collection of the blood sample, the test tube was gently inverted at least 5 times to ensure sufficient mixing and then placed on ice. The blood sample was centrifuged at 5000 rpm at 4° C. for 5 minutes to separate the plasma from the red blood cells. 100 μL of plasma was aspirated into a clean plastic centrifuge tube with a pipette, marking with the name of the compound and time point. Plasma was stored at −80° C. prior to analysis. The concentration of the compound of the present disclosure in plasma was determined by LC-MS/MS. The pharmacokinetic parameters were calculated based on the blood concentration of the drug for each animal at different time points.

(350) Experiments showed that the compounds of the present disclosure have better pharmacokinetic properties in vivo, and therefore have better pharmacodynamics and treatment effects. The pharmacokinetic experimental results of representative example compounds in rat are summarized in Table 2 and Table 3 below.

(351) TABLE-US-00002 TABLE 2 TPX-0005 T-1 T-2-A Dosage IV PO IV PO IV PO (mg/kg) 3 10 3 10 3 10 T.sub.max (h) 0.08 1.67 0.08 1.67 0.08 2.17 C.sub.max (h) 2185.8 731.8 2120.7 1863.8 2101.3 994.4 AUC.sub.last 3558.9 5474.1 6238.6 11543.3 6560.9 11522.3 (h*ng/mL) AUC.sub.INF_pred 3577.6 5586.6 6348.3 13062.9 6617.6 12905.9 (h*ng/mL) MRT.sub.INF_pred (h) 1.76 5.42 4.04 6.23 3.44 7.37 Vz_pred (L/kg) 4.46 15.81 3.28 10.05 2.67 6.70 Cl_pred (L/kg) 0.90 1.90 0.47 0.77 0.45 0.83 T.sub.1/2 (h) 5.77 0.76 9.05 0.36 5.59 4.84 F (%) 46.14 55.51 52.69

(352) TABLE-US-00003 TABLE 3 LOXO-195 L-1-A L-2-A Dosage IV PO IV PO IV PO (mg/kg) 3 10 3 10 3 10 T.sub.max (h) 0.08 0.33 0.08 1.58 0.08 0.50 C.sub.max (h) 5008.5 1663.8 2158.6 1492.0 1623.9 1405.3 AUC.sub.last 4666.0 4071.1 5005.1 11320.7 2476.3 4470.5 (h*ng/mL) AUC.sub.INF_pred 4669.6 4087.8 5028.5 11331.0 2477.8 4528.8 (h*ng/mL) MRT.sub.INF_pred (h) 0.54 2.31 2.18 4.90 1.20 2.80 Vz_pred (L/kg) 0.75 5.13 1.75 2.92 1.85 8.50 Cl_pred (L/kg) 0.71 2.45 0.60 0.92 1.23 2.44 T.sub.1/2 (h) 1.45 4.11 2.20 5.77 2.41 2.02 F (%) 26.17 67.86 54.16

(353) The above is a further detailed description of the present disclosure in conjunction with specific embodiments, and it cannot be assumed that the specific implementation of the present disclosure is limited to these descriptions. For ordinary artisan in the technical field to which the present disclosure belongs, without deviating from the concept of the present disclosure, various simple deductions or replacements may be made, which should be regarded as falling within the protection scope of the present disclosure.