Heterocyclic compounds and their application in medicine

Abstract

This invention relates to heterocyclic compounds and their use in medicine. In particular, the present invention discloses heterocyclic compound of formula I, or an isotopically labeled compound thereof, or an optical isomer thereof, a geometric isomer thereof, a tautomer thereof or a mixture of various isomers, or a pharmaceutically acceptable salt thereof, or a prodrug thereof. The invention also relates to the use of these compounds in medicine.

Claims

1. A compound of Formula I, ##STR00122## or an isotopically labeled compound thereof, or an optical isomer thereof, a geometric isomer thereof, a tautomer thereof or a mixture of various isomers, or a pharmaceutically acceptable salt thereof, or a prodrug thereof, in which R.sup.1 and R.sup.2 are each independently selected from the group consisting of hydrogen, halo, cyano, C1 to C4 alkyl and substituted alkyl; n is 1, 2, 3, or 4; R.sup.3, R.sup.4, R.sup.6 are each independently selected from the group consisting of hydrogen, C1 to C6 alkyl and substituted alkyl, aryl and heteroaryl; R.sup.5 is selected from the group consisting of C1 to C6 alkyl and substituted alkyl, aryl and heteroaryl; R.sup.7 is selected from the group consisting of C1 to C6 alkyl and substituted alkyl, C2 to C6 alkenyl and substituted alkenyl, C2 to C6 alkynyl and substituted alkynyl, and acyl having 1 to 6 carbon atoms; Z is selected from the group consisting of O, S, C(═O), C (R.sup.9)(R.sup.10), wherein, R.sup.9 and R.sup.10 are each independently selected from hydrogen, C1 to C6 alkyl and substituted alkyl; A is a saturated four to six membered ring, which is a full carbocyclic ring or a heterocyclic ring containing one oxygen atom, one nitrogen atom, or two nitrogen atoms as ring atoms.

2. The compound of Formula I, or an isotopically labeled compound thereof, or an optical isomer thereof, a geometric isomer thereof, a tautomer thereof or a mixture of various isomers, or a pharmaceutically acceptable salt thereof, or a prodrug thereof according to claim 1, wherein Z is selected from the group consisting of O, S, NH, C(═O), and CH.sub.2.

3. The compound of Formula I, or an isotopically labeled compound thereof, or an optical isomer thereof, a geometric isomer thereof, a tautomer thereof or a mixture of various isomers, or a pharmaceutically acceptable salt thereof, or a prodrug thereof according to claim 1, wherein Z is selected from O, S and NH.

4. The compound of Formula I, or an isotopically labeled compound thereof, or an optical isomer thereof, a geometric isomer thereof, a tautomer thereof or a mixture of various isomers, or a pharmaceutically acceptable salt thereof, or a prodrug thereof according to claim 1, wherein R.sup.6 is selected from the group consisting of hydrogen, C1 to C4 alkyl and substituted C1 to C4 alkyl and R.sup.5 is selected from the group consisting of C1 to C4 alkyl and substituted C1 to C4 alkyl.

5. The compound of Formula I, or an isotopically labeled compound thereof, or an optical isomer thereof, a geometric isomer thereof, a tautomer thereof or a mixture of various isomers, or a pharmaceutically acceptable salt thereof, or a prodrug thereof according to claim 1, wherein R.sup.6 is hydrogen, or methyl and R.sup.5 is methyl.

6. The compound of Formula I, or an isotopically labeled compound thereof, or an optical isomer thereof, a geometric isomer thereof, a tautomer thereof or a mixture of various isomers, or a pharmaceutically acceptable salt thereof, or a prodrug thereof according to claim 1, wherein R.sup.7 is selected from the group consisting of C1 to C6 alkyl, substituted C1 to C6 alkyl, and acyl having 1 to 6 carbon atoms.

7. The compound of Formula I, or an isotopically labeled compound thereof, or an optical isomer thereof, a geometric isomer thereof, a tautomer thereof or a mixture of various isomers, or a pharmaceutically acceptable salt thereof, or a prodrug thereof according to claim 1, wherein R.sup.7 is selected from the group consisting of propyl, butyl, pentyl, formyl, acetyl, propanoyl, butyryl, fluoro or difluoro propyl, and fluoro or difluoro butyl.

8. The compound of Formula I, or an isotopically labeled compound thereof, or an optical isomer thereof, a geometric isomer thereof, a tautomer thereof or a mixture of various isomers, or a pharmaceutically acceptable salt thereof, or a prodrug thereof according to claim 1, wherein R.sup.2 is hydrogen or C1 to C4 alkyl.

9. The compound of Formula I, or an isotopically labeled compound thereof, or an optical isomer thereof, a geometric isomer thereof, a tautomer thereof or a mixture of various isomers, or a pharmaceutically acceptable salt thereof, or a prodrug thereof according to claim 1, wherein R.sup.3 and R.sup.4 are each independently selected from the group consisting of hydrogen, C1 to C4 alkyl, and substituted C1 to C4 alkyl.

10. The compound of Formula I, or an isotopically labeled compound thereof, or an optical isomer thereof, a geometric isomer thereof, a tautomer thereof or a mixture of various isomers, or a pharmaceutically acceptable salt thereof, or a prodrug thereof according to claim 1, wherein one of R.sup.3 and R.sup.4 is hydrogen and the other is isobutyl or cyclopropylmethyl.

11. The compound of Formula I, or an isotopically labeled compound thereof, or an optical isomer thereof, a geometric isomer thereof, a tautomer thereof or a mixture of various isomers, or a pharmaceutically acceptable salt thereof, or a prodrug thereof according to claim 1, wherein R.sup.1 is hydrogen or halogen and n is 1 or 2.

12. The compound of Formula I, or an isotopically labeled compound thereof, or an optical isomer thereof, a geometric isomer thereof, a tautomer thereof or a mixture of various isomers, or a pharmaceutically acceptable salt thereof, or a prodrug thereof according to claim 1, wherein R.sup.1 is hydrogen or F and n is 1 or 2.

13. The compound of Formula I, or an isotopically labeled compound thereof, or an optical isomer thereof, a geometric isomer thereof, a tautomer thereof or a mixture of various isomers, or a pharmaceutically acceptable salt thereof, or a prodrug thereof according to claim 1, wherein A is a saturated four-membered carbocyclic ring or is a four-membered heterocyclic ring containing one oxygen atom, one nitrogen atom or two nitrogen atoms as ring atoms.

14. The compound of Formula I, or an isotopically labeled compound thereof, or an optical isomer thereof, a geometric isomer thereof, a tautomer thereof or a mixture of various isomers, or a pharmaceutically acceptable salt thereof, or a prodrug thereof according to claim 1, wherein A is an azetidine ring.

15. The compound of Formula I, or an isotopically labeled compound thereof, or an optical isomer thereof, a geometric isomer thereof, a tautomer thereof or a mixture of various isomers, or a pharmaceutically acceptable salt thereof, or a prodrug thereof according to claim 1, wherein the substituted alkyl is alkyl substituted with halogen.

16. The compound of Formula I, or an isotopically labeled compound thereof, or an optical isomer thereof, a geometric isomer thereof, a tautomer thereof or a mixture of various isomers, or a pharmaceutically acceptable salt thereof, or a prodrug thereof according to claim 1, wherein the substituted alkyl is alkyl substituted with one or two F.

17. A compound of Formula I, or an isotopically labeled compound thereof, or an optical isomer thereof, a geometric isomer thereof, a tautomer thereof or a mixture of various isomers, or a pharmaceutically acceptable salt thereof, or a prodrug thereof, wherein the compound is selected from the group consisting of: 1-(3-fluoropropyl)-N-(4-((6R, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinolin-6-yl) phenyl) azetidin-3-amine; N-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4 h] isoquinolin-6-yl) phenyl)-1-(3-fluoropropyl) azetidin-3-amine; N-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4 h] isoquinolin-6-yl) phenyl)-1-(2,3-difluoropropyl) azetidin-3-amine; N-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinolin-6-yl) phenyl)-1-(3,3,3-trifluoropropyl) azetidin-3-amine; N-(4-((6S, 7S)-7-cyclopropylmethyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinolin-6-yl)-3,5-difluorophenyl)-1-(3-fluoropropyl) azetidin-3-amine; N-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinolin-6-yl) phenyl)-1-propylazetidin-3-amine; 1-butyl-N-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinolin-6-yl) phenyl) azetidin-3-amine; N-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinolin-6-yl) phenyl)-1-isobutylazetidin-3-amine; N-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinolin-6-yl) phenyl)-1-pentylazetidin-3-amine; (6S, 7S)-6-(2,6-difluoro-4-((1-(3-fluoropropyl) azetidin-3-yl) thio) phenyl)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinoline; (6S, 7S)-6-(2,6-difluoro-4-((1-propyl) azetidin-3-yl) thio) phenyl)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinoline; (6S, 7S)-6-(4-((1-butylazetidin-3-yl) thio)-2,6-difluorophenyl)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinoline; (6S, 7S)-6-(2,6-difluoro-4-((1-isobutylazetidin-3-yl) thio) phenyl)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinoline; (6S, 7S)-6-(2,6-difluoro-4-((1-pentylazetidin-3-yl) thio) phenyl)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinoline; (6S, 7S)-6-(2,6-difluoro-4-((1-(3-fluoropropyl) azetidin-3-yl) oxy) phenyl)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinoline; (6S, 7S)-6-(2,6-difluoro-4-((1-propylazetidin-3-yl) oxy) phenyl)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinoline; (6S, 7S)-6-(2,6-difluoro-4-((1-isobutylazetidin-3-yl) oxy) phenyl)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinoline; (6S, 7S)-6-(4-((1-butylazetidin-3-yl) oxy) phenyl)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinoline; (6S, 7S)-6-(4-((1-pentylazetidin-3-yl) oxy) phenyl)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinoline; (6S, 7S)-7-(cyclopropylmethyl)-6-(2,6-difluoro-4-((1-(3-fluoropropyl) azetidin-3-yl) oxy) phenyl)-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinoline; (6S, 7S)-7-cyclopropylmethyl-6-(2,6-difluoro-4-((1-propylazetidin-3-yl) oxy) phenyl)-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinoline; (6S, 7S)-7-cyclopropylmethyl-6-(2,6-difluoro-4-((1-isobutylazetidin-3-yl) oxy) phenyl)-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinoline; (6S, 7S)-6-(4-((1-butylazetidin-3-yl) oxy)-2,6-difluorophenyl)-7-cyclopropylmethyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinoline; (6S, 7S)-7-cyclopropylmethyl-6-(2,6-difluoro-4-((1-pentylazetidin-3-yl) oxy) phenyl)-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinoline; (6S, 7S)-7-cyclopropylmethyl-6-(2,6-difluoro-4-((1-(3-fluoropropyl) azetidin-3-yl) thio) phenyl)-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinoline; (6S, 7S)-7-cyclopropylmethyl-6-(2,6-difluoro-4-((1-propylazetidin-3-yl) thio) phenyl)-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinoline; (6S, 7S)-6-(4-((1-butylazetidin-3-yl) thio)-2,6-difluorophenyl)-7-cyclopropylmethyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinoline; (6S, 7S)-6-(4-((1-isobutylazetidin-3-yl) thio)-2,6-difluorobenzene)-7-cyclopropylmethyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinoline; (6S, 7S)-7-cyclopropylmethyl-6-(2,6-difluoro-4-((1-pentylazetidin-3-yl) thio) phenyl)-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinoline; 3-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinolin-6-yl) phenoxy) azetidin-1-aldehyde; 1-(3-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinolin-6-yl) phenoxy) azetidin-1-yl) ethan-1-one; 1-(3-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinolin-6-yl) phenoxy) azetidin-1-yl) propan-1-one; 1-(3-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinolin-6-yl) phenoxy) azetidin-1-yl) butan-1-one; 1-(3-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinolin-6-yl) phenoxy) azetidin-1-yl)-2-methylpropan-1-one.

18. A pharmaceutical composition comprising a compound of formula I, or an isotopically labeled compound thereof, or an optical isomer thereof, a geometric isomer thereof, a tautomer thereof or a mixture of various isomers, or a pharmaceutically acceptable salt thereof, or a prodrug thereof according to claim 1, and one or more pharmaceutically acceptable carriers, adjuvants or excipients.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1, 2, and 3 show the immunoblot analysis results of compounds according to certain embodiments of the present invention, respectively.

DETAILED DESCRIPTION OF EMBODIMENTS

(2) The invention is further illustrated by the following examples; however, these examples do not limit the scope of the invention.

(3) The structures of compounds were determined by liquid chromatography-mass spectrometry (LCMS) or nuclear magnetic resonance (NMR). The NMR chemical shift (δ) is expressed in units of 10.sup.−δ (ppm). The NMR spectrawere measured by Bruker-500 nuclear magnetic resonance apparatus in which deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl.sub.3) and the like were used as solvent, and tetramethylsilane (TMS) was used as an internal standard. LCMS was determined using Shimadzu LCMS-2020.

(4) Silica gel plates Huanghai HSGF254 available from Yantai, Shandong, China or plates GF254 from Qingdao, Shandong, Chinawere used as thin-layer chromatography silica gel plates. Generally, Huanghaisilica gel with 200 to 300 mesh available from Yantai, Shandong was used as a carrier for column chromatography.

(5) All starting materials used in the present invention were purchased from chemical suppliers or can be synthesized by methods known in literatures.

(6) The abbreviations used in the description of this article are as follows:

(7) Boc: tert-butoxycarbonyl

(8) br: nuclear magnetic broad peak

(9) CAS: Chemical Abstracts Accession Number

(10) CDCl.sub.3: Deuterated chloroform

(11) d: nuclear magnetic double peak

(12) DIPEA: Diisopropylethylamine

(13) DMF: N, N-dimethylformamide

(14) DMSO-d6: dimethyl sulfoxide in which six hydrogen atoms are replaced by deuterium

(15) EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

(16) ESI: Electrospray ionization

(17) ER: Estrogen receptor

(18) LCMS: Liquid Chromatography-Mass Spectrometry

(19) mg: microgram

(20) ml: microliter

(21) mmol: micromole

(22) NMR: Nuclear Magnetic Resonance

(23) ppm: one part per million

(24) s: nuclear magnetic single peak

(25) t: nuclear magnetic triplet peak

(26) TFA: trifluoroacetic acid

(27) TMS: tetramethylsilane

(28) δ: chemical shift of nuclear magnetic resonance

Preparation of Intermediate Compounds

Preparation of Intermediate I-100′

(29) ##STR00005##

1-(2,3-difluoropropyl)azetidin-3-amine (I-100′-1)

(30) ##STR00006##

Step 1: 2-benzyloxymethyl oxide (I-100′-1-a)

(31) ##STR00007##

(32) NaOH (3.7 g), tetra-n-butylammonium iodide TBAI (3.43 g) and DMF (150 ml) were charged to a reaction flask and then cooled in an ice water bath. After that, ethylene oxide-2-ylmethanol (CAS: 52-5; 6.85 g) was dropped into the reaction flask and stirred for 2 hours; then benzyl bromide BnBr (17.3 g) was slowly added dropwise. After the addition was completed, the reaction was allowed to proceed at room temperature overnight. Then, water was added to quench the reaction, and the mixture was extracted with ethyl acetate. The ethyl acetate layer was collected and then washed twice with an aqueous diluted sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography to afford an oily product, I-100′-1-a (12 g). .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.41-7.26 (m, 5H), 4.53 (d, J=1.1 Hz, 2H), 3.76 (dd, J=11.5, 2.7 Hz, 1H), 3.30 (dd, J=11.5, 6.4 Hz, 1H), 3.15 (ddt, J=6.8, 4.2, 2.7 Hz, 1H), 2.74 (dd, J=5.1, 4.2 Hz, 1H), 2.57 (dd, J=5.1, 2.7 Hz, 1H).

Step 2: ((2,3-difluoropropoxy) methyl) benzene (I-100′-1-b)

(33) ##STR00008##

(34) I-100′-1-a (2 g) and Et.sub.3N—HF (1.24 g) were added to a sealed tube and reacted at 150° C. for 1.5 hours. After cooling, aqueous saturated sodium bicarbonate solution was to quench the reaction, and the mixture was extracted with ethyl acetate. Then, the collected ethyl acetate layer was washed twice with aqeuous diluted sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated, to afford crude product intermediate 3-benzyloxy-2-fluoro-1-propanol. Next, 20 ml of THF and DBU (2.04 g) were added to the above intermediate, which was cooled in an ice water bath, and then nonafluorobutylsulfonyl fluoride NfF (CAS: 375-72-4; 6.6 g) was added. The reaction was carried out for one hour. The reaction was quenched by the addition of an aqueous saturated sodium hydrogen carbonate solution, and the mixture was extracted with ethyl acetate. The collected ethyl acetate layer was washed twice with an aqueous diluted sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography to afford an oily product, I-100′-1-b (1.8 g). .sup.1H NMR (500 MHz, DMSO-d6) δ 7.41-7.25 (m, 5H), 5.05-4.84 (m, 1H), 4.80-4.55 (m, 2H), 4.54 (d, J=1.3 Hz, 2H), 3.76-3.59 (m, 2H).

Step 3: 2,3-difluoropropyl2-nitrobenzenesulfonate (I-100′-1-c)

(35) ##STR00009##

(36) I-100′-1-b (360 mg) and 10% palladium on carbon (76 mg) were mixed in 5 ml of THF, and the reaction was stirred overnight under a hydrogen blanket in a 40° C. oil bath to remove benzyl protection group. The reaction was then filtered to remove catalyst, and the filtrate was used directly in the next step. Then, to the filtrate, 5 ml of DCM was added and then Et.sub.3N (1.44 ml), DMAP (23 mg) and 2-nitrobenzenesulfonyl chloride (480 mg) were added. The reaction was carried out at room temperature overnight. The reaction was quenched by the addition of an aqueous saturated sodium hydrogen carbonate solution, and the mixture was extracted with ethyl acetate. The collected ethyl acetate layer was washed twice with an aqueous diluted sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to afford product I-100′-1-c (50 mg). .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.18 (ddd, J=9.3, 7.9, 1.3 Hz, 2H), 8.06 (td, J=7.8, 1.4 Hz, 1H), 7.98 (td, J=7.7, 1.3 Hz, 1H), 5.19-5.00 (m, 1H), 4.80-4.43 (m, 4H).

Step 4: benzyl (1-(2,3-difluoropropyl)azetidin-3-yl) carbamate (I-100′-1-d)

(37) ##STR00010##

(38) I-100′-1-c (100 mg), benzyl azetidin-3-ylcarbamate hydrochloride (CAS: 914348-04-2; 100 mg) and diisopropylethyl amine (138 mg) were mixed in 1 ml of DMF; the reaction was stirred at room temperature overnight. The reaction was quenched by adding an aqueous solution, and the mixture was extracted with ethyl acetate. The collected ethyl acetate layer was then washed twice with an aqueous diluted sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by a column chromatograph to afford product I-100′-1-d (98 mg). .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.78 (d, J=7.7 Hz, 1H), 7.41-7.27 (m, 5H), 5.00 (s, 2H), 4.81-4.42 (m, 3H), 4.14-4.00 (m, 1H), 3.54 (q, J=6.3 Hz, 2H), 2.93-2.84 (m, 2H), 2.65 (dd, J=22.8, 5.4 Hz, 2H).

Step 5: 1-(2,3-difluoropropyl)azetidin-3-amine (I-100′-1)

(39) ##STR00011##

(40) I-100′-1-d (3.5 mg) and 10% palladium on carbon (350 mg) were mixed in 40 ml of THF, and the reaction was stirred for 48 hours under a hydrogen blanket at room temperature to remove Cbz protection group. The reaction was then filtered to remove catalyst, and the filtrate was dried to affort product I-100′-1 (1.2 g). .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 4.78-4.41 (m, 3H), 3.51 (ddt, J=9.4, 5.0, 2.5 Hz, 2H), 3.34 (d, J=6.7 Hz, 1H), 2.65-2.58 (m, 4H), 1.85 (s, 2H).

(1-(3,3,3-trifluoropropyl)azetidin-3-amine (I-100′-2)

(41) ##STR00012##

Step 1: benzyl (1-(3,3,3-trifluoropropyl)azetidin-3-yl) carbamate (I-100′-2-d)

(42) ##STR00013##

(43) The synthetic route for I-100′-1-d was repeated, which starts from 1,1,1-trifluoro-3-iodopropane, benzyl azacyclbutan-3-ylcarbamate hydrochloride (CAS: 914348-04-2), and diisopropylethylamine (138 mg) to afford product I-100′-2-d. .sup.1H NMR (500 MHz, DMSO-d6, δ, ppm): 7.77 (d, J=8.0 Hz, 1H), 7.4-7.3 (m, 5H), 5.00 (s, 2H), 4.1-4.0 (m, 1H), 3.51 (t, J=7.0 Hz, 2H), 2.81 (t, J=7.0 Hz, 2H), 2.54 (t, J=7.5 Hz, 2H), 2.3-2.2 (m, 2H).

Step 2: (1-(3,3,3-trifluoropropyl)azetidin-3-amine (I-100′-2)

(44) ##STR00014##

(45) The synthetic route for I-100′-1 was repeated, which starts from I-100′-2-d to afford product I-100′-2. .sup.1H NMR (500 MHz, DMSO-d6, δ, ppm): 3.5-3.4 (m, 2H), 3.4-3.3 (m, 1H), 2.6-2.4 (m, 4H), 2.3-2.2 (m, 2H).

Preparation of Intermediate I-60

(46) ##STR00015##

(2S)-1-(4-bromophenyl)-4-methyl-2-(methylamino)pentane-1-olhydrochloride (I-60-1)

(47) ##STR00016##

Step 1: tert-butyl (S)-(1-(4-bromophenyl)-4-methyl-1-oxopent-2-yl) (methyl) carbamate (I-60-1-a)

(48) ##STR00017##

(49) Compound I-60-1-a was synthesized from the above two known compounds by the methods known in J. Am. Chem. Soc. 2005, vol 127, 6152-6153 and its ancillary materials. The above two known compounds were synthesized by the methods known in WO2004026293 and Org. Synth. 1969, 49, 66. .sup.1H NMR (500 MHz, DMSO-d6) δ (ppm): 7.83-7.72 (4H, m), 5.46-5.21 (1H, m), 2.60 (3H, d), 1.68-1.57 (2H, m), 1.57-1.46 (1H, m), 1.36 (9H, d, J=6.0 Hz), 0.99-0.92 (6H, m).

Step 2: tert-butyl ((2S)—1-(4-bromophenyl)-1-hydroxy-4-methylpent-2-yl) (methyl) carbamate (I-60-1-b)

(50) ##STR00018##

(51) At room temperature, compound I-60-1-a (1 g) was dissolved in 10 ml of methanol, and then sodium borohydride (197 mg) was added. The reaction mixture was stirred at room temperature overnight. Then, water was added to quench the reaction, and the mixture was extracted with ethyl acetate. The collected ethyl acetate layer was then washed twice with an aqueous diluted sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography to afford Product as a yellow oil, I-60-1-b (1 g). .sup.1H NMR (500 MHz, DMSO-d6) δ (ppm): 7.51-7.48 (2H, m), 7.27-7.23 (2H, m), 5.42-5.31 (1H, m), 4.55-4.53 (1H, m), 4.13-4.10 (1H, m), 2.68 (3H, d), 1.76-1.69 (1H, m), 1.78-1.34 (1H, m), 1.29 (3H, s), 1.24 (1H, s), 1.18 (6H, s), 0.87-0.76 (6H, m).

Step 3: (2S)-1-(4-bromophenyl)-4-methyl-2-(methylamino)pentane-1-olhydrochloride (I-60-1)

(52) ##STR00019##

(53) Compound I-60-1-b (1 g) was dissolved in 10 ml of methanol, to which 6 M aqueous hydrochloric acid solution (9 ml) was added at room temperature. The reaction was stirred at room temperature overnight and then, concentrated to afford crude product as a yellow oil, I-60-1 (839 mg). .sup.1H NMR (500 MHz, DMSO-d6) δ (ppm): 7.61-7.59 (2H, m), 7.41-7.38 (2H, m), 4.61 (1H, d, J=8.0 Hz), 3.23-3.19 (1H, m), 2.54-2.52 (3H, m), 1.47-1.42 (1H, m), 1.30-1.27 (3H, m), 1.26-1.12 (1H, m), 0.77 (3H, d, J=6.5 Hz), 0.62 (3H, d, J=6.5 Hz).

(2S)-1-(4-chloro-2,6-difluorophenyl)-4-methyl-2-(methylamino)pentane-1-ol hydrochloride (I-60-2)

(54) ##STR00020##

Step 1: tert-butyl (S)-(1-(4-chloro-2,6-difluorophenyl)-4-methyl-1-oxopent-2-yl) (methyl) carbamate (I-60-2-a)

(55) ##STR00021##

(56) The synthetic route for I-60-1-a was repeated, which starts from tert-butyl (S)-(1-(methoxy (methyl) amino)-4-methyl-1-oxopent-2-yl) (methyl) carbamate (CAS: 676628-64-1) and (4-chloro-2,6-difluorophenyl) magnesium bromide (see PCT 2016039404 for its synthesis) to afford product I-60-2-a. .sup.1H NMR (500 MHz, dmso-d6) δ (in ppm): 7.65-7.67 (1H, d, J=10.0 Hz), 7.57-7.58 (1H, d, J=8.0 Hz), 2.71-2.78 (3H, d, J=35.0 Hz), 2.22-2.23 (1H, d, J=5.0 Hz), 1.58-1.77 (4H, m), 1.32-1.35 (9H, d, J=15.0 Hz), 0.92-1.0 (18H, m).

Step 2: tert-butyl ((2S)-1-(4-chloro-2,6-difluorophenyl)-1-hydroxy-4-methylpent-2-yl) (methyl) carbamate (I-60-2-b)

(57) ##STR00022##

(58) The synthetic route for I-60-1-b was repeated, which starts from I-60-2-a to afford I-60-2-b: LCMS ESI (+): 378 (M+1).sup.+.

Step 3: (2S)-1-(4-chloro-2,6-difluorophenyl)-4-methyl-2-(methylamino)pentane-1-ol hydrochloride (I-60-2)

(59) ##STR00023##

(60) The synthetic route for I-60-1 was repeated, which starts from I-60-2-b to afford I-60-2: ESI (+): 278 (M+1).sup.+.

(2S)-1-(4-chloro-2,6-difluorophenyl)-3-cyclopropyl-2-(methylamino)propan-1-ol hydrochloride (I-60-3)

(61) ##STR00024##

Step 1: tert-butyl (S)-(1-(4-chloro-2,6-difluorophenyl)-3-cyclopropyl-1-oxopropan-2-yl) (methyl) carbamate (I-60-3-a)

(62) ##STR00025##

(63) The synthetic route for I-60-1-a was repeated, which starts from tert-butyl (S)-(3-cyclopropyl-1-(methoxy (methyl) amino)-1-oxopropan-2-yl) (methyl) carbamate (CAS: 676628-64-1) and (4-chloro-2,6-difluorophenyl) magnesium bromide (see PCT 2016039404 for its synthesis) to afford product I-60-3-a: .sup.1H NMR (500 MHz, dmso-d6) δ (in ppm): 7.49 (d, J=8.3 Hz, 1H), 7.39 (d, J=8.3 Hz, 1H), 4.66 (s, 0.5H), 4.36 (dd, J=9.5, 4.6 Hz, 0.5H), 2.79 (s, 1.5H), 2.68 (s, 1.5H), 1.81 (dt, J=14.2, 5.4 Hz, 0.5H), 1.68 (dt, J=12.6, 5.6 Hz, 0.5H), 1.64-1.58 (m, 0.5H), 1.54 (dt, J=14.2, 8.8 Hz, 0.5H), 1.19 (s, 4.5H), 1.11 (s, 4.5H), 0.64-0.53 (m, 1H), 0.35 (dtd, J=25.9, 8.7, 4.3 Hz, 2H), 0.06-−0.10 (m, 2H).

Preparation of tert-butyl (S)-(3-cyclopropyl-1-(methoxy (methyl) amino)-1-oxopropan-2-yl) (methyl) carbamate

(64) ##STR00026##

(65) NaH (132 mg) was suspended in 10 ml of DMF, and cooled with an ice-water bath. And then to the mixture, tert-butyl (S)-(3-cyclopropyl-1-(methoxy (methyl) amino)-1-oxopropan-2-yl) carbonate (0.9 g; CAS: 882004-10-6; prepared according to the literature Bioorganic & Medicinal Chemistry Letters, 2006, 16 (6), 1621-1627) in DMF (5 ml) was added dropwise and was stirred in an ice water bath for 2 hours. Then methyl iodide (563 mg) was added dropwise. The reaction was stirred at room temperature overnight. An aqueous solution was added to quench the reaction, and the mixture was extracted with ethyl acetate. The collected ethyl acetate layer was then washed twice with an aqueous diluted sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatographyto afford a pure product (570 mg). .sup.1H NMR (500 MHz, DMSO-d6) δ 4.92 (d, J=83.0 Hz, 1H), 3.59 (s, 3H), 3.03 (d, J=5.6 Hz, 3H), 2.65 (d, J=25.9 Hz, 3H), 1.59-1.45 (m, 1H), 1.33 (s, 10H), 0.51 (pd, J=7.2, 3.7 Hz, 1H), 0.39-0.24 (m, 2H), 0.05-0.0 (m, 2H).

Step 2: tert-butyl ((2S)-1-(4-chloro-2,6-difluorophenyl)-3-cyclopropyl-1-hydroxypropan-2-yl) (methyl) carbamate (I-60-3-b)

(66) ##STR00027##

(67) The synthetic route for I-60-1-b was repeated, which starts from I-60-2-a to afford I-60-2-b: LCMS ESI (+): 376 (M+1).sup.+.

Step 3: (2S)-1-(4-chloro-2,6-difluorophenyl)-3-cyclopropyl-2-(methylamino)propan-1-ol hydrochloride (I-60-3)

(68) ##STR00028##

(69) The synthetic route for I-60-1 was repeated, which starts from I-60-3-b to afford I-60-3: ESI (+): 276 (M+1).sup.+.

Example 1

1-(3-fluoropropyl)-N-(4-((6R, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinolin-6-yl) phenyl)azetidin-3-amine (1)

(70) ##STR00029##

Step 1: (2S)-2-(((1H-indazol-4-yl) methyl) (methyl) amino)-1-(4-bromophenyl)-4-methylpent-1-ol (1a)

(71) ##STR00030##

(72) Compound I-60-1 (839 mg), 4-(chloromethyl)-1H-indazole (ADV947321888, available from Advanced ChemBlocks Inc., 739 mg), potassium carbonate (1.8 g), and sodium iodide (39 mg) were mixed in anhydrous DMF at room temperature. The reaction was stirred at room temperature overnight. Then, the mixture was extracted with ethyl acetate and water; and the organic phase was separated. The separated organic phase was washed twice with an aqueous diluted sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography to afford a yellow solid product 1a (950 mg). .sup.1H NMR (500 MHz, DMSO-d6) δ (ppm): 13.00 (1H, s), 8.07 (1H, s), 7.50 (2H, d, J=8.0 Hz), 7.40 (1H, d, J=8.5 Hz), 7.31-7.25 (3H, m), 6.99 (1H, d, J=7.0 Hz), 5.18 (1H, s), 4.56-4.55 (1H, d, J=8.0 Hz), 4.16-4.07 (2H, m), 2.84-2.80 (1H, m), 2.26 (3H, s), 1.42-1.35 (2H, m), 0.88-0.84 (1H, m), 0.73 (3H, d, J=6.5 Hz), 0.59 (3H, d, J=6.5 Hz).

Step 2: (6R, 7S)-6-(4-bromophenyl)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] Isoquinoline (1b)

(73) ##STR00031##

(74) Compound 1a (950 mg) was dissolved in anhydrous dichloromethane (5 ml), and then aluminum chloride (913 mg) was added at room temperature. The reaction was stirred at room temperature for 3 hours, to which an aqueous saturated sodium carbonate solution was poured and then ethyl acetate was added for extraction. The organic phase was separated, and the separated organic phase was washed twice with an aqueous diluted sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography to afford a yellow solid product 1b (900 mg). .sup.1H NMR (500 MHz, DMSO-d6) δ (ppm): 13.02 (1H, s), 8.05 (1H, s), 7.41 (2H, d, J=8.5 Hz), 7.26 (1H, d, J=8.5 Hz), 7.12 (2H, d, J=8.5 Hz), 6.80 (1H, d, J=9.0 Hz), 4.22 (1H, d), 3.99 (1H, d, J=4.0 Hz), 3.84 (1H, d), 2.90-2.87 (1H, m), 2.32 (3H, s), 1.80-1.73 (1H, m), 1.48-1.43 (1H, m), 0.99-0.93 (1H, m), 0.89 (3H, d, J=6.5 Hz), 0.81 (3H, d, J=6.5 Hz).

Step 3: (6R, 7S)-6-(4-bromophenyl)-7-isobutyl-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (1c)

(75) ##STR00032##

(76) Compound 1b (398 mg) was dissolved in dichloromethane (5 ml), and 3,4-dihydro-2H-pyran (252 mg) and p-toluenesulfonic acid monohydrate (228 mg) were added at room temperature. The reaction was stirred at room temperature for 3 hours. Then it was poured into an aqueous sodium carbonate aqueous solution and ethyl acetate was added for extraction. The organic phase was separated, and the separated organic phase was washed twice with an aqeuous diluted sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography to afford a yellow oily Product 1c (350 mg). .sup.1H NMR (500 MHz, DMSO-d6) δ (ppm): 8.10 (1H, s), 7.45-7.41 (3H, m), 7.12-7.09 (2H, m), 6.86 (1H, d, J=8.5 Hz), 5.77 (1H, d, J=9.0 Hz), 4.25-4.20 (1H, m), 4.05-4.01 (2H, m), 3.87-3.82 (2H, m), 3.73-3.67 (1H, m), 2.90-2.85 (1H, m), 2.31 (3H, s), 2.04-1.99 (1H, m), 1.95-1.92 (1H, m), 1.78-1.71 (2H, m), 1.57-1.56 (2H, m), 1.46-1.42 (1H, m), 0.99-0.93 (1H, m), 0.90-0.89 (3H, m), 0.81-0.73 (3H, m).

Step 4: tert-butyl 3-((4-((6R, 7S)-7-isobutyl-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7,8,9-tetrahydro-3H-pyrazolo[3,4-h]isoquinolin-6-yl) phenyl) amino)azetidin-1-carboxylate (1d)

(77) ##STR00033##

(78) Compound 1c (260 mg) was dissolved in 1,4-dioxane (5 ml), followed by tert-butyl 3-aminoazetidin-1-carboxylate (139 mg), Pd.sub.2(dba).sub.3 (25 mg, CAS #: 51364-51-3), xantphos (47 mg, CAS #: 161265-03-8) and cesium carbonate (352 mg). The reaction was then refluxed under a nitrogen atmosphere overnight. Then, ethyl acetate and water were added for extraction. The organic phase was separated, and the separated organic phase was washed twice with an aqueous diluted sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography to afford a yellow oily product 1d (120 mg). .sup.1H NMR (500 MHz, DMSO-d6) δ (ppm): 8.15 (1H, s), 7.41-7.38 (1H, m), 6.86-6.83 (3H, m), 6.38-6.36 (2H, m), 6.05 (1H, d, J=8.0 Hz), 5.75 (1H, d, J=10.0 Hz), 4.23-4.13 (3H, m), 4.09-4.07 (1H, m), 3.86-3.79 (3H, m), 3.72-3.69 (1H, m), 3.67-3.57 (2H, m), 2.89-2.84 (1H, m), 2.42-2.37 (1H, m), 2.30 (3H, d, J=2.0 Hz), 2.04-2.01 (1H, m), 1.95-1.92 (1H, m), 1.76-1.70 (2H, m), 1.58-1.56 (2H, m), 1.37 (9H, s), 0.89-0.87 (3H, m), 0.79-0.77 (3H, m).

Step 5: N-(4-((6R, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline-6-yl) phenyl) azetidin-3-amine (1e)

(79) ##STR00034##

(80) Compound 1d (120 mg) was dissolved in dichloromethane (1.5 ml), and trifluoroacetic acid (0.5 ml) was added at room temperature. The reaction was performed at room temperature for 3 hours. Then it was poured into an aqueous saturated sodium carbonate solution and ethyl acetate was added for extraction. The organic phase was separated, and the separated organic phase was washed twice with an aqueous diluted sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product 1e (81 mg). LCMS ESI (+): 390 (M+1).sup.+.

Step 6: 1-(3-fluoropropyl)-N-(4-((6R, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinolin-6-yl) phenyl) azetidin-3-amine (1)

(81) ##STR00035##

(82) Compound 1e (81 mg) was dissolved in DMF (2 ml), followed by adding 1-bromo-3-fluoropropane (30 mg), diisopropylethylamine (54 mg), and sodium iodide (3 mg) at room temperature. The reaction was carried out at room temperature overnight. Then, ethyl acetate and water were added for extraction. The organic phase was separated, and the organic phase was washed twice with an aqueous diluted sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by preparative silica gel chromatography to afford a yellow oily product 1 (16 mg). .sup.1H NMR (500 MHz, DMSO-d6) δ (ppm): 13.00 (1H, s), 9.87 (1H, s), 8.03 (1H, s), 7.22 (1H, d, J=7.5 Hz), 6.89 (2H, d, J=7.5 Hz), 6.76 (1H, d, J=8.5 Hz), 6.41 (2H, d, J=8.0 Hz), 6.06 (1H, s), 4.55 (1H, t, J=6.0 Hz), 4.45 (1H, t, J=6.0 Hz), 4.25-4.11 (3H, m), 3.80 (2H, s), 2.95-2.88 (3H, m), 2.36-2.33 (3H, m), 1.85-1.71 (4H, m), 1.48-1.43 (1H, m), 1.31-1.29 (1H, m), 0.87 (3H, d, J=6.0 Hz), 0.77 (3H, s). LCMS ESI(+): 450 (M+1).

Example 2

N-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinolin-6-yl) phenyl)-1-(3-fluoropropyl) azetidin-3-amine (2)

(83) ##STR00036##

Step 1: (2S)-2-(((1H-indazol-4-yl) methyl) (methyl) amino)-1-(4-chloro-2,6-difluorophenyl)-4-methylpentane 1-ol (2a)

(84) ##STR00037##

(85) Compound I-60-2 (1130 mg), 4-(chloromethyl)-1H-indazole (ADV947321888, available from Advanced ChemBlocks Inc., 630 mg), potassium carbonate (2.5 g), and sodium iodide (54 Mg) were mixed in anhydrous DMF at room temperature. The reaction was stirred at room temperature overnight. Then, ethyl acetate and water were added for extraction. The organic phase was separated, and the separated organic phase was washed twice with an aqueous diluted sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography to afford a yellow solid product 2a (1660 mg). .sup.1H NMR (500 MHz, dmso-d6) δ (in ppm): 12.98 (1H, s), 8.03 (1H, s), 7.40-7.41 (1H, d, J=5.0 Hz), 7.25-6.30 (3H, m), 7.02-7.03 (1H, d, J=5.0 Hz), 5.43-5.44 (1H, d, J=5.0 Hz), 5.01-5.05 (1H, m), 1.54-1.60 (1H, m), 1.35-1.42 (1H, m), 0.72-0.73 (3H, d, J=5.0 Hz), 0.59-0.63 (3H, m), 0.56-0.57 (3H, d, J=5.0 Hz).

Step 2: (6S, 7S)-6-(4-chloro-2,6-difluorophenyl)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (2b)

(86) ##STR00038##

(87) The synthetic route for 1b was repeated, which starts from 2a. .sup.1H NMR (500 MHz, dmso-d6) δ (in ppm): 13.02 (1H, s), 8.06 (1H, s), 7.32-7.34 (2H, d, J=10.0 Hz), 7.24-7.25 (1H, d, J=5.0 Hz), 6.68-6.70 (1H, d, J=10.0 Hz), 4.31-4.33 (1H, d, J=10.0 Hz), 4.25-4.28 (1H, d, J=15.0 Hz), 4.09-4.12 (1H, d, J=15.0 Hz), 3.20-3.26 (1H, m), 2.30 (3H, s), 1.72-1.80 (1H, m), 1.52-1.60 (1H, m), 0.83-0.89 (4H, m), 0.71-0.72 (3H, d, J=5.0 Hz). LCMS ESI(+): 390 (M+H).

Step 3: (6S, 7S)-6-(4-chloro-2,6-difluorophenyl)-7-isobutyl-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (2c)

(88) ##STR00039##

(89) The synthetic route for 2c was repeated, which starts from 2b. LCMS ESI(+): 474 (M+H).

Step 4: N-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7, 8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinolin-6-yl) phenyl)-1-(3-fluoropropyl) azetidin-3-amine (2d)

(90) ##STR00040##

(91) Compound 2c (300 mg), 3-(3-fluoropropyl) azetidine (CAS: 1538772-53-0; 124 mg), Pd.sub.2 (dba).sub.3 (29 mg), t-buXphos (CAS: 564483-19-8; 54 mg) and cesium carbonate (821 mg) were suspended in anhydrous toluene (5 ml). The mixture was refluxed under a nitrogen atmosphere for 12 hours. After cooling, an aqueous solution was added to quench the reaction and ethyl acetate was added for extraction. The collected ethyl acetate layer was then wash twice with an aqueous diluted sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to afford 2d (210 mg). LCMS ESI (+): 570 (M+1).sup.+.

Step 5: N-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinolin-6-yl) phenyl)-1-(3-fluoropropyl) azetidin-3-amine (2)

(92) ##STR00041##

(93) Compound 2d (215 mg) was dissolved in 15 ml of methanol, and then 5 ml of concentrated hydrochloric acid was added. The mixture was stirred at room temperature for 2 hours. Then, an aqueous saturated sodium bicarbonate solution was added and ethyl acetate was added for extraction. The collected ethyl acetate layer was then washed twice with an aqueous diluted sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column to afford product 2 (113 Mg). .sup.1H NMR (500 MHz, dmso-d6) δ (in ppm): 12.96 (1H, s), 8.03 (1H, s), 7.21-7.23 (1H, d, J=10.0 Hz), 6.68-6.70 (1H, d, J=10.0 Hz), 6.59-6.60 (1H, d, J=5.0 Hz), 6.13-6.15 (2H, d, J=10.0 Hz), 4.50-4.52 (1H, t, J=5.0 Hz), 4.40-4.42 (1H, d, J=5.0 Hz), 4.21-4.24 (1H, m), 4.10-4.13 (1H, m), 3.93-3.80 (1H, m), 3.68 (2H, m), 3.51-3.55 (1H, m), 3.18 (1H, s), 2.82 (1H, s), 2.28 (3H, s), 1.91 (1H, s), 1.77 (1H, s), 1.63-1.72 (2H, m), 1.47-1.53 (1H, m), 0.89-0.95 (1H, m), 0.85-0.86 (3H, d, J=5.0 Hz), 0.71-0.72 (3H, d, J=5.0 Hz). LCMS ESI (+): 486 (M+1).sup.+.

Example 3

N-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinolin-6-yl) phenyl)-1-(2,3-difluoropropyl) azetidin-3-amine (3)

(94) ##STR00042##

Step 1: N-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7, 8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinolin-6-yl) phenyl)-1-(2,3-difluoropropyl) azetidin-3-amine (3a)

(95) ##STR00043##

(96) The synthetic route for 2d was repeated, which started from 2c and I-100′-1. .sup.1H NMR (500 MHz, DMSO-d6) δ 8.07 (s, 1H), 7.39 (dd, J=8.7, 4.7 Hz, 1H), 6.75 (d, J=8.7 Hz, 1H), 6.61 (d, J=6.6 Hz, 1H), 6.13 (d, J=12.4 Hz, 2H), 5.76 (dd, J=9.1, 3.6 Hz, 1H), 4.86-4.42 (m, 3H), 4.22 (t, J=13.8 Hz, 1H), 4.11 (t, J=13.8 Hz, 2H), 3.97 (q, J=6.6 Hz, 1H), 3.86 (d, J=11.6 Hz, 1H), 3.70 (d, J=8.7 Hz, 3H), 3.16 (s, 1H), 2.90 (s, 2H), 2.72 (dd, J=22.5, 5.4 Hz, 2H), 2.44-2.33 (m, 2H), 2.27 (s, 3H), 2.03 (d, J=12.3 Hz, 1H), 1.94 (d, J=13.0 Hz, 1H), 1.74 (s, 2H), 1.57 (s, 2H), 1.51 (d, J=11.8 Hz, 1H), 0.93 (s, 1H), 0.85 (d, J=6.7 Hz, 3H), 0.71 (d, J=6.5 Hz, 3H).

Step 2: N-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinolin-6-yl) phenyl)-1-(2,3-difluoropropyl) azetidin-3-amine (3)

(97) ##STR00044##

(98) The synthetic route for 2 was repeated, which started from 3a. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 12.98 (s, 1H), 8.02 (s, 1H), 7.21 (d, J=8.6 Hz, 1H), 6.69 (d, J=8.6 Hz, 1H), 6.62 (d, J=6.8 Hz, 1H), 6.13 (d, J=12.4 Hz, 2H), 4.82-4.44 (m, 3H), 4.22 (d, J=16.7 Hz, 1H), 4.13-4.05 (m, 2H), 3.96 (h, J=6.6 Hz, 1H), 3.72-3.66 (m, 2H), 3.18 (td, J=9.8, 3.8 Hz, 1H), 2.89 (t, J=6.6 Hz, 2H), 2.73 (d, J=5.4 Hz, 1H), 2.68 (d, J=5.4 Hz, 1H), 2.27 (s, 3H), 1.74 (dddd, J=13.1, 10.2, 6.3, 3.8 Hz, 1H), 1.49 (ddd, J=13.9, 9.7, 4.0 Hz, 1H), 0.91 (ddd, J=13.9, 9.7, 3.9 Hz, 1H), 0.85 (d, J=6.6 Hz, 3H), 0.71 (d, J=6.5 Hz, 3H). LCMS ESI (+): 504 (M+1).sup.+.

Example 4

N-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinolin-6-yl) phenyl)-1-(3,3,3-trifluoropropyl) azetidin-3-amine (4)

(99) ##STR00045##

Step 1: N-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7, 8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinolin-6-yl) phenyl)-1-(3,3,3-trifluoropropyl) azetidin-3-amine (4a)

(100) ##STR00046##

(101) The synthetic route for 2d was repeated, which started from 2c and I-100′-2. LCMS ESI (+): 606 (M+1).sup.+.

Step 2: N-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinolin-6-yl) phenyl)-1-(3,3,3-trifluoropropyl) azetidin-3-amine (4)

(102) ##STR00047##

(103) The synthetic route for 2 was repeated, which started from 4a. .sup.1H NMR (500 MHz, DMSO-d6, δ, ppm): 12.96 (s, 1H), 8.02 (s, 1H), 7.21 (d, J=8.5 Hz, 1H), 6.69 (d, J=8.5 Hz, 1H), 6.58 (d, J=5.0 Hz, 1H), 6.14 (d, J=12.0 Hz, 2H), 4.22 (d, J=16.5 Hz, 1H), 4.1-4.0 (m, 2H), 4.0-3.9 (m, 1H), 3.7-3.6 (m, 2H), 3.2-3.1 (m, 1H), 2.79 (t, J=6.5 Hz, 2H), 2.60 (t, J=7.5 Hz, 2H), 2.4-2.3 (m, 2H), 2.27 (s, 3H), 1.8-1.7 (m, 1H), 1.5-1.4 (m, 1H), 1.0-0.9 (m, 1H), 0.85 (d, J=6.5 Hz, 3H), 0.71 (d, J=6.5 Hz, 3H). LCMS ESI (+): 522 (M+1).sup.+.

Example 5

N-(4-((6S, 7S)-7-cyclopropylmethyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinolin-6-yl)-3,5-difluorophenyl)-1-(3-fluoropropyl) azetidin-3-amine (5)

(104) ##STR00048##

Step 1: (2S)-2-(((1H-indazol-4-yl) methyl) (methyl) amino)-1-(4-chloro-2,6-difluorophenyl)-3-cyclopropyl propan-1-ol (5a)

(105) ##STR00049##

(106) The synthetic route for 2a was repeated, which started from I-60-3. .sup.1H NMR (500 MHz, DMSO-d6) δ 13.24 (s, 1H), 8.34 (d, J=1.3 Hz, 1H), 7.64 (d, J=8.3 Hz, 1H), 7.49 (t, J=8.1 Hz, 3H), 7.24 (d, J=6.9 Hz, 1H), 5.55 (d, J=3.5 Hz, 1H), 5.20 (dd, J=9.1, 3.4 Hz, 1H), 4.38 (q, J=13.7 Hz, 2H), 3.46 (td, J=8.5, 5.4 Hz, 1H), 2.57 (s, 3H), 1.88-1.76 (m, 1H), 0.95 (ddd, J=13.7, 7.8, 5.3 Hz, 1H), 0.87-0.77 (m, 1H), 0.55 (tdd, J=8.9, 5.3, 3.9 Hz, 1H), 0.48 (tq, J=9.0, 4.1 Hz, 1H), 0.16 (dq, J=9.3, 4.8 Hz, 1H), −0.00 (dq, J=9.4, 4.8 Hz, 1H). LCMS ESI (+): 406 (M+1).sup.+.

Step 2: (6S, 7S)-6-(4-chloro-2,6-difluorophenyl)-7-cyclopropylmethyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo[3,4-h]isoquinoline (5b)

(107) ##STR00050##

(108) The synthetic route for 1b was repeated, which started from 5a. .sup.1H NMR (500 MHz, DMSO-d6) δ 13.30 (s, 1H), 8.31 (s, 1H), 7.54 (d, J=31.8 Hz, 3H), 6.96 (d, J=8.6 Hz, 1H), 4.71 (s, 1H), 4.50-4.23 (m, 2H), 3.36 (s, 1H), 2.70-2.50 (m, 3H), 1.89 (s, 1H), 1.23 (d, J=21.3 Hz, 1H), 1.03 (s, 1H), 0.61 (ddq, J=12.6, 8.6, 4.1 Hz, 2H), 0.25 (s, 1H), −0.01 (d, J=9.5 Hz, 1H). LCMS ESI (+): 388 (M+1).sup.+.

Step 3: (6S, 7S)-6-(4-chloro-2,6-difluorophenyl)-7-(cyclopropylmethyl-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (5c)

(109) ##STR00051##

(110) The synthetic route for 2c was repeated, which started from 5b. LCMS ESI (+): 472 (M+H).

Step 4: N-(4-((6S, 7S)-7-cyclopropylmethyl-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinolin-6-yl)-3,5-difluorophenyl)-1-(3-fluoropropyl) azetidin-3-amine (5d)

(111) ##STR00052##

(112) The synthetic route for 2d was repeated, which started from 5c and 3-(3-fluoropropyl) azetidine (CAS: 1538772-53-0). LCMS ESI(+): 568 (M+H).

Step 5: N-(4-((6S, 7S)-7-cyclopropylmethyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinolin-6-yl)-3,5-difluorophenyl)-1-(3-fluoropropyl) azetidin-3-amine (5)

(113) ##STR00053##

(114) The synthetic route for 2 was repeated, which started from 5d. .sup.1H NMR (500 MHz, DMSO-d6) δ 12.96 (s, 1H), 8.02 (s, 1H), 7.22 (d, J=8.8 Hz, 1H), 6.67 (d, J=8.6 Hz, 1H), 6.13 (d, J=12.3 Hz, 2H), 5.33 (dd, J=5.5, 4.2 Hz, 1H), 4.51 (t, J=6.1 Hz, 1H), 4.41 (t, J=6.1 Hz, 1H), 4.25 (d, J=9.4 Hz, 1H), 4.14 (d, J=15.9 Hz, 1H), 3.93 (p, J=6.5 Hz, 1H), 3.86 (d, J=16.1 Hz, 1H), 3.65 (d, J=6.7 Hz, 2H), 2.77 (s, 3H), 2.37 (d, J=5.7 Hz, 3H), 1.69 (p, J=6.3 Hz, 1H), 1.64 (t, J=6.5 Hz, 1H), 1.47 (d, J=7.9 Hz, 2H), 1.30 (td, J=7.2, 4.4 Hz, 3H), 0.88-0.83 (m, 2H). LCMS ESI(+): 484 (M+H).

Example 6

N-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinolin-6-yl) phenyl)-1-propylazetidin-3-amine (6)

(115) ##STR00054##

Step 1: tert-butyl 3-((3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinolin-6-yl) phenyl) amino) azetidin-1-carboxylate (6a)

(116) ##STR00055##

(117) The synthetic route for 1d was repeated, which started from 2c and tert-butyl 3-aminoazetidin-1-carboxylate (CAS: 193269-78-2). LCMS ESI (+): 610 (M+1).sup.+.

Step 2: N-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinolin-6-yl) phenyl) azetidin-3-amine (6b)

(118) ##STR00056##

(119) The synthetic route for 1e was repeated, which started from 6a. LCMS ESI (+): 426 (M+1).sup.+.

Step 3: N-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinolin-6-yl) phenyl)-1-propylazetidin-3-amine (6)

(120) ##STR00057##

(121) The synthetic route for 1 was repeated, which started from 6b and 1-bromopropane. LCMS ESI (+): 468 (M+1).sup.+.

Example 7

1-butyl-N-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinolin-6-yl) phenyl) azetidin-3-amine (7)

(122) ##STR00058##

Step 1: 1-butyl-N-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinolin-6-yl) phenyl) azetidin-3-amine (7)

(123) ##STR00059##

(124) The synthetic route for 6 was repeated, which started from 6b and 1-bromobutane. LCMS ESI (+): 482 (M+1).sup.+.

Example 8

N-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinolin-6-yl) phenyl)-1-isobutylazetidin-3-amine (8)

(125) ##STR00060##

Step 1: N-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinolin-6-yl) phenyl)-1-isobutylazetidin-3-amine (8)

(126) ##STR00061##

(127) The synthetic route for 6 was repeated, which started from 6b and 1-bromo-2-methylpropane. LCMS ESI (+): 482 (M+1).sup.+.

Example 9

N-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinolin-6-yl) phenyl)-1-pentylazetidin-3-amine (9)

(128) ##STR00062##

Step 1: N-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinolin-6-yl) phenyl)-1-pentylazetidin-3-amine (9)

(129) ##STR00063##

(130) The synthetic route for 6 was repeated, which started from 6b and 1-bromo-pentane. LCMS ESI (+): 496 (M+1).sup.+.

Example 10

(6S, 7S)-6-(2,6-difluoro-4-((1-(3-fluoropropyl) azetidin-3-yl) thio) phenyl)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (10)

(131) ##STR00064##

Step 1: tert-butyl 3-((3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinolin-6-yl) phenyl) thio) azetidin-1-carboxylate (10a)

(132) ##STR00065##

(133) Coupling method according to the literature Org. Lett. 2004, vol. 6, 4587-5590: Compound 2c, tert-butyl 3-mercaptoazetidin-1-carboxylate (CAS: 941585-25-7), Pd.sub.2 (dba).sub.3, Xantphos and DIPEA were refluxed in anhydrous 1,6-dioxane. The reaction was then cooled. Water was added and then the mixture was extracted with ethyl acetate. The ethyl acetate layer was collected, then washed twice with an aqueous diluted sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatograph to afford product 10a. LCMS ESI (+): 627 (M+1).sup.+.

Step 2: (6S, 7S)-6-(4-(azetidin-3-ylthio)-2,6-difluorophenyl)-7-isobutyl-8-methyl-6,7, 8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (10b)

(134) ##STR00066##

(135) The synthetic route for 1e was repeated, which started from 10a. LCMS ESI (+): 443 (M+1).sup.+.

Step 3: (6S, 7S)-6-(2,6-difluoro-4-((1-(3-fluoropropyl) azetidin-3-yl) thio) phenyl)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (10)

(136) ##STR00067##

(137) The synthetic route for 1 was repeated, which started from 10b and 1-bromo-3-fluoropropane. LCMS ESI (+): 503 (M+1).sup.+.

Example 11

(6S, 7S)-6-(2,6-difluoro-4-((1-propyl) azetidin-3-yl) thio) phenyl)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (11)

(138) ##STR00068##

Step 1: (6S, 7S)-6-(2,6-difluoro-4-((1-propyl) azetidin-3-yl) thio) phenyl)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (11)

(139) ##STR00069##

(140) The synthetic route for 1 was repeated, which started from 10b and 1-bromo-propane. LCMS ESI (+): 485 (M+1).sup.+.

Example 12

(6S, 7S)-6-(4-((1-butylazetidin-3-yl) thio)-2,6-difluorophenyl)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (12)

(141) ##STR00070##

Step 1: (6S, 7S)-6-(4-((1-butylazetidin-3-yl) thio)-2,6-difluorophenyl)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (12)

(142) ##STR00071##

(143) The synthetic route for 11 was repeated, which started from 10b and 1-bromobutane. LCMS ESI (+): 499 (M+1).sup.+.

Example 13

(6S, 7S)-6-(2,6-difluoro-4-((1-isobutylazetidin-3-yl) thio) phenyl)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (13)

(144) ##STR00072##

Step 1: (6S, 7S)-6-(2,6-difluoro-4-((1-isobutylazetidin-3-yl) thio) phenyl)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (13)

(145) ##STR00073##

(146) The synthetic route for 11 was repeated, which started from 10b and 1-bromo-2-methylpropane. LCMS ESI (+): 499 (M+1).sup.+.

Example 14

(6S, 7S)-6-(2,6-difluoro-4-((1-pentylazetidin-3-yl) thio) phenyl)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (14)

(147) ##STR00074##

Step 1: (6S, 7S)-6-(2,6-difluoro-4-((1-pentylazetidin-3-yl) thio) phenyl)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (14)

(148) ##STR00075##

(149) The synthetic route for 11 was repeated, which started from 10b and 1-bromo-pentane. LCMS ESI (+): 513 (M+1).sup.+.

Example 15

(6S, 7S)-6-(2,6-difluoro-4-((1-(3-fluoropropyl) azetidin-3-yl) oxy) phenyl)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (15)

(150) ##STR00076##

Step 1: tert-butyl 3-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinolin-6-yl) phenoxy) azetidin-1-carboxylate (15a)

(151) ##STR00077##

(152) Compound 2c (100 mg), tert-butyl 3-hydroxyazetidin-1-carboxylate (CAS: 141699-55-0; 73 mg), Pd.sub.2(dba).sub.3 (10 mg), t-buXphos (CAS: 564483-19-8; 9 mg) and cesium carbonate (202 mg) were suspended in anhydrous toluene (3 ml). The mixture was refluxed under a nitrogen atmosphere overnight. After cooling; water was added to quench the reaction and the mixture was extracted with ethyl acetate. The collected ethyl acetate layer was then washed twice with an aqueous diluted sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified to afford product 15a (63 mg). LCMS ESI (+): 611 (M+1).sup.+.

Step 2: (6S, 7S)-6-(4-(azetidin-3-yloxy)-2,6-difluorophenyl)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (15b)

(153) ##STR00078##

(154) The synthetic route for 1e was repeated, which started from 15a. LCMS ESI (+): 427 (M+1).sup.+.

Step 3: (6S, 7S)-6-(2,6-difluoro-4-((1-(3-fluoropropyl) azetidin-3-yl) oxy) phenyl)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (15)

(155) ##STR00079##

(156) The synthetic route for 1 was repeated, which started from 15b and 1-bromo-3-fluoropropane. .sup.1H NMR (500 MHz, dmso-d6) δ (in ppm): 12.99 (1H, s), 8.05 (1H, s), 7.22-7.24 (1H, d, J=10.0 Hz), 6.66-6.68 (1H, d, J=10.0 Hz), 6.59-6.61 (2H, d, J=10.0 Hz), 4.79-4.85 (1H, m), 4.49-4.52 (1H, t, J=5.0 Hz), 4.40-4.42 (1H, t, J=5.0 Hz), 4.20-4.26 (2H, m), 4.07-4.12 (1H, m), 3.70-3.74 (2H, m), 3.20-3.22 (1H, m), 3.17-3.18 (2H, d, J=5.0 Hz), 2.94-2.97 (2H, m), 2.29 (3H, s), 1.60-1.80 (3H, m), 1.49-1.56 (1H, m), 0.88-0.90 (1H, m), 0.84-0.86 (3H, d, J=10.0 Hz), 0.70-0.71 (3H, d, J=5.0 Hz). LCMS ESI (+): 487 (M+1).sup.+.

Example 16

(6S, 7S)-6-(2,6-difluoro-4-((1-propylazetidin-3-yl)oxy) phenyl)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (16)

(157) ##STR00080##

Step 1: (6S, 7S)-6-(2,6-difluoro-4-((1-propylazetidin-3-yl)oxy) phenyl)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (16)

(158) ##STR00081##

(159) The synthetic route for 1 was repeated, which started from 15b and 1-bromopropane. .sup.1H NMR (500 MHz, dmso-d6) δ (in ppm): 12.99 (1H, s), 8.05 (1H, s), 7.22-7.24 (1H, d, J=10.0 Hz), 6.66-6.68 (1H, d, J=10.0 Hz), 6.60-6.61 (2H, d, J=5.0 Hz), 4.78-4.83 (1H, m), 4.20-4.26 (2H, m), 4.08-4.11 (1H, m), 4.20-4.26 (2H, m), 3.69-3.73 (2H, m), 3.20-3.22 (1H, m), 3.17-3.22 (1H, m), 2.90-2.93 (2H, m), 2.36-2.39 (2H, m), 2.29 (3H, s), 1.70-1.78 (1H, m), 1.49-1.56 (1H, m), 1.26-1.34 (2H, m), 0.85-0.90 (1H, m), 0.83-0.85 (3H, d, J=10.0 Hz), 0.70-0.71 (3H, d, J=5.0 Hz). LCMS ESI (+): 469 (M+1).sup.+.

Example 17

(6S, 7S)-6-(2,6-difluoro-4-((1-isobutylazetidin-3-yl)oxy) phenyl)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (17)

(160) ##STR00082##

Step 1: (6S, 7S)-6-(2,6-difluoro-4-((1-isobutylazetidin-3-yl)oxy) phenyl)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (17)

(161) ##STR00083##

(162) The synthetic route for 1 was repeated, which started from 15b and 1-bromo-2-methylpropane. .sup.1H NMR (500 MHz, dmso-d6) δ (in ppm): 11.96 (1H, s), 8.00 (1H, s), 7.34-7.36 (1H, d, J=10.0 Hz), 6.75-6.77 (1H, d, J=10.0 Hz), 6.33-6.36 (2H, d, J=10.0 Hz), 4.94 (1H, s), 4.68 (1H, m), 4.41-4.45 (1H, m), 4.25 (1H, m), 3.60 (1H, m), 3.41 (2H, m), 3.04-3.14 (6H, m), 2.55-2.64 (4H, m), 1.71-1.84 (3H, m), 1.40 (9H, m), 0.96-0.98 (6H, d, J=10.0 Hz), 0.88-0.90 (3H, d, J=10.0 Hz), 0.75-0.76 (3H, d, J=5.0 Hz). LCMS ESI (+): 483 (M+1).sup.+.

Example 18

(6S, 7S)-6-(4-((1-butylazetidin-3-yl) oxy) phenyl)-7-isobutyl-8-methyl-6,7,8, 9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (18)

(163) ##STR00084##

Step 1: (6S, 7S)-6-(4-((1-butylazetidin-3-yl) oxy) phenyl)-7-isobutyl-8-methyl-6,7,8, 9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (18)

(164) ##STR00085##

(165) The synthetic route for 1 was repeated, which started from 15b and 1-bromo-butane. .sup.1H NMR (500 MHz, dmso-d6) δ (in ppm): 12.99 (1H, s), 8.04 (1H, s), 7.22-7.24 (1H, d, J=10.0 Hz), 6.65-6.67 (1H, d, J=10.0 Hz), 6.60-6.62 (2H, d, J=10.0 Hz), 4.80-4.87 (1H, m), 4.20-4.28 (2H, m), 4.08-4.11 (1H, m), 3.87 (2H, m), 3.20 (3H, m), 2.55 (2H, m), 2.29 (3H, s), 1.71-1.76 (1H, m), 1.50-1.54 (1H, m), 1.29-1.32 (4H, m), 0.83-0.87 (6H, d, J=10.0 Hz), 0.70-0.71 (3H, d, J=5.0 Hz). LCMS ESI (+): 483 (M+1).sup.+.

Example 19

(6S, 7S)-6-(4-((1-pentylazetidin-3-yl) oxy) phenyl)-7-isobutyl-8-methyl-6,7,8, 9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (19)

(166) ##STR00086##

Step 1: (6S, 7S)-6-(4-((1-pentylazetidin-3-yl) oxy) phenyl)-7-isobutyl-8-methyl-6,7,8, 9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (19)

(167) ##STR00087##

(168) The synthetic route for 1 was repeated, which started from 15b and 1-bromo-pentane. .sup.1H NMR (500 MHz, dmso-d6) δ (in ppm): 12.08 (1H, s), 8.01 (1H, s), 7.28-7.30 (1H, d, J=10.0 Hz), 6.78-6.79 (1H, d, J=5.0 Hz), 6.32-6.34 (2H, d, J=10.0 Hz), 4.95 (1H, s), 4.52-4.68 (1H, m), 4.22-4.44 (4H, m), 3.38-3.57 (3H, m), 2.80-2.86 (1H, m), 2.53 (3H, s), 2.04 (2H, s), 1.50-1.85 (4H, m), 1.31-1.35 (4H, m), 1.24-1.28 (4H, m), 0.86-0.92 (6H, m), 0.76-0.77 (3H, d, J=5.0 Hz). LCMS ESI (+): 497 (M+1).sup.+.

Example 20

(6S, 7S)-7-(cyclopropylmethyl)-6-(2,6-difluoro-4-((1-(3-fluoropropyl) azetidin-3-yl)oxy) phenyl)-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (20)

(169) ##STR00088##

Step 1: tert-butyl 3-(4-((6S, 7S)-7-(isopropylmethyl)-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinolin-6-yl)-3,5-difluorophenoxy) azetidin-1-carboxylate (20a)

(170) ##STR00089##

(171) The synthetic route for 15a was repeated, which started from 5c and tert-butyl 3-hydroxylazetidin-1-carboxylate. LCMS ESI (+): 609 (M+1).sup.+.

Step 2: (6S, 7S)-6-(4-(azetidin-3-yloxy)-2,6-difluorophenyl)-7-cyclopropylmethyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (20b)

(172) ##STR00090##

(173) The synthetic route for 1e was repeated, which started from 20a. LCMS ESI (+): 425 (M+1).sup.+.

Step 3: (6S, 7S)-7-(cyclopropylmethyl)-6-(2,6-difluoro-4-((1-(3-fluoropropyl) azetidin-3-yl)oxy) phenyl)-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinoline (20)

(174) ##STR00091##

(175) The synthetic route for 1 was repeated, which started from 20b and 1-bromo-3-fluoropropane. .sup.1H NMR (500 MHz, DMSO-d6) δ 12.99 (s, 1H), 8.04 (s, 1H), 7.23 (dd, J=8.8, 4.2 Hz, 1H), 6.71-6.55 (m, 3H), 5.52-5.36 (m, 2H), 4.81 (td, J=5.7, 3.6 Hz, 1H), 4.50 (t, J=6.0 Hz, 1H), 4.40 (t, J=6.1 Hz, 1H), 4.35 (d, J=9.0 Hz, 1H), 4.18-4.07 (m, 1H), 3.90 (d, J=16.2 Hz, 1H), 3.72 (qd, J=5.9, 4.9, 2.3 Hz, 2H), 2.97-2.87 (m, 3H), 2.38 (d, J=3.5 Hz, 3H), 2.23-2.16 (m, 1H), 2.05 (dt, J=13.7, 5.8 Hz, 1H), 1.68 (p, J=6.4 Hz, 1H), 1.63 (t, J=6.5 Hz, 1H), 1.60 (dd, J=6.1, 1.3 Hz, 2H), 1.02-0.95 (m, 0.5H), 0.80 (d, J=6.8 Hz, 0.5H), 0.38 (ddd, J=23.9, 8.8, 4.4 Hz, 1H), 0.00 (dt, J=9.3, 4.4 Hz, 0.5H), −0.28 (dt, J=9.3, 4.4 Hz, 0.5H). LCMS ESI (+): 485 (M+1).sup.+.

Example 21

(6S, 7S)-7-cyclopropylmethyl-6-(2,6-difluoro-4-((1-propylazetidin-3-yl) oxy) phenyl)-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (21)

(176) ##STR00092##

Step 1: (6S, 7S)-7-cyclopropylmethyl-6-(2,6-difluoro-4-((1-propylazetidin-3-yl) oxy) phenyl)-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (21)

(177) ##STR00093##

(178) The synthetic route for 1 was repeated, which started from 20b and 1-bromopropane. .sup.1H NMR (500 MHz, DMSO-d6) δ 12.99 (s, 1H), 8.03 (s, 1H), 7.24 (dd, J=13.4, 8.8 Hz, 1H), 6.61 (dd, J=25.0, 9.9 Hz, 3H), 5.51-5.38 (m, 2H), 5.32 (t, J=4.7 Hz, 1H), 5.26 (d, J=3.1 Hz, 1H), 4.80 (q, J=5.5, 5.1 Hz, 1H), 4.35 (d, J=9.2 Hz, 1H), 4.16 (d, J=6.7 Hz, 1H), 4.12 (s, 1H), 3.90 (d, J=15.8 Hz, 1H), 3.80 (d, J=16.9 Hz, 1H), 3.70 (t, J=6.3 Hz, 2H), 2.90 (tt, J=5.5, 2.8 Hz, 2H), 2.40-2.34 (m, 4H), 2.18 (s, 1H), 2.09-2.02 (m, 1H), 2.01-1.95 (m, 1H), 1.60 (dd, J=5.9, 1.3 Hz, 1H), 1.29 (q, J=7.1 Hz, 2H), 1.16-1.11 (m, 1H), 0.89-0.83 (m, 3H). LCMS ESI (+): 467 (M+1).sup.+.

Example 22

(6S, 7S)-7-cyclopropylmethyl-6-(2,6-difluoro-4-((1-isobutylazetidin-3-yl) oxy) phenyl)-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (22)

(179) ##STR00094##

Step 1: (6S, 7S)-7-cyclopropylmethyl-6-(2,6-difluoro-4-((1-isobutylazetidin-3-yl) oxy) phenyl)-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (22)

(180) ##STR00095##

(181) The synthetic route for 1 was repeated, which started from 20b and 1-bromo-2-methylpropane. LCMS ESI (+): 481 (M+1).sup.+.

Example 23

(6S, 7S)-6-(4-((1-butylazetidin-3-yl) oxy)-2,6-difluorophenyl)-7-cyclopropylmethyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (23)

(182) ##STR00096##

Step 1: (6S, 7S)-6-(4-((1-butylazetidin-3-yl) oxy)-2,6-difluorophenyl)-7-cyclopropylmethyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (23)

(183) ##STR00097##

(184) The synthetic route for 1 was repeated, which started from 20b and 1-bromo-butane. LCMS ESI (+): 481 (M+1).sup.+.

Example 24

(6S, 7S)-7-cyclopropylmethyl-6-(2,6-difluoro-4-((1-pentylazetidin-3-yl) oxy) phenyl)-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (24)

(185) ##STR00098##

Step 1: (6S, 7S)-7-cyclopropylmethyl-6-(2,6-difluoro-4-((1-pentylazetidin-3-yl) oxy) phenyl)-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (24)

(186) ##STR00099##

(187) The synthetic route for 1 was repeated, which started from 20b and 1-bromo-pentane. LCMS ESI (+): 495 (M+1).sup.+.

Example 25

(6S, 7S)-7-cyclopropylmethyl-6-(2,6-difluoro-4-((1-(3-fluoropropyl) azetidin-3-yl) thio)) phenyl)-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (25)

(188) ##STR00100##

Step 1: tert-butyl 3-((4-((6S, 7S)-7-cyclopropylmethyl-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinolin-6-yl)-3,5-difluorophenyl) thio) azetidin-1-carboxylate (25a)

(189) ##STR00101##

(190) Coupling method according to the literature Org. Lett. 2004, vol. 6, 4587-5590: Compound 5c, tert-butyl 3-mercaptoazetidin-1-carboxylate (CAS: 941585-25-7), Pd.sub.2 (dba).sub.3, Xantphos and DIPEA were refluxed in anhydrous 1,6-dioxane. Ater cooling, water was added and then the mixture was extracted with ethyl acetate. The ethyl acetate layer was collected, then washed twice with an aqueous diluted sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatograph to afford product 25a. LCMS ESI (+): 625 (M+1).sup.+.

Step 2: (6S, 7S)-6-(4-(azetidin-3-ylthio)-2,6-difluorophenyl)-7-cyclopropylmethyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (25b)

(191) ##STR00102##

(192) The synthetic route for 1e was repeated, which started from 25a. LCMS ESI (+): 441 (M+1).sup.+.

Step 3: (6S, 7S)-7-cyclopropylmethyl-6-(2,6-difluoro-4-((1-(3-fluoropropyl) azetidin-3-yl) thio)) phenyl)-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (25)

(193) ##STR00103##

(194) The synthetic route for 1 was repeated, which started from 25b and 1-bromo-3-fluoropropane. LCMS ESI (+): 501 (M+1).sup.+.

Example 26

(6S, 7S)-7-cyclopropylmethyl-6-(2,6-difluoro-4-((1-propylazetidin-3-yl) thio) phenyl)-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (26)

(195) ##STR00104##

Step 1: (6S, 7S)-7-cyclopropylmethyl-6-(2,6-difluoro-4-((1-propylazetidin-3-yl) thio) phenyl)-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (26)

(196) ##STR00105##

(197) The synthetic route for 1 was repeated, which started from 25b and 1-bromopropane. LCMS ESI (+): 483 (M+1).sup.+.

Example 27

(6S, 7S)-6-(4-((1-butylazetidin-3-yl)thio)-2,6-difluorophenyl)-7-cyclopropylmethyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (27)

(198) ##STR00106##

Step 1: (6S, 7S)-6-(4-((1-butylazetidin-3-yl)thio)-2,6-difluorophenyl)-7-cyclopropylmethyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (27)

(199) ##STR00107##

(200) The synthetic route for 11 was repeated, which started from 25b and 1-bromobutane. LCMS ESI (+): 497 (M+1).sup.+.

Example 28

(6S, 7S)-6-(4-((1-isobutylazetidin-3-yl) thio)-2,6-difluorophenyl)-7-cyclopropylmethyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (28)

(201) ##STR00108##

Step 1: (6S, 7S)-6-(4-((1-isobutylazetidin-3-yl) thio)-2,6-difluorophenyl)-7-cyclopropylmethyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (28)

(202) ##STR00109##

(203) The synthetic route for 11 was repeated, which started from 25b and 1-bromo-2-methylpropane. LCMS ESI (+): 497 (M+1).sup.+.

Example 29

(6S, 7S)-7-cyclopropylmethyl-6-(2,6-difluoro-4-((1-pentylazetidin-3-yl) thio) phenyl)-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (29)

(204) ##STR00110##

Step 1: (6S, 7S)-7-cyclopropylmethyl-6-(2,6-difluoro-4-((1-pentylazetidin-3-yl) thio) phenyl)-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinoline (29)

(205) ##STR00111##

(206) The synthetic route for 1 was repeated, which started from 25b and 1-bromo-pentane. LCMS ESI (+): 511 (M+1).sup.+.

Example 30

3-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinolin-6-yl) phenoxy) azetidin-1-aldehyde (30)

(207) ##STR00112##

Step 1: 3-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h]isoquinolin-6-yl) phenoxy) azetidin-1-aldehyde (30)

(208) ##STR00113##

(209) 15b (727 mg), DIPEA (1.04 ml) and DMF (10 ml) were stirred at room temperature overnight. Water was added and the mixture was extracted with ethyl acetate. The ethyl acetate layer was collected, washed twice with an aqueous diluted sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified through a column chromatograph to afford 240 mg of product 30. .sup.1H NMR (500 MHz, dmso-d6) δ (in ppm): 12.99 (1H, s), 8.04 (1H, s), 8.02 (1H, s), 7.22-7.24 (1H, d, J=10.0 Hz), 6.65-6.69 (3H, m), 5.11-5.16 (1H, m), 4.57-4.62 (1H, m), 4.33-4.38 (1H, m), 4.20-4.26 (2H, m), 4.07-4.11 (2H, m), 3.80-3.82 (2H, d, J=10.0 Hz), 2.29 (3H, s), 1.71-1.77 (1H, m), 1.49-1.56 (1H, m), 0.82-0.90 (4H, m), 0.70-0.71 (3H, d, J=5.0 Hz). LCMS ESI (+): 455 (M+1).sup.+.

Example 31

1-(3-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinolin-6-yl) phenoxy) azetidin-1-yl) ethan-1-one (31)

(210) ##STR00114##

Step 1: 1-(3-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinolin-6-yl) phenoxy) azetidin-1-yl) ethan-1-one (31)

(211) ##STR00115##

(212) 15b (1 equivalent), acetic anhydride (1.2 equivalents), DIPEA (3 equivalents), and DMF (20 volumes; relative to 15b) were stirred at room temperature overnight. Water was added and the mixture was extracted with ethyl acetate. The ethyl acetate layer was collected, washed twice with dilute sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to afford product 31. LCMS ESI (+): 469 (M+1).sup.+.

Example 32

1-(3-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinolin-6-yl) phenoxy) azetidin-1-yl) propan-1-one (32)

(213) ##STR00116##

Step 1: 1-(3-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinolin-6-yl) phenoxy) azetidin-1-yl) propan-1-one (32)

(214) ##STR00117##

(215) The synthetic route for 31 was repeated, which started from 15b and propionic anhydride. LCMS ESI (+): 483 (M+1).sup.+.

Example 33

1-(3-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinolin-6-yl) phenoxy) azetidin-1-yl) butan-1-one (33)

(216) ##STR00118##

Step 1: 1-(3-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinolin-6-yl) phenoxy) azetidin-1-yl) butan-1-one (33)

(217) ##STR00119##

(218) The synthetic route for 31 was repeated, which started from 15b and but anionic anhydride. LCMS ESI (+): 497 (M+1).sup.+.

Example 34

1-(3-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinolin-6-yl) phenoxy) azetidin-1-yl)-2-methylpropan-1-one (34)

(219) ##STR00120##

Step 1: 1-(3-(3,5-difluoro-4-((6S, 7S)-7-isobutyl-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo [3,4-h] isoquinolin-6-yl) phenoxy) azetidin-1-yl)-2-methylpropan-1-one (1)

(220) ##STR00121##

(221) The synthetic route for 31 was repeated, which started from 15b and isobutanionic anhydride. LCMS ESI (+): 497 (M+1).sup.+.

Evaluation of Pharmacological Activity

(222) Assay 1. Effect of compounds of the present invention on degradation efficiency of estrogen receptors in MCF-7 and MCF-7/TAMR1 cells as measured by immunoblotting (Western Blot)

(223) 1. Experimental Materials:

(224) 1) Reagents: RIPA Lysate (ThermoFisher #89901), Protease Inhibitor (ThermoFisher #78438), BCA Quantitation Kit (ThermoFisher #23225), 4×LDS Sample Buffer (ThermoFisher #NP0007), 4-12% gradient precast gel (ThermoFisher #NW04120BOX), electrophoresis solution (ThermoFisher #B0002), transfer solution (ThermoFisher #BT00061), NC membrane (Merck #HATF00010), estrogen receptor (Cell Signaling Technology #8644), (3-actin (Cell Signaling Technology #4970), HRP-labeled murine secondary antibody (ThermoFisher #31430), HRP-labeled rabbit secondary antibody (ThermoFisher #31460), substrate color development kit (ThermoFisher #34076), TBST, PBS, skim milk powder

(225) 2) Instruments: Running glue tank (ThermoFisher #B1000), film transfer tank (ThermoFisher #NW2000), power supply (ThermoFisher #PS0301)

(226) 3) Cells: Human breast cancer cell lines MCF-7 and MCF-7/TAMR1 that are from cell bank of the Chinese Academy of Sciences and used directly.

(227) 2. Experimental Procedures:

(228) 1) 2 ml of MCF-7 or MCF-7/TAMR1 cells was added to a medium in a 6-well plate at a density of 0.6*10.sup.6 cells/ml in which the medium is DMEM high glucose medium supplemented with 10% FBS. The plate was incubated at 37° C. for 24 hours in a 5% CO.sub.2 cell incubator.

(229) 2) To each culture, β-actin was added as an internal standard. Then different concentrations of corresponding compounds (10 nm or 100 nm of compounds of the present invention or 0.1% DMSO as a control) were added to the 6-well plate for incubating MCF-7 cells. The plate was incubated for 8 hours in a 5% CO.sub.2 cell incubator.

(230) 3) Cells were harvested and extracted with with a volume volume of RIPA lysate that is 3-5 times of the volume of cells containing 1*protease inhibitor for total cellular proteins.

(231) 4) Immunoblotting analysis was carried out using equal amounts of cellular proteins.

(232) Experimental Results

(233) The experimental results were shown in FIGS. 1 to 3.

(234) Assay 2: Inhibitory effect of compounds of the present invention on the growth of MCF-7 cells by a cell drug inhibition experiment

(235) 1. Experimental Materials

(236) 1) Reagents: DMEM high glucose medium (Lonza #12-604F), FBS (BI #04-00F1ACS), antibiotics (Thermofisher #15070063), bovine insulin (Western reagent #11070-73-8), trypsin (Thermofisher #25200-056), CellTiter-Glo reagent (Promega #G7571)

(237) 2) Instruments: American MD-M5 microplate reader 5;

(238) 3) Cells: as above.

(239) 2. Experimental Procedures:

(240) 1) 100 μl of MCF-7 cells was added to a medium in a white opaque 96-well plate at a density of 5000 cells/ml in which the medium is DMEM high-glucose medium supplemented with 10% FBS. The cells were cultured in a 5% CO.sub.2 cell incubator at 37° C. for 24 hours.

(241) 2) Different concentrations of corresponding compounds were added to the 96-well plate for incubating MCF-7 cells. The cells were incubated for 10 days in a 5% CO.sub.2 cell incubator.

(242) 3) To each well of MCF-7 cells, 100 μl of CellTiter-Glo reagent was added and then set aside at room temperature for 10 minutes. The chemiluminescence signal was read using an MD-M5 microplate reader, and the data was processed using GraphPad Prism to calculate IC.sub.50 value.

(243) 3. Experimental Results:

(244) TABLE-US-00001 Examples IC.sub.50 value Fulvestrant +++ CAS: 129453- 61- 8 (positive control) Example 1 ++++ Example 2 +++++ Example 3 ++ Example 4 ++ Example 5 ++ Example 6 ++ Example 7 ++ Example 8 ++ Example 9 ++ Example 10 +++++ Example 11 +++++ Example 12 +++ Example 13 +++ Example 14 ++ Example 15 +++++ Example 16 +++++ Example 17 ++++ Example 18 ++ Example 19 ++ Example 20 ++ Example 21 ++ Example 22 ++ Example 23 ++ Example 24 ++ Example 25 +++ Example 26 +++ Example 27 ++ Example 28 ++ Example 29 ++ Example 30 +++++ Example 31 +++++ Example 32 +++++ Example 33 +++ Example 34 ++++ In the table: ++ represents: 1 μM < IC.sub.50 +++ represensts: 0.01 μM < IC.sub.50 ≤ 1 μM ++++ represensts: 1 nM < IC.sub.50 ≤ 0.01 μM +++++ represensts: IC.sub.50 ≤ 1 nM

(245) As can be seen from the IC.sub.50 value of compounds of above examples, compounds of the examples may inhibit proliferation of human breast cancer MCF-7 cells at extremely low concentrations. Some of compounds had a higher IC.sub.50 (IC.sub.50 value in the range of ++++ to +++++) activity than known SERD molecules (Cancer Research, 2016, 76: 3307).

(246) It was shown from the above experimental results involving the degradation of estrogen receptors in MCF-7 and the inhibitory effects on growth of MCF-7 cells that the compounds of the present invention can effectively degrade estrogen receptors and inhibit the proliferation of human breast cancer MCF-7 cells and thus they can be used for the treatment or preventation of various diseases associated with estrogen by degrading estrogen receptor, such as cancer (breast cancer, ovarian cancer, colorectal cancer, prostate cancer, endometrial cancer), osteoporosis, neurodegenerative diseases, cardiovascular diseases, insulin resistance, lupus erythematosus, endometriosis, and obesity.

(247) While embodiments of the present invention have been illustrated and described, it is not intended that all possible embodiments of the invention have been illustrated and described. Rather, the words used in the specification are merely illustrative and not restrictive, and it shall be understood that various changes may be made without departing from the spirit and scope of the invention.