The present application claims the priority of the following applications: patent application No. 202010614735.9 entitled “Tetrahydroisoquinoline Compounds and Use Thereof” and filed with the China National Intellectual Property Administration on Jun. 30, 2020 and patent application No. 202110296535.8 entitled “Tetrahydroisoquinoline Compounds and Use Thereof” and filed with the China National Intellectual Property Administration on Mar. 19, 2021, which are incorporated into the present application in their entireties by reference.

TECHNICAL FIELD

The present disclosure relates to tetrahydroisoquinoline compounds as PRMT5 inhibitors, a preparation method thereof, pharmaceutical compositions comprising the compounds and the use thereof in the treatment of diseases mediated by PRMT5 or associated with PRMT5.

BACKGROUND

Epigenetic alterations are key mediators driving and maintaining the malignant phenotype of tumors. DNA methylation, histone acetylation and methylation, non-coding RNAs, and changes in post-translational modification, rather than changes in DNA sequences, are all epigenetic drivers of cancer development. Arginine methylation is an important class of post-translational modifications that affect cell growth and proliferation, apoptosis, angiogenesis and metastasis by regulating transcription and post-transcriptional RNA processing. There are three types of methylarginines: ω-NG-monomethylarginine (MMA), ω-NG,N′G-asymmetric dimethylarginine (ADMA), and ω-NG,N′G-symmetric dimethylarginine (SDMA). Such modifications are to transfer a methyl group from S-adenosylmethionine (AdoMet) to the arginine side chain of histone and nonhistone under the catalysis of the protein arginine methyltransferase (PRMT) family. Nine PRMT genes are annotated in the human genome and based on the methylarginine types produced, these genes are grouped into type I (PRMT1, 2, 3, 4, 6 and 8), type II (PRMT5 and PRMT9), and type III enzymes (PRMT7). PRMT5 is a major type II enzyme, which can catalyze the symmetric dimethylation of arginine. PRMT5 was first discovered in two-hybrid experiment for detecting proteins having interactions with the Janus tyrosine kinase (Jak2).

PRMT5 is a general transcriptional factor and can form complexes with other transcriptional factors, including BRG1 and hBRM, Blimp1 and Snail. PRMT5 is implicated with many different cellular biological processes through methylating many substrates within the cytoplasm and nucleus, including residue Arg3 of histone H4 (H4R3) and residue Arg8 of H3 (H3R8). H4R3 methylation is associated with transcriptional repression, while H3R8 methylation is considered to be associated with both transcriptional activation and transcriptional repression. In addition to directly inducing inhibitory histone labeling, the role of the enzyme PRMT5 in gene silencing is also mediated by forming multiple repressive protein complexes, including NuRD components, HDACs, MDB proteins, and DNA methyltransferases. The substrate specificity of PRMT5 is affected through its interaction with some binding proteins. The core component of such protein complexes is MEP50. MEP50 is necessary to the enzymatic activity of PRMT5. Studies have found that PRMT5 can methylate proteins involved in RNA splicing, such as SmD3, which hence can be used to monitor the chemical activity of PRMT5 in cell organism.

PRMT5 plays an important role in tumorigenesis. Studies have found that the expression of PRMT5 is up-regulated in many tumors, including lymphoma, lung cancer, breast cancer and colorectal cancer. Furthermore, the expression of PRMT5 is increased in the samples from mantle cell lymphoma (MCL) patients, while PRMT5 knockout can inhibit the cell proliferation of MCL, indicating that PRMT5 plays an important role in MCL. PRMT5 over-expression promotes cell proliferation, and in cell lines of melanoma, breast cancer and lung cancer, PRMT5 knockout can inhibit the proliferation of the cells. Therefore, PRMT5 is a potential target for cancer treatment.

Loss of methylthioadenosine phosphorylase (MTAP) endows cells with selective dependence on PRMT5 and its binding protein WDR77. MTAP is often lost due to its proximity to the normally deleted tumor suppressor gene CDKN2A. Cells carrying MTAP deletion have an increased concentration of intracellular methylthioadenosine (MTA, a metabolite cleaved by MTAP). Furthermore, MTA specifically inhibits the enzyme activity of PRMT5. MTA or small-molecule inhibitors of PRMT5 significantly inhibit the cell viability of cancer cell lines having MTAP deletion relative to cells expressing MTAP.

Therefore, there is a need in the art to develop active small-molecule compounds that can inhibit the activity of PRMT5 and can treat various diseases associated with PRMT5.

SUMMARY

The present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof,

[Image Omitted]

wherein

X is selected from CR⁸R⁹, NR¹, or O;

R¹is selected from H, C₁-C₁₀alkyl, C₂-C₁₀alkenyl, C₂-C₁₀alkynyl, C₆-C₁₀aryl, 5- to 10-membered heteroaryl, C₃-C₁₀cycloalkyl, or 3- to 10-membered heterocyclyl, wherein C₁-C₁₀alkyl, C₂-C₁₀alkenyl, C₂-C₁₀alkynyl, C₆-C₁₀aryl, 5- to 10-membered heteroaryl, C₃-C₁₀cycloalkyl, or 3- to 10-membered heterocyclyl is optionally substituted with one or more R^a;

R²and R⁵are independently selected from H, halogen, CN, or the following groups which are optionally substituted with one or more R^b: NH₂, C₁-C₁₀alkyl, C₁-C₁₀alkoxy, C₃-C₁₀cycloalkyl, or 3- to 10-membered heterocyclyl;

R³and R⁴are independently selected from H, deuterium, halogen, CN, or the following groups which are optionally substituted with one or more R^b: NH₂, C₁-C₁₀alkyl, C₁-C₁₀alkoxy, C₃-C₁₀cycloalkyl, or 3- to 10-membered heterocyclyl;

or, R³and R⁴together with the C to which they are attached form C₃-C₁₀cycloalkyl or 3- to 10-membered heterocyclyl, wherein the C₃-C₁₀cycloalkyl or 3- to 10-membered heterocyclyl is optionally substituted with one or more R^e;

R⁸and R⁹are independently selected from H, OH, CN, NO₂, C₁-C₁₀alkyl, C₁-C₁₀alkoxy, C₃-C₁₀cycloalkyl, or 3- to 10-membered heterocyclyl, wherein the C₁-C₁₀alkyl, C₁-C₁₀alkoxy, C₃-C₁₀cycloalkyl, or 3- to 10-membered heterocyclyl is optionally substituted with one or more R^f;

m is selected from 0, 1, 2, 3 or 4;

R^ais selected from halogen, ═O, OH, CN, NO₂, C₁-C₁₀alkyl, C₁-C₁₀alkoxy, C₆-C₁₀aryl, 5- to 10-membered heteroaryl, C₃-C₁₀cycloalkyl, or 3- to 10-membered heterocyclyl;

R^bis selected from halogen, OH, CN, ═O, NO₂, C₁-C₁₀alkyl, C₃-C₁₀cycloalkyl, 3- to 10-membered heterocyclyl, C₁-C₁₀alkoxy, C₃-C₁₀cycloalkyloxy, 3- to 10-membered heterocyclyloxy, C₂-C₁₀alkenyl, C₂-C₁₀alkynyl, C₆-C₁₀aryl, 5- to 10-membered heteroaryl, C₆-C₁₀aryloxy, or 5- to 10-membered heteroaryloxy;

R^eand R^fare independently selected from halogen, ═O, OH, CN, NO₂, C₁-C₁₀alkyl, or C₁-C₁₀alkoxy;

R⁶and R⁷are independently selected from H, C₁-C₁₀alkyl, C₆-C₁₀aryl, 5- to 10-membered heteroaryl, C₃-C₁₀cycloalkyl, or 3- to 10-membered heterocyclyl, wherein the C₁-C₁₀alkyl, C₆-C₁₀aryl, 5- to 10-membered heteroaryl, C₃-C₁₀cycloalkyl, or 3- to 10-membered heterocyclyl is optionally substituted with one or more R^c;

R^cis selected from halogen, OH, CN, ═O, C₁-C₁₀alkoxy, C₆-C₁₀aryl, C₆-C₁₀aryloxy, 5- to 10-membered heteroaryl, 5- to 10-membered heteroaryloxy, C₃-C₁₀cycloalkyl, C₃-C₁₀cycloalkyloxy, 3- to 10-membered heterocyclyl, or 3- to 10-membered heterocyclyloxy, wherein the C₁-C₁₀alkoxy, C₆-C₁₀aryl, C₆-C₁₀aryloxy, 5- to 10-membered heteroaryl, 5- to 10-membered heteroaryloxy, C₃-C₁₀cycloalkyl, C₃-C₁₀cycloalkyloxy, 3- to 10-membered heterocyclyl, or 3- to 10-membered heterocyclyloxy is optionally substituted with one or more R^c1;

R^c1is selected from halogen, OH, ═O, CN, NO₂, C₁-C₁₀alkyl, or C₁-C₁₀alkoxy; or, R⁶and R⁷together with the N to which they are attached form 6- to 13-membered spiroheterocycloalkyl, 6- to 13-membered fused heterocycloalkyl, 6- to 13-membered bridged heterocycloalkyl, 3- to 8-membered monocyclic heterocycloalkyl, or 5- to 10-membered heteroaryl, wherein the 6- to 13-membered spiroheterocycloalkyl, 6- to 13-membered fused heterocycloalkyl, 6- to 13-membered bridged heterocycloalkyl, 3- to 8-membered monocyclic heterocycloalkyl, or 5- to 10-membered heteroaryl is optionally substituted with one or more R^d;

R^dis selected from halogen, OH, ═O, CN, NO₂, C₁-C₁₀alkyl, C₁-C₁₀alkoxy, C₁-C₁₀alkyl-NH—, (C₁-C₁₀alkyl)₂-N—, C₆-C₁₀aryl, 5- to 10-membered heteroaryl, C₃-C₁₀cycloalkyl, C₃-C₁₀cycloalkyloxy, C₃-C₁₀cycloalkyl-NH—, (C₃-C₁₀cycloalkyl)₂-N—, or 3- to 10-membered heterocyclyl, wherein the C₁-C₁₀alkyl, C₁-C₁₀alkoxy, C₁-C₁₀alkyl-NH—, (C₁-C₁₀alkyl)₂-N—, C₆-C₁₀aryl, 5- to 10-membered heteroaryl, C₃-C₁₀cycloalkyl, C₃-C₁₀cycloalkyloxy, C₃-C₁₀cycloalkyl-NH—, (C₃-C₁₀cycloalkyl)₂-N—, or 3- to 10-membered heterocyclyl is optionally substituted with one or more R^d1;

R^d1is selected from deuterium, halogen, OH, ═O, CN, NO₂, or C₁-C₁₀alkoxy.

In some embodiments, R⁶and R⁷are independently selected from H, C₁-C₁₀alkyl, C₆-C₁₀aryl, 5- to 10-membered heteroaryl, C₃-C₁₀cycloalkyl, or 3- to 10-membered heterocyclyl, wherein the C₁-C₁₀alkyl, C₆-C₁₀aryl, 5- to 10-membered heteroaryl, C₃-C₁₀cycloalkyl, or 3- to 10-membered heterocyclyl is optionally substituted with one or more R^c; R^cis selected from halogen, OH, CN, ═O, C₁-C₁₀alkoxy, C₆-C₁₀aryl, C₆-C₁₀aryloxy, 5- to 10-membered heteroaryl, 5- to 10-membered heteroaryloxy, C₃-C₁₀cycloalkyl, C₃-C₁₀cycloalkyloxy, 3- to 10-membered heterocyclyl, or 3- to 10-membered heterocyclyloxy, wherein the C₁-C₁₀alkoxy, C₆-C₁₀aryl, C₆-C₁₀aryloxy, 5- to 10-membered heteroaryl, 5- to 10-membered heteroaryloxy, C₃-C₁₀cycloalkyl, C₃-C₁₀cycloalkyloxy, 3- to 10-membered heterocyclyl, or 3- to 10-membered heterocyclyloxy is optionally substituted with one or more R^c1; R^c1is selected from halogen, OH, ═O, CN, NO₂, C₁-C₁₀alkyl, or C₁-C₁₀alkoxy; or, R⁶and R⁷together with the N to which they are attached form 6- to 13-membered spiroheterocycloalkyl, 6- to 13-membered bridged heterocycloalkyl, 3- to 8-membered monocyclic heterocycloalkyl, or 5- to 10-membered heteroaryl, wherein the 6- to 13-membered spiroheterocycloalkyl, 6- to 13-membered bridged heterocycloalkyl, 3- to 8-membered monocyclic heterocycloalkyl, or 5- to 10-membered heteroaryl is optionally substituted with one or more R^d; R^dis selected from halogen, OH, ═O, CN, NO₂, C₁-C₁₀alkyl, C₁-C₁₀alkoxy, C₁-C₁₀alkyl-NH—, (C₁-C₁₀alkyl)₂-N—, C₆-C₁₀aryl, 5- to 10-membered heteroaryl, C₃-C₁₀cycloalkyl, C₃-C₁₀cycloalkyloxy, C₃-C₁₀cycloalkyl-NH—, (C₃-C₁₀cycloalkyl)₂-N—, or 3- to 10-membered heterocyclyl, wherein the C₁-C₁₀alkyl, C₁-C₁₀alkoxy, C₆-C₁₀aryl, 5- to 10-membered heteroaryl, C₃-C₁₀cycloalkyl, or 3- to 10-membered heterocyclyl is optionally substituted with one or more R^d1; R^d1is selected from halogen, OH, ═O, CN, NO₂, or C₁-C₁₀alkoxy.

In some embodiments, R¹is selected from H, C₁-C₁₀alkyl, C₆-C₁₀aryl, 5- to 10-membered heteroaryl, C₃-C₁₀cycloalkyl or 3- to 10-membered heterocyclyl, wherein the C₁-C₁₀alkyl, C₆-C₁₀aryl, 5- to 10-membered heteroaryl, C₃-C₁₀cycloalkyl, or 3- to 10-membered heterocyclyl is optionally substituted with one or more R^a.

In some embodiments, R¹is selected from H, and C₁-C₁₀alkyl optionally substituted with one or more R^a.

In some embodiments, R¹is selected from H, CH₃, or CH₂CH₃.

In some embodiments, R¹is selected from CH₃or CH₂CH₃.

In some embodiments, R², R³, R⁴and R⁵are independently selected from H, halogen, CN, or the following groups which are optionally substituted with one or more R^b: NH₂, C₁-C₁₀alkyl, C₁-C₁₀alkoxy, C₃-C₁₀cycloalkyl, or 3- to 10-membered heterocyclyl; or, R³and R⁴together with the C to which they are attached form C₃-C₁₀cycloalkyl or 3- to 10-membered heterocyclyl, wherein the C₃-C₁₀cycloalkyl or 3- to 10-membered heterocyclyl is optionally substituted with one or more R^e.

In some embodiments, R², R³, R⁴and R⁵are independently selected from H, halogen, CN, C₁-C₁₀alkyl, C₁-C₁₀alkoxy, C₃-C₁₀cycloalkyl, or 3- to 10-membered heterocyclyl, wherein the C₁-C₁₀alkyl, C₁-C₁₀alkoxy, C₃-C₁₀cycloalkyl, or 3- to 10-membered heterocyclyl is optionally substituted with one or more R^b.

In some embodiments, R², R³, R⁴and R⁵are independently selected from H, halogen, CN, C₁-C₁₀alkyl, or C₁-C₁₀alkoxy.

In some embodiments, R², R³, R⁴and R⁵are all H.

In some embodiments, R⁸and R⁹are independently selected from H, OH, CN, NO₂, C₁-C₁₀alkyl, or C₁-C₁₀alkoxy.

In some embodiments, X is selected from NR¹or O.

In some embodiments, X is selected from NCH₃, NCH₂CH₃, or O.

In some embodiments, m is selected from 0, 1 or 2.

In some embodiments, m is selected from 0 or 1.

In some embodiments, m is selected from 0.

In some embodiments, R⁶and R⁷are independently selected from H, C₁-C₁₀alkyl, C₃-C₁₀cycloalkyl, or 3- to 10-membered heterocyclyl, wherein the C₁-C₁₀alkyl, C₃-C₁₀cycloalkyl, or 3- to 10-membered heterocyclyl is optionally substituted with one or more R^c.

In some embodiments, R⁶and R⁷are independently selected from H, or C₁-C₁₀alkyl optionally substituted with one or more R^c.

In some embodiments, R^cis selected from halogen, OH, C₆-C₁₀aryl, 5- to 10-membered heteroaryl, C₃-C₁₀cycloalkyl, or 3- to 10-membered heterocyclyl, wherein the C₆-C₁₀aryl, 5- to 10-membered heteroaryl, C₃-C₁₀cycloalkyl, or 3- to 10-membered heterocyclyl is optionally substituted with one or more R^c1.

In some embodiments, R^cis selected from halogen, OH, C₆-C₁₀aryl, or 5- to 10-membered heteroaryl, wherein the C₆-C₁₀aryl, or 5- to 10-membered heteroaryl is optionally substituted with one or more R^c1.

In some embodiments, R^cis selected from C₆-C₁₀aryl, or 5- to 10-membered heteroaryl, wherein the C₆-C₁₀aryl, or 5- to 10-membered heteroaryl is optionally substituted with one or more R^c1.

In some embodiments, R^cis selected from phenyl or pyridyl optionally substituted with one or more R^c1.

In some embodiments, R^c1is selected from halogen, OH, ═O, CN, or C₁-C₁₀alkyl.

In some embodiments, R^c1is selected from halogen.

In some embodiments, R^c1is selected from F.

In some embodiments, R⁶and R⁷are independently selected from CH₃, CH₂CH₃, benzyl, 4-fluorophenethyl, or pyridine-3-ethyl.

In some embodiments, R⁶and R⁷together with the N to which they are attached form 6- to 13-membered spiroheterocycloalkyl, 6- to 13-membered fused heterocycloalkyl, 6- to 13-membered bridged heterocycloalkyl, 3- to 8-membered monocyclic heterocycloalkyl, or 5- to 10-membered heteroaryl, wherein the 6- to 13-membered spiroheterocycloalkyl, 6- to 13-membered bridged heterocycloalkyl, 3- to 8-membered monocyclic heterocycloalkyl, or 5- to 10-membered heteroaryl is optionally substituted with one or more R^d.

In some embodiments, R^dis selected from halogen, OH, ═O, CN, NO₂, C₁-C₁₀alkyl, C₁-C₁₀alkoxy, C₁-C₁₀alkyl-NH—, (C₁-C₁₀alkyl)₂-N—, C₆-C₁₀aryl, 5- to 10-membered heteroaryl, C₃-C₁₀cycloalkyl, C₃-C₁₀cycloalkyloxy, C₃-C₁₀cycloalkyl-NH—, (C₃-C₁₀cycloalkyl)₂-N—, or 3- to 10-membered heterocyclyl, wherein the C₁-C₁₀alkyl, C₁-C₁₀alkoxy, C₆-C₁₀aryl, 5- to 10-membered heteroaryl, C₃-C₁₀cycloalkyl, or 3- to 10-membered heterocyclyl is optionally substituted with one or more R^d1.

In some embodiments, R^dis selected from halogen, OH, ═O, NO₂, CN, C₁-C₁₀alkyl, C₁-C₁₀alkoxy, C₁-C₁₀alkyl-NH—, or (C₁-C₁₀alkyl)₂-N—, wherein the C₁-C₁₀alkyl, C₁-C₁₀alkoxy, C₁-C₁₀alkyl-NH—, or (C₁-C₁₀alkyl)₂-N— is optionally substituted with one or more R^d1.

In some embodiments, R^dis selected from halogen, OH, NO₂, CN, C₁-C₁₀alkyl, C₁-C₁₀alkoxy, C₁-C₁₀alkyl-NH—, or (C₁-C₁₀alkyl)₂-N—, wherein the C₁-C₁₀alkyl, C₁-C₁₀alkoxy, C₁-C₁₀alkyl-NH—, or (C₁-C₁₀alkyl)₂-N— is optionally substituted with one or more R^d1.

In some embodiments, R^dis selected from halogen, OH, ═O, CN, C₁-C₁₀alkyl, C₁-C₁₀alkoxy, C₁-C₁₀alkyl-NH—, or (C₁-C₁₀alkyl)₂-N—, wherein the C₁-C₁₀alkyl, C₁-C₁₀alkoxy, C₁-C₁₀alkyl-NH—, or (C₁-C₁₀alkyl)₂-N— is optionally substituted with one or more R^d1.

In some embodiments, R^dis selected from halogen, CN, C₁-C₁₀alkyl, or C₁-C₁₀alkoxy, wherein the C₁-C₁₀alkyl, or C₁-C₁₀alkoxy is optionally substituted with one or more R^d1.

In some embodiments, R^d1is selected from halogen, OH, ═O, CN, NO₂, or C₁-C₁₀alkoxy.

In some embodiments, R^d1is selected from halogen, OH, ═O, CN, or NO₂.

In some embodiments, R^d1is selected from halogen or ═O.

In some embodiments, R^d1is selected from deuterium or ═O.

In some embodiments, R^dis selected from halogen, OH, ═O, NO₂, CN, C₁-C₁₀alkyl, C₁-C₁₀alkoxy, C₁-C₁₀alkyl-NH—, or (C₁-C₁₀alkyl)₂-N—.

In some embodiments, R^dis selected from halogen, OH, NO₂, CN, C₁-C₁₀alkyl, C₁-C₁₀alkoxy, C₁-C₁₀alkyl-NH—, or (C₁-C₁₀alkyl)₂-N—.

In some embodiments, R^dis selected from halogen, OH, ═O, CN, C₁-C₁₀alkyl, C₁-C₁₀alkoxy, C₁-C₁₀alkyl-NH—, or (C₁-C₁₀alkyl)₂-N—.

In some embodiments, R^dis selected from halogen, CN, C₁-C₁₀alkyl, or C₁-C₁₀alkoxy.

In some embodiments, R^dis selected from F, ═O, OH, N(CH₃)₂, CN, C(O)CH₃, CH₂CH₃, OCH₃, or OCD₃.

In some embodiments, R^dis selected from F, ═O, OH, N(CH₃)₂, CN, C(O)CH₃, OCH₃, or OCD₃.

In some embodiments, R^dis selected from F, ═O, OH, N(CH₃)₂, CN, C(O)CH₃, or OCH₃.

In some embodiments, R^dis selected from F, ═O, OH, N(CH₃)₂, CN, CH₂CH₃, or OCH₃.

In some embodiments, R^dis selected from F, CN, C(═O)CH₃, CH₂CH₃, or OCH₃.

In some embodiments, R^dis selected from F, CN, CH₂CH₃, or OCH₃.

In some embodiments, R⁶and R⁷together with the N to which they are attached form 6- to 13-membered spiroheterocycloalkyl, wherein the 6- to 13-membered spiroheterocycloalkyl is optionally substituted with one or more R^d.

In some embodiments R⁶and R⁷together with the N to which they are attached form

[Image Omitted]

optionally substituted with one or more R^d, wherein:

n, n′, p, and p′ are independently selected from 1, 2, 3, or 4, and n+n′+p+p′≤10;

W and Y are independently selected from CH₂, NH, or O; and

Z is selected from CH₂, NH, O, or a bond.

In some embodiments, n, n′, p, and p′ are independently selected from 1, 2 or 3, and n+n′+p+p′≤10.

In some embodiments, n, n′, p, and p′ are independently selected from 1 or 2, and n+n′+p+p′≤8.

In some embodiments, W and Y are selected from CH₂.

In some embodiments, Z is selected from O, CH₂, or a bond.

In some embodiments, Z is selected from CH₂, or a bond.

In some embodiments, R⁶and R⁷together with the N to which they are attached form the following groups which are optionally substituted with one or more R^d:

[Image Omitted]

In some embodiments, R⁶and R⁷together with the N to which they are attached form the following groups which are optionally substituted with one or more R^d:

[Image Omitted]

In some embodiments, R⁶and R⁷together with the N to which they are attached form the following groups:

[Image Omitted]

In some embodiments, R⁶and R⁷together with the N to which they are attached form the following groups:

[Image Omitted]

In some embodiments, R⁶and R⁷together with the N to which they are attached form the following groups:

[Image Omitted]

In some embodiments, R⁶and R⁷together with the N to which they are attached form 6- to 13-membered bridged heterocycloalkyl, wherein the 6- to 13-membered bridged heterocycloalkyl is optionally substituted with one or more R^d.

In some embodiments, R⁶and R⁷together with the N to which they are attached form

[Image Omitted]

optionally substituted with one or more R^d, wherein:

q, q′, and k are independently selected from 1, 2 or 3; and

Q is selected from CH₂, NH, O, or a bond.

In some embodiments, q and q′ are independently selected from 1 or 2, and k is selected from 1, 2 or 3.

In some embodiments, q, q′ and k are independently selected from 1 or 2.

In some embodiments, Q is selected from CH₂or O.

In some embodiments, R⁶and R⁷together with the N to which they are attached form the following groups which are optionally substituted with one or more R^d.

[Image Omitted]

In some embodiments, R⁶and R⁷together with the N to which they are attached form the following groups which are optionally substituted with one or more R^d:

[Image Omitted]

In some embodiments, R⁶and R⁷together with the N to which they are attached form

[Image Omitted]

In some embodiments, R⁶and R⁷together with the N to which they are attached form

[Image Omitted]

In some embodiments, R⁶and R⁷together with the N to which they are attached form 6- to 13-membered fused heterocycloalkyl, 3- to 8-membered monocyclic heterocycloalkyl, or 5- to 10-membered heteroaryl optionally substituted with one or more R^d.

In some embodiments, R⁶and R⁷together with the N to which they are attached form 3- to 8-membered monocyclic heterocycloalkyl or 5- to 10-membered heteroaryl optionally substituted with one or more R^d.

In some embodiments, R⁶and R⁷together with the N to which they are attached form 5- to 6-membered monocyclic heterocycloalkyl or 5- to 6-membered heteroaryl optionally substituted with one or more R^d.

In some embodiments, R⁶and R⁷together with the N to which they are attached form 6- to 13-membered fused heterocycloalkyl or 5- to 6-membered monocyclic heterocycloalkyl optionally substituted with one or more R^d.

In some embodiments, R⁶and R⁷together with the N to which they are attached form 5- to 6-membered monocyclic heterocycloalkyl optionally substituted with one or more R^d.

In some embodiments, R⁶and R⁷together with the N to which they are attached form

[Image Omitted]

a morpholine ring or a piperazine ring optionally substituted with one or more R^d.

In some embodiments, R⁶and R⁷together with the N to which they are attached form a morpholine ring or a piperazine ring optionally substituted with one or more R^d.

In some embodiments, R⁶and R⁷together with the N to which they are attached form

[Image Omitted]

In some embodiments, R⁶and R⁷together with the N to which they are attached form

[Image Omitted]

In some embodiments, the compound of formula (I) or the pharmaceutically acceptable salt thereof according to the present disclosure is selected from a compound of formula (II), or a pharmaceutically acceptable salt thereof:

[Image Omitted]

wherein X, R², R³, R⁴, R⁵, R⁶, R⁷, and m are as defined above.

In some embodiments, the compound of formula (I) or the pharmaceutically acceptable salt thereof according to the present disclosure is selected from a compound of formula (III), or a pharmaceutically acceptable salt thereof:

[Image Omitted]

wherein X, R², R³, R⁴, R⁵, R⁶, R⁷, and m are as defined above.

In some embodiments, the compound of formula (I) or the pharmaceutically acceptable salt thereof according to the present disclosure is selected from the following compounds or pharmaceutically acceptable salts thereof:

[Image Omitted]

In some embodiments, the pharmaceutically acceptable salt of the compound of formula (I) is selected from a hydrochloride.

In another aspect, the present disclosure relates to a pharmaceutical composition comprising the compound of formula (I) or the pharmaceutically acceptable salt thereof according to the present disclosure.

In some embodiments, the pharmaceutical composition of the present disclosure also comprises a pharmaceutically acceptable adjuvant.

In another aspect, the present disclosure relates to a method of treating a disease mediated by PRMT5 in a mammal, comprising administering a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same to a mammal, preferably a human in need of the treatment.

In another aspect, the present disclosure relates to the use of the compound of formula (I) or the pharmaceutically acceptable salt thereof or the pharmaceutical composition comprising the same in the preparation of a medicament for preventing or treating a disease mediated by PRMT5.

In another aspect, the present disclosure relates to the use of the compound of formula (I) or the pharmaceutically acceptable salt thereof or the pharmaceutical composition comprising the same in the prevention or treatment of a disease mediated by PRMT5.

In another aspect, the present disclosure relates to the compound of formula (I) or the pharmaceutically acceptable salt thereof or the pharmaceutical composition comprising the same for preventing or treating a disease mediated by PRMT5.

In another aspect, the present disclosure relates to the compound of formula (I) or the pharmaceutically acceptable salt thereof or the pharmaceutical composition comprising the same that prevents or treats a disease mediated by PRMT5.

In some embodiments, the disease mediated by PRMT5 is cancer.

Definition and Description of Terminology

Unless otherwise stated, the following terms used in the present disclosure have the following meanings. A specific term without specifical definition should not be considered uncertain or unclear, and it should be understood in its ordinary meaning in the art. When a trade name appears herein, it is intended to refer to the corresponding commodity or an active ingredient thereof.

Unless otherwise stated, the definitions of groups and terms described in the specification and claims of the present disclosure, including their definitions as examples, exemplary definitions, preferred definitions, definitions listed in tables, and definitions of specific compounds in the examples, etc., may be arbitrarily combined or incorporated with one another. The group definitions and compound structures derived from such combinations and incorporations should fall within the scope described in the specification of the present disclosure.

In the present disclosure,

[Image Omitted]

denotes a linking site.

The term “substituted” means that any one or more hydrogen atoms on the designated atom are replaced with a substituent, provided that the valence state of the designated atom is normal, and the substituted compound is stable. When the substituent is oxo (i.e., ═O), it means that two hydrogen atoms are substituted, which would not occur on aromatic groups.

The term “optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and the description includes the occurrence and the non-occurrence of the event or circumstance. For example, the expression “ethyl is optionally substituted with halogen” means that ethyl may be unsubstituted (CH₂CH₃), mono-substituted (such as CH₂CH₂F), polysubstituted (such as CHFCH₂F, CH₂CHF₂), or completely substituted (CF₂CF₃). With respect to any group containing one or more substituents, it will be understood by those skilled in the art that any substitution or substitution patterns that are sterically impractical and/or synthetically non-feasible are not intended to be introduced into such groups.

C_m-C_nherein means that the moiety has an integer number of carbon atoms, wherein the integer number is within the range m-n. For example, “C₁-C₁₀” means that the group may have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms.

Where any variable (such as R) appears more than once in the composition or structure of a compound, its definition in each case is independent. For example, if a group is substituted with two Rs, then each R has an independent option.

When the bonds of a substituent are cross-linked to two atoms on a ring, the substituent can be bonded with any atom on the ring. For example, the structural unit

[Image Omitted]

means that the substitution with R⁵may occur on any position of the phenyl ring.

The term “halo” or “halogen” refers to fluoro, chloro, bromo and iodo.

The term “hydroxy” refers to —OH group.

The term “cyano” refers to —CN group.

The term “amino” refers to —NH₂group.

The term “alkyl” refers to a hydrocarbyl group of general formula C_nH_2n+1. The alkyl group can be linear or branched. For example, the term “C₁-C₁₀alkyl” is to be understood as denoting a linear or branched, saturated monovalent hydrocarbyl having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. Said alkyl is such as methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, and the like; and the term “C_1-6alkyl” means alkyl that contains 1 to 6 carbon atoms (such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, neopentyl, hexyl, 2-methylpentyl, and the like). Similarly, the alkyl moiety (i.e., alkyl) in alkoxy, alkylamino, and dialkylamino has the same definition as that described above.

The term “alkoxy” refers to —O-alkyl. Preferably, “C₁-C₁₀alkoxy” may include “C₁-C₆alkoxy”.

The term “alkylamino” refers to —NH-alkyl.

The term “dialkylamino” refers to —N(alkyl)₂.

The term “C₂-C₁₀alkenyl” is to be understood as preferably meaning a linear or branched, monovalent hydrocarbyl, which contains one or more double bonds and has 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, preferably “C₂-C₆alkenyl”, further preferably “C₂-C₄alkenyl”, and more further preferably, C₂alkenyl or C₃alkenyl. It is to be understood that in the case in which the alkenyl group contains more than one double bond, then the double bonds may be isolated from, or conjugated with, each other. Said alkenyl is such as vinyl, allyl, (E)-2-methylvinyl, (Z)-2-methylvinyl, (E)-but-2-enyl, (Z)-but-2-enyl, (E)-but-1-enyl, (Z)-but-1-enyl, isopropenyl, 2-methylprop-2-enyl, 1-methylprop-2-enyl, 2-methylprop-1-enyl, (E)-1-methylprop-1-enyl, (Z)-1-methylprop-1-enyl.

The term “C₂-C₁₀alkynyl” is to be understood as preferably meaning a linear or branched, monovalent hydrocarbyl which contains one or more triple bonds and has 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, preferably “C₂-C₆alkynyl”, further preferably “C₂-C₄alkynyl”, and more further preferably, C₂alkynyl or C₃alkynyl. Said alkynyl is such as ethynyl, prop-1-ynyl, prop-2-ynyl, but-1-ynyl, but-2-ynyl, but-3-ynyl, 1-methylprop-2-ynyl.

The term “cycloalkyl” refers to a carbocyclic group that is fully saturated and can exist as a monocyclic ring, fused ring, bridged ring or spirocyclic ring. Unless otherwise indicated, the carbocyclic group is typically a 3- to 10-membered ring. Non-limiting examples of cycloalkyl include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl (bicyclo[2.2.1]heptyl), bicyclo[2.2.2]octyl, adamantyl, spiro[4.5]decane, and the like. Spirocycloalkyl refers to cycloalkyl that exists as a spirocyclic ring. Preferably, “C₃-C₁₀cycloalkyl” is further preferably “C₃-C₆cycloalkyl”.

The term “cycloalkyloxy” can be understood as “cycloalkyl-O—”, preferably, “C₃-C₁₀cycloalkyloxy” may include “C₃-C₆cycloalkyloxy”.

The term “heterocycloalkyl” refers to a cyclic group that is fully saturated and can exist as a monocyclic ring, fused ring, bridged ring or spirocyclic ring. Unless indicated otherwise, the heterocycle is typically a 3 to 20-membered ring containing 1 to 3 heteroatoms (preferably 1 or 2 heteroatoms) independently selected from sulfur, oxygen and/or nitrogen. “3- to 8-membered monocyclic heterocycloalkyl” refers to a fully saturated cyclic group having 3, 4, 5, 6, 7, or 8 ring atoms, existing as a monocyclic ring, and containing 1 to 3 heteroatoms (preferably, 1 or 2 heteroatoms) independently selected from sulfur, oxygen and/or nitrogen. Examples of 3-membered heterocycloalkyl include but are not limited to oxiranyl, thiiranyl, and aziranyl; non-limiting examples of 4-membered heterocycloalkyl include but are not limited to azetidinyl, oxetanyl, and thietanyl; examples of 5-membered heterocycloalkyl include but are not limited to tetrahydrofuryl, tetrahydrothienyl, pyrrolidinyl, isoxazolidinyl, oxazolidinyl, isothiazolidinyl, thiazolidinyl, imidazolidinyl, and tetrahydropyrazolyl; examples of 6-membered heterocycloalkyl include but are not limited to piperidyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, piperazinyl, 1,4-thioxanyl, 1,4-dioxanyl, thiomorpholinyl, 1,3-dithianyl, 1,4-dithianyl; and examples of 7-membered heterocycloalkyl include but are not limited to azepanyl, oxepanyl, and thiepanyl. Monocyclic heterocycloalkyl is preferably monocyclic heterocycloalkyl having 5 or 6 ring atoms. Spiroheterocycloalkyl refers to heterocycloalkyl that exists as a spirocyclic ring.

The term “6- to 13-membered spiroheterocycloalkyl” refers to a fully saturated cyclic group having 6, 7, 8, 9, 10, 11, 12, or 13 ring atoms, existing as a spirocyclic ring, and containing 1 to 3 heteroatoms (preferably, 1 or 2 heteroatoms) independently selected from sulfur, oxygen and/or nitrogen.

The term “6- to 13-membered fused heterocycloalkyl” refers to a fully saturated cyclic group having 6, 7, 8, 9, 10, 11, 12, or 13 ring atoms, existing as a fused ring/by means of fusing, and containing 1 to 3 heteroatoms (preferably, 1 or 2 heteroatoms) independently selected from sulfur, oxygen and/or nitrogen.

The term “6- to 13-membered bridged heterocycloalkyl” refers to a fully saturated cyclic group having 6, 7, 8, 9, 10, 11, 12, or 13 ring atoms, existing as a bridged ring, and containing 1 to 3 heteroatoms (preferably, 1 or 2 heteroatoms) independently selected from sulfur, oxygen and/or nitrogen.

The term “heterocyclyl” refers to a monocyclic heterocyclyl and fused heterocyclyl system, wherein a fused heterocycle includes fused heterocyclyl, spiro heterocyclyl, and bridged heterocyclyl, and can be saturated, partially saturated, or unsaturated but cannot be aromatic.

The term “3- to 10-membered heterocyclyl” means a saturated or partially saturated monovalent monocyclic or bicyclic hydrocarbyl comprising 1-5, preferably 1-3 heteroatoms selected from N, O and S. In particular, the heterocyclyl can include but is not limited to: a 4-membered ring such as azetidinyl, and oxetanyl; a 5-membered ring such as tetrahydrofuryl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, and pyrrolinyl; or a 6-membered ring such as tetrahydropyranyl, piperidyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, or trithianyl; or a partially saturated 6-membered ring such as tetrahydropyridyl; or a 7-membered ring such as diazepanyl. Optionally, the 3- to 10-membered heterocyclyl can be 8- to 10-membered benzo-fused heterocyclyl or 8- to 10-membered heteroaryl-fused heterocyclyl, including but not limited to such as benzopiperidinyl, pyridopiperidyl, or pyrimidopiperidyl, and the like; and the heterocyclyl, i.e., 3- to 10-membered heterocyclyl can also include, but is not limited to a 5,5-membered ring, e.g., a hexahydrocyclopenta[c]pyrrol-2(1H)-yl, or a 5,6-membered bicyclic ring, e.g., a hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl. A nitrogen-containing ring can be partially unsaturated, i.e., it can contain one or more double bonds, such as, without being limited thereto, a 2,5-dihydro-1H-pyrrolyl, 4H-[1,3,4]thiadiazinyl, 4,5-dihydrooxazolyl, or 4H-[1,4]thiazinyl, or, it may be benzo-fused, such as, without being limited thereto, a dihydroisoquinolinyl. According to the present disclosure, the heterocyclyl is not aromatic.

The term “3- to 10-membered heterocyclyloxy” is to be understood as “3- to 10-membered heterocyclyl-O—”.

The term “C₆-C₁₀aryl” is understood to preferably denote a monovalent aromatic or partially aromatic monocyclic or bicyclic hydrocarbyl having 6 to 10 carbon atoms, especially a ring having 6 carbon atoms (“C₆aryl”), such as phenyl; or a ring having 9 carbon atoms (“C₉aryl”), such as indanyl or indenyl, or a ring having 10 carbon atoms (“C₁₀aryl”), such as naphthyl.

The term “C₆-C₁₀aryloxy” is to be understood as “C₆-C₁₀aryl-O—”.

The term “5- to 10-membered heteroaryl” is understood to include monovalent monocyclic or bicyclic aromatic ring systems having 5, 6, 7, 8, 9, or 10 ring atoms, especially 5 or 6 or 9 or 10 ring atoms, and containing 1-5, preferably 1-3, heteroatoms each independently selected from N, O and S, and in addition, may be benzo-fused in each case. In particular, the heteroaryl is selected from thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, etc. and the benzo derivatives thereof, such as benzofuryl, benzothienyl, benzothiazolyl, benzoxazolyl, benzisoxazolyl, benzoimidazolyl, benzotriazolyl, indazolyl, indolyl, isoindolyl, and the like; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and the like, and the benzo derivatives thereof such as quinolyl, quinazolinyl, isoquinolinyl, and the like; or azocinyl, indolizinyl, purinyl, and the like and the benzo derivatives thereof; or cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, and the like.

The term “5- to 10-membered heteroaryloxy” is to be understood as “5- to 10-membered heteroaryl-O”.

The term “treatment” refers to the administration of the compounds or preparations of the present disclosure for preventing, ameliorating or eliminating diseases or one or more symptoms associated with the diseases and comprises:

(i) prophylaxis of occurrence of diseases or conditions in mammals, particularly when the mammals are susceptible to the conditions, but have not been diagnosed with the conditions;

(ii) inhibition of diseases or conditions, i.e., restraining their development; or (iii) relief of diseases or conditions, i.e., resolution of the diseases or conditions.

The term “therapeutically effective amount” means an amount of a compound of the present disclosure used to (i) treat or prevent a particular disease, condition, or disorder, (ii) attenuate, ameliorate, or eliminate one or more symptoms of a particular disease, condition, or disorder, or (iii) prevent or delay the onset of one or more symptoms of a particular disease, condition, or disorder described herein. The amount of a compound of the present disclosure which constitutes a “therapeutically effective amount” will vary depending on the compound, the conditions and their severity, the manner of administration, and the age of the mammal to be treated, but can be determined routinely by those skilled in the art according to their own knowledge and the present disclosure.

The term “pharmaceutically acceptable” refers to compounds, materials, compositions and/or dosage forms, which are, within the scope of sound medical judgment, suitable for use in contact with human and animal tissues, without excessive toxicity, irritation, allergic reactions or other problems or complications, which is commensurate with a reasonable benefit/risk ratio.

As pharmaceutically acceptable salts, for example, the following examples may be mentioned: metal salts, ammonium salts, salts formed with organic bases, inorganic acids, organic acids, basic or acidic amino acids, and the like.

The term “pharmaceutical composition” refers to a mixture of one or more of the compounds or salts thereof according to the present disclosure and a pharmaceutically acceptable adjuvant. An object of the pharmaceutical composition is to facilitate administering the compound according to the present disclosure to an organism.

The term “pharmaceutically acceptable adjuvant” refers to those adjuvants which have no significant irritating effect on the organism and do not impair the bioactivity and properties of the active compound. Suitable adjuvants are well known to those skilled in the art, and are such as a carbohydrate, a wax, a water-soluble and/or water-swellable polymer, a hydrophilic or hydrophobic material, gelatin, an oil, a solvent, water, and the like.

The word “comprise” and its variants such as “comprises” or “comprising” is to be understood as an open, non-exclusive meaning, i.e., “including but not limited to”.

The compounds and intermediates of the present disclosure may also exist in different tautomeric forms, and all such forms are embraced within the scope of the present disclosure. The term “tautomer” or “tautomeric form” refers to structural isomers of different energies which are interconvertible via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via migration of a proton, such as keto-enol and imine-enamine isomerizations. A specific example of a proton tautomer is the imidazole moiety where the proton may migrate between the two ring nitrogens. Valence tautomers include interconversions by reorganization of some of the bonding electrons.

The present disclosure also includes isotopically-labeled compounds of the present disclosure which are identical to those recited herein, but have one or more atoms replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ³¹P ³²P, ³⁵S, ¹⁸F, ¹²³I, ¹²⁵I and ³⁶Cl, respectively.

Certain isotopically-labeled compounds of the present disclosure (e.g., those labeled with ³H and ¹⁴C) are useful in tissue distribution assays of compounds and/or substrates. Tritiated (i.e., ³H) and carbon-14 (i.e., ¹⁴C) isotopes are particularly preferred for their ease of preparation and detectability. Positron emitting isotopes such as ¹⁵O, ¹³N, ¹¹C, and ¹⁸F are useful for positron emission tomography (PET) studies to determine substrate occupancy. The isotopically-labeled compounds of the present disclosure can generally be prepared according to following procedures analogous to those disclosed in the Schemes and/or in the Examples herein below by substituting a non-isotopically-labeled reagent with an isotopically-labeled reagent.

Furthermore, substitution with heavier isotopes (such as deuterium, i.e., ²H) may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances, wherein the deuterium substitution may be partial or complete, and partial deuterium substitution means that at least one hydrogen is substituted with at least one deuterium.

All isotopic variations of the compounds of the present disclosure, whether radioactive or not, are intended to be encompassed within the scope of the present disclosure.

The compound of the present disclosure may be asymmetric, such as, having one or more stereoisomers. Stereoisomer refers to isomers created by a different spatial arrangement of atoms in molecules; and unless otherwise stated, all stereoisomers are included, such as cis and trans isomers, conformational isomers, enantiomers and diastereomers. The compound comprising asymmetric carbon atoms of the present disclosure can be isolated in optically active-pure or racemic forms. The optically active-pure form can be resolved from the racemic mixture or synthesized by utilizing chiral raw materials or chiral reagents. The diagrammatic presentation of the racemate or enantiomerically pure compound herein is from Maehr, J. Chem. Ed. 1985, 62: 114-120. Unless otherwise stated, the wedge-shaped bond and the dashed bond

[Image Omitted]

are used to represent the absolute configuration of a stereocenter. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are encompassed within the scope of the present disclosure.

Non-limiting examples of stereoisomers include, but are not limited to:

[Image Omitted]

The pharmaceutical composition of the present disclosure may be prepared by combining the compound of the present disclosure with an appropriate pharmaceutically acceptable adjuvant. For example, the pharmaceutical composition of the present disclosure may be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres, and aerosols, and the like.

Typical administration routes of the compound or the pharmaceutically acceptable salt thereof, or the pharmaceutical composition thereof according to the present disclosure include, but are not limited to, oral administration, rectal administration, topical administration, administration by inhalation, parenteral administration, sublingual administration, intravaginal administration, intranasal administration, intraocular administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, and intravenous administration.

The pharmaceutical composition of the present disclosure can be manufactured by using well-known methods in the art, such as conventional mixing method, dissolution method, granulation method, dragee manufacture method, grinding method, emulsification method, and freeze-drying method, and the like.

In some embodiments, the pharmaceutical composition is in oral form. For oral administration, the pharmaceutical composition may be formulated by mixing the active compound with a pharmaceutically acceptable adjuvant well-known in the art. Such adjuvants enable the compounds of the present disclosure to be formulated into tablets, pills, lozenges, dragees, capsules, liquids, gels, syrups, suspensions and the like, for oral administration to patients.

A solid oral composition can be prepared by a conventional mixing, filling or tabletting method. For example, it can be obtained by the following method: mixing the active compound with a solid adjuvant, optionally grinding the resulting mixture, adding other suitable adjuvants, if necessary, and then processing the mixture into granules to obtain cores of tablets or dragees. Suitable adjuvants include, but are not limited to, binders, diluents, disintegrants, lubricants, glidants, sweeting agents, or flavoring agents, and the like.

The pharmaceutical composition can also be suitable for parenteral administration, such as sterile solutions, suspensions or lyophilized products in a suitable unit dosage form.

The daily administration dose of the compound of general formula I in all the administration manners described herein is from 0.01 mg/kg body weight to 100 mg/kg body weight, preferably from 0.05 mg/kg body weight to 50 mg/kg body weight, and more preferably from 0.1 mg/kg body weight to 30 mg/kg body weight, in the form of a single dose or divided doses.

The compounds of the present disclosure can be prepared by various synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by the combination with other chemical synthesis methods, and equivalent alternative embodiments well known to those skilled in the art, wherein the preferred embodiments include but are not limited to the examples of the present disclosure.

The chemical reactions described in the specific embodiments of the present disclosure are completed in a suitable solvent, wherein the solvent must be suitable for the chemical changes of the present disclosure and the reagents and materials required thereby. In order to obtain the compounds of the present disclosure, sometimes a person skilled in the art needs to modify or select synthesis steps or reaction schemes based on the existing embodiments.

An important consideration in the design of a synthetic route in the art is the selection of a suitable protecting group for a reactive functional group, such as an amino group in the present disclosure. For example, reference may be made to Greene's Protective Groups in Organic Synthesis (4th Ed)., Hoboken, N.J.: John Wiley & Sons, Inc. All references cited herein are incorporated into the present disclosure in their entireties.

In some embodiments, the compound of general formula (I) of the present disclosure may be prepared by a person skilled in the field of organic synthesis via route 1:

[Image Omitted]

reacting compounds M1 and M2 in the presence of a base to produce M3; reacting M3 in the presence of a palladium catalyst/base/ethanol/CO to produce M4; subjecting M4 to carboxyl deprotection in the presence of a base and producing M5 in the presence of Boc-anhydride; and reacting M5 and R⁶R7NH in the presence of a condensing agent and further removing the amino protecting group to produce the compound of formula (I).

In some embodiments, the compound of general formula (I) of the present disclosure may be prepared by a person skilled in the field of organic synthesis via route 2:

[Image Omitted]

reacting compound M in the presence of MsCl M and a base to produce M7: reacting M7 in the presence of a base to produce M8; reacting M8 in the presence of a palladium catalyst/base/ethanol/CO to produce M9; subjecting M9 to a hydrolysis reaction in the presence of a base to obtain M10; and reacting M10 and R⁶R⁷NH in the presence of a condensing agent and further removing the amino protecting group to produce the compound of formula (I).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the ball-and-stick representation of the single crystal of compound 1-8.

FIG. 2 is the tumor growth curve of mice receiving the test compound in Z-138 subcutaneous tumor models.

FIG. 3 is the body weight change curve of mice receiving the test compound in Z-138 subcutaneous tumor models.

DETAILED DESCRIPTION

For clarity, the present disclosure is further illustrated by the following examples, but the examples are not intended to limit the scope of the present disclosure. All reagents used in the present disclosure are commercially available and can be used without further purification.

Example 1. Preparation of tert-butyl (S)-3-((S)-oxiran-2-yl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (Intermediate 1)

[Image Omitted]

- Intermediate 1

Preparation of Compound b:

[Image Omitted]

At room temperature, (S)-2-(tert-butoxycarbonyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (a) (55 g, 200 mmol), dimethylhydroxylamine hydrochloride (29.4 g, 300 mmol), and 2-(7-azabenzotriazole)-N,N,N′,N′-tetramethylurea hexafluorophosphate (91 g, 240 mmol) were added into a 1 L single-necked flask, and then anhydrous N,N-dimethylformamide (500 mL) was added. Under nitrogen, the reaction was cooled in ice bath and then N,N-diisopropylethylamine (104 mL, 600 mmol) was added dropwise. The reaction solution was reacted at room temperature for 4 hours. Upon completion of reaction, excess N,N-diisopropylethylamine and N,N-dimethylformamide were removed by rotary evaporation and then the resulting mixture was cooled under ice bath, diluted with saturated brine (1 L), and extracted with ethyl acetate (200 mL×2). The organic phases were combined and washed with 5% aqueous sodium carbonate solution (500 mL×2), and then washed with saturated brine (500 mL). The resulting mixture was dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure and then purified by silica gel column chromatography (eluent gradient: petroleum ether/ethyl acetate=2/1) to afford the target intermediate tert-butyl (S)-3-(methoxy(methyl)carbamoyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (b) (62 g, yield: 97%).

LCMS: Rt: 1.76 min; MS m/z (ESI): 321.3 [M+H].

Chiral HPLC: Rt: 3.159 min.

Preparation of Compound c:

[Image Omitted]

At room temperature, tert-butyl (S)-3-(methoxy(methyl)carbamoyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (b) (20 g, 62.5 mmol) was weighed into a 500 mL three-necked flask, and anhydrous tetrahydrofuran (200 mL) was added. The mixture was cooled to −70° C. and a solution of diisobutylaluminum hydride (DIBAL-H) in toluene (1.5 mol/L, 83 mL, 125 mmol) was slowly added dropwise. The reaction solution was stirred at −70° C. for 1 hour. Upon completion of reaction, saturated ammonium chloride solution (100 mL) was slowly added at −70° C. to quench the reaction and then 0.5 mol/L hydrochloric acid aqueous solution (200 mL) was added to dilute the reaction. After layering, the organic phase was washed with saturated sodium chloride aqueous solution (200 mL×2), then dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent gradient: petroleum ether/ethyl acetate=8/1) to afford the intermediate tert-butyl (S)-3-formyl-3,4-dihydroisoquinoline-2(1H)-carboxylate (c) (15 g, yield: 92%).

LCMS: Rt: 1.93 min; MS m/z (ESI): 206.1 [M-56+H].

Chiral HPLC: Rt: 2.018 min.

Preparation of Compound d:

[Image Omitted]

Methyltriphenylphosphonium bromide (238 g, 0.67 mol) was dispersed into anhydrous tetrahydrofuran (1.5 L). Under nitrogen, the mixture was cooled to −70° C. Sodium bis(trimethylsilyl)amide (334 mL, 0.67 mol) was slowly added dropwise whilst the temperature was controlled below −50° C. After the addition was completed, the reaction was slowly warmed to room temperature and stirred for 2 hours. The reaction was cooled to −70° C. again. A solution of tert-butyl (S)-3-formyl-3,4-dihydroisoquinoline-2(1H)-carboxylate (c) (87 g, 0.33 moL) in tetrahydrofuran solution (200 mL) was slowly added dropwise whilst the temperature was controlled below −50° C. After the addition was completed, the reaction was slowly warmed to room temperature overnight. After the reaction was complete as detected by TLC, the reaction was cooled to 0° C. and quenched with saturated ammonium chloride solution. The 1 mol/L hydrochloric acid aqueous solution was slowly added to adjust the pH to 3-4, and ethyl acetate (500 mL) was added for extraction. The organic phase was washed with saturated brine (200 mL×2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was recrystallized by adding mixed solvent (ethyl acetate/petroleum ether=1/4) and then filtered to remove precipitated triphenyl phosphine oxide. The filtrate was concentrated and then subjected to silica gel column chromatography (ethyl acetate/petroleum ether=1/8) to afford the target product tert-butyl (S)-3-vinyl-3,4-dihydroisoquinoline-2(1H)-carboxylate (d) (85 g, yield: 98%).

Chiral HPLC: Rt: 1.883 min.

Preparation of Compound e:

[Image Omitted]

Tert-butyl (S)-3-vinyl-3,4-dihydroisoquinoline-2(1H)-carboxylate (d) (25.9 g, 0.1 mol) was dissolved in ethyl acetate/acetonitrile (500 mL/500 mL) solution and the mixture was cooled to 0° C. An aqueous solution of sodium periodate (32.1 g, 0.15 mol) and ruthenium trichloride hydrate (1.6 g, 7.7 mmol) was added within 10 minutes and the reaction solution was stirred at 0° C. for 10 minutes. The reaction was complete as detected by TLC. Saturated sodium thiosulfate solution (150 mL) was added to quench the reaction. The resulting mixture was stirred for 30 minutes and layered, and the organic phase was washed with saturated brine (200 mL×2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (eluents: ethyl acetate/petroleum ether=1/2-1/1) to afford the product tert-butyl (S)-3-((S)-1,2-dihydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (e) (less-polar product, 14 g, yield: 48%). The polarity of the eluent (ethyl acetate/petroleum ether=2/1-1/0) was increased to afford the product tert-butyl (S)-3-((R)-1,2-dihydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (e-1) (more-polar product, 8 g, yield: 28%).

Preparation of Compound f:

[Image Omitted]

Tert-butyl (S)-3-((S)-1,2-dihydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (e) (14 g, 0.047 mol) was dissolved in dichloromethane (150 mL). Then p-toluenesulfonyl chloride (10.0 g, 0.052 mol) was added in batches under stirring following the addition of triethylamine (9.90 mL, 0.072 mol). The reaction solution was stirred at 40° C. overnight. The reaction solution was cooled to room temperature and washed with saturated brine (100 mL×2). The organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (ethyl acetate/petroleum ether=1/4) to afford the target product tert-butyl (S)-3-((S)-1-hydroxy-2-(tosyloxy)ethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (f) (12.6 g, yield: 59%).

Preparation of Compound Intermediate 1:

[Image Omitted]

Tert-butyl (S)-3-((S)-1-hydroxy-2-(tosyloxy)ethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (f) (12.6 g, 28.2 mmol) was dissolved in N,N-dimethylformamide (150 mL). Under nitrogen, sodium hydride (1.70 g, 42.3 mmol) was added in batches, and the reaction solution was stirred at 40° C. for 1 hour. The reaction was complete as detected by TLC. The reaction was cooled to 0° C. and quenched by adding saturated brine dropwise. The reaction solution was directly purified using a reverse-phase silica gel chromatographic column (water/acetonitrile=50/10). The resulting solution from the column chromatography was extracted with ethyl acetate (200 mL×3). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford the intermediate tert-butyl (S)-3-((S)-oxiran-2-yl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (intermediate 1) (6.5 g, yield: 72%).

Example 2. Preparation of 8-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one hydrochloride (Compound 001)

[Image Omitted]

Preparation of Compound 1-2:

[Image Omitted]

At room temperature, 4-bromo-2-fluorobenzoic acid (1-1) (7.5 g, 34.2 mmol) and N-tert-butoxycarbonyl-1,2-ethylenediamine (16.4 g, 102.7 mmol) were added to N-methyl pyrrolidone (30 mL). Upon completion of addition, the mixture was warmed to 120° C. and reacted for 16 hours. Upon completion of reaction, the reaction solution was slowly added to water (150 mL), adjusted with 2 mol/L dilute hydrochloric acid to the pH of 5-6, and then extracted with ethyl acetate (150 mL×2). The organic phases were combined, washed with saturated brine (150 mL×2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford the target intermediate 4-bromo-2-((2-((tert-butoxycarbonyl)amino)ethyl)amino)benzoic acid (1-2) (crude, 11.0 g, yield: 89%).

LCMS: Rt: 1.836 min; MS m/z (ESI): 359.0 [M+H].

Preparation of Compound 1-3:

[Image Omitted]

At room temperature, 4-bromo-2-((2-((tert-butoxycarbonyl)amino)ethyl)amino)benzoic acid (1-2) (11.0 g, 30.6 mmol) was added to 4 mol/L hydrochloric acid/dioxane solution (30 mL) and then the mixture was reacted at room temperature for 1 hour. Upon completion of reaction, the reaction solution was concentrated under reduced pressure to afford the target intermediate 2-((2-aminoethyl)amino)-4-bromobenzoic acid (1-3) (crude, 8 g, yield: 100%).

LCMS: Rt: 0.688 min; MS m/z (ESI): 259.0 [M+H].

Preparation of Compound 1-4:

[Image Omitted]

At room temperature, 2-((2-aminoethyl)amino)-4-bromobenzoic acid (1-3) (3 g, 11.6 mmol), 2-(7-azabenzotriazole)-N,N,N′,N′-tetramethylurea hexafluorophosphate (8.8 g, 23.2 mmol) and triethylamine (4.7 g, 46.4 mmol) were added to ultradry N,N-dimethylformamide (20 mL) and the mixture was reacted at room temperature for 1.5 hours. Upon completion of reaction, saturated brine (100 mL) was added, and the resulting mixture was extracted with ethyl acetate (100 mL×2). The organic phase was washed with saturated brine (50 mL×2), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified using a normal phase silica gel chromatographic column (eluent gradient: dichloromethane/methanol=20/1) to afford the target intermediate 8-bromo-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one (1-4) (1.6 g, yield: 57%).

LCMS: Rt: 1.173 min; MS m/z (ESI): 241.0 [M+H].

Preparation of Compound 1-5:

[Image Omitted]

At room temperature, 8-bromo-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one (1-4) (1.6 g, 6.6 mmol), sodium cyanoborohydride (834 mg, 13.3 mmol) and acetaldehyde (584 mg, 13.3 mmol) were added to a mixed solution of acetic acid (2 mL) and methanol (20 mL). The mixture was reacted at room temperature for 1 hour. Upon completion of reaction, 2 mol/L dilute hydrochloric acid (1 mL) and water (50.0 mL) were added and the mixture was extracted with ethyl acetate (50.0 mL×2). The organic phases were combined, washed with saturated brine (50 mL×2), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified using a normal phase silica gel chromatographic column (eluent gradient: petroleum ether/ethyl acetate=1/2) to afford the target intermediate 8-bromo-1-ethyl-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one (1-5) (1.1 g, yield: 62%).

LCMS: Rt: 1.499 min; MS m/z (ESI): 269.0 [M+H].

Preparation of Compound 1-6:

[Image Omitted]

At room temperature, 8-bromo-1-ethyl-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one (1-5) (400 mg, 1.52 mmol) was added to ultradry N,N-dimethylformamide (20 mL) and then sodium hydride (90.9 mg, 2.27 mmol) was added. After the addition, the mixture was heated to 40° C. and stirred for 1 h. Then intermediate 1 (500 mg, 1.82 mmol) prepared in example 1 was added, and the resulting mixture was reacted for 16 hours. Upon completion of reaction, water (100 mL) was added, and the mixture was extracted with ethyl acetate (100 mL×2). The organic phases were combined, washed with saturated brine (50 mL×2), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified using a normal phase silica gel chromatographic column (eluent gradient: dichloromethane/methanol=20/1) to afford the target intermediate (1R,10aS)-1-((8-bromo-1-ethyl-5-oxo-1,2,3,5-tetrahydro-4H-benzo[e][1,4]diazepin-4-yl)methyl)-1,5,10,10a-tetrahydro-3H-oxazolo[3,4-b]isoquinolin-3-one (1-6) (150 mg, yield: 21%).

LCMS: Rt: 1.872 min; MS m/z (ESI): 470.1 [M+H].

Preparation of Compound 1-7:

[Image Omitted]

At room temperature, the intermediate (1R,10aS)-1-((8-bromo-1-ethyl-5-oxo-1,2,3,5-tetrahydro-4H-benzo[e][1,4]diazepin-4-yl)methyl)-1,5,10,10a-tetrahydro-3H-oxazolo[3,4-b]isoquinolin-3-one (1-6) (150 mg, 0.32 mmol), [1,1′-bis(diphenylphosphine)ferrocene]palladium dichloride (11.7 mg, 0.02 mmol) and potassium acetate (94 mg, 0.96 mmol) were added to anhydrous ethanol (10 mL). The mixture was subjected to CO replacement three times, heated to 70° C. and reacted for 3.0 hours. Upon completion of reaction, the reaction was cooled to room temperature, and the reaction solution was concentrated under reduced pressure. Saturated brine (50 mL) was added and then the mixture was extracted with ethyl acetate (50 mL×2). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was purified using a normal phase silica gel chromatographic column (eluent gradient: petroleum ether/ethyl acetate=1/2) to afford the target intermediate ethyl 1-ethyl-5-oxo-4-(((1R,10aS)-3-carbonyl-1,5,10,10a-tetrahydro-3H-oxazolo[3,4-b]isoquinolin-1-yl)methyl)-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepine-8-carboxylate (1-7) (130 mg, yield: 87%).

LCMS: Rt: 1.784 min; MS m/z (ESI): 464.1 [M+H].

Preparation of Compound 1-8:

[Image Omitted]

At room temperature, ethyl 1-ethyl-5-oxo-4-(((1R,10aS)-3-carbonyl-1,5,10,10a-tetrahydro-3H-oxazolo[3,4-b]isoquinolin-1-yl)methyl)-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepine-8-carboxylate (1-7) (130 mg, 0.28 mmol) was added to a mixed solution of methanol (4 mL) and water (4 mL), and sodium hydroxide (90 mg, 2.24 mmol) was added then. The mixture was heated to 70° C. and reacted for 16.0 hours. Upon completion of reaction, the reaction system was cooled to room temperature and Boc anhydride (122 mg, 0.56 mmol) was added. The resulting mixture was reacted for 1.5 hours. Upon completion of reaction, the reaction system was cooled to 0° C. The reaction solution was adjusted with 1 mol/L hydrochloric acid aqueous solution to the pH of 5.0 and then extracted with ethyl acetate (30 mL×3). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was subjected to a reverse-phase silica gel chromatographic column (eluent gradient: acetonitrile/water=43%) to afford the target intermediate 4-((R)-2-((S)-2-(tert-butoxycarbonyl)-1,2,3,4-tetrahydroisoquinolin-3-yl)-2-hydroxyethyl)-1-ethyl-5-oxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepine-8-carboxylic acid (1-8) (crude, 60 mg, yield: 42%).

LCMS: Rt: 1.722 min; MS m/z (ESI): 510.3 [M+H].

Single Crystal X-Ray Structure Determination and Single Crystal X-Ray Analysis for Compound 1-8:

Method for preparing the single crystal: Compound 1-8 (10.0 mg) was weighed and placed into a 3 mL screw neck glass bottle and methanol (2 mL) was added. After the mixture was stirred for 5 minutes, the solid was dissolved and the mixture was clarified. 0.5 mL of water was added into the glass bottle and the mixture was further stirred for 5 minutes. The solution was filtered through 0.22 μm microporous filter membrane into a 3 mL screw neck glass bottle and the glass bottle was covered with a preservative film. 8 small holes were made by a needle on the film covering the bottle. The bottle was placed at room temperature for 7 days to obtain the above-mentioned single crystal of the compound.

X-ray analysis was carried out on the single crystal sample obtained. The test results are shown in Table 1 and FIG. 1.

TABLE 1

Single crystal sample of compound 1-8 and crystal data

Chemical formula
C₂₈H₃₅N₃O₆•H₂O

Chemical formula weight
527.60

Crystal system
Monoclinic form

Space group
P1 21 1

a, Å
a = 6.0902(12)

b, Å
b = 9.601(2)

c, Å
c = 23.026(5)

α, deg
90

β, deg
90.334(6)

γ, deg
90

Volume, Å³
1346.4(5)

Z
2

D_calc, g cm⁻³
1.301

temperature, K
100(2)

Radiation (wavelength)
Mo Kα (0.71073)

Instruments
BRUKER D8 VENTURE

PHOTON II

h, k, l ranges
−8 <= h <= 7, −12 <= k <= 12,

−30 <= l <= 30

θ range
2.298-28.383

Program
SHELXL-2014

Data acquired
28356

Unique data
6667

R (F_o)
0.0676

Rw (F_o²)
0.1957

Goodness of fit
1.018

Absolute configuration determination
Flack parameter (0.0(2))

From the above-mentioned X-ray crystal diffraction experiment, it can be determined that the chemical structure and absolute configuration of compound 1-8 is as follows:

[Image Omitted]

Preparation of Compound 1-9:

[Image Omitted]

At room temperature, 4-((R)-2-((S)-2-(tert-butoxycarbonyl)-1,2,3,4-tetrahydroisoquinolin-3-yl)-2-hydroxyethyl)-1-ethyl-5-oxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepine-8-carboxylic acid (1-8) (60 mg, 0.12 mmol), 2-(7-azabenzotriazole)-N,N,N′,N′-tetramethylurea hexafluorophosphate (89.4 mg, 0.24 mmol) and triethylamine (47.5 mg, 0.47 mmol) were added to ultradry N,N-dimethylformamide (5.0 mL), then 3-oxa-8-azabicyclo[3.2.1]octane hydrochloride (26.3 mg, 0.18 mmol) was added, and the mixture was reacted at room temperature for 1.5 hours. Upon completion of reaction, saturated brine (30 mL) was added, and the resulting mixture was extracted with ethyl acetate (30 mL×2). The organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was purified using a normal phase silica gel chromatographic column (eluent gradient: petroleum ether/ethyl acetate=1/2) to afford the target intermediate tert-butyl (3S)-3-((1R)-2-(8-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-1-ethyl-5-oxo-1,2,3,5-tetrahydro-4H-benzo[e][1,4]diazepin-4-yl)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (1-9) (20 mg, yield: 28%).

LCMS: Rt: 1.779 min; MS m/z (ESI): 605.2 [M+H].

Preparation of Compound 001:

[Image Omitted]

At 0° C., 4 mol/L hydrochloric acid/dioxane solution (1.0 mL) was added to tert-butyl (3S)-3-((1R)-2-(8-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-1-ethyl-5-oxo-1,2,3,5-tetrahydro-4H-benzo[e][1,4]diazepin-4-yl)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (1-9) (20 mg, 0.033 mmol), and the mixture was reacted for 1 hour. Upon completion of reaction, the reaction solution was concentrated under reduced pressure. The residue was purified by high-performance liquid preparative chromatography (eluent gradient:

Instruments
Waters 2767

Mobile phase
A: 0.1% HCl in H₂O, B: CH₃CN

Chromatographic column
Waters sunfire C18 19*250 mm 10 μm

Injection volume
500 μL

Table of running gradients
Time (min)
Mobile phase A (%)
Mobile phase B (%)

0
90
5

1
80
20

11
60
40

11.2
5
95

13
5
95

13.2
95
5

15
95
5

to afford the target compound 8-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one hydrochloride (compound 001) (3.65 mg, yield: 20%).

¹H NMR (400 MHz, CD₃OD): δ 7.65-7.63 (m, 1H), 7.31-7.16 (m, 6H), 4.64 (s, 1H), 4.50-4.36 (m, 2H), 4.33-4.29 (m, 1H), 3.99-3.96 (m, 2H), 3.84-3.77 (m, 1H), 3.79-3.57 (m, 8H), 3.47-3.46 (m, 1H), 3.34-3.30 (m, 2H), 3.29-3.25 (m, 2H), 2.05-1.99 (m, 4H), 1.18-1.14 (m, 3H).

LCMS: Rt: 0.798 min; MS m/z (ESI): 505.2 [M+H].

Example 3. Preparation of 1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-8-(2-methoxy-7-azaspiro[3.5]nonane-7-carbonyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one hydrochloride (Compound 002)

[Image Omitted]

Preparation of Compound 2-2:

[Image Omitted]

At room temperature, 4-((R)-2-((S)-2-(tert-butoxycarbonyl)-1,2,3,4-tetrahydroisoquinolin-3-yl)-2-hydroxyethyl)-1-ethyl-5-oxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepine-8-carboxylic acid (1-8) (100 mg, 0.196 mmol), 2-(7-azabenzotriazole)-N,N,N′,N′-tetramethylurea hexafluorophosphate (231 mg, 0.608 mmol) and N,N-diisopropylethylamine (157 mg, 1.216 mmol) were added to ultradry N,N-dimethylformamide (2.5 mL), then 2-methoxy-7-azaspiro[3.5]nonane hydrochloride (53 mg, 0.275 mmol) was added, and the mixture was stirred at room temperature for 1.0 hour. Upon completion of reaction, water (30 mL) was added and then the resulting mixture was extracted with ethyl acetate (30 mL×3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was purified using a normal phase silica gel chromatographic column (eluents: dichloromethane/methanol=20/1) to afford the target intermediate tert-butyl (S)-3-((R)-2-(1-ethyl-8-(2-methoxy-7-azaspiro[3.5]nonane-7-carbonyl)-5-oxo-1,2,3,5-tetrahydro-4H-benzo[e][1,4]diazepin-4-yl)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (2-2) (110 mg, yield: 86.7%).

LCMS: Rt: 2.062 min; MS m/z (ESI): 647.3 [M+H].

Preparation of Compound 002:

[Image Omitted]

At room temperature, tert-butyl (S)-3-((R)-2-(1-ethyl-8-(2-methoxy-7-azaspiro[3.5]nonane-7-carbonyl)-5-oxo-1,2,3,5-tetrahydro-4H-benzo[e][1,4]diazepin-4-yl)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (2-2) (110 mg, 0.170 mmol) was added to a solution of 4 mol/L hydrochloric acid/dioxane (2.5 mL). The mixture was stirred and reacted at room temperature for 1.0 hour. Upon completion of reaction, the reaction solution was concentrated under reduced pressure and the residue was purified by high-performance liquid preparative chromatography (refer to example 2 for the eluents and gradient) to afford the target compound 1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-8-(2-methoxy-7-azaspiro[3.5]nonane-7-carbonyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one hydrochloride (compound 002) (39.81 mg, yield: 42.8%).

¹H NMR (400 MHz, CD₃OD): δ 7.64 (d, J=7.6 Hz, 1H), 7.35-7.22 (m, 4H), 7.12 (d, J=8 Hz, 2H), 4.51-4.36 (m, 2H), 4.33-4.30 (m, 1H), 4.04-3.89 (m, 2H), 3.73-3.61 (m, 7H), 3.51-3.50 (m, 1H), 3.38-3.34 (m, 4H), 3.31-3.30 (m, 2H), 3.22 (s, 3H), 2.29-2.23 (m, 2H), 1.71-1.68 (m, 4H), 1.55 (s, 2H), 1.17 (t, J=7.0 Hz, 3H).

LCMS: Rt: 1.355 min; MS m/z (ESI): 547.4 [M+H].

Example 4. Preparation of 7-(1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-5-oxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepine-8-carbonyl)-7-azaspiro[3.5]nonane-2-carbonitrile (Compound 003)

[Image Omitted]

Preparation of Compound 3-2:

[Image Omitted]

At room temperature, tert-butyl 2-cyano-7-azaspiro[3.5]nonane-7-carboxylate (3-1) (600 mg, 2.4 mmol) was dissolved in dichloromethane (2 mL), trifluoroacetic acid (1 mL) was slowly added, and the reaction solution was stirred at room temperature for 1 hour. Upon completion of reaction, the reaction solution was concentrated under reduced pressure to afford the intermediate 7-azaspiro[3.5]nonane-2-carbonitrile trifluoroacetate (3-2) (crude, 590 mg, yield: 100%).

Reference can be made to the synthetic method in example 2 for the other steps, and 7-azaspiro[3.5]nonane-2-carbonitrile trifluoroacetate (3-2) was used in place of 3-oxa-8-azabicyclo[3.2.1]octane hydrochloride. The final hydrochloride product prepared was dissolved in dichloromethane and neutralized and washed with saturated sodium bicarbonate aqueous solution. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford the target product 7-(1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-5-oxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepine-8-carbonyl)-7-azaspiro[3.5]nonane-2-carbonitrile (compound 003).

¹H NMR (400 MHz, CD₃OD): δ 7.55 (d, J=7.7 Hz, 1H), 7.13-7.09 (m, 3H), 7.04-7.02 (m, 1H), 6.98-6.95 (m, 2H), 4.06-3.87 (m, 4H), 3.80-3.75 (m, 1H), 3.65-3.60 (m, 4H), 3.51-3.49 (m, 1H), 3.37-3.31 (m, 3H), 3.29-3.18 (m, 3H), 3.02-2.83 (m, 3H), 2.34-2.32 (m, 2H), 2.19-2.16 (m, 2H), 1.75-1.62 (m, 4H), 1.11 (t, J=7.2 Hz, 3H).

LCMS: Rt: 1.324 min; MS m/z (ESI): 542.3 [M+H].

Example 5. Preparation of 1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-8-(3-methoxy-8-azabicyclo[3.2.1]octane-8-carbonyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one hydrochloride (Compound 004)

[Image Omitted]

Reference can be made to the synthetic method in example 2 and 3-methoxy-8-azabicyclo[3.2.1]octane hydrochloride was used in place of 3-oxa-8-azabicyclo[3.2.1]octane hydrochloride to afford the target product 1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-8-(3-methoxy-8-azabicyclo[3.2.1]octane-8-carbonyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one hydrochloride (compound 004).

¹H NMR (400 MHz, CD₃OD): δ 7.68 (d, J=7.6 Hz, 1H), 7.30-7.22 (m, 6H), 4.68 (s, 1H), 4.51-4.37 (m, 2H), 4.33-4.31 (m, 1H), 4.03-3.98 (m, 2H), 3.74-3.62 (m, 5H), 3.60-3.50 (m, 2H), 3.44-3.32 (m, 2H), 3.30 (s, 3H), 3.28-3.22 (m, 2H), 2.25-2.07 (m, 4H), 2.01-1.89 (m, 4H), 1.19 (t, J=6.8 Hz, 3H).

LCMS: Rt: 1.347 min; MS m/z (ESI): 533.2 [M+H].

Example 6. Preparation of 4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-8-(3-methoxy-8-azabicyclo[3.2.1]octane-8-carbonyl)-1-methyl-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one hydrochloride (Compound 005)

[Image Omitted]

Preparation of Compound 5-2:

[Image Omitted]

At room temperature, 8-bromo-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one (1-4) (500 mg, 2.07 mmol), sodium cyanoborohydride (260.7 mg, 4.15 mmol) and paraformaldehyde (125 mg, 4.15 mmol) were added to a mixed solution of acetic acid (1 mL) and methanol (10 mL). The mixture was reacted at room temperature for 1 hour. Upon completion of reaction, 2 mol/L dilute hydrochloric acid (1 mL) and water (50 mL) were added and the mixture was extracted with ethyl acetate (50 mL×2). The organic phases were combined, washed with saturated brine (40 mL×3), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified using a normal phase silica gel chromatographic column (eluent gradient: petroleum ether/ethyl acetate=1/2) to afford the target intermediate 8-bromo-1-methyl-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one (5-2) (400 mg, yield: 76%).

LCMS: Rt: 1.359 min; MS m/z (ESI): 255.0 [M+H].

Preparation of Compound 5-3:

[Image Omitted]

At room temperature, 8-bromo-1-methyl-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one (5-2) (400 mg, 1.57 mmol) was added to ultradry N,N-dimethylformamide (40 mL) and then sodium hydride (75.3 mg, 1.88 mmol) was added. After the addition, the mixture was heated to 40° C. and stirred for 1 hour. Then tert-butyl (S)-3-((S)-oxiran-2-yl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (intermediate 1) (518 mg, 1.88 mmol) was added, and the resulting mixture was reacted for 16 hours. Upon completion of reaction, water (120 mL) was added, and the reaction was extracted with ethyl acetate (100 mL×2). The organic phases were combined, washed with saturated brine (50 mL×2), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified using normal phase silica gel chromatographic column (eluent gradient: petroleum ether/ethyl acetate=1/1) to afford the target intermediate (1R,10aS)-1-((8-bromo-1-methyl-5-oxo-1,2,3,5-tetrahydro-4H-benzo[e][1,4]diazepin-4-yl)methyl)-1,5,10,10a-tetrahydro-3H-oxazolo[3,4-b]isoquinolin-3-one (5-3) (520 mg, yield: 73%).

LCMS: Rt: 1.749 min; MS m/z (ESI): 456.0 [M+H].

Preparation of Compound 5-4:

[Image Omitted]

At room temperature, (1R,10aS)-1-((8-bromo-1-methyl-5-oxo-1,2,3,5-tetrahydro-4H-benzo[e][1,4]diazepin-4-yl)methyl)-1,5,10,10a-tetrahydro-3H-oxazolo[3,4-b]isoquinolin-3-one (5-3) (520 mg, 1.14 mmol) was added to a mixed solution of methanol (5 mL) and water (5 mL), and sodium hydroxide (274 mg, 6.84 mmol) was added. The mixture was heated to 70° C. and reacted for 16.0 hours. Upon completion of reaction, the reaction system was cooled to room temperature and Boc anhydride (497 mg, 2.28 mmol) was added. The resulting mixture was reacted for 1.5 hours. Upon completion of reaction, the mixture was extracted with ethyl acetate (30 mL×3), and the organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was purified using a normal phase silica gel chromatographic column (eluent gradient: petroleum ether/ethyl acetate=1/1) to afford the target intermediate tert-butyl (S)-3-((R)-2-(8-bromo-1-methyl-5-oxo-1,2,3,5-tetrahydro-4H-benzo[e][1,4]diazepin-4-yl)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (5-4) (530 mg, yield: 88%).

LCMS: Rt: 2.088 min; MS m/z (ESI): 530.1 [M+H].

Preparation of Compound 5-5:

[Image Omitted]

At room temperature, tert-butyl (S)-3-((R)-2-(8-bromo-1-methyl-5-oxo-1,2,3,5-tetrahydro-4H-benzo[e][1,4]diazepin-4-yl)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (5-4) (530 mg, 1 mmol), [1,1′-bis(diphenylphosphine)ferrocene]palladium dichloride dichloromethane complex (38 mg, 0.05 mmol) and potassium acetate (294 mg, 3 mmol) were added to anhydrous ethanol (15 mL). The mixture was subjected to CO replacement three times, heated to 70° C. and reacted for 3.0 hours. Upon completion of reaction, the reaction was cooled to room temperature, and the reaction solution was concentrated under reduced pressure. Saturated brine (50 mL) was added and then the mixture was extracted with ethyl acetate (50 mL×2). The organic phase was dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified using a normal phase silica gel chromatographic column (eluent gradient: petroleum ether/ethyl acetate=1/1) to afford the target intermediate ethyl 4-((R)-2-((S)-2-(tert-butoxycarbonyl)-1,2,3,4-tetrahydroisoquinolin-3-yl)-2-hydroxyethyl)-1-methyl-5-oxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepine-8-carboxylate (5-5) (500 mg, yield: 96%).

LCMS: Rt: 1.965 min; MS m/z (ESI): 524.2 [M+H].

Preparation of Compound 5-6:

[Image Omitted]

At room temperature, the intermediate ethyl 4-((R)-2-((S)-2-(tert-butoxycarbonyl)-1,2,3,4-tetrahydroisoquinolin-3-yl)-2-hydroxyethyl)-1-methyl-5-oxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepine-8-carboxylate (5-5) (500 mg, 0.96 mmol) was added to a mixed solution of methanol (5 mL) and water (5 mL), and lithium hydroxide monohydrate (161 mg, 3.82 mmol) was added. The mixture was reacted at room temperature for 2 hours. Upon completion of reaction, the reaction solution was adjusted with 1 mol/L hydrochloric acid aqueous solution to the pH of 5.0 and extracted with ethyl acetate (30 mL×3), and the organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford the target intermediate 4-((R)-2-((S)-2-(tert-butoxycarbonyl)-1,2,3,4-tetrahydroisoquinolin-3-yl)-2-hydroxyethyl)-1-methyl-5-oxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepine-8-carboxylic acid (5-6) (420 mg, yield: 88%).

LCMS: Rt: 1.686 min; MS m/z (ESI): 496.2 [M+H].

Preparation of Compound 5-7:

[Image Omitted]

At room temperature, 4-((R)-2-((S)-2-(tert-butoxycarbonyl)-1,2,3,4-tetrahydroisoquinolin-3-yl)-2-hydroxyethyl)-1-methyl-5-oxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepine-8-carboxylic acid (5-6) (70 mg, 0.14 mmol), 2-(7-azabenzotriazole)-N,N,N′,N′-tetramethylurea hexafluorophosphate (106 mg, 0.28 mmol) and triethylamine (57 mg, 0.56 mmol) were added to ultradry N,N-dimethylformamide (5.0 mL), and 3-methoxy-8-azabicyclo[3.2.1]octane hydrochloride (30 mg, 0.17 mmol) was added. The mixture was reacted at room temperature for 1.5 hours. Upon completion of reaction, saturated brine (30 mL) was added, and the resulting mixture was extracted with ethyl acetate (30 mL×2). The organic phase was washed with saturated brine (30 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was purified using a normal phase silica gel chromatographic column (eluent: ethyl acetate) to afford the target intermediate tert-butyl (3S)-3-((1R)-1-hydroxyl-2-(8-(3-methoxy-8-azabicyclo[3.2.1]octane-8-carbonyl)-1-methyl-5-oxo-1,2,3,5-tetrahydro-4H-benzo[e][1,4]diazepin-4-yl)ethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (5-7) (65 mg, yield: 75%).

LCMS: Rt: 1.831 min; MS m/z (ESI): 619.3 [M+H].

Preparation of Compound 005:

[Image Omitted]

At room temperature, tert-butyl (3S)-3-((1R)-1-hydroxyl-2-(8-(3-methoxy-8-azabicyclo[3.2.1]octane-8-carbonyl)-1-methyl-5-oxo-1,2,3,5-tetrahydro-4H-benzo[e][1,4]diazepin-4-yl)ethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (5-7) (65 mg, 0.1 mmol) was added to dichloromethane (2 mL), then trifluoroacetic acid (1 ml) was added. The mixture was reacted at room temperature for 1 hour. Upon completion of reaction, the reaction solution was concentrated under reduced pressure. The residue was purified by high-performance liquid preparative chromatography (referring to example 2 for the eluents and gradient) to afford the target compound 4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-8-(3-methoxy-8-azabicyclo[3.2.1]octane-8-carbonyl)-1-methyl-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one hydrochloride (compound 5) (20.33 mg, yield: 37%).

¹H NMR (400 MHz, CD₃OD): δ 7.67-7.65 (m, 1H), 7.31-7.19 (m, 6H), 4.69 (s, 1H), 4.50-4.33 (m, 3H), 4.05-4.00 (m, 2H), 3.70-3.60 (m, 4H), 3.57-3.52 (m, 3H), 3.35-3.32 (m, 1H), 3.28-3.22 (m, 4H), 2.98 (s, 3H), 2.23-2.06 (m, 4H), 2.00-1.90 (m, 4H).

LCMS: Rt: 1.271 min; MS m/z (ESI): 519.2 [M+H].

Example 7. Preparation of 1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-8-(9-methoxy-3-azaspiro[5.5]undecane-3-carbonyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one hydrochloride (Compound 006)

[Image Omitted]

Preparation of Compound 6-2:

[Image Omitted]

At room temperature, tert-butyl 9-oxo-3-azaspiro[5.5]undecane-3-carboxylate (6-1) (400 mg, 1.5 mmol) was added to methanol (10 mL), and at 0° C., sodium borohydride (171 mg, 4.5 mmol) was added. The mixture was reacted at room temperature for 1.5 hours. The reaction was quenched by adding an aqueous ammonium chloride solution (10 mL) and the reaction solution was extracted with ethyl acetate (50 mL×2). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified using a normal phase silica gel chromatographic column (petroleum ether/ethyl acetate=2/1) to afford tert-butyl 9-hydroxy-3-azaspiro[5.5]undccane-3-carboxylatc (6-2) (305 mg, yield: 76%).

LCMS: Rt: 1.570 min; MS m/z (ESI): 270.0 [M+H].

Preparation of Compound 6-3:

[Image Omitted]

Tert-butyl 9-hydroxy-3-azaspiro[5.5]undecane-3-carboxylate (6-2) (540 mg, 2 mmol) was dissolved in N,N-dimethylformamide (10 mL) and the mixture was cooled to 0° C. Sodium hydride (320 mg, 8 mmol) was added in batches and then the mixture was reacted at 0° C. for 1.0 hour. After iodomethane (568 mg, 4 mmol) was added, the resulting reaction solution was stirred at room temperature overnight. Upon completion of reaction, the reaction was quenched by adding saturated ammonium chloride solution (10 mL). The resulting reaction solution was diluted with saturated brine (50 mL) and extracted with ethyl acetate (20 mL×2). The organic phase was washed with saturated brine (30 mL×2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford the target intermediate tert-butyl 9-methoxy-3-azaspiro[5.5]undecane-3-carboxylate (6-3) (crude, 560 mg, yield: 100%).

Preparation of Compound 6-4:

[Image Omitted]

Tert-butyl 9-methoxy-3-azaspiro[5.5]undecane-3-carboxylate (6-3) (560 mg, 2 mmol) was dissolved in dioxane (1 mL) and the mixture was cooled to 0° C. 4 mol/L hydrochloric acid/dioxane solution (4 mL) was added and the mixture was reacted at 0° C. for 1.0 hour. Upon completion of reaction, the reaction solution was concentrated under reduced pressure. The residue was slurried with methyl tert-butyl ether and then filtered to afford the target intermediate 9-methoxy-3-azaspiro[5.5]undecane hydrochloride (6-4) (192 mg, yield: 44%).

LCMS: Rt: 0.415 min; MS m/z (ESI): 184.2 [M+H].

Reference can be made to the synthetic method in example 2 for the other steps and 9-methoxy-3-azaspiro[5.5]undecane hydrochloride (6-4) was used in place of 3-oxa-8-azabicyclo[3.2.1]octane hydrochloride to afford the target product 1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-8-(9-methoxy-3-azaspiro[5.5]undecane-3-carbonyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one hydrochloride (compound 006).

¹H NMR (400 MHz, CD₃OD): δ 7.62-7.60 (m, 1H), 7.31-7.21 (m, 4H), 7.08-7.03 (m, 2H), 4.47-4.38 (m, 2H), 4.30-4.27 (m, 1H), 3.99-3.95 (m, 1H), 3.74-3.56 (m, 7H), 3.43-3.38 (m, 3H), 3.32-3.31 (m, 3H), 3.27-3.22 (m, 5H), 1.78-1.70 (m, 4H), 1.63-1.57 (m, 1H), 1.51-1.38 (m, 5H), 1.32-1.23 (m, 2H), 1.15 (t, J=7 Hz, 3H).

LCMS: Rt: 1.347 min; MS m/z (ESI): 575.3 [M+H].

Example 8. Preparation of 8-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-3,4-dihydrobenzo[f][1,4]oxazepin-5(2H)-one (Compound 007)

[Image Omitted]

Preparation of Compound 7-2:

[Image Omitted]

At room temperature, 4-bromo-2-hydroxybenzoic acid (7-1) (2.5 g, 11.5 mmol) and N-hydroxybutyldicarboxamide (2.65 g, 23.0 mmol) were added to ultradry tetrahydrofuran (75.0 mL), and then N,N-diisopropylcarbodiimide (2.9 g, 23.0 mmol) was added. The mixture was reacted at room temperature for 4.0 hours. Upon completion of reaction, the reaction solution was concentrated under reduced pressure. The residue was purified using a normal phase silica gel chromatographic column (eluent gradient: petroleum ether/ethyl acetate=2/1) to afford the target intermediate 2,5-dicarbonylpyrrolidin-1-yl 4-bromo-2-hydroxybenzoate (7-2) (3.45 g, yield: 96%).

LCMS: Rt: 1.546 min; MS m/z (ESI): 311.8 [M−H].

Preparation of Compound 7-4:

[Image Omitted]

At room temperature, tert-butyl (S)-3-((R)-2-amino-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (7-3, referring to U.S. Ser. No. 10/494,376 B2 for the synthetic method) (1.0 g, 3.42 mmol) and 1,8-diazabicycloundec-7-ene (2.1 g, 13.68 mmol) were added to acetonitrile (30.0 mL), then at 0° C., tert-butyldimethylsilyl chloride (1.03 g, 6.85 mmol) was added. The mixture was reacted at room temperature for 5.0 hours. Upon completion of reaction, water (50 mL) was added, and the mixture was extracted with ethyl acetate (50 mL×2). The organic phase was washed with saturated brine (30 mL×3), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was purified using a normal phase silica gel chromatographic column (eluent gradient: dichloromethane/methanol=50/1) to afford the target intermediate tert-butyl (S)-3-((R)-2-amino-1-((tert-butyldimethylsilyl)oxy)ethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (7-4) (1.21 g, yield: 87%).

LCMS: Rt: 1.321 min; MS m/z (ESI): 407.1 [M+H].

Preparation of Compound 7-5:

[Image Omitted]

At room temperature, 2,5-dicarbonylpyrrolidin-1-yl 4-bromo-2-hydroxybenzoate (7-2) (773.0 mg, 2.46 mmol) and tert-butyl (S)-3-((R)-2-amino-1-((tert-butyldimethylsilyl)oxy)ethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (7-4) (1.1 g, 2.71 mmol) were added to ultradry N,N-dimethylformamide (15.0 mL), and then triethylamine (1.8 mL) was added. The mixture was reacted at room temperature for 16.0 hours. Upon completion of reaction, water (100 mL) was added, and the mixture was extracted with ethyl acetate (100 mL×2). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was purified using a normal phase silica gel chromatographic column (eluent gradient: petroleum ether/ethyl acetate=5/1) to afford the target intermediate tert-butyl (S)-3-((R)-2-(4-bromo-2-hydroxybenzamido)-1-((tert-butyl dimethylsilyl)oxy)ethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (7-5) (1.1 g, yield: 74%).

LCMS: Rt: 1.814 min; MS m/z (ESI): 605.1 [M+H].

Preparation of Compound 7-6:

[Image Omitted]

At room temperature, tert-butyl (S)-3-((R)-2-(4-bromo-2-hydroxybenzamido)-1-((tert-butyl dimethylsilyl)oxy)ethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (7-5) (500.0 mg, 0.825 mmol) and 2-bromoethanol (231.0 mg, 1.85 mmol) were added to N,N-dimethylformamide (8.0 mL), and then potassium carbonate (340.0 mg, 2.46 mmol) was added. The mixture was warmed to 60° C. and reacted for 8.0 hours. Upon completion of reaction, water (50 mL) was added, and the mixture was extracted with ethyl acetate (50 mL×2). The organic phase was washed with saturated brine (30 mL×3), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was purified using a normal phase silica gel chromatographic column (eluent gradient: petroleum ether/ethyl acetate=3/1) to afford the target intermediate (S)-3-((R)-2-(4-bromo-2-(2-hydroxyethoxy)benzamido)-1-((tert-butyl dimethylsilyl)oxy)ethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (7-6) (454 mg, yield: 85%).

LCMS: Rt: 2.40 min; MS m/z (ESI): 649.1 [M+H].

Preparation of Compound 7-7:

[Image Omitted]

At room temperature, (S)-3-((R)-2-(4-bromo-2-(2-hydroxyethoxy)benzamido)-1-((tert-butyl dimethylsilyl)oxy)ethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (7-6) (454 mg, 0.7 mmol) and triethylamine (212.0 mg, 2.1 mmol) were added to dichloromethane (10.0 mL), and the mixture was cooled to 0° C. Then methyl sulfonyl chloride (160.0 mg, 1.4 mmol) was added and the mixture was reacted at room temperature for 2.0 hours. Upon completion of reaction, water (50 mL) was added, and the mixture was extracted with dichloromethane (50 mL×3). The organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was purified using a normal phase silica gel chromatographic column (eluent gradient: petroleum ether/ethyl acetate=2/1) to afford the target intermediate tert-butyl (S)-3-((R)-2-(4-bromo-2-(2-((methanesulfonyl)oxy)ethoxy)benzamido)-1-((tert-butyl dimethylsilyl)oxy)ethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (7-7) (510 mg, yield: 100%).

LCMS: Rt: 2.123 min; MS m/z (ESI): 727.1 [M+H].

Preparation of Compound 7-8:

[Image Omitted]

At room temperature, tert-butyl (S)-3-((R)-2-(4-bromo-2-(2-((methanesulfonyl)oxy)ethoxy)benzamido)-1-((tert-butyl dimethylsilyl)oxy)ethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (7-7) (510 mg, 0.7 mmol) was added to ultradry N,N-dimethylformamide (10.0 mL), and then sodium hydride (36.4 mg, 0.91 mmol) was added. The mixture was reacted at 50° C. for 16.0 hours. Upon completion of reaction, water (50 mL) was added, and the mixture was extracted with ethyl acetate (50 mL×3). The organic phase was washed with saturated brine (50 mL×3), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was purified using a normal phase silica gel chromatographic column (eluent gradient: petroleum ether/ethyl acetate=10/1) to afford the target intermediate tert-butyl (S)-3-((R)-2-(8-bromo-5-carbonyl-2,3-dihydrobenzo[f][1,4]oxazepin-4(5H)-yl)-1-((tert-butyl dimethylsilyl)oxy)ethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (7-8) (341 mg, yield: 77%).

LCMS: Rt: 2.405 min; MS m/z (ESI): 631.1 [M+H].

Preparation of Compound 7-9:

[Image Omitted]

At room temperature, tert-butyl (S)-3-((R)-2-(8-bromo-5-carbonyl-2,3-dihydrobenzo[f][1,4]oxazepin-4(5H)-yl)-1-((tert-butyl dimethylsilyl)oxy)ethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (7-8) (291 mg, 0.46 mmol), [1,1′-bis(diphenylphosphine)ferrocene]palladium dichloride (17 mg, 0.023 mmol) and potassium acetate (135 mg, 1.38 mmol) were added to anhydrous ethanol (17.0 mL). The mixture was subjected to CO replacement three times, heated to 70° C. and reacted for 4.0 hours. Upon completion of reaction, the reaction solution was cooled to room temperature and concentrated under reduced pressure. The residue was purified using a normal phase silica gel chromatographic column (eluent gradient: petroleum ether/ethyl acetate=5/1) to afford the target intermediate ethyl 4-((R)-2-((S)-2-(tert-butoxycarbonyl)-1,2,3,4-tetrahydroisoquinolin-3-yl)-2-((tert-butyl dimethylsilyl)oxy)ethyl)-5-oxo-2,3,4,5-tetrahydrobenzo[f][1,4]oxazepine-8-carboxylate (7-9) (261 mg, yield: 91%).

LCMS: Rt: 1.532 min; MS m/z (ESI): 625.0 [M+H].

Preparation of Compound 7-10:

[Image Omitted]

At room temperature, ethyl 4-((R)-2-((S)-2-(tert-butoxycarbonyl)-1,2,3,4-tetrahydroisoquinolin-3-yl)-2-((tert-butyl dimethylsilyl)oxy)ethyl)-5-oxo-2,3,4,5-tetrahydrobenzo[f][1,4]oxazepine-8-carboxylate (7-9) (261 mg, 0.42 mmol) was added to a mixed solution of methanol (5.0 mL), tetrahydrofuran (5.0 mL) and water (5.0 mL). Then lithium hydroxide monohydrate (140.0 mg, 3.34 mmol) was added, and the mixture was reacted at room temperature for 3.0 hours. Upon completion of reaction, the reaction system was cooled to 0° C. and the reaction solution was adjusted with 1 N hydrochloric acid aqueous solution to the pH of 5.0 and then extracted with ethyl acetate (30 mL×2). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to afford the target intermediate 4-((R)-2-((S)-2-(tert-butoxycarbonyl)-1,2,3,4-tetrahydroisoquinolin-3-yl)-2-((tert-butyl dimethylsilyl)oxy)ethyl)-5-oxo-2,3,4,5-tetrahydrobenzo[f][1,4]oxazepine-8-carboxylic acid (7-10) (241 mg, yield: 97%).

LCMS: Rt: 0.788 min; MS m/z (ESI): 597.2 [M+H].

Preparation of Compound 7-11:

[Image Omitted]

At room temperature, 4-((R)-2-((S)-2-(tert-butoxycarbonyl)-1,2,3,4-tetrahydroisoquinolin-3-yl)-2-((tert-butyl dimethylsilyl)oxy)ethyl)-5-oxo-2,3,4,5-tetrahydrobenzo[f][1,4]oxazepine-8-carboxylic acid (7-10) (20 mg, 0.033 mmol), 2-(7-azabenzotriazole)-N,N,N′,N′-tetramethylurea hexafluorophosphate (25.5 mg, 0.067 mmol) and N,N-diisopropylethylamine (17.0 mg, 0.134 mmol) were added to ultradry N,N-dimethylformamide (1.0 mL). Then 3-oxa-8-azabicyclo[3.2.1]octane hydrochloride (6.5 mg, 0.044 mmol) was added, and the mixture was reacted at room temperature for 2.0 hours. Upon completion of reaction, saturated brine (30 mL) was added, and the resulting mixture was extracted with ethyl acetate (30 mL×2). The organic phase was washed with saturated brine (30 mL×2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by Prep-TLC (developing agent: petroleum ether/ethyl acetate=1/1) to afford the target intermediate tert-butyl (3S)-3-((1R)-2-(8-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-5-oxo-2,3-dihydrobenzo[f][1,4]oxazepin-4(5H)-yl)-1-((tert-butyl dimethylsilyl)oxy)ethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (7-11) (20.1 mg, yield: 87%).

LCMS: Rt: 1.813 min; MS m/z (ESI): 692.4 [M+H].

Preparation of Compound 007:

[Image Omitted]

At 0° C., tert-butyl (3S)-3-((1R)-2-(8-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-5-oxo-2,3-dihydrobenzo[f][1,4]oxazepin-4(5H)-yl)-1-((tert-butyl dimethylsilyl)oxy)ethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (7-11) (20.1 mg) was added to a hydrochloric acid/dioxane solution (4 mol/L, 5.0 mL), and the reaction solution was stirred at room temperature for 20 hours. Upon completion of reaction, the mixture was concentrated under reduced pressure. The residue was purified by high-performance liquid preparative chromatography using the eluent gradient below:

Instruments
Waters 2767

Mobile phase
A: 0.1% NH₄HCO₃in H₂O B: CH₃CN

Chromatographic column
Waters sunfire C18 19*250 mm 10 μm

Injection volume
500 μL

Table of running gradients
Time (min)
Mobile phase A (%)
Mobile phase B (%)

0
90
5

1
80
20

11
60
40

11.2
5
95

13
5
95

13.2
95
5

15
95
5

to afford or the target compound 8-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-3,4-dihydrobenzo[f][1,4]oxazepin-5(2H)-one (compound 007) (7.09 mg, yield: 51%).

¹H NMR (400 MHz, CD₃OD): δ 7.79 (d, J=8.0 Hz, 1H), 7.31-7.26 (m, 1H), 7.18-7.08 (m, 4H), 7.08-7.02 (m, 1H), 4.63 (s, 1H), 4.56-4.47 (m, 2H), 4.10-3.94 (m, 5H), 3.84-3.63 (m, 6H), 3.62-3.52 (m, 1H), 2.99-2.82 (m, 3H), 2.12-1.92 (m, 4H).

LCMS: Rt: 1.525 min; MS n/z (ESI): 478.1 [M+H].

Example 9. Preparation of 4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-8-(3-methoxy-8-azabicyclo[3.2.1]octane-8-carbonyl)-3,4-dihydrobenzo[f][1,4]oxazepin-5(2H)-one (Compound 008)

[Image Omitted]

Reference can be made to the synthetic method in example 8 and 3-methoxy-8-azabicyclo[3.2.1]octane hydrochloride was used in place of 3-oxa-8-azabicyclo[3.2.1]octane hydrochloride to afford the target product 4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-8-(3-methoxy-8-azabicyclo[3.2.1]octane-8-carbonyl)-3,4-dihydrobenzo[f][1,4]oxazepin-5(2H)-one (compound 008).

¹H NMR (400 MHz, CD₃OD): δ 7.78 (d, J=8.0 Hz, 1H), 7.24 (dd, J=8.0 Hz, 1.6 Hz, 1H), 7.13-7.03 (m, 5H), 4.66 (s, 1H), 4.53-4.50 (m, 2H), 4.07-3.94 (m, 5H), 3.76-3.66 (m, 3H), 3.55 (s, 1H), 3.29 (s, 3H), 2.95-2.85 (m, 3H), 2.19-2.07 (m, 4H), 1.95-1.86 (m, 4H).

LCMS: Rt: 1.305 min; MS m/z (ESI): 506.3 [M+H].

Example 10. Preparation of 4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-8-(2-methoxy-7-azaspiro[3.5]nonane-7-carbonyl)-3,4-dihydrobenzo[f][1,4]oxazepin-5(2H)-one (Compound 009)

[Image Omitted]

Reference can be made to the synthetic method in example 8 and 2-methoxy-7-azaspiro[3.5]nonane hydrochloride was used in place of 3-oxa-8-azabicyclo[3.2.1]octane hydrochloride to afford the target compound 4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-8-(2-methoxy-7-azaspiro[3.5]nonane-7-carbonyl)-3,4-dihydrobenzo[f][1,4]oxazepin-5(2H)-one (compound 009).

¹H NMR (400 MHz, CD₃OD): δ 7.77 (d, J=7.9 Hz, 1H), 7.16 (d, J=7.9 Hz, 1H), 7.15-7.07 (m, 3H), 7.07-6.99 (m, 2H), 4.57-4.45 (m, 2H), 4.10-3.85 (m, 5H), 3.80-3.54 (m, 5H), 3.36-3.30 (m, 2H), 3.21 (s, 3H), 3.00-2.82 (m, 3H), 2.31-2.18 (m, 2H), 1.79-1.60 (m, 4H), 1.54 (s, 2H).

LCMS: Rt: 1.306 min; MS m/z (ESI): 520.3 [M+H].

Example 11. Preparation of 1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-8-(8-oxa-2-azaspiro[4.5]decane-2-carbonyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one hydrochloride (Compound 010)

[Image Omitted]

Reference can be made to the synthetic method in example 2 and 8-oxa-2-azaspiro[4.5]decane was used in place of 3-oxa-8-azabicyclo[3.2.1]octane hydrochloride to afford the target product 1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-8-(8-oxa-2-azaspiro[4.5]decane-2-carbonyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one hydrochloride (compound 010).

¹H NMR (400 MHz, CD₃OD): δ 7.71 (dd, J=7.9, 2.0 Hz, 1H), 7.42-7.19 (m, 6H), 4.52-4.31 (m, 3H), 4.07-3.97 (m, 1H), 3.80-3.62 (m, 9H), 3.62-3.49 (m, 4H), 3.49-3.33 (m, 3H), 3.29-3.20 (m, 2H), 1.96 (t, J=7.3 Hz, 1H), 1.89 (t, J=6.9 Hz, 1H), 1.72-1.63 (m, 2H), 1.59-1.49 (m, 2H), 1.20 (t, J=7.0 Hz, 3H).

LCMS: Rt: 1.230 min; MS m/z (ESI): 533.2 [M+H].

Example 12. Preparation of 1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-8-(7-methoxy-2-azaspiro[3.5]nonane-2-carbonyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one hydrochloride (Compound 011)

[Image Omitted]

Preparation of Compound 011-2:

[Image Omitted]

At room temperature, tert-butyl 7-hydroxy-2-azaspiro[3.5]nonane-2-carboxylate (50 mg, 0.207 mmol) was added to ultradry N,N-dimethylformamide (1.5 mL), and then sodium hydride (20 mg, 0.498 mmol) was added under ice bath. The mixture was reacted at room temperature for 0.5 hours. Then iodomethane (59 mg, 0.414 mmol) was added, and the resulting mixture was reacted at room temperature for 1.5 hours. Upon completion of reaction, the reaction was quenched with saturated aqueous ammonium chloride solution and extracted with ethyl acetate (30 mL×2). The organic phases were combined, washed with saturated brine (50 mL×3), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford the target intermediate tert-butyl 7-methoxy-2-azaspiro[3.5]nonane-2-carboxylate (011-2) (51 mg).

LCMS: Rt: 1.800 min; MS m/z (ESI): 256.0 [M+H].

Preparation of Compound 011-3:

[Image Omitted]

At room temperature, tert-butyl 7-methoxy-2-azaspiro[3.5]nonane-2-carboxylate (011-2) (51 mg, 0.2 mmol) was added to a hydrochloric acid-dioxane solution (4 N, 3.0 mL) and the mixture was reacted at room temperature for 1 hour. Upon completion of reaction, the reaction solution was concentrated under reduced pressure to afford the intermediate 7-methoxy-2-azaspiro[3.5]nonane hydrochloride (011-3) (33.7 mg).

LCMS: Rt: 0.391 min; MS m/z (ESI): 156.3 [M+H].

Reference can be made to the synthetic method in example 2 for the other steps and 7-methoxy-2-azaspiro[3.5]nonane hydrochloride (011-3) was used in place of 3-oxa-8-azabicyclo[3.2.1]octane hydrochloride to afford the target product 1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-8-(7-methoxy-2-azaspiro[3.5]nonane-2-carbonyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one hydrochloride (compound 011).

¹H NMR (400 MHz, CD₃OD): δ 7.70 (d, J=8.0 Hz, 1H), 7.51-7.46 (m, 2H), 7.31-7.22 (m, 4H), 4.49-4.33 (m, 3H), 4.05-3.88 (m, 3H), 3.85 (s, 2H), 3.72-3.58 (m, 6H), 3.47-3.40 (m, 2H), 3.35-3.31 (m, 3H), 3.30-3.21 (m, 3H), 1.94-1.38 (m, 4H), 1.62-1.56 (m, 2H), 1.44-1.38 (m, 2H), 1.20 (t, J=7.2 Hz, 3H).

LCMS: Rt: 1.302 min; MS m/z (ESI): 547.2 [M+H].

Example 13. Preparation of 8-(4,4-difluoro-6-azaspiro[2.5]octane-6-carbonyl)-1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one hydrochloride (Compound 012)

[Image Omitted]

Reference can be made to the synthetic method in example 2 and 4,4-difluoro-6-azaspiro[2.5]octane hydrochloride was used in place of 3-oxa-8-azabicyclo[3.2.1]octane hydrochloride to afford the target product 8-(4,4-difluoro-6-azaspiro[2.5]octane-6-carbonyl)-1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one hydrochloride (compound 012).

¹H NMR (400 MHz, CD₃OD): δ7.74 (d, J=7.6 Hz, 1H), 7.31-7.22 (m, 6H), 4.50-4.35 (m, 3H), 4.04-4.00 (m, 2H), 3.87 (s, 1H), 3.74-3.63 (m, 7H), 3.55-3.35 (m, 3H), 3.28-3.22 (m, 2H), 1.73-1.66 (m, 2H), 1.21 (t, J=6.8 Hz, 3H), 0.91 (s, 2H), 0.57 (s, 2H).

LCMS: Rt: 1.361 min; MS m/z (ESI): 539.3 [M+H].

Example 14. Preparation of 8-(1,1-difluoro-6-azaspiro[2.5]octane-6-carbonyl)-1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one hydrochloride (Compound 013)

[Image Omitted]

Reference can be made to the synthetic method in example 2 and 1,1-difluoro-6-azaspiro[2.5]octane hydrochloride was used in place of 3-oxa-8-azabicyclo[3.2.1]octane hydrochloride to afford the target product 8-(1,1-difluoro-6-azaspiro[2.5]octane-6-carbonyl)-1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one hydrochloride (compound 013).

¹H NMR (400 MHz, CD₃OD): δ7.70 (d, J=8.0 Hz, 1H), 7.31-7.22 (m, 6H), 4.49-4.33 (m, 3H), 4.01 (dd, J=4.4 Hz, 14.0 Hz, 1H), 3.80 (s, 2H), 3.73-3.58 (m, 6H), 3.48-3.35 (m, 4H), 3.27-3.21 (m, 2H), 1.78-1.611 (m, 4H), 1.26-1.25 (m, 2H), 1.20 (t, J=6.8 Hz, 3H).

LCMS: Rt: 1.330 min; MS m/z (ESI): 539.3 [M+H].

Example 15. Preparation of 1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-8-(morpholine-4-carbonyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one hydrochloride (Compound 014)

[Image Omitted]

Reference can be made to the synthetic method in example 2 and morpholine was used in place of 3-oxa-8-azabicyclo[3.2.1]octane hydrochloride to afford the target product 1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-8-(morpholine-4-carbonyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one hydrochloride (compound 014).

¹H NMR (400 MHz, CD₃OD): δ 7.62 (d, J=7.7 Hz, 1H), 7.32-7.20 (m, 4H), 7.12-7.06 (m, 2H), 4.50-4.37 (m, 2H), 4.34-4.26 (m, 1H), 3.98 (dd, J=14.2, 4.5 Hz, 1H), 3.84-3.53 (m, 12H), 3.50-3.39 (m, 3H), 3.30-3.20 (m, 3H), 1.16 (t, J=7.0 Hz, 3H).

LCMS: Rt: 1.336 min; MS m/z (ESI): 479.1 [M+H].

Example 16. Preparation of 8-(4-acetylpiperazine-1-carbonyl)-1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one hydrochloride (Compound 015)

[Image Omitted]

Reference can be made to the synthetic method in example 2 and 1-acetylpiperazine was used in place of 3-oxa-8-azabicyclo[3.2.1]octane hydrochloride to afford the target product 8-(4-acetylpiperazine-1-carbonyl)-1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one hydrochloride (compound 015).

¹H NMR (400 MHz, CD₃OD): δ 7.61 (d, J=8.0 Hz, 1H), 7.31-7.20 (m, 4H), 7.10-7.03 (m, 2H), 4.50-4.36 (m, 2H), 4.33-4.22 (m, 1H), 3.97 (dd, J=14.2, 4.4 Hz, 1H), 3.86-3.35 (m, 15H), 3.28-3.11 (m, 3H), 2.21-2.04 (m, 3H), 1.15 (t, J=6.8 Hz, 3H).

LCMS: Rt: 1.574 min; MS m/z (ESI): 520.3 [M+H].

Example 17. Preparation of N-benzyl-1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-N-methyl-5-oxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diaza-8-carboxamide hydrochloride (Compound 016)

[Image Omitted]

Reference can be made to the synthetic method in example 2 and N-methyl-1-phenylmethanamine was used in place of 3-oxa-8-azabicyclo[3.2.1]octane hydrochloride to afford the target product N-benzyl-1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-N-methyl-5-oxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diaza-8-carboxamide hydrochloride (compound 016).

¹H NMR (400 MHz, CD₃OD): δ 7.67-7.57 (m, 1H), 7.42-7.17 (m, 9H), 7.16-6.99 (m, 2H), 4.76 (s, 11H), 4.55 (s, 11H), 4.47-4.36 (m, 2H), 4.33-4.24 (m, 11H), 4.04-3.87 (m, 1H), 3.76-3.34 (m, 7H), 3.27-3.16 (m, 2H), 3.11-2.86 (m, 4H), 1.23-0.90 (m, 3H).

LCMS: Rt: 1.460 min; MS m/z (ESI): 513.1 [M+H].

Example 18. Preparation of 1-ethyl-N-(4-fluorophenethyl)-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-N-methyl-5-oxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepine-8-carboxamide hydrochloride (Compound 017)

[Image Omitted]

Reference can be made to the synthetic method in example 2 and 2-(4-fluorophenyl)-N-methylethylamine was used in place of 3-oxa-8-azabicyclo[3.2.1]octane hydrochloride to afford the target product 1-ethyl-N-(4-fluorophenethyl)-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-N-methyl-5-oxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepine-8-carboxamide hydrochloride (compound 017).

¹H NMR (400 MHz, CD₃OD): δ 7.56 (dd, J=35.7, 7.8 Hz, 1H), 7.38-7.16 (m, 5H), 7.09-6.84 (m, 4H), 6.77-6.67 (m, 1H), 4.52-4.38 (m, 2H), 4.35-4.26 (m, 4.5 Hz, 1H), 3.98 (dd, J=14.2, 4.4 Hz, 1H), 3.87-3.50 (m, 7H), 3.49-3.38 (m, 1H), 3.30-3.09 (m, 6H), 3.06-2.95 (m, 1H), 2.89-2.79 (m, 2H), 1.20-1.06 (m, 3H).

LCMS: Rt: 1.274 min; MS m/z (ESI): 545.3 [M+H].

Example 19. Preparation of 1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-N-methyl-5-oxo-N-(2-(pyridin-3-yl)ethyl)-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepine-8-carboxamide hydrochloride (Compound 018)

[Image Omitted]

Reference can be made to the synthetic method in example 2 and N-methyl-2-(pyridin-3-yl)ethan-1-amine was used in place of 3-oxa-8-azabicyclo[3.2.1]octane hydrochloride to afford the target product 1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-N-methyl-5-oxo-N-(2-(pyridin-3-yl)ethyl)-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepine-8-carboxamide hydrochloride (compound 018).

¹H NMR (400 MHz, CD₃OD): δ 9.07-8.56 (m, 3H), 8.19-7.96 (m, 1H), 7.77-7.52 (m, 1H), 7.39-7.18 (m, 4H), 7.17-6.52 (m, 2H), 4.53-4.28 (m, 3H), 4.13-3.82 (m, 3H), 3.79-3.42 (m, 6H), 3.41-3.31 (m, 3H), 3.29-2.93 (m, 6H), 1.17 (s, 3H).

LCMS: Rt: 1.654 min; MS m/z (ESI): 528.3 [M+H].

Example 20. Preparation of N,N,1-triethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-5-oxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepine-8-carboxamide hydrochloride (Compound 019)

[Image Omitted]

Reference can be made to the synthetic method in example 2 and diethylamine was used in place of 3-oxa-8-azabicyclo[3.2.1]octane hydrochloride to afford the target product N,N,1-triethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-5-oxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepine-8-carboxamide hydrochloride (compound 019).

¹H NMR (400 MHz, CD₃OD): δ 7.69 (d, J=7.7 Hz, 1H), 7.34-7.21 (m, 4H), 7.20-7.12 (m, 2H), 4.51-4.38 (m, 2H), 4.38-4.31 (m, 1H), 4.06-3.96 (m, 1H), 3.75-3.61 (m, 5H), 3.60-3.49 (m, 3H), 3.47-3.33 (m, 3H), 3.29-3.20 (m, 3H), 1.26 (t, J=7.1 Hz, 3H), 1.22-1.10 (m, 6H).

LCMS: Rt: 1.274 min; MS m/z (ESI): 465.3 [M+H].

Example 21. Preparation of 8-(3-oxa-9-azabicyclo[3,3,1]nonane-9-carbonyl)-1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one hydrochloride (Compound 020)

[Image Omitted]

Reference can be made to the synthetic method in example 2 and 3-oxa-9-azabicyclo[3.3.1]nonane hydrochloride was used in place of 3-oxa-8-azabicyclo[3.2.1]octane hydrochloride to afford the target product 8-(3-oxa-9-azabicyclo[3.3.1]nonane-9-carbonyl)-1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one hydrochloride (compound 020).

¹H NMR (400 MHz, CD₃OD): δ 7.63 (d, J=8.0 Hz, 1H), 7.28-7.21 (m, 4H), 7.11 (t, J=6.4 Hz, 2H), 4.50 (s, 1H), 4.42-4.31 (m, 2H), 4.30-4.29 (m, 1H), 4.03-3.95 (m, 2H), 3.87-3.85 (m, 2H), 3.78-3.66 (m, 7H), 3.58 (s, 1H), 3.33-3.30 (m, 2H), 3.29-3.25 (m, 2H), 2.69-2.55 (m, 1H), 1.99-1.83 (m, 4H) 1.66-1.64 (m, 11H), 1.15 (t, J=7.0 Hz, 3H).

LCMS: Rt: 1.382 min; MS m/z (ESI): 519.5 [M+H].

Example 22. Preparation of 8-(2-(dimethylamino)-7-azaspiro[3.5]nonane-7-carbonyl)-1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one hydrochloride (Compound 021)

[Image Omitted]

Preparation of Compound 021-2:

[Image Omitted]

At room temperature, tert-butyl 2-oxo-7-azaspiro[3.5]nonane-7-carboxylate (1 g, 4.2 mmol) was added to methanol (10 mL) and then dissolved. Then acetic acid (1.5 mL, 20.9 mmol) and dimethylamine (10.5 mL, 20.9 mmol) were added, and the resulting mixture was reacted at room temperature for 0.5 hours. Subsequently, sodium cyanoborohydride (1.3 g, 20.9 mmol) was added. Upon completion of reaction, the reaction solution was concentrated under reduced pressure to remove methanol, ethyl acetate (50 mL) was added for dissolution, and the mixture was washed with a sodium chloride solution (10 mL×2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford tert-butyl 2-(dimethylamino)-7-azaspiro[3.5]nonane-7-carboxylate (021-2) (800 mg).

LCMS: Rt: 1.054 min; MS m/z (ESI): 269.1 [M+H].

Preparation of Compound 021-3

[Image Omitted]

At room temperature, tert-butyl 2-(dimethylamino)-7-azaspiro[3.5]nonane-7-carboxylate (021-2) (800 mg, 2.9 mmol) was added to a hydrogen chloride-dioxane (2.5 mL, 4 mol/L) solution. The mixture was stirred and reacted at room temperature for 1.0 hour. Upon completion of reaction, the reaction solution was concentrated under reduced pressure and then slurried with ethyl acetate (3 mL). The product was collected to afford a hydrochloride (700 mg) of N,N-dimethyl-7-azaspiro[3.5]nonane-2-amine (021-3).

LCMS: Rt: 0.263 min; MS m/z (ESI): 169.1 [M+H].

Reference can be made to the synthesis in example 2 for the other steps and the hydrochloride of N,N-dimethyl-7-azaspiro[3.5]nonane-2-amine (021-3) was used in place of 3-oxa-8-azabicyclo[3.2.1]octane hydrochloride to afford the target product 8-(2-(dimethylamino)-7-azaspiro[3.5]nonane-7-carbonyl)-1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one hydrochloride (compound 021).

¹H NMR (400 MHz, CD₃OD): δ 7.60 (d, J=8.0 Hz, 1H), 7.33-7.19 (m, 4H), 7.07-6.99 (m, 2H), 4.49-4.36 (m, 2H), 4.34-4.27 (m, 1H), 3.98 (dd, J=14.2, 4.6 Hz, 1H), 3.80-3.60 (m, 6H), 3.60-3.52 (m, 3H), 3.48-3.37 (m, 2H), 3.29-3.20 (m, 4H), 2.78 (s, 6H), 2.38 (s, 2H), 2.06 (t, J=10.3 Hz, 2H), 1.80-1.56 (m, 4H), 1.14 (t, J=7.0 Hz, 3H).

LCMS: Rt: 0.517 min; MS m/z (ESI): 560.6 [M+H].

Example 23. Preparation of 1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-8-(3-hydroxy-8-azabicyclo[3.2.1]octane-8-carbonyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one hydrochloride (Compound 022)

[Image Omitted]

Reference can be made to the synthetic method in example 2 and 8-azabicyclo[3.2.1]octane-3-ol was used in place of 3-oxa-8-azabicyclo[3.2.1]octane hydrochloride to afford the target compound 1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-8-(3-hydroxy-8-azabicyclo[3.2.1]octane-8-carbonyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one hydrochloride (compound 022).

¹H NMR (400 MHz, CD₃OD): δ 7.68 (d, J=7.7 Hz, 1H), 7.37-7.17 (m, 6H), 4.70 (s, 1H), 4.51-4.37 (m, 2H), 4.37-4.28 (m, 1H), 4.15-3.93 (m, 3H), 3.76-3.50 (m, 6H), 3.47-3.33 (m, 2H), 3.28-3.15 (m, 2H), 2.47-2.12 (m, 2H), 2.08-1.87 (m, 4H), 1.79 (d, J=14.4 Hz, 1H), 1.39-1.24 (m, 1H), 1.18 (t, J=7.0 Hz, 3H).

LCMS: Rt: 1.310 min; MS m/z (ESI): 519.5 [M+H].

Example 24. Preparation of 1-ethyl-8-(hexahydro-1H-furo[3,4-c]pyrrole-5-carbonyl)-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one hydrochloride (Compound 023)

[Image Omitted]

Reference can be made to the synthetic method in example 2 and hexahydro-1H-furo[3,4-c]pyrrole was used in place of 3-oxa-8-azabicyclo[3.2.1]octane hydrochloride to afford the target compound 1-ethyl-8-(hexahydro-1H-furo[3,4-c]pyrrole-5-carbonyl)-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one hydrochloride (compound 023).

¹H NMR (400 MHz, CD₃OD): δ 7.63 (d, J=8.0 Hz, 1H), 7.30-7.17 (m, 6H), 4.43-4.42 (m, 2H), 4.31-4.28 (m, 1H), 4.00-3.97 (m, 1H), 3.95-3.85 (m, 3H), 3.81-3.68 (m, 8H), 3.66-3.60 (m, 3H), 3.37-3.34 (m, 2H), 3.30-3.25 (m, 2H), 3.03-2.99 (m, 2H), 1.16 (t, J=7.0 Hz 3H).

LCMS: Rt: 1.316 min; MS m/z (ESI): 505.5 [M+H].

Example 25. Preparation of 1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-8-(2-hydroxy-7-azaspiro[3.5]nonane-7-carbonyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one (Compound 024)

[Image Omitted]

Reference can be made to the synthetic method in example 2 and 7-azaspiro[3.5]nonane-2-ol hydrochloride was used in place of 3-oxa-8-azabicyclo[3.2.1]octane hydrochloride to finally prepare a hydrochloride of the target compound 024. The resulting hydrochloride was dissolved with water and then adjusted with saturated sodium bicarbonate to the pH of 8. The aqueous phase was extracted with ethyl acetate 3 times, and the organic phase was washed with saturated sodium chloride solution 3 times and then dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated and then lyophilized following the addition of pure water to afford the target product 1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-8-(2-hydroxy-7-azaspiro[3.5]nonane-7-carbonyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one (compound 024).

¹H NMR (400 MHz, CD₃OD): δ 7.56 (d, J=7.7 Hz, 1H), 7.15-7.06 (m, 3H), 7.06-7.00 (m, 1H), 6.99-6.92 (m, 2H), 4.31-4.16 (m, 11H), 4.09-3.85 (m, 4H), 3.78 (dd, J=14.0, 6.4 Hz, 11H), 3.71-3.56 (m, 4H), 3.55-3.46 (m, 1H), 3.40-3.31 (m, 2H), 3.28-3.11 (m, 3H), 3.04-2.91 (m, 2H), 2.91-2.80 (m, 1H), 2.36-2.21 (m, 2H), 1.78-1.59 (m, 4H), 1.53 (s, 2H), 1.11 (t, J=7.1 Hz, 3H).

LCMS: Rt: 1.016 min; MS m/z (ESI): 533.2 [M+H].

Example 26. Preparation of 1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-8-(2-oxa-6-azaspiro[3.3]heptane-6-carbonyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one (Compound 025)

[Image Omitted]

Reference can be made to the synthetic method in example 2 and 2-oxa-6-azaspiro[3.3]heptane was used in place of 3-oxa-8-azabicyclo[3.2.1]octane hydrochloride, and the final compound obtained was purified by preparative chromatography (Waters sunfire C18 column, 19*250 mm 10 μm; using a mixture of water (containing 0.1% aqueous ammonia) and acetonitrile with a decreasing polarity as the eluent) to afford the target product 1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-8-(2-oxa-6-azaspiro[3.3]heptane-6-carbonyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one (compound 025).

¹H NMR (400 MHz, CD₃OD): δ 7.56 (d, J=7.8 Hz, 1H), 7.25-7.18 (m, 2H), 7.19-7.02 (m, 4H), 4.83-4.75 (m, 4H), 4.51 (s, 2H), 4.32 (s, 2H), 4.14-3.95 (m, 3H), 3.90 (dd, J=14.1, 3.8 Hz, 1H), 3.77 (dd, J=14.1, 6.5 Hz, 1H), 3.65-3.56 (m, 2H), 3.55-3.44 (m, 1H), 3.40-3.32 (m, 1H), 3.28-3.15 (m, 2H), 3.15-2.86 (m, 3H), 1.13 (t, J=7.0 Hz, 3H).

LCMS: Rt: 1.346 min; MS m/z (ESI): 491.4 [M+H].

Example 27. Preparation of 1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-8-(2-oxo-7-azaspiro[3.5]nonane-7-carbonyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one (Compound 026)

[Image Omitted]

Reference can be made to the synthetic method in example 2 and 7-azaspiro[3.5]nonane-2-one hydrochloride was used in place of 3-oxa-8-azabicyclo[3.2.1]octane hydrochloride. The final compound was prepared and purified through a chromatographic column (referring to example 1 for the eluents and gradient). The resulting eluate was adjusted with saturated sodium bicarbonate to the pH of 8-9 and extracted with ethyl acetate 3 times. The organic phase was washed with saturated sodium chloride solution 3 times and then dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated and then lyophilized following the addition of pure water to afford the target product 1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-8-(2-oxo-7-azaspiro[3.5]nonane-7-carbonyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one (compound 026).

¹H NMR (400 MHz, CD₃OD): δ 7.57 (d, J=7.6 Hz, 1H), 7.13-6.99 (m, 6H), 4.07-3.87 (m, 4H), 3.81-3.61 (m, 5H), 3.55-3.33 (m, 4H), 3.27-3.16 (m, 3H), 3.03-2.93 (m, 2H), 2.90-2.83 (m, 4H), 1.857 (s, 2H), 1.734 (s, 2H), 1.12 (t, J=7.1 Hz, 3H).

LCMS: Rt: 1.067 min; MS m/z (ESI): 531.2 [M+H].

Example 28. Preparation of 1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-8-(2-(methoxy-ds)-7-azaspiro[3.5]nonane-7-carbonyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one hydrochloride (Compound-02

[Image Omitted]

Preparation of Compound 027-2:

[Image Omitted]

At room temperature, tert-butyl 2-hydroxy-7-azaspiro[3.5]nonane-7-carboxylate (1.5 g) was dissolved in N,N-dimethylformamide (15 mL). At 0° C., sodium hydride (300 mg) was added. The mixture was reacted at 0° C. for 0.5 hours and then deuterated iodomethane (1 g) was added. The reaction solution was reacted at room temperature for 16 hours. The reaction was complete as detected by TLC and then quenched with aqueous ammonia (5 mL). The resulting mixture was diluted by adding water (15 mL), and the aqueous phase was extracted with ethyl acetate (20 mL) twice. The organic phase of the extraction solution was washed with saturated sodium chloride aqueous solution (20 mL) twice, then dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified using a normal phase silica gel chromatographic column (petroleum ether:ethyl acetate=10:1) to collect the product, which was concentrated to afford tert-butyl 2-(methoxy-d₃)-7-azaspiro[3.5]nonane-7-carboxylate (027-2) (1.5 g).

Preparation of Compound 027-3:

[Image Omitted]

At room temperature, tert-butyl 2-(methoxy-d₃)-7-azaspiro[3.5]nonane-7-carboxylate (027-2) (1.5 g) was dissolved in a hydrogen chloride-dioxane (15 mL, 4M) solution, and then the mixture was reacted and stirred at room temperature for 2 hours. The reaction was complete as detected by TLC. The reaction solution was directly concentrated under reduced pressure. The residue was slurried with ethyl acetate (5 mL) and filtered. The solid was dried to afford 2-(methoxy-d₃)-7-azaspiro[3.5]nonane hydrochloride (027-3) (1.0 g).

Reference can be made to the synthetic method of compound 002 in example 3 for the other steps and 2-(methoxy-d₃)-7-azaspiro[3.5]nonane hydrochloride (027-3) was used in place of 2-methoxy-7-azaspiro[3.5]nonane hydrochloride to afford the target product 1-ethyl-4-((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-8-(2-(methoxy-d₃)-7-azaspiro[3.5]nonane-7-carbonyl)-1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one hydrochloride (compound 027).

¹H NMR (400 MHz, CD₃OD): δ 7.67 (d, J=7.6 Hz, 1H), 7.29-7.23 (m, 4H), 7.21-714 (m, 2H), 4.48-4.32 (m, 3H), 4.00-3.91 (m, 2H), 3.73-3.61 (m, 7H), 3.53-3.42 (m, 1H), 3.34-3.29 (m, 4H), 3.27-3.21 (m, 2H), 2.26-2.24 (m, 2H), 1.68-1.55 (m, 6H), 1.19 (t, J=7.0 Hz, 3H).

LCMS: Rt: 1.469 min; MS m/z (ESI): 550.4 [M+H].

TEST EXAMPLES OF BIOLOGICAL ACTIVITY AND RELATED PROPERTIES
Test Example 1: PRMT5 Enzymatic Activity Inhibition Experiment

Materials: PRMT5/MEP50 proteins were purchased from BPS Bioscience Inc. (USA); Histone H4 Peptide substrate was purchased from Sangon Biotech (Shanghai) Co., Ltd.; Anti-Histone H4 (symmetric dimethyl R3) antibody—ChIP Grade was purchased from Abcam PLC. (USA); S-(5′-Adenosyl)-L-methionine chloride dihydrochloride was purchased from Sigma PLC. (USA); 384-well plate, AlphaScreen Streptavidin Donor beads, AlphaScreen Protein A Acceptor beads, and Envision 2104 multi-label Reader were purchased from Perkin-Elmer Instruments (USA) Corporation; and Echo 550 Liquid Handler was purchased from Labcyte Inc. (USA).

Enzymatic activity assay: The compounds were added into a 384-well plate by using the Echo 550 Liquid Handler with the final concentration of 0-1000 nM (initial concentration: 1000 nM, 3-fold dilution, for 10 points), and the DMSO content was 0.5%. 10 μL of a 2× PRMT5/MEP50 solution was added into each well and incubated at room temperature for 30 minutes. 10 μL of 2× PRMT5/MEP50 substrate solution was added to each well to start the reaction and incubated at room temperature for 60 minutes. A 6× detection reagent containing AlphaScreen Protein A Acceptor beads and Anti-Histone H4 (symmetric dimethyl R3) antibodies was prepared and 5 μL was added to each well. The plate was incubated at room temperature for 60 minutes. A 6× detection reagent containing AlphaScreen Streptavidin Donor beads was prepared and 5 μL was added to each well. The plate was incubated at room temperature for 60 minutes. The signal values were detected by using the Envision. The test results are shown in Table 2.

Test Example 2: Inhibitory Activity Experiment of the Compounds on Tumor Cell Proliferation

Cell culture: Z-138 cells were cultured in an IMDM culture solution containing 10% Horse serum+1% penicillin−streptomycin at 37° C., 5% CO₂. Cells in logarithmic growth phase were suitable for the experiment.

Detection of cell proliferation activity: The inhibitory activities of the compounds on the proliferation of Z-138 cells were detected by using the Cell-Titer Glo reagent. The concentrations of the cells were adjusted, and 180 μL was inoculated in each well of the 96-well plate (500/well) and equilibrated for 10-15 minutes at 37° C., 5% CO₂. 20 μL of a culture solution containing the compounds was added into each well with the final concentration reaching 0-300 nM (initial concentration: 300 nM, 3-fold dilution, for 10 points), and the DMSO content was 0.1%. The cell plate was placed at 37° C., 5% CO₂and incubated for 8 days. Culture solution was replaced at day 4: 100 μL of the supernatant was slowly aspirated and 100 μL of a fresh culture solution containing the compounds was provided as a supplement while the concentrations of the compounds were held constant. The cell viability was detected by using the Cell-Titer Glo reagent. The test results are shown in Table 2.

Test Example 3: Inhibitory Activity Experiment of Compounds on SDMA

Materials and cells: Z-138 cells were purchased from ATCC (USA); IMDM medium and penicillin-streptomycin were purchased from Sigma PLC. (USA); horse serum was purchased from Hyclone (USA); Hoechst antibodies were purchased from Invitrogen (USA); Alexa Fluor 488 goat anti-rabbit IgG antibodies were purchased from Invitrogen (USA); Anti-dimethyl-Arginine symmetric (SYM11) antibodies were purchased from Merck & Co., Inc. (USA); DPBS was purchased from Gibco (USA); Nonfat Dry milk was purchased from Cell Signaling Technology (USA); paraformaldehyde was purchased from Beijing Solarbio Science & Technology Co., Ltd.; 384-well plate and Echo 550 Liquid Handler were purchased from Labcyte Inc. (USA); and an ImageXpress Nano was purchased from Molecular Devices (USA).

Immunofluorescent assay: The effects of the compounds on SDMA in Z-138 cells were detected by using immunofluorescence. The cells were adjusted to the concentration of 1*10⁵/mL and 40 μL was inoculated in each well of the 384-well plate (4000/well) and equilibrated for 10-15 minutes at 37° C., 5% CO₂. The compound was added into a 384-well plate by using the Echo 550 Liquid Handler with the final concentration of 0-300 nM (initial concentration: 300 nM, 3-fold dilution, for 10 points), and the DMSO content was 0.1%. The cell plate was placed at 37° C., 5% CO₂and incubated for 2 days. 40 μL of 8% paraformaldehyde was added into each well and incubated at room temperature for 30 minutes. The supernatant was discarded, the plate was washed with DPBS, and 40 μL of 0.5% PBST (phosphate buffer with Tween) was added into each well and incubated at room temperature for 60 minutes. The supernatant was discarded, the plate was washed with 0.05% PBST, and 40 μL of a blocking solution (1% Nonfat milk in 0.05% PBST) was added into each well and incubated at room temperature for 60 minutes. The supernatant was discarded, and 20 μL of the primary antibody (SYM11, diluted with a blocking solution at 1:500) was added into each well and maintained at 4° C. overnight. The supernatant was discarded, the plate was washed with 0.05% PBST, 20 μL of the secondary antibody (Alexa Fluor 488 goat anti-rabbit and Hoechst, diluted with a blocking solution at 1:1000 and 1:5000, respectively) was added to each well and incubated at room temperature for 60 minutes. The supernatant was discarded, the plate was washed with 0.05% PBST, and the fluorescent intensity was detected with ImageXpress Nano. The test results are shown in the table below Table 2

TABLE 2

Inhibition of
Inhibition of cell
Inhibition of SDMA

enzymatic activity
proliferation
in cells

Compounds
IC₅₀(nM)
Z-138 IC₅₀(nM)
IC₅₀(nM)

001
4.56
3.47
3.80

002
4.05
2.19
1.40

003
2.02
1.21
0.80

004
4.53
1.87
1.40

005
6.73
4.27
1.50

006
3.62
4.80
1.80

007
7.66
7.55
NA

008
12.24
6.04
NA

009
4.96
2.97
6.10

010
NA
1.93
NA

011
NA
2.20
NA

012
NA
4.09
NA

013
NA
2.37
NA

014
NA
11.69
NA

015
NA
11.14
NA

016
NA
45.10
NA

017
NA
27.59
NA

018
NA
25.99
NA

019
NA
20.44
NA

020
NA
3.71
NA

021
NA
8.35
NA

022
NA
14.72
NA

023
NA
16.78
NA

024
NA
10.61
NA

025
NA
138.47
NA

026
NA
5.62
NA

027
NA
2.36
NA

Notes:

“NA” means that no test is performed.

Test Example 4: Pharmacokinetic Experiment in Mice

Experimental materials: GB17-SCID mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.; DMSO, HP-β-CD (Hydroxypropyl-β-cyclodextrin), MC (methyl cellulose) and acetonitrile were purchased from Merck & Co., Inc. (USA), and K₂EDTA anticoagulation tubes were purchased from Jiangsu Xinkang Medical Instrument Co., Ltd.

Experiment method: Six female CB17-SCID mice (20-30 g, 4-6 weeks) were randomized into 2 groups, 3 mice/group. Group 1 was given compound 002 via tail vein injection at a dose of 2 mg/kg, and the vehicle was 5% of DMSO+95% of 10% HP-β-CD aqueous solution; and group 2 was given compound 002 orally at a dose of 10 mg/kg, and the vehicle was 0.5% MC aqueous solution. Prior to the experiment, the animals were given food and water as usual. At pre-dose and 0.083 (only for the vein injection group), 0.25, 0.5, 1, 2, 4, 6, 8 and 24 h post-dose, blood was sampled from the mice in each group from the vein. The whole blood sample collected was placed in a K₂EDTA anticoagulation tube and centrifuged for 5 min (4000 rpm, 4° C.) and then the plasma was taken for detection.

10 μL of the mouse plasma sample was taken and 150 μL of acetonitrile solvent (wherein internal standard compound was contained) was added to precipitate proteins. After the mixture was vortexed for 0.5 min, centrifugation (4700 rpm, 4° C.) was performed for 15 min. The supernatant was diluted 2-fold with water containing 0.05% (v/v) formic acid and 3 μL was injected into the LC-MS/MS system (AB Sciex Triple Quad 6500+) for the quantitative detection. The concentrations of the samples were determined with the present of plasma standard curve (linear range: 0.5-1000 ng/mL) of CB17-SCID mice and quality control samples. For the preparation of 10-fold diluted samples, 2 μL of mouse plasma sample was taken and 18 μL of blank plasma was added. After the mixture was vortexed for 0.5 min, 300 μL of acetonitrile solvent (wherein internal standard compound was contained) was added to precipitate proteins. The other processing steps were identical to those for the samples not diluted.

Pharmacokinetic test results are as shown in Table 3.

TABLE 3

Results of pharmacokinetic tests in mice

PK parameters
Compound 002

Mice IV
Cl_obs (mL/min/kg)
61.1 ± 6.2

(2 mpk)
T_1/2(h)
3.56 ± 0.55

V_ss—obs (L/kg)
8.20 ± 0.60

Mice PO (10 mpk)
T_max(h)
0.33 ± 0.14

C_max(ng/mL)
827 ± 79

T_1/2(h)
2.68 ± 0.20

AUC_last(h*ng/mL)
2646 ± 563

F (%)
96.5 ± 20.5

Test Example 5: Hepatocellular Metabolic Stability Test

Experimental materials: Human hepatocytes were purchased from Biopredic; mouse hepatocytes were purchased from BioIVT; acetonitrile and methanol were purchased from Merck & Co., Inc.; AOPI dyes were purchased from Nexcelom; dexamethasone was purchased from NIFDC; DMSO was purchased from Beijing Solarbio Science & Technology Co., Ltd.; DPBS (10×), GlutaMAX™-1 (100×) and recombinant human insulin were purchased form Gibco by Life Technologies; fetal bovine serum was purchased from Corning; formic acid was purchased from DIKMAPURE; Isotonic Percoll was purchased from GE Healthcare; alprazolam was purchased from Supelco; caffeine was purchased from ChromaDex.inc; and HEPES, tolbutamide and Williams' Medium E were purchased from Sigma.

Experiment Preparation:

The powder of a test substance was formulated with DMSO into a stock solution with a high concentration, which was diluted to a working solution of 100 μM with acetonitrile prior to use. The final concentration of the test substance was 1 μM.

The specific preparation information of the hepatocyte thawing media is shown in Table 4 below. 49.5 mL of Williams'E Medium and 0.5 mL of GlutaMAX were mixed as an incubation media. Hepatocyte thawing media and incubation media were placed in a 37° C. water-bath and pre-heated for at least 15 minutes prior to use. A tube of hepatocytes stored at an ultra-low temperature was taken and the hepatocytes were ensured to be at a frozen state at a low temperature prior to thawing. The hepatocytes were rapidly placed in a 37° C. water-bath and gently shaken until all the ice crystals were dispersed, and then after 70% ethanol was sprayed, the hepatocytes were transferred to a biosafety cabinet. The content of the tube was poured into a centrifugal tube containing 50 mL of thawing media and centrifuged at 100 g for 10 minutes. After centrifugation, the thawing media was aspirated, and a sufficient amount of incubation media was added to obtain a cell suspension with a cell density of about 1.5×10⁶cells/mL. The hepatocytes were counted, and the viable cell density was determined using Cellometer Vision, and the survival rate of the hepatocytes must be higher than 75%. The hepatocyte suspension was diluted by using the incubation media to a viable cell density of 0.5×10⁶viable cells/mL.

TABLE 4

Preparation of hepatocyte thawing media

Initial
Final
Volume of

Reagents
concentration
concentration
reagent

Williams' Medium E
—
—
31.2
mL

Isotonic Percoll
—
30%
13.5
mL

DPBS (10x)
—
—
1.5
mL

GlutaMAX ™-1 (100x)
200 mM/100x
2
mM
500
μL

HEPES
1
M
15
mM
750
μL

FBS
—
5%
2.5
mL

Recombinant human insulin
4
mg/mL
4
μg/mL
50
μL

Dexamethasone
10
mM
1
μM
5
μL

Experiment Method:

247.5 μL of viable cell (human hepatocytes or mouse hepatocytes) suspension or media were transferred into a 96-well deep-well plate, and the deep-well plate was placed in a vortex incubator and pre-heated for 10 minutes. Incubation of all the samples was performed in parallel duplicate. Test substance (2.5 μL, 100 μM) was added into each well to start the reaction and the deep-well plate was placed in the vortex incubator again. The samples were incubated, and 25 μL of the suspension was taken at 0, 15, 30, 60, 90 and 120 minutes, respectively. 125 μL of acetonitrile containing internal standard (100 nM alprazolam, 200 nM caffeine, and 100 nM tolbutamide) was added to terminate the reaction. The mixture was vortexed for 10 minutes and centrifuged at 3220 g, 4° C. for 30 minutes. After centrifugation, 100 μL of the supernatant was transferred into the loading plate and 150 μL of pure water was added and mixed uniformly for LC-MS/MS analysis.

All the data calculation was performed by the Microsoft Excel software. The peak area was detected by extracting the ion chromatogram. The in vitro half-life (t_1/2) of the parent drug was detected by performing linear fitting of the natural logarithm of elimination percent of the parent drug and time.

The in vitro half-life (t_1/2) was calculated by means of the slope:

in vitro t_1/2=0.693/k.

The experimental results are as shown in Table 5.

TABLE 5

Results of hepatocellular metabolic stability test

Compounds
Human t_1/2(min)
Mouse t_1/2(min)

001
1471.30
122.34

002
252.37
94.62

003
7862.70
118.05

004
671.53
52.26

005
336.63
77.84

024
772.45
35.29

Test Example 6: In Vivo Drug Efficacy Experiment in Mice

Experimental materials: Z138 cells were purchased from ATCC; IMDM culture solution, penicillin-streptomycin and 0.25% trypsin-EDTA were purchased from Gibco; horse serum and PBS were purchased from Hyclone; and Matrigel was purchased from Corning.

Animal information: CB17-SCID mice (female, 6-7 weeks, body weight: about 14-20 g) were purchased from Shanghai Lingchang Biotechnology Co., Ltd. The mice were fed in SPF environment and each cage position was individually ventilated. All animals had free access to standard certified commercial laboratory food and water.

Experiment Method:

Cell culture: Human mantle cell lymphoma Z-138 cell lines were cultured in vitro and the culture conditions involve: IMDM (cell culture solution) supplemented with 10% horse serum and 1% penicillin-streptomycin solution, 37° C., 5% CO₂incubator. Conventional digestion treatment with 0.25% trypsin-EDTA digestion solution was carried out twice a week for passage. When the cell saturation was 85%-90%, and the number reached the requirement, the cells were collected and counted.

Cell inoculation: 0.1 ml/(containing 1×10⁷) Z-138 cell suspension (PBS Matrigel=1:1) was inoculated subcutaneously on the right back of each mouse. At day 18 post inoculation, when the average tumor volume measured has reached about 125 mm³, grouping and administration were performed by using the randomized stratification and grouping method based on the tumor volume and animal body weight. PBS was a phosphate buffer free of Ca and Mg ions, and Matrigel was a matrix gel.

Administration: Compound 002 was administered at the doses of 1.5 mg/kg, 5 mg/kg, or 15 mg/kg, PO, twice daily (BID)×3 weeks. 6 mice per group.

Tumor Measurement and Experimental Index:

The diameter of the tumor was measured with vernier calipers twice a week. The calculation formula of tumor volume was V=0.5a×b2, wherein a and b represented the long and short diameters of the tumor, respectively. The body weights of the mice were weighed twice a week.

The tumor-inhibiting efficacy of the compound was evaluated using tumor growth inhibition (TGI) (%).

TGI (%)=[(1−(average tumor volume at the end of administration in a certain treatment group−average tumor volume at the beginning of administration in the treatment group)/(average tumor volume at the end of administration in the solvent control group−average tumor volume at the beginning of administration in the solvent control group)]×100%.

Experimental Results:

See table 6, FIG. 2 and FIG. 3. No morbidity or death of the mice occurred during the experiment.

TABLE 6

Tumor volumes of Z-138 subcutaneous tumor models

Test

Frequency of
Average tumor volume (mm³)
TGI

Groups
compounds
Dose
administration
0 Day
4 Day
7 Day
11 Day
14 Day
18 Day
21 Day
(%)

Group
Solvent
/
BID
125
216
286
422
543
741
1007
/

1
control

Group
Compound
1.5
BID
125
151
203
374
436
567
689
36.0%

2
002
mg/kg

Group
Compound
5
BID
124
157
197
281
310
452
585
47.8%

3
002
mg/kg

Group
Compound
15
BID
126
140
113
59
34
18
16
112.5%

4
002
mg/kg

Experiment Conclusion:

In Z-138 models of mice with subcutaneous transplantation tumor, compound 002 of the present disclosure has significant inhibitory effects on tumor growth when it is administered twice daily at 1.5 mg/kg, 5 mg/kg and 15 mg/kg and shows a good dose-response relationship. When administered twice daily at 15 mg/kg, the compound shows an effect of shrinking tumor. In this efficacy experiment, the tested doses of compound 002 do not significantly affect the body weights of the mice, nor did they cause any death in mice, and the mice can tolerate the doses.

The embodiments of the present disclosure are illustrated as above. However, the present disclosure is not limited to the above-mentioned embodiments. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present disclosure shall be included within the scope of protection of the present disclosure.