Patent Description:
Disclosed herein are compounds for inhibiting Bcl-<NUM> and treating disease associated with undesirable bcl-<NUM> activity (Bcl-<NUM> related diseases), a method of using the compounds disclosed herein for treating dysregulated apoptotic diseases including neurodegenerative conditions, e.g., Alzheimer's disease; and proliferative diseases, e.g., cancers, autoimmune diseases and pro-thrombotic conditions, and a pharmaceutical composition comprising the same. The only Example forming part of the invention is Example F43 (see [<NUM>] on page <NUM>). All other Examples are reference examples only.

Programmed cell death or apoptosis occurs in multicellular organisms to dispose damaged or unwanted cells, which is critical for normal tissue homeostasis. However defective apoptotic processes have been implicated in a wide variety of diseases. Excessive apoptosis causes atrophy, whereas an insufficient amount results in uncontrolled cell proliferation, such as cancer (<NPL>). Resistance to apoptotic cell death is a hallmark of cancer and contributes to chemoresistance (Nat Med. <NUM>, <NUM>, <NUM>-<NUM>). Several key pathways controlling apoptosis are commonly altered in cancer. Some factors like Fas receptors and caspases promote apoptosis, while some members of the B-cell lymphoma <NUM> (Bcl-<NUM>) family of proteins inhibit apoptosis. Negative regulation of apoptosis inhibits cell death signaling pathways, helping tumors to evade cell death and developing drug resistance.

There are two distinct apoptosis pathways including the extrinsic pathway and the intrinsic pathway. The extrinsic pathway is activated in response to the binding of death-inducing ligands to cell-surface death receptors (<NPL>). The B cell lymphoma <NUM> (BCL-<NUM>) gene family, a group of proteins homologous to the Bcl-<NUM> protein, encodes more than <NUM> proteins that regulate the intrinsic apoptosis pathway. Bcl-<NUM> family proteins are characterized by containing at least one of four conserved Bcl-<NUM> homology (BH) domains (BH1, BH2, BH3 and BH4) (<NPL>; <NPL>; <NPL>). Bcl-<NUM> family proteins, consisting of pro-apoptotic and anti-apoptotic molecules, can be classified into the following three subfamilies according to sequence homology within four BH domains: (<NUM>) a subfamily shares sequence homology within all four BH domains, such as Bcl-<NUM>, Bcl-XL and Bcl-w which are anti-apoptotic; (<NUM>) a subfamily shares sequence homology within BH1, BH2 and BH4, such as Bax and Bak which are pro-apoptotic; (<NUM>) a subfamily shares sequence homology only within BH3, such as Bik, Bid and HRK which are pro-apoptotic. One of the unique features of Bcl-<NUM> family proteins is heterodimerization between anti-apoptotic and pro-apoptotic proteins, which is considered to inhibit the biological activity of their partners. This heterodimerization is mediated by the insertion of a BH3 region of a pro-apoptotic protein into a hydrophobic cleft composed of BH1, BH2 and BH3 from an anti-apoptotic protein. In addition to the BH1 and BH2, the BH4 domain is required for anti-apoptotic activity. In contrast, BH3 domain is essential and, itself, sufficient for pro-apoptotic activity.

Similar to oncogene addiction, in which tumor cells rely on a single dominant gene for survival, tumor cells may also become dependent on Bcl-<NUM> in order to survive. Bcl-<NUM> overexpress is found frequently in acute myeloid leukemia (AML), acute l7ymphocytic leukemia (ALL), relapsed/refractory chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), non-Hodgkin lymphoma (NHL) and solid tumors such as pancreatic, prostate, breast, and small cell and non-small cell lung cancers (<NPL>; <NPL>; <NPL>; <NPL>). Dysregulated apoptotic pathways have also been implicated in the pathology of other significant diseases such as neurodegenerative conditions (up-regulated apoptosis), e.g., Alzheimer's disease; and proliferative diseases (down-regulated apoptosis), e.g., cancers, autoimmune diseases and pro-thrombotic conditions. Target to either Bcl-<NUM> or Bcl-xL, a number of small-molecule BH3 mimetics have been reported in (Recent Patents on Anti<NPL>; <NPL>; <NPL>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>). Some of the Bcl-<NUM> small molecule inhibitors have been investigated at various stages of drug development: the Bcl-<NUM>/Bcl-xL inhibitor ABT-<NUM> (navitoclax, <CIT>) has shown promising clinical activity in lymphoid malignancies such as chronic lymphocytic leukemia. However, its efficacy in these settings is limited by platelet death and attendant thrombocytopenia caused by Bcl-xL inhibition (<NPL>; <NPL>; <NPL>). The new generation of the BCL-<NUM> selective inhibitor venetoclax (ABT-<NUM>/GDC-<NUM>) was proceeded, which demonstrated robust activity in these cancers but also spared platelets (<NPL>; <NPL>). S55746 (also known as BCL201), APG-<NUM>, APG-<NUM> are being studied at clinical trial stage. Currently, Venetoclax (formerly ABT-<NUM>) is the only Bcl-<NUM> selective inhibitor approved by FDA for the treatment of patients who have relapsed or refractory chronic lymphocytic leukemia (CLL) with the 17p deletion. Recently, however, a novel Gly101Val mutation in BCL2 was identified after the patients were treated with the Bcl-<NUM> inhibitor venetoclax (ABT-<NUM>) for <NUM> to <NUM> months (<NPL>). This mutation dramatically reduced the binding affinity of Bcl-<NUM> for Venetoclax (ABT-<NUM>) by about <NUM>-fold in cell based assay.

Therefore, there is a need of new small molecules that selectively inhibit Bcl-<NUM> proteins for the treatment of dysregulated apoptotic diseases such as cancers, autoimmune diseases and pro-thrombotic conditions. Unexpectedly, the inventors of the present application found some compounds disclosed herein show not only much higher potency and selectivity but also much lower CYP2C9 inhibition, indicating potential better efficacy and lower potential risk of drug-drug interaction (DDI). Also, the inventors of the present application found that the compounds disclosed herein exhibit inhibitory activity against both Bcl-<NUM> wild type and Bcl-<NUM> G101V mutation type, suggesting a type of new potential Bcl-<NUM> inhibitors without resistance concern.

In one aspect the invention provides a compound that is: <NUM>-((<NUM>-pyrrolo[<NUM>,<NUM>-b]pyridin-<NUM>-yl)oxy)-N-((<NUM>-((((1r,4r)-<NUM>-hydroxy-<NUM>-methylcyclohexyl)methyl)amino)-<NUM>-nitrophenyl)sulfonyl)-<NUM>-(<NUM>-((S)-<NUM>-(<NUM>-isopropylphenyl)pyrrolidin-<NUM>-yl)-<NUM>-azaspiro[<NUM>]nonan-<NUM>-yl)benzamide:
<CHM>
or a pharmaceutically acceptable salt thereof.

A compound defined by the claimed invention can be used in a method for treating dysregulated apoptotic diseases, comprising administering a subject in need thereof a therapeutically effective amount of the compound, or a pharmaceutically acceptable salt thereof. In one aspect, the invention provides a compound of the invention or a pharmaceutically acceptable salt thereof, for use in a method of treating cancer, such as, bladder cancer, brain cancer, breast cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, spleen cancer published in <CIT> and <CIT>.

In one embodiment, the dysregulated apoptotic disease is autoimmune disease, such as, Systemic Lupus Erythematosus (SLE).

In one aspect, the invention provides a pharmaceutical composition comprising a compound of the invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

References to methods of treatment by therapy or surgery or in vivo diagnosis methods in this description, are to be interpreted as references to compounds of the present invention for use in those methods.

The following terms have the indicated meanings throughout the specification:.

As used herein, including the appended claims, the singular forms of words such as "a", "an", and "the", include their corresponding plural references unless the context clearly dictates otherwise.

The term "or" is used to mean, and is used interchangeably with, the term "and/or" unless the context clearly dictates otherwise.

The term "alkyl" refers to a hydrocarbon group selected from linear and branched saturated hydrocarbon groups comprising from <NUM> to <NUM>, such as from <NUM> to <NUM>, further such as from <NUM> to <NUM>, more further such as from <NUM> to <NUM>, or from <NUM> to <NUM>, or from <NUM> to <NUM>, carbon atoms. Examples of alkyl groups comprising from <NUM> to <NUM> carbon atoms (i.e., C<NUM>-<NUM> alkyl) include, but not limited to, methyl, ethyl, <NUM>-propyl or n-propyl ("n-Pr"), <NUM>-propyl or isopropyl ("i-Pr"), <NUM>-butyl or n-butyl ("n-Bu"), <NUM>-methyl-<NUM>-propyl or isobutyl ("i-Bu"), <NUM>-methylpropyl or s-butyl ("s-Bu"), <NUM>,<NUM>-dimethylethyl or t-butyl ("t-Bu"), <NUM>-pentyl, <NUM>-pentyl, <NUM>-pentyl, <NUM>-methyl-<NUM>-butyl, <NUM>-methyl-<NUM>-butyl, <NUM>-methyl-<NUM>-butyl, <NUM>-methyl-<NUM>-butyl, <NUM>-hexyl, <NUM>-hexyl, <NUM>-hexyl, <NUM>-methyl-<NUM>-pentyl, <NUM>-methyl-<NUM>-pentyl, <NUM>-methyl-<NUM>-pentyl, <NUM>-methyl-<NUM>-pentyl, <NUM>-methyl-<NUM>-pentyl, <NUM>,<NUM>-dimethyl-<NUM>-butyl and <NUM>,<NUM>-dimethyl-<NUM>-butyl groups. The alkyl group can be optionally enriched in deuterium, e.g., -CD<NUM>, -CD<NUM>CD<NUM> and the like.

The term "halogen" refers to fluoro (F), chloro (Cl), bromo (Br) and iodo (I).

The term "haloalkyl" refers to an alkyl group in which one or more hydrogen is/are replaced by one or more halogen atoms such as fluoro, chloro, bromo, and iodo. Examples of the haloalkyl include haloC<NUM>-<NUM>alkyl, haloC<NUM>-<NUM>alkyl or halo C<NUM>-<NUM>alkyl, but not limited to -CF<NUM>, - CH<NUM>Cl, -CH<NUM>CF<NUM>, -CCl<NUM>, CF<NUM>, and the like.

The term "alkenyl" refers to a hydrocarbon group selected from linear and branched hydrocarbon groups comprising at least one C=C double bond and from <NUM> to <NUM>, such as from <NUM> to <NUM>, further such as from <NUM> to <NUM>, carbon atoms. Examples of the alkenyl group, e.g., C<NUM>-<NUM> alkenyl, include, but not limited to ethenyl or vinyl, prop-<NUM>-enyl, prop-<NUM>-enyl, <NUM>-methylprop-<NUM>-enyl, but-<NUM>-enyl, but-<NUM>-enyl, but-<NUM>-enyl, buta-<NUM>,<NUM>-dienyl, <NUM>-methylbuta-<NUM>,<NUM>-dienyl, hex-<NUM>-enyl, hex-<NUM>-enyl, hex-<NUM>-enyl, hex-<NUM>-enyl, and hexa-<NUM>,<NUM>-dienyl groups.

The term "alkynyl" refers to a hydrocarbon group selected from linear and branched hydrocarbon group, comprising at least one C=C triple bond and from <NUM> to <NUM>, such as <NUM> to <NUM>, further such as from <NUM> to <NUM>, carbon atoms. Examples of the alkynyl group, e.g., C<NUM>-<NUM> alkynyl, include, but not limited to ethynyl, <NUM>-propynyl, <NUM>-propynyl (propargyl), <NUM>-butynyl, <NUM>-butynyl, and <NUM>-butynyl groups.

The term "alkyloxy" or "alkoxy" refers to an alkyl group as defined above attached to the parent molecular moiety through an oxygen atom. Examples of an alkyloxy, e.g., C<NUM>-<NUM>alkyloxy or C<NUM>-<NUM> alkyloxy includes, but not limited to, methoxy, ethoxy, isopropoxy, propoxy, n-butoxy, tert-butoxy, pentoxy and hexoxy and the like.

The term "cycloalkyl" refers to a hydrocarbon group selected from saturated cyclic hydrocarbon groups, comprising monocyclic and polycyclic (e.g., bicyclic and tricyclic) groups including fused, bridged or spiro cycloalkyl.

For example, the cycloalkyl group may comprise from <NUM> to <NUM>, such as from <NUM> to <NUM>, further such as <NUM> to <NUM>, further such as <NUM> to <NUM>, <NUM> to <NUM>, or <NUM> to <NUM> carbon atoms. Even further for example, the cycloalkyl group may be selected from monocyclic group comprising from <NUM> to <NUM>, such as from <NUM> to <NUM>, further such as <NUM> to <NUM>, <NUM> to <NUM> carbon atoms. Examples of the monocyclic cycloalkyl group include cyclopropyl, cyclobutyl, cyclopentyl, <NUM>-cyclopent-<NUM>-enyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, and cyclododecyl groups. In particular, Examples of the saturated monocyclic cycloalkyl group, e.g., C<NUM>-<NUM> cycloalkyl, include, but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In a preferred embedment, the cycloalkyl is a monocyclic ring comprising <NUM> to <NUM> carbon atoms (abbreviated as C<NUM>-<NUM> cycloalkyl), including but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Examples of the bicyclic cycloalkyl groups include those having from <NUM> to <NUM> ring atoms arranged as a fused bicyclic ring selected from [<NUM>,<NUM>], [<NUM>,<NUM>], [<NUM>,<NUM>], [<NUM>,<NUM>] and [<NUM>,<NUM>] ring systems, or as a bridged bicyclic ring selected from bicyclo[<NUM>. <NUM>]heptane, bicyclo[<NUM>. <NUM>]octane, and bicyclo[<NUM>. <NUM>]nonane. Further Examples of the bicyclic cycloalkyl groups include those arranged as a bicyclic ring selected from [<NUM>,<NUM>] and [<NUM>,<NUM>] ring systems, such as
<CHM>
wherein the wavy lines indicate the points of attachment. The ring may be saturated or have at least one double bond (i.e. partially unsaturated), but is not fully conjugated, and is not aromatic, as aromatic is defined herein.

The term "spiro cycloalkyl" refers to a cyclic structure which contains carbon atoms and is formed by at least two rings sharing one atom. The term "<NUM> to <NUM> membered spiro cycloalkyl" refers to a cyclic structure which contains <NUM> to <NUM> carbon atoms and is formed by at least two rings sharing one atom.

The term "fused cycloalkyl" refers to a fused ring which contains carbon atoms and is formed by two or more rings sharing two adjacent atoms. The term "<NUM> to <NUM> membered fused cycloalkyl" refers to a fused ring which contains <NUM> to <NUM> ring carbon atoms and is formed by two or more rings sharing two adjacent atoms.

Examples include but are not limited to bicyclo[<NUM>. <NUM>]butyl, bicyclo[<NUM>. <NUM>]pentyl, bicyclo[<NUM>. <NUM>]hexyl, bicyclo[<NUM>. <NUM>]heptyl, bicyclo[<NUM>. <NUM>]octyl, bicyclo[<NUM>. <NUM>]octyl, decalin, as well as benzo <NUM> to <NUM> membered cycloalkyl, benzo C<NUM>-<NUM> cycloalkenyl, <NUM>,<NUM>-dihydro-<NUM>-indenyl, <NUM>-indenyl, <NUM>,<NUM>,<NUM>,<NUM>-tetralyl, <NUM>,<NUM>-dihydronaphthyl, etc. Preferred embodiments are <NUM> to <NUM> membered fused cyclyl, which refer to cyclic structures containing <NUM> to <NUM> ring atoms within the above examples.

The term "bridged cycloalkyl" refers to a cyclic structure which contains carbon atoms and is formed by two rings sharing two atoms which are not adjacent to each other. The term "<NUM> to <NUM> membered bridged cycloalkyl" refers to a cyclic structure which contains <NUM> to <NUM> carbon atoms and is formed by two rings sharing two atoms which are not adjacent to each other.

The term "cycloalkenyl" refers to non-aromatic cyclic alkyl groups of from <NUM> to <NUM> carbon atoms having single or multiple rings and having at least one double bond and preferably from <NUM> to <NUM> double bonds. In one embodiment, the cycloalkenyl is cyclopentenyl or cyclohexenyl, preferably cyclohexenyl.

The term "cycloalkynyl" refers to non-aromatic cycloalkyl groups of from <NUM> to <NUM> carbon atoms having single or multiple rings and having at least one triple bond.

The term "aryl" used alone or in combination with other terms refers to a group selected from:.

The terms "aromatic hydrocarbon ring" and "aryl" are used interchangeable throughout the disclosure herein. In some embodiments, a monocyclic or bicyclic aromatic hydrocarbon ring has <NUM> to <NUM> ring-forming carbon atoms (i.e., C<NUM>-<NUM> aryl). Examples of a monocyclic or bicyclic aromatic hydrocarbon ring includes, but not limited to, phenyl, naphth-<NUM>-yl, naphth-<NUM>-yl, anthracenyl, phenanthrenyl, and the like. In some embodiments, the aromatic hydrocarbon ring is a naphthalene ring (naphth-<NUM>-yl or naphth-<NUM>-yl) or phenyl ring. In some embodiments, the aromatic hydrocarbon ring is a phenyl ring.

The term "heteroaryl" refers to a group selected from:.

When the total number of S and O atoms in the heteroaryl group exceeds <NUM>, those heteroatoms are not adjacent to one another. In some embodiments, the total number of S and O atoms in the heteroaryl group is not more than <NUM>. In some embodiments, the total number of S and O atoms in the aromatic heterocycle is not more than <NUM>. When the heteroaryl group contains more than one heteroatom ring member, the heteroatoms may be the same or different. The nitrogen atoms in the ring(s) of the heteroaryl group can be oxidized to form N-oxides. The term "C-linked heteroaryl" as used herein means that the heteroaryl group is connected to the core molecule by a bond from a C-atom of the heteroaryl ring.

The terms "aromatic heterocyclic ring" and "heteroaryl" are used interchangeable throughout the disclosure herein. In some embodiments, a monocyclic or bicyclic aromatic heterocyclic ring has <NUM>-, <NUM>-, <NUM>-, <NUM>-, <NUM>- or <NUM>-ring forming members with <NUM>, <NUM>, <NUM>, or <NUM> heteroatom ring members independently selected from nitrogen (N), sulfur (S) and oxygen (O) and the remaining ring members being carbon. In some embodiments, the monocyclic or bicyclic aromatic heterocyclic ring is a monocyclic or bicyclic ring comprising <NUM> or <NUM> heteroatom ring members independently selected from nitrogen (N), sulfur (S) and oxygen (O). In some embodiments, the monocyclic or bicyclic aromatic heterocyclic ring is a <NUM>- to <NUM>-membered heteroaryl ring, which is monocyclic and which has <NUM> or <NUM> heteroatom ring members independently selected from nitrogen (N), sulfur (S) and oxygen (O). In some embodiments, the monocyclic or bicyclic aromatic heterocyclic ring is a <NUM>- to <NUM>-membered heteroaryl ring, which is bicyclic and which has <NUM> or <NUM> heteroatom ring members independently selected from nitrogen, sulfur and oxygen.

Examples of the heteroaryl group or the monocyclic or bicyclic aromatic heterocyclic ring include, but are not limited to, (as numbered from the linkage position assigned priority <NUM>) pyridyl (such as <NUM>-pyridyl, <NUM>-pyridyl, or <NUM>-pyridyl), cinnolinyl, pyrazinyl, <NUM>,<NUM>-pyrimidinyl, <NUM>,<NUM>-pyrimidinyl, <NUM>,<NUM>-imidazolyl, imidazopyridinyl, isoxazolyl, oxazolyl, thiazolyl, isothiazolyl, thiadiazolyl (such as <NUM>,<NUM>,<NUM>-thiadiazolyl, <NUM>,<NUM>,<NUM>-thiadiazolyl, or <NUM>,<NUM>,<NUM>-thiadiazolyl), tetrazolyl, thienyl (such as thien-<NUM>-yl, thien-<NUM>-yl), triazinyl, benzothienyl, furyl or furanyl, benzofuryl, benzoimidazolyl, indolyl, isoindolyl, indolinyl, oxadiazolyl (such as <NUM>,<NUM>,<NUM>-oxadiazolyl, <NUM>,<NUM>,<NUM>-oxadiazolyl, or <NUM>,<NUM>,<NUM>-oxadiazolyl), phthalazinyl, pyrazinyl, pyridazinyl, pyrrolyl, triazolyl (such as <NUM>,<NUM>,<NUM>-triazolyl, <NUM>,<NUM>,<NUM>-triazolyl, or <NUM>,<NUM>,<NUM>-triazolyl), quinolinyl, isoquinolinyl, pyrazolyl, pyrrolopyridinyl (such as <NUM>-pyrrolo[<NUM>,<NUM>-b]pyridin-<NUM>-yl), pyrazolopyridinyl (such as <NUM>-pyrazolo[<NUM>,<NUM>-b]pyridin-<NUM>-yl), benzofuranyl, benzoxazolyl (such as benzo[d]oxazol-<NUM>-yl), pteridinyl, purinyl, <NUM>-oxa-<NUM>,<NUM>-diazolyl, <NUM>-oxa-<NUM>,<NUM>-diazolyl, <NUM>-oxa-<NUM>,<NUM>-diazolyl, <NUM>-oxa-<NUM>,<NUM>-diazolyl, <NUM>-thia-<NUM>,<NUM>-diazolyl, <NUM>-thia-<NUM>,<NUM>-diazolyl, <NUM>-thia-<NUM>,<NUM>-diazolyl, <NUM>-thia-<NUM>,<NUM>-diazolyl, furazanyl (such as furazan-<NUM>-yl, furazan-<NUM>-yl), benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, furopyridinyl, benzothiazolyl (such as benzo[d]thiazol-<NUM>-yl), indazolyl (such as <NUM>-indazol-<NUM>-yl) and <NUM>,<NUM>,<NUM>,<NUM>-tetrahydroisoquinoline.

"Heterocyclyl", "heterocycle" or "heterocyclic" are interchangeable and refer to a non-aromatic heterocyclyl group comprising one or more heteroatoms selected from the group consisting of NH, O, S, SO or SO<NUM> heteroatoms as ring members, with the remaining ring members being carbon, including monocyclic, fused, bridged, and spiro ring, i.e., containing monocyclic heterocyclyl, bridged heterocyclyl, spiro heterocyclyl, and fused heterocyclic groups.

The term "monocyclic heterocyclyl" refers to monocyclic groups in which at least one ring member is a heteroatom selected from the group consisting of NH, O, S, SO or SO<NUM>. A heterocycle may be saturated or partially saturated.

Exemplary monocyclic <NUM> to <NUM>-membered heterocyclyl groups include, but not limited to, (as numbered from the linkage position assigned priority <NUM>) pyrrolidin-<NUM>-yl, pyrrolidin-<NUM>-yl, pyrrolidin-<NUM>-yl, imidazolidin-<NUM>-yl, imidazolidin-<NUM>-yl , pyrazolidin-<NUM>-yl, pyrazolidin-<NUM>-yl, piperidin-<NUM>-yl, piperidin-<NUM>-yl, piperidin-<NUM>-yl, piperidin-<NUM>-yl, <NUM>,<NUM>-piperazinyl, pyranyl, morpholinyl, morpholino, morpholin-<NUM>-yl, morpholin-<NUM>-yl, oxiranyl, aziridin-<NUM>-yl, aziridin-<NUM>-yl, azocan-<NUM>-yl, azocan-<NUM>-yl, azocan-<NUM>-yl, azocan-<NUM>-yl, azocan-<NUM>-yl, thiiranyl, azetidin-<NUM>-yl, azetidin-<NUM>-yl, azetidin-<NUM>-yl, oxetanyl, thietanyl, <NUM>,<NUM>-dithietanyl, <NUM>,<NUM>-dithietanyl, dihydropyridinyl, tetrahydropyridinyl, thiomorpholinyl, thioxanyl, piperazinyl, homopiperazinyl, homopiperidinyl, azepan-<NUM>-yl, azepan-<NUM>-yl, azepan-<NUM>-yl, azepan-<NUM>-yl, oxepanyl, thiepanyl, <NUM>,<NUM>-oxathianyl, <NUM>,<NUM>-dioxepanyl, <NUM>,<NUM>-oxathiepanyl, <NUM>,<NUM>-oxaazepanyl, <NUM>,<NUM>-dithiepanyl, <NUM>,<NUM>-thiazepanyl and <NUM>,<NUM>-diazepanyl, <NUM>,<NUM>-dithianyl, <NUM>,<NUM>-azathianyl, oxazepinyl, diazepinyl, thiazepinyl, dihydrothienyl, dihydropyranyl, dihydrofuranyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, tetrahydrothiopyranyl, <NUM>-pyrrolinyl, <NUM>-pyrrolinyl, <NUM>-pyrrolinyl, indolinyl, <NUM>-pyranyl, <NUM>-pyranyl, <NUM>,<NUM>-dioxanyl, <NUM>,<NUM>-dioxolanyl, pyrazolinyl, pyrazolidinyl, dithianyl, dithiolanyl, pyrazolidinyl, imidazolinyl, pyrimidinonyl, or <NUM>,<NUM>-dioxo-thiomorpholinyl.

The term "spiro heterocyclyl" or "heterospirocyclyl" refers to a <NUM> to <NUM>-membered polycyclic heterocyclyl with rings connected through one common carbon atom (called a spiro atom), comprising one or more heteroatoms selected from the group consisting of NH, O, S, SO or SO<NUM> heteroatoms as ring members, with the remaining ring members being carbon. One or more rings of a spiro heterocyclyl group may contain one or more double bonds, but none of the rings has a completely conjugated pi-electron system. Preferably a spiro heterocyclyl is <NUM> to <NUM>-membered, and more preferably <NUM> to <NUM>-membered. According to the number of common spiro atoms, a spiro heterocyclyl is divided into mono-spiro heterocyclyl, di-spiro heterocyclyl, or poly-spiro heterocyclyl, and preferably refers to mono-spiro heterocyclyl or di-spiro heterocyclyl, and more preferably <NUM>-membered/<NUM>-membered, <NUM>-membered/<NUM>-membered, <NUM>-membered/<NUM>-membered, <NUM>-membered/<NUM>-membered, <NUM>-membered/<NUM>-membered, or <NUM>-membered/<NUM>-membered mono-spiro heterocyclyl. Representative examples of spiro heterocyclyls include, but not limited to the following groups: <NUM>,<NUM>-dihydrospiro[indene-<NUM>,<NUM>'-pyrrolidine] (e.g., <NUM>,<NUM>-dihydrospiro[indene-<NUM>,<NUM>'-pyrrolidine]-<NUM>'-yl), <NUM>,<NUM>-dihydrospiro[indene-<NUM>,<NUM>'-pyrrolidine] (e.g., <NUM>,<NUM>-dihydrospiro[indene-<NUM>,<NUM>'-pyrrolidine]-<NUM>'-yl), azaspiro[<NUM>]heptane (e.g., <NUM>-azaspiro[<NUM>]heptane-<NUM>-yl), azaspiro[<NUM>]octane (e.g., <NUM>-azaspiro[<NUM>]octane-<NUM>- yl), <NUM>-oxa-<NUM>-azaspiro[<NUM>]octane (e.g., <NUM>-oxa-<NUM>-azaspiro[<NUM>]octane-<NUM>-yl), azaspiro[<NUM>]octane (e.g., <NUM>-azaspiro[<NUM>]octan-<NUM>-yl), azaspiro[<NUM>]octane (e.g., <NUM>-azaspiro[<NUM>]octan-<NUM>-yl), <NUM>-azaspiro[<NUM>]nonane (e.g., <NUM>-azaspiro[<NUM>]nonan-<NUM>-yl), <NUM>-azaspiro[<NUM>]nonane (e.g., <NUM>-azaspiro[<NUM>]nonan-<NUM>-yl),<NUM>,<NUM>-dioxaspiro[<NUM>]decane, <NUM>-oxa-<NUM>-aza-spiro[<NUM>]nonane (e.g., <NUM>-oxa-<NUM>-aza-spiro[<NUM>]non-<NUM>-yl), <NUM>-oxa-spiro[<NUM>]nonyl and <NUM>-oxa-spiro[<NUM>]heptyl.

The term "fused heterocyclic group" refers to a <NUM> to <NUM>-membered polycyclic heterocyclyl group, wherein each ring in the system shares an adjacent pair of atoms (carbon and carbon atoms or carbon and nitrogen atoms) with another ring, comprising one or more heteroatoms selected from the group consisting of NH, O, S, SO or SO<NUM> heteroatoms as ring members, with the remaining ring members being carbon. One or more rings of a fused heterocyclic group may contain one or more double bonds, but none of the rings has a completely conjugated pi-electron system. Preferably, a fused heterocyclyl is <NUM> to <NUM>-membered, and more preferably <NUM> to <NUM>-membered. According to the number of membered rings, a fused heterocyclyl is divided into bicyclic, tricyclic, tetracyclic, or polycyclic fused heterocyclyl, preferably refers to bicyclic or tricyclic fused heterocyclyl, and more preferably <NUM>-membered/<NUM>-membered, or <NUM>-membered/<NUM>-membered bicyclic fused heterocyclyl. Representative examples of fused heterocycles include, but not limited to, the following groups octahydrocyclopenta[c]pyrrole (e.g., octahydrocyclopenta[c]pyrrol-<NUM>-yl), octahydropyrrolo[<NUM>,<NUM>-c]pyrrolyl, octahydroisoindolyl, isoindolinyl (e.g., isoindoline-<NUM>-yl), octahydro-benzo[b] [<NUM>,<NUM>] dioxin, dihydrobenzofuranyl, benzo[d][<NUM>,<NUM>]dioxolyl.

The term "bridged heterocyclyl" refers to a <NUM> to <NUM>-membered polycyclic heterocyclic alkyl group, wherein every two rings in the system share two disconnected atoms, comprising one or more heteroatoms selected from the group consisting of NH, O, S, SO or SO<NUM> heteroatoms as ring members, with the remaining ring members being carbon. One or more rings of a bridged heterocyclyl group may contain one or more double bonds, but none of the rings has a completely conjugated pi-electron system. Preferably, a bridged heterocyclyl is <NUM> to <NUM>-membered, and more preferably <NUM> to <NUM>-membered. According to the number of membered rings, a bridged heterocyclyl is divided into bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocyclyl, and preferably refers to bicyclic, tricyclic or tetracyclic bridged heterocyclyl, and more preferably bicyclic or tricyclic bridged heterocyclyl. Representative examples of bridged heterocyclyls include, but not limited to, the following groups: <NUM>-azabicyclo[<NUM>. <NUM>]heptyl, azabicyclo[<NUM>. <NUM>]hexyl, <NUM>-azabicyclo[<NUM>. <NUM>]octyl and <NUM>-azabicyclo[<NUM>. <NUM>]decyl.

The heterocyclyl ring may be fused to aryl, heteroaryl or cycloalkyl ring, wherein the ring structure is connected to the parent heterocyclic group together.

"C-linked heterocyclyl" as used refers to a heterocyclyl group which is connected to the other part of the molecule by a direct bond from a carbon atom of the heterocyclyl ring.

"N-linked heterocyclyl" as used refers to a heterocyclyl group which is connected to the other part of the molecule by a direct bond from a nitrogen atom of the heterocyclyl ring.

Compounds disclosed herein may contain an asymmetric center and may thus exist as enantiomers. "Enantiomers" refer to two stereoisomers of a compound which are non-superimposable mirror images of one another. Where the compounds disclosed herein possess two or more asymmetric centers, they may additionally exist as diastereomers. Enantiomers and diastereomers fall within the broader class of stereoisomers. All such possible stereoisomers as substantially pure resolved enantiomers, racemic mixtures thereof, as well as mixtures of diastereomers are intended to be included. All stereoisomers of the compounds disclosed herein and /or pharmaceutically acceptable salts thereof are intended to be included. Unless specifically mentioned otherwise, reference to one isomer applies to any of the possible isomers. Whenever the isomeric composition is unspecified, all possible isomers are included.

The term "substantially pure" as used herein means that the target stereoisomer contains no more than <NUM>%, such as no more than <NUM>%, further such as no more than <NUM>%, even further such as no more than <NUM>%, by weight of any other stereoisomer(s). In some embodiments, the term "substantially pure" means that the target stereoisomer contains no more than <NUM>%, for example, no more than <NUM>%, such as no more than <NUM>%, by weight of any other stereoisomer(s).

When compounds disclosed herein contain olefinic double bonds, unless specified otherwise, such double bonds are meant to include both E and Z geometric isomers.

When compounds disclosed herein contain a di-substituted cyclohexyl or cyclobutyl group, substituents found on cyclohexyl or cyclobutyl ring may adopt cis and trans formations. Cis formation means that both substituents are found on the upper side of the <NUM> substituent placements on the carbon, while trans would mean that they were on opposing sides.

It may be advantageous to separate reaction products from one another and /or from starting materials. The desired products of each step or series of steps is separated and /or purified (hereinafter separated) to the desired degree of homogeneity by the techniques common in the art. Typically such separations involve multiphase extraction, crystallization from a solvent or solvent mixture, distillation, sublimation, or chromatography. Chromatography can involve any number of methods including, for example: reverse-phase and normal phase; size exclusion; ion exchange; high, medium and low pressure liquid chromatography methods and apparatus; small scale analytical; simulated moving bed ("SMB") and preparative thin or thick layer chromatography, as well as techniques of small scale thin layer and flash chromatography. One skilled in the art will apply techniques most likely to achieve the desired separation.

"Diastereomers" refers to stereoisomers of a compound with two or more chiral centers but which are not mirror images of one another. Diastereomeric mixtures can be separated into their individual diastereomers on the basis of their physical chemical differences by methods well known to those skilled in the art, such as by chromatography and /or fractional crystallization. Enantiomers can be separated by converting the enantiomeric mixture into a diastereomeric mixture by reaction with an appropriate optically active compound (e.g., chiral auxiliary such as a chiral alcohol or Mosher's acid chloride), separating the diastereomers and converting (e.g., hydrolyzing) the individual diastereoisomers to the corresponding pure enantiomers. Enantiomers can also be separated by use of a chiral HPLC column.

A single stereoisomer, e.g., a substantially pure enantiomer, may be obtained by resolution of the racemic mixture using a method such as formation of diastereomers using optically active resolving agents (<NPL>; <NPL>). Racemic mixtures of chiral compounds of the invention can be separated and isolated by any suitable method, including: (<NUM>) formation of ionic, diastereomeric salts with chiral compounds and separation by fractional crystallization or other methods, (<NUM>) formation of diastereomeric compounds with chiral derivatizing reagents, separation of the diastereomers, and conversion to the pure stereoisomers, and (<NUM>) separation of the substantially pure or enriched stereoisomers directly under chiral conditions. See: <NPL>.

"Pharmaceutically acceptable salts" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. A pharmaceutically acceptable salt may be prepared in situ during the final isolation and purification of the compounds disclosed herein, or separately by reacting the free base function with a suitable organic acid or by reacting the acidic group with a suitable base.

In addition, if a compound disclosed herein is obtained as an acid addition salt, the free base can be obtained by basifying a solution of the acid salt. Conversely, if the product is a free base, an addition salt, such as a pharmaceutically acceptable addition salt, may be produced by dissolving the free base in a suitable organic solvent and treating the solution with an acid, in accordance with conventional procedures for preparing acid addition salts from base compounds. Those skilled in the art will recognize various synthetic methodologies that may be used without undue experimentation to prepare non-toxic pharmaceutically acceptable addition salts.

As defined herein, "a pharmaceutically acceptable salt thereof' include salts of at least one compound of Formula (I), and salts of the stereoisomers of the compound of Formula (I), such as salts of enantiomers, and /or salts of diastereomers.

The terms "administration", "administering", "treating" and "treatment" herein, when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, mean contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition to the animal, human, subject, cell, tissue, organ, or biological fluid. Treatment of a cell encompasses contact of a reagent to the cell, as well as contact of a reagent to a fluid, where the fluid is in contact with the cell. The term "administration" and "treatment" also means in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding compound, or by another cell. The term "subject" herein includes any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit) and most preferably a human.

The term "effective amount" or "therapeutically effective amount" refers to an amount of the active ingredient, such as compound that, when administered to a subject for treating a disease, or at least one of the clinical symptoms of a disease or disorder, is sufficient to affect such treatment for the disease, disorder, or symptom. The "therapeutically effective amount" can vary with the compound, the disease, disorder, and/or symptoms of the disease or disorder, severity of the disease, disorder, and/or symptoms of the disease or disorder, the age of the subject to be treated, and/or the weight of the subject to be treated. An appropriate amount in any given instance can be apparent to those skilled in the art or can be determined by routine experiments. In some embodiments, "therapeutically effective amount" is an amount of at least one compound and /or at least one stereoisomer thereof, and /or at least one pharmaceutically acceptable salt thereof disclosed herein effective to "treat" as defined above, a disease or disorder in a subject. In the case of combination therapy, the "therapeutically effective amount" refers to the total amount of the combination objects for the effective treatment of a disease, a disorder or a condition.

The pharmaceutical composition comprising the compound disclosed herein can be administrated via oral, inhalation, rectal, parenteral or topical administration to a subject in need thereof. For oral administration, the pharmaceutical composition may be a regular solid formulation such as tablets, powder, granule, capsules and the like, a liquid formulation such as water or oil suspension or other liquid formulation such as syrup, solution, suspension or the like; for parenteral administration, the pharmaceutical composition may be solution, water solution, oil suspension concentrate, lyophilized powder or the like. Preferably, the formulation of the pharmaceutical composition is selected from tablet, coated tablet, capsule, suppository, nasal spray or injection, more preferably tablet or capsule. The pharmaceutical composition can be a single unit administration with an accurate dosage. In addition, the pharmaceutical composition may further comprise additional active ingredients.

All formulations of the pharmaceutical composition disclosed herein can be produced by the conventional methods in the pharmaceutical field. For example, the active ingredient can be mixed with one or more excipients, then to make the desired formulation. The "pharmaceutically acceptable excipient" refers to conventional pharmaceutical carriers suitable for the desired pharmaceutical formulation, for example: a diluent, a vehicle such as water, various organic solvents, etc, a filler such as starch, sucrose, etc a binder such as cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone (PVP); a wetting agent such as glycerol; a disintegrating agent such as agar, calcium carbonate and sodium bicarbonate; an absorption enhancer such as quaternary ammonium compound; a surfactant such as hexadecanol; an absorption carrier such as Kaolin and soap clay; a lubricant such as talc, calcium stearate, magnesium stearate, polyethylene glycol, etc. In addition, the pharmaceutical composition further comprises other pharmaceutically acceptable excipients such as a decentralized agent, a stabilizer, a thickener, a complexing agent, a buffering agent, a permeation enhancer, a polymer, aromatics, a sweetener, and a dye.

The term "disease" refers to any disease, discomfort, illness, symptoms or indications, and can be interchangeable with the term "disorder" or "condition".

Throughout this specification and the claims which follow, unless the context requires otherwise, the term "comprise", and variations such as "comprises" and "comprising" are intended to specify the presence of the features thereafter, but do not exclude the presence or addition of one or more other features. When used herein the term "comprising" can be substituted with the term "containing", "including" or sometimes "having".

Throughout this specification and the claims which follow, the term "Cn-m" indicates a range which includes the endpoints, wherein n and m are integers and indicate the number of carbons. Examples include C<NUM>-<NUM>, C<NUM>-<NUM>, and the like.

Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

A compound defined by the claims is disclosed as "Example F43" below. Other reference compounds are disclosed below for the purposes of better understanding the claimed invention.

The examples below are intended to be purely exemplary and should not be considered to be limiting in any way. Efforts have been made to ensure accuracy with respect to numbers used (for example, amounts, temperature, etc.), but some experimental errors and deviations should be accounted for. Unless indicated otherwise, temperature is in degrees Centigrade. Reagents were purchased from commercial suppliers such as Sigma-Aldrich, Alfa Aesar, or TCI, and were used without further purification unless indicated otherwise.

Unless indicated otherwise, the reactions set forth below were performed under a positive pressure of nitrogen or argon or with a drying tube in anhydrous solvents; the reaction flasks were fitted with rubber septa for the introduction of substrates and reagents via syringe; and glassware was oven dried and/or heat dried.

<NUM>H NMR spectra were recorded on a Agilent instrument operating at <NUM>. <NUM>HNMR spectra were obtained using CDCl<NUM>, CD<NUM>Cl<NUM>, CD<NUM>OD, D<NUM>O, d<NUM>-DMSO, d<NUM>-acetone or (CD<NUM>)<NUM>CO as solvent and tetramethylsilane (<NUM> ppm) or residual solvent (CDCl<NUM>: <NUM> ppm; CD<NUM>OD: <NUM> ppm; D<NUM>O: <NUM> ppm; d<NUM>-DMSO: <NUM> ppm; d6-acetone: <NUM>; (CD<NUM>)<NUM>CO: <NUM>) as the reference standard. When peak multiplicities are reported, the following abbreviations are used: s (singlet), d (doublet), t (triplet), q (quartet), qn (quintuplet), sx (sextuplet), m (multiplet), br (broadened), dd (doublet of doublets), dt (doublet of triplets). Coupling constants, when given, are reported in Hertz (Hz).

LC-MS spectrometer (Agilent <NUM>) Detector: MWD (<NUM>-<NUM>), Mass detector: <NUM> SQ.

Mobile phase: A: acetonitrile with <NUM>% Formic acid, B: water with <NUM>% Formic acid
Column: Poroshell <NUM> EC-C18, <NUM>×<NUM>, <NUM>
Gradient method: Flow: <NUM>/min.

Preparative HPLC was conducted on a column (<NUM> × <NUM> ID, <NUM>, Gemini NX- C18) at a different flow rate and injection volume, at room temperature and UV Detection at <NUM> and <NUM>.

In the following examples, the abbreviations below are used:.

A mixture of methyl <NUM>-bromo-<NUM>-fluorobenzoate (<NUM>, <NUM> mol), <NUM>-pyrrolo[<NUM>,<NUM>-b]pyridin-<NUM>-ol (<NUM>, <NUM> mol) and K<NUM>CO<NUM> (<NUM>, <NUM> mol) in DMF (<NUM>) was heated at <NUM> for about <NUM>. The reaction mixture was cooled to ambient temperature, filtered and the filtrate was diluted with DCM (<NUM>). The resulting solution was washed with H<NUM>O (<NUM> × <NUM>) and concentrated. The residue was recrystallized from EA (<NUM>) and PE (<NUM>), the cake (<NUM>) was collected as the first batch. The filtrate was concentrated and dissolved in EA (<NUM>). The solution was washed with H<NUM>O (<NUM> × <NUM>), concentrated, and slurried with EA (<NUM>) and PE (<NUM>) at reflux for <NUM>, cooled to ambient temperature, filtered to give the product (<NUM>) as the second batch. The two batches of product were combined to afford the product (<NUM>, <NUM> %) as a brown solid. MS (ESI, m/e) [M+<NUM>]+ <NUM>, <NUM>.

tert-butyl <NUM>-(<NUM>-(prop-<NUM>-en-<NUM>-yl)phenyl)pyrrolidine-<NUM>-carboxylate was prepared using the similar procedure as tert-butyl <NUM>-(<NUM>-cyclopropylphenyl)pyrrolidine-<NUM>-carboxylate. <NUM>H NMR (<NUM>, DMSO-d<NUM>) δ ppm: <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>). MS (ESI, m/e) [M+<NUM>]+<NUM>.

A mixture of tert-butyl <NUM>-(<NUM>-(prop-<NUM>-en-<NUM>-yl)phenyl)pyrrolidine-<NUM>-carboxylate (<NUM>, <NUM> mmol) and Pd(OH)<NUM>/C (<NUM>) in MeOH (<NUM>) was stirred overnight at room temperature under a balloon of H<NUM>. Then the reaction mixture was filtered and concentrated to give the desired product as a colorless oil (<NUM>, <NUM>%) without further purification for the next deprotection step with TFA. <NUM>H NMR (<NUM>, DMSO-d<NUM>) δ ppm: <NUM> (d, J = <NUM>, <NUM>), <NUM> (t, J = <NUM>, <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>).

A solution of tert-butyl <NUM>-(<NUM>-isopropylphenyl)pyrrolidine-<NUM>-carboxylate (<NUM>, <NUM> mmol) in DCM (<NUM>) and TFA (<NUM>) was stirred at r. After solvents were removed, the resulted residue was dissolved with DCM (<NUM>) and washed with aq. NaHCO<NUM> (<NUM> × <NUM>). The organic layer was collected and dried over anhydrous Na<NUM>SO<NUM>, filtered and concentrated to give the desired product as a colorless oil (<NUM>). <NUM>H NMR (<NUM>, DMSO-d<NUM>) δ ppm: <NUM> (d, J = <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>). MS (ESI, m/e) [M+<NUM>]+ <NUM>.

To a stirred solution of CH<NUM>MgBr (<NUM>, <NUM> mol, <NUM> in Et<NUM>O ) in dried toluene (<NUM>) was added <NUM>,<NUM>-dioxaspiro[<NUM>]decan-<NUM>-one (<NUM>, <NUM> mol) solution in <NUM> dried toluene dropwise. The resulting mixture was stirred at <NUM>~<NUM> for <NUM> hours. The mixture was poured into saturated aq. NH<NUM>Cl solution (<NUM>) and extracted with EtOAc(<NUM> × <NUM>). The combined organic phase was washed with brine(<NUM>), dried over Na<NUM>SO<NUM> and concentrated to afford the <NUM>-methyl-<NUM>,<NUM>-dioxaspiro[<NUM>]decan-<NUM>-ol (<NUM>, crude) as a white solid.

To a stirred solution of <NUM> N HCl (<NUM>) was added <NUM>-methyl-<NUM>,<NUM>-dioxaspiro[<NUM>]decan-<NUM>-ol (<NUM>, <NUM> mol). The mixture was stirred at <NUM> for <NUM> hours. The resulting mixture was cooled to room temperature and added NaCl solid to saturation, then extracted with EtOAc (<NUM> × <NUM>). The combined organic phase was dried over Na<NUM>SO<NUM> and concentrated to give the <NUM>-hydroxy-<NUM>-methylcyclohexan-<NUM>-one (<NUM>, crude) as a yellow oil.

To a stirred solution of <NUM>-hydroxy-<NUM>-methylcyclohexan-<NUM>-one (<NUM>, <NUM> mol) in CH<NUM>NO<NUM> (<NUM>) was added N<NUM>,N<NUM>-dimethylethane-<NUM>,<NUM>-diamine (<NUM>, <NUM> mol). The mixture was stirred for <NUM> hours at <NUM> under nitrogen atmosphere. After cooled to room temperature, the reaction mixture was concentrated and purified by silica gel column chromatography eluted with EA/PE = <NUM>/<NUM> to afford the (S)-<NUM>-methyl-<NUM>-(nitromethyl)cyclohex-<NUM>-en-<NUM>-ol (<NUM>) as a yellow oil.

To a stirred solution of (S)-<NUM>-methyl-<NUM>-(nitromethyl)cyclohex-<NUM>-en-<NUM>-ol (<NUM>, <NUM> mol) in DCM (<NUM>) was added Crabtree's catalyst (<NUM>, <NUM> mmol). The mixture was stirred at <NUM> for overnight under H<NUM> (<NUM> atm) atmosphere. After cooled to room temperature, the reaction mixture was filtrated and concentrated to afford (1r,4r)-<NUM>-methyl-<NUM>-(nitromethyl)cyclohexan-<NUM>-ol (<NUM>, crude) as a yellow oil.

To a stirred solution of (1r,4r)-<NUM>-methyl-<NUM>-(nitromethyl)cyclohexan-<NUM>-ol (<NUM>, <NUM> mol) in MeOH(<NUM>) was added <NUM>% wet Pd/C (<NUM>). The mixture was stirred for overnight at <NUM> under H<NUM> (<NUM> atm) atmosphere. After cooled to room temperature, the reaction mixture was filtrated and concentrated to afford (1r,4r)-<NUM>-(aminomethyl)-<NUM>-methylcyclohexan-<NUM>-ol (<NUM>, crude) as a brown solid. <NUM>H NMR (<NUM>, Methanol-d<NUM>) δ ppm: <NUM> (d, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> -<NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>).

To a stirred solution of (1r,4r)-<NUM>-(aminomethyl)-<NUM>-methylcyclohexan-<NUM>-ol (<NUM>, <NUM> mol) in THF (<NUM>) was added <NUM>-fluoro-<NUM>-nitrobenzenesulfonamide (<NUM>, <NUM> mol) and TEA (<NUM>, <NUM> mol). The mixture was stirred at room temperature for overnight. The resulting mixture was diluted with water (<NUM>) and extracted with EtOAc (<NUM> × <NUM>). The combined organic phase was washed with brine (<NUM>), dried over anhydrous Na<NUM>SO<NUM> and concentrated. The residue was purified by slurry in EtOAc (<NUM>) for three times to afford <NUM>-((((1r,4r)-<NUM>-hydroxy-<NUM>-methylcyclohexyl)methyl)amino)-<NUM>-nitrobenzenesulfonamide (<NUM>) as a yellow solid. <NUM> NMR (<NUM>, DMSO-d<NUM>) δ ppm: <NUM> (t, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (td, J = <NUM>, <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>). MS (ESI, m/e) [M+<NUM>]+ <NUM>.

To the solution of tert-butyl <NUM>-oxo-<NUM>-azaspiro[<NUM>]nonane-<NUM>-carboxylate (<NUM>, <NUM> mol) in MeOH (<NUM>) and EA (<NUM>) was added conc. HCl acid (<NUM>, <NUM> mol) at room temperature and stirred for <NUM> hours. After concentrated in vacuum, MeOH (<NUM>) was added into the residue and then the resulting mixture was concentrated in vacuum (repeated this work-up twice). The brown residue was suspended in EA (<NUM>) and stirred for <NUM> hour. The solid precipitation was filtered and dried in vacuum to afford the tittle product as an off-white powder (<NUM>, yield: <NUM>%). <NUM>H NMR (<NUM>, DMSO-d<NUM>) δ ppm: <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>). MS (ESI, m/e) [M+<NUM>]+ <NUM>.

The mixture of methyl <NUM>-((<NUM>-pyrrolo[<NUM>,<NUM>-b]pyridin-<NUM>-yl)oxy)-<NUM>-fluorobenzoate (<NUM>), <NUM>,<NUM>-dimethoxy-<NUM>-azaspiro[<NUM>]nonane hydrochloride (<NUM>, <NUM> eq. ) and DBU (<NUM>, <NUM> eq. ) in NMP (<NUM>) was stirred for <NUM> hours at <NUM>. After the reaction was completed, the mixture was cooled to <NUM> ± <NUM> and citric acid in water (<NUM>%, <NUM>) was added drop-wise into the system under stirring. After filtered, the cake was collected and dissolved with DCM (<NUM>). The solution of crude product was washed with citric acid in water (<NUM>%, <NUM>), saturated aq. NaHCO<NUM> (<NUM>) and <NUM>% aq. NaCl (<NUM>), and then dried over anhydrous Na<NUM>SO<NUM>. Silica gel (<NUM>) was added into the solution of crude product under stirring and then filtered. The filtrate was concentrated to <NUM>. MTBE (<NUM>) was poured into the system. After stirred for <NUM> hours, the cake was collected after filtration and was dried in vacuum to give an off-white solid (<NUM>, yield: <NUM>%). <NUM>H NMR (<NUM>, DMSO-d<NUM>) δ ppm: <NUM> (s, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (dd, J= <NUM>, J= <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>). MS (ESI, m/e) [M+<NUM>]+ <NUM>.

To the solution of methyl <NUM>-((<NUM>-pyrrolo[<NUM>,<NUM>-b]pyridin-<NUM>-yl)oxy)-<NUM>-(<NUM>,<NUM>-dimethoxy-<NUM>-azaspiro[<NUM>]nonan-<NUM>-yl)benzoate (<NUM>, <NUM> mol) in DCM (<NUM>) was added diluted HCl acid (<NUM>, <NUM>) and stirred for overnight. After the reaction was completed, the mixture was cooled to <NUM> and was adjusted to pH = <NUM>-<NUM> with aqueous NaOH solution (<NUM>) under stirring. The organic phase was separated and washed with <NUM>% aq. NaCl (<NUM>), then washed with H<NUM>O (<NUM>). After the organic phase was concentrated to <NUM>, MTBE (<NUM>) was poured into the solution and then the system was concentrated to <NUM> (repeated this work-up <NUM> times). The resulting system was stirred for <NUM> hour. After filtration, the cake was collected and then dried in vacuum to obtain the tittle product as a white solid (<NUM>, yield: <NUM>%). <NUM>H NMR (<NUM>, DMSO-d<NUM>) δ ppm: <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (t, J= <NUM>, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM> (dd, J= <NUM>, J= <NUM>, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>). MS (ESI, m/e) [M+<NUM>]+ <NUM>.

To a mixture of (S)-tert-butyl <NUM>-(<NUM>-bromophenyl)pyrrolidine-<NUM>-carboxylate (<NUM>, <NUM> mmol) and <NUM>,<NUM>,<NUM>,<NUM>-tetramethyl-<NUM>-(prop-<NUM>-en-<NUM>-yl)-<NUM>,<NUM>,<NUM>-dioxaborolane (<NUM>, <NUM> mmol) in dioxane (<NUM>) and H<NUM>O (<NUM>) was added Cs<NUM>CO<NUM> (<NUM>, <NUM> mmol) and Pd(dppf)Cl<NUM> (<NUM>, <NUM> mmol). The mixture was stirred at <NUM> for <NUM> hours. TLC showed the reaction was completed. The mixture was concentrated in vacuum. The residue was purified by column chromatography on silica gel (eluent: PE/EA (v/v) = <NUM>/<NUM> to <NUM>/<NUM>) to obtain (S)-tert-butyl <NUM>-(<NUM>-(prop-<NUM>-en-<NUM>-yl)phenyl)pyrrolidine-<NUM>-carboxylate (<NUM>, crude). The crude product was used directly in next step.

To a solution of (S)-tert-butyl <NUM>-(<NUM>-(prop-<NUM>-en-<NUM>-yl)phenyl)pyrrolidine-<NUM>-carboxylate (<NUM>, <NUM> mmol) in MeOH (<NUM>) was added Pd/C (<NUM>, <NUM>%) and the mixture was stirred at <NUM> under H<NUM> (<NUM> Psi) for <NUM> hours. TLC showed the reaction was completed. The mixture was filtered and the filtrate was concentrated in vacuum to give (S)-tert-butyl <NUM>-(<NUM>-isopropylphenyl)pyrrolidine-<NUM>-carboxylate (<NUM>, crude), which was used in next step without further purification. <NUM>H NMR (<NUM>, CDCl<NUM>) δ ppm: <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>).

To a solution of tert-butyl <NUM>-(<NUM>-isopropylphenyl)pyrrolidine-<NUM>-carboxylate (<NUM>, <NUM> mmol) in DCM (<NUM>) was added HCl in <NUM>,<NUM>-dioxane (<NUM>, <NUM>, <NUM> mmol) dropwise at room temperature. The mixture was stirred at room temperature for overnight. The mixture was concentrated in vacuum. The resulting residue was slurried with EA (<NUM>) and then filtered, dried in vacuum to give (S)-<NUM>-(<NUM>-isopropylphenyl)pyrrolidine hydrochloride <NUM> (yield: <NUM>%). <NUM>H NMR (<NUM>, DMSO-d<NUM>) δ ppm: <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>). MS (ESI, m/e) [M+<NUM>]+ <NUM>.

A mixture of (S)-<NUM>-(<NUM>-isopropylphenyl)pyrrolidine hydrochloride (<NUM>, <NUM> mole) and methyl <NUM>-((<NUM>-pyrrolo[<NUM>,<NUM>-b]pyridin-<NUM>-yl)oxy)-<NUM>-(<NUM>-oxo-<NUM>-azaspiro[<NUM>]nonan-<NUM>-yl)benzoate (<NUM>, <NUM> mole) in DCM (<NUM>) was charged into a reactor. The temperature was controlled blow <NUM> and NaBH(OAc)<NUM> (<NUM>, <NUM> mole) was added into the reactor in <NUM>-<NUM> portions. Then the reaction mixture was stirred at room temperature and monitored by TLC. After the starting material ketone was consumed completely, the mixture was adjusted to pH = <NUM>~<NUM> with diluted HCl acid (<NUM>). The separated organic phase was washed with H<NUM>O (<NUM> × <NUM>) and then washed with aq. NaHCO<NUM> (<NUM> × <NUM>), saturated aq. NaCl (<NUM>). The organic phase was collected, then dried over anhydrous Na<NUM>SO<NUM> and concentrated. <NUM> off-white solid was obtained as crude product, which was used in next step directly. MS (ESI, m/e) [M+<NUM>]+ <NUM>.

To a solution of methyl (S)-<NUM>-((<NUM>-pyrrolo[<NUM>,<NUM>-b]pyridin-<NUM>-yl)oxy)-<NUM>-(<NUM>-(<NUM>-(<NUM>-isopropylphenyl)pyrrolidin-<NUM>-yl)-<NUM>-azaspiro[<NUM>]nonan-<NUM>-yl)benzoate (<NUM>, <NUM> mmol) in THF (<NUM>) and MeOH (<NUM>) was added aq. NaOH (<NUM>). It was stirred at room temperature overnight. After THF and MeOH were removed in vacuum, <NUM> of water was added into the residue. The resulting mixture was adjusted to pH = <NUM>~<NUM> with <NUM> N HCl acid at room temperature with stirring. The precipitate was filtered and dried in vacuum to give the product as a white solid (<NUM>, yield: <NUM>%). <NUM>H NMR (<NUM>, DMSO-d<NUM>) δ ppm: <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> -<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>). MS (ESI, m/e) [M+<NUM>]+ <NUM>.

A mixture of (S)-<NUM>-((<NUM>-pyrrolo[<NUM>,<NUM>-b]pyridin-<NUM>-yl)oxy)-<NUM>-(<NUM>-(<NUM>-(<NUM>-isopropylphenyl)pyrrolidin-<NUM>-yl)-<NUM>-azaspiro[<NUM>]nonan-<NUM>-yl)benzoic acid (<NUM>, <NUM> mmol), <NUM>-((((1r,4r)-<NUM>-hydroxy-<NUM>-methylcyclohexyl)methyl)amino)-<NUM> -nitrobenzenesulfonamide (<NUM>, <NUM> mmol), TEA (<NUM>, <NUM> mmol), EDCI (<NUM>, <NUM> mmol) and DMAP (<NUM>, <NUM> mmol) in anhydrous DCM (<NUM>) was stirred overnight at room temperature. The reaction was monitored by HPLC. After starting material of (S)-<NUM>-((<NUM>-pyrrolo[<NUM>,<NUM>-b]pyridin-<NUM>-yl)oxy)-<NUM>-(<NUM>-(<NUM>-(<NUM>-isopropylphenyl)pyrrolidin-<NUM>-yl)-<NUM>-azaspiro[<NUM>]nonan-<NUM>-yl)benzoic acid was consumed completely, the reaction mixture was heated to ~<NUM> and N<NUM>,N<NUM>-dimethylethane-<NUM>,<NUM>-diamine (<NUM>, <NUM> mmol) was added in one portion. The reaction was stirred for another <NUM> hours. The mixture was washed twice with <NUM> wt % aq. AcOH solution (<NUM> × <NUM>) and then washed with saturated aq. NaHCO<NUM> (<NUM>× <NUM>). The organic layer was collected and concentrated to about <NUM>. <NUM> of silica gel was added and stirred for <NUM> hours. After filtration, <NUM> EA was added into the filtrate at reflux and further stirred for <NUM> hours. After the mixture was cooled to room temperature, the precipitate was filtered and then the wet cake was washed twice with EA (<NUM>). After drying in vacuum at <NUM>-<NUM>, the desired compound was obtained (<NUM>, yield: <NUM>%). <NUM>H NMR (DMSO-d<NUM>) δ ppm: <NUM> (s, <NUM>), <NUM> (br, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM> (s, <NUM>). MS (ESI, m/e) [M+<NUM>]+ <NUM>.

Two enantiomers G8-a (faster isomer) and G8-b (slower isomer) were separated by chiral preparative HPLC. The chiral separation conditions are shown below. The faster enantiomer was eluted at retention time of <NUM> to give G8-a. The slower enantiomer was eluted at retention time of <NUM> to give G8-b.

Example G8-a: <NUM>H NMR (<NUM>, DMSO-d<NUM>) δ ppm: <NUM> (br, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>). MS (ESI, m/e) [M+<NUM>]+ <NUM>. Example G8-b: <NUM>H NMR (<NUM>, DMSO-d<NUM>) δ ppm: <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>). MS (ESI, m/e) [M+<NUM>]+ <NUM>.

Two enantiomers G10b-a (faster isomer) and G10b-b (slower isomer) of G10b-S were separated by chiral preparative HPLC.

Example GlOb-a: <NUM>H NMR (<NUM>, DMSO-d<NUM>) δ ppm: <NUM> (br, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>),<NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>),<NUM> (s, <NUM>), <NUM>-<NUM> (m,<NUM>), <NUM>-<NUM> (m,<NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>). MS (ESI, m/e) [M+<NUM>]+ <NUM>. Example G10b-b: <NUM>H NMR (<NUM>, DMSO-d<NUM>) δ ppm: <NUM> (br, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), , <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>). MS (ESI, m/e) [M+<NUM>]+ <NUM>.

Two enantiomers G24b-a (faster isomer) and G24b-b (slower isomer) of (trans- or cis-)<NUM>-((<NUM>-pyrrolo[<NUM>,<NUM>-b]pyridin-<NUM>-yl)oxy)-<NUM>'-(<NUM>-(S)-(<NUM>-cyclopropylphenyl)pyrrolidin-<NUM>-yl)-N-((<NUM>-(((<NUM>-hydroxy-<NUM>-methylcyclohexyl)methyl)amino)-<NUM>-nitrophenyl)sulfonyl)-<NUM>',<NUM>',<NUM>',<NUM>'-tetrahydro-[<NUM>,<NUM>'-biphenyl]-<NUM>-carboxamide were separated by chiral preparative HPLC.

Example G24b-a: <NUM>H NMR (<NUM>, DMSO-d<NUM>) δ ppm: <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>),<NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> -<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM>(m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>). MS (ESI, m/e) [M+<NUM>]+ <NUM>. Example G24b-b: <NUM>H NMR (<NUM>, DMSO-d<NUM>) δ ppm: <NUM>-<NUM>(m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM>(m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>). MS (ESI, m/e) [M+<NUM>]+ <NUM>.

Two enantiomers G30-a (faster isomer) and G30-b (slower isomer) of G30-S were separated by chiral preparative HPLC.

Example G30-a: <NUM>H NMR (<NUM>, DMSO-d<NUM>) δ ppm: <NUM> (br, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>),<NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>). [M+<NUM>]+ <NUM>. Example G30-b: <NUM>H NMR (<NUM>, DMSO-d<NUM>) δ ppm: <NUM>-<NUM> (m, <NUM>),<NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>),<NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM>(m,<NUM>). (MS (ESI, m/e) [M+<NUM>]+ <NUM>.

Two enantiomers G35-a (faster isomer) and G35-b (slower isomer) were separated by chiral preparative HPLC.

Example G35-a : <NUM>H NMR (<NUM>, DMSO-d<NUM>) δ ppm: <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>). MS (ESI, m/e) [M+<NUM>]+ <NUM>. Example G35-b: <NUM>H NMR (<NUM>, DMSO-d<NUM>) δ ppm: <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>). MS (ESI, m/e) [M+<NUM>]+ <NUM>.

Two enantiomers G75-a (faster isomer) and G75-b (slower isomer) of <NUM>-((<NUM>-pyrrolo[<NUM>,<NUM>-b]pyridin-<NUM>-yl)oxy)-<NUM>'-((S)-<NUM>-(<NUM>-cyclopropylphenyl)pyrrolidin-<NUM>-yl)-N-((<NUM>-nitro-<NUM>-(((<NUM>-(oxetan-<NUM>-yl)piperidin-<NUM>-yl)methyl)amino)phenyl)sulfonyl)-<NUM>',<NUM>',<NUM>',<NUM>'-tetrahydro-[<NUM>,<NUM>'-biphenyl]-<NUM>-carboxamide were separated by chiral preparative HPLC.

Example G75-a: <NUM>H NMR (DMSO-d<NUM>) δ ppm: <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>). MS (ESI, m/e) [M+<NUM>]+ <NUM>. Example G75-b: <NUM>H NMR (CDCl3-d<NUM>) δ ppm: <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>). MS (ESI, m/e) [M+<NUM>]+ <NUM>.

After deprotection of tert-butyldimethyl-silanyl for G80a, Example G81a: (cis- or trans-) <NUM>-((<NUM>-pyrrolo[<NUM>,<NUM>-b]pyridin-<NUM>-yl)oxy)-<NUM>'-((S)-<NUM>-(<NUM>-cyclopropylphenyl)pyrrolidin-<NUM>-yl)-N-((<NUM>-((<NUM>-hydroxycyclohexyl)methoxy)-<NUM>-nitrophenyl)sulfonyl)-<NUM>',<NUM>',<NUM>',<NUM>'-tetrahydro-[<NUM>,<NUM>'-biphenyl]-<NUM>-carboxamide was obtained.

After deprotection of tert-butyldimethyl-silanyl for G80b, Example G81b: (trans- or cis-) <NUM>-((<NUM>-pyrrolo[<NUM>,<NUM>-b]pyridin-<NUM>-yl)oxy)-<NUM>'-((S)-<NUM>-(<NUM>-cyclopropylphenyl)pyrrolidin-<NUM>-yl)-N-((<NUM>-((<NUM>-hydroxycyclohexyl)methoxy)-<NUM>-nitrophenyl)sulfonyl)-<NUM>',<NUM>',<NUM>',<NUM>'-tetrahydro-[<NUM>,<NUM>'-biphenyl]-<NUM>-carboxamide was obtained. <NUM>H NMR (<NUM>, DMSO-d<NUM>) δ ppm: <NUM> (br, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM>(m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM>(m, <NUM>). MS (ESI, m/e) [M+<NUM>]+ <NUM>.

Two enantiomers G90-a (faster isomer) and G90-b (slower isomer) were separated by chiral preparative HPLC.

Example G90-a: <NUM>H NMR (DMSO-d<NUM>) δ ppm: <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (t, J= <NUM>, <NUM>). MS (ESI, m/e) [M+<NUM>]+ <NUM>. Example G90-b: <NUM>H NMR (DMSO-d<NUM>) δ ppm: <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (t, J = <NUM>, <NUM>). MS (ESI, m/e) [M+<NUM>]+ <NUM>.

Two enantiomers G110b-a (faster isomer) and G110b-b (slower isomer) of G110b were separated by chiral preparative HPLC. The chiral separation conditions are shown below.

Example G110b-a: <NUM>H NMR (<NUM>, DMSO-d<NUM>) δ ppm: <NUM> (br, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), MS (ESI, m/e) [M+<NUM>]+ <NUM>. Example G110b-b: <NUM>H NMR (<NUM>, DMSO-d<NUM>) δ ppm: <NUM> (br, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM>(m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m,<NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>). MS (ESI, m/e) [M+<NUM>]+ <NUM>.

Compounds disclosed herein were tested for blocking of Bcl-<NUM>/Bcl-xl protein with its ligand in an assay based on fluorescence polarization methodology. Recombinant human <NUM> Bcl-<NUM>/ <NUM> Bcl-xl protein was pre-incubated with a serial dilution of compounds disclosed herein (maximum concentration is <NUM> for Bcl-<NUM> assay, and <NUM> or <NUM> for Bcl-xl assay, <NUM>-fold serially diluted, <NUM> points) at room temperature for <NUM> hour in an assay buffer containing <NUM> potassium phosphate buffer, pH <NUM>, <NUM> NaCl, <NUM> EDTA, <NUM>% Tween-<NUM>, <NUM>% BSA. Then the FITC labeled Bak peptide Ac-GQVGRQLAIIGDK(FITC)INR-amide (<NUM> for Bcl-<NUM>, <NUM> for Bcl-xl) was added to plate and further incubated at room temperature for <NUM>. The FP signals (<NUM>-<NUM>-<NUM>) were read on BMG PHERAstar FS or BMG PHERAstar FSX instrument. The inhibition percentage of Bcl-<NUM>/Bcl-xl interaction with its ligand in presence of increasing concentrations of compounds was calculated based on the FP signals. The IC50 for each compound was derived from fitting the data to the four-parameter logistic equation by Graphpad Prism software.

Compounds disclosed herein were tested for blocking of Bcl-<NUM>/Bcl-X protein with its ligand in an assay based on Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) methodology. Recombinant human <NUM> Bcl-<NUM>/<NUM> Bcl-X protein was pre-incubated with a serial dilution of compounds disclosed herein (maximum concentration is <NUM> for Bcl-<NUM> assay, and <NUM> for Bcl-xl assay, <NUM>-fold serially diluted, <NUM> points; or maximum concentration is <NUM> for Bcl-<NUM> assay, and <NUM> for Bcl-xl assay, <NUM>-fold serially diluted, <NUM> points) at room temperature for <NUM> hour in an assay buffer containing <NUM> potassium phosphate buffer, pH <NUM>, <NUM> NaCl, <NUM> EDTA, <NUM>% Tween-<NUM>, <NUM>% BSA. Then the FITC labeled Bak peptide Ac-GQVGRQLAIIGDK(FITC)INR-amide (<NUM> for Bcl-<NUM>, <NUM> for Bcl-xl) and MAb Anti 6His Tb cryptate Gold were added to plate and further incubated at room temperature for <NUM> hour. The TR-FRET signals (<NUM>-<NUM>-<NUM>) were read on BMG PHERAstar FSX instrument. The inhibition percentage of Bcl-<NUM>/Bcl-X interaction with its ligand in presence of increasing concentrations of compounds was calculated based on the TR-FRET signals. The IC<NUM> for each compound was derived from fitting the data to the four-parameter logistic equation by Graphpad Prism software. To improve the assay sensitivity and test more potent compounds in the present application, the bcl-<NUM> concentration was reduced in method B.

The Bcl-<NUM> family proteins are central regulators of apoptosis. Bcl-<NUM> and Bcl-XL are antiapoptotic factors within this family. In our cell proliferation assay, the Bcl-<NUM> dependent acute lymphoblastic leukemia (ALL) cell line, RS4;<NUM>, was used to study the cellular potency of Bcl-<NUM> inhibitors. The cells (ATCC, CRL-<NUM>) were cultured in RPMI-<NUM> complete medium (RPMI-<NUM> medium, HEPES (Gibco, <NUM>-<NUM>) supplemented with <NUM>% fetal bovine serum (FBS) (Gibco, <NUM>-<NUM>), <NUM> unit/ml penicillin and <NUM>µg/ml streptomycin (Gibco, <NUM>)) and maintained in a humidified chamber at <NUM> containing <NUM>% CO<NUM>. Each compound was serially diluted with <NUM> as the maximum concentration. To test the apoptotic effect of the compounds, the cells were seeded at <NUM>,<NUM> in <NUM>µl per well in <NUM>-well plates and treated with <NUM>-point dilution series of each compound for <NUM> hours at <NUM>. Cell viability was assessed after the treatment using CellTiter-GLO luminescent assay (Promega) according to the manufacturer's recommendations. Briefly, <NUM>µl of CellTiter-GLO reagent was added into <NUM>µl of cell culture. Mixture was agitated on an orbital shaker for <NUM> minutes to ensure cell lysis followed by <NUM> mins incubation at room temperature to allow development and stabilization of luminescent signals, which corresponded to quantity of ATP and thus the quantity of metabolically active cells. Luminescent signals were measured using PHERAstar FS reader (BMG). Mean IC<NUM> values for cell viability were determined with GraphPad Prism software. The Bcl-XL-dependent ALL cell line, Molt-<NUM> (ATCC, CRL-<NUM>) was also used in cell proliferation assay to further evaluate the specificity of these inhibitors. Similarly, the cells were cultured in RPMI-<NUM> complete medium (RPMI-<NUM> medium, HEPES (Gibco, <NUM>-<NUM>) supplemented with <NUM>% fetal bovine serum (FBS) (Gibco, <NUM>-<NUM>), <NUM> unit/ml penicillin and <NUM>µg/ml streptomycin (Gibco, <NUM>) and <NUM>×GlutaMAX (Gibco, <NUM>-<NUM>)) and maintained in a humidified chamber at <NUM> containing <NUM>% CO<NUM>. The antiproliferative IC<NUM>s of these compounds were similarly determined as a percentage of viable cells upon treatment compared to the untreated control using CellTiter-GLO luminescent assay.

Selected compounds disclosed herein were tested for blocking of Bcl-<NUM>-G101 protein with its ligand in an assay based on time-resolved fluorescence resonance energy transfer methodology. <NUM> of Recombinant human Bcl-<NUM>-G101V protein was pre-incubated with a serial dilution of compounds disclosed herein (maximum concentration is <NUM>, <NUM>-fold serially diluted, <NUM> points; or maximum concentration is <NUM>, <NUM>-fold serially diluted, <NUM> points) at room temperature for <NUM> hour in an assay buffer containing <NUM> potassium phosphate buffer, pH <NUM>, <NUM> NaCl, <NUM> EDTA, <NUM>% Tween-<NUM>, <NUM>% BSA. Then <NUM> of the FITC labeled Bak peptide Ac-GQVGRQLAIIGDK(FITC)INR-amide and Mab Anti-6His Tb cryptate Gold was added to plate and further incubated at room temperature for <NUM> hour. The TR-FRET signals (ex337nm, em490nm/<NUM>) were read on BMG PHERAstar FSX instrument. The inhibition percentage of Bcl-<NUM>-G101V interaction with its ligand in presence of increasing concentrations of compounds was calculated based on the ratio of fluorescence at <NUM> to that at <NUM>. The IC<NUM> for each compound was derived from fitting the data to the four-parameter logistic equation by Graphpad Prism software or Dotmatics. The data was shown in Table <NUM>-C.

To further assess the compound's binding affinity to Bcl-<NUM> Gly101Val mutant, selected compounds in Table <NUM>-C together withABT-<NUM> were examined in biochemical assay. These compounds were confirmed to be unexpectedly more potent than ABT-<NUM> (<NUM>), which indicates these compounds may overcome the BCL2 resistant mutant.

TABLE <NUM> describes ABT-<NUM>, its structurally similar analogs and their activity in both of biochemical assay and cellular assay. As can be seen from the table, these analogs exhibit a dramatic trend of decreasing activity (at least more than <NUM> fold) for Bcl-<NUM> compared with ABT-<NUM>. For example, the most similar analog B6 shows about <NUM> fold less potent in biochemical assay and more than <NUM> fold less potent in RS4;<NUM> cellular proliferation assay. The decrease in activity of ABT-<NUM> analogs from B1 to B5 ranges from <NUM> fold to greater than <NUM> fold in biochemical assay, and the drop potency in RS4;<NUM> cellular proliferation assay are all greater than <NUM> fold.

TABLE 3A describes selected compounds without the carbon atom between two rings A and B and their activity or potency in both of biochemical assay and cellular assay. Compounds in the present patent show unexpected structure-activity relationship (SAR). When ring A is phenyl or spiro ring, compounds (F5, F55, A4, A8) with an ortho-substituent (e.g., Cl atom or cyclopropyl) on the phenyl group are much more potent (> <NUM> fold) compared to those compounds with the same substituent on other positions of the phenyl group. However, the above SAR with respect to the change of the substitution positions on the phenyl group were not found when the ring A is hexane or hexene group.

Table 3B describes some examples with different ring A and their activity or potency in both of biochemical assay and cellular assay. No -CH<NUM>- between Ring A and B. Surprisingly, examples F21, F22, F23,F24, F25, F26, F34, F37, F38, F40, F41, F43-F48, F62-F64, F90, F91b, F92, F99, F104, F106, F109, F111, F120, F126, F130 and F132b with spiro ring as ring A have significantly increased activity in both of biochemical assay and cellular assay, compared to examples with other rings as ring A (i.e., examples A8a, G92-R, G94-R, G95-R and G96-R with phenyl rings as ring A, and C3, G30-a, G30-b, G10b-a, G10b-b, G24b-a, G24b-b, G9-a, G9-b, G8-a, G8-b, G107-a, G107-b, G90-a, and G90-b with hexene rings as ring A, and D2b-S and G76-S, G77-S with hexane rings as ring A). Compounds in the current patent show unexpected SAR, which can be further explained by an additional sulfur-pi interaction with Met115 in co-crystal of compound F22 having a spiro ring as ring A with bcl-<NUM> protein compared to those of compounds G10b-a, G10b-b having hexene rings as ring A with bcl-<NUM> protein.

Examples F21, F22, F23, F24, F25, F26, F34, F37, F38, F40, F41, F43-F48, F62-F64, F90, F91b, F92, F99, F104, F106, F109, F111, F120, F126, F130 and F132b with spiro rings as ring A are <NUM> to > <NUM> fold more potent than ABT-<NUM> and Example <NUM> from <CIT> in biochemical assay using method B, and ><NUM> to ><NUM> fold more potent than ABT-<NUM> in cellular assay. Moreover, the selectivity of examples with spiro ring as ring A against Bcl-xl is better than that of ABT-<NUM> in biochemical assay or cellular assay. Further, the unexpected SAR also happened on the chiral center of pyrrolidine ring. The more potent isomer in example with phenyl ring as ring A has R configuration (i.e., examples G92-R, G94-R, G95-R, G96-R, G118, G122 and G124), while the more potent isomer in example with hexane, hexane or spiro ring as ring A has S configuration.

Table 3C describes compounds with spiro ring as ring A and their activity in both of biochemical assay (using method B) and cellular assay. As can be seen from the table, inserting -CH<NUM>- between ring A (spiro ring) and ring B (pyrrolidine ring) dramatically reduced the potency, which is consistent with the unexpected structure-activity relationship (SAR). For example, the F115 shows > <NUM> folds and <NUM> folds less potent than its analog F23 in biochemical assay and cellular assay, respectively. F113 and F114 show <NUM> to <NUM> folds and <NUM> to <NUM> folds less potent than their analog F34 in biochemical assay and cellular assay, respectively.

Moreover, all these compounds are much more potent than Example <NUM> from <CIT> and F133 in biochemical assay, which may be attributed to the optimum combination of the spiro moiety and the <NUM>-(<NUM>-substituted phenyl)pyrrolidin-<NUM>-yl moiety or <NUM>-(<NUM>-substituted phenyl)-<NUM>-alkylpiperazin-<NUM>-yl moiety of the compounds disclosed herein.

Recombinant Bcl-<NUM> protein with GST tag was expressed in E. coli BL21 (DE3), induced with <NUM> IPTG for <NUM> at <NUM>. The cells were harvested by centrifugation at <NUM>,<NUM> for <NUM>, re-suspended in lysis buffer containing <NUM> Tris, pH <NUM> and <NUM> NaCl, and lysed by sonication. After centrifugation at <NUM>,<NUM> for <NUM>, the supernatant was incubated with Glutathione S-transferase resin at <NUM> for <NUM>. The resin was rinsed three times with the lysis buffer, followed by treatment with PreScission protease at <NUM> overnight. The flow through was concentrated and sequentially applied to a size-exclusion chromatography column (Superdex-<NUM>, GE Healthcare) in a buffer containing <NUM> Tris, pH <NUM> and <NUM> NaCl. The peak was collected and concentrated to approximately <NUM>/ml. Protein solution was incubated with A4a for <NUM> at <NUM>, and then mixed with a reservoir solution containing <NUM> Bis-Tris, pH <NUM> and <NUM>% PEG <NUM>,<NUM>. Co-crystals of Bcl-<NUM> with A4a were obtained by vapor diffusion from hanging drops cultured at <NUM>.

Nylon loops were used to harvest the co-crystals and then immersed the crystals in the reservoir solution supplemented with <NUM>% glycerol for <NUM> sec. Diffraction data were collected on Eiger <NUM> detector at BL17U1, Shanghai Synchrotron Radiation Facility, and were processed with XDS program. The phase was solved with program PHASER using the Bcl-<NUM> crystal structure (PDB code 4MAN) as the molecular replacement searching model. refine was used to perform rigid body, TLS, restrained refinement against X-ray data, followed by manually adjustment in COOT program and further refinement in Phenix. refine program.

Values in parentheses refer to the highest resolution shell.

The absolute stereochemistry of the more potent compound A4a in enzymatic and cellular assays is assigned as (S)-configuration on the chiral carbon atom based on its co-crystal structure with Bcl-<NUM> protein. The binding pose of A4a is distinct from that of ABT-<NUM> analog (compound structure see table <NUM>, PDB code: 4MAN) to Bcl2 protein. Compared to ABT-<NUM> analog, <NUM>-(<NUM>-chlorophenyl)-pyrrolidinyl moiety of A4a induces a different conformation of the residues around p2 pocket of Bcl-<NUM>, such as Phe112, Met115, Glu136 and Phe153, which results in a larger and flatter pocket on the surface of the protein.

As shown in <FIG>. , binding pose of F22 is distinct from that of ABT-<NUM> analog (PDB code: 4MAN). Compared to ABT-<NUM> analog, <NUM>-(<NUM>-cyclopropylphenyl)-pyrrolidinyl moiety of F22 induces a different conformation of the residues around p2 pocket of Bcl-<NUM>, such as Asp111, Phe112 and Met115, which create an extra sub-pocket. Hydrophobic interaction between <NUM>-cyclopropylphenyl with the surrounding residues contributes to the better potency of F22.

As shown in <FIG>, water bridge is observed between nitrogen atom of F22 pyrrolidinyl ring and backbone carbonyl of Vall <NUM> through <NUM> water molecules in the crystal structure. This water bridge contributes to a more stable interaction between F22 and Bcl-<NUM> protein, while no such water bridge can be observed between ABT-<NUM> analog and Bcl-<NUM>. As shown in <FIG>, optimal sulfur-π interaction between Met115 and <NUM>-cyclopropylphenyl of F22 is observed in the crystal structure. Similar interaction can also be observed in the crystal structure of ABT-<NUM> analog (PDB code: 4MAN), but the interaction is not optimal in that crystal structure.

In summary, based on the crystal structure of F22, hydrophobic interaction between the cyclopropyl group and induced sub-pocket, water bridge with Val133 and sulfur-π interaction with Met115 all contribute to the better potency of F22.

Protein was purified as described previously. Protein solution was incubated with F22 by a molar ratio <NUM>:<NUM> for <NUM> at <NUM>, and then mixed with a reservoir solution containing <NUM> ammonium acetate, <NUM> Bis-Tris, pH <NUM> and <NUM>% PEG <NUM>,<NUM>. Co-crystals were obtained by vapor diffusion from hanging drops cultured at <NUM>.

Nylon loops were used to harvest the co-crystals and then immersed the crystals in the reservoir solution supplemented with <NUM>% glycerol for <NUM> sec. Diffraction data were collected at home lab diffractometer, and were processed with XDS program. The phase was solved with program PHASER using the Bcl-2_G10B-a in house crystal structure as the molecular replacement searching model. refine was used to perform rigid body, TLS, restrained refinement against X-ray data, followed by manually adjustment in COOT program and further refinement in Phenix. refine program.

Method: The five isoform-selective probe substrate (in a cocktail manner) was used as a measure of activity for the individual cytochrome P450 (CYPs) in a pool of human liver microsomes, i.e., phenacetin for CYP1A2, diclofenac for CYP2C9, S-Mephenytoin for CYP2C <NUM>, dextromethorphan for CYP2D6, midazolam for CYP3A. Test compounds, at <NUM> concentration levels including zero, were incubated in human liver microsomes (HLM) together with the <NUM> probe substrate (in a cocktail manner). IC<NUM> was determined by monitoring the reduction of the CYP activity as a function of test compound concentration and quantified by product formation using LC-MS/MS. Ketoconazole for CYP3A was included as quality control. All incubations were performed in singlet. The final incubation conditions are listed below.

Data Analysis: The uninhibited fraction of CYP activity (remaining activity fraction) will be calculated as <MAT>, where AM and AIS denote the peak areas of the probe metabolites and IS, respectively, and "I" and "<NUM>" represent the incubations in the presence and absence of the test compound, respectively. The IC<NUM> value of test compound will be determined as appropriate by fitting a curve of uninhibited fraction versus concentration of the test compound, using the following four-parameter model (Hill equation): <MAT>
where Top, Bottom, S, x and y donate the experimentally maximum remaining enzyme activity (%), the experimentally minimum remaining enzyme activity (%), the slope factor, the test compound concentration, and the uninhibited fraction (%), respectively.

In the case that no significant inhibition is observed over the concentration range (the uninhibited fraction does not reach <NUM>% even at the highest test compound concentration), the IC<NUM> will not be calculated.

The general criteria to evaluate the potential risk of drug-drug interaction (DDI) is as followed.

Compared with Compound ABT-<NUM> (Venetoclax) and ABT-<NUM> (Navitoclax) showing high CYP 2C9 inhibition, representative compounds disclosed herein, for example, Compounds G2, G12, G10b-b, G24b-b, G35b, G77-S, F21, F26, F37, F43, F44, F45, F106, F107, G122 and G124 showed much lower CYP 2C9 inhibition, indicating the compounds disclosed herein have lower potential risk of drug-drug interaction (DDI).

It is to be understood that, if any prior art publication is referred to herein; such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art in any country.

Claim 1:
A compound that is: <NUM>-((<NUM>-pyrrolo[<NUM>,<NUM>-b]pyridin-<NUM>-yl)oxy)-N-((<NUM>-((((1r,4r)-<NUM>-hydroxy-<NUM>-methylcyclohexyl)methyl)amino)-<NUM>-nitrophenyl)sulfonyl)-<NUM>-(<NUM>-((S)-<NUM>-(<NUM>-isopropylphenyl)pyrrolidin-<NUM>-yl)-<NUM>-azaspiro[<NUM>]nonan-<NUM>-yl)benzamide:
<CHM>
or a pharmaceutically acceptable salt thereof.