EA048753B1 - PYRIDINYL-SUBSTITUTED OXOISOINDOLINE COMPOUNDS - Google Patents

Description

Cross reference

This application claims the benefit of Indian Provisional Application Serial No. 202111016193, filed on April 6, 2021, and Indian Provisional Application Serial No. 202111022098, filed on May 17, 2021, the contents of each of which are incorporated herein in their entirety.

Description

The present invention generally relates to pyridinyl-substituted oxoisoindoline compounds that inhibit the Helios protein. Provided herein are pyridinyl-substituted oxoisoindoline compounds, compositions containing such compounds, and methods of using them. The invention also relates to pharmaceutical compositions containing at least one compound of the invention that are useful for treating proliferative diseases such as cancer and viral infections.

State of the art

Regulatory T cells (Treg) play an important role in the control of self-tolerance and immune homeostasis by maintaining inhibitory activity and anergy in the face of active immune and inflammatory responses. By maintaining a stable, anergic, and suppressive phenotype, Tregs attenuate excessive immune responses and prevent or attenuate autoimmunity. Several reports have documented the presence of Tregs in human tumor tissues. Studies have demonstrated a clear negative correlation between Treg numbers and T cell infiltration into tumors and survival (Curiel et al., 2004, Nat. Med. 10: 942-949; Viguier et al., 2004, J Immuno. 1173:1444-1453; Beyer et al., 2006, Blood 108: 804-811; Zou et al., 2006, Nat. Rev. Immunol. 6: 295-307), suggesting a potentially critical role for Tregs in preventing the development of effective antitumor immunity. Accumulating evidence suggests that Foxp3+CD25+CD4+Treg preferentially infiltrate tumors and appear to interfere with the immune response to tumor cells in rodents and humans. Following activation by specific antigen, Tregs suppress responding T cells in an antigen-nonspecific and bystander manner in vitro (Takahashi et al., 1998, Int Immunol. 10:1969–80; Thornton et al., 1998, J Exp. Med. 188:287–96). Foxp3+CD25+CD4+Treg appear to be able to suppress a broad spectrum of antitumor immune responses involving CD4+ helper T cells, CD8+ T cells, natural killer cells, and natural killer T cells (Tanaka et al., 2017, Cell Research 27:109–118). Intratumoral depletion of CD25+CD4+Treg cells caused regression of established tumors with altered cytokine milieu at tumor sites (Yu et al., 2005, J Exp Med. 201: 779–91). Furthermore, transfer of Treg-depleted CD4+ T cells markedly enhanced antitumor immune responses compared with transfer of Treg-containing T cells (Antony et al., 2005, J Immunol 174:2591–601). Tumor-infiltrating Tregs activated by either tumor-derived autoantigens or tumor-associated antigens can similarly suppress specific antitumor immune responses. Modulation of the activity of key Treg differentiation control factors may represent a potential therapeutic strategy for the treatment of several diseases, including cancer and viral infections.

FoxP3+CD4 Tregs are remarkably stable. Research is ongoing to understand the genetic mechanisms that ensure their phenotypic stability after expansion during inflammation, infection, or autoimmunity. Transcription factors (TFs) responsible for maintaining a stable immunosuppressive Treg phenotype likely contribute to this process. The Helios gene (IKZF2), a member of the Ikaros TF family, differs from other Ikaros family members by its selective expression on thymocytes undergoing negative selection, as well as on regulatory CD4 and CD8 T cell lines. Helios is expressed by two regulatory T cell lines, FoxP3+CD4+ and Ly49+CD8+ Tregs, which are required for maintenance of self-tolerance (Kim et al., 2015, Science 350:334–339; Sebastian et al., 2016, J Immunol 196:144–155). Interestingly, recent studies show that although Helios is largely dispensable for Treg activity at steady state, control of the FoxP3+ CD4 Treg genetic program by Helios under inflammatory conditions is required to maintain a stable phenotype and enhance suppressive function (Thornton et al., 2010, J Immunol. 184:3433–3441; Kim et al., 2015). Helios expression by Tregs has been shown to be critical for their ability to maintain a suppressive and anergic phenotype in the face of intense inflammatory response. Activation of the IL-2Ra-STAT5 pathway has been shown to be a key factor in Treg survival and stability (Kim et al., 2015). Helios plays an indispensable role in maintaining the FoxP3+ CD4 Tregs phenotype by exerting lymphocyte-like dominant inhibition to prevent autoimmunity in the presence of highly activated self-reactive T cells from scurfy mice lacking the fork head domain of FoxP3. Bone marrow chimeras (BM) reconstituted with Helios-/-/Scurfy, but not Helios+/+/Scurfy, BM cells rapidly developed autoimmunity (Kim et al., 2015). These observations indicate a critical contribution of Helios to the selection, differentiation, and function of self-reactive T cells. Immune suppression,

- 1 048753 mediated by Tregs may interfere with the antitumor immune response. Selective Helios deficiency in FoxP3+ CD4 Tregs results in increased Treg instability and conversion of intratumoral CD4 Tregs to effector T cells (Teff). Intratumoral Treg instability may increase the number of Teff cells in the tumor as a result of Treg conversion and decreased suppressive Treg activity. Furthermore, defective IL-2 responses were observed in Helios-deficient intratumoral Tregs, which results in decreased numbers of activated Tregs and may also contribute to increased intratumoral Teff activity. The interaction of tumor cells with infiltrating immune cells leads to the secretion of inflammatory mediators, including TNF-α, IL-6, IL-17, IL-1, and TGF-β, and the formation of a local inflammatory environment (Kim et al., 2015).

Instability of the Helios-deficient Treg lineage is also accompanied by a decrease in FoxP3 expression and leads to the acquisition of an effector phenotype due to the production of proinflammatory cytokines. Conversion of Helios-deficient Tregs to effector cells in the tumor tissue microenvironment is associated with increased expression of genes controlling the Teff phenotype (Yates et al., 2018, PNAS, 2018, 115: 2162-2167). Acquisition of an unstable phenotype in Helios deficiency occurs only within the tumor microenvironment (TME), but not in peripheral lymphoid organs (Nakagawa et al., 2016, PNAS 113: 6248-6253). In the chronic inflammatory TME, Helios deficiency in Tregs can dramatically attenuate the suppressed genetic programs associated with T helper cell differentiation by upregulating TFs and T helper cell-associated effector cytokines. These genetic alterations of Helios-deficient Tregs are most evident in the Treg subset with high affinity for self-antigens, as evidenced by increased GITR/PD-1 expression and increased sensitivity to self-antigens. Their combined effects may promote the phenotypic conversion of Tregs to Teff within the TME with increased T-cell receptor (TCR) engagement and costimulatory receptor expression by Tregs, suggesting that the gene expression changes that are central to Treg conversion are dependent on the immune environment (Yates et al., 2018).

Reduced Helios expression in FoxP3+ CD4 Tregs may enable the conversion of memory Tregs to Teff cells expressing autoreactive T cell receptors with tumor antigen specificity. An altered Treg signature may be selectively induced in the chronic inflammatory setting of growing tumors. Helios-deficient Tregs may exhibit a TCR repertoire biased toward high affinity for self-peptides/MHC, which may facilitate potent activation in the TME (Yates et al., 2018). Given the increased TCR autoreactivity in CD4 Tregs compared to conventional T cells, Treg conversion may result in the generation of highly effective effector CD4 T cells with attenuated Treg-mediated suppression in the TME. A more effective strategy may depend on approaches that selectively convert intratumoral Tregs to Teff cells without affecting the systemic Treg population. As a key player in maintaining the number and functional stability of Tregs in response to various immunological insults, pharmacological intervention of Helios may be relevant for strategies that enhance current tumor immunotherapy. Since Treg to Teff conversion can only occur in the inflammatory intratumor microenvironment, antibody- or small-molecule-based approaches targeting Helios may lead to improved Treg-dependent cancer immunotherapy. Importantly, the conversion of Helios-deficient Tregs occurs only in the local inflammatory tumor environment. Such an approach may not cause autoimmune side effects associated with systemic Treg depletion. Thus, strategies aimed at exploiting Helios-dependent control of the intratumoral Treg phenotype offer great promise for improving cancer immunotherapy. Furthermore, deletion of Foxp3+Treg has been reported to enhance vaccine-induced antitumor T cell responses (Nishikawa et al., 2010, Int. J. Cancer 127: 759–767), suggesting that downregulation of Helios may be beneficial for enhancing the efficacy of tumor vaccines.

Beyond antitumor immunotherapy, in viral infections, Treg cells can limit the immunopathology caused by excessive inflammation, but potentially suppress effective antiviral T cell responses and promote viral persistence (Schmitz et al., 2013, PLOS Pathogens 9: el003362). Chronic, but not acute, lymphocytic choriomeningitis virus infection in mice results in a marked increase in Foxp3+ Tregs, suggesting a potential mechanism by which some infectious agents can evade the host immune response by activating and expanding Tregs (Punkosdy et al., 2011, PNAS 108: 3677-3682). In the context of chronic viral infections, treatment benefits may be achieved by reducing Helios levels in activated Tregs.

There is a need for compounds that are useful as inhibitors of the Helios protein.

The essence of the invention

The present invention provides pyridinyl substituted oxoisoindoline compounds of formula (I) or salts thereof, which are useful for reducing Helios protein levels, reducing levels

- 2 048753 Helios activity and/or inhibition of Helios expression levels in cells.

The present invention also provides pharmaceutical compositions comprising a compound of formula (I) and/or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.

The present invention also provides a method of treating a disease or disorder by reducing the activity of the Helios protein, the method comprising administering to a patient a compound of formula (I) and/or a pharmaceutically acceptable salt thereof.

The present invention also provides methods and intermediates for preparing compounds of formula (I) and/or salts thereof.

The present invention also provides a compound of formula (I) and/or a pharmaceutically acceptable salt thereof for use in therapy.

The present invention also provides the use of compounds of formula (I) and/or pharmaceutically acceptable salts thereof for the manufacture of a medicament for reducing Helios protein levels, reducing Helios activity levels and/or inhibiting Helios expression levels in cells to control Treg differentiation, for the treatment of certain diseases, including cancer and viral infections.

The compounds of formula (I) and compositions containing the compounds of formula (I) can be used for the treatment, prevention or treatment of viral infections and various proliferative diseases, such as cancer. Pharmaceutical compositions containing these compounds are useful for the treatment, prevention or slowing down the progression of diseases and disorders in various therapeutic areas, such as viral infections and cancer.

These and other features of the invention will be set out in detail as the invention is further described.

Detailed description

The applicants have discovered substituted oxoisoindoline compounds that inhibit the Helios protein by facilitating the interaction of the Helios protein and the corresponding E3 ubiquitin ligase complex (Cullin4-Cereblon, CUL4-CRBN). These compounds reduce the level of the Helios protein, reduce the activity level

Helios and/or inhibit the expression level of Helios in cells to control Treg differentiation. These compounds are useful for the treatment of several diseases, including cancer and viral infections. The compounds are proposed for use as pharmaceuticals with desirable stability, bioavailability, therapeutic index and toxicity values that are important for their medicinal ability.

The first aspect of the present invention provides at least one compound of formula (I)

or its salt, where R1 is -NH2 or -NH(CHs);

each R2 is independently F, Cl, -CN, C1.4 alkyl, -CH2F, -CHF2, -CF3, -OCH3, or cyclopropyl; each R4 is independently F, Cl, -CH3, -CH2F, -CHF2, -CF3, or -OCH3;

R6 is hydrogen, _Cb2alkyl , or _{Cb2fluoroalkyl} ; m is zero, 1, 2, or 3; and n is zero, 1, 2, or 3; provided that if R is hydrogen, then m is 1, 2, or 3.

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein R1 is -NH ₂ .

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein R ₁ is -NH(CH3).

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein each R2 is independently F, Cl, -CN, -CH3, -CH2CH3, -CH(CH3)2, -CH2CH2CH2CH3, -CH2F, -CHF2, _-CF3 , _-OCH3 , or cyclopropyl.

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein each R2 is independently F, Cl, -CN, -CH3, -CH2CH3, -CH(CH3)2, -CH2CH2CH2CH3, -CH2F, -CHF2, or _-CF3 .

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein each R2 is independently F, Cl, -CN, -CH3, -CH2CH3, -CH2F, -CHF2 or -CF3.

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In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein each R2 is independently F, Cl, -CN, -CH3, or -CF3.

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein each R ₂ is independently -CN, -CH ₃ or -CF ₃ .

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein each R ₂ is independently F, -CN, -CH ₃ or -CF ₃ .

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein each R ₂ is independently F, -CN or -CH ₃ .

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein n is zero, 1, or 2.

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein n is zero or 1.

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein n is 1 or 2.

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein n is zero.

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein n is 1.

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein n is 2.

In one embodiment, there is provided a compound of formula (I) or a salt thereof, wherein each R2 is independently -CN, -CH3, or -CF3; and n is zero, 1, or 2. Included in this embodiment are compounds in which n is 1 or 2.

In one embodiment, there is provided a compound of formula (I) or a salt thereof, wherein each R2 is independently -CN, -CH3, or -CF3; and n is zero or 1. Included in this embodiment are compounds in which n is 1.

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein each R ₂ is independently -CN, -CH ₃ or -CF ₃ , and n is 2.

In one embodiment, there is provided a compound of formula (I) or a salt thereof, wherein each R ₂ is independently -CN or -CH ₃ ; and n is 1 or 2. Included in this embodiment are compounds in which n is 1. Also included in this embodiment are compounds in which n is 2.

In one embodiment, there is provided a compound of formula (I) or a salt thereof, wherein each R2 is -CH3; and n is 1 or 2. Included in this embodiment are compounds in which n is 1. Also included in this embodiment are compounds in which n is 2.

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein each R ₄ is independently F, Cl, -CH3, -CH2F, -CHF2, or -CF ₃ .

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein each R ₄ is independently F, -CH ₃ , -CH ₂ F, -CHF ₂ or -CF ₃ .

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein each R ₄ is independently F, -CH ₃ , -CHF ₂ or -CF ₃ .

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein each R4 is independently F or -CH3.

In one embodiment, there is provided a compound of formula (I) or a salt thereof, wherein each R ₄ is F3. Included in this embodiment are compounds in which m is 1. Also included in this embodiment are compounds in which m is 2.

In one embodiment, there is provided a compound of formula (I) or a salt thereof, wherein each R4 is -CH3. Included in this embodiment are compounds in which m is 1. Also included in this embodiment are compounds in which m is 2.

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein m is zero, 1, or 2.

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein m is zero or 1.

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein m is 1 or 2.

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein m is 1, 2 or 3.

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein m is 1.

In one embodiment, there is provided a compound of formula (I) or a salt thereof, wherein R6 is hydrogen; and m is 1, 2, or 3.

In one embodiment, there is provided a compound of formula (I) or a salt thereof, wherein R ₆ is hydrogen; and m is 1 or 2.

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In one embodiment, there is provided a compound of formula (I) or a salt thereof, wherein R ₆ is hydrogen; and m is 1.

In one embodiment, there is provided a compound of formula (I) or a salt thereof, wherein R ₆ is hydrogen; and m is 2.

In one embodiment, there is provided a compound of formula (I) or a salt thereof, wherein R ₆ is hydrogen; and m is 3.

In one embodiment, there is provided a compound of formula (I) or a salt thereof, wherein R ₆ is hydrogen; m is 1; and R ₄ is F, Cl, -CH ₃ , -CH ₂ F, -CHF2, or -CF ₃ . Included in this embodiment are compounds in which R 4 is F, -CH ₃ , -CHF2, or -CF ₃ . Also included in this embodiment are compounds in which R ₄ is F, -CH ₃ , or -CF3. Furthermore, included in this embodiment are compounds in which R 4 is F or -CH3.

In one embodiment, there is provided a compound of formula (I) or a salt thereof, wherein R ₆ is hydrogen; m is 1; and R4 is F.

In one embodiment, there is provided a compound of formula (I) or a salt thereof, wherein R ₆ is hydrogen; m is 1; and R ₄ is -CH ₃ .

In one embodiment, there is provided a compound of formula (I) or a salt thereof, wherein R ₆ is hydrogen; m is 1 or 2; and R ₄ is F or -CH ₃ .

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein R ₆ is C _1-2 alkyl or C _1-2 fluoroalkyl.

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein R ₆ is C _1-2 alkyl, -CH2F, -CF2H, -CF3 or -CH2CF3.

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein R ₆ is C _1-2 alkyl, -CH2F, -CF2H or -CF3.

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein R ₆ is C _1-2 alkyl or -CF3.

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein R ₆ is C _1-2 alkyl.

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein R ₆ is -CH ₃ .

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein R ₆ is -CH2F, -CF2H, -CF3 or -CH2CF3.

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein R ₆ is -CH2F, -CF2H or -CF3.

In one embodiment, there is provided a compound of formula (I) or a salt thereof, wherein R ₆ is C _1-2 alkyl or C _1-2 fluoroalkyl; and m is zero.

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein R ₆ is C _1-2 alkyl, -CH2F, -CF2H, -CF3, or -CH2CF3; and m is zero.

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein R ₆ is C _1-2 alkyl, -CH2F, -CF2H, or -CF3; and m is zero.

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein R ₆ is C _1-2 alkyl or -CF3; and m is zero.

In one embodiment, there is provided a compound of formula (I) or a salt thereof, wherein R ₆ is C _1-2 alkyl; and m is zero.

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein R ₆ is -CH3; and m is zero.

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein R ₆ is -CH2F, -CF2H, -CF3, or -CH2CF3; and m is zero.

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein R ₆ is -CH ₂ F, -CF ₂ H, or -CF ₃ ; and m is zero.

In one embodiment, there is provided a compound of formula (I) or a salt thereof, wherein R1 is -NH2 or -NH( _CH3 ); each R2 is independently -CN, _-CH3 or -CF3; each _R4 is independently F or _-CH3 ; _R6 is hydrogen or _-CH3 ; m is zero or 1; and n is 1 or 2; provided that when _R6 is hydrogen, m is 1.

In one embodiment, there is provided a compound of formula (I) or a salt thereof, wherein Ri is -NH2; each R2 is independently -CN, -CH3, or -CF3; R4 is F or -CH3; R6 is hydrogen or -CH3; m is zero or 1; and n is 1 or 2; provided that when R is hydrogen, m is 1.

In one embodiment, there is provided a compound of formula (I) or a salt thereof, wherein R1 is -NH( _CH3 ); each R2 is independently -CN or _-CH3 ; R4 is F or _-CH3 ; _R6 is hydrogen or _-CH3 ; m is zero or 1; and n is 1 or 2; provided that when _R6 is hydrogen, m is 1.

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In one embodiment, there is provided a compound of formula (I) or a salt thereof, wherein R1 is -NH2; each R2 is independently -CN, -CH3, or -CF3; each R4 is independently F or -CH3; _R6 is -CH3; m is 1; and n is 1 or 2. Included in this embodiment are compounds in which each R2 is -CH3.

In one embodiment, there is provided a compound of formula (I) or a salt thereof, wherein R ₁ is -NH2; each R2 is independently -CN or -CH3; R4 is F or -CH3; R ₆ is hydrogen; m is 1; and n is 1 or 2.

In one embodiment, there is provided a compound of formula (I) or a salt thereof, wherein R ₁ is -NH2 or -NH(CH ₃ ); each R2 is independently -CH3; R4 is F; R6 is hydrogen or -CH3; m is zero or 1; and n is 1 or 2; provided that when R6 is hydrogen, m is 1.

In one structure:

from the embodiments there is provided a compound of formula (I) or a salt thereof having

In one structure:

from

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from

In one of the structures:

embodiments provide a compound of formula (I)

embodiments provide a compound of formula (I)

embodiments provide a compound of formula (I)

embodiments provide a compound of formula (I)

or or or his his his his salt, salt, salt, salt, having having having having

- 6 048753

In one structure:

from the embodiments there is provided a compound of formula (I) or a salt thereof having

In one structure:

from

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embodiments provide a compound of formula (I)

embodiments provide a compound of formula (I)

embodiments provide a compound of formula (I)

embodiments provide a compound of formula (I)

embodiments provide a compound of formula (I)

or or or or his his his his his salt, salt, salt, salt, salt, having having having having having

- 7 048753

In one embodiment, a compound of formula (I) or a salt thereof is provided having the structure:

In one embodiment, a compound of formula (I) or a salt thereof is provided having the structure:

In one embodiment, there is provided a compound of formula (I) or a salt thereof, wherein said compound is: 2-amino-6-(2-(2,6-dioxopiperidin-3-yl)-4-methyl-1oxoisoindolin-5-yl)-4-methylnicotinonitrile (1); 2-amino-6-(2-(2,6-dioxopiperidin-3-yl)-6-methyl-1oxoisoindolin-5-yl)-4-methylnicotinonitrile (2); 2-amino-6-(2-(2,6-dioxopiperidin-3-yl)-4-fluoro-1oxoisoindolin-5-yl)-4-methylnicotinonitrile (3); 3-(5-(6-amino-4-methylpyridin-2-yl)-4-fluoro-1oxoisoindolin-2-yl)piperidine-2,6-dione (4); 3-(5-(6-amino-4-(trifluoroethyl)pyridin-2-yl)-4-fluoro-1oxoisoindolin-2-yl)piperidine-2,6-dione (5); 3-(4-fluoro-5-(4-methyl-6-(methylamino)pyridin-2-yl)-1oxoisoindolin-2-yl)piperidine-2,6-dione (6); 3-(5-(6-amino-4,5-dimethylpyridin-2-yl)-4-fluoro-1oxoisoindolin-2-yl)piperidine-2,6-dione (7); 3-(^)-5-(6-amino-4,5-dimethylpyridin-2-yl)-3-methyl-1oxoisoindolin-2-yl)piperidine-2,6-dione (8); 3-(^)-5-(6-amino-4,5-dimethylpyridin-2-yl)-3-methyl-1oxoisoindolin-2-yl)piperidine-2,6-dione (9); 2-amino-6-((3S)-2-(2,6-dioxopiperidin-3-yl)-3-methyl-1oxoisoindolin-5-yl)-4-methylnicotinonitrile (10); 2-amino-6-((3R)-2-(2,6-dioxopiperidin-3-yl)-3methyl-1-oxoisoindolin-5-yl)-4-methylnicotinonitrile (11); 3-(^)-5-(4,5-dimethyl-6-(methylamino)pyridin-2-yl)-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione (12); 3-(^)-5-(4,5-dimethyl-6-(methylamino)pyridin-2-yl)-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione (13); 6-((3S)-2-(2,6-dioxopiperidin-3-yl)-3-methyl-1-oxoisoindolin-5-yl)-4-methyl-2-(methylamino)nicotinonitrile (14);

6-((3R)-2-(2,6-dioxopiperidin-3-yl)-3-methyl-1-oxoisoindolin-5-yl)-4-methyl-2-(methylamino)nicotinonitrile (15); 3-(^)-5-(6-amino-4,5-dimethylpyridin-2-yl)-4-fluoro-3-methyl-1-oxoisoindolin-2yl)piperidine-2,6-dione (16);

3-(^)-5-(6-amino-4,5-dimethylpyridin-2-yl)-4-fluoro-3-methyl-1oxoisoindolin-2-yl)piperidin-2,6-dione oxoisoindolin-2-yl )piperidin-2,6-dione oxoisoindolin-2-yl)piperidin-2,6-dione oxoisoindolin-2-yl)piperidin-2,6-dione (17); 3-(5-(6-amino-3-methylpyridin-2-yl)-4-fluoro-1 (18); 3-(5-(6-amino-5-fluoro-4-methylpyridin-2-yl) -4-fluoro-1 (19); 3 -(5-(6-amino-3 -fluoro-4-methylpyridin-2-yl)-4-fluoro-1 (20); 3-(5-(6- amino-5-fluoro-4-methylpyridin-2-yl)-4-fluoro-3methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione (21-22); 2-amino-6-(( (3R)-2-(2,6-dioxopiperidin-3yl)-4-fluoro-3-methyl-1-oxoisoindolin-5-yl)-4-methylnicotinonitrile (23); 3-(^)-5-(6-amino-4-methylpyridin-2-yl)-4-fluoro-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione (24); 3-( (R)-5-(6-amino-4-(trifluoromethyl)pyridin-2-yl)-4-fluoro-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione (25); 3- (^)-5-(6-amino-3-methylpyridin-2-yl)-4-fluoro-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione (26); 3-(^)-5-(6-aminopyridin-2-yl)-4-fluoro-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione (27); ^)-3- (^)-5-(6-amino-4,5-dimethylpyridin-2-yl)-4-fluoro-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6dione 28); 3-(^ )-5-(6-amino-5-fluoro-4-methylpyridin-2-yl)-4-fluoro-3-methyl-1-oxoisoindolin-2yl)piperidine-2,6-dione (29); or 3-(^)-5-(6-amino-5-fluoro-4-methylpyridin-2-yl)-4-fluoro-3-methyl-1oxoisoindolin-2-yl)piperidine-2,6-dione (30 ).

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein said compound is 3-(^)-5-(6-amino-5-fluoro-4-methylpyridin-2-yl)-3-methyl-1oxoisoindolin-2-yl)piperidine-2,6-dione.

In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein said compound is 3-(^)-5-(6-amino-5-fluoro-4-methylpyridin-2-yl)-3-methyl-1oxoisoindolin-2-yl)piperidine-2,6-dione.

The present invention may be embodied in other specific forms without departing from its spirit and essential features. The present invention includes all combinations of aspects and/or embodiments specified herein. It should be understood that any and all embodiments of the present invention may be taken in combination with any other embodiment.

- 8 048 753 embodiments or embodiment variants to describe additional embodiments. It should also be understood that each individual element of the embodiments is intended to be combined with any other elements of any embodiment to describe an additional embodiment.

The features and advantages of the invention may be more clearly understood by those skilled in the art after reading the following detailed description. It should be understood that some features of the invention, which for clarity are described above and below in the context of separate embodiments, may also be combined in a single embodiment. Conversely, various features of the invention, which for brevity are described in the context of a single embodiment, may also be combined so as to form subcombinations thereof. The embodiments referred to herein as exemplary or preferred are illustrative and not limiting.

Unless otherwise stated herein, references made in the singular may also include the plural. For example, a and an may refer to one or one or more.

As used herein, the phrase compounds and/or salts thereof refers to at least one compound, at least one salt of the compound, or a combination thereof. For example, compounds of formula (I) and/or salts thereof include a compound of formula (I); two compounds of formula (I); a salt of a compound of formula (I); a compound of formula (I) and one or more salts of a compound of formula (I); and two or more salts of a compound of formula (I).

Unless otherwise stated, any atom with unsatisfied valences is assumed to have sufficient hydrogen atoms to satisfy the valences.

The definitions provided herein take precedence over the definitions provided in any patent, patent application and/or patent application publication incorporated herein by reference.

Below are definitions of various terms used in describing the present invention. These definitions apply to terms used throughout the specification (unless otherwise limited in specific instances), either individually or as part of a larger group.

Throughout the description, groups and their substituents can be selected by one skilled in the art to obtain stable fragments and compounds.

According to the convention used in this field of technology,

used here in structural formulas to denote the bond that is the point of attachment of a molecule or substituent to the core or main chain structure.

The terms halo and halogen as used herein refer to F, Cl, Br, and I.

The term cyano refers to the -CN group.

The term amino refers to the -NH2 group.

The term oxo refers to the =O group.

The term "alkyl" as used herein refers to both branched and straight chain saturated aliphatic hydrocarbon groups having, for example, 1 to 12 carbon atoms, 1 to 6 carbon atoms, and 1 to 4 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (e.g., n-propyl and i-propyl), butyl (e.g., n-butyl, i-butyl, sec-butyl, and t-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), n-hexyl, 2-methylpentyl, 2-ethylbutyl, 3-methylpentyl, and 4-methylpentyl. When numbers appear in a subscript following the symbol C, the subscript more precisely identifies the number of carbon atoms that a particular group may contain. For example, C1-4 alkyl refers to straight and branched chain alkyl groups containing one to four carbon atoms.

The term fluoroalkyl, as used herein, includes both branched and straight chain saturated aliphatic hydrocarbon groups substituted with one or more fluorine atoms. For example, C1-4 fluoroalkyl includes C1, _C2 , _C3 , and _C4 alkyl groups substituted with one or more fluorine atoms. Representative examples of fluoroalkyl groups include, but are not limited to, -CF3 and -CH2CF3.

The compounds of the present invention include all isotopes of the atoms occurring in the present compounds. Isotopes include atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include deuterium (D) and tritium (T). Isotopes of carbon include ¹³ C and ¹⁴ C. Isotopically labeled compounds of the present invention can be prepared by conventional methods known to those skilled in the art or by methods similar to those described herein, using a suitable isotopically labeled reagent in place of the unlabeled reagent otherwise used.

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The phrase pharmaceutically acceptable is used herein to mean those compounds, materials, compositions and/or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reaction or other problems or complications commensurate with a reasonable benefit/risk ratio.

The compounds of formula (I) may form salts which are also within the scope of the present invention. Unless otherwise indicated, reference to a compound of the invention means reference to one or more salts thereof. The term salt(s) means acidic and/or basic salt(s) formed with inorganic and/or organic acids and bases. Furthermore, the term salt(s) may include zwitterions (inner salts), for example when a compound of formula (I) contains both a basic group such as an amine or pyridine or imidazole ring and an acidic group, for example a carboxylic acid. Pharmaceutically acceptable (i.e. non-toxic, physiologically acceptable) salts are preferred, such as, for example, acceptable metal and amine salts in which the cation does not contribute significantly to the toxicity or biological activity of the salt. However, other salts may also be useful, for example in isolation or purification steps which may be used in the preparation, and are thus within the scope of the invention. Salts of the compounds of formula (I) can be prepared, for example, by reacting a compound of formula (I) with an equivalent amount of an acid or base in a medium such as a medium in which the salt precipitates or in an aqueous medium, followed by lyophilization.

Examples of acid addition salts include acetates (e.g., those formed with acetic acid or trihaloacetic acid, such as trifluoroacetic acid), adipates, alginates, ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, digluconates, dodecyl sulfates, ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides (formed with hydrochloric acid), hydrobromides (formed with hydrogen bromide), hydroiodides, maleates (formed with maleic acid), 2-hydroxyethanesulfonates, lactates, methanesulfonates (formed with methanesulfonic acid), 2-naphthalenesulfonates, nicotinates, nitrates, oxalates, pectinates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, salicylates, succinates, sulfates (e.g. formed with sulfuric acid), sulfonates (e.g. mentioned herein), tartrates, thiocyanates, toluenesulfonates such as tosylates, undecanoates, etc.

Examples of basic salts include ammonium salts, alkali metal salts such as sodium, lithium and potassium salts; alkaline earth metal salts such as calcium and magnesium salts; barium, zinc and aluminum salts; salts of organic bases (e.g. organic amines) such as trialkylamines, for example, triethylamine, procaine, dibenzylamine, N-benzyl-b-phenethylamine, 1-ephenamine, N,N'dibenzylethylenediamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, dicyclohexylamine or similar pharmaceutically acceptable amine salts with amino acids such as arginine, lysine and the like. The basic nitrogen-containing groups can be quaternized by such means as lower alkyl halides (e.g. methyl, ethyl, propyl and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g. dimethyl, diethyl, dibutyl and diamyl sulfates), long-chain halides (e.g. decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides), aralkyl halides (e.g. benzyl and phenethyl bromides) and others.

The compounds of formula (I) may be provided as amorphous or crystalline solids. Lyophilization may be used to obtain solid compounds of formula (I).

It should also be understood that solvates (e.g., hydrates) of the compounds of formula (I) are also within the scope of the present invention. The term solvate means a physical association of a compound of formula (I) with one or more solvent molecules, both organic and inorganic. Such physical association includes hydrogen bonding. In some cases, the solvate is isolable, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. The term solvate encompasses both solution-phase solvates and solvates that are isolable. Typical solvates include hydrates, ethanolates, methanolates, isopropanolates, acetonitrile solvates, and ethyl acetate solvates. Methods of solvation are known in the art.

Various forms of prodrugs are well known in the art and are described in Rautio, J. et al., Nature Review Drug Discovery, 17, 559-587 (2018).

In addition, the compounds of formula (I) after their preparation can be isolated and purified to obtain a composition containing an amount by weight equal to or greater than 99% of the compound of formula (I) (substantially pure), which is then used or formulated as described herein. Such substantially pure compounds of formula (I) are also considered herein as part of the present invention.

By stable compound and stable structure is meant a compound that is sufficiently robust to withstand isolation from a reaction mixture with a useful degree of purity and transformation into an effective therapeutic agent. The present invention is intended to realize stable compounds.

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The term Helios inhibitor refers to an agent that is capable of decreasing the level of Helios protein, decreasing the level of Helios activity, and/or inhibiting the level of Helios expression in cells to control Treg differentiation. A Helios inhibitor may be a reversible or irreversible inhibitor.

As used herein, Helios protein refers to a protein that belongs to the Ikaros family of zinc finger proteins. In humans, Helios is encoded by the IKZF2 gene. Helios is also known as IKAROS family zinc finger protein 2, ANF1A2, ZNF1A2, ZNFN1A2, zinc finger protein subfamily 1A, 2, and Ikaros family zinc finger protein 2. Members of this protein family include Ikaros, Helios, Aiolos, Eos, and Pegasus. As used herein, Helios protein includes various isoforms, which include isoforms 1-5, listed below.

Isoform 1 (UniProt Q9UKS7-1)

METEAIDGYITCDNELSPEREHSNMAIDLTSSTPNGQHASPSHMTSTNSVKLEMQSDEEC DRKPLSREDEIRGHDEGSSLEEPLIESSEVADNRKVQELQGEGGIRLPNGKLKCDVCGMV СIGPNVLMVHKRSΗТGERPFHCNQCGA.SFTQKGNLLRHIKLHSGEKPFKCPFCS YACRRR DALTGHLRTHSVGKPHKCNYCGRSYKQRSSLEEHKERCHNYLQNVSMEAAGQVMSHHVPP MEDCKEQEPIMDNNISLVPFERPAVIEKLTGNMGKRKSSTPQKFVGEKLMRFSYPDIHFD MNLTYEKEAELMQSHMMDQAINNAITYLGAEALHPLMQHPPSTIAEVAPVISSAYSQVYH PNRIERPISRETADSHENNMDGPISLIRPKSRPQEREASPSNSCLDSTDSESSHDDHQSY QGHPALNPKRKQSPAYMKEDVKALDTTKAPKGSLKDIYKVFNGEGEQIRAFKCEHCRVLF LDHVMYTIHMGCHGYRDPLECNICGYRSQDRYEFSSHIVRGEHTFH (SEQ ID NO: 1)

Isoform 2 (UniProt Q9UKS7-2)

METEAIDGYITCDNELSPEREHSNMAIDLTSSTPNGQHASPSHMTSTNSVKLEMQSDEEC DRKPLSREDEIRGHDEGSSLEEPLIESSEVADNRKVQELQGEGGIRLPNGERPFHCNQCG

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ASFTQKGNLLRHIKLHSGEKPFKCPFCSYACRRRDALTGHLRTHSVGKPHKCNYCGRSYK QRSSLEEHKERCHNYLQNVSMEAAGQVMSHHVPPMEDCKEQEPIMDNNISLVPFERPAVI EKLTGNMGKRKSSTPQKFVGEKLMRFSYPDIHFDMNLTYEKEAELMQSHMMDQAINNAIT YLGAEALHPLMQHPPSTIAEVAPVISSAYSQVYHPNRIERPISRETADSHENNMDGPISL IRPKSRPQEREASPSNSCLDSTDSESSHDDHQSYQGHPALNPKRKQSPAYMKEDVKALDT TKAPKGSLKDIYKVFNGEGEQIRAFKCEHCRVLFLDHVMYTIHMGCHGYRDPLECNICGY RSQDRYEFSSHIVRGEHTFH (SEQ ID NO: 2)

Isoform 4 (UniProt Q9UKS7-4)

METEAIDGYITCDNELSPEREHSNMAIDLTSSTPNGQHASPSHMTSTNSVKLEMQSDEEC DRKPLSREDEIRGHDEGSSLEEPLIESSEVADNRKVQELQGEGGIRLPNGERPFHCNQCG ASFTQKGNLLRHIKLHSGEKPFKCPFCSYACRRRDALTGHLRTHSVGKPHKCNYCGRSYK QRSSLEEHKERCHNYLQNVSMEAAGQVMSHHGEKLMRFSYPDIHFDMNLTYEKEAELMQS HMMDQAINNAITYLGAEALHPLMQHPPS TIAEVAPVIS SAYSQVYHPNRIERPISRETAD SHENNMDGPISLIRPKSRPQEREASPSNSCLDSTDSESSHDDHQSYQGHPALNPKRKQSP AYMKEDVKALDTTKAPKGSLKDIYKVFNGEGEQIRAFKCEHCRVLFLDHVMYTIHMGCHG YRDPLECNICGYRSQDRYEFSSHIVRGEHTFH (SEQ ID NO: 3)

Isoform 6 (UniProt Q9UKS7-6)

METEAIDGYITCDNELSPEREHSNMAIDLTSSTPNGQHASPSHMTSTNSVKLEMQ SDEECDRKPLSREDEIRGHDEGSSLEEPLIESSEVADNRKVQELQGEGGIRLPNGKLKCD VC GMVCIGPNVLMVHKRS ΗT GERPFHCNQCGASFTQKGNLLRHIKLHSGEKPFKCPFCSY ACRRDALTGHLRTHSVGKP

Isoform 7 (UniProt Q9UKS7-7)

METEAIDGYITCDNELSPEREHSNMAIDLTSSTPNGQHASPSHMTSTNSVKLEMQSDEEC DRKPLSREDEIRGHDEGSSLEEPLIESSEVADNRKVQELQGEGGIRLPNGERPFHCNQCG ASFTQKGNLLRHIKLHSGEKPFKCPFCSYACRRRDALTGHLRTHSVPPMEDCKEQEPIMD NNISLVPFERPAVIEKLTGNMGKRKSSTPQKFVGEKLMRFSYPDIHFDMNLTYEKEAELM QSHMMDQAINNAITYLGAEALHPLMQHPPSTIAEVAPVISSAYSQVYHPNRIERPISRET ADSHENNMDGPISLIRPKSRPQEREASPSNSCLDSTDSESSHDDHQSYQGHPALNPKRKQ SPAYMKEDVKALDTTKAPKGSLKDIYKVFNGEGEQIRAFKCEHCRVLFLDHVMYTIHMGC HGYRDPLECNICGYRSQDRYEFSSHIVRGEHTFH (SEQ ID NO: 5)

The Helios 1, 2, 4, 6, and 7 isoforms listed above contain the degron FHCNQCGASFTQKGNLLRHIKLH (SEQ ID NO: 6) (bold and underlined). The degron is a portion of the protein that plays a role in regulating the rate of protein degradation.

In this document, the Eos protein is encoded by the IKZF4 gene and is also known as IKAROS family zinc finger 4, ZNFN1A4, zinc finger protein subfamily 1A, 4, Ikaros family zinc finger protein 4, and KIAA1782. The Eos protein includes isoforms encoded by the following two human isoforms 1 (Q9H2S9-1) and 2 (Q9H2S9-2).

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Isoform 1 (UniProt Q9H2S9-1)

MHTPPALPRRFQGGGRVRTPGSHRQGKDNLERDPSGGCVPDFLPQAQDSNHFIMESLFCE SSGDSSLEKEFLGAPVGPSVSTPNSQHSSPSRSLSANSIKVEMYSDEESSRLLGPDERLL EKDDSVIVEDSLSEPPLGYCDGSGPEPHSPGIRLPNGKLKCDVCGMVCIGPNVLMVHKRS HTGERPFHCNQCGASFTQKGNLLRHIKLHSGEKPFKCPFCNYACRRRDALTGHLRTHSVS SPTVGKPYKCNYCGRSYKQQSTLEEHKERCHNYLQSLSTEAQALAGQPGDEIRDLEMVPD SMLHSSSERPTFIDRLANSLTKRKRSTPQKFVGEKQMRFSLSSDLPYDVNSGGYEKDVELV AHHSLEPGFGSSLAFVGAEHLRPLRLPPTNCISELTPVISSVYTQMQPLPGRLELPGSRE AGEGPEDLADGGPLLYRPRGPLTDPGASPSNGCQDSTDTESNHEDRVAGWSLPQGPPPQ PPPTIWGRHSPAYAKEDPKPQEGLLRGTPGPSKEVLRWGESGEPVKAFKCEHCRILFL DHVMFTIHMGCHGFRDPFECNICGYHSQDRYEFSSHIVRGEHKVG (SEQ ID NO: 7)

Isoform 2 (UniProt Q9H2S9-2)

MDSRYLQLQLYLPSCSLLQGSGDSSLEKEFLGAPVGPSVSTPNSQHSSPSRSLSANSIKV EMYSDEESSRLLGPDERLLEKDDSVIVEDSLSEPLGYCDGSGPEPHSPGIRLPNGKLKC DVCGMVCIGPNVLMVHKRSHTTGERPFHCNQCGASFTQKGNLLRHIKLHSGEKPFKCPFCN YACRRRDALTGHLRTHSVSSPTVGKPYKCNYCGRSYKQQSTLEEHKERCHNYLQSLSTEA QALAGQPGDEIRDLEMVPDSMLHSSSERPTFIDRLANSLTKRKRSTPQKFVGEKQMRFSL SDLPYDVNSGGYEKDVELVAHHSLEPGFGSSLAFVGAEHLRPLRLPPTNCISELTPVISS VYTQMQPLPGRLELPGSREAGEGPEDLADGGPLLYRPRGPLTDPGASPSNGCQDSTDTES NHEDRVAGWSLPQGPPPQPPPTIWGRHSPAYAKEDPKPQEGLLRGTPGPSKEVLRWG ESGEPVKAFKCEHCRILFLDHVMFTIHMGCHGFRDPFECNICGYHSQDRYEFSSHIVRGE HKVG (SEQ ID NO: 8)

The above Eos 1 and 2 protein isoforms include the degron FHCNQCGASFTQKGNLLRHIKLH (SEQ ID NO: 6) (bold and underlined), which is identical to the degron for the Helios protein.

As used herein, the Ikaros protein is encoded by the IKZF1 gene. Ikaros is also known as IKAROS family zinc finger 1, ZNFN1A1, zinc finger protein subfamily 1A, 1, Ikaros family zinc finger protein 1, IK1, lymphoid transcription factor LyF-1, Hs.54452, PPP1R92, protein phosphatase 1 regulatory subunit 92, PRO0758, CVID13, and CLL-associated antigen KW-6. The Ikaros protein includes isoforms encoded by the amino acid sequences Q13422-1, Q13422-2, Q13422-3, Q13422-4, Q13422-7, and Q13422-8. The Ikaros protein also includes isoforms encoded by the amino acid sequences Q13422-5 and Q13422-6.

As used herein, the Aiolos protein is encoded by the IKZF3 gene. The Aiolos protein is also known as IKAROS family zinc finger protein 3, ZNFN1 A3, zinc finger protein subfamily 1 A, 3, Ikaros family zinc finger protein 3, and AIO. The Aiolos protein includes isoforms encoded by the amino acid sequences Q9UKT9-1, Q9UKT9-3, Q9UKT9-4, Q9UKT96, Q9UKT9-7, Q9UKT9-8, Q9UKT9-9, and Q9UKT9-14. The Aiolos protein also includes isoforms encoded by the amino acid sequences Q9UKT9-2, Q9UKT9-5, Q9UKT9-10, Q9UKT9-11, Q9UKT9-12 and Q9UKT9-13, Q9UKT9-15 and Q9UKT9-16.

As used herein, Pegasus protein is also known as IKAROS family zinc finger protein 5, ZNFN1A5, zinc finger protein subfamily 1A, 5, and Ikaros family zinc finger protein 5. Pegasus is encoded by the IKZF5 gene.

As used herein, the term contacting refers to combining the compounds in an in vitro or in vivo system. For example, contacting a Helios protein with a compound of formula (I) includes administering a compound of the present invention to an individual or patient, such as a human, having a Helios protein, as well as, for example, administering a compound of formula (I) to a sample containing a cellular or purified preparation containing a Helios protein.

The terms treat, cure, and therapy as used herein refer to any type of intervention or process performed on a subject or the administration of an active agent to a subject for the purpose of reversing, alleviating, improving, suppressing, slowing, or preventing the progression, development, severity, or recurrence of a symptom, complication, condition, or biochemical feature associated with a disease. In contrast, prophylaxis or prevention means administration to a subject not suffering from a disease to prevent the occurrence of the disease. The terms treat, cure, and therapy do not include prophylaxis or prevention.

By therapeutically effective amount is meant the amount of a compound of the present invention alone or the amount of the claimed combination of compounds or the amount of a compound of the present invention in combination with other active ingredients, effective

- 13 048753 for reducing Helios protein levels, reducing Helios activity levels and/or inhibiting Helios expression levels in cells or effective for the treatment or prevention of viral infections and proliferative diseases such as cancer.

As used herein, the term cell refers to cells in vitro, ex vivo, or in vivo. In some embodiments, an ex vivo cell may be part of a tissue sample taken from an organism, such as a mammal. In some embodiments, an in vitro cell may be a cell in a cell culture. In some embodiments, an in vivo cell is a cell living in an organism, such as a mammal.

The term patient includes humans and other mammals receiving either therapeutic or prophylactic treatment.

The term subject includes any human or non-human animal. For example, the methods and compositions disclosed herein can be used to treat a subject suffering from cancer. A non-human animal includes all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc. In one embodiment, the subject is a human.

The phrase pharmaceutically acceptable carrier, as used herein, means a pharmaceutically acceptable material, composition, or vehicle such as a liquid or solid filler, diluent, excipient, processing aid (e.g., lubricant, talc, magnesium, calcium, or zinc stearate, or stearic acid), or encapsulating solvent that participates in the transport or delivery of the subject compound from one organ or body part to another organ or body part. Each carrier must be acceptable in the sense of being compatible with the other ingredients of the formulation, including the adjuvant, excipient, or vehicle such as diluents, preservatives, fillers, flow control agents, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, perfumes, antibacterial agents, antifungal agents, lubricating agents, and dispensing agents, depending on the nature of the route of administration and the dosage forms; and not cause harm to the patient.

The term pharmaceutical composition means a composition comprising a compound of the invention in combination with at least one additional pharmaceutically acceptable carrier.

Utility

Compounds of formula (I) are useful for the treatment of cancer.

In one embodiment, the present invention provides a combination preparation of a compound of formula (I) and/or a pharmaceutically acceptable salt thereof, a stereoisomer thereof or a tautomer thereof and an additional therapeutic agent(s) for simultaneous, separate or sequential use in the treatment and/or prevention of multiple diseases or disorders associated with Helios protein activity. The combination preparation can be used to reduce the level of Helios protein, the level of Helios activity and/or the level of Helios expression in cells to control Treg differentiation.

The compounds of formula (I) and pharmaceutical compositions comprising at least one compound of formula (I) are useful for the treatment or prevention of any diseases or conditions that are associated with the activity of the Helios protein. These include viral and other infections (e.g. skin infections, gastrointestinal infections, urinary tract infections, genitourinary infections, systemic infections) and proliferative diseases (e.g. cancer). The compounds of formula (I) and pharmaceutical compositions comprising at least one compound of formula (I) can be administered to animals, preferably mammals (e.g. pets, cats, dogs, mice, rats), and more preferably humans. Any route of administration can be used to deliver the compound or pharmaceutical composition to the patient. In some embodiments, the compound of formula (I) or a pharmaceutical composition comprising at least a compound of formula (I) is administered orally. In other embodiments, the compound of formula (I) or a pharmaceutical composition comprising at least a compound of formula (I) is administered parenterally.

Compounds of formula (I) can selectively reduce Helios protein levels, reduce Helios activity levels, and/or inhibit Helios expression levels in cells to control Treg differentiation. For example, compounds of formula (I) can be used to selectively reduce Helios activity levels and/or inhibit Helios expression levels in cells to control Treg differentiation in a cell or in an individual in need of reducing Helios protein levels, reducing Helios activity levels, and/or inhibiting Helios expression levels by administering an inhibitory amount of a compound of formula (I) or a salt thereof.

In one aspect, the compound(s) of formula (I) are sequentially administered prior to administration of the immuno-oncology drug. In another aspect, the compound(s) of formula (I) are administered simultaneously with the immuno-oncology drug. In another aspect, the compound(s) of formula (I) are sequentially administered following administration of the immuno-oncology drug.

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In another aspect, the compounds of formula (I) may be formulated together with an immuno-oncology drug.

Immuno-oncology preparations include, for example, a small molecule drug, an antibody, or another biological or small molecule. Examples of biological immuno-oncology preparations include, but are not limited to, cancer vaccines, antibodies, and cytokines. In one aspect, the antibody is a monoclonal antibody. In another aspect, the monoclonal antibody is humanized or human.

In one aspect, the immuno-oncology agent is (i) an agonist of a stimulatory (including costimulatory) receptor or (ii) an antagonist of an inhibitory (including coinhibitory) signal on T cells, both of which result in enhanced antigen-specific T cell responses (often referred to as immune checkpoint regulators).

Some of the stimulatory and inhibitory molecules belong to the immunoglobulin superfamily (IgSF). One important family of membrane-bound ligands that bind to costimulatory or coinhibitory receptors is the B7 family, which includes B7-1, B7-2, B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6. Another family of membrane-bound ligands that bind to costimulatory or coinhibitory receptors is the TNF family of molecules that bind to cognitive members of the TNF receptor family, which includes CD40 and CD40L, OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 41BBL, CD137 (4-1BB), TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR/Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTeR, LIGHT, DcR3, HVEM, VEGI/TL1A, TRAMP/DR3, EDAR, EDA1, XEDAR, EDA2, TNFR1, lymphotoxin α/TNFe, TNFR2, TNFa, LTeR, lymphotoxin α 1β2, FAS, FASL, RELT, DR6, TROY, NGFR.

In one aspect, T cell responses can be stimulated by a combination of a compound of formula (I) and one or more of (i) antagonists of a protein that inhibits T cell activation (e.g., immune checkpoint inhibitors), such as CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, Galectin 9, CEACAM-1, BTLA, CD69, Galectin-1, TIGIT, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, and TIM-4, and (ii) an agonist of a protein that stimulates T cell activation, such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3 and CD28H.

Other agents that can be combined with the compounds of formula (I) for the treatment of cancer include antagonists of inhibitory receptors on NK cells or agonists of activating receptors on NK cells. For example, the compounds of formula (I) can be combined with KIR antagonists such as lirilumab.

Other combination therapy agents include agents that inhibit or deplete macrophages or monocytes, including, but not limited to, CSF-1R antagonists such as CSF-1R antagonist antibodies including RG7155 (WO11/70024, WO11/107553, WO11/131407, WO13/87699, WO13/119716, WO13/132044) or FPA-008 (WO11/140249; WO13169264; WO14/036357).

In another aspect, the compounds of formula (I) can be used with one or more agonist agents that ligate positive costimulatory receptors, blocking agents that attenuate signaling through inhibitory receptors, antagonists and one or more agents that systemically increase the frequency of anti-tumor T cells, agents that overcome various immunosuppressive pathways in the tumor microenvironment (e.g., block the interaction of inhibitory receptors (e.g., PD-L1/PD-1), deplete or suppress Tregs (e.g., with a monoclonal antibody against CD25 (e.g., daclizumab) or by ex vivo depletion with anti-CD25 beads), inhibit metabolic enzymes such as IDO or reverse/prevent T cell anergy or exhaustion), and agents that induce activation of innate immunity and/or inflammation at tumor sites.

In one aspect, the immuno-oncology drug is a CTLA-4 antagonist, such as an antagonist antibody to CTLA-4. Suitable antibodies to CTLA-4 include, for example, YERVOY (ipilimumab) or tremelimumab.

In another aspect, the immuno-oncology product is a PD-1 antagonist, such as an antagonist PD-1 antibody. Suitable PD-1 antibodies include, for example, OPDIVO™ (nivolumab), KEYTRUDA™ (pembrolizumab), or MEDI-0680 (AMP-514; WO2012/145493). The immuno-oncology product may also include pidilizumab (CT-011), although its specificity for binding PD-1 has been questioned. Another approach to targeting the PD-1 receptor is to use a recombinant protein consisting of the extracellular domain of PD-L2 (B7DC) fused to the Fc portion of IgG1, called AMP-224.

In another aspect, the immuno-oncology drug is a PD-L1 antagonist, such as an antagonist antibody to PD-L1. Suitable antibodies to PD-L1 include, for example, MPDL3280A (RG7446; WO2010/077634), durvalumab (MEDI4736), BMS-936559 (WO207/005874), and MSB0010718C (WO2013/79174).

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In another aspect, the immuno-oncology drug is a LAG-3 antagonist, such as an anti-LAG-3 antagonist antibody. Suitable anti-LAG3 antibodies include, for example, BMS986016 (WO10/19570, WO14/08218) or IMP-731 or IMP-321 (WO08/132601, WO09/44273).

In another aspect, the immuno-oncology drug is a CD137 (4-1BB) agonist, such as an anti-CD137 agonist antibody. Suitable anti-CD137 antibodies include, for example, urelumab and PF-05082566 (WO12/32433).

In another aspect, the immuno-oncology drug is a GITR agonist, such as an agonist antibody to GITR. Suitable antibodies to GITR include, for example, BMS-986153, BMS-986156, TRX-518 (WO06/105021, WO09/009116), and MK-4166 (WO11/028683).

In another aspect, the immuno-oncology drug is an IDO antagonist. Suitable IDO antagonists are, for example, INCB-024360 (WO206/122150, WO07/75598, WO08/36653, WO08/36642), indoximod or NLG-919 (WO09/73620, WO09/1156652, WO11/56652, WO12/142237).

In another aspect, the immuno-oncology drug is an OX40 agonist, such as an OX40 agonist antibody. Suitable OX40 antibodies include, for example, MEDI-6383 or MEDI-6469.

In another aspect, the immuno-oncology drug is an OX40L antagonist, such as an OX40 antagonist antibody. Suitable OX40L antagonists include, for example, RG7888 (WO06/029879).

In another aspect, the immuno-oncology drug is a CD40 agonist, such as an anti-CD40 agonist antibody. In another embodiment, the immuno-oncology drug is a CD40 antagonist, such as an anti-CD40 antagonist antibody. Suitable anti-CD40 antibodies include, for example, lucatumumab or dacetuzumab.

In another aspect, the immuno-oncology drug is a CD27 agonist, such as an anti-CD27 agonist antibody. Suitable anti-CD27 antibodies include, for example, varlilumab.

In another aspect, the immuno-oncology drug is MGA271 (to B7H3) (WO11/109400).

Combination therapy includes both sequential administration of the therapeutic agents, i.e., administration of each therapeutic agent at different times, and substantially simultaneous administration of the therapeutic agents or at least two of them. Substantially simultaneous administration can be accomplished, for example, by administering to the subject a single dosage form with a fixed ratio of each therapeutic agent or several single dosage forms for each of the therapeutic agents. Sequential or substantially simultaneous administration of each therapeutic agent can be accomplished by any suitable route, including, but not limited to, oral, intravenous, intramuscular, and direct absorption through mucosal tissues. The therapeutic agents can be administered by the same route or by different routes. For example, the first therapeutic agent of a selected combination can be administered by intravenous injection, and the other therapeutic agents of the combination can be administered orally. Alternatively, for example, all of the therapeutic agents can be administered orally or all of the therapeutic agents can be administered by intravenous injection. Combination therapy may also include the administration of the above-described therapeutic agents in additional combination with other biologically active ingredients and non-drug treatments (e.g., surgery or radiation therapy). If the combination therapy includes non-drug treatment, the non-drug treatment may be administered at any appropriate time as long as the beneficial effect of the combined action of the therapeutic agents and the non-drug treatment is achieved. For example, in appropriate cases, a beneficial effect may also be achieved by temporarily discontinuing the non-drug treatment, perhaps for several days or even weeks, while continuing to administer the therapeutic agents.

Types of cancer that can be treated with the compound of formula (I) include, but are not limited to, brain cancer, skin cancer, bladder cancer, ovarian cancer, breast cancer, stomach cancer, pancreatic cancer, prostate cancer, colon cancer, blood cancer, lung cancer, and bone cancer. Examples of such types of cancer include neuroblastoma, intestinal carcinoma such as rectal carcinoma, colon carcinoma, familial adenomatous polyposis carcinoma and hereditary nonpolyposis colorectal cancer, esophageal carcinoma, lip carcinoma, laryngeal carcinoma, hypopharynx carcinoma, tongue carcinoma, salivary gland carcinoma, gastric carcinoma, adenocarcinoma, thyroid carcinoma, papillary thyroid carcinoma, renal carcinoma, renal parenchymal carcinoma, ovarian carcinoma, cervical carcinoma, uterine carcinoma, endometrial carcinoma, chorionic carcinoma, pancreatic carcinoma, prostate carcinoma, testicular carcinoma, breast carcinoma, urinary tract carcinoma, melanoma, brain tumors such as glioblastoma, astrocytoma, meningioma, medulloblastoma and peripheral neuroectodermal tumors, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), T-cell

- 16 048753 adult leukemia lymphoma, diffuse large B-cell lymphoma (DLBCL), hepatocellular carcinoma, gallbladder carcinoma, bronchial carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, multiple myeloma, basal cell carcinoma, teratoma, retinoblastoma, choroidal melanoma, seminoma, rhabdomyosarcoma, craniopharyngioma, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma, Ewing's sarcoma and plasmacytoma.

One or more additional pharmaceutical agents or treatment methods, such as, for example, antiviral agents, chemotherapeutic or other anticancer agents, immunoenhancers, immunosuppressants, radiation, antitumor and antiviral vaccines, cytokine therapy (e.g., IL2 and GM-CSF) and/or tyrosine kinase inhibitors, may optionally be used in combination with the compounds of formula (I) to treat diseases, disorders or conditions associated with the Helios protein. These agents may either be combined with the present compounds in a single dosage form, or the agents may be administered simultaneously or sequentially as separate dosage forms.

Suitable chemotherapeutic or other anticancer agents include, for example, alkylating agents (including, but not limited to, nitrogen mustards, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas, and triazenes) such as uracil mustard, chlormethine, cyclophosphamide (CYTOXAN™), ifosfamide, melphalan, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, carmustine, lomustine, streptozocin, dacarbazine, and temozolomide.

In the treatment of melanoma, suitable agents for use in combination with the compounds of formula (I) are: dacarbazine (DTIC), optionally together with other chemotherapeutic agents such as carmustine (BCNU) and cisplatin; the Dartmouth regimen consisting of DTIC, BCNU, cisplatin and tamoxifen; a combination of cisplatin, vinblastine and DTIC, temozolomide or YERVOY™. The compounds of formula (I) can also be combined with immunotherapy agents, including cytokines such as interferon alpha, interleukin 2 and tumor necrosis factor (TNF), in the treatment of melanoma.

The compounds of formula (I) may also be used in combination with vaccine therapy in the treatment of melanoma. Melanoma vaccines are in some ways similar to antiviral vaccines that are used to prevent diseases caused by viruses such as polio, measles, and mumps. Weakened melanoma cells or parts of melanoma cells, called antigens, may be injected into the patient to stimulate the body's immune system to destroy the melanoma cells.

Melanomas localized on the arms or legs can also be treated with a combination of agents including one or more compounds of formula (I) using the hyperthermic isolated limb perfusion technique. In this case, the blood circulation in the affected limb is temporarily separated from the rest of the body and high doses of chemotherapy are injected into the artery supplying the limb, thus providing high doses to the tumor area without exposing the internal organs to these doses, which could otherwise cause severe side effects. Typically, the fluid is heated to between 38.9°C and 40°C. The chemotherapy drug most often used is melphalan. It may be administered together with another drug called tumor necrosis factor (TNF).

Suitable chemotherapeutic or other anticancer agents include, for example, antimetabolites (including, but not limited to, folate antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors) such as methotrexate, 5-fluorouracil, floxuridine, citrabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatin, and gemcitabine.

Suitable chemotherapeutic or other anticancer agents further include, for example, certain natural products and their derivatives (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines and epipodophyllotoxins), such as vinblastine, vincristine, vindesine, bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, ara-C, paclitaxel (Taxol), mithramycin, deoxycoformycin, mitomycin-C, L-asparaginase, interferons (especially IFN-a), etoposide and teniposide.

Other cytotoxic drugs include navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxafine, and droloxafine.

Also suitable are cytotoxic agents such as epidophyllotoxin; antineoplastic enzyme; topoisomerase inhibitor; procarbazine; mitoxantrone; platinum coordination complexes such as cisplatin and carboplatin; biological response modifiers; growth inhibitors; antihormonal therapeutic agents; leucovorin; tegafur; hematopoietic growth factors.

Other anticancer drugs include antibodies such as trastuzumab (HERCEPTIN®), antibodies to costimulatory molecules such as CTLA-4, 4-1BB, and PD-1, or antibodies to cytokines (IL-1O or TGF-β).

Other anticancer drugs also include agents that block the migration of immune cells, such as chemokine receptor antagonists, including CCR2 and CCR4.

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Other anticancer drugs also include drugs that boost the immune system, such as adjuvants or adoptive T-cell transfer.

Cancer vaccines include dendritic cells, synthetic peptides, DNA vaccines and recombinant viruses.

The pharmaceutical composition of the invention may further comprise at least one signal transduction inhibitor (STI). A signal transduction inhibitor is an agent that selectively inhibits one or more vital steps in signaling pathways during normal functioning of cancer cells, thereby leading to apoptosis. Suitable STIs include, but are not limited to: (i) bcr/abl kinase inhibitors, such as, for example, STI 571 (GLEEVEC™); (ii) epidermal growth factor (EGF) receptor inhibitors, such as, for example, kinase inhibitors (IRESSA™, SSI-774) and antibodies (Imclone: C225 [Goldstein et al., Clin. Cancer Res., 1:1311-1318 (1995)], and Abgenix: ABX-EGF); (iii) her-2/neu receptor inhibitors, such as farnesyl transferase inhibitors (FTIs), e.g. L-744,832 (Kohl et al., Nat. Med, l(8):792-797 (1995)); (iv) inhibitors of the Akt family of kinases or the Akt pathway, such as, e.g., rapamycin (see, e.g., Sekulic et al., Cancer Res., 60:3504-3513 (200)); (v) cell cycle kinase inhibitors, such as, e.g., flavopiridol and UCN-O1 (see, e.g., Sausville, Curr. Med. Chem. Anti-Cane. Agents, 3:47-56 (203)); and (vi) phosphatidyl inositol kinase inhibitors such as, for example, LY294002 (see, for example, Vlahos et al., J. Biol. Chem., 269:5241-5248 (1994)). Alternatively, the at least one STI and the at least one compound of formula (I) may be in separate pharmaceutical compositions. In a particular embodiment of the present invention, the at least one compound of formula (I) and the at least one STI may be administered to the patient simultaneously or sequentially. In other words, the at least one compound of formula (I) may be administered first, the at least one STI may be administered first, or the at least one compound of formula (I) and the at least one STI may be administered simultaneously. Furthermore, when more than one compound of formula (I) and/or STI is used, the compounds may be administered in any order.

The present invention also further provides a pharmaceutical composition for treating a chronic viral infection in a patient, comprising at least one compound of formula (I), optionally at least one chemotherapeutic drug and, optionally, at least one antiviral agent, in a pharmaceutically acceptable carrier.

A method for treating a chronic viral infection in a patient by administering an effective amount of said pharmaceutical composition is also provided.

In a particular embodiment of the present invention, at least one compound of formula (I) and at least one chemotherapeutic drug are administered to the patient simultaneously or sequentially. In other words, at least one compound of formula (I) may be administered first, followed by at least one chemotherapeutic drug, or at least one compound of formula (I) and at least one STI may be administered simultaneously. Furthermore, when more than one compound of formula (I) and/or chemotherapeutic agent are used, the compounds may be administered in any order. Likewise, any antiviral agent or STI may also be administered at any time compared to the administration of the compound of formula (I).

Chronic viral infections that can be treated using this combination therapy include, in particular, diseases caused by: hepatitis C virus (HCV), human papillomavirus (HPV), cytomegalovirus (CMV), herpes simplex virus (HSV), Epstein-Barr virus (EBV), varicella zoster virus, Coxsackie virus, human immunodeficiency virus (HIV).

Suitable antiviral agents contemplated for use in combination with a compound of formula (I) may include nucleoside and nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors and other antiviral drugs.

Examples of suitable NRTIs include zidovudine (AZT); didanosine (ddl); zalcitabine (ddC); stavudine (d4T); lamivudine (3TC); abacavir (1592U89); adefovir dipivoxil [bis-(POM)-PMEA]; lobucavir (BMS-180194); BCH-I0652; emtricitabine [(-)-FTC]; beta-L-RO4 (also called be'ga-k-O4C and called beta-B-2',3'-dichlorooxy-5-fluoro-cytidene); DAPD, ((-)-beta-O-2,6-diaminopurinedioxolane); and lodenosine (FddA). Typical suitable NNRTIs include nevirapine (BI-RG-587); delaviradine (VNAR, U-90152); efavirenz (DMP-266); PNU-142721; AG-1549; MKC-442 (1-(ethoxymethyl)-5-(1methylethyl)-6-(phenylmethyl)-(2,4(III,3H)-pyrimidine); (+)-calanolide A (NSC-675451) and B. Typical protease inhibitors include saquinavir (Ro 31-8959); ritonavir (ABT-538); indinavir (MK639); nelfnavir (AG-1343); amprenavir (141W94); lasinavir (BMS-234475); DMP-450; BMS-2322623; ABT-378; AG-1549. Other antivirals include hydroxyurea, ribavirin, IL-2, IL-12, pentafuside, and Yissum Project No. 11607.

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Combination therapy includes both sequential administration of the therapeutic agents, i.e., administration of each therapeutic agent at different times, and substantially simultaneous administration of the therapeutic agents or at least two of them. Substantially simultaneous administration can be accomplished, for example, by administering to the subject a unit dosage form with a fixed ratio of each therapeutic agent or several unit dosage forms for each of the therapeutic agents. Sequential or substantially simultaneous administration of each therapeutic agent can be accomplished by any suitable route, including, but not limited to, oral, intravenous, intramuscular, and direct absorption through mucosal tissues. The therapeutic agents can be administered by the same route or by different routes. For example, the first therapeutic agent of a selected combination can be administered by intravenous injection, and the other therapeutic agents of the combination can be administered orally. Alternatively, for example, all of the therapeutic agents can be administered orally or all of the therapeutic agents can be administered by intravenous injection. Combination therapy may also include the administration of the above-described therapeutic agents in additional combination with other biologically active ingredients and non-drug treatments (e.g., surgery or radiation therapy). If the combination therapy additionally includes non-drug treatment, the non-drug treatment may be administered at any appropriate time as long as a beneficial effect from the combined action of the combination of therapeutic agents and the non-drug treatment is achieved. For example, in appropriate cases, a beneficial effect is achieved even when the non-drug treatment is temporarily discontinued, perhaps for several days or even weeks, while the therapeutic agents are continued.

Pharmaceutical compositions

The invention also provides pharmaceutical compositions that contain a therapeutically effective amount of one or more compounds of formula (I) formulated with one or more pharmaceutically acceptable carriers (additives) and/or diluents, and, optionally, one or more additional therapeutic agents described above.

The compounds of formula (I) may be administered by any suitable route, preferably in the form of a pharmaceutical composition adapted thereto and at a dose effective for the intended treatment. The compounds and compositions of the compounds of formula (I) may be administered for any of the uses described herein by any suitable route, for example, orally, in the form of tablets, capsules (each including extended-release or slow-release formulations), pills, powders, granules, elixirs, tinctures, suspensions (including nanosuspensions, microsuspensions, spray-dried dispersions), syrups and emulsions; sublingually, buccally, parenterally, for example by subcutaneous, intravenous, intramuscular, intrasternal injection or infusion routes (e.g. as sterile injectable aqueous or non-aqueous solutions or suspensions); nasally, including administration into the nasal membranes, such as by an inhalation spray; topically, such as a cream or ointment; or rectally, such as a suppository. They may be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

For oral administration, the pharmaceutical composition may be, for example, in the form of a tablet, capsule, liquid capsule, suspension or liquid. The pharmaceutical composition is preferably available as a dosage unit containing a certain amount of the active ingredient. For example, the pharmaceutical composition may be presented as a tablet or capsule containing an amount of the active ingredient in the range of about 0.1 to 1000 mg, preferably about 0.25 to 250 mg, and more preferably about 0.5 to 100 mg. A suitable daily dose for a human or other mammal may vary widely depending on the condition of the patient and other factors, but can be determined using conventional methods.

Any pharmaceutical composition contemplated herein can be delivered, for example, orally by any acceptable and suitable oral preparations. Typical oral preparations include, but are not limited to, for example, tablets, sticks, lozenges, aqueous and oily suspensions, dispersible powders or granules, emulsions, hard and soft capsules, liquid capsules, syrups and elixirs. Pharmaceutical compositions intended for oral administration can be prepared according to any methods known in the art for the manufacture of pharmaceutical compositions intended for oral administration. In order to obtain pharmaceutically palatable preparations, the pharmaceutical composition according to the invention can contain at least one agent selected from sweeteners, flavoring agents, coloring agents, softening agents, antioxidants and preservatives.

For example, a tablet can be prepared by mixing at least one compound of formula (I) and/or at least one pharmaceutically acceptable salt thereof with at least one non-toxic pharmaceutically acceptable excipient suitable for the manufacture of tablets. Typical excipients include, but are not limited to,

- 19 048753 inert diluents such as, for example, calcium carbonate, sodium carbonate, lactose, calcium phosphate and sodium phosphate; granulating and disintegrating agents such as microcrystalline cellulose, sodium croscarmellose, corn starch and alginic acid; binders such as starch, gelatin, polyvinylpyrrolidone and gum arabic; and lubricating agents such as magnesium stearate, stearic acid and talc. In addition, tablets can be either uncoated or coated, which is applied by known methods to mask the unpleasant taste of the drug or to slow down the disintegration and absorption of the active ingredient in the gastrointestinal tract, thereby maintaining the action of the active ingredient for a longer period of time. Typical water-soluble taste-masking materials include, but are not limited to, hydroxypropyl methylcellulose and hydroxypropyl cellulose. Examples of time delay materials include, but are not limited to, ethyl cellulose and cellulose acetate butyrate.

Hard gelatin capsules can be prepared, for example, by mixing at least one compound of formula (I) and/or at least one salt thereof with at least one inert solid diluent, such as, for example, calcium carbonate, calcium phosphate, kaolin.

Soft gelatin capsules can be prepared, for example, by mixing at least one compound of formula (I) and/or at least one pharmaceutically acceptable salt thereof with at least one water-soluble carrier, such as, for example, polyethylene glycol; and at least one oil carrier, such as, for example, peanut oil, liquid paraffin, olive oil.

An aqueous suspension can be prepared, for example, by mixing at least one compound of formula (I) and/or at least one pharmaceutically acceptable salt thereof with at least one auxiliary substance suitable for the preparation of an aqueous suspension. Exemplary auxiliary substances suitable for the preparation of an aqueous suspension include, but are not limited to, suspending agents such as, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, alginic acid, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as, for example, a phosphatide of natural origin, for example lecithin; condensation products of alkylene oxide with fatty acids, such as, for example, polyoxyethylene stearate; condensation products of ethylene oxide with long-chain aliphatic alcohols, such as, for example, heptadecaethyleneoxycetanol; condensation products of ethylene oxide with partial esters of fatty acids and hexitol, such as polyoxyethylene sorbitol monooleate; and condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, such as, for example, polyethylene sorbitan monooleate. The aqueous suspension may also contain at least one preservative, such as, for example, ethyl and n-propyl p-hydroxybenzoate; at least one coloring agent; at least one flavoring agent; and/or at least one sweetener, including, but not limited to, sucrose, saccharin and aspartame.

Oil suspensions can be prepared, for example, by suspending at least one compound of formula (I) and/or at least one pharmaceutically acceptable salt thereof in a vegetable oil, such as, for example, peanut oil; olive oil; sesame oil; and coconut oil; or in a mineral oil, such as, for example, liquid paraffin. The oil suspension can also contain at least one thickener, for example, beeswax, hard paraffin, cetyl alcohol. To impart a pleasant taste to the oil suspension, at least one of the sweeteners already described above and/or at least one flavoring agent can be added thereto. The oil suspension can also contain at least one preservative, including, but not limited to, an antioxidant, such as, for example, butylated hydroxyanisole and alpha-tocopherol.

Dispersible powders and granules can be prepared, for example, by mixing at least one compound of formula (I) and/or at least one pharmaceutically acceptable salt thereof with at least one dispersing and/or wetting agent; at least one suspending agent; and/or at least one preservative. Suitable dispersing agents, wetting agents and suspending agents have already been described above. Typical preservatives include, but are not limited to, for example, antioxidants, such as ascorbic acid. In addition, dispersible powders and granules can contain at least one auxiliary component, including, but not limited to, for example, sweeteners, flavorings and colorings.

An emulsion containing at least one compound of formula (I) and/or at least one pharmaceutically acceptable salt thereof can be prepared, for example, in the form of an oil-in-water emulsion. The oil phase of the emulsions containing compounds of formula (I) can be prepared from known ingredients in a known manner. The oil phase can be provided by, but is not limited to, a vegetable oil, such as, for example, olive or peanut oil, a mineral oil, such as, for example, liquid paraffin, and mixtures thereof. Although the phase can contain only an emulsifier, it can contain a mixture of at least one emulsifier with a fat or oil or with a fat and oil. Suitable emulsifiers include, but are not limited to, natural phosphatides,

- 20 048753 emulsifiers, such as soya bean lecithin; esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate; condensation products of partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. Preferably, the hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabilizer. Preferably, both an oil and a fat are also included. Together, the emulsifier(s) with or without the stabilizer(s) form a so-called emulsifying wax, and the wax together with the oil and fat form a so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations. The emulsion may also contain a sweetener, a flavoring, a preservative and/or an antioxidant. Emulsifiers and emulsion stabilizers suitable for use in the composition of the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate, sodium lauryl sulfate, glyceryl distearate alone or with a wax, or other materials known in the art.

The compounds of formula (I) and/or at least one pharmaceutically acceptable salt thereof can, for example, also be administered intravenously, subcutaneously and/or intramuscularly in any pharmaceutically acceptable and suitable injection form. Typical injection forms include, but are not limited to, for example, sterile aqueous solutions containing acceptable carriers and solvents, such as, for example, water, Ringer's solution and isotonic sodium chloride solution; sterile oil-in-water microemulsions; and aqueous or oily suspensions.

Formulations for parenteral administration may be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. These solutions and suspensions may be prepared from sterile powders or granules using one or more of the carriers or diluents mentioned for use in formulations for oral administration or using other suitable dispersing or wetting agents and suspending agents. The compounds may be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, tragacanth and/or various buffers. Other excipients and routes of administration are well and widely known in pharmaceutical practice. The active ingredient may also be administered by injection as a composition with suitable carriers including saline, dextrose or water or with a cyclodextrin (i.e. Captisol), solubilization with a co-solvent (e.g. propylene glycol) or micellar solubilization (e.g. Tween 80).

The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, 1,3-butanediol solution. Water, Ringer's solution and isotonic sodium chloride solution can be used as acceptable carriers and solvents. In addition, sterile fixed oils are commonly used as a solvent or suspending medium. Any ordinary fixed oil can be used for this purpose, including synthetic mono- or diglycerides. In addition, fatty acids, for example, oleic acid, are used for the preparation of injectables.

A sterile injection oil-in-water microemulsion can be prepared, for example, by: 1) dissolving at least one compound of formula (I) in an oil phase, such as, for example, a mixture of soybean oil and lecithin; 2) combining the oil phase containing formula (I) with a mixture of water and glycerol; and 3) treating this mixture to obtain a microemulsion.

A sterile aqueous or oleaginous suspension can be prepared according to methods already known in the art. For example, a sterile aqueous solution or suspension can be prepared using a non-toxic parenterally acceptable diluent or solvent, such as, for example, 1,3-butanediol; and a sterile oleaginous suspension can be prepared using a sterile non-toxic acceptable solvent or suspending medium, such as, for example, sterile fixed oils, for example, synthetic mono- or diglycerides; and fatty acids, such as, for example, oleic acid.

Pharmaceutically acceptable carriers are formulated taking into account a number of factors that are within the skill of the art. These include, but are not limited to: the type and nature of the active agent included; the subject to whom the composition containing the agent is to be administered; the intended route of administration of the composition; and the therapeutic indication. Pharmaceutically acceptable carriers include both aqueous and non-aqueous liquid media, as well as various solid and semi-solid dosage forms. Such carriers may include a number of different ingredients and additives in addition to the active agent, and such additional ingredients are included in the composition for a variety of reasons, such as to stabilize the active agent, as binders, etc., as is well known to those skilled in the art. Descriptions of suitable pharmaceutically acceptable carriers and factors involved in their selection can be found in a variety of readily available sources, such as, for example, Allen, L.V. Jr. et al. Remington: The Science and Practice of Pharmacy (2 volumes), 22nd edition (2012), Pharmaceutical Press.

Pharmaceutically acceptable carriers, excipients and vehicles that may be used in the pharmaceutical compositions of the present invention include, but are not limited to,

- 21 048753 including but not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-alpha-tocopherol polyethyleneglycol 1000 succinate, surfactants used in dosage forms such as Tween, polyethoxylated castor oil, e.g. CREMOPHOR™ surfactant (BASF) or other similar polymer delivery matrices, whey proteins such as human serum albumin, buffering agents such as phosphates, glycine, sorbic acid, potassium sorbate, mixtures of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, trisilicate magnesium, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and wool fat. Cyclodextrins such as alpha-, beta- and gamma-cyclodextrin or chemically modified derivatives such as hydroxyalkyl cyclodextrins, including 2- and 3-hydroxypropyl cyclodextrins, or other solubilized derivatives can also be advantageously used to improve the delivery of the compounds of the formulae described herein.

The pharmaceutically active compounds of the present invention can be processed according to conventional pharmaceutical methods to obtain medicaments intended for administration to patients, including humans and other mammals. The pharmaceutical compositions can be subjected to conventional pharmaceutical operations, such as sterilization, and/or can contain conventional auxiliary substances, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, etc. Tablets and pills can be further prepared with an enteric coating. Such compositions can also contain auxiliary substances, such as wetting, sweetening, flavoring and perfume substances.

For therapeutic purposes, the active compounds of the present invention are typically combined with one or more excipients appropriate to the said route of administration. For oral administration, the compounds can be mixed with lactose, sucrose, starch powder, alkanoic acid esters, alkyl ethers of cellulose, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia, sodium alginate, polyvinylpyrrolidone and/or polyvinyl alcohol, and then tabletted or encapsulated for ease of administration. Such capsules or tablets can contain a controlled release formulation, which can be provided as a dispersion of the active compound in hydroxypropyl methylcellulose.

The amounts of compounds administered and the dosing regimen in the treatment of a disease with the compounds and/or compositions of the present invention depend on a variety of factors including the age, weight, sex, health condition of the subject, type of disease, severity of the disease, route and frequency of administration, and the particular compound used. Thus, the dosing regimen can vary widely, but can be determined by standard methods. The daily dose can be from about 0.001 to 100 mg/kg body weight, preferably from about 0.0025 to about 50 mg/kg body weight, and most preferably from about 0.005 to 10 mg/kg body weight. The daily dose can be administered from one to four doses per day. Other dosing regimens include one dose per week and one dose per two-day cycle.

The pharmaceutical compositions of the present invention comprise at least one compound of formula (I) and/or at least one pharmaceutically acceptable salt thereof and optionally an additional agent selected from any pharmaceutically acceptable carrier, excipient and vehicle. Alternative compositions of the present invention comprise a compound of formula (I) as described herein or a prodrug thereof and a pharmaceutically acceptable carrier, excipient or vehicle.

The present invention also includes pharmaceutical kits useful, for example, for the treatment or prevention of diseases or disorders associated with the Helios protein and other diseases mentioned herein, which include one or more containers containing a pharmaceutical composition containing a therapeutically effective amount of a compound of formula (I). Such kits may further include, if desired, one or more different components of conventional pharmaceutical kits, such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers, as will be readily apparent to those skilled in the art. The kit may also include instructions in the form of inserts or labels indicating the amount of components to be administered, recommendations for administration and/or recommendations for mixing the components.

The dosage regimen of the compounds of the present invention will, of course, vary with known factors such as the pharmacodynamic characteristics of the particular drug, the route and mode of administration; the species, age, sex, health, health status and weight of the patient; the nature and severity of the symptoms; the type of concomitant treatment; the frequency of treatment; the route of administration, the renal and hepatic function of the patient and the desired effect.

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As a general guide, the daily oral dose of each active ingredient when used to achieve the stated effects will be from about 0.001 to about 5000 mg per day, preferably from about 0.01 to about 1000 mg per day, and most preferably from about 0.1 to about 250 mg per day. When administered intravenously, the most preferred doses are from about 0.01 to about 10 mg/kg/min by constant rate infusion. The compounds of formula (I) may be administered as a single daily dose, or the total daily dose may be administered in divided doses two, three, or four times daily.

The compounds are typically administered in admixture with suitable pharmaceutical diluents, excipients or carriers (hereinafter collectively referred to as pharmaceutical carriers) which are selected taking into account the intended form of administration, such as oral tablets, capsules, elixirs and syrups, and in accordance with standard pharmaceutical practice.

Dosage forms (pharmaceutical compositions) suitable for administration may contain from about 1 mg to about 200 mg of active ingredient per dosage unit. In such pharmaceutical compositions, the active ingredient is typically present in an amount of from about 0.1 to 95% by weight, based on the total weight of the composition.

A typical capsule for oral administration contains at least one of the compounds of formula (I) (250 mg), lactose (75 mg) and magnesium stearate (15 mg). The mixture is passed through a 60 mesh sieve and packed into a No. 1 gelatin capsule.

A typical injection preparation is prepared by aseptically placing at least one of the compounds of formula (I) (250 mg) in a vial, aseptically freeze-drying and sealing. For use, the contents of the vial are mixed with 2 ml of physiological saline to obtain an injection preparation.

The present invention includes pharmaceutical compositions containing as an active ingredient a therapeutically effective amount of at least one of the compounds of formula (I), alone or in combination with a pharmaceutical carrier. Optionally, the compounds of formula (I) can be used alone, in combination with other compounds of formula (I) or in combination with one or more other therapeutic agents, for example, an anticancer agent or another pharmaceutically active substance.

Regardless of the route of administration chosen, the compounds of formula (I), which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient effective to achieve a therapeutic response for a particular patient, composition and route of administration, while not being toxic to the patient.

The dosage level selected will depend upon a variety of factors, including the activity of the particular compound of formula (I), ester, salt or amide thereof, the route of administration, the time of administration, the rate of elimination or metabolism of the particular compound, the rate and extent of absorption, the duration of treatment, other drugs, compounds and/or materials used in combination with the particular compound, the age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in the medical arts.

A physician or veterinarian having sufficient skill in the art can easily determine and prescribe the necessary effective amount of the pharmaceutical composition. For example, a physician or veterinarian can start the dosage of the compounds of formula (I) included in the pharmaceutical composition at levels lower than required to achieve the therapeutic effect and gradually increase the dosage until the effect is achieved.

In general, a suitable daily dose of a compound of formula (I) will be that amount of the compound which is the smallest effective dose to produce a therapeutic effect. Such effective dose will generally depend on the factors described above. Generally, oral, intravenous, intracerebroventricular and subcutaneous doses of compounds of formula (I) to a patient are from about 0.01 to about 50 mg per kilogram of body weight per day.

If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally in unit dosage forms. In some aspects of the invention, the dosage is one dose per day.

Although the compound of formula (I) can be administered alone, it is preferably administered in the form of a pharmaceutical formulation (composition).

The above-mentioned other therapeutic agents used in combination with the compounds of formula (I) can be used, for example, in amounts specified in the Physicians' Desk Reference (PDR) or otherwise determined by one skilled in the art. In the methods of the present invention, the other therapeutic agents can be administered before, simultaneously with, or after administration of the compounds of the invention.

Methods of obtaining

The compounds of the present invention can be prepared by a variety of methods well known to those skilled in the art of organic synthesis. The compounds of the present invention can be synthesized using the methods described below, together with synthetic methods known in the art of synthetic organic chemistry, or variations thereof, which will be appreciated by those skilled in the art. Preferred methods include, but are not limited to, the methods described below. All references cited herein are incorporated by reference in their entireties.

The compounds of the present invention can be prepared using the reactions and procedures described in this section. The reactions are carried out in solvents appropriate to the reagents and materials employed and suitable for the transformations being effected. Furthermore, in describing the synthetic routes given below, it is to be understood that all suggested reaction conditions, including the choice of solvent, reaction atmosphere, reaction temperature, experimental duration, and work-up procedures, are chosen as standard conditions for the given reaction, as should be readily recognized by one skilled in the art. One skilled in the art of organic synthesis will appreciate that the functional groups present on the various portions of the molecule must be compatible with the proposed reagents and reactions. Such limitations on the substituents that are compatible with the reaction conditions will be readily apparent to those skilled in the art, and alternative routes must then be used. Sometimes, it will be necessary to change the order of synthetic steps or to select one particular process scheme over another to prepare a compound of the invention. It should also be recognized that another important factor in planning any synthetic route in this area is the judicious choice of the protecting group used to protect the reactive functional groups present in the compounds described in the present invention. An authoritative study describing the numerous alternatives available to the skilled artisan is provided by Greene and Wuts (Protective Groups In Hydrogenous Synthesis, Fourth Edition, Wiley and Sons, 207).

Compounds of formula (I) can be prepared by the methods illustrated in the following schemes. As shown in the schemes, the final product is a compound having the same structural formula as formula (I). It should be understood that any compound of formula (I) can be prepared according to the schemes by appropriate selection of reactants with appropriate substitution. Solvents, temperature, pressure and other reaction conditions can be easily selected by a person skilled in the art. Starting materials are commercially available or are easily prepared by a person skilled in the art. The constituents of the compounds correspond to the definitions given herein or elsewhere in the description.

General routes to the compounds described in the invention are illustrated in Schemes 1-2, where R1, _R2 , R4 and R6 are as defined previously in the text or a functional group that can be converted to the final substituent. Substituent X is a leaving group, for example, a halide (preferably I, Br or Cl) or a triflate. Substituent M is a suitable coupling partner, such as a boronic acid, a boronic ester or a stannane. Substituent R is a carboxylic acid protecting group, for example, tert-butyl, methyl, ethyl or benzyl. As shown in Scheme 1, the general procedure for preparing compounds of the invention begins with a suitable substituted aryl fluoride 1. Upon treatment with a suitable nucleophile, such as intermediate 2, where M can be MgBr or Li, substituted aryl halides such as 3 can be obtained. Halogenation, preferably bromination, can be accomplished by treating 3 with reagents such as N-bromosuccinimide to afford bromide 4. Treatment of 4 with amine 5 under basic conditions, such as potassium carbonate, results in initial displacement of the secondary bromide, followed by cyclization to afford lactam 6.

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Scheme 1

If M is stannane, then compound 6 can be coupled with a suitable substituted heterocycle 7 in a Stille coupling reaction using a suitable catalyst system (e.g. Pd( _PPh3 ) ₄ or bis(triphenylphosphine)dichloropalladium(II)/CuI) to afford 8. Alternatively, compound 6 can be converted to the boronic acid or boronic ester 9 under conditions well known to those skilled in the art. Boronic acid or boronic ester 9 can be coupled with a suitable substituted heterocycle 10 in a Suzuki-Miyaura coupling reaction using a suitable palladium catalyst (e.g. Pd( _PPh3 ) ₄ or 1,N-bis(diphenylphosphino)ferrocene]dichloropalladium(II)) in the presence of a suitable base (e.g. cesium carbonate, potassium phosphate or sodium bicarbonate) to afford 8.

Depending on the particular choice of acid protecting group R in intermediate 8, different conditions may be required for its conversion to compound 11 (Scheme 2). For example, if R is methyl, ethyl, or benzyl, then base-induced cyclization of 8 may be preferred to directly convert 8 to 11 using a suitable base (e.g., LiHMDS) in a suitable solvent (e.g., tetrahydrofuran). In the case where R is tert-butyl, acidic cyclization 12 may be preferred to directly convert 8 to 11 using a suitable acid (e.g., benzenesulfonic acid) in a suitable solvent (e.g., acetonitrile). In some cases, it may be preferable to use a two-step procedure in which the free carboxylic acid corresponding to 8 is first released under conditions appropriate for the particular acid protecting group R. Such methods are well known to those skilled in the art of organic synthesis. For example, if R is tert-butyl, acidic hydrolysis using a suitable acid (e.g., trifluoroacetic acid or hydrochloric acid) may be preferred. If R is methyl, ethyl, or benzyl, basic hydrolysis using a suitable base (e.g., LiOH) may be preferred. In other cases, when R is benzyl, deprotection by palladium-catalyzed hydrogenolysis may be preferred. Once released, the carboxylic acid can be activated for intramolecular attack by the pendant primary amide using thionyl chloride/dimethylformamide or carbonyldiimidazole/dimethylaminopyridine to afford 11.

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Scheme 2

Examples

The following examples illustrate specific embodiments of the present invention and do not limit the scope of the present invention. Chemical abbreviations and symbols, like scientific abbreviations and symbols, have their usual and customary meanings unless otherwise indicated. Additional abbreviations used in the examples and elsewhere in this application are defined above. Common intermediates are typically used to prepare more than one example. Example compounds are identified by example and the step in which they were prepared (e.g., 1-A refers to example 1, step A) or by example only if the compound is the title compound of the example (e.g., 1 refers to the title compound of example 1). In some cases, alternative preparations of intermediates or example compounds are described. Often, chemists skilled in the art of synthesis can develop alternative preparation methods that may be desirable based on one or more considerations such as shorter reaction times, less expensive starting materials, ease of operation or isolation, improved yield, the possibility of catalysis, the absence of toxic reagents, the availability of specialized equipment and a reduction in the number of linear steps, etc. The purpose of describing alternative preparation methods is to further enable the preparation of the examples of the present invention. In some cases, certain functional groups in the present examples and claims may be replaced by well-known bioisosteric substitutions known in the art, for example, the replacement of a carboxylic acid group with a tetrazole or phosphate moiety.

Abbreviations.

ACN acetonitrile

AIBN 2,2-azobisiosbutyronitrile n-BuLi p-butyl lithium

DCE dichloroethane

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DCM dichloromethane DIPEA N,N-d iisopr opylethyl amine DMF dimethylformamide DMSO dimetil sulph oxide dppf bis(diphenylphosphino)ferrocene EtOH ethanol EtOAc ethyl acetate Hex hexanes

H-Glu(OtBu)-NH2 HC1 hydrochloride tert-butyl-(48)-4,5-diamino-5 -

oxopentanoate HPLC high performance liquid chromatography Foundation of Hunig LiHMDS N,N-diisopropylethylamine lithium bis(trimethylsilyl)amide MeCN acetonitrile min minute(s) ml milliliter(s) NBS p-bromosuccinimide Pd(dppf)2C12 [1,G- bis(diphenylphosphino)ferrocene]dichloropalladium(II) Pd(dtbpf)C12 [1,G-bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II) Pd _{( PPh3} ) _4PhSO3H tetrakis(triphenylphosphine)palladium benzenesulfonic acid PTSOH para-toluenesulfonic acid TEA triethylamine THF tetrahydrofuran XPhos Pd G2 chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(P)

Method 1 preparative HPLC: XBridge C18, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; gradient: 0 min hold at 15% B, 15-50% B for 25 min, then 6 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fractions were collected based on MS signals.

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Example 1. 2-Amino-6-(2-(2,6-dioxopiperidin-3-yl)-4-methyl-1-oxoisoindolin-5-yl)-4methylnicotinonitrile

Preparation 1A: tert-butyl ((3)-5-amino-4-(5-bromo-4-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate.

To a suspension of tert-butyl ((D)-4,5-diamino-5-oxopentanoate hydrochloride (2.162 g, 6.06 mmol) in acetonitrile (30 mL) at 0 °C was added DIPEA (3.018 mL, 17.28 mmol). After stirring for 20 min, the reaction mixture was treated with a solution of methyl 4-bromo-2-(bromomethyl)-3-methylbenzoate (2.650 g, 8.22 mmol) in MeCN (10 mL). The reaction mixture was stirred at 0 °C for 5 min. After this time, the ice bath was removed and the reaction mixture was allowed to warm to room temperature and stirred at room temperature for 1 h. The reaction mixture was heated to 60 °C and maintained at this temperature overnight. The reaction mixture was concentrated, dissolved in EtOAc, washed twice with water, then _with 1.5 M _K2HPO4 , brine, dried over _MgSO4 , filtered and concentrated. The material was purified using silica gel and eluted with 30-100% EtOAc/Hex. The resulting material was triturated with 8 mL of ether to give 33.7% yield of the title product. 11 NMR (400 MHz, CHLOROFORM-d) δ 7.70 (d, J=8.1 Hz, 1H), 7.56 (d, J=8.1 Hz, 1H), 6.23 (br s, 1H), 5.31 (br s, 1H), 4.92 (dd, J=8.6, 6.4 Hz, 1H), 4.55-4.47 (m, 1H), 4.45-4.36 (m, 1H), 2.41 (s, 3H), 2.41-2.14 (m, 4H), 1.45 (s, 9H).

Preparation 1B: tert-butyl ((^)-5-amino-4-(4-methyl-1-oxo-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan2-yl)isoindolin-2-yl)-5-oxopentanoate.

In a dry flask were placed tert-butyl ((D))-5-amino-4-(5-bromo-4-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate (300 mg, 0.730 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxaborolane) (278 mg, 1.094 mmol), and potassium acetate (214 mg, 2.188 mmol) and flushed with nitrogen. The solid was suspended in dioxane (10 mL) and degassed with nitrogen flow for 5 min with stirring. Pd(dppf) _Cl2 (16.02 mg, 21.88 μmol) was added to the reaction mixture. The flask was degassed for 5 min, sealed, and heated to 60°C for 18 h under nitrogen. The reaction mixture was diluted with EtOAc, washed with brine and dried over MgSO4. The product was concentrated and purified on a 40 g silica gel column by the ISCO method eluting with 0-40% B/DCM (where B = 15% EtOH/EtOAc + 0.1% TEA) to give the product in 99% yield. MS (ES): m/z = 459.3 [M+H]+.

Example 1.

A 2 mL microwave vial was charged with 2-amino-6-bromo-4-methylnicotinonitrile (12 mg, 0.057 mmol, tert-butyl-(4-methyl-1-oxo-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2yl)isoindolin-2-yl)-5-oxopentanoate (28.5 mg, 0.062 mmol, Pd(dtbpf) _Cl2 (1.106 mg, 1.698 μmol), dioxane (1.5 mL), and aqueous _K3PO4 (0.075 mL, 0.226 mmol ₎ . The vial was sealed and the air was replaced with nitrogen. The reaction mixture was microwaved for 15 min at 120 °C. After cooling to room temperature, the reaction mixture was heated to 120 °C. At 20°C, the reaction mixture was diluted with EtOAc, washed with brine, and the organic layer was separated and concentrated. The resulting residue was dissolved in 1 mL of PhSO3H in MeCN (1.44 g/40 mL) and heated in the microwave for 10 min at 120°C. The material was concentrated to dryness, the residue was dissolved in 1.9 mL of DMSO and purified by preparative HPLC method 1 to give the title compound in 49% yield. MS (ES): m/z = 390.3 [M+H]+. Ή NMR (500 MHz, DMSO-d ₆ ) δ ppm 11.03 (s, 1H) 7.66 (d, J=7.63 Hz, 1H) 7.53 (d, J=7.93 Hz, 1H) 6.91 (s, 2H) 6.78 (s, 1H) 5.18 (br dd, J=13.43, 5.19 Hz, 1H) 4.52 (br d, J=17.09 Hz, 1H) 4.35 (br d, J=17.40 Hz, 1H) 2.91-3.01 (m, 1H) 2.65 (br d, J=16.78 Hz, 1H) 2.47 (br dd, J=13.12, 4.27 Hz, 1H) 2.43 (s, 3H) 2.33 (s, 3H) 2.03-2.10 (m, 1H).

Example 2. 2-Amino-6-(2-(2,6-dioxopiperidin-3-yl)-6-methyl-1-oxoisoindolin-5-yl)-4methylnicotinonitrile

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Preparation 2A: methyl 4-bromo-2-(bromomethyl)-5-methylbenzoate.

To a solution of 4-bromo-2,5-dimethylbenzoic acid (5.0 g, 21.82 mmol) was added SOCl ₂ (31.66 mL, 436 mmol) at room temperature and stirred for 2 h. LCMS was used to confirm the formation of the acid chloride, followed by concentration to dryness. Methanol (30 mL) was added at room temperature, stirred for 0.5 h, and concentrated again to dryness. To the resulting methyl ester was added CCl ₄ (180 mL), followed by 1-bromopyrrolidine-2,5-dione (4.08 g, 22.92 mmol), and then AIBN (0.108 g, 0.654 mmol). The resulting mixture was heated to 80 °C with stirring overnight. After cooling to room temperature, the mixture was concentrated, dissolved in EtOAc, washed with brine and dried over MgSO ₄ . LCMS showed two major peaks. The resulting mixture of regioisomers (5.4 g) was used for the next step.

Preparation 2B: tert-butyl ((3)-5-amino-4-(5-bromo-6-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate.

To a suspension of methyl 4-bromo-2-(bromomethyl)-5-methylbenzoate (5.4 g, 16.76 mmol) in acetonitrile (100 mL) was added tert-butyl ((3-bromomethyl)-4,5-diamino-5-oxopentanoate, HCl (4.00 g, 16.76 mmol) followed by Hunig's base (5.84 mL, 33.52 mmol). After stirring for 1 h at room temperature, the reaction mixture was placed in a 40 °C bath and stirred at this temperature for 6 days. The reaction mixture was cooled to room temperature, diluted with EtOAc, washed with brine, dried over MgSO ₄ , filtered, and concentrated. The residue was purified by ISCO method [120 g column] eluting with EtOAc/DCM from 0-100% to give 2.80 g of the desired compound. In addition, two other by-products (methyl (N)-5-(((1-amino-5(tert-butoxy)-1,5-dioxopentan-2-yl)amino)methyl)-4-bromo-2-methylbenzoate and tert-butyl (N)-5-amino-4(((2-(N)-1-amino-5-(tert-butoxy)-1,5-dioxopentan-2-yl)-6-bromo-3-oxoisoindolin-5-yl)methyl)amino)5-oxopentanoate) were isolated. MS (ES): m/z = 411.0 [M+H]+. 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.70 (d, J=6.44 Hz, 2H) 6.31 (br s, 1H) 5.36 (br s, 1H) 4.90 (dd, J=8.68, 6.34 Hz, 1H) 4.51 (d, J=16.98 Hz, 1H) 4.41 (d, J=16.98 Hz, 1H) 2.51 (s, 3H) 2.21-2.43 (m, 3H) 2.12-2.19 (m, 1H) 1.44 (s, 9H).

Preparation 2C: tert-butyl (^)-5-amino-4-(6-methyl-1-oxo-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan2-yl)isoindolin-2-yl)-5-oxopentanoate.

A 40 mL pressure vial was charged with tert-butyl ((3)-5-amino-4-(5-bromo-6-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate (600 mg, 1.458 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxaborolane) (556 mg, 2.188 mmol), potassium acetate (430 mg, 4.376 mmol) and flushed with nitrogen. The solid was suspended in dioxane (20 mL) and degassed with nitrogen stream for 5 min with stirring. The reaction mixture was treated with Pd(dppf) _Cl2 (32.02 mg, 0.044 mmol), degassed for 5 min. The tube was sealed and heated to 95 °C for 3 h under nitrogen. The reaction mixture was cooled to room temperature, diluted with EtOAc, washed with brine and dried over MgSO ₄ . The filtrate was concentrated and purified on a 40 g silica gel column by the ISCO method eluting (0%-40% B/DCM, where B = 15% EtOH/EtOAc + 0.1% TEA) to give the title compound in 95% yield. MS (ES): m/z = 459.3 [M+H]+.

Example 2.

A 2 mL microwave vial was charged with 2-amino-6-bromo-4-methylnicotinonitrile (12 mg, 0.057 mmol), tert-butyl ((3)-5-amino-4-(6-methyl-1-oxo-5-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)isoindolin-2-yl)-5-oxopentanoate (28.5 mg, 0.062 mmol), Pd(dtbpf) _Cl2 (1.106 mg, 1.698 _μmol ), dioxane (1.5 mL), and aqueous _K3PO4 (0.075 mL, 0.226 mmol). The vial was sealed and the air was replaced with nitrogen. The reaction mixture was heated in the microwave for 15 min at 120 °C. After cooling to room temperature, the reaction mixture was diluted with EtOAc, washed with brine, the organic layer was separated and concentrated. The resulting residue was dissolved in 1 mL of _PhSO3H in MeCN (1.44 g/40 mL) and heated in a microwave oven for 10 min at 120 °C. The mixture was concentrated to dryness, the residue was dissolved in 1.7 mL of DMSO and purified by preparative HPLC method 1 to afford the title compound in 35% yield. MS (ES): m/z=390.3 [M+H]+. 1H NMR (500 MHz, DMSO-d6) δ ppm 10.97-11.02 (m, 1H) 7.65 (s, 1H) 7.55 (s, 1H) 6.76 (s, 1H) 5.13 (dd, J=13.31, 5.18 Hz, 1H) 4.39-4.51 (m, 1H) 4.28-4.39 (m, 1H) 2.87-2.98 (m, 1H) 2.58-2.65 (m, 1H) 2.51 (d, J=7.74 Hz, 3H) 2.42-2.46 (m, 1H) 2.41 (br s, 3H) 2.39-2.40 (m, 1H) 1.99-2.06 (m, 1H).

Example 3. 2-Amino-6-(2-(2,6-dioxopiperidin-3-yl)-4-fluoro-1-oxoisoindolin-5-yl)-4methylnicotinonitrile

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Intermediate compound 3A. 5-Bromo-4-fluoro-3-hydroxyisobenzofuran-1(3H)-one.

To a stirred solution of 2,2,6,6-tetramethylpiperidine (7.07 mL, 41.6 mmol) in THF (150 mL) was added 2.5 M n-BuLi solution in hexanes (16 mL, 40.0 mmol) at 0 °C. The reaction mixture was stirred for 30 min at 0 °C. To the reaction mixture was added dropwise a solution of 4-bromo-3-fluorobenzoic acid (3.5 g, 15.98 mmol) in anhydrous THF (100 mL) at -50 °C. The reaction mixture was stirred for 3 h at the same temperature. Anhydrous DMF (2.48 mL, 32.0 mmol) was added at -50 °C and the reaction mixture was warmed to room temperature and stirred for 16 h. The reaction was quenched with 1.5 N HCl (100 mL). The reaction mixture was extracted with ethyl acetate (3 x 30 mL). The combined organic layer was washed with brine, dried over anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The residue was purified by flash chromatography (SiO ₂ column, 120 g, 0-50% EtOAc/pet ether) to give 5-bromo-4-fluoro-3-hydroxyisobenzofuran-1(3H)-one (1.0 g, 23% yield) as a yellow solid. LCMS (method A): retention time 0.48 min, [M+H]+ 245.1, 247.1; ^1H NMR (400 MHz, acetonitrile^) δ 7.93 (dd, J=8.0, 5.5 Hz, 1H), 7.59 (d, J=8.0 Hz, 1H), 6.74 (br s, 1H), 5.94 (br s, 1H).

Intermediate 3B. tert-Butyl ((3)-5-amino-4-(5-bromo-4-fluoro-1-oxoisoindolin-2-yl)5-oxopentanoate.

To a stirred solution of 5-bromo-4-fluoro-3-hydroxyisobenzofuran-1(3H)-one (1.7 g, 6.88 mmol) and tert-butyl ((3H))-4,5-diamino-5-oxopentanoate HCl (1.67 g, 8.26 mmol) in DMF (30 mL) at 0°C was added sodium triacetoxyborohydride (3.65 g, 17.21 mmol). The reaction mixture was warmed to room temperature and stirred for 48 h. The reaction mixture was diluted with ice water (50 mL) and the resulting white solid was filtered and dried under reduced pressure to give tert-butyl ((3)-5-amino-4-(5-bromo-4-fluoro-1-oxoisoindolin-2-yl)-5-oxopentanoate (1.6 g, 50% yield) as a white solid. LCMS (method A): retention time 1.39 min, [M+H]+ 413.9, 415.3; 1H NMR (400 MHz, DMSO46) δ 7.85 (dd, J=8.0, 6.0 Hz, 1H), 7.59 (br s, 1H), 7.51 (d, J= 8.0 Hz, 1H), 7.23 (br s, 1H), 4.77-4.59 (m, 3H), 2.26-2.13 (m, 3H), 2.08-1.96 (m, 1H), 1.34 (s, 9H).

Intermediate compound 3C. tert-Butyl-((3-yl)-5-amino-4-(5-(6-amino-5-cyano-4-methylpyridin-2yl)-4-fluoro-1-oxoisoindolin-2-yl)-5-oxopentanoate.

A stirred solution of 2-amino-6-chloro-4-methylnicotinonitrile (50 mg, 0.30 mmol) and hexamethylditin (0.093 mL, 0.45 mmol) in toluene (2 mL) was purged with argon for five minutes, after which [1,N-bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II) (9.72 mg, 0.015 mmol) was added. The reaction mixture was stirred for 2 h at 100 °C, cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure to give the crude product. [M+H]+ 298.2. To a solution of the crude product in dioxane (2 ml) was added tert-butyl ((3)-5-amino-4-(5-bromo-4-fluoro-1-oxoisoindolin-2-yl)-5-oxopentanoate (98 mg, 0.24 mmol). The reaction mixture was purged with argon for five minutes, bis(triphenylphosphine)palladium(II) chloride (16.60 mg, 0.024 mmol) was added and heated at 100 °C for 16 h. The reaction mixture was cooled to room temperature, filtered and the filtrate was concentrated under reduced pressure to give the crude product, which was purified by flash chromatography (SiO2 column, 80 g, 0-100% B (B = 15% EtOH in EtOAc, 0.5% TEA)/chloroform) to give tert-butyl ((3))-5-amino-4-(5-(6-amino-5-cyano-4-methylpyridin-2-yl)-4-fluoro-1-oxoisoindolin-2-yl)-5-oxopentanoate (60 mg, 40.7% yield). LCMS (method A): retention time 1.35 min, [M+H]+ 468.3.

Example 3.

To a stirred solution of intermediate 3C (60 mg, 0.13 mmol) in acetic acid (2 mL) was added benzenesulfonic acid (20.30 mg, 0.13 mmol). The reaction mixture was heated to 150 °C under microwave irradiation for 10 min. The volatiles were removed under reduced pressure and the resulting crude product was purified by preparative LCMS (column: Waters XBridge C18, 19 x 150 mm, 5 μm particles; mobile phase A: 0.1% trifluoroacetic acid in water; mobile phase B: acetonitrile; gradient: 10-40% B over 20 min, then a 5 min hold at 100% B; flow rate: 15 mL/min). The desired fractions were lyophilized to dryness to give 2-amino-6-(2(2,6-dioxopiperidin-3-yl)-4-fluoro-1-oxoisoindolin-5-yl)-4-methylnicotinonitrile (15 mg, 29% yield). LCMS (method G): retention time 1.62 min, [M+H]+ 394.2; 1H NMR (400 MHz, DMSO-d ₆ ) δ 11.01 (br s, 1H), 8.01 (t, J=7.3 Hz, 1H), 7.70 (d, J=7.9 Hz, 1H), 7.06 (d, J=1.5 Hz, 1H), 6.99 (s, 2H), 5.14 (dd, J=13.3, 5.1 Hz, 1H), 4.74-4.38 (m, 2H), 3.05-2.85 (m, 1H), 2.75-2.57 (m, 2H), 2.46 (s, 3H), 2.13-1.95 (m, 1H).

General procedure 2: A stirred solution of intermediate 3B (1 equiv.) and arylstannane reagent (1 equiv.) in dioxane (5 mL/mmol) was purged with argon for 5 min. Bis(triphenylphosphine)palladium(II) chloride (0.1 equiv.) was added and the reaction mixture was heated at 100 °C for 16 h. The reaction mixture was cooled to room temperature, filtered through a celite pad and the filtrate was concentrated under reduced pressure. The crude material was transferred to a microwave vial, _PhSO3H (2 equiv.) and acetic acid (5 mL/mmol) were added and the mixture was heated at 150 °C for 10 min in the microwave reactor. The mixture was concentrated and the residue was purified by preparative HPLC.

- 30 048753

Example 4 3-(5-(6-Amino-4-methylpyridin-2-yl)-4-fluoro-1-oxoisoindolin-2-yl)piperidin-2,6dione

Intermediate 4A. 4-Methyl-6-(trimethylstannyl)pyridin-2-amine.

A stirred solution of 6-bromo-4-methylpyridin-2-amine (75 mg, 0.382 mmol) and hexamethylditin (0.119 mL, 0.57 mmol) in toluene (5 mL) was purged with argon for five minutes, after which [1,N-bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II) (12.4 mg, 0.02 mmol) was added. The reaction mixture was stirred for 2 h at 100 °C, cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure to give 4-methyl-6(trimethylstannyl)pyridin-2-amine (130 mg, 89% yield). LCMS (method A): retention time 1.62 min, [M+H]+ 273.1.

Example 4.

The Stille coupling reaction and cyclization were carried out according to general procedure 2 using preparation 4A and preparation 3B. MS (ES): m/z = [M+H]+ 369.1; 1H NMR (400 MHz, DMSO-de) δ = 11.02 (s, 1H), 8.02 (t, J= 7.2 Hz, 1H), 7.65 (d, J= 7.8 Hz, 1H), 6.84 (s, 1H), 6.34 (s, 1H), 6.03 (s, 2H), 5.15 (dd, J= 5.0, 13.3 Hz, 1H), 4.66-4.57 (m, h), 4.49-4.38 (m, 1H), 3.02-2.85 (m, 1H), 2.62 (br d, J= 17.4 Hz, 1H), 2.49-2.40 (m, 1H), 2.22 (s, 3H), 2.07-1.98 (m, 1H).

Example

5.

-(5 -(6-Amino-4-(trifluoroethyl)pyridin-2-yl)-4-fluoro-1-oxoisoindolin-2 yl)piperidine-2,6-dione

Intermediate connection 5A.

A stirred solution of 6-chloro-4-(trifluoromethyl)pyridin-2-amine (75 mg, 0.382 mmol) and hexamethylditin (0.119 mL, 0.57 mmol) in toluene (5 mL) was purged with argon for five minutes, after which [1,N-bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II) (12.4 mg, 0.02 mmol) was added. The reaction mixture was stirred for 2 h at 100 °C, cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure to give 4-(trifluoromethyl)-6(trimethylstannyl)pyridin-2-amine (130 mg, 89% yield). LCMS (method A): retention time 1.92 min, [M+H]+ 327.1.

Example 5.

Stille coupling and cyclization were carried out according to General Procedure 1 using intermediate 5A and intermediate 3B. MS (ES): m/z = [M+H]+ 423.3; ¹ H

NMR (400 MHz, DMSO-d6) δ = 11.02 (s, 1H), 8.05 (t, J= 7.3 Hz, 1H), 7.69 (d, J= 7.8 Hz, 1H ), 7.15 (s, 1H), 6.78 (d, J= 11.8 Hz, 3H), 5.15 (dd, J= 5.0, 13.3 Hz, 1H), 4.68-4.57 (m, 1H), 4.52-4.39 (m, 1H), 2.99-2.82 (m, 1H), 2.67-2.58 (m, 1H), 2.48-2.39 (m, 1H), 2.12-1.93 (m, 1H).

Example 6. 3-(4-Fluoro-5-(4-methyl-6-(methylamino)pyridin-2-yl)-1-oxoisoindolin-2-yl)piperidine2,6-dione

Intermediate 6A. ^4-Dimethyl-6-(trimethylstannyl)pyridin-2-amine A stirred solution of 6-chloro-N,4-dimethylpyridin-2-amine (100 mg, 0.64 mmol) and hexamethylditin (0.199 mL, 0.96 mmol) in toluene (10 mL) was purged with argon for five minutes, after which [1,1'-bis-31 048753 (di-tert-butylphosphino)ferrocene]dichloropalladium(II) (20.8 mg, 0.032 mmol) was added. The reaction mixture was stirred for 2 h at 100 °C, cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure to give y4-dimethyl-6-(trimethylstannyl)pyridin-2amine (250 mg, 55.0% yield). LCMS (method A): retention time 1.29 min, [M+H]+ 287.1.

Example 6.

The Stille coupling reaction and cyclization were carried out according to General Procedure 2 using intermediate 6A and intermediate 3B. MS (ES): m/z = [M+H]+ 383.1; ¹ H NMR (400 MHz, D\lSO-d6) δ 11.03 (s, 1H), 8.02 (s, 1H), 7.72 (d, J=7.5 Hz, 1H), 6.93 (s, 1H), 6.58 (br s, 1H), 5.16 (dd, J=13.1, 5.0 Hz, 1H), 4.82-4.40 (m, 3H), 2.89 (s, 3H), 2.75-2.63 (m, 1H), 2.64-2.57 (m, 1H), 2.46-2.41 (m, 1H), 2.36-2.28 (m, 3H), 2.06-2.00 (m, 1H). 19/19

Example 7. 3-(5-(6-Amino-4,5-dimethylpyridin-2-yl)-4-fluoro-1-oxoisoindolin-2-yl)piperidine-2,6 dione

H ₃ C n ₃ with αν <Г ^Н nh ₂ (7)

Intermediate 7A. 3,4-Dimethyl-6-(trimethylstannyl)pyridin-2-amine.

A stirred solution of 6-chloro-3,4-dimethylpyridin-2-amine (75 mg, 0.48 mmol) and hexamethylditin (0.149 mL, 0.72 mmol) in toluene (3 mL) was purged with argon for five minutes, after which [1,1'-bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II) (15.6 mg, 0.024 mmol) was added. The reaction mixture was stirred for 2 h at 100 °C, cooled to room temperature, and filtered. The filtrate was concentrated under reduced pressure to give 3,4-dimethyl-6(trimethylstannyl)pyridin-2-amine (150 mg, 55.0% yield). LCMS (method A): retention time 1.16 min, [M+H]+ 287.2.

Example 7.

The Stille coupling reaction and cyclization were carried out according to General Procedure 1 using intermediate 7A and intermediate 3B. MS (ES): m/z = [M+H]+ 383.0; ¹ H NMR (400 MHz, DMSO-de) δ 11.01 (br s, 1H), 8.05 (t, J=7.3 Hz, 1H), 7.65 (d, J=7.9 Hz, 1H), 6.93 (d, J=1.5 Hz, 1H), 5.81 (s, 2H), 5.14 (dd, J=13.3, 5.1 Hz, 1H), 4.71-4.34 (m, 2H), 3.01-2.88 (m, 1H), 2.67-2.58 (m, 2H), 2.462.41 (m, 1H), 2.23 (s, 3H), 2.04 (s, 3H).

Examples 8 and 9. 3-(^)-5-(6-Amino-4,5-dimethylpyridin-2-yl)-3-methyl-1-oxoisoindolin-2yl)piperidine-2,6-dione and 3-(^)-5-(6-amino-4,5-dimethylpyridin-2-yl)-3-methyl-1-oxoisoindolin-2yl)piperidine-2,6-dione

Intermediate 8A. 4-Bromo-2-ethylbenzoic acid.

To a solution of 4-bromo-2-fluorobenzoic acid (5 g, 22.83 mmol) in anhydrous THF (100 mL) was added 1 M ethylmagnesium bromide in THF (22.83 mL, 68.5 mmol) at -78 °C over 15 min. The reaction mixture was slowly warmed to room temperature and stirred under nitrogen for 12 h. The reaction mixture was cooled to 0 °C. The reaction was quenched by dropwise addition of MeOH (15 mL). The reaction mixture was concentrated under reduced pressure.

The resulting residue was partitioned between EtOAc and 2 M aqueous HCl. The layers were separated and the aqueous layer was extracted twice with EtOAc. The combined organic layer was washed with brine, dried over _{anhydrous Na2SO4} , filtered and concentrated under reduced pressure to give the crude product. The crude product was purified by flash chromatography ( _SiO2 column, 80 g, 0-30% EtOAc/pet ether) to give 4-bromo-2-ethylbenzoic acid (4 g, 76% yield). LCMS (method D): retention time 2.49 min, [M+H]+ 228.8, 230.0; 1H NMR (400 MHz, DMSOd6) δ 13.04 (br s, 1H), 7.71 (d, J=8.5 Hz, 1H), 7.55 (d, J=2.0 Hz, 1H), 7.49 (dd, J=8.0, 2.0 Hz, 1H), 2.92 (q, J=7.2 Hz, 2H), 1.15 (t, J=7.5 Hz, 3H).

Intermediate 8B. Methyl 4-bromo-2-ethylbenzoate.

To a stirred mixture of 4-bromo-2-ethylbenzoic acid (4.0 g, 17.46 mmol) and _Cs2CO3 (11.38 g, 34.9 mmol) in DMF (40 _mL ) was added methyl iodide (2.18 mL, 34.9 mmol). The reaction mixture was

- 32 048753 was stirred at room temperature for 14 h, filtered through a pad of celite and concentrated under reduced pressure. The residue was purified by flash chromatography (SiO2 column, 80 g, 0-50% EtOAc/pet ether) to give methyl 4-bromo-2-ethyl benzoate (3.3 g, 78% yield) as a colorless oil. 1H NMR (400 MHz, CHLOROFORM-d) δ 7.76 (d, J= 8.5 Hz, 1H), 7.46 (d, J=1.5 Hz, 1H), 7.40 (dd, J=8.5, 2.0 Hz, 1H), 3.91 (s, 3H), 2.99 (q, J=7.5 Hz, 2H), 1.26 (t, J=7.5 Hz, 3H).

Intermediate 8C. Methyl 4-bromo-2-(1-bromoethyl)benzoate.

To a stirred solution of methyl 4-bromo-2-ethyl benzoate (3.0 g, 12.34 mmol) in benzene (40 mL) was added NBS (3.29 g, 18.51 mmol) followed by AIBN (0.405 g, 2.47 mmol). The reaction mixture was heated at 85 °C for 15 h. The reaction mixture was cooled to room temperature, diluted with EtOAc, washed with saturated 10% sodium thiosulfate solution and brine. The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered, and concentrated under reduced pressure. The residue was purified by flash chromatography ( _SiO2 column, 40 g, 0-30% EtOAc/pet ether) to give methyl 4-bromo-2-(1-bromoethyl)benzoate (2.1 g, 53% yield) as a white solid. ¹ H NMR (400 MHz, chloroform-d) δ 7.96 (d, J=2.0 Hz, 1H), 7.73 (s, 1H), 7.49 (dd, J=8.5, 2.0 Hz, 1H), 6.28 (q, J=7.0 Hz, 1H), 3.95 (s, 3H).

Intermediate 8D. tert-Butyl (4S)-5-amino-4-(5-bromo-3-methyl-1-oxoisoindolin-2yl)-5-oxopentanoate.

To a stirred solution of methyl 4-bromo-2-(1-bromoethyl)benzoate (1.0 g, 3.11 mmol) and HGlu(OtBu)-NH ₂ HCl (0.754 g, 3.73 mmol) in acetonitrile (30 mL) was added DIPEA (2.71 mL, 15.53 mmol). The resulting reaction mixture was heated at 85 °C for 15 h. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was purified by flash chromatography ( _SiO2 column, 40 g, 0-10% MeOH/DCM) to give tert-butyl ((3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate (650 mg, 51% yield) as a semi-solid. LCMS (method A): retention time 1.45 min, [M+H]+ 411.3, 413.3.

Intermediate 8E. tert-Butyl 5-amino-4-(5-(6-amino-4,5-dimethylpyridin-2-yl)-3methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate.

A stirred solution of 6-chloro-3,4-dimethylpyridin-2-amine (200 mg, 1.28 mmol) and hexamethylditin (544 mg, 1.66 mmol) in toluene (6 mL) was purged with argon for five minutes, after which [1,N-bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II) (83 mg, 0.13 mmol) was added. The reaction mixture was stirred for 2 h at 100 °C, cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure to give the crude product. [M+H]+ 287.0. To a solution of this crude product in dioxane (4 ml) was added tert-butyl 5-amino-4-(5-bromo-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate (0.289 g, 0.70 mmol). The reaction mixture was purged with argon for five minutes. Bis(1,2-bis(diphenylphosphino)ethane)palladium(0) (0.063 g, 0.07 mmol) was added and heated at 100 °C for 16 h. The reaction mixture was cooled to room temperature, filtered and the filtrate was concentrated under reduced pressure to give the crude product, which was purified by flash chromatography (SiO ₂ column, 80 g, 0-100% B (B = 15% EtOH in EtOAc + 0.5% TEA)/chloroform) to give tert-butyl 5-amino-4-(5-(6-amino-4,5-dimethylpyridin-2-yl)-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate (110 mg, 34.6% yield). LCMS (method A): retention time 1.40 min, [M+H]+ 453.3.

Intermediate 8F. 3-(5-(6-Amino-4,5-dimethylpyridin-2-yl)-3-methyl-1oxoisoindolin-2-yl)piperidine-2,6-dione.

To a stirred solution of tert-butyl 5-amino-4-(5-(6-amino-4,5-dimethylpyridin-2-yl)-3methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate (110 mg, 0.243 mmol, 34.6% yield) in acetonitrile (5 mL) was added p-toluenesulfonic acid (84 mg, 0.442 mmol). The reaction mixture was heated to 120 °C under microwave irradiation for 30 min. The volatiles were removed under reduced pressure and the crude product was purified by preparative HPLC (Column: Hypersil gold c18 (19 X 250 mm), 5 μm Mobile phase A - 10 mM ammonium acetate in water, Mobile phase B: ACNFLOW: 20 mL T/B%: 0/20, 18/80, 19/100, 21/100, 22/20, 24/20). The desired fractions were lyophilized to dryness to give 3-(5-(6-amino-4,5-dimethylpyridin-2-yl)-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione. LCMS (Method D): retention time 2.15 min, [M+H]+ 379.0.

Examples 8 and 9.

3-(5-(6-Amino-4,5-dimethylpyridin-2-yl)-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione (100 mg) was separated by SFC method (column Welk-01(R,R)(250*4.6) mm, 5 μm; % _CO2 : 45%; % cosolvent: 5 mM ammonium acetate in methanol and acetonitrile (1:1); flow rate: 4 g/min; temperature: 30°C; UV: 237 nm), the fractions of the first peak eluted at a retention time of 2.64 min were concentrated and lyophilized to obtain 3-(^)-5-(6-amino-4,5-dimethylpyridin-2-yl)-3-methyl-1oxoisoindolin-2-yl)piperidine-2,6-dione (12 mg, yield 15%). LCMS (method D): retention time 2.112 min, [M+H]+ 379.0; 1H NMR (400 MHz, DMSO-d ₆ ) δ 11.10-10.71 (s, 1H), 8.19 (s, 1H), 8.12 (d, J=7.9 Hz, 1H), 7.69 (d, J=8.0 Hz, 1H), 7.14 (s, 1H), 5.73 (s, 2H), 4.93-4.67 (m, 2H), 2.85-2.71 (m, 2H), 2.25 (s, 3H), 2.04 (s, 3H), 1.51 (d, J=6.6 Hz, 3H) and the second peak fractions eluted at retention time 4.03 min,

- 33 048753 was concentrated and lyophilized to give 3-(^)-5-(6-amino-4,5-dimethylpyridin-2-yl)-3methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione (8 mg, 10% yield). LCMS (method D): retention time 2.162 min, [M+H]+ 379.0; 1H NMR (400 MHz, DMSO-de) δ 11.00-10.89 (s, 1H), 8.18 (s, 1H), 8.13 (d, J=8.0 Hz, 1H), 7.70 (d, J=8.0 Hz, 1H), 7.13 (s, 1H), 5.73 (s, 2H), 4.84-4.68 (m, 2H), 2.91-2.65 (m, 3H), 2.25 (s, 3H), 2.04 (s, 3H), 1.51-1.46 (m, 3H).

Examples 10 and 11. 2-Amino-6-((3S)-2-(2,6-dioxopiperidin-3-yl)-3-methyl-1-oxoisoindolin-5-yl)4-methylnicotinonitrile and 2-amino-6-((3R)-2-(2,6-dioxopiperidin-3-yl)-3-methyl-1-oxoisoindolin-5yl)-4-methylnicotinonitrile

Intermediate connections 10A and 11A.

A stirred solution of 2-amino-6-chloro-4-methylnicotinonitrile (300 mg, 1.790 mmol) and 1,1,1,2,2,2-hexamethyldistannane (762 mg, 2.327 mmol) in toluene (10 mL) was purged with argon for five minutes, after which [1,N-bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II) (117 mg, 0.179 mmol) was added. The reaction mixture was stirred for 2 h at 100 °C, cooled to room temperature, and filtered. The filtrate was concentrated under reduced pressure to give the crude product. [M+H]+ 298.0 The crude material was dissolved in dioxane and intermediate 8D (0.695 g, 1.689 mmol) was added. The reaction mixture was purged with argon for five minutes and bis(diphenylphosphino)ethane)palladium(0) (0.153 g, 0.169 mmol) was added. The reaction mixture was heated to 100 °C and stirred for 16 h. The reaction mixture was cooled to room temperature, filtered and the filtrate was concentrated under reduced pressure to give the crude product, which was purified by flash chromatography (SiO ₂ column, 80 g, 0-100% B (B = 15% EtOH in EtOAc, 0.5% TEA)/chloroform) to give tert-butyl 5-amino-4-(5-(6-amino-5-cyano-4-methylpyridin-2-yl)-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate (200 mg, 0.431 mmol, 25.5% yield). LCMS (method A): retention time 1.47 min, [M+H]+ 464.3.

Examples 10 and 11.

To a stirred solution of tert-butyl 5-amino-4-(5-(6-amino-5-cyano-4-methylpyridin-2-yl)-3methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate (200 mg, 0.431 mmol) in acetonitrile (10 mL) was added p-toluenesulfonic acid (164 mg, 0.863 mmol). The reaction mixture was heated to 120 °C under microwave irradiation for 30 min. The volatiles were removed under reduced pressure and the resulting crude product was purified by preparative HPLC (Column: Hypersil gold c18 (19 X 250 mm), 5 μm Mobile phase A - 10 mM ammonium acetate in water. Mobile phase B: ACN Flow rate: 20 mL S/W%: 0/20, 18/80, 19/100, 21/100, 22/20, 24/20). The desired fractions were lyophilized to dryness to give 2-amino-6-(2-(2,6-dioxopiperidin-3-yl)-3-methyl-1-oxoisoindolin-5-yl)-4methylnicotinonitrile. LCMS (Method D): retention time 1.66 min, [M+H]+ 390.2 as a mixture of diastereomers. The diastereomers were separated by the SFC method (column Welk-01(R,R)(250*4.6) mm, 5 μm; % _CO2 : 45%; % cosolvent: 5 mM ammonium acetate in methanol and acetonitrile (1:1); flow rate: 4 g/min; temperature: 30°C; UV: 237 nm), the first eluting isomer fractions at a retention time of 2.28 min were concentrated to dryness and lyophilized to obtain 2-amino-6-((3S)-2-(2,6dioxopiperidin-3-yl)-3-methyl-1-oxoisoindolin-5-yl)-4-methylnicotinonitrile (20 mg, yield 12%). LCMS (method D): retention time 1.64 min, [M+H]+ 390.0; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.0110.72 (s, 1H), 8.28 (s, 1H), 8.19 (dd, J=8.0, 1.3 Hz, 1H), 7.76 (d, J=8.0 Hz, 1H), 7.35 (s, 1H), 6.92 (s, 2H), 4.944.68 (m, 2H), 2.65-2.58 (m, 2H), 2.44 (s, 3H), 2.14-1.91 (m, 2H), 1.51 (d, J=6.6 Hz, 3H); and the second eluting isomer fractions at a retention time of 3.25 min were concentrated to dryness and lyophilized to give 2-amino-6-((3R)-2-(2,6-dioxopiperidin-3-yl)-3-methyl-1-oxoisoindolin-5-yl)-4methylnicotinonitrile (20 mg, yield 12%). LCMS (method D): retention time 1.66 min, [M+H]+ 390.0; 1H NMR (400 MHz, DMSO-d ₆ ) δ 11.01-10.72 (s, 1H), 8.27 (s, 1H), 8.20 (d, J=8.0 Hz, 1H), 7.78 (d, J=8.0 Hz, 1H), 7.34 (s, 1H), 6.90 (s, 2H), 4.84-4.70 (m, 2H), 2.84-2.67 (m, 2H), 2.44 (s, 3H), 2.14-1.91 (m, 2H), 1.49 (br d, J=6.6 Hz, 3H).

- 34 048753

Examples 12 and 13. 3-(^)-5-(4,5-Dimethyl-6-(methylamino)pyridin-2-yl)-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione and 3-(^)-5-(4,5-dimethyl-6-(methylamino)pyridin-2-yl)-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione

Intermediate connections 12A and 13A.

To a solution of intermediate 8D (350 mg, 0.764 mmol) in dioxane (8 mL) were added 6-chloro-H,3,4-trimethylpyridin-2-amine (130 mg, 0.764 mmol) and 3 M aqueous dibasic potassium phosphate (0.764 mL, 2.291 mmol). The reaction mixture was purged with nitrogen for 15 min at room temperature. _PdCl2 (dppf) _-CH2Cl2 adduct (62.4 mg, 0.076 mmol ₎ was added under nitrogen and the reaction mixture was heated at 100 °C for 12 h. The reaction mixture was cooled to room temperature, filtered through a pad of celite and concentrated under reduced pressure. The residue was purified by flash chromatography ( _SiO2 column, 24 g, 0-10% MeOHVDCM) to give tert-butyl ((3-methyl-6-(methylamino)pyridin-2-yl)-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate (230 mg, 0.493 mmol, 64.6% yield) as a colorless solid. LCMS (method A): retention time 1.81 min, [M+H]+ 467.4.

Examples 12 and 13.

To a stirred solution of tert-butyl 5-amino-4-(5-(4,5-dimethyl-6-(methylamino)pyridin-2-yl)3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate (200 mg, 0.429 mmol) in acetic acid (1 mL) was added benzenesulfonic acid (67.8 mg, 0.429 mmol). The reaction mixture was heated to 120 °C under microwave irradiation for 30 min. The volatiles were removed under reduced pressure and the obtained crude product was purified by preparative HPLC (Column: X Select CSH C18 (250*20 mm) 5 μm Mobile phase: A: 10 mM ammonium acetate B: CAN T/B: 0/20, 18/85, 20/100, 21/20, 23/20 Flow rate: 20 ml/min). The desired fractions were lyophilized to dryness to give 3-(5-(4,5-dimethyl-6(methylamino)pyridin-2-yl)-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione. LCMS (Method A): retention time 1.47 min, [M+H]+ 393.4 as a mixture of diastereomers. The diastereomers were separated by the SFC method (Chiralapak IC column (250*4.6) mm, 5 μm; % CO2: 45%; % cosolvent: 5 mM ammonium acetate in methanol and acetonitrile (1:1); flow rate: 4 g/min; temperature: 30°C; UV: 237 nm), the first eluting isomer fractions at a retention time of 8.65 min were concentrated to dryness and lyophilized to give 3-(^)-5-(4,5-dimethyl-6-(methylamino)pyridin-2-yl)-3-methyl-1oxoisoindolin-2-yl)piperidine-2,6-dione (3 mg, yield 2%), LCMS (method D): retention time 1.743 min, [M+H]+ 393.4; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.07-10.81 (m, 1H), 8.35-8.12 (m, 2H), 7.71 (d, J=8.0 Hz, 1H), 7.14 (s, 1H), 6.03 (br d, J=4.0 Hz, 1H), 4.88-4.69 (m, 2H), 2.96 (d, J=4.5 Hz, 3H), 2.78-2.63 (m, 3H), 2.26 (s, 3H), 2.03 (s, 4H), 1.49 (d, J=6.5 Hz, 3H), and the second peak fractions eluting at a retention time of 11.18 min were concentrated and lyophilized to obtain 3-((P)-5-(4.5-dimethyl-6(methylamino)pyridin-2-yl)-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione (3 mg, yield 2%). LCMS (method D): retention time 1.669 min, [M+H]+ 393.4; 1H NMR (400 MHz, DMSO-d ₆ ) δ 11.0710.81 (m, 1H), 8.35-8.12 (m, 2H), 7.71 (d, J=8.0 Hz, 1H), 7.14 (s, 1H), 6.03 (br d, J=4.0 Hz, 1H), 4.88-4.69 (m, 2H), 2.96 (d, J=4.5 Hz, 3H), 2.78-2.63 (m, 3H), 2.26 (s, 3H), 2.03 (s, 4H), 1.49 (d, J=6.5 Hz, 3H).

Examples 14 and 15. 6-((3S)-2-(2,6-Dioxopiperidin-3-yl)-3-methyl-1-oxoisoindolin-5-yl)-4-methyl2-(methylamino)nicotinonitrile and 6-((3R)-2-(2,6-dioxopiperidin-3-yl)-3-methyl-1-oxoisoindolin-5yl)-4-methyl-2-(methylamino)nicotinonitrile

Intermediate connections 14A and 15A.

To a solution of intermediate 8D (200 mg, 0.436 mmol) in dioxane (8 mL) was added 6-chloro-4-methyl-2-(methylamino)nicotinonitrile (79 mg, 0.436 mmol) followed by K ₃ PO ₄ (0.291 mL, 0.873 mmol of a 3 M aqueous solution). The reaction mixture was purged with nitrogen for 15 min at room temperature.

- 35 048753 type. Xphos Pd G4 (37.6 mg, 0.044 mmol) was added under nitrogen atmosphere and heated at 80 °C for 12 h. The reaction mixture was cooled to room temperature, filtered through celite and concentrated under reduced pressure. The residue was purified by flash chromatography (SiO ₂ column, 24 g, 0-10% MeOH\DCM) to give tert-butyl (4S)-5-αmino-4-(5-(5-cyano-4-methyl-6-(methylamino)pyridin-2yl)-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate (75 mg, 0.157 mmol, 36.0% yield) as a colorless solid. LCMS (method A): retention time 1.59 min, [M+H]+ 478.2.

Examples 14 and 15:

To a solution of tert-butyl (4S)-5-amino-4-(5-(5-cyano-4-methyl-6-(methylamino)pyridin-2-yl)-3-methyl1-oxoisoindolin-2-yl)-5-oxopentanoate (60 mg, 0.126 mmol) in acetonitrile (3 mL) was added benzenesulfonic acid (19.87 mg, 0.126 mmol) at room temperature. The reaction mixture was heated to 120 °C for 45 min in a microwave reactor. The reaction mixture was cooled to room temperature and concentrated under reduced pressure to give the crude product. The crude product was purified by flash chromatography ( _SiO2 column, 24 g, 0-10% MeOH/DCM) to give 6-(2-(2,6-dioxopiperidin-3-yl)-3-methyl-1-oxoisoindolin-5-yl)-4-methyl-2-(methylamino)nicotinonitrile as a colorless solid as a mixture of diastereomers. LCMS (method A): retention time 1.22 min, [M+H]+ 404.1. Diastereomers were separated by the SFC method (column Welk01(R,R)(250*4.6) mm, 5 μm; % _CO2 : 45%; % cosolvent: 5 mM ammonium acetate in methanol and acetonitrile (1:1); flow rate: 4 g/min; temperature: 30°C; UV: 237 nm), the first eluting isomer fractions at a retention time of 2.91 min were concentrated to dryness and lyophilized to obtain 6-((3S)-2(2,6-dioxopiperidin-3-yl)-3-methyl-1-oxoisoindolin-5-yl)-4-methyl-2-(methylamino)nicotinonitrile (6 mg, yield 12%), LCMS (method D): retention time 2.071 min, [M+H]+ 404.2; ¹ H NMR (400 MHz, DMSO-d6) δ 11.27-10.74 (m, 1H), 8.35 (s, 1H), 8.28 (d, J=8.0 Hz, 1H), 7.77 (d, J=8.5 Hz, 1H), 7.37 (s, 1H), 7.12 (br d, J=4.5 Hz, 1H), 4.87 (q, J=6.2 Hz, 1H), 4.81-4.71 (m, 1H), 3.00 (d, J=4.5 Hz, 3H), 2.68 (s, 1H), 2.44 (s, 3H), 1.72 (s, 3H), 1.52 (d, J=7.0 Hz, 3H) and fractions of the second peak eluted at a retention time of 4.74 min, concentrated and lyophilized to give 6-((3R)-2-(2,6-dioxopiperidin-3-yl)-3methyl-1-oxoisoindolin-5-yl)-4-methyl-2-(methylamino)nicotinonitrile (8 mg, 16% yield). LCMS (method D): retention time 2.063 min, [M+H]+ 404.2; 1H NMR (400 MHz, DMSO-d ₆ ) δ 11.08-10.87 (m, 1H), 8.34 (s, 1H), 8.28 (d, J=8.3 Hz, 1H), 7.77 (d, J=8.0 Hz, 1H), 7.36 (s, 1H), 7.19-7.05 (m, 1H), 4.91-4.71 (m, 2H), 2.99 (d, J=4.5 Hz, 3H), 2.76-2.57 (m, 3H), 2.44 (s, 3H), 1.71 (s, 1H), 1.49 (d, J=6.8 Hz, 3H).

Examples 16 and 17. 3-(^)-5-(6-Amino-4,5-dimethylpyridin-2-yl)-4-fluoro-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione and 3-(^)-5-(6-amino-4,5-dimethylpyridin-2-yl)-4-fluoro-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione

Intermediate 16A. 4-Bromo-2-ethyl-3-fluorobenzoic acid.

A solution of 4-bromo-2,3-difluorobenzoic acid (2.0 g, 8.44 mmol) in tetrahydrofuran (40 mL) was cooled to -78 °C and 1 M ethyl magnesium bromide in THF (8.44 mL, 25.3 mmol) was added dropwise. The reaction mixture was allowed to reach room temperature and stirred under nitrogen for 12 h. The reaction was quenched by adding MeOH (15 mL) dropwise at 0 °C. The volatiles were removed under reduced pressure and the residue was partitioned between EtOAc and 2 M aqueous HCl. The layers were separated and extracted with EtOAc. The combined organic layer was washed with brine, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give the crude product. The crude product was purified by flash chromatography ( _SiO2 column, 40 g, 0.80% EtOAc/pet ether) to afford 4-bromo-2-ethyl-3-fluorobenzoic acid (1 g, 48% yield). LCMS (method A): retention time 0.69 min, [M-H] ⁺ 245.1, 247.1; 1H NMR (300 MHz, DMSO- _d6 ) δ 13.36 (s, 1H), 7.68-7.53 (m, 2H), 2.95 (qd, J = 7.4, 2.6 Hz, 2H), 1.24-1.06 (m, 3H).

Intermediate 16B. Methyl 4-bromo-2-ethyl-3-fluorobenzoate.

To a stirred mixture of 4-bromo-2-ethyl-3-fluorobenzoic acid (0.7 g, 2.83 mmol) and K2CO3 (0.783 g, 5.67 mmol) in acetone (15 mL) at room temperature was added dimethyl sulfate (0.541 mL, 5.67 mmol). The reaction mixture was stirred at 50 °C for 14 h and filtered through a celite pad. The filtrate was concentrated in vacuo and purified by flash chromatography (SiCh column, 24 g, 0-50% EtOAc/pet ether) to give methyl 4-bromo-2-ethyl-3-fluorobenzoate (0.51 g, 69% yield) as a colorless oil. 1H NMR (300 MHz, CHLOROFORM-d) δ 7.58-7.51 (m, 1H), 7.49-7.37 (m, 1H), 3.90 (s, 3H), 3.01 (qd, J = 7.4, 2.6 Hz, 2H), 1.23 (t, J = 7.4 Hz, 3H).

- 36 048753

Intermediate 16C. Methyl 4-bromo-2-(1-bromoethyl)-3-fluorobenzoate.

To a stirred solution of methyl 4-bromo-2-ethyl-3-fluorobenzoate (0.515 g, 1.972 mmol) in DCE (10 mL) was added NBS (0.386 g, 2.170 mmol) followed by AIBN (0.065 g, 0.394 mmol). The reaction mixture was heated at 85 °C for 15 h. The reaction mixture was diluted with ethyl acetate, washed with saturated 10% sodium thiosulfate solution and brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The resulting residue was purified by flash chromatography ( _SiO2 column, 24 g, 0-30% EtOAc/pet ether) to give methyl 4-bromo-2-(1-bromoethyl)-3-fluorobenzoate (0.6 g, 89% yield) as a white solid. ¹ H NMR (300 MHz, CHLOROFORM-d) δ 7.70-7.53 (m, 1H), 7.52-7.44 (m, 1H), 6.16-5.87 (m, 1H), 3.93 (s, 3H), 1.95 (dd, J = 7.0, 1.3 Hz, 3H).

Intermediate 16D. tert-Butyl (4S)-5-amino-4-(5-bromo-4-fluoro-3-methyl-1oxoisoindolin-2-yl)-5-oxopentanoate.

To a stirred solution of methyl 4-bromo-2-(1-bromoethyl)-3-fluorobenzoate (0.86 g, 2.53 mmol) and H-Glu(OtBu)-NH ₂ HCl (0.845 g, 3.54 mmol) in acetonitrile (15 mL) was added DIPEA (1.325 mL, 7.59 mmol). The reaction mixture was heated at 85 °C for 15 h. The volatiles were removed under reduced pressure and purified by flash chromatography (SiO ₂ column, 40 g, 0-10% MeOH/DCM) to give tert-butyl (4S)-5-amino-4-(5-bromo-4-fluoro-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate (0.23 g, 21% yield) as a pale yellow solid. LCMS (method A): retention time 1.19 min, [M+23H]+ 451.3, 453.4.

Intermediate 16E. tert-Butyl-(4S)-5-amino-4-(5-(6-amino-4,5-dimethylpyridin-2-yl)4-fluoro-3-methyl-1-oxoisoindolin-2-yl)-5 -oxopentanoate.

To a stirred solution of tert-butyl ((3-dimethyl)-5-amino-4-(5-bromo-4-fluoro-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate (350 mg, 0.815 mmol) in 1,4-dioxane (20 mL) was added 3,4-dimethyl-6-(trimethylstannyl)pyridin-2-amine (232 mg, 0.815 mmol). The mixture was purged with argon for five minutes and bis(triphenylphosphine)palladium(II) chloride (57.2 mg, 0.082 mmol) was added. The reaction mixture was heated at 100 °C for 16 h, cooled to room temperature and diluted with ethyl acetate. The mixture was washed with brine, the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give the crude compound. The crude compound was purified by flash chromatography ( _SiO2 column, 24 g, 0-5% MeOH/DCM), the combined product fractions were concentrated under reduced pressure to give tert-butyl (4S)-5-amino4-(5-(6-amino-4,5-dimethylpyridin-2-yl)-4-fluoro-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate (270 mg, 70.4% yield) as a pale brown solid. LCMS (method A): retention time 1.64 min, [M+H]+ 471.2 ^1H NMR (400 MHz, DMSO-d6) δ 7.99 (t, J=6.9 Hz, 1H), 7.59 (s, 1H), 7.47 (br s, 1H), 7.30-7.13 (m, 2H), 6.90 (s, 1H), 5.74 (br d, J=5.3 Hz, 2H), 4.89 (q, J=6.1 Hz, 1H), 4.50-4.37 (m, 1H), 2.31-2.19 (m, 6H), 2.04 (s, 3H), 1.55-1.45 (m, 3H), 1.37 (d,J=2.9 Hz, 9H).

Examples 16 and 17.

To a stirred solution of tert-butyl (4S)-5-amino-4-(5-(6-amino-4,5-dimethylpyridin-2-yl)-4fluoro-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate (270 mg, 0.574 mmol) in acetonitrile (10 mL) was added PTSOH (218 mg, 1.148 mmol). The reaction mixture was heated at 120 °C under microwave irradiation for 30 min. The volatiles were removed under reduced pressure and the resulting crude product was purified by preparative HPLC using polar organics (CELLULOSE-2 [250 x 4.6 mm], 10 mM ammonium acetate in MeOH, flow: 23 ml/min (isocratic gradient)), the first eluting isomer fractions at a retention time of 9.73 min were concentrated to dryness and lyophilized to give 3-(^)-5-(6-amino-4,5-dimethylpyridin-2-yl)-4fluoro-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione (60 mg, yield 27%). LCMS (method G): retention time 1.83 min, [M+H]+397.1; 1H NMR (400 MHz, DMSO-d ₆ ) δ 11.10-10.87 (m, 1H), 8.01 (t, J=7.3 Hz, 1H), 7.57 (d, J=8.0 Hz, 1H), 6.92 (d, J=2.0 Hz, 1H), 5.78 (s, 2H), 5.01 (q, J=6.5 Hz, 1H), 4.73 (br dd, J=12.3, 4.8 Hz, 1H), 2.82-2.67 (m, 1H), 2.64-2.55 (m, 2H), 2.23 (s, 3H), 2.04 (s, 3H), 1.54 (d, J=7.0 Hz, 3H). The fractions of the second eluted peak at a retention time of 13.96 min were concentrated to dryness and lyophilized to give 3-((R)-5-(6-amino-4,5-dimethylpyridin-2-yl)-4-fluoro-3-methyl-1-oxoisoindolin2-yl)piperidine-2,6-dione (70 mg, 31% yield). LCMS (method G): retention time 1.84 min, [M+H]+ 397.1; ^1H NMR (400 MHz, DMSO-d6) δ 11.07-10.89 (m, 1H), 8.01 (t, J=7.1 Hz, 1H), 7.58 (d, J=8.0 Hz, 1H), 6.91 (s, 1H), 5.79 (s, 2H), 4.89 (q, J=6.5 Hz, 1H), 4.80 (dd, J=12.6, 5.1 Hz, 1H), 2.94-2.78 (m, 1H), 2.73-2.59 (m, 2H), 2.22 (s, 3H), 2.03 (s, 3H), 1.51 (d, >6.8 Hz, 3H).

Method A: ACQUITY UPLC® VEN C18 (3.0 x 50 mm) 1.7 µm. Mobile phase A: 95:5 water:acetonitrile with 2.5 mM NH ₄ OAc. Mobile phase B: 5:95 water:acetonitrile with 2.5 mM NH ₄ OAc; temperature: 40°C; gradient: 20% B to 100% B in 2 min; flow rate: 0.7 mL/min; detection: MS and UV (220 nm).

Method B: Column: XBridge BEH XP C18 (50 x 2.1) mm, 2.5 μm. Mobile phase A: 95:5 water:acetonitrile with 10 mM NH4OAc. Mobile phase B: 5:95 water:acetonitrile with 10 mM _NH4OAc ; temperature: 50°C; gradient: 0% B to 100% B in 3 min; flow rate: 1.1 mL/min; detection: MS and UV (220 nm).

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Method D: Column-Kinetex XB-C18 (75 X 3 mm-2.6 μm). Mobile phase A: 5 mM NH ₄ COOH in water. Mobile phase B: acetonitrile; gradient: 10% B to 50% B over 3 min, flow: 1.0 mL/min; 50% B to 100% B for 4.1 min, flow: 1.0 mL/min; delay up to 4.5 min; from 4.5 min to 5.0 min 90% B flow: 1.5 mL/min; detection: MS and UV (220 nm).

Method G: Column: Kinetex XB-C18 (75 x 3 mm-2.6 μm). Mobile phase A: 5 mM NH ₄ CO ₂ H in water. Mobile phase B: acetonitrile; gradient: 20% B to 100% B in 4 min, flow: 1.0 mL/min; retention up to 4.6 min, flow: 1.5 mL/min; retention up to 4.7 min; from 4.7 min to 5.0 min 20% B, flow: 1.0 mL/min; detection: MS and UV (220 nm).

Example 18 3-(5-(6-Amino-3-methylpyridin-2-yl)-4-fluoro-1-oxoisoindolin-2-yl)piperidin-2,6dione

Example 18 was synthesized from 6-chloropyridin-2-amine and intermediate 3B according to General Procedure 2. The crude product was purified by preparative LCMS (column: Waters XBridge C18, 150 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; mobile phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; gradient: 0 min hold at 10% B, 10-30% B for 20 min, then 5 min hold at 100% B; flow rate: 20 mL/min. LCMS (method B): retention time 0.98 min, [M+H]+ 369.1; 1H NMR (400 MHz, DMSO-d ₆ ) δ 11.04 (s, 1H), 7.77 (br d, J= 7.6 Hz, 2H), 7.74-7.68 (m, 1H), 6.93-6.87 (m, 1H), 5.17 (dd, J= 13.3, 5.1 Hz, 1H), 4.67 (d, J= 17.6 Hz, 1H), 4.55-4.47 (m, 1H), 3.01-2.89 (m, 2H), 2.67-2.61 (m, 1H), 2.47-2.39 (m, 1H), 2.04 (s, 3H).

General procedure 4.

A mixture of aryl halide (1 equiv), aryl boronic acid pinacol ester (1.0 equiv), potassium carbonate (1.5 equiv), dioxane (4 mL/mmol), and water (0.4 mL/mmol) was purged with argon for 5 min at room temperature. [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloromethane complex was added and the reaction mixture was heated at 110 °C for 2 h. The reaction mixture was cooled to room temperature, diluted with EtOAc, and filtered through a pad of celite. The filtrate was washed with brine, dried over anhydrous Na ₂ SO ₄ , filtered, and concentrated under reduced pressure. The residue was purified by flash chromatography. The isolated product was dissolved in acetonitrile, pTSA- _H2O (2 equiv) was added and the mixture was heated at 120°C for 1.5 h in a microwave reactor. The reaction mixture was cooled to room temperature, concentrated under reduced pressure and the crude product was purified by preparative HPLC to afford the desired product.

Example

19.

yl)piperidine-2,6-dione

-(5 -(6-Amino-5-fluoro-4-methylpyridin-2-yl)-4-fluoro-1-oxoisoindolin-2-

Intermediates 19A and 19B. 6-Bromo-3-fluoro-4-methylpyridin-2-amine and 6-bromo-5-fluoro-4-methylpyridin-2-amine.

To a solution of 6-bromo-4-methylpyridin-2-amine (2.6 g, 13.90 mmol) in chloroform (100 mL) and water (100 mL) was added selectfluoro (2.462 g, 6.95 mmol). The reaction mixture was stirred at room temperature for 48 h. The reaction mixture was diluted with DCM (200 mL), washed with brine, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (SiO ₂ column, 80 g, 0-30%

EtOAc/petroleum ether) to give intermediate 19A: 6-bromo-3-fluoro-4-methylpyridin-2-amine (600 mg, 19.6% yield); LCMS (method A): retention time 1.21 min, [M+H]+ 207.1. 1H NMR (300 MHz, DMSO-d ₆ ) δ 6.60 (d, J=4.2 Hz, 1H), 6.55 (s, 2H), 2.13 (d, J=1.9 Hz, 3H); and intermediate 19B: 6-bromo-5-fluoro-4-methylpyridin-2-amine (500 mg, 4.4% yield); LCMS (method A): retention time 1.14, [M+H]+ 207.1; ¹ H NMR (300 MHz, CHLOROFORM-d) δ 6.25 (d, J=4.2 Hz, 1H), 4.52-4.22 (m, 2H), 2.23 (d, J=1.1 Hz, 3H).

- 38 048753

Example 19.

Example 19 was synthesized according to General Procedure 4 using 6-bromo-3-fluoro-4-methylpyridin-2-amine and tert-butyl ((4-fluoro-1-oxo-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoindolin-2-yl)-5-oxopentanoate (synthesized according to the procedure given in Example 2, starting from 4-bromo-3-fluoro-2-methylbenzoic acid). The crude product was purified by preparative LCMS (column: YMC EXRS 250 mm x 21 mm, mobile phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; mobile phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; gradient: 0 min hold at 10% B, 10-30% B for 20 min, then 5 min hold at 100% B; flow rate: 20 mL/min. LCMS (method D): retention time 1.56 min, [M+H]+ 387.15; ¹ H NMR (400 MHz, DMSO-de) δ 11.02 (s, 1H), 8.09-7.92 (m, 1H), 7.66 (d, J=8.0 Hz, 1H), 7.32-6.78 (m, 1H), 5.14 (dd, J=13.8, 5.8 Hz, 1H), 4.74-4.33 (m, 2H), 2.98-2.86 (m, 1H), 2.68-2.56 (m, 2H), 2.24 (s, 3H), 22.09-2.05 (m, 1H).

Example ²⁰

Example 20 was synthesized according to General Procedure 4 using 6-bromo-5-fluoro-4-methylpyridin-2-amine and tert-butyl ((4-fluoro-1-oxo-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoindolin-2-yl)-5-oxopentanoate (synthesized according to the procedure given in Example 2, starting from 4-bromo-3-fluoro-2-methylbenzoic acid). The crude product was purified by preparative LCMS (column: Waters XBridge C18, 150 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; mobile phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; gradient: 0 min hold at 10% B, 10-30% B for 20 min, then 5 min hold at 100% B; flow rate: 20 mL/min. LCMS (method B): retention time 1.03 min, [M+H]+ 387.1; 1H NMR (400 MHz, DMSO-d6) δ 11.04 (s, 1H), 7.74-7.57 (m, 2H), 6.46 (d, J=4.5 Hz, 1H), 5.97 (s, 2H), 5.16 (dd, J=5.0, 13.3 Hz, 1H), 4.65 (d, J=17.5 Hz, 1H), 4.52-4.42 (m, 1H), 3.032.86 (m, 1H), 2.68-2.59 (m, 1H), 2.49-2.41 (m, 1H), 2.21 (s, 3H), 2.12-1.99 (m, 1H).

Examples 21 and 22. 3-(5-(6-Amino-5-fluoro-4-methylpyridin-2-yl)-4-fluoro-3-methyl-1-oxoisoindolin2-yl)piperidine-2,6-dione

Intermediate 21A. tert-Butyl (4S)-5-amino-4-(3-methyl-1-oxo-5-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)isoindolin-2-yl)-5-oxopentanoate.

A mixture of intermediate 8D (1.0 g, 2.43 mmol), potassium acetate (0.239 g, 2.43 mmol), and bispin (0.617 g, 2.43 mmol) in anhydrous DME (15 mL) was purged with _argon for 10 min at room temperature. Under argon atmosphere, the adduct _PdCl2 (dppf) _-CH2Cl2 (0.159 g, 0.195 mmol) was added. The tube was sealed and the mixture was heated at 90 °C for 12 h. The reaction mixture was cooled to room temperature, filtered through a pad of celite, and the filtrate was concentrated under reduced pressure. The residue was dissolved in diethyl ether and filtered through a pad of celite. The filtrate was concentrated under reduced pressure to give tert-butyl (4S)-5-amino-4(3-methyl-1-oxo-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoindolin-2-yl)-5-oxopentanoate (1.0 g, yield 90%). LCMS (method A): retention time 1.70 min, [M+H]+ 459.1.

Getting 21V and 22V. tert-Butyl-(4S)-5-amino-4-(5-(6-amino-5-fluoro-4-methylpyridin-2-yl)-3methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate.

To a solution of tert-butyl ((D))-5-amino-4-(3-methyl-1-oxo-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2yl)isoindolin-2-yl)-5-oxopentanoate (0.241 g, 0.527 mmol) in dioxane (10 mL) were added 6-bromo-3-fluoro-4-methylpyridin-2-amine (0.09 g, 0.44 mmol) and sodium bicarbonate (0.5 M solution, 2.195 mL, 1.097 mmol). The reaction mixture was purged with nitrogen for 15 min at room temperature, bis(triphenylphosphine)palladium(II) chloride (0.031 g, 0.044 mmol) was added under nitrogen and heated at 100 °C for

- 39 048753 for 12 h. The reaction mixture was cooled to room temperature, filtered through a pad of celite and concentrated under reduced pressure. The residue was purified by flash chromatography (S1O2 column, 24 g, 50-100% EtOAc/DCM) to give tert-butyl (4S)-5-amino-4-(5-(6-amino-5-fluoro-4-methylpyridin-2-yl)-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate (150 mg) as a mixture of diastereomers. Separation of diastereomers was performed by the SFC method (column Chiral Pak IG (250*4.6) mm, 5 μm; % CO2: 45%; % cosolvent: 5 mM ammonium acetate in methanol and acetonitrile (1:1); flow rate: 4 g/min; temperature: 30°C; UV: 237 nm), the first eluted isomer fractions at a retention time of 3.4 min were concentrated to dryness to obtain tert-butyl (4S)-5-amino-4-(5-(6-amino-5-fluoro-4-methylpyridin-2-yl)-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate (40 mg, yield 20%). LCMS (method A): retention time 1.41, [M+H]+ 457.1. The fractions of the second peak eluting at a retention time of 4.6 min were concentrated to give tert-butyl (4S)-5-amino-4-(5-(6-amino-5-fluoro-4-methylpyridin-2-yl)-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate (50 mg, yield 25%). LCMS (method A): retention time 1.40, [M+H]+ 457.4.

Example 21.

To a stirred solution of (4S)-5-amino-4-(5-(6-amino-5-fluoro-4-methylpyridin-2-yl)-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate (0.04 g, 0.088 mmol) in acetonitrile (10 mL) was added benzenesulfonic acid (0.028 g, 0.175 mmol). The reaction mixture was heated at 120 °C under microwave irradiation for 30 min. The reaction mixture was cooled to room temperature, concentrated under reduced pressure and purified by preparative LCMS (column: Waters XBridge C18, 150 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; mobile phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; gradient: 0 min hold at 10% B, 10-30% B for 20 min, then 5 min hold at 100% B; flow rate: 20 mL/min. LCMS (method B): retention time 1.19 min, [M+H]+ 383.1; ¹ H NMR (400 MHz, DMSO-d6) δ 10.95 (s, 1H), 8.14 (s, 1H), 8.07 (dd, J=1.1, 8.1 Hz, 1H), 7.72 (d, J=8.0 Hz, 1H), 7.17 (d, J=4.4 Hz, 1H), 4.82-4.67 (m, 2H), 2.93-2.79 (m, 1H), 2.71-2.59 (m, 2H), 2.26 (s, 3H), 2.05-1.96 (m, 1H), 1.48 (d, J=6.6 Hz, 3H).

Example 22.

To a stirred solution of (4S)-5-amino-4-(5-(6-amino-5-fluoro-4-methylpyridin-2-yl)-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate (0.04 g, 0.088 mmol) in acetonitrile (10 mL) was added benzenesulfonic acid (0.028 g, 0.175 mmol). The reaction mixture was heated at 120 °C under microwave irradiation for 30 min. The reaction mixture was cooled to room temperature, concentrated under reduced pressure and the residue was purified by preparative LCMS (column: Waters XBridge C18, 150 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; mobile phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; gradient: 0 min hold at 10% B, 10-30% B for 20 min, then 5 min hold at 100% B; flow rate: 20 mL/min. LCMS (method B): retention time 1.19 min, [M+H]+ 383.1; 1H NMR (400 MHz, DMSO-d6) δ 10.92 (s, 1H), 8.16 (s, 1H), 8.07 (dd, J=1.1, 8.0 Hz, 1H), 7.70 (d, J=8.0 Hz, 1H), 7.17 (d, J=4.3 Hz, 1H), 6.26 (br s, 2H), 4.83 (q, J=6.6 Hz, 1H), 4.74 (br dd, J=4.3, 11.3 Hz, 1H), 2.83-2.71 (m, 1H), 2.642.55 (m, 2H), 2.25 (d, J=1.5 Hz, 3H), 2.09-1.96 (m, 1H), 1.50 (d, J=6.6 Hz, 3H).

Example 23. 2-Amino-6-((3R)-2-(2,6-dioxopiperidin-3-yl)-4-fluoro-3-methyl-1-oxoisoindolin-5yl)-4-methylnicotinonitrile

Oh oh

nh ₂

Example 23 was synthesized from 2-amino-6-chloro-4-methylnicotinonitrile and tert-butyl ((3)-5-amino-4(3)-4-fluoro-3-methyl-1-oxo-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoindolin-2-yl)-5-oxopentanoate (synthesized according to the procedure given in Example 2, starting from 4-bromo-3-fluoro-2-methylbenzoic acid) by General Procedure 4. The crude product was purified by preparative LCMS (column: Waters XBridge C18, 150 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; mobile phase B: 95:5 Acetonitrile: water with 0.1% trifluoroacetic acid; gradient: 0 min hold at 10% B, 10-30% B for 20 min, then 5 min hold at 100% B; flow rate: 20 mL/min. LCMS (method B): retention time 1.19 min, [M+H]+ 408.0; 1H NMR (400 MHz, DMSO-d6) δ 11.00 (s, 1H), 8.17-7.85 (m, 1H), 7.73-7.52 (m, 1H), 7.05 (s, 1H), 6.99 (s, 2H), 5.08-4.89 (m, 1H), 4.86-4.72 (m, 1H), 2.90-2.75 (m, 1H), 2.74-2.58 (m, 2H), 2.42 (s, 3H), 2.12-2.00 (m, 1H), 1.57-1.47 (m, 3H).

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Example 24. yl)piperidine-2,6-dione

3-(^)-5-(6-Amino-4-methylpyridin-2-yl)-4-fluoro-3-methyl-1-oxoisoindolin-2-

Example 24 was synthesized from 6-chloro-4-methylpyridin-2-amine and tert-butyl ((3)-5-amino-4-(3)-4fluoro-3-methyl-1-oxo-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoindolin-2-yl)-5-oxopentanoate (synthesized according to the procedure given in Example 2, starting from 4-bromo-3-fluoro-2-methylbenzoic acid) by General Procedure 4. The crude product was purified by preparative LCMS (column: Waters XBridge C18, 150 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; mobile phase B: 95:5 Acetonitrile: water with 0.1% trifluoroacetic acid; gradient: 0 min hold at 10% B, 10-30% B for 20 min, then 5 min hold at 100% B; flow rate: 20 mL/min. LCMS (method B): retention time 1.20 min, [M+H]+ 383.2; 1H NMR (400 MHz, DMSO-d6) δ 11.00 (d, J=0.8 Hz, 1H), 8.01-7.84 (m, 1H), 7.71 (d, J=7.8 Hz, 1H), 6.96 (br d, J=1.0 Hz, 1H), 6.71-6.54 (m, 1H), 4.99-4.91 (m, 1H), 4.87-4.78 (m, 1H), 2.86 (br dd, J=4.4, 3.4 Hz, 1H), 2.752.62 (m, 2H), 2.47-2.44 (m, 1H), 2.34 (s, 4H), 1.94 (d, J=16.0 Hz, 1H), 1.53 (d, J=6.8 Hz, 3H).

Example 25. 3-(^)-5-(6-Amino-4-(trifluoromethyl)pyridin-2-yl)-4-fluoro-3-methyl-1-oxoisoindolin2-yl)piperidine-2,6-dione

Example 25 was synthesized from 6-chloro-4-(trifluoromethyl)pyridin-2-amine and tert-butyl ((3)-5-amino-4(3)-4-fluoro-3-methyl-1-oxo-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoindolin-2-yl)-5-oxopentanoate (synthesized according to the procedure given in Example 2, starting from 4-bromo-3-fluoro-2-methylbenzoic acid) by General Procedure 4. The crude product was purified by preparative LCMS (column: Waters XBridge C18, 150 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; mobile phase B: 95:5 Acetonitrile: water with 0.1% trifluoroacetic acid; gradient: 0 min hold at 10% B, 10-30% B for 20 min, then 5 min hold at 100% B; flow rate: 20 mL/min. LCMS (method B): retention time 1.39 min, [M+H]+ 438.1; 1H NMR (400 MHz, DMSO-d6) δ 10.99 (s, 1H), 8.13-7.95 (m, 1H), 7.65 (d, J=7.8 Hz, 1H), 7.15 (s, 1H), 6.79 (s, 2H), 4.98-4.88 (m, 1H), 4.86-4.77 (m, 1H), 2.85 (br s, 1H), 2.93-2.78 (m, 1H), 2.72-2.59 (m, 2H), 2.08-2.00 (m, 1H), 1.52 (d, J=6.8 Hz, 3H).

Example 26. 3-(^)-5-(6-Amino-3-methylpyridin-2-yl)-4-fluoro-3-methyl-1-oxoisoindolin-2yl)piperidine-2,6-dione

Example 26 was synthesized from 6-chloro-5-methylpyridin-2-amine and tert-butyl ((3)-5-amino-4-((3)-4fluoro-3-methyl-1-oxo-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoindolin-2-yl)-5-oxopentanoate (synthesized according to the procedure given in Example 2, starting from 4-bromo-3-fluoro-2-methylbenzoic acid) by General Procedure 4. The crude product was purified by preparative LCMS (column: Waters XBridge C18, 150 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; mobile phase B: 95:5 Acetonitrile: water with 0.1% trifluoroacetic acid; gradient: 0 min hold at 10% B, 10-30% B for 20 min, then 5 min hold at 100% B; flow rate: 20 mL/min. LCMS (method B): retention time 1.07 min, [M+H]+ 383.1; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.01 (s, 1H), 7.90-7.77 (m, 1H), 7.74-7.61 (m, 2H), 6.86 (br dd, J=5.5, 3.9 Hz,

- 41 048753

1H), 4.96 (q, J=6.5 Hz, 1H), 4.84 (dd, J=12.6, 5.1 Hz, 1H), 3.01-2.82 (m, 2H ), 2.71-2.60 (m, 2H), 2.12-2.01 (m, 5H), 1.51 (d, J=6.6 Hz, 3H).

Example 27. 3-(CH)-5-(6-Aminopyridin-2-yl)-4-fluoro-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione

Example 27 was synthesized from 6-chloropyridin-2-amine and tert-butyl ((3-amino)-4-(1H)-4-fluoro-3methyl-1-oxo-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoindolin-2-yl)-5-oxopentanoate (synthesized according to the procedure of Example 2, starting from 4-bromo-3-fluoro-2-methylbenzoic acid) according to General Procedure 4. The crude product was purified by preparative LCMS (Column: Waters XBridge C18,

150 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; mobile phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; gradient: 0 min hold at 10% B, 10-30% B for 20 min, then 5 min hold at 100% B; flow rate: 20 mL/min. LCMS (method B): retention time 1.01 min, [M+H]+ 369.1; ¹ H NMR(400 MHz, DMSO-d6) δ 7.95 (t, J=7.1 Hz, 1H), 7.81-7.62 (m, 2H), 7.22 (s, 1H), 7.09 (s, 1H), 7.04 (br d, J=7.0 Hz, 1H), 6.97 (s, 1H), 6.78-6.72 (m, 1H), 4.93 (q, J=6.5 Hz, 1H), 4.82 (dd, J=12.4, 5.3 Hz, 1H), 2.91-2.83 (m, 1H), 2.75-2.60 (m, 2H), 2.36-2.32 (m, 1H), 1.52 (d, J=6.8 Hz, 3H).

Example 28 dio n

To a stirred solution of Example 17 (250 mg, 0.531 mmol) in acetonitrile (4 mL) was added TFA (1 mL, 13.81 mmol). The reaction mixture was heated at 90 °C for 2 h. The reaction mixture was concentrated under reduced pressure and the crude product was purified by preparative SFC (Chiral Pak IC (250 X 50) mm, 5 μm; % _CO2 : 50%; % cosolvent: 50% 5 mM ammonium acetate in ACN:MEOH (50:50); flow rate: 300.0 g/min; temperature: 40 °C; UV: 240 nm), the first eluting isomer fractions at a retention time of 6.9 min were concentrated to dryness and lyophilized to afford (N)-3-(N)-5-(6-amino-4,5-dimethylpyridin-2-yl)-4-fluoro-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione (15 mg, yield 7%). LCMS (method D): retention time 1.28 min, [M+H]+ 397.1; 1H NMR (400 MHz, DMSO-d ₆ ) δ 10.97-10.89 (m, 1H), 7.99 (br d, J=6.8 Hz, 1H), 7.57 (d, J=8.0 Hz, 1H), 6.91 (s, 1H), 5.80 (s, 2H), 5.00 (q, J=6.7 Hz, 1H), 4.76-4.67 (m, 1H), 2.82-2.70 (m, 1H), 2.67-2.55 (m, 2H), 2.22 (s, 3H), 2.03 (s, 4H), 1.53 (d, J=6.8 Hz, 3H).

Example 29 n

Example 29 was synthesized from 6-chloro-3-fluoro-4-methylpyridin-2-amine and tert-butyl ((3-fluoro)-5-amino4-(1H)-4-fluoro-3-methyl-1-oxo-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoindolin-2-yl)-5-oxopentanoate (synthesized according to the procedure given in Example 2, starting from 4-bromo-3-fluoro-2-methylbenzoic acid) by General Procedure 4. The crude product was purified by preparative LCMS (column: Waters XBridge C18, 150 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; mobile phase B: 95:5 acetonitrile: water with 0.1% trifluoroacetic acid; gradient: 0-min hold at 10% B, 10-30% B for 20 min, then 5-min hold at 100% B; flow rate: 20 mL/min. LCMS (method B): retention time 1.18 min,

- 42 048753

[M+H]+ 401.3; 1H NMR (400 MHz, DMSO-d ₆ ) δ 10.98 (s, 1H), 7.95 (t, J=7.1 Hz, 1H), 7.60 (d, J=7.8 Hz, 1H), 6.93 (d, J=3.3 Hz, 1H), 4.90 (q, J=6.6 Hz, 1H), 4.80 (dd, J=4.9, 12.9 Hz, 1H), 2.89-2.78 (m, 1H), 2.70-2.60 (m, 2H), 2.24 (s, 3H), 2.07-1.97 (m, 1H), 1.51 (d, J=6.5 Hz, 3H).

Example 30. 3-((I)-5-(6-Amino-5-fluoro-4-methylpyridin-2-yl)-4-fluoro-3-methyl-1-oxoisoindolin-2yl)piperidine-2,6-dione

Preparation of 30A. 3-Fluoro-4-methyl-6-(trimethylstannyl)pyridin-2-amine.

A stirred solution of 6-bromo-3-fluoro-4-methylpyridin-2-amine (0.05 g, 0.24 mmol) and hexamethylditin (0.076 mL, 0.366 mmol) in toluene (3 mL) was purged with argon for five minutes, after which [1,G-bis-(di-tert-butylphosphino)ferrocene]dichloropalladium(II) (0.016 g, 0.024 mmol) was added. The reaction mixture was stirred for 15 h at 100 °C, cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure to give 3-(trimethyl-4-methyl-6-(trimethylstannyl)pyridin-2-amine (69 mg, 82% yield). LCMS (method A): retention time 1.68, [M+H]+ 289.2.

Example 30.

The Stille coupling reaction and cyclization were carried out according to General Procedure 2 using intermediate 30A and intermediate 16D. LCMS (Method D): retention time 1.17 min, [M+H] ⁺ 401.3; 1H NMR (400 MHz, DMSO-d6) δ 10.94 (s, 1H), 7.94 (t, J=7.3 Hz, 1H), 7.58 (d, J=8.0 Hz, 1H), 6.93 (d, J=3.3 Hz, 1H), 6.31 (br s, 2H), 5.01 (q, J=6.6 Hz, 1H), 4.72 (dd, J=4.9, 11.9 Hz, 1H), 2.79-2.67 (m, 1H), 2.64-2.55 (m, 2H), 2.24 (s, 3H), 2.07-1.98 (m, 1H), 1.53 (d, J=6.8 Hz, 3H).

Biological tests.

The pharmacological properties of the compounds of the present invention can be confirmed by a number of biological assays. The following examples of biological assays were carried out with the compounds of the invention.

Helios Cellular Degradation Assay.

Jurkat cells were seeded at 80,000 cells/well in 40 µl RPMI + 10% FBS in a 384-well cell culture plate before using acoustic dosing technology to add the compound of interest. Cell cultures were incubated for 72 h at 37°C and 5% _CO2 . To facilitate analysis, cell cultures were centrifuged at 200 rpm for 5 min and the supernatant was discarded. After shaking the plate to remove cell pellets, cells were resuspended in 50 µl fixation buffer (eBioScience FoxP3 Buffer Kit 00-5523-00) for 60 min at room temperature. After centrifugation and discarding the supernatant, the cells were permeabilized in 50 µl permeabilization buffer (eBioScience FoxP3 buffer set 00-552300) for 10 min at room temperature. After permeabilization, the cells were centrifuged and the supernatant was replaced with 20 μl of fluorescently labeled antibodies against Helios, Ikaros, and Aiolos or the corresponding isotope controls in 1x permeabilization buffer (Ikaros-Alexa488 [Biolegend, Cat #368408, 1:50], Helios-PE [CST, Cat #29360, 1:50], Aiolos-Alexa647 [Biolegend, Cat #371106Biolegend, 1:25]), and the staining reactions were incubated for 1 h at room temperature, protected from light. Then, 30 μl of 1x permeabilization buffer was added before centrifugation of the cells and the supernatant was discarded. Stained cells were resuspended in 25 µl flow cytometry buffer (PBS + 0.2% BSA) and analyzed on an Intellicyt Ique Plus flow cytometer.

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Table 4

No. example Helios Jurkat IC50 (µM) Ikaros Jurkat IC50 (µM) 1 1,4 >10 3 0,003 >10 7 0,001 >10 9 0,003 >10 10 0.31 >10 AND 0,006 >10 12 0,033 >10 13 2.8 >10 14 1,0 >10

15 0,057 >10 16 0.56 >10 17 0,007 >10 19 0,0035 >10 20 490 >10 21 0,0058 >10 22 0,20 >10 23 0,035 >10 24 0.16 >10 25 1,3 >10 29 0,024 >10 30 0,70 >10

Claims (15)

1. Compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R ₁ is -NH2; each R2 independently represents F, -CN, -СН3, -CF3; each R ₄ independently represents F or -CH3; R ₆ represents hydrogen or -CH3; - 44 048753 m is 1 and n is 0, 1 or 2. 2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein I ₆ is -CH3. 3. A compound according to any one of claims 1, 2 or a pharmaceutically acceptable salt thereof, wherein each R4 is independently F. 4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R ₆ is hydrogen. 5. A compound or a pharmaceutically acceptable salt thereof, wherein said compound is 2-amino-6-(2-(2,6-dioxopiperidin-3-yl)-4-methyl-1-oxoisoindolin-5-yl)-4-methylnicotinonitrile (1); 2-amino-6-(2-(2,6-dioxopiperidin-3-yl)-6-methyl-1-oxoisoindolin-5-yl)-4-methylnicotinonitrile (2); 2-amino-6-(2-(2,6-dioxopiperidin-3-yl)-4-fluoro-1-oxoisoindolin-5-yl)-4-methylnicotinonitrile (3); 3-(5-(6-amino-4-methylpyridin-2-yl)-4-fluoro-1-oxoisoindolin-2-yl)piperidine-2,6-dione (4); 3-(5-(6-amino-4-(trifluoroethyl)pyridin-2-yl)-4-fluoro-1-oxoisoindolin-2-yl)piperidin-2,6-dione (5); 3-(5-(6-amino-4,5-dimethylpyridin-2-yl)-4-fluoro-1-oxoisoindolin-2-yl)piperidine-2,6-dione (7); 3-(^)-5-(4,5-dimethyl-6-(methylamino)pyridin-2-yl)-3-methyl-1-oxoisoindolin-2-yl)piperidin2,6-dione (12); 3-((I)-5-(4,5-dimethyl-6-(methylamino)pyridin-2-yl)-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione (13); 6-((3S)-2-(2,6-dioxopiperidin-3-yl)-3-methyl-1-oxoisoindolin-5-yl)-4-methyl-2-(methylamino)nicotinonitrile (14); 6-((3I)-2-(2,6-dioxopiperidin-3 -yl)-3 -methyl-1-oxoisoindolin-5 -yl)-4-methyl-2-(methylamino)nicotinonitrile (15); 3-(^)-5-(6-amino-4,5-dimethylpyridin-2-yl)-4-fluoro-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione (16); 3-((I)-5-(6-amino-4,5-dimethylpyridin-2-yl)-4-fluoro-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione (17); 3-(5-(6-amino-3-methylpyridin-2-yl)-4-fluoro-1-oxoisoindolin-2-yl)piperidine-2,6-dione (18); 3-(5-(6-amino-5-fluoro-4-methylpyridin-2-yl)-4-fluoro-1-oxoisoindolin-2-yl)piperidine-2,6-dione (19); 3-(5-(6-amino-3-fluoro-4-methylpyridin-2-yl)-4-fluoro-1-oxoisoindolin-2-yl)piperidine-2,6-dione (20); 2-amino-6-((3I)-2-(2,6-dioxopiperidin-3-yl)-4-fluoro-3-methyl-1-oxoisoindolin-5-yl)-4-methylnicotinonitrile (23); 3-{(I)-5-(6-amino-4-methylpyridin-2-yl)-4-fluoro-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione (24); 3-((I)-5-(6-amino-4-(trifluoromethyl)pyridin-2-yl)-4-fluoro-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione (25); 3-{(R)-5-(6-amino-3-methylpyridin-2-yl)-4-fluoro-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione (26); 3-{(I)-5-(6-aminopyridin-2-yl)-4-fluoro-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione (27); (I)-3-((I)-5-(6-amino-4,5-dimethylpyridin-2-yl)-4-fluoro-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione (28); 3-((I)-5-(6-amino-5-fluoro-4-methylpyridin-2-yl)-4-fluoro-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione (29) or 3-(^)-5-(6-amino-5-fluoro-4-methylpyridin-2-yl)-4-fluoro-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione (30). 6. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, having the structure NF ^SN Z _NH2 7. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, having the structure O O. NF ^SN Z - 45 048753 8. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, having the structure 12. A pharmaceutical composition comprising a compound according to any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. 13. Use of a compound according to any one of claims 1 to 11 for the treatment of cancer. 14. The use according to claim 13, wherein said cancer is selected from colon cancer, stomach cancer, pancreatic cancer, breast cancer, prostate cancer, lung cancer, ovarian cancer, cervical cancer, kidney cancer, head and neck cancer, lymphoma, leukemia and melanoma. 15. A method for reducing Helios protein levels, Helios activity levels or Helios expression levels in cells, comprising contacting said Helios protein with a compound according to any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof. 16. The method according to claim 15, wherein the Helios protein is an amino acid sequence encoded by SEQ ID NO: 1, 2, 3, 4 or 5. 17. A method for reducing Eos protein levels, Eos activity levels, or Eos expression levels in cells, comprising contacting said Eos protein with a compound according to any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof. 18. The method according to claim 17, wherein the Eos protein is an amino acid sequence encoded by SEQ ID NO: 7 or 8.