WO2020033285A1 - Prmt5 inhibitors - Google Patents

Abstract

The present invention provides a compound of formula (I) Formula (I) and the pharmaceutically acceptable salts, esters, and prodrugs thereof, are PRMT5 inhibitors. Also provided are methods of making compounds of formula (I), pharmaceutical compositions comprising compounds of formula (I), and methods of using these compounds to treat cancer, sickle cell, and hereditary persistence of foetal hemoglobin (HPFH) mutations.

Description

TITLE OF THE INVENTION

PRMT5 INHIBITORS BACKGROUND OF THE INVENTION PRMT5 (aka JBP1, SKB1, lBP72, SKB1his and HRMTIL5) is a Type II arginine methyltransferase, and was first identified in a two-hybrid search for proteins interacting with the Janus tyrosine kinase (Jak2) (Pollack et al., 1999). PRMT5 plays a significant role in control and modulation of gene transcription. Inter alia, PRMT5 is known to methylate histone H3 at Arg-8 (a site distinct from that methylated by PRMT4) and histone H4 at Arg-3 (the same site methylated by PRMT1) as part of a complex with human SWI/SNF chromatin remodeling components BRG1 and BRM. PRMT5 has been reported to perform diverse roles including but not limited to impacting cell viability, stemness, DNA damage repair and RNA splicing (Clarke et al., Mol Cell (2017), Chiang et al., Cell Rep (2017), Gerhart et al., Sci Rep (2018)).

Specifically, inhibition of PRMT5 induces alternative splicing of the negative regulator of p53, MDM4 resulting in increased expression of the short isoform of MDM4 (MDM4-S), decreased expression of the full-length isoform (MDM4-FL) and increased p53 activity (Gerhart et al., Sci Rep (2018)).

PRMT5 is involved in the methylation and functional modulation of the tumor suppressor protein p53. (See Berger, 2008; Durant et al., 2009; Jansson et al., 2008; Scoumanne et al., 2009). Most of the physiological functions of p53 are attributable to its role as a transcriptional activator, responding to agents that damage DNA. p53 status is wild type in approximately half of human cancer cases. These include 94% in cervix, 87% in blood malignancies, 85% in bones and endocrine glands, and 75% of primary breast cancer.

Restoration of p53 in cancer cells harbouring wild type p53, by way of inhibiting mechanisms that suppress its function leads to growth arrest and apoptosis, and is regarded as a potentially effective means of tumor suppression.

The role of PRMT5 and p53 arginine methylation on cell cycle regulation and DNA damage response have been explored by both Jansson et al. and Scoumanne et al. (Jansson et al., 2008; Scoumanne et al., 2009). Although some differences are evident between the results from the two groups concerning cell cycle regulation in unperturbed cells (which may be ascribed to cell type specific effects and/or the actual nature of the experimental arrangements), both groups report similar results with respect to the DNA damage response.

In response to DNA damage caused by a variety of agents, including doxorubicin, camptothecin and UV light, and also in response to treatment with Nutlin-3, knockdown of PRMT5 results in an increase in sub-G1 population and concomitant reduction in G1 cells and, in the presence of p53, a significant increase in apoptosis. Knockdown of PRMT5 also resulted in a reduced level of p21, a key p53 target gene that regulates cell cycle arrest during the p53 response and MDM2, a p53 E3 ubiquitin ligase, but not PUMA, NOXA, AlP1 & APAF1, p53 target genes linked to apoptosis.

Knockdown of PRMT5 (but not PRMT1 or CARM1/PRMT4) results in decreased p53 stabilisation, decreased basal p53 levels, decreased p53 oligomerisation, and also decreased expression of elF4E a major component of translational machinery involved in ribosome binding to mRNA. Indeed, elF4E is a potent oncogene, which has been shown to promote malignant transformation in vitro and human cancer formation.

Knockdown of PRMT5 would be expected to lead to a reduction in the level of arginine methylated p53. Consistent with arginine methylation status of p53 influencing the p53 response (reduced arginine methylation biasing the response to proapoptotic), Jannson et al. showed that a p53 mutant in which each of the three critical arginine residues were substituted with lysine (p53KKK) retained the ability to induce apoptosis but its cell cycle arrest activity was significantly compromised.

Moreover, pS3KKK also has a significantly reduced ability to induce transcription of p21, by contrast with APAF1. The promoter binding specificity of wild-type p53 to key target genes is also significantly affected by arginine methylation status: Knockdown of PRMT5 results in decreased p53 binding to the promoter regions of the p21 and (intriguingly) PUMA genes, but does not affect p53 binding to the promoter regions of NOXA or APAF1.

The role of PRMT5 in the DNA damage response has been explored with groups reporting a role for PRMT5 in regulating high fidelity homologous recombination mediated DNA repair in both solid (Clarke et al., Mol Cell (2017) and hematological tumor models (Hamard et al., Cell Rep (2018)).

PRMT5 is aberrantly expressed in around half of human cancer cases, further linking this mechanism to cancers. PRMT5 overexpression has been observed in patient tissue samples and cell lines of Prostate cancer (Gu et al., 2012), Lung cancer (Zhongping et al., 2012), Melanoma cancer (Nicholas et al., 2012), Breast cancer (Powers et al., 2011), Colorectal cancer (Cho et al., 2012), Gastric cancer (Kim et al., 2005), Esophagus and Lung carcinoma (Aggarwal et al., 2010) and B-Cell lymphomas and leukemia (Wang, 2008). Moreover, elevated expression of PRMT5 in Melanoma, Breast and Colorectal cancers has been demonstrated to correlate with a poor prognosis.

Lymphoid malignancies including chronic lymphocytic leukemia (CLL) are associated with over-expression of PRMT5. PRMT5 is over-expressed (at the protein level) in the nucleus and cytosol in a number of patient derived Burkitt's lymphoma; mantle cell lymphoma (MCL); in vitro EBV-transformed lymphoma; leukemia cell lines; and B-CLL cell lines, relative to normal CD19+ B lymphocytes (Pal et al., 2007; Wang et al., 2008). Intriguingly, despite elevated levels of PRMT5 protein in these tumor cells, the levels of PRMT5 mRNA are reduced (by a factor of 2 - 5). Translation of PRMT5 mRNA is, however, enhanced in lymphoma cells, resulting in increased levels of PRMT5 (Pal et al., 2007; Wang et al., 2008).

In addition to genomic changes, CLL, like almost all cancers, has aberrant epigenetic abnormalities characterised by global hypomethylation and hot-spots of repressive hypermethylation of promoters including tumor suppressor genes. While the role of epigenetics in the origin and progression of CLL remains unclear, epigenetic changes appear to occur early in the disease and specific patterns of DNA methylation are associated with worse prognosis (Chen et al., 2009; Kanduri et al., 2010). Global symmetric methylation of histones H3R8 and H4R3 is increased in transformed lymphoid cell lines and MCL clinical samples (Pal et al., 2007), correlating with the overexpression of PRMT5 observed in a wide variety of lymphoid cancer cell lines and MCL clinical samples.

PRMT5 is therefore a target for the identification of novel cancer therapeutics. Hemoglobin is a major protein in red blood cells and is essential for the transport of oxygen from the lungs to the tissues. In adult humans, the most common hemoglobin type is a tetramer called hemoglobin A, consisting of two a and two b subunits. In human infants, the hemoglobin molecule is made up of two a and two ү chains. The gamma chains are gradually replaced by b subunits as the infant grows. The developmental switch in human ß-like globin gene subtype from foetal (ү) to adult (ß) that begins at birth heralds the onset of the

hemoglobinopathies ß-thalassemia or Sickle cell disease (SCD). In ß-thalassemia the adult chains are not produced. In SCD, a point mutation in the coding sequence in the ß globin gene leads to the production of a protein with altered polymerisation properties. The observation that increased adult ү-globin gene expression (in the setting of hereditary persistence of foetal hemoglobin (HPFH) mutations) significantly ameliorates the clinical severity of ß-thalassemia and SCD has prompted the search for therapeutic strategies to reverse ү-globin gene silencing. To date, this has been achieved through pharmacological induction, using compounds that broadly influence epigenetic modifications, including DNA methylation and histone

deacetylation. The development of more targeted therapies is dependent on the identification of the molecular mechanisms underpinning foetal globin gene silencing. These mechanisms have remained elusive, despite exhaustive study of the HPFH mutations, and considerable progress in many other aspects of globin gene regulation.

PRMT5 plays a critical role in triggering coordinated repressive epigenetic events that initiate with dimethylation of histone H4 Arginine 3 (H4R3me2s), and culminate in DNA methylation and transcriptional silencing of the ү-genes (Rank et al., 2010). Integral to the synchronous establishment of the repressive markers is the assembly of a PRMT5-dependent complex containing the DNA methyltransferase DNMT3A, and other repressor proteins (Rank et al., 2010). DNMT3A is directly recruited to bind to the PRMT5-induced H4R3me2s mark, and loss of this mark through shRNA-mediated knock-down of PRMT5, or enforced expression of a mutant form of PRMT5 lacking methyltransferase activity leads to marked upregulation of ү- gene expression, and complete abrogation of DNA methylation at the ү-promoter. Treatment of human erythroid progenitors with non-specific methyltransferase inhibitors (Adox and MTA) also resulted in upregulation of ү-gene expression (He Y, 2013). Inhibitors of PRMT5 thus have potential as therapeutics for hemoglobinopathies such as ß-thalassemia or Sickle Cell Disease (SCD).

The present inventors have developed compounds that inhibit the activity of PRMT5 and therefore may be of use in treating conditions ameliorated by the inhibition of the activity of PRMT5. SUMMARY OF THE INVENTION

Compounds of formula I

and the pharmaceutically acceptable salts, esters, and prodrugs thereof, are PRMT5 inhibitors. Also provided are methods of making compounds of formula I, pharmaceutical compositions comprising compounds of formula I, and methods of using these compounds to treat cancer, sickle cell, and hereditary persistence of foetal hemoglobin (HPFH) mutations. DETAILED DESCRIPTION OF THE INVENTION

The invention is a compound of the formula

or pharmaceutically acceptable salts thereof, wherein

X is CH₂ or O;

Y is CR⁹R⁹, CR⁹OR⁹, C(CFH2)(R⁹), C(CF2H)(R⁹), C(CF3)(R⁹), C(CFH2)(OR⁹), C(CF2H)(OR⁹), C(CF₃)(OR⁹), CHF, CF₂, C(CH)(Z), CH(Z), CH(CH₂)Z, or CH(CH₂)₂Z;

Z is NR⁹R⁹, or OR⁹;

A¹ is CH, CR⁹, or CNH₂;

A² is CH, N, or CF;

Z¹ is N, SR⁴, CR⁴, or O;

Z ² is N, SR⁵, CR⁵, or O;

Z ³ is N, SR⁶, CR⁶, or O;

Z ⁴ is N, SR⁷, CR⁷, or O;

Z ⁵ is N, SR⁸, CR⁸, or O;

R¹ is H, C1-6alkyl optionally substituted with one to four halogens, or CH=CH2;

R² is H, C_1-6alkyl optionally substituted with one to four halogens, or CH=CH₂;

provided that if R¹ is H, then R² cannot be H; and if R² is H, then R¹ cannot be H;

R³ is H, CH₃, NH₂, or NHCH₃;

R⁴ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, OC_1-6alkyl, or taken together with R⁵ to form a 5 or 6 membered ring comprising one N atom;

R⁵ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, OC_1-6alkyl, or taken together with R⁴ or R⁶ to form a 5 or 6 membered ring comprising one N atom;

R⁶ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, OC_1-6alkyl, or taken together with R⁵ or R⁷ to form a 5 or 6 membered ring comprising one N atom;

R⁷ is H, halogen, C1-6alkyl optionally substituted with one to four halogens, OC1-6alkyl, or taken together with R⁸ to form a 5 or 6 membered ring comprising one N atom; R⁸ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, OC_1-6alkyl, or taken together with R⁷ to form a 5 or 6 membered ring comprising one N atom; and

each R⁹, when present, is independently H or C_1-6alkyl. The invention is a compound of the formula I, or pharmaceutically acceptable salts thereof, wherein

X is CH₂ or O;

Y is CR⁹R⁹, CR⁹OR⁹, C(CFH₂)(R⁹), C(CF₂H)(R⁹), C(CF₃)(R⁹), C(CFH₂)(OR⁹), C(CF₂H)(OR⁹), C(CF₃)(OR⁹), CHF, or CF₂;

A¹ is CH, CR⁹, or CNH₂;

A² is CH, N, or CF;

Z¹ is N, or CR⁴;

Z ² is N, or CR⁵;

Z ³ is N, or CR⁶;

Z ⁴ is N, or CR⁷;

Z ⁵ is N, or CR⁸;

R¹ is H, C_1-6alkyl optionally substituted with one to four halogens, or CH=CH₂;

R² is H, C_1-6alkyl optionally substituted with one to four halogens, or CH=CH₂;

provided that if R¹ is H, then R² cannot be H; and if R² is H, then R¹ cannot be H;

R³ is H, CH₃, NH₂, or NHCH₃;

R⁴ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, OC_1-6alkyl, or taken together with R⁵ to form a 5 or 6 membered ring comprising one N atom;

R⁵ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, OC_1-6alkyl, or taken together with R⁴ or R⁶ to form a 5 or 6 membered ring comprising one N atom;

R⁶ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, OC_1-6alkyl, or taken together with R⁵ or R⁷ to form a 5 or 6 membered ring comprising one N atom;

R⁷ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, OC_1-6alkyl, or taken together with R⁸ to form a 5 or 6 membered ring comprising one N atom;

R⁸ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, OC_1-6alkyl, or taken together with R⁷ to form a 5 or 6 membered ring comprising one N atom; and

each R⁹, when present, is independently H or C_1-6alkyl. An embodiment of the invention of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is the compound of formula (Ia);

. In a subembodiment of the invention of formula Ia, X is CH₂ or O.

In a subembodiment of the invention of formula Ia, Y is CR⁹R⁹, CR⁹OR⁹, C(CFH₂)(R⁹), C(CF₂H)(R⁹), C(CF₃)(R⁹), C(CFH₂)(OR⁹), C(CF₂H)(OR⁹), C(CF₃)(OR⁹), CHF, or CF₂.

In a further subembodiment of the invention of formula I or Ia, Y is CH₂, CH(CH₃), CH(OH), or CCH3(OH).

In a subembodiment of the invention of formula Ia, Z¹ is N, SR⁴, CR⁴, or O.

In a subembodiment of the invention of formula Ia, Z² is N, SR⁵, CR⁵, or O.

In a subembodiment of the invention of formula Ia, Z³ is N, SR⁶, CR⁶, or O.

In a subembodiment of the invention of formula Ia, Z⁴ is N, SR⁷, CR⁷, or O.

In a subembodiment of the invention of formula Ia, Z⁵ is N, SR⁸, CR⁸, or O.

In a subembodiment of the invention of formula Ia, Z¹ is N, or CR⁴.

In a subembodiment of the invention of formula Ia, Z² is N, or CR⁵.

In a subembodiment of the invention of formula Ia, Z³ is N, or CR⁶.

In a subembodiment of the invention of formula Ia, Z⁴ is N, or CR⁷.

In a subembodiment of the invention of formula Ia, Z⁵ is N, or CR⁸.

In a subembodiment of the invention of formula Ia, R¹ is H, C_1-6alkyl optionally substituted with one to four halogens, or CH=CH₂.

In a subembodiment of the invention of formula I or Ia, R¹ is H, CH₃, CH₂F, CHF₂, CF₃ or CH=CH₂.

In a further subembodiment of the invention of formula I or Ia, R¹ is CH₃.

In a subembodiment of the invention of formula Ia, R² is H, C_1-6alkyl optionally substituted with one to four halogens, or CH=CH₂.

In a subembodiment of the invention of formula I or Ia, R² is H, CH₃, CH₂F, CHF₂, CF₃, or CH=CH₂.

In a further subembodiment of the invention of formula I or Ia, R² is H.

In a subembodiment of the invention of formula Ia, if R¹ is H, then R² cannot be H; and if R² is H, then R¹ cannot be H.

In a subembodiment of the invention of formula I or Ia, R¹ is CH3 and R² is H.

In a subembodiment of the invention of formula Ia, R³ is H, CH₃, NH₂, or NHCH₃.

In a further subembodiment of the invention of formula I or Ia, R³ is NH₂.

In a subembodiment of the invention of formula Ia, R⁴ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, or OC_1-6alkyl.

In a subembodiment of the invention of formula I or Ia, R⁴ is H, halogen, CH₃, C₂H₅, C₃H₇, OCH₃, OC₂H₅, OC₃H₇, CF₃, CF₂H, or CFH₂.

In a further subembodiment of the invention of formula I or Ia, R⁴ is H, halogen, CH₃, OCH_3, CF₃, or CF₂H.

In a further subembodiment of the invention of formula I or Ia, R⁴ is H, CH₃, F, or Cl.

In a subembodiment of the invention of formula Ia, R⁴ together with R⁵ forms a 5 or 6 membered ring comprising one N atom.

In a subembodiment of the invention of formula Ia, R⁵ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, or OC_1-6alkyl.

In a subembodiment of the invention of formula I or Ia, R⁵ is H, halogen, CH₃, C₂H₅, C₃H₇, OCH₃, OC₂H₅, OC₃H₇, CF₃, CF₂H, or CFH₂.

In a further subembodiment of the invention of formula I or Ia, R⁵ is H, halogen, CH₃, OCH_3, CF₃, or CF₂H.

In a further subembodiment of the invention of formula I or Ia, R⁵ is H, F, or Cl.

In a further subembodiment of the invention of formula I or Ia, R⁵ is F.

In a further subembodiment of the invention of formula I or Ia, R⁵ is CHF2.In a subembodiment of the invention of formula Ia, R⁵ together with R⁴ or R⁶ forms a 5 or 6 membered ring comprising one N atom.

In a subembodiment of the invention of formula Ia, R⁶ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, or OC_1-6alkyl.

In a further subembodiment of the invention of formula I or Ia, R⁶ is H, halogen, CH₃, C₂H₅, C₃H₇, OCH₃, OC₂H₅, OC₃H₇, CF₃, CF₂H, or CFH₂.

In a further subembodiment of the invention of formula I or Ia, R⁶ is H, halogen, CH₃, OCH_3, CF₃, or CF₂H.

In a further subembodiment of the invention of formula I or Ia, R⁶ is H, F, or Cl.

In a subembodiment of the invention of formula Ia, R⁶ together with R⁵ or R⁷ forms a 5 or 6 membered ring comprising one N atom.

In a subembodiment of the invention of formula Ia, R⁷ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, or OC_1-6alkyl.

In a subembodiment of the invention of formula I or Ia, R⁷ is H, halogen, CH3, C2H5, C3H7, OCH₃, OC₂H₅, OC₃H₇, CF₃, CF₂H, or CFH₂.

In a further subembodiment of the invention of formula I or Ia, R⁷ is H, halogen, CH₃, OCH_3, CF₃, or CF₂H.

In a further subembodiment of the invention of formula I or Ia, R⁷ is H, F, or Cl.

In a further subembodiment of the invention of formula I or Ia, R⁷ is F.

In a further subembodiment of the invention of formula I or Ia, R⁷ is CHF₂.

In a subembodiment of the invention of formula Ia, R⁷ together with R⁸ forms a 5 or 6 membered ring comprising one N atom.

In a subembodiment of the invention of formula Ia, R⁸ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, or OC1-6alkyl.

In a subembodiment of the invention of formula I or Ia, R⁸ is H, halogen, CH₃, C₂H₅, C₃H₇, OCH₃, OC₂H₅, OC₃H₇, CF₃, CF₂H, or CFH₂.

In a further subembodiment of the invention of formula I or Ia, R⁸ is H, halogen, CH₃, OCH₃, CF₃, or CF₂H.

In a further subembodiment of the invention of formula I or Ia, R⁸ is H, CH₃, F, or Cl.

In a subembodiment of the invention of formula Ia, R⁸ together with R⁷ forms a 5 or 6 membered ring comprising one N atom.

In a subembodiment of the invention of formula Ia, each R⁹, when present, is independently H or C_1-6alkyl.

In a further subembodiment of the invention of formula Ia, R⁹ is H. An embodiment of the invention of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is the compound of formula (Ib);

. In a subembodiment of the invention of formula Ib, Z¹ is N, SR⁴, CR⁴, or O. In a subembodiment of the invention of formula Ib, Z² is N, SR⁵, CR⁵, or O.

In a subembodiment of the invention of formula Ib, Z³ is N, SR⁶, CR⁶, or O.

In a subembodiment of the invention of formula Ib, Z⁴ is N, SR⁷, CR⁷, or O.

In a subembodiment of the invention of formula Ib, Z⁵ is N, SR⁸, CR⁸, or O.

In a subembodiment of the invention of formula Ib, Z¹ is N, or CR⁴.

In a subembodiment of the invention of formula Ib, Z² is N, or CR⁵.

In a subembodiment of the invention of formula Ib, Z³ is N, or CR⁶.

In a subembodiment of the invention of formula Ib, Z⁴ is N, or CR⁷.

In a subembodiment of the invention of formula Ib, Z⁵ is N, or CR⁸. In a subembodiment of the invention of formula Ib, R¹ is H, C_1-6alkyl optionally substituted with one to four halogens, or CH=CH2.

In a subembodiment of the invention of formula Ib, R¹ is H, CH₃, CH₂F, CHF₂, CF₃ or CH=CH₂. In a further subembodiment of the invention of formula Ib, R¹ is CH₃.

In a subembodiment of the invention of formula Ib, R² is H, C_1-6alkyl optionally substituted with one to four halogens, or CH=CH₂.

In a subembodiment of the invention of formula Ib, R² is H, CH₃, CH₂F, CHF₂, CF₃, or CH=CH₂. In a further subembodiment of the invention of formula Ib, R² is H.

In a subembodiment of the invention of formula Ib, if R¹ is H, then R² cannot be H; and if R² is H, then R¹ cannot be H.

In a subembodiment of the invention of formula Ib, R¹ is CH₃ and R² is H.

In a subembodiment of the invention of formula Ib, R³ is H, CH3, NH2, or NHCH3.

In a further subembodiment of the invention of formula Ib, R³ is NH₂.

In a subembodiment of the invention of formula Ib, R⁴ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, or OC_1-6alkyl.

In a subembodiment of the invention of formula Ib, R⁴ is H, halogen, CH₃, C₂H₅, C₃H₇, OCH₃, OC₂H₅, OC₃H₇, CF₃, CF₂H, or CFH₂.

In a further subembodiment of the invention of formula Ib, R⁴ is H, halogen, CH₃, OCH_3, CF₃, or CF₂H.

In a further subembodiment of the invention of formula Ib, R⁴ is H, CH₃, F, or Cl.

In a subembodiment of the invention of formula Ib, R⁴ together with R⁵ forms a 5 or 6 membered ring comprising one N atom.

In a subembodiment of the invention of formula Ib, R⁵ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, or OC_1-6alkyl. In a subembodiment of the invention of formula Ib, R⁵ is H, halogen, CH₃, C₂H₅, C₃H₇, OCH₃, OC2H5, OC3H7, CF3, CF2H, or CFH2.

In a further subembodiment of the invention of formula Ib, R⁵ is H, halogen, CH₃, OCH_3, CF₃, or CF₂H.

In a further subembodiment of the invention of formula Ib, R⁵ is H, F, or Cl.

In a further subembodiment of the invention of formula Ib, R⁵ is F.

In a further subembodiment of the invention of formula Ib, R⁵ is CHF₂.

In a subembodiment of the invention of formula Ib, R⁵ together with R⁴ or R⁶ forms a 5 or 6 membered ring comprising one N atom.

In a subembodiment of the invention of formula Ib, R⁶ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, or OC_1-6alkyl.

In a subembodiment of the invention of formula Ib, R⁶ is H, halogen, CH3, C2H5, C3H7, OCH3, OC₂H₅, OC₃H₇, CF₃, CF₂H, or CFH₂.

In a further subembodiment of the invention of formula Ib, R⁶ is H, halogen, CH₃, OCH_3, CF₃, or CF₂H.

In a further subembodiment of the invention of formula Ib, R⁶ is H, F, or Cl.

In a subembodiment of the invention of formula Ib, R⁶ together with R⁵ or R⁷ forms a 5 or 6 membered ring comprising one N atom.

In a subembodiment of the invention of formula Ib, R⁷ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, or OC_1-6alkyl.

In a subembodiment of the invention of formula Ib, R⁷ is H, halogen, CH₃, C₂H₅, C₃H₇, OCH₃, OC2H5, OC3H7, CF3, CF2H, or CFH2.

In a further subembodiment of the invention of formula Ib, R⁷ is H, halogen, CH₃, OCH_3, CF₃, or CF₂H.

In a further subembodiment of the invention of formula Ib, R⁷ is H, F, or Cl.

In a further subembodiment of the invention of formula Ib, R⁷ is F.

In a further subembodiment of the invention of formula Ib, R⁷ is CHF₂.

In a subembodiment of the invention of formula Ia, R⁷ together with R⁸ forms a 5 or 6 membered ring comprising one N atom.

In a subembodiment of the invention of formula Ib, R⁸ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, or OC_1-6alkyl.

In a subembodiment of the invention of formula Ib, R⁸ is H, halogen, CH3, C2H5, C3H7, OCH3, OC₂H₅, OC₃H₇, CF₃, CF₂H, or CFH₂.

In a further subembodiment of the invention of formula Ib, R⁸ is H, halogen, CH₃, OCH_3, CF₃, or CF₂H.

In a further subembodiment of the invention of formula Ib, R⁸ is H, CH3, F, or Cl.

In a subembodiment of the invention of formula Ia, R⁸ together with R⁷ forms a 5 or 6 membered ring comprising one N atom. An embodiment of the invention of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is the compound of formula (Ic);

.

In a subembodiment of the invention of formula Ic, R³ is H, CH3, NH2, or NHCH3.

In a further subembodiment of the invention of formula Ic, R³ is NH₂.

In a subembodiment of the invention of formula Ic, R⁴ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, or OC_1-6alkyl.

In a subembodiment of the invention of formula Ic, R⁴ is H, halogen, CH₃, C₂H₅, C₃H₇, OCH₃, OC₂H₅, OC₃H₇, CF₃, CF₂H, or CFH₂.

In a further subembodiment of the invention of formula Ic, R⁴ is H, halogen, CH₃, OCH_3, CF₃, or CF₂H.

In a further subembodiment of the invention of formula Ic, R⁴ is H, CH₃, F, or Cl.

In a subembodiment of the invention of formula Ic, R⁴ together with R⁵ forms a 5 or 6 membered ring comprising one N atom.

In a subembodiment of the invention of formula Ic, R⁵ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, or OC_1-6alkyl.

In a subembodiment of the invention of formula Ic, R⁵ is H, halogen, CH₃, C₂H₅, C₃H₇, OCH₃, OC₂H₅, OC₃H₇, CF₃, CF₂H, or CFH₂.

In a further subembodiment of the invention of formula Ic, R⁵ is H, halogen, CH₃, OCH_3, CF₃, or CF₂H.

In a further subembodiment of the invention of formula Ic, R⁵ is H, F, or Cl.

In a further subembodiment of the invention of formula Ic, R⁵ is F. In a further subembodiment of the invention of formula Ic, R⁵ is CHF₂.

In a subembodiment of the invention of formula Ic, R⁵ together with R⁴ or R⁶ forms a 5 or 6 membered ring comprising one N atom.

In a subembodiment of the invention of formula Ic, R⁶ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, or OC_1-6alkyl.

In a further subembodiment of the invention of formula Ic, R⁶ is H, halogen, CH₃, C₂H₅, C₃H₇, OCH₃, OC₂H₅, OC₃H₇, CF₃, CF₂H, or CFH₂.

In a further subembodiment of the invention of formula Ic, R⁶ is H, halogen, CH₃, OCH_3, CF₃, or CF₂H.

In a further subembodiment of the invention of formula Ic, R⁶ is H, F, or Cl.

In a subembodiment of the invention of formula Ic, R⁶ together with R⁵ or R⁷ forms a 5 or 6 membered ring comprising one N atom.

In a subembodiment of the invention of formula Ic, R⁷ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, or OC_1-6alkyl.

In a subembodiment of the invention of formula Ic, R⁷ is H, halogen, CH₃, C₂H₅, C₃H₇, OCH₃, OC₂H₅, OC₃H₇, CF₃, CF₂H, or CFH₂.

In a further subembodiment of the invention of formula Ic, R⁷ is H, halogen, CH₃, OCH_3, CF₃, or CF₂H.

In a further subembodiment of the invention of formula Ic, R⁷ is H, F, or Cl.

In a further subembodiment of the invention of formula Ic, R⁷ is F.

In a further subembodiment of the invention of formula Ic, R⁷ is CHF₂.

In a subembodiment of the invention of formula Ic, R⁷ together with R⁸ forms a 5 or 6 membered ring comprising one N atom.

In a subembodiment of the invention of formula Ic, R⁸ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, or OC_1-6alkyl.

In a subembodiment of the invention of formula Ic, R⁸ is H, halogen, CH₃, C₂H₅, C₃H₇, OCH₃, OC₂H₅, OC₃H₇, CF₃, CF₂H, or CFH₂.

In a further subembodiment of the invention of formula Ic, R⁸ is H, halogen, CH₃, OCH₃, CF₃, or CF₂H.

In a further subembodiment of the invention of formula Ic, R⁸ is H, CH₃, F, or Cl.

In a subembodiment of the invention of formula Ic, R⁸ together with R⁷ forms a 5 or 6 membered ring comprising one N atom.

In an embodiment of the invention, the compound of formula I is

,

,

NH₂ NH₂ Cl

Cl Cl

F

Cl

Cl

F

F

F

H H ,

F F F

F F F

NH₂ F F

F F

H N

H N

H N

F F H N

F

F

or a

Reference to compounds of formula I encompasses reference formula Ia, Ib, and Ic, and all subembodiments thereof, in the following.

In one embodiment, the present invention is a composition for treating cancer comprising a compound of formula I, Ia, Ib, or Ic, or a pharmaceutically acceptable salt thereof.

In one embodiment, the present invention is a composition for treating cancer comprising a compound of formula I, Ia, Ib, or Ic, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

In one embodiment, the present invention is a composition for treating hemoglobinopathies such as ß-thalassemia or Sickle Cell Disease (SCD), comprising a compound of formula I, Ia, Ib, or Ic, or a pharmaceutically acceptable salt thereof.

In one embodiment, the present invention is a composition for treating hemoglobinopathies such as ß-thalassemia or Sickle Cell Disease (SCD), comprising a compound of formula I, Ia, Ib, or Ic, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

In another embodiment, the present invention is a method of treating cancer comprising administering to a patient in need thereof a compound of formula I, Ia, Ib, or Ic, or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention is a method of treating hemoglobinopathies such as ß-thalassemia or Sickle Cell Disease (SCD), comprising administering to a patient in need thereof a compound of formula I, Ia, Ib, or Ic, or a

pharmaceutically acceptable salt thereof.

In another embodiment, the present invention is a method of treating cancer comprising administering to a patient in need thereof a compound of formula I, Ia, Ib, or Ic, or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention is a method of treating cancer comprising administering to a patient in need thereof a compound of formula I, Ia, Ib, or Ic, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

In another embodiment, the present invention is a method of treating hemoglobinopathies such as ß-thalassemia or Sickle Cell Disease (SCD), comprising administering to a patient in need thereof a compound of formula I, Ia, Ib, or Ic, or a

pharmaceutically acceptable salt thereof.

In another embodiment, the present invention is a method of treating hemoglobinopathies such as ß-thalassemia or Sickle Cell Disease (SCD), comprising administering to a patient in need thereof a compound of formula I, Ia, Ib, or Ic, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In another embodiment, the present invention is a method of treating cancer comprising administering to a patient in need thereof a composition comprising a compound of formula I, Ia, Ib, or Ic, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

In another embodiment, the present invention is a method of treating hemoglobinopathies such as ß-thalassemia or Sickle Cell Disease (SCD), comprising

administering to a patient in need thereof a composition comprising a compound of formula I, Ia, Ib, or Ic, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

In another embodiment of the present invention is the use of a compound of formula I, Ia, Ib, or Ic, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating cancer or hemoglobinopathies such as ß-thalassemia or Sickle Cell Disease (SCD).

In another embodiment, the present invention includes the use of compounds of formula I, Ia, Ib, or Ic for the preparation of a medicament for the treatment of cancer. In another embodiment, the present invention includes the use of compounds of formula I, Ia, Ib, or Ic for the preparation of a medicament for the treatment of hemoglobinopathies such as ß-thalassemia or Sickle Cell Disease (SCD). In a subembodiment, the cancer is cardiac cancer, lung cancer, gastrointestinal cancer, genitourinary tract cancer, liver cancer, bone cancer, nervous system cancer, gynecological cancer, hematological cancer, skin cancer, or adrenal cancer.

In another embodiment, the present invention includes compounds of formula I, Ia, Ib, or Ic for use in the treatment of cancer or hemoglobinopathies such as ß-thalassemia or Sickle Cell Disease (SCD). In another embodiment, the present invention includes compounds of formula I, Ia, Ib or Ic for use in the treatment of cardiac cancer, lung cancer, gastrointestinal cancer, genitourinary tract cancer, liver cancer, bone cancer, nervous system cancer,

gynecological cancer, hematological cancer, skin cancer, or adrenal cancer.

In one embodiment, the compound disclosed herein is selected from the group consisting of the compounds exemplified herein, for example, in Examples 1– 93, or a pharmaceutically acceptable salt thereof.

The term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. The term “anti-cancer agent” means a drug (medicament or pharmaceutically active ingredient) for treating cancer. The term“antineoplastic agent” means a drug (medicament or pharmaceutically active ingredient) for treating cancer (i.e., a chemotherapeutic agent). The term“at least one” means one or more than one. The meaning of“at least one” with reference to the number of compounds of the invention is independent of the meaning with reference to the number of chemotherapeutic agents. The term“chemotherapeutic agent” means a drug (medicament or pharmaceutically active ingredient) for treating cancer (i.e., an antineoplastic agent). The term “compound” with reference to the antineoplastic agents, includes the agents that are antibodies. The term“consecutively” means one following the other. The term“effective amount” means a “therapeutically effective amount”. The term "therapeutically effective amount" means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician. Thus, for example, in the methods of treating cancer described herein“effective amount” (or“therapeutically effective amount”) means, the amount of the compound (or drug), or radiation, that results in: (a) the reduction, alleviation or disappearance of one or more symptoms caused by the cancer, (b) the reduction of tumor size, (c) the elimination of the tumor, and/or (d) long-term disease stabilization (growth arrest) of the tumor. Also, for example, an effective amount, or a therapeutically effective amount of the PRMT5 inhibitor (i.e., a compound of the invention) is that amount which results in the reduction in PRMT5 activity. The term“treating cancer” or“treatment of cancer” refers to administration to a mammal afflicted with a cancerous condition and refers to an effect that alleviates the cancerous condition by killing the cancerous cells, and also refers to an effect that results in the inhibition of growth and/or metastasis of the cancer.

The invention also provides a pharmaceutical composition comprising an effective amount of at least one compound of formula I, Ia, Ib, or Ic, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. The invention also provides a pharmaceutical composition comprising an effective amount of at least one compound of formula I, Ia, Ib, or Ic, and an effective amount of at least one other pharmaceutically active ingredient (such as, for example, a chemotherapeutic agent), and a pharmaceutically acceptable carrier.

The invention also provides a method of inhibiting PRMT5 in a patient in need of such treatment comprising administering to said patient an effective amount of at least one compound of formula I, Ia, Ib, or Ic or a pharmaceutically acceptable salt thereof. The invention also provides a method for treating cancer in a patient in need of such treatment, said method comprising administering to said patient an effective amount of at least one compound of formula I, Ia, Ib, or Ic, or a pharmaceutically acceptable salt thereof. The invention also provides a method for treating cancer in a patient in need of such treatment, said method comprising administering to said patient an effective amount of at least one compound of formula I, Ia, Ib, or Ic, or a pharmaceutically acceptable salt thereof , in combination with an effective amount of at least one chemotherapeutic agent. The methods of the invention include the administration of a pharmaceutical composition comprising at least one compound of the invention and a

pharmaceutically acceptable carrier. The invention also provides any of the above methods of treating cancer wherein the cancer is colorectal. The invention also provides any of the above methods of treating cancer wherein the cancer is melanoma. The methods of treating cancers described herein can optionally include the administration of an effective amount of radiation (i.e., the methods of treating cancers described herein optionally include the administration of radiation therapy).

The methods of treating cancer described herein include methods of treating cancer that comprise administering a therapeutically effective amount of a compound of the instant invention in combination with radiation therapy and/or in combination with a second compound selected from: an estrogen receptor modulator, an androgen receptor modulator, a retinoid receptor modulator, a cytotoxicytostatic agent, an antiproliferative agent, a prenyl- protein transferase inhibitor, an HMG-CoA reductase inhibitor, an HIV protease inhibitor, a reverse transcriptase inhibitor, an angiogenesis inhibitor, PPAR-g agonists, PPAR-d agonists, an inhibitor of inherent multidrug resistance, an anti-emetic agent, an agent useful in the treatment of anemia, an agent useful in the treatment of neutropenia, an immunologic-enhancing drug, an inhibitor of cell proliferation and survival signaling, a bisphosphonate, an aromatase inhibitor, an siRNA therapeutic, g-secretase and/or NOTCH inhibitors, agents that interfere with receptor tyrosine kinases (RTKs), an agent that interferes with a cell cycle checkpoint, and any of the therapeutic agents listed herein.

In any of the methods of treating cancer described herein, unless stated otherwise, the methods can optionally include the administration of an effective amount of radiation therapy. For radiation therapy, g-radiation is preferred.

Thus, another example of the invention is directed to a method of treating cancer in a patient in need of such treatment, said method comprising administering an effective amount of a compound of formula I, Ia, Ib, or Ic. Another example of the invention is directed to a method of treating cancer in a patient in need of such treatment, said method comprising administering to said patient an effective amount of a compound of formula I, Ia, Ib, or Ic, and an effective amount of at least one chemotherapeutic agent. Methods for the safe and effective administration of most of these chemotherapeutic agents are known to those skilled in the art. In addition, their administration is described in the standard literature. For example, the administration of many of the

chemotherapeutic agents is described in the“Physicians’ Desk Reference” (PDR), e.g., 1996 edition (Medical Economics Company, Montvale, NJ 07645-1742, USA), the Physicians’ Desk Reference, 56^th Edition, 2002 (published by Medical Economics company, Inc. Montvale, NJ 07645-1742), the Physicians’ Desk Reference, 57^th Edition, 2003 (published by Thompson PDR, Montvale, NJ 07645-1742), the Physicians’ Desk Reference, 60^th Edition, 2006 (published by Thompson PDR, Montvale, NJ 07645-1742), and the Physicians’ Desk Reference, 64^th Edition, 2010 (published by PDR Network, LLC at Montvale, NJ 07645-1725), presently accessible through www.pdr.net; the disclosures of which are incorporated herein by reference thereto.

If the patient is responding, or is stable, after completion of the therapy cycle, the therapy cycle can be repeated according to the judgment of the skilled clinician. Upon completion of the therapy cycles, the patient can be continued on the compounds of the invention at the same dose that was administered in the treatment protocol. This maintenance dose can be continued until the patient progresses or can no longer tolerate the dose (in which case the dose can be reduced, and the patient can be continued on the reduced dose).

Those skilled in the art will recognize that the actual dosages and protocols for administration employed in the methods of the invention may be varied according to the judgment of the skilled clinician. The actual dosage employed may be varied depending upon the requirements of the patient and the severity of the condition being treated. Determination of the proper dosage for a particular situation is within the skill of the art. A determination to vary the dosages and protocols for administration may be made after the skilled clinician considers such factors as the patient’s age, condition and size, as well as the severity of the cancer being treated and the response of the patient to the treatment.

The amount and frequency of administration of the compound of formula I, Ia, Ib, or Ic and the chemotherapeutic agents will be regulated according to the judgment of the attending clinician (physician) considering such factors as age, condition and size of the patient as well as severity of the cancer being treated.

The chemotherapeutic agent can be administered according to therapeutic protocols well known in the art. It will be apparent to those skilled in the art that the

administration of the chemotherapeutic agent can be varied depending on the cancer being treated and the known effects of the chemotherapeutic agent on that disease. Also, in accordance with the knowledge of the skilled clinician, the therapeutic protocols (e.g., dosage amounts and times of administration) can be varied in view of the observed effects of the administered therapeutic agents on the patient, and in view of the observed responses of the cancer to the administered therapeutic agents.

The initial administration can be made according to established protocols known in the art, and then, based upon the observed effects, the dosage, modes of administration and times of administration can be modified by the skilled clinician.

The particular choice of chemotherapeutic agent will depend upon the diagnosis of the attending physicians and their judgement of the condition of the patient and the appropriate treatment protocol.

The determination of the order of administration, and the number of repetitions of administration of the chemotherapeutic agent during a treatment protocol, is well within the knowledge of the skilled physician after evaluation of the cancer being treated and the condition of the patient.

Thus, in accordance with experience and knowledge, the practicing physician can modify each protocol for the administration of a chemotherapeutic agent according to the individual patient’s needs, as the treatment proceeds. All such modifications are within the scope of the present invention.

The attending clinician, in judging whether treatment is effective at the dosage administered, will consider the general well-being of the patient as well as more definite signs such as relief of cancer-related symptoms (e.g., pain), inhibition of tumor growth, actual shrinkage of the tumor, or inhibition of metastasis. Size of the tumor can be measured by standard methods such as radiological studies, e.g., CAT or MRI scan, and successive measurements can be used to judge whether or not growth of the tumor has been retarded or even reversed. Relief of disease-related symptoms such as pain, and improvement in overall condition can also be used to help judge effectiveness of treatment.

The compounds, compositions and methods provided herein are useful for the treatment of cancer. Cancers that may be treated by the compounds, compositions and methods disclosed herein include, but are not limited to: (1) Cardiac: sarcoma (angiosarcoma,

fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma; (2) Lung: bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma, non-small cell; (3) Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma), colon, colorectal, rectal; (4) Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate

(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); (5) Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; (6) Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing’s sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors; (7) Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma,

schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma, meningioma, glioma, sarcoma); (8) Gynecological: uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecal cell tumors, Sertoli- Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tubes (carcinoma), breast; (9) Hematologic: blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelomonocytic (CMML), myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome),

Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma]; (10) Skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis; and (11) Adrenal glands:

neuroblastoma. Examples of cancer that may be treated by the compounds, compositions and methods of the invention include thyroid cancer, anaplastic thyroid carcinoma, epidermal cancer, head and neck cancer (e.g., squamous cell cancer of the head and neck), sarcoma, tetracarcinoma, hepatoma and multiple myeloma. Thus, the term "cancerous cell" as provided herein, includes a cell afflicted by any one of the above-identified conditions.

In the treatment of breast cancer (e.g., postmenopausal and premenopausal breast cancer, e.g., hormone-dependent breast cancer) the compound of formula I, Ia, Ib, or Ic, or a pharmaceutically acceptable salt thereof, may be used with an effective amount of at least one antihormonal agent selected from the group consisting of: (a) aromatase inhibitors, (b) antiestrogens, and (c) LHRH analogues; and optionally an effective amount of at least one chemotherapeutic agent. Examples of aromatase inhibitors include but are not limited to:

Anastrozole (e.g., Arimidex), Letrozole (e.g., Femara), Exemestane (Aromasin), Fadrozole and Formestane (e.g., Lentaron). Examples of antiestrogens include but are not limited to: Tamoxifen (e.g., Nolvadex), Fulvestrant (e.g., Faslodex), Raloxifene (e.g., Evista), and Acolbifene.

Examples of LHRH analogues include but are not limited to: Goserelin (e.g., Zoladex) and Leuprolide (e.g., Leuprolide Acetate, such as Lupron or Lupron Depot). Examples of

chemotherapeutic agents include but are not limited to: Trastuzumab (e.g., Herceptin), Gefitinib (e.g., Iressa), Erlotinib (e.g., Erlotinib HCl, such as Tarceva), Bevacizumab (e.g., Avastin), Cetuximab (e.g., Erbitux), and Bortezomib (e.g., Velcade).

In one example of the invention the cancer treated is colo-rectal cancer (such as, for example, colon adenocarcinoma and colon adenoma). Thus, another example of the invention is directed to a method of treating colo-rectal cancer in a patient in need of such treatment, said method comprising administering an effective of a compound of formula I, Ia, Ib, or Ic, or a pharmaceutically acceptable salt thereof, to said patient. Another example of the invention is directed to a method of treating colo-rectal cancer in a patient in need of such treatment, said method comprising administering to said patient an effective amount of a compound of formula I, Ia, Ib, or Ic, or a pharmaceutically acceptable salt thereof, and an effective amount of at least one chemotherapeutic agent.

In one example of the invention the cancer treated is melanoma. Thus, another example of the invention is directed to a method of treating melanoma in a patient in need of such treatment, said method comprising administering an effective amount of a compound of formula I, Ia, Ib, or Ic, or a pharmaceutically acceptable salt thereof, to said patient. Another example of the invention is directed to a method of treating melanoma in a patient in need of such treatment, said method comprising administering to said patient an effective amount of a compound of formula I, Ia, Ib, or Ic, or a pharmaceutically acceptable salt thereof, and an effective amount of at least one chemotherapeutic agent.

The compounds of the invention are also useful in preparing a medicament that is useful in treating cancer. The instant compounds are also useful in combination with therapeutic, chemotherapeutic and anti-cancer agents. Combinations of the presently disclosed compounds with therapeutic, chemotherapeutic and anti-cancer agents are within the scope of the invention. Examples of such agents can be found in Cancer Principles and Practice of Oncology by V.T. Devita and S. Hellman (editors), 9^th edition (May 16, 2011), Lippincott Williams & Wilkins Publishers. A person of ordinary skill in the art would be able to discern which combinations of agents would be useful based on the particular characteristics of the drugs and the cancer involved. Such agents include the following: estrogen receptor modulators, programmed cell death protein 1 (PD-1) inhibitors, programmed death-ligand 1 (PD-L1) inhibitors, androgen receptor modulators, retinoid receptor modulators, cytotoxic/cytostatic agents, antiproliferative agents, prenyl-protein transferase inhibitors, HMG-CoA reductase inhibitors and other angiogenesis inhibitors, HIV protease inhibitors, reverse transcriptase inhibitors, inhibitors of cell proliferation and survival signaling, bisphosphonates, aromatase inhibitors, siRNA therapeutics, g-secretase inhibitors, agents that interfere with receptor tyrosine kinases (RTKs) and agents that interfere with cell cycle checkpoints. The instant compounds are particularly useful when co-administered with radiation therapy.

“Estrogen receptor modulators” refers to compounds that interfere with or inhibit the binding of estrogen to the receptor, regardless of mechanism. Examples of estrogen receptor modulators include, but are not limited to, tamoxifen, raloxifene, idoxifene, LY353381,

LY117081, toremifene, fulvestrant, 4-[7-(2,2-dimethyl-1-oxopropoxy-4-methyl-2-[4-[2-(1- piperidinyl)ethoxy]phenyl]-2H-1-benzopyran-3-yl]-phenyl-2,2-dimethylpropanoate, 4,4’- dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone, and SH646.

PD-1 inhibitors include pembrolizumab (lambrolizumab), nivolumab and MPDL3280A. PDL- inhibitors include atezolizumab, avelumab, and durvalumab.

“Androgen receptor modulators” refers to compounds which interfere or inhibit the binding of androgens to the receptor, regardless of mechanism. Examples of androgen receptor modulators include finasteride and other 5a-reductase inhibitors, nilutamide, flutamide, bicalutamide, liarozole, and abiraterone acetate.

“Retinoid receptor modulators” refers to compounds which interfere or inhibit the binding of retinoids to the receptor, regardless of mechanism. Examples of such retinoid receptor modulators include bexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoic acid, a- difluoromethylornithine, ILX23-7553, trans-N-(4’-hydroxyphenyl) retinamide, and N-4- carboxyphenyl retinamide.

“Cytotoxic/cytostatic agents” refer to compounds which cause cell death or inhibit cell proliferation primarily by interfering directly with the cell’s functioning or inhibit or interfere with cell myosis, including alkylating agents, tumor necrosis factors, intercalators, hypoxia activatable compounds, microtubule inhibitors/microtubule-stabilizing agents, inhibitors of mitotic kinesins, histone deacetylase inhibitors, inhibitors of kinases involved in mitotic progression, inhibitors of kinases involved in growth factor and cytokine signal transduction pathways, antimetabolites, biological response modifiers, hormonal/anti-hormonal therapeutic agents, haematopoietic growth factors, monoclonal antibody targeted therapeutic agents, topoisomerase inhibitors, proteosome inhibitors, ubiquitin ligase inhibitors, and aurora kinase inhibitors.

Examples of cytotoxic/cytostatic agents include, but are not limited to, sertenef, cachectin, ifosfamide, tasonermin, lonidamine, carboplatin, altretamine, prednimustine, dibromodulcitol, ranimustine, fotemustine, nedaplatin, oxaliplatin, temozolomide, heptaplatin, estramustine, improsulfan tosilate, trofosfamide, nimustine, dibrospidium chloride, pumitepa, lobaplatin, satraplatin, profiromycin, cisplatin, irofulven, dexifosfamide, cis-aminedichloro(2- methyl-pyridine)platinum, benzylguanine, glufosfamide, GPX100, (trans, trans, trans)-bis-mu- (hexane-1,6-diamine)-mu-[diamine-platinum(II)]bis[diamine(chloro)platinum (II)]tetrachloride, diarizidinylspermine, arsenic trioxide, 1-(11-dodecylamino-10-hydroxyundecyl)-3,7- dimethylxanthine, zorubicin, idarubicin, daunorubicin, bisantrene, mitoxantrone, pirarubicin, pinafide, valrubicin, amrubicin, antineoplaston, 3’-deamino-3’-morpholino-13-deoxo-10- hydroxycarminomycin, annamycin, galarubicin, elinafide, MEN10755, 4-demethoxy-3-deamino- 3-aziridinyl-4-methylsulphonyl-daunorubicin (see WO 00/50032), Raf kinase inhibitors (such as Bay43-9006) and mTOR inhibitors (such as Wyeth’s CCI-779).

An example of a hypoxia activatable compound is tirapazamine.

Examples of proteosome inhibitors include but are not limited to lactacystin and MLN-341 (Velcade).

Examples of microtubule inhibitors/microtubule-stabilising agents include paclitaxel, vindesine sulfate, 3’,4’-didehydro-4’-deoxy-8’-norvincaleukoblastine, docetaxol, rhizoxin, dolastatin, mivobulin isethionate, auristatin, cemadotin, RPR109881, BMS184476, vinflunine, cryptophycin, 2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl) benzene sulfonamide, anhydrovinblastine, TDX258, the epothilones (see for example U.S. Pat. Nos. 6,284,781 and 6,288,237) and BMS188797. In an example the epothilones are not included in the microtubule inhibitors/microtubule-stabilising agents.

Some examples of topoisomerase inhibitors are topotecan, hycaptamine, irinotecan, rubitecan, 6-ethoxypropionyl-3’,4’-O-exo-benzylidene-chartreusin, 9-methoxy-N,N- dimethyl-5-nitropyrazolo[3,4,5-kl]acridine-2-(6H) propanamine, 1-amino-9-ethyl-5-fluoro-2,3- dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3’,4’:b,7]-indolizino[1,2b]quinoline- 10,13(9H,15H)dione, lurtotecan, 7-[2-(N-isopropylamino)ethyl]-(20S)camptothecin, BNP1350, BNPI1100, BN80915, BN80942, etoposide phosphate, teniposide, sobuzoxane, 2’- dimethylamino-2’-deoxy-etoposide, GL331, N-[2-(dimethylamino)ethyl]-9-hydroxy-5,6- dimethyl-6H-pyrido[4,3-b]carbazole-1-carboxamide, asulacrine, (5a, 5aB, 8aa,9b)-9-[2-[N-[2- (dimethylamino)ethyl]-N-methylamino]ethyl]-5-[4-hydro0xy-3,5-dimethoxyphenyl]- 5,5a,6,8,8a,9-hexohydrofuro(3’,4’:6,7)naphtho(2,3-d)-1,3-dioxol-6-one, 2,3-(methylenedioxy)-5- methyl-7-hydroxy-8-methoxybenzo[c]-phenanthridinium, 6,9-bis[(2- aminoethyl)amino]benzo[g]isoguinoline-5,10-dione, 5-(3-aminopropylamino)-7,10-dihydroxy-2- (2-hydroxyethylaminomethyl)-6H-pyrazolo[4,5,1-de]acridin-6-one, N-[1- [2(diethylamino)ethylamino]-7-methoxy-9-oxo-9H-thioxanthen-4-ylmethyl]formamide, N-(2- (dimethylamino)ethyl)acridine-4-carboxamide, 6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy- 7H-indeno[2,1-c] quinolin-7-one, and dimesna.

Examples of inhibitors of mitotic kinesins, and in particular the human mitotic kinesin KSP, are described in Publications WO03/039460, WO03/050064, WO03/050122, WO03/049527, WO03/049679, WO03/049678, WO04/039774, WO03/079973, WO03/099211, WO03/105855, WO03/106417, WO04/037171, WO04/058148, WO04/058700, WO04/126699, WO05/018638, WO05/019206, WO05/019205, WO05/018547, WO05/017190,

US2005/0176776. In an example inhibitors of mitotic kinesins include, but are not limited to inhibitors of KSP, inhibitors of MKLP1, inhibitors of CENP-E, inhibitors of MCAK and inhibitors of Rab6-KIFL.

Examples of“histone deacetylase inhibitors” include, but are not limited to, SAHA, TSA, oxamflatin, PXD101, MG98 and scriptaid. Further reference to other histone deacetylase inhibitors may be found in the following manuscript; Miller, T.A. et al., J. Med.

Chem.46(24):5097-5116 (2003).

“Inhibitors of kinases involved in mitotic progression” include, but are not limited to, inhibitors of aurora kinase, inhibitors of Polo-like kinases (PLK; in particular inhibitors of PLK-1), inhibitors of bub-1 and inhibitors of bub-R1. An example of an“aurora kinase inhibitor” is VX-680 (tozasertib).

“Antiproliferative agents” includes antisense RNA and DNA oligonucleotides such as G3139, ODN698, GEM231, and INX3001, and antimetabolites such as enocitabine, carmofur, tegafur, pentostatin, doxifluridine, trimetrexate, fludarabine, capecitabine, galocitabine, cytarabine ocfosfate, fosteabine sodium hydrate, raltitrexed, paltitrexid, emitefur, tiazofurin, decitabine, nolatrexed, pemetrexed, nelzarabine, 2’-deoxy-2’-methylidenecytidine, 2’- fluoromethylene-2’-deoxycytidine, N-[5-(2,3-dihydro-benzofuryl)sulfonyl]-N’-(3,4- dichlorophenyl)urea, N6-[4-deoxy-4-[N2-[2(E),4(E)-tetradecadienoyl]glycylamino]-L-glycero- B-L-manno-heptopyranosyl]adenine, aplidine, ecteinascidin, troxacitabine, 4-[2-amino-4-oxo- 4,6,7,8-tetrahydro-3H-pyrimidino[5,4-b][1,4]thiazin-6-yl-(S)-ethyl]-2,5-thienoyl-L-glutamic acid, aminopterin, 5-flurouracil, alanosine, 11-acetyl-8-(carbamoyloxymethyl)-4-formyl-6-methoxy- 14-oxa-1,11-diazatetracyclo(7.4.1.0.0)-tetradeca-2,4,6-trien-9-yl acetic acid ester, swainsonine, lometrexol, dexrazoxane, methioninase, 2’-cyano-2’-deoxy-N4-palmitoyl-1-B-D-arabino furanosyl cytosine, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone and trastuzumab.

Examples of monoclonal antibody targeted therapeutic agents include those therapeutic agents which have cytotoxic agents or radioisotopes attached to a cancer cell specific or target cell specific monoclonal antibody. Examples include Bexxar.

“HMG-CoA reductase inhibitors” refers to inhibitors of 3-hydroxy-3- methylglutaryl-CoA reductase. Examples of HMG-CoA reductase inhibitors that may be used _{include but are not limited to lovastatin (MEVACOR} ^® _{; see U.S. Patent Nos. 4,231,938,}

4_{,294,926 and 4,319,039), simvastatin (ZOCOR} ^® _{; see U.S. Patent Nos. 4,444,784, 4,820,850 and 4,916,239), pravastatin (PRAVACHOL} ^® _{; see U.S. Patent Nos. 4,346,227, 4,537,859, 4,410,629, 5,030,447 and 5,180,589), fluvastatin (LESCOL} ^® _{; see U.S. Patent Nos. 5,354,772, 4,911,165, 4,929,437, 5,189,164, 5,118,853, 5,290,946 and 5,356,896), atorvastatin (LIPITOR} ^® _{; see U.S. Patent Nos. 5,273,995, 4,681,893, 5,489,691 and 5,342,952), rosuvastatin (CRESTOR} ^® _{U.S. Reissue Patent RE37,314) and cerivastatin (also known as rivastatin and BAYCHOL} ^® _{; see} US Patent No.5,177,080). The structural formulas of these and additional HMG-CoA reductase inhibitors that may be used in the instant methods are described at page 87 of M. Yalpani, "Cholesterol Lowering Drugs", Chemistry & Industry, pp.85-89 (5 February 1996) and US Patent Nos.4,782,084 and 4,885,314. The term HMG-CoA reductase inhibitor as used herein includes all pharmaceutically acceptable lactone and open-acid forms (i.e., where the lactone ring is opened to form the free acid) as well as salt and ester forms of compounds which have HMG-CoA reductase inhibitory activity, and therefore the use of such salts, esters, open-acid and lactone forms is included within the scope of the invention.

“Prenyl-protein transferase inhibitor” refers to a compound which inhibits any one or any combination of the prenyl-protein transferase enzymes, including farnesyl-protein transferase (FPTase), geranylgeranyl-protein transferase type I (GGPTase-I), and geranylgeranyl- protein transferase type-II (GGPTase-II, also called Rab GGPTase). Examples of prenyl-protein transferase inhibitors can be found in the following publications and patents: WO 96/30343, WO 97/18813, WO 97/21701, WO 97/23478, WO 97/38665, WO 98/28980, WO 98/29119, WO 95/32987, U.S. Patent Nos.5,420,245, 5,523,430, 5,532,359, 5,510,510, 5,589,485, and 5,602,098, European Patent Publ.0618221, European Patent Publ.0675112, European Patent Publ.0604181, European Patent Publ.0696593, WO 94/19357, WO 95/08542, WO 95/11917, WO 95/12612, WO 95/12572, WO 95/10514, U.S. Patent No.5,661,152, WO 95/10515, WO 95/10516, WO 95/24612, WO 95/34535,

WO 95/25086, WO 96/05529, WO 96/06138, WO 96/06193, WO 96/16443, WO 96/21701, WO 96/21456, WO 96/22278, WO 96/24611, WO 96/24612, WO 96/05168, WO 96/05169, WO 96/00736, U.S. Patent No.5,571,792, WO 96/17861, WO 96/33159, WO 96/34850, WO

96/34851, WO 96/30017, WO 96/30018, WO 96/30362, WO 96/30363, WO 96/31111, WO 96/31477, WO 96/31478, WO 96/31501, WO 97/00252, WO 97/03047, WO 97/03050,

WO 97/04785, WO 97/02920, WO 97/17070, WO 97/23478, WO 97/26246, WO 97/30053, WO 97/44350, WO 98/02436, and U.S. Patent No.5,532,359. For an example of the role of a prenyl- protein transferase inhibitor on angiogenesis see European J. of Cancer, Vol.35, No.9, pp.1394- 1401 (1999).

“Angiogenesis inhibitors” refers to compounds that inhibit the formation of new blood vessels, regardless of mechanism. Examples of angiogenesis inhibitors include, but are not limited to, tyrosine kinase inhibitors, such as inhibitors of the tyrosine kinase receptors Flt-1 (VEGFR1) and Flk-1/KDR (VEGFR2), inhibitors of epidermal-derived, fibroblast-derived, or platelet derived growth factors, MMP (matrix metalloprotease) inhibitors, integrin blockers, interferon-a, interleukin-12, pentosan polysulfate, cyclooxygenase inhibitors, including nonsteroidal anti-inflammatories (NSAIDs) like aspirin and ibuprofen as well as selective cyclooxy-genase-2 inhibitors like celecoxib and rofecoxib (PNAS, Vol.89, p.7384 (1992); JNCI, Vol.69, p.475 (1982); Arch. Opthalmol., Vol.108, p.573 (1990); Anat. Rec., Vol.238, p.68 (1994); FEBS Letters, Vol.372, p.83 (1995); Clin, Orthop. Vol.313, p.76 (1995); J. Mol.

Endocrinol., Vol.16, p.107 (1996); Jpn. J. Pharmacol., Vol.75, p.105 (1997); Cancer Res., Vol. 57, p.1625 (1997); Cell, Vol.93, p.705 (1998); Intl. J. Mol. Med., Vol.2, p.715 (1998); J. Biol. Chem., Vol.274, p.9116 (1999)), steroidal anti-inflammatories (such as corticosteroids, mineralocorticoids, dexamethasone, prednisone, prednisolone, methylpred, betamethasone), carboxyamidotriazole, combretastatin A-4, squalamine, 6-O-chloroacetyl-carbonyl)-fumagillol, thalidomide, angiostatin, troponin-1, angiotensin II antagonists (see Fernandez et al., J. Lab. Clin. Med.105:141-145 (1985)), and antibodies to VEGF (see, Nature Biotechnology, Vol.17, pp.963- 968 (October 1999); Kim et al., Nature, 362, 841-844 (1993); WO 00/44777; and WO 00/61186). Other therapeutic agents that modulate or inhibit angiogenesis and may also be used in combination with the compounds of the instant invention include agents that modulate or inhibit the coagulation and fibrinolysis systems (see review in Clin. Chem. La. Med.38:679-692 (2000)). Examples of such agents that modulate or inhibit the coagulation and fibrinolysis pathways include, but are not limited to, heparin (see Thromb. Haemost.80:10-23 (1998)), low molecular weight heparins and carboxypeptidase U inhibitors (also known as inhibitors of active thrombin activatable fibrinolysis inhibitor [TAFIa]) (see Thrombosis Res.101:329-354 (2001)). TAFIa inhibitors have been described in U.S. Ser. Nos.60/310,927 (filed August 8, 2001) and 60/349,925 (filed January 18, 2002).

“Agents that interfere with cell cycle checkpoints” refer to compounds that inhibit protein kinases that transduce cell cycle checkpoint signals, thereby sensitizing the cancer cell to DNA damaging agents. Such agents include inhibitors of ATR, ATM, the CHK1 and CHK2 kinases and cdk and cdc kinase inhibitors and are specifically exemplified by 7- hydroxystaurosporin, flavopiridol, CYC202 (Cyclacel) and BMS-387032.

“Agents that interfere with receptor tyrosine kinases (RTKs)” refer to compounds that inhibit RTKs and therefore mechanisms involved in oncogenesis and tumor progression. Such agents include inhibitors of c-Kit, Eph, PDGF, Flt3 and c-Met. Further agents include inhibitors of RTKs as described by Bume-Jensen and Hunter, Nature, 411:355-365, 2001.

“Inhibitors of cell proliferation and survival signalling pathway” refer to compounds that inhibit signal transduction cascades downstream of cell surface receptors. Such agents include inhibitors of serine/threonine kinases (including but not limited to inhibitors of Akt such as described in WO 02/083064, WO 02/083139, WO 02/083140, US 2004-0116432, WO 02/083138, US 2004-0102360, WO 03/086404, WO 03/086279, WO 03/086394, WO 03/084473, WO 03/086403, WO 2004/041162, WO 2004/096131, WO 2004/096129, WO 2004/096135, WO 2004/096130, WO 2005/100356, WO 2005/100344, US 2005/029941, US 2005/44294, US 2005/43361, 60/734188, 60/652737, 60/670469), inhibitors of Raf kinase (for example BAY-43-9006 ), inhibitors of MEK (for example CI-1040 and PD-098059), inhibitors of mTOR (for example Wyeth CCI-779), and inhibitors of PI3K (for example LY294002).

As described above, the combinations with NSAID’s are directed to the use of NSAID's which are potent COX-2 inhibiting agents. For purposes of the specification an NSAID is potent if it possesses an IC₅₀ for the inhibition of COX-2 of 1µM or less as measured by cell or microsomal assays.

The invention also encompasses combinations with NSAID’s which are selective COX-2 inhibitors. For purposes of the specification NSAID’s which are selective inhibitors of COX-2 are defined as those which possess a specificity for inhibiting COX-2 over COX-1 of at least 100 fold as measured by the ratio of IC50 for COX-2 over IC50 for COX-1 evaluated by cell or microsomal assays. Such compounds include, but are not limited to those disclosed in U.S. Patent 5,474,995, U.S. Patent 5,861,419, U.S. Patent 6,001,843, U.S. Patent 6,020,343, U.S. Patent 5,409,944, U.S. Patent 5,436,265, U.S. Patent 5,536,752, U.S. Patent 5,550,142, U.S. Patent 5,604,260, U.S.5,698,584, U.S. Patent 5,710,140, WO 94/15932, U.S. Patent 5,344,991, U.S. Patent 5,134,142, U.S. Patent 5,380,738, U.S. Patent 5,393,790, U.S. Patent 5,466,823, U.S. Patent 5,633,272 and U.S. Patent 5,932,598, all of which are hereby incorporated by reference.

Inhibitors of COX-2 that are particularly useful in the instant method of treatment are: 3-phenyl-4-(4-(methylsulfonyl)phenyl)-2-(5H)-furanone; and 5-chloro-3-(4- methylsulfonyl)-phenyl-2-(2-methyl-5-pyridinyl)pyridine; or a pharmaceutically acceptable salt thereof.

Compounds that have been described as specific inhibitors of COX-2 and are therefore useful in the present invention include, but are not limited to, the following: rofecoxib, etoricoxib, parecoxib, BEXTRA® and CELEBREX® or a pharmaceutically acceptable salt thereof.

Other examples of angiogenesis inhibitors include, but are not limited to, endostatin, ukrain, ranpirnase, IM862, 5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]- 1-oxaspiro[2,5]oct-6-yl(chloroacetyl)carbamate, acetyldinanaline, 5-amino-1-[[3,5-dichloro-4- (4-chlorobenzoyl)phenyl]methyl]-1H-1,2,3-triazole-4-carboxamide,CM101, squalamine, combretastatin, RPI4610, NX31838, sulfated mannopentaose phosphate, 7,7-(carbonyl- bis[imino-N-methyl-4,2-pyrrolocarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino]-bis-(1,3- naphthalene disulfonate), and 3-[(2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone (SU5416).

As used above,“integrin blockers” refers to compounds which selectively ^{antagonize, inhibit or counteract binding of a physiological ligand to the a} _v ^b ₃ ^{integrin, to} compounds which selectively antagonize, inhibit or counteract binding of a physiological ligand to the avb5 integrin, to compounds which antagonize, inhibit or counteract binding of a ^{physiological ligand to both the a} _v ^b ₃ ^{integrin and the a} _v ^b ₅ ^{integrin, and to compounds which} antagonize, inhibit or counteract the activity of the particular integrin(s) expressed on capillary ^{endothelial cells. The term also refers to antagonists of the a} _v ^b ₆ ^{, a} _v ^b ₈ ^{, a} ₁ ^b ₁ ^{, a} ₂ ^b ₁ ^{, a} ₅ ^b ₁ ^{, a} ₆ ^b ₁ ^{and a} ₆ ^b ₄ ^{integrins. The term also refers to antagonists of any combination of a} _v ^b ₃ ^{, a} _v ^b ₅ ^{, a} _v ^b ₆ ^{, a} _v ^b ₈ ^{, a} ₁ ^b ₁ ^{, a} ₂ ^b ₁ ^{, a} ₅ ^b ₁ ^{, a} ₆ ^b ₁ ^{and a} ₆ ^b ₄ ^integrins.

Some specific examples of tyrosine kinase inhibitors include N- (trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide, 3-[(2,4-dimethylpyrrol-5- yl)methylidenyl)indolin-2-one, 17-(allylamino)-17-demethoxygeldanamycin, 4-(3-chloro-4- fluorophenylamino)-7-methoxy-6-[3-(4-morpholinyl)propoxyl]quinazoline, N-(3- ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine, BIBX1382, 2,3,9,10,11,12- hexahydro-10-(hydroxymethyl)-10-hydroxy-9-methyl-9,12-epoxy-1H-diindolo[1,2,3-fg:3’,2’,1’- kl]pyrrolo[3,4-i][1,6]benzodiazocin-1-one, SH268, genistein, STI571, CEP2563, 4-(3- chlorophenylamino)-5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidinemethane sulfonate, 4-(3-bromo- 4-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, 4-(4’-hydroxyphenyl)amino-6,7- dimethoxyquinazoline, SU6668, STI571A, N-4-chlorophenyl-4-(4-pyridylmethyl)-1- phthalazinamine, and EMD121974.

Combinations with compounds other than anti-cancer compounds are also encompassed in the instant methods. For example, combinations of the instantly claimed compounds with PPAR-g (i.e., PPAR-gamma) agonists and PPAR-d (i.e., PPAR-delta) agonists are useful in the treatment of certain malignancies. PPAR-g and PPAR-d are the nuclear peroxisome proliferator-activated receptors g and d. The expression of PPAR-g on endothelial cells and its involvement in angiogenesis has been reported in the literature (see J. Cardiovasc. Pharmacol.1998; 31:909-913; J. Biol. Chem.1999; 274:9116-9121; Invest. Ophthalmol Vis. Sci. 2000; 41:2309-2317). More recently, PPAR-g agonists have been shown to inhibit the angiogenic response to VEGF in vitro; both troglitazone and rosiglitazone maleate inhibit the development of retinal neovascularization in mice (Arch. Ophthamol.2001; 119:709-717). Examples of PPAR-g agonists and PPAR- g/a agonists include, but are not limited to, thiazolidinediones (such as DRF2725, CS-011, troglitazone, rosiglitazone, and pioglitazone), fenofibrate, gemfibrozil, clofibrate, GW2570, SB219994, AR-H039242, JTT-501, MCC-555, GW2331, GW409544, NN2344, KRP297, NP0110, DRF4158, NN622, GI262570, PNU182716, DRF552926, 2-[(5,7- dipropyl-3-trifluoromethyl-1,2-benzisoxazol-6-yl)oxy]-2-methylpropionic acid (disclosed in USSN 09/782,856), and 2(R)-7-(3-(2-chloro-4-(4-fluorophenoxy) phenoxy)propoxy)-2- ethylchromane-2-carboxylic acid (disclosed in USSN 60/235,708 and 60/244,697).

Another example of the instant invention is the use of the presently disclosed compounds in combination with gene therapy for the treatment of cancer. For an overview of genetic strategies to treating cancer see Hall et al., (Am. J. Hum. Genet.61:785-789, 1997) and Kufe et al., (Cancer Medicⁱⁿe, 5th Ed, pp 876-889, BC Decker, Hamilton 2000). Gene therapy can be used to deliver any tumor suppressing gene. Examples of such genes include, but are not limited to, p53, which can be delivered via recombinant virus-mediated gene transfer (see U.S. Patent No.6,069,134, for example), a uPA/uPAR antagonist ("Adenovirus-Mediated Delivery of a uPA/uPAR Antagonist Suppresses Angiogenesis-Dependent Tumor Growth and Dissemination in”Mice," Gene Therapy, August 1998;5(8):1105-13), and interferon gamma (J. Immunol.

2000;164:217-222).

The compounds of the instant invention may also be administered in combination with an inhibitor of inherent multidrug resistance (MDR), in particular MDR associated with high levels of expression of transporter proteins. Such MDR inhibitors include inhibitors of p- glycoprotein (P-gp), such as LY335979, XR9576, OC144-093, R101922, VX853 and PSC833 (valspodar).

A compound of the present invention may be employed in conjunction with anti- emetic agents to treat nausea or emesis, including acute, delayed, late-phase, and anticipatory emesis, which may result from the use of a compound of the present invention, alone or with radiation therapy. For the prevention or treatment of emesis, a compound of the present invention may be used in conjunction with other anti-emetic agents, especially neurokinin-1 receptor antagonists, 5HT3 receptor antagonists, such as ondansetron, granisetron, tropisetron, and zatisetron, GABAB receptor agonists, such as baclofen, a corticosteroid such as Decadron (dexamethasone), Kenalog, Aristocort, Nasalide, Preferid, Benecorten or others such as disclosed in U.S.Patent Nos.2,789,118, 2,990,401, 3,048,581, 3,126,375, 3,929,768, 3,996,359, 3,928,326 and 3,749,712, an antidopaminergic, such as the phenothiazines (for example prochlorperazine, fluphenazine, thioridazine and mesoridazine), metoclopramide or dronabinol. In another example, conjunctive therapy with an anti-emesis agent selected from a neurokinin-1 receptor antagonist, a 5HT3 receptor antagonist and a corticosteroid is disclosed for the treatment or prevention of emesis that may result upon administration of the instant compounds.

Neurokinin-1 receptor antagonists of use in conjunction with the compounds of the present invention are fully described, for example, in U.S. Patent Nos.5,162,339, 5,232,929, 5,242,930, 5,373,003, 5,387,595, 5,459,270, 5,494,926, 5,496,833, 5,637,699, 5,719,147;

European Patent Publication Nos. EP 0360390, 0394989, 0428434, 0429366, 0430771, 0 436334, 0443132, 0482539, 0498069, 0499313, 0512901, 0512902, 0514273, 0514 274, 0514275, 0514276, 0515681, 0517589, 0520555, 0522808, 0528495, 0532456, 0 533280, 0536817, 0545478, 0558156, 0577394, 0585913,0590152, 0599538, 0610 793, 0634402, 0686629, 0693489, 0694535, 0699655, 0699674, 0707006, 0708101, 0 709375, 0709376, 0714891, 0723959, 0733632 and 0776893; PCT International Patent Publication Nos. WO 90/05525, 90/05729, 91/09844, 91/18899, 92/01688, 92/06079, 92/12151, 92/15585, 92/17449, 92/20661, 92/20676, 92/21677, 92/22569, 93/00330, 93/00331, 93/01159, 93/01165, 93/01169, 93/01170, 93/06099, 93/09116, 93/10073, 93/14084, 93/14113, 93/18023, 93/19064, 93/21155, 93/21181, 93/23380, 93/24465, 94/00440, 94/01402, 94/02461, 94/02595, 94/03429, 94/03445, 94/04494, 94/04496, 94/05625, 94/07843, 94/08997, 94/10165, 94/10167, 94/10168, 94/10170, 94/11368, 94/13639, 94/13663, 94/14767, 94/15903, 94/19320, 94/19323, 94/20500, 94/26735, 94/26740, 94/29309, 95/02595, 95/04040, 95/04042, 95/06645, 95/07886, 95/07908, 95/08549, 95/11880, 95/14017, 95/15311, 95/16679, 95/17382, 95/18124, 95/18129, 95/19344, 95/20575, 95/21819, 95/22525, 95/23798, 95/26338, 95/28418, 95/30674, 95/30687, 95/33744, 96/05181, 96/05193, 96/05203, 96/06094, 96/07649, 96/10562, 96/16939, 96/18643, 96/20197, 96/21661, 96/29304, 96/29317, 96/29326, 96/29328, 96/31214, 96/32385, 96/37489, 97/01553, 97/01554, 97/03066, 97/08144, 97/14671, 97/17362, 97/18206, 97/19084, 97/19942 and 97/21702; and in British Patent Publication Nos.2266529, 2268931, 2269170, 2269590, 2271774, 2292144, 2293168, 2293169, and 2302689. The preparation of such compounds is fully described in the aforementioned patents and publications, which are incorporated herein by reference.

In an example, the neurokinin-1 receptor antagonist for use in conjunction with the compounds of the present invention is selected from: 2-(R)-(1-(R)-(3,5- bis(trifluoromethyl)phenyl)ethoxy)-3-(S)-(4-fluorophenyl)-4-(3-(5-oxo-1H,4H-1,2,4- triazolo)methyl)morpholine, or a pharmaceutically acceptable salt thereof, which is described in U.S. Patent No.5,719,147.

A compound of the instant invention may also be administered with an agent useful in the treatment of anemia. Such an anemia treatment agent is, for example, a continuous erythropoiesis receptor activator (such as epoetin alfa).

A compound of the instant invention may also be administered with an agent useful in the treatment of neutropenia. Such a neutropenia treatment agent is, for example, a hematopoietic growth factor which regulates the production and function of neutrophils such as a human granulocyte colony stimulating factor, (G-CSF). Examples of a G-CSF include filgrastim.

A compound of the instant invention may also be administered with an immunologic-enhancing drug, such as levamisole, isoprinosine and Zadaxin.

A compound of the instant invention may also be useful for treating or preventing cancer in combination with P450 inhibitors including: xenobiotics, quinidine, tyramine, ketoconazole, testosterone, quinine, methyrapone, caffeine, phenelzine, doxorubicin,

troleandomycin, cyclobenzaprine, erythromycin, cocaine, furafyline, cimetidine,

dextromethorphan, ritonavir, indinavir, amprenavir, diltiazem, terfenadine, verapamil, cortisol, itraconazole, mibefradil, nefazodone and nelfinavir. A compound of the instant invention may also be useful for treating or preventing cancer in combination with Pgp and/or BCRP inhibitors including: cyclosporin A, PSC833, GF120918, cremophorEL, fumitremorgin C, Ko132, Ko134, Iressa, Imatnib mesylate, EKI-785, Cl1033, novobiocin, diethylstilbestrol, tamoxifen, resperpine, VX-710, tryprostatin A, flavonoids, ritonavir, saquinavir, nelfinavir, omeprazole, quinidine, verapamil, terfenadine, ketoconazole, nifidepine, FK506, amiodarone, XR9576, indinavir, amprenavir, cortisol, testosterone,

LY335979, OC144-093, erythromycin, vincristine, digoxin and talinolol.

A compound of the instant invention may also be useful for treating or preventing cancer, including bone cancer, in combination with bisphosphonates (understood to include bisphosphonates, diphosphonates, bisphosphonic acids and diphosphonic acids). Examples of bisphosphonates include but are not limited to: etidronate (Didronel), pamidronate (Aredia), alendronate (Fosamax), risedronate (Actonel), zoledronate (Zometa), ibandronate (Boniva), incadronate or cimadronate, clodronate, EB-1053, minodronate, neridronate, piridronate and tiludronate including any and all pharmaceutically acceptable salts, derivatives, hydrates and mixtures thereof.

A compound of the instant invention may also be useful for treating or preventing breast cancer in combination with aromatase inhibitors. Examples of aromatase inhibitors include but are not limited to: anastrozole, letrozole and exemestane.

A compound of the instant invention may also be useful for treating or preventing cancer in combination with siRNA therapeutics.

The compounds of the instant invention may also be administered in combination with g-secretase inhibitors and/or inhibitors of NOTCH signaling. Such inhibitors include compounds described in WO 01/90084, WO 02/30912, WO 01/70677, WO 03/013506, WO 02/36555, WO 03/093252, WO 03/093264, WO 03/093251, WO 03/093253, WO 2004/039800, WO 2004/039370, WO 2005/030731, WO 2005/014553, USSN 10/957,251, WO 2004/089911, WO 02/081435, WO 02/081433, WO 03/018543, WO 2004/031137, WO 2004/031139, WO 2004/031138, WO 2004/101538, WO 2004/101539 and WO 02/47671 (including LY-450139).

A compound of the instant invention may also be useful for treating or preventing cancer in combination with PARP inhibitors.

A compound of the instant invention may also be useful for treating cancer in combination with the following therapeutic agents: pembrolizumab (Keytruda^®), abarelix _{(Plenaxis depot} ^® _{); aldesleukin (Prokine} ^® _{); Aldesleukin (Proleukin} ^® _{); Alemtuzumabb}

(_Campath ^® _{); alitretinoin (Panretin} ^® _{); allopurinol (Zyloprim} ^® _{); altretamine (Hexalen} ^® _{); amifostine (Ethyol} ^® _{); anastrozole (Arimidex} ^® _{); arsenic trioxide (Trisenox} ^® _{); asparaginase (Elspar} ^® _{); azacitidine (Vidaza} ^® _{); bevacuzimab (Avastin} ^® _{); bexarotene capsules (Targretin} ^® _{); bexarotene gel (Targretin} ^® _{); bleomycin (Blenoxane} ^® _{); bortezomib (Velcade} ^® _{); busulfan intravenous (Busulfex} ^® _{); busulfan oral (Myleran} ^® _{); calusterone (Methosarb} ^® _{); capecitabine (Xeloda} ^® _{); carboplatin (Paraplatin} ^® _{); carmustine (BCNU} ^® _{, BiCNU} ^® _{); carmustine (Gliadel} ^® _{); carmustine with Polifeprosan 20 Implant (Gliadel Wafer} ^® _{); celecoxib (Celebrex} ^® _{); cetuximab (Erbitux} ^® _{); chlorambucil (Leukeran} ^® _{); cisplatin (Platinol} ^® _{); cladribine (Leustatin} ^® _{, 2-CdA} ^® _{); clofarabine (Clolar} ^® _{); cyclophosphamide (Cytoxan} ^® _{, Neosar} ^® _{); cyclophosphamide (Cytoxan Injection} ^® _{); cyclophosphamide (Cytoxan Tablet} ^® _{); cytarabine (Cytosar-U} ^® _{); cytarabine liposomal (DepoCyt} ^® _{); dacarbazine (DTIC-Dome} ^® _{); dactinomycin, actinomycin D}

(_Cosmegen ^® _{); Darbepoetin alfa (Aranesp} ^® _{); daunorubicin liposomal (DanuoXome} ^® _);

d_{aunorubicin, daunomycin (Daunorubicin} ^® _{); daunorubicin, daunomycin (Cerubidine} ^® _);

D_{enileukin diftitox (Ontak} ^® _{); dexrazoxane (Zinecard} ^® _{); docetaxel (Taxotere} ^® _{); doxorubicin (Adriamycin PFS} ^® _{); doxorubicin (Adriamycin} ^® _{, Rubex} ^® _{); doxorubicin (Adriamycin PFS Injection} ^® _{); doxorubicin liposomal (Doxil} ^® _{); dromostanolone propionate (Dromostanolone} ^® _{); dromostanolone propionate (Masterone injection} ^® _{); Elliott's B Solution (Elliott's B Solution} ^® _{); epirubicin (Ellence} ^® _{); Epoetin alfa (epogen} ^® _{); erlotinib (Tarceva} ^® _{); estramustine (Emcyt} ^® _{); etoposide phosphate (Etopophos} ^® _{); etoposide, VP-16 (Vepesid} ^® _{); exemestane (Aromasin} ^® _{); Filgrastim (Neupogen} ^® _{); floxuridine (intraarterial) (FUDR} ^® _{); fludarabine (Fludara} ^® _);

f_{luorouracil, 5-FU (Adrucil} ^® _{); fulvestrant (Faslodex} ^® _{); gefitinib (Iressa} ^® _{); gemcitabine (Gemzar} ^® _{); gemtuzumab ozogamicin (Mylotarg} ^® _{); goserelin acetate (Zoladex Implant} ^® _{); goserelin acetate (Zoladex} ^® _{); histrelin acetate (Histrelin implant} ^® _{); hydroxyurea (Hydrea} ^® _{); Ibritumomab Tiuxetan (Zevalin} ^® _{); idarubicin (Idamycin} ^® _{); ifosfamide (IFEX} ^® _{); imatinib mesylate (Gleevec} ^® _{); interferon alfa 2a (Roferon A} ^® _{); Interferon alfa-2b (Intron A} ^® _{); irinotecan (Camptosar} ^® _{); lenalidomide (Revlimid} ^® _{); letrozole (Femara} ^® _{); leucovorin (Wellcovorin} ^® _{, Leucovorin} ^® _{); Leuprolide Acetate (Eligard} ^® _{); levamisole (Ergamisol} ^® _{); lomustine, CCNU (CeeBU} ^® _{); meclorethamine, nitrogen mustard (Mustargen} ^® _{); megestrol acetate (Megace} ^® _{); melphalan, L-PAM (Alkeran} ^® _{); mercaptopurine, 6-MP (Purinethol} ^® _{); mesna (Mesnex} ^® _{); mesna (Mesnex tabs} ^® _{); methotrexate (Methotrexate} ^® _{); methoxsalen (Uvadex} ^® _{); mitomycin C}

(_Mutamycin ^® _{); mitotane (Lysodren} ^® _{); mitoxantrone (Novantrone} ^® _{); nandrolone}

p_{henpropionate (Durabolin-50} ^® _{); nelarabine (Arranon} ^® _{); Nofetumomab (Verluma} ^® _); Oprelvekin (Neumega^®); oxaliplatin (Eloxatin^®); paclitaxel (Paxene^®); paclitaxel (Taxol^®); paclitaxel protein-bound particles (Abraxane^®); palifermin (Kepivance^®); pamidronate

(Aredia^®); pegademase (Adagen (Pegademase Bovine)^®); pegaspargase (Oncaspar^®);

Pegfilgrastim (Neulasta^®); pemetrexed disodium (Alimta^®); pentostatin (Nipent^®); pipobroman (Vercyte^®); plicamycin, mithramycin (Mithracin^®); porfimer sodium (Photofrin^®);

procarbazine (Matulane^®); quinacrine (Atabrine^®); Rasburicase (Elitek^®); Rituximab

(Rituxan^®); Ridaforolimus; sargramostim (Leukine^®); Sargramostim (Prokine^®); sorafenib (Nexavar^®); streptozocin (Zanosar^®); sunitinib maleate (Sutent^®); talc (Sclerosol^®); tamoxifen (Nolvadex^®); temozolomide (Temodar^®); teniposide, VM-26 (Vumon^®); testolactone

(Teslac^®); thioguanine, 6-TG (Thioguanine^®); thiotepa (Thioplex^®); topotecan (Hycamtin^®); toremifene (Fareston^®); Tositumomab (Bexxar^®); Tositumomab/I-131 tositumomab (Bexxar^®); Trastuzumab (Herceptin^®); tretinoin, ATRA (Vesanoid^®); Uracil Mustard (Uracil Mustard Capsules^®); valrubicin (Valstar^®); vinblastine (Velban^®); vincristine (Oncovin^®); vinorelbine (Navelbine^®); vorinostat (Zolinza^®) and zoledronate (Zometa^®).

In an example, the angiogenesis inhibitor to be used as the second compound is selected from a tyrosine kinase inhibitor, an inhibitor of epidermal-derived growth factor, an inhibitor of fibroblast-derived growth factor, an inhibitor of platelet derived growth factor, an MMP (matrix metalloprotease) inhibitor, an integrin blocker, interferon-a, interleukin-12, pentosan polysulfate, a cyclooxygenase inhibitor, carboxyamidotriazole, combretastatin A-4, squalamine, 6-O-chloroacetyl-carbonyl)-fumagillol, thalidomide, angiostatin, troponin-1, or an antibody to VEGF. In an example, the estrogen receptor modulator is tamoxifen or raloxifene.

Thus, the scope of the instant invention encompasses the use of the instantly claimed compounds in combination with a second compound selected from: an estrogen receptor modulator, an androgen receptor modulator, a retinoid receptor modulator, a cytotoxic/cytostatic agent, an antiproliferative agent, a prenyl-protein transferase inhibitor, an HMG-CoA reductase inhibitor, an HIV protease inhibitor, a reverse transcriptase inhibitor, an angiogenesis inhibitor, PPAR-g agonists, PPAR-d agonists, an inhibitor of inherent multidrug resistance, an anti-emetic agent, an agent useful in the treatment of anemia, an agent useful in the treatment of neutropenia, an immunologic-enhancing drug, an inhibitor of cell proliferation and survival signaling, a bisphosphonate, an aromatase inhibitor, an siRNA therapeutic, g-secretase and/or NOTCH inhibitors, agents that interfere with receptor tyrosine kinases (RTKs), an agent that interferes with a cell cycle checkpoint, and any of the therapeutic agents listed above. Also included in the scope of the claims is a method of treating cancer that comprises administering a therapeutically effective amount of a compound of the instant invention in combination with radiation therapy and/or in combination with a second compound selected from: an estrogen receptor modulator, an androgen receptor modulator, a retinoid receptor modulator, a cytotoxiccytostatic agent, an antiproliferative agent, a prenyl-protein transferase inhibitor, an HMG-CoA reductase inhibitor, an HIV protease inhibitor, a reverse transcriptase inhibitor, an angiogenesis inhibitor, PPAR-g agonists, PPAR-d agonists, an inhibitor of inherent multidrug resistance, an anti-emetic agent, an agent useful in the treatment of anemia, an agent useful in the treatment of neutropenia, an immunologic-enhancing drug, an inhibitor of cell proliferation and survival signaling, a bisphosphonate, an aromatase inhibitor, an siRNA therapeutic, g-secretase and/or NOTCH inhibitors, agents that interfere with receptor tyrosine kinases (RTKs), an agent that interferes with a cell cycle checkpoint, and any of the therapeutic agents listed above.

And yet another example of the invention is a method of treating cancer that comprises administering a therapeutically effective amount of a compound of the instant invention in combination with paclitaxel or trastuzumab.

The invention further encompasses a method of treating or preventing cancer that comprises administering a therapeutically effective amount of a compound of the instant invention in combination with a COX-2 inhibitor.

The therapeutic combination disclosed herein may be used in combination with one or more other active agents, including but not limited to, other anti-cancer agents that are used in the prevention, treatment, control, amelioration, or reduction of risk of a particular disease or condition (e.g., cell-proliferation disorders). In one embodiment, a compound disclosed herein is combined with one or more other anti-cancer agents for use in the prevention, treatment, control amelioration, or reduction of risk of a particular disease or condition for which the compounds disclosed herein are useful. Such other active agents may be administered, by a route and in an amount commonly used therefor, prior to, contemporaneously, or sequentially with a compound of the present disclosure.

The instant invention also includes a pharmaceutical composition useful for treating or preventing cancer that comprises a therapeutically effective amount of a compound of the instant invention and a second compound selected from: an estrogen receptor modulator, an androgen receptor modulator, a retinoid receptor modulator, a cytotoxic/cytostatic agent, an antiproliferative agent, a prenyl-protein transferase inhibitor, an HMG-CoA reductase inhibitor, an HIV protease inhibitor, a reverse transcriptase inhibitor, an angiogenesis inhibitor, a PPAR-g agonist, a PPAR-d agonist, an inhibitor of cell proliferation and survival signaling, a bisphosphonate, an aromatase inhibitor, an siRNA therapeutic, g-secretase and/or NOTCH inhibitors, agents that interfere with receptor tyrosine kinases (RTKs), an agent that interferes with a cell cycle checkpoint, and any of the therapeutic agents listed above.

When any variable occurs more than one time in any constituent, its definition on each occurrence is independent at every other occurrence. Also, combinations of substituents and variables are permissible only if such combinations result in stable compounds.

It is understood that substituents and substitution patterns on the compounds of the instant invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results. Also,“optionally substituted” means either unsubstituted or substituted with the specified groups, radicals or moieties.

The present invention includes compounds of structural formula I, Ia, Ib, or Ic, as well as the pharmaceutically acceptable salts thereof, and also salts that are not pharmaceutically acceptable when they are used as precursors to the free compounds or their pharmaceutically acceptable salts or in other synthetic manipulations.

The compounds of the present invention may be administered in the form of a pharmaceutically acceptable salt. The term "pharmaceutically acceptable salt" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids. Salts of basic compounds encompassed within the term "pharmaceutically acceptable salt" refer to non-toxic salts of the compounds of the invention which are generally prepared by reacting the free base with a suitable organic or inorganic acid. Representative salts of basic compounds of the present invention include, but are not limited to, the following: acetate, ascorbate, adipate, alginate, aspirate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, 4-bromobenzenesulfonate, butyrate, camphorate, camphorsulfonate, camsylate, carbonate, chloride, clavulanate, citrate,

cyclohexylamidosulfonate, cyclopentane propionate, diethylacetic, digluconate, dihydrochloride, dodecylsulfanate, edetate, edisylate, estolate, esylate, ethanesulfonate, formic, fumarate, gluceptate, glucoheptanoate, gluconate, glucuonate, glutamate, glycerophosphate,

glycollylarsanilate, hemisulfate, heptanoate, hexanoate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, 2-hydroxyethanesulfonate, hydroxynaphthoate, iodide, isonicotinic, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, methanesulfonate, mucate, 2-naphthalenesulfonate, napsylate, nicotinate, nitrate, N-methylglucamine ammonium salt, oleate, oxalate, pamoate (embonate), palmitate, pantothenate, pectinate, persulfate, phosphate/diphosphate, pimelic, phenylpropionic, polygalacturonate, propionate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, teoclate, thiocyanate, tosylate, triethiodide, trifluoroacetate, trifluoromethylsulfonate, p-toluenesulfonate, undeconate, valerate and the like.

Furthermore, where the compounds of the invention carry an acidic moiety, suitable pharmaceutically acceptable salts thereof include, but are not limited to, salts derived from inorganic bases including aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, mangamous, potassium, sodium, zinc, and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium, and sodium salts.

With basic reagents such as hydroxides, carbonates, hydrogencarbonates, alkoxides and ammonia, organic bases or alternatively basic amino acids the compounds of the formula I, Ia, Ib, or Ic, form stable alkali metal, alkaline earth metal or optionally substituted ammonium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, cyclic amines, dicyclohexyl amines and basic ion-exchange resins, such as arginine, betaine, caffeine, choline, N,N- dibenzylethylenediamine, diethanolamine, diethylamine, 2-diethylaminoethanol, 2- dimethylaminoethanol, ethanolamine, ethylamine, ethylenediamine, N-ethylmorpholine, N- ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, ornithine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethanolamine, triethylamine, trimethylamine, tripropylamine, trometamol, tromethamine, and the like. Also, included are the basic nitrogen-containing groups may be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl; and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides and others.

The preparation of pharmacologically acceptable salts from compounds of the formula I, Ia, Ib or Ic capable of salt formation, including their stereoisomeric forms, is carried out by known methods, for example, by mixing a compound of the present invention with an equivalent amount and a solution containing a desired acid, base, or the like, and then collecting the desired salt by filtering the salt or distilling off the solvent. The compounds of the present invention and salts thereof may form solvates with a solvent such as water, ethanol, or glycerol. The compounds of the present invention may form an acid addition salt and a salt with a base at the same time according to the type of substituent of the side chain.

The present invention encompasses all stereoisomeric forms of the compounds of formula I, Ia, Ib, or Ic. Centers of asymmetry that are present in the compounds of formula I, Ia, Ib, or Ic can all independently of one another have (R) configuration or (S) configuration. When bonds to the chiral carbon are depicted as straight lines in the structural formulas of the invention, it is understood that both the (R) and (S) configurations of the chiral carbon, and hence both enantiomers and mixtures thereof, are embraced within the formula. Similarly, when a compound name is recited without a chiral designation for a chiral carbon, it is understood that both the (R) and (S) configurations of the chiral carbon, and hence individual enantiomers and mixtures thereof, are embraced by the name. The production of specific stereoisomers or mixtures thereof may be identified in the Examples where such stereoisomers or mixtures were obtained, but this in no way limits the inclusion of all stereoisomers and mixtures thereof from being within the scope of the invention.

The invention includes all possible enantiomers and diastereomers and mixtures of two or more stereoisomers, for example mixtures of enantiomers and/or diastereomers, in all ratios. Thus, enantiomers are a subject of the invention in enantiomerically pure form, both as levorotatory and as dextrorotatory antipodes, in the form of racemates and in the form of mixtures of the two enantiomers in all ratios. In the case of a cis/trans isomerism the invention includes both the cis form and the trans form as well as mixtures of these forms in all ratios. The preparation of individual stereoisomers can be carried out, if desired, by separation of a mixture by customary methods, for example by chromatography or crystallization, by the use of stereochemically uniform starting materials for the synthesis or by stereoselective synthesis. Optionally a derivatization can be carried out before a separation of stereoisomers. The separation of a mixture of stereoisomers can be carried out at an intermediate step during the synthesis of a compound of formula I, Ia, Ib, or Ic, or it can be done on a final racemic product. Absolute stereochemistry may be determined by X-ray crystallography of crystalline products or crystalline intermediates which are derivatized, if necessary, with a reagent containing a stereogenic center of known configuration. Where compounds of the invention are capable of tautomerization, all individual tautomers as well as mixtures thereof are included in the scope of the invention. The present invention includes all such isomers, as well as salts, solvates

(including hydrates) and solvated salts of such racemates, enantiomers, diastereomers and tautomers and mixtures thereof.

In the compounds of the invention, the atoms may exhibit their natural isotopic abundances, or one or more of the atoms may be artificially enriched in a particular isotope having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number predominantly found in nature. The present invention is meant to include all suitable isotopic variations of the specifically and generically described compounds. For _{example, different isotopic forms of hydrogen (H) include protium (} ¹ _{H) and deuterium (} ² _H). Protium is the predominant hydrogen isotope found in nature. Enriching for deuterium may afford certain therapeutic advantages, such as increasing in vivo half-life or reducing dosage requirements, or may provide a compound useful as a standard for characterization of biological samples. Isotopically-enriched compounds can be prepared without undue experimentation by conventional techniques well known to those skilled in the art or by processes analogous to those described in the general process schemes and examples herein using appropriate isotopically- enriched reagents and/or intermediates.

Furthermore, compounds of the present invention may exist in amorphous form and/or one or more crystalline forms, and as such all amorphous and crystalline forms and mixtures thereof of the compounds of formula I, Ia, Ib, or Ic are intended to be included within the scope of the present invention. In addition, some of the compounds of the instant invention may form solvates with water (i.e., a hydrate) or common organic solvents. Such solvates and hydrates, particularly the pharmaceutically acceptable solvates and hydrates, of the instant compounds are likewise encompassed within the scope of the invention, along with un-solvated and anhydrous forms.

The present invention includes compounds of structural formula I, Ia, Ib, or Ic, or any other generic structural formula or specific compound described or claimed herein, and is intended to encompass the specific compound or compounds falling within the scope of the formula or embodiment.

The present invention includes compounds of structural formula I, Ia, Ib, or Ic as well as salts thereof, particularly pharmaceutically acceptable salts, solvates of such compounds and solvated salt forms thereof, where such forms are possible unless specified otherwise.

Except where noted herein, "alkyl" is intended to include both branched- and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. Commonly used abbreviations for alkyl groups are used throughout the specification, e.g. methyl may be represented by conventional abbreviations including“Me” or CH₃ or a symbol

" _"

that is an extended bond as the terminal group, e.g. , ethyl may be represented by“Et” or CH₂CH₃, propyl may be represented by“Pr” or CH₂CH₂CH₃, butyl may be represented by“Bu” or CH₂CH₂CH₂CH₃ , etc.“C_1-4 alkyl” (or“C₁-C₄ alkyl”) for example, means linear or branched chain alkyl groups, including all isomers, having the specified number of carbon atoms. For example, the structures

^a

have equivalent meanings. C_1-4 alkyl includes n-, iso-, sec- and t-butyl, n- and isopropyl, ethyl and methyl. If no number is specified, 1-4 carbon atoms are intended for linear or branched alkyl groups.

Also, in the case of a carboxylic acid (-COOH) or alcohol group being present in the compounds of the present invention, pharmaceutically acceptable esters of carboxylic acid derivatives, such as methyl, ethyl, or pivaloyloxymethyl, or acyl derivatives of alcohols, such as O-acetyl, O-pivaloyl, O-benzoyl, and O-aminoacyl, can be employed. Included are those esters and acyl groups known in the art for modifying the solubility or hydrolysis characteristics for use as sustained-release or prodrug formulations.

"Aryl" refers to an aromatic monocyclic or multicyclic ring moiety comprising 6 to 14 ring carbon atoms, or more specifically, 6 to 10 ring carbon atoms. Monocyclic aryl rings include, but are not limited to, phenyl and naphthyl. Multicyclic rings include, but are not limited to, naphthyl and bicyclic rings wherein phenyl is fused to a C5-7cycloalkyl or C5-7cycloalkenyl ring. Aryl groups may be optionally substituted with one or more substituents as defined herein. Bonding can be through any of the carbon atoms of any ring.

“Optionally substituted” refers to“unsubstituted or substituted,” and therefore, the generic structural formulas described herein encompass compounds containing the specified optional substituent(s) as well as compounds that do not contain the optional substituent(s). Each substituent is independently defined each time it occurs within the generic structural formula definitions.

If the compounds of formula I, Ia, Ib, or Ic simultaneously contain acidic and basic groups in the molecule the invention also includes, in addition to the salt forms mentioned, inner salts or betaines (zwitterions). Salts can be obtained from the compounds of formula I, Ia, Ib, or Ic by customary methods which are known to the person skilled in the art, for example by combination with an organic or inorganic acid or base in a solvent or dispersant, or by anion exchange or cation exchange from other salts. The present invention also includes all salts of the compounds of formula I, Ia, Ib, and Ic which, owing to low physiological compatibility, are not directly suitable for use in pharmaceuticals but which can be used, for example, as intermediates for chemical reactions or for the preparation of physiologically acceptable salts. The invention also includes derivatives of the compound of formula I, Ia, or Ib, acting as prodrugs and solvates. Any pharmaceutically acceptable pro-drug modification of a compound of the invention which results in conversion in vivo to a compound within the scope of the invention is also within the scope of the invention. Prodrugs, following administration to the patient, are converted in the body by normal metabolic or chemical processes, such as through hydrolysis in the blood, to the compound of formula I, Ia, or Ib. Such prodrugs include those that demonstrate enhanced bioavailability, tissue specificity, and/or cellular delivery, to improve drug absorption of the compound of formula I, Ia, or Ib. The effect of such prodrugs may result from modification of physicochemical properties such as lipophilicity, molecular weight, charge, and other physicochemical properties that determine the permeation properties of the drug. For example, esters can optionally be made by esterification of an available carboxylic acid group or by formation of an ester on an available hydroxy group in a compound. Similarly, labile amides can be made. Pharmaceutically acceptable esters or amides of the compounds of the invention may be prepared to act as pro-drugs which can be hydrolyzed ba–k to an acid (or - COO- depending on the pH of the fluid or tissue where conversion takes place) or hydroxy form particularly in vivo and as such are encompassed within the scope of the invention. Examples of pharmaceutically acceptable pro-drug modifications include, but are not limited to, -C_1-6alkyl esters and–C_1-6alkyl substituted with phenyl esters.

When any variable occurs more than one time in any constituent or in formula I, Ia, Ib, or Ic, its definition on each occurrence is independent of its definition at every other occurrence. Also, combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.

Except where noted, the term "halogen" means fluorine, chlorine, bromine or iodine.

Where ring atoms are represented by variables such as“X”, e.g,

the variables are defined by indicating the atom located at the variable ring position without depicting the ring bonds associated with the atom. For example, when X in the above ring is nitrogen, the definition will show“N” and will not depict the bonds associated with it, e.g., will not show“=N-“. Likewise, when X is a carbon atom that is substituted with bromide, the definition will show“C-Br” and will not depict the bonds associated with it, e.g., will not show ^" _"

.

The invention also includes derivatives of the compound of formula I, Ia, Ib, or Ic acting as prodrugs and solvates. Prodrugs, following administration to the patient, are converted in the body by normal metabolic or chemical processes, such as through hydrolysis in the blood, to the compound of formula I, Ia, Ib, or Ic. Such prodrugs include those that demonstrate enhanced bioavailability, tissue specificity, and/or cellular delivery, to improve drug absorption of the compound of formula I, Ia, Ib, or Ic. The effect of such prodrugs may result from modification of physicochemical properties such as lipophilicity, molecular weight, charge, and other physicochemical properties that determine the permeation properties of the drug.

The invention also relates to medicaments containing at least one compound of the formula I, Ia, Ib or Ic and/or of a pharmaceutically acceptable salt of the compound of the formula I, Ia, Ib or Ic and/or an optionally stereoisomeric form of the compound of the formula I, Ia, Ib or Ic or a pharmaceutically acceptable salt of the stereoisomeric form of the compound of formula I, Ia, Ib or Ic together with a pharmaceutically acceptable carrier, additive and/or other active substances and auxiliaries.

The medicaments according to the invention can be administered by oral, inhalative, rectal or transdermal administration or by subcutaneous, intraarticular, intraperitoneal or intravenous injection. Oral administration is preferred. Coating of stents with compounds of the formula I, Ia, Ib or Ic and other surfaces which come into contact with blood in the body is possible.

The invention also relates to a process for the production of a medicament, which comprises bringing at least one compound of the formula I, Ia, Ib or Ic into a suitable

administration form using a pharmaceutically acceptable carrier and optionally further suitable active substances, additives or auxiliaries.

Suitable solid or galenical preparation forms are, for example, granules, powders, coated tablets, tablets, (micro)capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions and preparations having prolonged release of active substance, in whose preparation customary excipients such as vehicles, disintegrants, binders, coating agents, swelling agents, glidants or lubricants, flavorings, sweeteners and solubilizers are used.

Frequently used auxiliaries which may be mentioned are magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, lactose, gelatin, starch, cellulose and its derivatives, animal and plant oils such as cod liver oil, sunflower, peanut or sesame oil, polyethylene glycol and solvents such as, for example, sterile water and mono- or polyhydric alcohols such as glycerol.

The dosage regimen utilizing the compounds is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed. An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter, or arrest the progress of the condition.

Oral dosages of the compounds, when used for the indicated effects, will range between about 0.01 mg per kg of body weight per day (mg/kg/day) to about 30 mg/kg/day, preferably 0.025-7.5 mg/kg/day, more preferably 0.1-2.5 mg/kg/day, and most preferably 0.1-0.5 mg/kg/day (unless specified otherwise, amounts of active ingredients are on free base basis). For example, an 80 kg patient would receive between about 0.8 mg/day and 2.4 g/day, preferably 2- 600 mg/day, more preferably 8-200 mg/day, and most preferably 8-40 mg/kg/day. A suitably prepared medicament for once a day administration would thus contain between 0.8 mg and 2.4 g, preferably between 2 mg and 600 mg, more preferably between 8 mg and 200 mg, and most preferably 8 mg and 40 mg, e.g., 8 mg, 10 mg, 20 mg and 40 mg. Advantageously, the compounds may be administered in divided doses of two, three, or four times daily. For administration twice a day, a suitably prepared medicament would contain between 0.4 mg and 4 g, preferably between 1 mg and 300 mg, more preferably between 4 mg and 100 mg, and most preferably 4 mg and 20 mg, e.g., 4 mg, 5 mg, 10 mg and 20 mg.

Intravenously, the patient would receive the active ingredient in quantities sufficient to deliver about 0.01 mg per kg of body weight per day (mg/kg/day) to about 30 mg/kg/day, preferably 0.025-7.5 mg/kg/day, more preferably 0.1-2.5 mg/kg/day, and even more preferably 0.1-0.5 mg/kg/day. Such quantities may be administered in a number of suitable ways, e.g. large volumes of low concentrations of active ingredient during one extended period of time or several times a day, low volumes of high concentrations of active ingredient during a short period of time, e.g. once a day. Typically, a conventional intravenous formulation may be prepared which contains a concentration of active ingredient of between about 0.01-1.0 mg/ml, e.g.0.1 mg/ml, 0.3 mg/ml, and 0.6 mg/ml, and administered in amounts per day of between 0.01 ml/kg patient weight and 10.0 ml/kg patient weight, e.g.0.1 ml/kg, 0.2 ml/kg, 0.5 ml/kg. In one example, an 80 kg patient, receiving 8 ml twice a day of an intravenous formulation having a concentration of active ingredient of 0.5 mg/ml, receives 8 mg of active ingredient per day.

Glucuronic acid, L-lactic acid, acetic acid, citric acid or any pharmaceutically acceptable acid/conjugate base with reasonable buffering capacity in the pH range acceptable for intravenous administration may be used as buffers. The choice of appropriate buffer and pH of a formulation, depending on solubility of the drug to be administered, is readily made by a person having ordinary skill in the art.

The compounds of the invention may be prepared by employing reactions as shown in the following Reaction Schemes, in addition to other standard manipulations that are known in the literature or exemplified in the experimental procedures. The illustrative Reaction Schemes below, therefore, are not limited by the compounds listed or by any particular substituents employed for illustrative purposes. Substituent numbering as shown in the Reaction Schemes do not necessarily correlate to that used in the claims and often, for clarity, a single substituent is shown attached to the compound where multiple substituents are optionally allowed under the definitions of formula I, Ia, Ib, or Ic hereinabove. Methods for Making the Compounds of Present Invention

General Methods

The compounds of the present invention can be readily produced from known compounds or commercially available compounds by, for example, known processes described in published documents, and produced by production processes described below. The present invention is not limited to the production processes described below. The invention also includes processes for the preparation of compounds of the invention.

It should be noted that, when a compound of structural formula I, Ia, Ib, or Ic has a reactive group such as hydroxy group, amino group, carboxyl group, or thiol group as its substituent, such group may be adequately protected with a protective group in each reaction step and the protective group may be removed at an adequate stage. The process of such introduction and removal of the protective group may be adequately determined depending on the group to be protected and the type of the protective group, and such introduction and removal are conducted, for example, by the process described in the review section of Greene, T.W., et. al.,“Protective Groups in Organic Synthesis”, 2007, 4th Ed., Wiley, New York, or Kocienski, P.,“Protecting Groups” 1994, Thieme.

It should be noted that, if a discrepancy between the chemical name and structure exists, the structure is understood to dominate.

The present invention is not limited in scope by the specific embodiments disclosed in the examples which are intended as illustrations of a few aspects of the invention and any embodiments that are functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the relevant art and are intended to fall within the scope of the appended claim.

All solvents used were commercially available and were used without further purification. Reactions were typically run using anhydrous solvents under an inert atmosphere of nitrogen.

Starting materials used were either available from commercial sources or prepared according to literature procedures and had experimental data in accordance with those reported. Abbreviations used are those conventional in the art of the following.

ACN acetonitrile

AcOH acetic acid

aq. aqueous

9-BBN 9-Borabicyclo(3.3.1)nonane

BSA Bis(trimethylsilyl)acetamide

ºC degree Celsius

conc. concentration

DCM dichloromethane

DIBAL-H diisobutylaluminium hydride

DIEA N,N-diisopropylethylamine

DMSO dimethylsulfoxide

DTT dithiothreitol

EDC N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide

Equiv equivalents (molar)

EtOAc ethyl acetate

EtOH ethanol

g gram

h hour(s)

HPLC high pressure liquid chromatography

K-Selectride potassium tri-sec-butylborohydride

LCMS liquid chromatography and mass spectrometry

M molar

MeOH methanol

MS mass spectrometry

mmol millimole

mg milligram min minutes

mL milliliter(s)

N normal

ND Not determined

nM nanomolar

NMR nuclear magnetic resonance

OTIPS triisopropylsilyl ether

PdCl₂(dppf) [1,1-bis(diphenylphosphine)ferrocene]dichloropalladium(II)

Pd(PPh₃)₄ tetrakis(triphenylphosphine)palladium(0)

POCl₃ phosphorus(V) oxychloride

psi pound per square inch

rt room temperature

SFC Supercritical fluid chromatography

SOCl₂ thionyl chloride

TBAF tetrabutylammonium fluoride

TBDPSCl tert-butyl(chloro)diphenylsilane

TBDPSO tert-butyldiphenylsilyl ether

TFA trifluoroacetic acid

THF tetrahydrofuran

TLC thin layer chromatography

TMSOTf Trimethylsilyl trifluoromethanesulfonate

TPAP Tetrapropylammonium perruthenate

TsOH p-toluenesulfonic acid

mL microliter

General Synthetic Schemes

While the present invention has been described in conjunction with the specific examples set forth above, many alternatives, modifications and variations thereof will be apparent to those of ordinary skill in the art. In some cases the order of carrying out the steps of the reaction schemes may be varied to facilitate the reaction or to avoid unwanted reaction products. All such alternatives, modifications and variations are intended to fall within the spirit and scope of the present invention. Starting materials and intermediates are purchased from commercial sources, made from known procedures, or are otherwise illustrated.

Unless otherwise indicated, all variables are as previously defined. Scheme 1:

Compounds of formula 4 and 5 can be formed in the above sequence. Addition of an organomagnesium or organolithium reagent to 1 generates the corresponding ketone 2 (R³ = Ac or H) which can be reduced with a number of hydride sources to provide alcohol 3. Alcohol 3 was treated with ammonia to give compounds of formula 4. When alcohol 4 has a Boc protected amine present in the R¹ group, it is treated with TFA to give the corresponding free amine derivative of formula 5. Scheme 2:

In Scheme

Compounds of formula 7 and 8 can be formed in the following sequence below. Addition of an organomagnesium or organolithium reagent to 1 generates the corresponding ketone 2 (R³ = Ac or H) which was further treated with a Grignard reagent to generate the corresponding tertiary alcohol 6. Tertiary alcohol 6 was treated with ammonia to give compounds of formula 7. When compound 7 has a Boc protected amine present in the R¹ group, it is treated with TFA to give the corresponding free amine derivative of formula 8. Scheme 3:

Compounds of formula 11 and 12 can be formed in the above sequence. Addition of an organomagnesium or organolithium reagent to 1 generates the corresponding ketone 2 which was further treated with methylmagnesium bromide to yield the corresponding tertiary alcohol 9. Dehydration of 9 with Martin sulfurane gives the corresponding olefin 10, which was then treated with ammonia to give compounds of formula 11. Hydrogenation of olefin 11 gives compounds of formula 12. Scheme 4:

Compounds of formula 17 and 18 can be formed in the above sequence. Addition of an organomagnesium or organolithium reagent to 1 generates the corresponding ketone 13. Diol 13 is protected to give acetonide 14 which is then treated with Tebbe reagent to give olefin 15. Olefin 15 is treated with dimethylzinc and Pd(PPh3)4 to give compound 16, which is then treated with TFA to give compounds of formula 17. Hydrogenation of olefin 17 gives compounds of formula 18. Scheme 5:

e

In Scheme

Compounds of formula 20 and 21 can be formed in the above sequence. Addition of an organomagnesium or organolithium reagent to 1 generates the corresponding ketone 13 which is further treated with a Grignard reagent to give the corresponding tertiary alcohol 19. Alcohol 19 is treated with Me₂Zn and Pd(PPh₃)₄ to give compounds of formula 20. When compound 20 has a Boc protected amine present in the R¹ group, it is treated with TFA to give the corresponding free amine derivative of formula 21. Scheme 6:

25

In Scheme 6, R is either F, CHF₂ or H.

Compounds of formula 25 can be formed in the above sequence. Ketone 22 is treated with methylmagnesium bromide to give the corresponding tertiary alcohol 23. Alcohol 23 is treated with TFA to give the corresponding amine 24 which is then treated with ammonia to give compounds of formula 25. Scheme 7:

29

In Scheme 7, R is either F, CHF₂ or H.

Compounds 29 can be formed in the above sequence. Ketone 22 is treated with

methylmagnesium bromide to give the corresponding tertiary alcohol 23. Alcohol 23 is treated with Martin sulfurane to give olefin 26 which is then treated with ammonia to give diol 27. Olefin 27 is hydrogenated to afford compound 28 and then treated with TFA to afford compound 29. Scheme 8:

In Scheme 8, R is either F, CHF₂ or H.

Compounds 32 can be formed in the above sequence. Ketone 30 is reduced with NaBH4 to give alcohol 31 which is then treated with TFA to afford compound 32. SYNTHESIS OF INTERMEDIATES

Synthetic scheme of Intermediate 1

Step 1

Step 2 Step 3

H ^SO

AcO OAc

Intermediate 1

Intermediate 1: (2S,3S,4R,5R)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2- (methoxy(methyl)carbamoyl)-3-methyltetrahydrofuran-3,4-diyl diacetate

Step 1: A solution of (3R,4R,5R)-5-(hydroxymethyl)oxolane-2,3,4-triol (750 g, 5 mol) in acetone (7 L), sulfuric acid (75 mL) and dioxo(sulfonylidene)copper (1503 g, 9.42 mol) was placed into a 10-L 4-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen. The resulting solution was stirred for 48 hours at room temperature. The mixture was filtered, and then concentrated NH₄OH (25%, 200 mL) was added. The mixture was filtered, dried over Na₂SO₄, and concentrated under reduced pressure. The residue was diluted with methanol (4.5 L) and HCl 0.1 N, 1.3 L) was added. The reaction was stirred at 40 °C for 2 hours and quenched with NaHCO₃ (189 g until neutral pH). The mixture was filtered and concentrated under reduced pressure. The residue was co-evaporated with EtOH/toluene, then diluted with DCM, dried over Na2SO4, and concentrated under reduced pressure affording (3aR,5R,6S,6aR)- 5-(hydroxymethyl)-2,2-dimethyl-tetrahydro-2H-furo[2,3-d][1,3]dioxol-6-ol as an oil. The material was used crude without further purification.

Step 2: Into a 10L 4-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen was added a solution of (3aR,5R,6S,6aR)-5-(hydroxymethyl)-2,2-dimethyl- tetrahydro-2H-furo[2,3-d][1,3]dioxol-6-ol (951 g, 5 mol) in pyridine (7 L) and tert- butyl(chloro)diphenylsilane (1404 g, 5.1 mol). The resulting solution was stirred overnight at room temperature. To the solution was added 600 mL of MeOH. The resulting mixture was concentrated under reduced pressure. The residue was solubilized in EtOAc, washed with HCl (0.5 M), saturated NaHCO₃ and brine. The organic layer was dried over Na₂SO₄, and

concentrated under reduced pressure to give (3aR,5R,6S,6aR)-5-[[(tert- butyldiphenylsilyl)oxy]methyl]-2,2-dimethyl-tetrahydro-2H-furo[2,3-d][1,3]dioxol-6-ol as an oil. MS: 451 [M+Na]⁺.

Step 3: Into a 20-L 4-necked round-bottom flask, purged and maintained with an inert

atmosphere of nitrogen, was placed a solution of (3aR,5R,6S,6aR)-5-[[(tert- butyldiphenylsilyl)oxy]methyl]-2,2-dimethyl-tetrahydro-2H-furo[2,3-d][1,3]dioxol-6-ol (2142 g, 5 mol) in dichloromethane (15 L) and Dess-Martin reagent (3183 g, 7.50 mol) was added. The resulting solution was stirred overnight at room temperature. The reaction was then quenched by the addition of saturated NaHCO₃ and concentrated under reduced pressure. Et₂O was added, and the solid was removed by filtration. The filtrate was concentrated under reduced pressure to provide (3aR,5R,6aS)-5-[[(tert-butyldiphenylsilyl)oxy]methyl]-2,2-dimethyl-tetrahydro-2H- furo[2,3-d][1,3]dioxol-6-one as an oil. The material was used crude without further purification. Step 4: Into a 20-L 4-necked round-bottom flask purged and maintained with an inert

atmosphere of nitrogen, was placed a solution of (3aR,5R,6aS)-5-[[(tert- butyldiphenylsilyl)oxy]methyl]-2,2-dimethyl-tetrahydro-2H-furo[2,3-d][1,3]dioxol-6-one (2133 g, 5 mol) in tetrahydrofuran (10 L). This was followed by the dropwise addition of

chloro(methyl)magnesium (1836 mL, 5.5 mol, 3.0 M in THF) with stirring at 0 °C. The resulting solution was stirred for 1 hour at room temperature. The reaction was then quenched by the addition of saturated NH₄Cl. The organic layer (THF) was washed with brine. The mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure affording

(3aR,5R,6R,6aR)-5-[[(tert-butyldiphenylsilyl)oxy]methyl]-2,2,6-trimethyl-tetrahydro-2H- furo[2,3-d][1,3]dioxol-6-ol as a an oil. MS: 465 [M+Na]⁺. Step 5: Into a 20-L 4-necked round-bottom flask, purged and maintained with an inert atmosphere of nitrogen, was placed a solution of (3aR,5R,6R,6aR)-5-[[(tert- butyldiphenylsilyl)oxy]methyl]-2,2,6-trimethyl-tetrahydro-2H-furo[2,3-d][1,3]dioxol-6-ol (2220 g, 5 mol) in AcOH (10 L). This was followed by the dropwise addition of sulfuric acid (49.2 g, 502 mmol) with stirring at 10 °C. Acetyl acetate (2045 g, 20.0 mol) was added dropwise to the reaction mixture with stirring at 10 °C. The resulting solution was stirred for 3 hours at room temperature. After completion of the reaction, the mixture was concentrated under reduced pressure and diluted with EtOAc. The resulting mixture was washed with saturated NaHCO₃ and brine. The mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified via column chromatography on silica with ethyl

acetate/petroleum ether (1:1) affording (2R,3R,4R,5R)-2,4-bis(acetyloxy)-5-[[(tert- butyldiphenylsilyl)oxy]methyl]-4-methyloxolan-3-yl acetate as an oil. MS: 551 [M+Na]⁺.

Step 6: Into a 5-L 4-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed 4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (420 g, 1503 mmol, 1.00 equiv), BSA (305 g, 1.5 mol), a solution of (2R,3R,4R,5R)-2,4-bis(acetyloxy)-5-[[(tert- butyldiphenylsilyl)oxy]methyl]-4-methyloxolan-3-yl acetate (795 g, 1.50 mol) in MeCN (7 L), and trimethylsilyl trifluoromethanesulfonate (668 g, 3.00 mol). The resulting solution was stirred for 6 hours at 80 ^oC. After completion of the reaction, the mixture was concentrated under reduced pressure, and diluted with EtOAc. The resulting mixture was washed with saturated NaHCO₃ and brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified via column chromatography on silica with ethyl acetate/petroleum ether (1:1) affording (2R,3R,4R,5R)-4-(acetyloxy)-5-[[(tert- butyldiphenylsilyl)oxy]methyl]-2-[4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl]-4- methyloxolan-3-yl acetate as an oil. MS: 748 [M+H]⁺.

Step 7: To a solution of (2R,3R,4R,5R)-2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-(4-chloro-5- iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3-methyltetrahydrofuran-3,4-diyl diacetate (590 g, 0.79 mol) in dry THF (7.5 L) was added dropwise iPrMgCl-LiCl (1.3 M in THF, 790 mL, 1.03 mol) under N₂ below -50 ^oC. After the addition, the reaction mixture was stirred at this temperature for 2 h. This cold reaction mixture was poured into saturated aq. NH₄Cl (4000 mL), and then extracted with EtOAc (1500 mL x3). The combined organic layers were washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give (2R,3R,4R,5R)-2-(((tert- butyldiphenylsilyl)oxy)methyl)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3- methyltetrahydrofuran-3,4-diyl diacetate (480 g, 0.77 mol) as an oil, which was used directly for the next step. MS: 644 [M+Na]⁺.

Step 8: Into a 5-L 3-necked round-bottom flask, purged and maintained with an inert atmosphere of nitrogen, was placed a solution of (2R,3R,4R,5R)-4-(acetyloxy)-5-[[(tert- butyldiphenylsilyl)oxy]methyl]-2-[4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl]-4-methyloxolan- 3-yl acetate (186 g, 299 mmol) in tetrahydrofuran (1.8 L) and AcOH (90 g, 1.50 mol, ). This was followed by the dropwise addition of TBAF (600 mL of 1 M solution in THF, 600 mmol) with stirring at room temperature. The resulting solution was stirred for 2 hours at room temperature. The reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (eluting with 0 to 10% MeOH/DCM) to afford

(2R,3R,4R,5R)-4-(acetyloxy)-2-[4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl]-5-(hydroxymethyl)- 4-methyloxolan-3-yl acetate as a solid. MS: 384 [M+H]⁺.

Step 9: To a solution of (2R,3R,4R,5R)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2- (hydroxymethyl)-3-methyltetrahydrofuran-3,4-diyl diacetate (17 g, 44.3 mmol) in acetonitrile (200 mL) was added 4-methylmorpholine N-oxide monohydrate (59.9 g, 443 mmol) and TPAP (1.56 g, 4.43 mmol). The reaction mixture was stirred at room temperature for 2 hours and the resulting mixture was concentrated under reduced pressure. The crude was purified by flash column chromatography (methanol in DCM, 0 to 15%) to yield (2S,3S,4R,5R)-3,4-diacetoxy-5- (4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3-methyltetrahydrofuran-2-carboxylic acid as a solid. MS: 398 [M+H]⁺.

Step 10: To a solution of (2S,3S,4R,5R)-3,4-diacetoxy-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin- 7-yl)-3-methyltetrahydrofuran-2-carboxylic acid (16.0 g, 40.3 mmol), 1-hydroxy-7- azabenzotriazole (17 mL, 121 mmol), and N,O-dimethylhydroxylamine hydrochloride (11.8 g, 121 mmol) in DCM (300 mL) were added DIEA (42.3 mL, 242 mmol) and EDC (23.2 g, 121 mmol). The resulting mixture was stirred for 3 hours. The reaction mixture was concentrated under reduced pressure and the crude was directly purified by flash column chromatography (EtOAc/EtOH (3:1) in hexane, 0 to 100%) to yield (2S,3S,4R,5R)-5-(4-chloro-7H-pyrrolo[2,3- d]pyrimidin-7-yl)-2-(methoxy(methyl)carbamoyl)-3-methyltetrahydrofuran-3,4-diyl diacetate as a solid. MS: 441 [M+H]⁺. Intermediate 2: Preparation of non-commercially available Grignard reagent

To a solution of magnesium (15 equiv) and lithium chloride (7.5 equiv) in THF (0.57 M for lithium chloride) were added DIBAL-H (0.06 equiv). After 5 minutes at room temperature, the reaction mixture was cooled to 0 °C and aryl halide (6.0 equiv) was added at 0 °C. The resulting mixture was stirred at 0 °C for 1 hour and then used as a THF solution of the corresponding Grignard reagent.

Synthetic scheme of Intermediate 3

Step 1 Step 2

Intermediate 3 Intermediate 3: tert-butyl 6-(difluoromethyl)-5-fluoro-8-iodo-3,4-dihydroisoquinoline-2 1H)-

carboxylate

Step 1: To a solution of tert-butyl 6-(difluoromethyl)-5-fluoro-8-hydroxy-3,4- dihydroisoquinoline-2(1H)-carboxylate (2 g, 6.30 mmol) in DCM (60 ml) were added triethylamine (0.966 ml, 6.93 mmol) and trifluoromethanesulfonic anhydride (1.12 ml, 6.62 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 30 min and then the reaction was quenched with methanol. The resulting mixture was concentrated in vacuo and the crude material was purified by flash column chromatography (EtOAc in hexane, 5 to 100%) to yield tert-butyl 6-(difluoromethyl)-5-fluoro-8-(((trifluoromethyl)sulfonyl)oxy)-3,4- dihydroisoquinoline-2(1H)-carboxylate as an oil. MS: 472 [M+Na]⁺.

Step 2: To a solution of tert-butyl 6-(difluoromethyl)-5-fluoro-8- (((trifluoromethyl)sulfonyl)oxy)-3,4-dihydroisoquinoline-2(1H)-carboxylate (1.1 g, 2.5 mmol) and sodium iodide (1.1 g, 7.50 mmol) in 1,3-dimethyl-2-imidazolidinone (10 ml) was added tris(acetonitrile)pentamethylcyclopentadienylruthenium(II) hexafluorophosphate (126 mg, 0.250 mmol), and the reaction mixture was stirred at 120 °C for 3 h. The reaction mixture was cooled to rt and diluted with ethyl acetate. The organic solution was washed with water twice and then dried over Na₂SO₄. The organic solvent was removed under reduced pressure and the crude material was purified by flash column chromatography (EtOAc in hexanes, 5 to 100%) to yield tert-butyl 6-(difluoromethyl)-5-fluoro-8-iodo-3,4-dihydroisoquinoline-2(1H)-carboxylate as an oil. MS: 450 [M+Na]⁺. Synthetic scheme of Intermediates 4 and 5

I

I^{ntermediate 4 Intermediate 5} Intermediate 4 and Intermediate 5: (2S,3S,4R,5R)-2-(2-(tert-butoxycarbonyl)-6- (difluoromethyl)-5-fluoro-1 tetrahydroisoquinoline-8-carbonyl)-3-methyl-5-(4-methyl-7H-

pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-3,4-diyl diacetate (Intermediate 4)

tert-butyl 6-(difluoromethyl)-8- 3S,4R,5R)-3,4-dihydroxy-3-methyl-5-(4-methyl-7H-

pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-2-carbonyl)-5-fluoro-3,4-dihydroisoquinoline- 2(1H)-carboxylate (Intermediate 5)

Step 1: A vial containing (2S,3S,4R,5R)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2- (methoxy(methyl)carbamoyl)-3-methyltetrahydrofuran-3,4-diyl diacetate (1.8 g, 4.08 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.118 g, 0.102 mmol) was sealed and purged with argon gas. THF (16.3 ml) was added to the reaction vial at room temperature, then

trimethylaluminum (4.08 ml, 8.17 mmol, 2.0 M in hexanes) was added. The reaction vial was heated to 80 °C and stirred for 4 h. The reaction mixture was cooled to 0 °C and quenched with saturated aqueous sodium bicarbonate. The mixture was diluted with chloroform and filtered through a Celite pad. The filtrate was dried over Na₂SO₄ and then the organic solvent was removed under reduced pressure. The crude material was purified by flash column

chromatography (EtOAc/EtOH 3:1 in hexanes, 0 to 80%) to afford (2S,3S,4R,5R)-2- (methoxy(methyl)carbamoyl)-3-methyl-5-(4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7- yl)tetrahydrofuran-3,4-diyl diacetate as an amorphous solid. MS: 421 [M+H]⁺.

Step 2: To a solution of tert-butyl 6-(difluoromethyl)-5-fluoro-8-iodo-3,4-dihydroisoquinoline- 2(1H)-carboxylate (366 mg, 0.857 mmol) in THF (6 ml) was added isopropylmagnesium chloride lithium chloride complex (791 µl, 1.03 mmol, 1.3 M in THF) at 0 °C, and the resulting mixture was stirred for 40 min. This solution was added to a separate solution of (2S,3S,4R,5R)- 2-(methoxy(methyl)carbamoyl)-3-methyl-5-(4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7- yl)tetrahydrofuran-3,4-diyl diacetate (396 mg, 0.942 mmol) in THF (3.2 ml) at 0 °C. The reaction mixture was stirred at 0 °C for 5 h and then quenched with acetic acid (196 µl, 3.43 mmol). The mixture was poured into saturated aqueous NH₄Cl and extracted with EtOAc (3x). The organic layers were combined, dried over sodium sulfate, and then filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by flash column chromatography (EtOAc/EtOH 3:1 in hexanes 0 to 40%) to afford (2S,3S,4R,5R)-2-(2-(tert- butoxycarbonyl)-6-(difluoromethyl)-5-fluoro-1,2,3,4-tetrahydroisoquinoline-8-carbonyl)-3- methyl-5-(4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-3,4-diyl diacetate (Intermediate 4) and tert-butyl 6-(difluoromethyl)-8-((2S,3S,4R,5R)-3,4-dihydroxy-3-methyl-5- (4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-2-carbonyl)-5-fluoro-3,4- dihydroisoquinoline-2(1H)-carboxylate (Intermediate 5), both as oils.

Intermediate 4: MS: 661 [M+H]⁺.

Intermediate 5: MS: 577 [M+H]⁺. EXAMPLES

The following experimental procedures detail the preparation of specific examples of the instant disclosure. The examples are for illustrative purposes only and are not intended to limit the scope of the instant disclosure in any way.

Examples 1 and 2

(2R,3S,4R,5R)-5-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-

difluorophenyl)(hydroxy)methyl)-3-methyltetrahydrofuran-3,4-diol (Example 1)

(2R,3S,4R,5R)-5-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-

difluorophenyl)(hydroxy)methyl)-3-methyltetrahydrofuran-3,4-diol (Example 2)

E^{xample 1 Example 2} Step 1: To a solution of (2S,3S,4R,5R)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2- (methoxy(methyl)carbamoyl)-3-methyltetrahydrofuran-3,4-diyl diacetate (1.2 g, 2.72 mmol) in THF (30 ml) was added 3,4-difluorophenylmagnesium bromide (0.5 M in THF, 21.8 mL, 10.9 mmol) at 0 °C (Grignard reagents that are not commercially available were prepared following the procedure described in intermediate 2 starting from the corresponding commercially available aryl halides). The reaction mixture was stirred at 0 °C for 90 min and then quenched with acetic acid (1.56 mL, 27.2 mmol). The resulting mixture was concentrated under reduced pressure and then the residue was dissolved in ethyl acetate. The organic solution was washed with 1 N citric acid followed by saturated aqueous NaHCO₃. The organic layer was dried over Na₂SO₄ and filtered. The organic solvent was removed under reduced pressure and the crude was purified by flash column chromatography (EtOAc/EtOH (3:1) in hexane, 5 to 100%) to yield (2S,3S,4R,5R)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-(3,4-difluorobenzoyl)-3- methyltetrahydrofuran-3,4-diyl diacetate as a solid. MS: 494 [M+H]⁺.

Step 2: To a solution of (2S,3S,4R,5R)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-(3,4- difluorobenzoyl)-3-methyltetrahydrofuran-3,4-diyl diacetate (500 mg, 1.01 mmol) in methanol (10 mL) was added sodium borohydride (115 mg, 3.04 mmol), and the reaction mixture was stirred at 0 °C for 15 minutes. The reaction was quenched with acetone (10 mL) and then acetic acid (522 µL, 9.11 mmol) at 0 °C, and the resulting mixture was concentrated under reduced pressure. The resulting crude was purified by flash column chromatography (EtOAc/EtOH (3:1) in hexane, 5 to 100%) to yield (2R,3R,4R,5R)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2- ((3,4-difluorophenyl)(hydroxy)methyl)-3-methyltetrahydrofuran-3,4-diyl diacetate as a solid. MS: 496 [M+H]⁺.

Step 3: A solution of (2R,3R,4R,5R)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((3,4- difluorophenyl)(hydroxy)methyl)-3-methyltetrahydrofuran-3,4-diyl diacetate (380 mg, 0.766 mmol) in 7 N ammonia in methanol (3.0 mL) was stirred at 130 °C for 4 hours in a microwave reactor. The reaction mixture was concentrated under reduced pressure and then purified by chiral SFC (GreenSep Basic column, 25%/75% methanol/CO₂) to yield (2R,3S,4R,5R)-5-(4- amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((R)-(3,4-difluorophenyl)(hydroxy)methyl)-3- methyltetrahydrofuran-3,4-diol (Example 1) as a solid and (2R,3S,4R,5R)-5-(4-amino-7H- pyrrolo[2,3-d]pyrimidin-7-yl)-2-((S)-(3,4-difluorophenyl)(hydroxy)methyl)-3- methyltetrahydrofuran-3,4-diol (Example 2) as a solid.

Example 1: MS: 393 [M+H]⁺.¹H-NMR (500 MHz, CD₃OD) d 8.11 (s, 1H), 7.45– 7.36 (m, 1H), 7.32– 7.15 (m, 3H), 6.62 (d, J = 3.6 Hz, 1H), 5.86 (d, J = 7.9 Hz, 1H), 5.02 (d, J = 3.6 Hz, 1H), 4.66 (d, J = 7.9 Hz, 1H), 4.34 (d, J = 4.0 Hz, 1H), 1.18 (s, 3H). Example 2: MS: 393 [M+H]⁺.¹H-NMR (500 MHz, CD₃OD) d 8.15 (s, 1H), 7.43 (d, J = 3.6 Hz, 1H), 7.29– 7.23 (m, 1H), 7.18– 7.09 (m, 2H), 6.64 (d, J = 3.6 Hz, 1H), 5.86 (d, J = 8.0 Hz, 1H), 4.86 (d, J = 1.0 Hz, 1H), 4.68 (d, J = 8.0 Hz, 1H), 4.14 (d, J = 1.0 Hz, 1H), 1.6 (s, 3H). Example 3

(2R,3S,4R,5R)-5-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((R)-1-(3,4-difluorophenyl)-1- hydroxyethyl)-3-methyltetrahydrofuran-3,4-diol

Step 1: To a solution of (2S,3S,4R,5R)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2- (methoxy(methyl)carbamoyl)-3-methyltetrahydrofuran-3,4-diyl diacetate (1.2 g, 2.72 mmol) in THF (30 mL) was added 3,4-difluorophenylmagnesium bromide (0.5 M in THF, 21.8 mL, 10.9 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 90 minutes and then quenched with acetic acid (1.56 mL, 27.2 mmol). The resulting mixture was concentrated under reduced pressure and the residue was dissolved in ethyl acetate. The organic solution was washed with 1N citric acid and saturated aqueous NaHCO₃, and the organic layer was dried over Na₂SO₄. The organic solvent was removed under reduced pressure and the crude was purified by flash column chromatography (EtOAc/EtOH (3:1) in hexane, 5 to 100%) to yield (2S,3S,4R,5R)-5-(4-chloro- 7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-(3,4-difluorobenzoyl)-3-methyltetrahydrofuran-3,4-diyl diacetate as a solid. MS: 494 [M+H]⁺.

Step 2: To a solution of (2S,3S,4R,5R)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-(3,4- difluorobenzoyl)-3-methyltetrahydrofuran-3,4-diyl diacetate (300 mg, 0.607 mmol) in THF (6.0 mL) was added methylmagnesium bromide (3.0 M in diethyl ether, 607 µL, 1.822 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 15 minutes and then quenched with acetic acid (278 µL, 4.86 mmol). The resulting mixture was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate. The organic solution was washed with 1 N citric acid, and then the organic layer was dried over Na₂SO₄. The organic solvent was removed under reduced pressure, and the crude material was purified by flash column chromatography (methanol in DCM, 0 to 15 %) to yield (2R,3R,4R,5R)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((R)- 1-(3,4-difluorophenyl)-1-hydroxyethyl)-3-methyltetrahydrofuran-3,4-diyl diacetate as a solid. MS: 510 [M+H]⁺. Step 3: A solution of (2R,3R,4R,5R)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((R)-1- (3,4-difluorophenyl)-1-hydroxyethyl)-3-methyltetrahydrofuran-3,4-diyl diacetate (257.3 mg, 0.505 mmol) in 7 N ammonia in methanol (3.0 ml) was stirred at 130 °C for 4 hours in a microwave reactor. The reaction mixture was concentrated under reduced pressure and then purified by chiral SFC (ES Basic column, 25%/75% methanol/CO₂) to yield (2R,3S,4R,5R)-5- (4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((R)-1-(3,4-difluorophenyl)-1-hydroxyethyl)-3- methyltetrahydrofuran-3,4-diol as a solid. MS: 407 [M+H]⁺.¹H-NMR (500 MHz, CD₃OD) d 8.14 (s, 1H), 7.56– 7.43 (m, 1H), 7.36– 7.19 (m, 3H), 6.61 (d, J = 3.7 Hz, 1H), 5.78 (d, J = 8.1 Hz, 1H), 4.67 (d, J = 8.1 Hz, 1H), 4.48 (s, 1H), 1.44 (s, 3H), 0.86 (s, 3H).

Examples 4 and 5

(2R,3S,4R,5R)-5-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((R)-(3-(difluoromethyl)-5- fluorophenyl)(hydroxy)methyl)-3-methyltetrahydrofuran-3,4-diol (Example 4)

(2R,3S,4R,5R)-5-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((S)-(3-(difluoromethyl)-5- fluorophenyl)(hydroxy)methyl)-3-methyltetrahydrofuran-3,4-diol (Example 5)

E^{xample 4 Example 5} Step 1: To a solution of magnesium (165 mg, 6.81 mmol) and lithium chloride (144 mg, 3.40 mmol) in THF (6.0 mL) was added DIBAL-H (27.2 µL, 0.027 mmol). After 5 minutes at room temperature, the reaction mixture was cooled to 0 °C, and 1-bromo-3-(difluoromethyl)-5- fluorobenzene (612 mg, 2.72 mmol) was added at 0 °C. The resulting mixture was stirred at 0 °C for 1 hour, and then the mixture was added to a solution of (2S,3S,4R,5R)-5-(4-chloro-7H- pyrrolo[2,3-d]pyrimidin-7-yl)-2-(methoxy(methyl)carbamoyl)-3-methyltetrahydrofuran-3,4-diyl diacetate (200 mg, 0.454 mmol) in THF (3.0 mL) at 0 °C. The resulting mixture was stirred for 10 minutes at 0 °C. The reaction was quenched with acetic acid (390 µL, 6.81 mmol), and the resulting mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate and then washed with 1 N citric acid. The organic layer was dried over Na₂SO₄, and the organic solvent was removed under reduced pressure. The crude material was then purified by flash column chromatography (EtOAc/EtOH (3:1) in hexanes, 5 to 100%) to yield (2S,3S,4R,5R)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-(3-(difluoromethyl)-5- fluorobenzoyl)-3-methyltetrahydrofuran-3,4-diyl diacetate as a solid. MS: 526 [M+H]⁺.

For step 2, either one of the following two conditions were used (Step 2-A or Step 2-B).

Step 2-A: To a solution of (2S,3S,4R,5R)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-(3- (difluoromethyl)-5-fluorobenzoyl)-3-methyltetrahydrofuran-3,4-diyl diacetate (266 mg, 0.505 mmol) in methanol (4.5 mL) was added sodium borohydride (57.4 mg, 1.52 mmol) at 0 °C, and the reaction mixture was stirred at 0 °C for 15 minutes. The reaction was quenched with acetone (3 mL) followed by acetic acid (260 µl, 4.55 mmol) at 0 °C, and the resulting mixture was concentrated under reduced pressure. The crude was purified by flash column chromatography (EtOAc/EtOH (3:1) in hexanes, 5 to 100%) to yield (2R,3R,4R,5R)-5-(4-chloro-7H-pyrrolo[2,3- d]pyrimidin-7-yl)-2-((3-(difluoromethyl)-5-fluorophenyl)(hydroxy)methyl)-3- methyltetrahydrofuran-3,4-diyl diacetate as a solid. MS: 528 [M+H]⁺.

Step 2-B: A mixture of dichloro(pentamethylcyclopentadienyl)iridium(III) dimer (25.5 mg, 0.032 mmol) and (1R,2R)-(-)-N-(4-toluenesulfonyl)-1,2-diphenylethylenediamine (23.5 mg, 0.064 mmol) in 1:1 DCM/water (6.0 mL) was stirred at 40 °C for 1h. The mixture was cooled to room temperature, ammonium formate (1.52 g, 24.1 mmol) was added, and the mixture was stirred for 5 min. Then, a solution of (2S,3S,4R,5R)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7- yl)-2-(3-(difluoromethyl)-5-fluorobenzoyl)-3-methyltetrahydrofuran-3,4-diyl diacetate (843 mg, 1.60 mmol) in DCM (3.0 mL) was added and the reaction was stirred at room temperature overnight. The reaction was quenched with brine (10 mL) and extracted with DCM (3 x 10 mL). The combined organic fractions were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel

(EtOAc/isohexane, 0%-100%) to yield (2R,3R,4R,5R)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin- 7-yl)-2-((3-(difluoromethyl)-5-fluorophenyl)(hydroxy)methyl)-3-methyltetrahydrofuran-3,4-diyl diacetate as a solid. MS: 528 [M+H]⁺.

Step 3: A solution of (2R,3R,4R,5R)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((3- (difluoromethyl)-5-fluorophenyl)(hydroxy)methyl)-3-methyltetrahydrofuran-3,4-diyl diacetate (197 mg, 0.373 mmol) in 7 N ammonia in methanol (3.0 mL) was stirred at 130 °C for 4 hours in a microwave reactor. The reaction mixture was concentrated under reduced pressure and then purified by reverse-phase HPLC (ACN/water with 0.1% TFA modifier) to yield (2R,3R,4R,5R)- 5-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((R)-(3-(difluoromethyl)-5- fluorophenyl)(hydroxy)methyl)-3-methyltetrahydrofuran-3,4-diol, TFA salt (Example 4) as a solid and (2R 3R 4R 5R)-5-(4-amino-7H-pyrrolo[23-d]pyrimidin-7-yl)-2-((S)-(3- (difluoromethyl)-5-fluorophenyl)(hydroxy)methyl)-3-methyltetrahydrofuran-3,4-diol TFA salt (Example 5) as a solid.

Example 4: MS: 425 [M+H]⁺.¹H-NMR (500 MHz, CD₃OD) d 8.27 (s, 1H), 7.74 (d, J = 3.8 Hz, 1H), 7.45 (s, 1H), 7.34 (d, J = 9.7 Hz, 1H), 7.14 (d, J = 8.7 Hz, 1H), 6.93 (d, J = 3.8 Hz, 1H), 6.73 (t, J = 56.2 Hz, 1H), 6.17 (d, J = 7.8 Hz, 1H), 4.97 (d, J = 5.9 Hz, 1H), 4.52 (d, J = 7.8 Hz, 1H), 4.25 (d, J = 5.9 Hz, 1H), 1.32 (s, 3H).

Example 5: MS: 425 [M+H]⁺.¹H-NMR (500 MHz, CD₃OD) d 8.25 (s, 1H), 8.20 (d, J = 3.8 Hz, 1H), 7.39 (s, 1H), 7.27 (d, J = 9.5 Hz, 1H), 7.11 (d, J = 8.9 Hz, 1H), 7.00 (d, J = 3.8 Hz, 1H), 6.70 (t, J = 56.1 Hz, 1H), 6.30 (d, J = 7.8 Hz, 1H), 4.96 (s, 1H), 4.60 (d, J = 7.8 Hz, 1H), 4.12 (d, J = 1.1 Hz, 1H), 1.60 (s, 3H). Examples 6 and 7

(2R,3S,4R,5R)-5-(4-amino- midin-7-yl)-2-((R)-(3-(difluoromethyl)-2,5-

difluorophenyl)(hydroxy)methyl)-3-methyltetrahydrofuran-3,4-diol (Example 6) (2R,3S,4R,5R)-5-(4-amino- rimidin-7-yl)-2-((S)-(3-(difluoromethyl)-2,5-

difluorophenyl)(hydroxy)methyl)-3-methyltetrahydrofuran-3,4-diol (Example 7)

Example 6 Example 7 Step 1: To a solution of 1-bromo-3-(difluoromethyl)-2,5-difluorobenzene (882 mg, 3.63 mmol) in diethyl ether (10 ml) was added n-butyllithium (2268 µl, 3.63 mmol, 1.6 M in hexane) at - 78 °C. The resulting mixture was stirred at -78 °C for 40 min. Then, a solution of (2S,3S,4R,5R)- 5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-(methoxy(methyl)carbamoyl)-3- methyltetrahydrofuran-3,4-diyl diacetate (400 mg, 0.907 mmol) in diethyl ether and THF (5.0 ml and 2.0 ml, respectively) was added, and the resulting mixture was stirred at -78 °C for 3 h. The reaction was quenched with acetic acid (312 µl, 5.44 mmol), and the resulting mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate and then washed with 1 N citric acid. The organic layer was dried over Na₂SO₄ and then organic solvent was removed under reduced pressure. The crude was purified by flash column chromatography (EtOH/ethyl acetate (1:3) in hexanes, 5 to 100%) to yield ((2S,3S,4R,5R)-5-(4-chloro-7H- pyrrolo[2,3-d]pyrimidin-7-yl)-3,4-dihydroxy-3-methyltetrahydrofuran-2-yl)(3-(difluoromethyl)- 2,5-difluorophenyl)methanone as an amorphous solid. MS: 460 [M+H]⁺. Step 2: To a solution of ((2S,3S,4R,5R)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3,4- dihydroxy-3-methyltetrahydrofuran-2-yl)(3-(difluoromethyl)-2,5-difluorophenyl)methanone (333.5 mg, 0.725 mmol) in THF (8.0 ml) was added K-Selectride (3627 µl, 3.63 mmol, 1 M in THF) at -78 °C, and the resulting mixture was stirred at -78 °C for 30 min. The reaction was quenched with acetone (4.0 ml) and then the resulting mixture was concentrated under reduced pressure. The residue was purified by flash column chromatography (EtOH/ethyl acetate (1:3) in hexanes, 5 to 100%) to yield (2R,3S,4R,5R)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2- ((3-(difluoromethyl)-2,5-difluorophenyl)(hydroxy)methyl)-3-methyltetrahydrofuran-3,4-diol as an amorphous solid. MS: 462 [M+H]⁺. Step 3: A solution of (2R,3S,4R,5R)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((R)-(3- (difluoromethyl)-2,5-difluorophenyl)(hydroxy)methyl)-3-methyltetrahydrofuran-3,4-diol (188.6 mg, 0.408 mmol) in 7 N ammonia in methanol (3.0 ml) was stirred at 130 °C for 4 h in a microwave reactor. The reaction mixture was concentrated under reduced pressure and then purified by reverse-phase HPLC (ACN/water with 0.1% NH₄OH modifier) to yield

(2R,3S,4R,5R)-5-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((R)-(3-(difluoromethyl)-2,5- difluorophenyl)(hydroxy)methyl)-3-methyltetrahydrofuran-3,4-diol (Example 6) and

(2R,3S,4R,5R)-5-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((S)-(3-(difluoromethyl)-2,5- difluorophenyl)(hydroxy)methyl)-3-methyltetrahydrofuran-3,4-diol (Example 7) as amorphous solids. Example 6: MS: 443 [M+H]⁺.¹H-NMR (500 MHz, CD₃OD) d 8.24 (s, 1H), 7.68 (d, J = 3.8 Hz, 1H), 7.53– 7.47 (m, 1H), 7.26– 7.20 (m, 1H), 6.94 (d, J = 3.8 Hz, 1H), 6.92 (t, J = 54.6 Hz, 1H), 6.21 (d, J = 7.9 Hz, 1H), 5.17 (d, J = 6.4 Hz, 1H), 4.45 (d, J = 7.9 Hz, 1H), 4.24 (d, J = 6.4 Hz, 1H), 1.41 (s, 3H). Example 7: MS: 443 [M+H]⁺.¹H-NMR (500 MHz, CD₃OD) d 8.25 (s, 1H), 8.18 (d, J = 3.8 Hz, 1H), 7.37– 7.31 (m, 1H), 7.23– 7.17 (m, 1H), 7.02 (d, J = 3.8 Hz, 1H), 6.98 (t, J = 54.5 Hz, 1H), 6.33 (d, J = 7.8 Hz, 1H), 5.23 (s, 1H), 4.59 (d, J = 7.8 Hz, 1H), 4.12 (s, 1H), 1.60 (s, 3H). Examples 8 and 9 (2R,3S,4R,5R)-5-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((R)-(6-(difluoromethyl)-5- fluoro-1 tetrahydroisoquinolin-8-yl)(hydroxy)methyl)-3-methyltetrahydrofuran-3,4-diol

(Example 8)

(2R,3S,4R,5R)-5-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((S)-(6-(difluoromethyl)-5- fluoro-1 tetrahydroisoquinolin-8-yl)(hydroxy)methyl)-3-methyltetrahydrofuran-3,4-diol

(Example 9)

E^{xample 8 Example 9} Step 1: To a solution of tert-butyl 6-(difluoromethyl)-5-fluoro-8-iodo-3,4-dihydroisoquinoline- 2(1H)-carboxylate (332 mg, 0.777 mmol) in THF (6.0 ml) was added isopropylmagnesium chloride lithium chloride complex (717 µl, 0.933 mmol, 1.3 M in THF) at 0 °C. After 2 h at 0 °C, the resulting mixture was added to a solution of (2S,3S,4R,5R)-5-(4-chloro-7H-pyrrolo[2,3- d]pyrimidin-7-yl)-2-(methoxy(methyl)carbamoyl)-3-methyltetrahydrofuran-3,4-diyl diacetate (377 mg, 0.855 mmol) in THF (3.0 ml) at 0 °C. The resulting mixture was stirred for 5 h, gradually increasing the temperature to rt. The reaction was quenched with acetic acid (178 µl, 3.11 mmol), and the resulting mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate and then washed with 1 N citric acid. The organic layer was dried over Na₂SO₄ and the organic solvent was removed under reduced pressure. The crude material was purified by flash column chromatography (EtOH : ethyl acetate (1:3) in hexanes, 5 to 100%) to yield (2S,3S,4R,5R)-2-(2-(tert-butoxycarbonyl)-6-(difluoromethyl)-5-fluoro-1,2,3,4- tetrahydroisoquinoline-8-carbonyl)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3- methyltetrahydrofuran-3,4-diyl diacetate as an amorphous solid. MS: 681 [M+H]⁺. Step 2: To a solution of (2S,3S,4R,5R)-2-(2-(tert-butoxycarbonyl)-6-(difluoromethyl)-5-fluoro- 1,2,3,4-tetrahydroisoquinoline-8-carbonyl)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3- methyltetrahydrofuran-3,4-diyl diacetate (49.4 mg, 0.073 mmol) in methanol (4.5 ml) was added sodium borohydride (8.23 mg, 0.218 mmol) at 0 °C, and the reaction mixture was stirred at 0 °C for 15 min. The reaction was quenched with acetone (3 ml) and then acetic acid (37.4 µl, 0.653 mmol) at 0 °C, and the resulting mixture was concentrated under reduced pressure. The crude was purified by flash column chromatography (EtOH : ethyl acetate (1:3) in hexanes, 5 to 100%) to yield (2R,3R,4R,5R)-2-((2-(tert-butoxycarbonyl)-6-(difluoromethyl)-5-fluoro-1,2,3,4- tetrahydroisoquinolin-8-yl)(hydroxy)methyl)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3- methyltetrahydrofuran-3,4-diyl diacetate as an amorphous solid. MS: 683 [M+H]⁺. Step 3: A solution of (2R,3R,4R,5R)-2-((2-(tert-butoxycarbonyl)-6-(difluoromethyl)-5-fluoro- 1,2,3,4-tetrahydroisoquinolin-8-yl)(hydroxy)methyl)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7- yl)-3-methyltetrahydrofuran-3,4-diyl diacetate (37.3 mg, 0.055 mmol) in 7 N ammonia in methanol (4.0 ml) was stirred at 130 °C for 4 h in a microwave reactor. The reaction mixture was concentrated under reduced pressure to yield tert-butyl 8-(((2R,3S,4R,5R)-5-(4-amino-7H- pyrrolo[2,3-d]pyrimidin-7-yl)-3,4-dihydroxy-3-methyltetrahydrofuran-2-yl)(hydroxy)methyl)-6- (difluoromethyl)-5-fluoro-3,4-dihydroisoquinoline-2(1H)-carboxylate, which was used for the next step without further purification. MS: 580 [M+H]⁺. Step 4: To a solution of tert-butyl 8-(((2R,3S,4R,5R)-5-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7- yl)-3,4-dihydroxy-3-methyltetrahydrofuran-2-yl)(hydroxy)methyl)-6-(difluoromethyl)-5-fluoro- 3,4-dihydroisoquinoline-2(1H)-carboxylate (31.9 mg, 0.055 mmol) in DCM (2.0 ml) was added TFA (1.0 ml) at 0 °C. The resulting mixture was stirred at 0 °C for 30 min. The reaction mixture was concentrated under reduced pressure and the resulting residue was purified by reverse-phase HPLC (ACN/water with 0.1% TFA modifier) to yield (2R,3S,4R,5R)-5-(4-amino-7H- pyrrolo[2,3-d]pyrimidin-7-yl)-2-((R)-(6-(difluoromethyl)-5-fluoro-1,2,3,4-tetrahydroisoquinolin- 8-yl)(hydroxy)methyl)-3-methyltetrahydrofuran-3,4-diol, bis TFA salt (Example 8) and

(2R,3S,4R,5R)-5-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((S)-(6-(difluoromethyl)-5- fluoro-1,2,3,4-tetrahydroisoquinolin-8-yl)(hydroxy)methyl)-3-methyltetrahydrofuran-3,4-diol, bis TFA salt (Example 9), both as solids. Example 8: MS: 480 [M+H]⁺.¹H-NMR (500 MHz, CD₃OD) d 8.26 (s, 1H), 7.66 (d, J = 6.9 Hz, 1H), 7.56 (d, J = 3.8 Hz, 1H), 6.94 (t, J = 54.7 Hz, 1H), 6.88 (d, J = 3.8 Hz, 1H), 6.05 (d, J = 7.8 Hz, 1H), 4.96 (d, J = 8.8 Hz, 1H), 4.74 (d, J = 16.5 Hz, 1H), 4.56 (d, J = 7.8 Hz, 1H), 4.52 (d, J = 16.5 Hz, 1H), 4.14 (d, J = 8.8 Hz, 1H), 3.51– 3.40 (m, 2H), 3.08– 3.01 (m, 2H), 1.59 (s, 3H). Example 9: MS: 480 [M+H]⁺.¹H-NMR (500 MHz, CD₃OD) d 8.24 (s, 1H), 8.07 (d, J = 3.7 Hz, 1H), 7.69 (d, J = 7.1 Hz, 1H), 6.99 (d, J = 3.7 Hz, 1H), 6.87 (t, J = 54.8 Hz, 1H), 6.28 (d, J = 7.9 Hz, 1H), 5.01 (s, 1H), 4.65 (d, J = 16.2 Hz, 1H), 4.63 (d, J = 7.9 Hz, 1H), 4.40 (d, J = 16.2 Hz, 1H), 4.04 (s, 1H), 3.58– 3.43 (m, 2H), 3.13– 3.05 (m, 2H), 1.62 (s, 3H). Example 10 (2R,3S,4R,5R)-5-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2- -1-(6-(difluoromethyl)-5-

fluoro-1,2,3,4-tetrahydroisoquinolin-8-yl)-1-hydroxyethyl)-3-methyltetrahydrofuran-3,4-diol

Step 1: To a solution of tert-butyl 6-(difluoromethyl)-5-fluoro-8-iodo-3,4-dihydroisoquinoline- 2(1H)-carboxylate (332 mg, 0.777 mmol) in THF (6.0 ml) was added isopropylmagnesium chloride lithium chloride complex (717 µl, 0.933 mmol, 1.3 M in THF) at 0 °C. After 2 h at 0 °C, the resulting mixture was added to a solution of (2S,3S,4R,5R)-5-(4-chloro-7H-pyrrolo[2,3- d]pyrimidin-7-yl)-2-(methoxy(methyl)carbamoyl)-3-methyltetrahydrofuran-3,4-diyl diacetate (377 mg, 0.855 mmol) in THF (3.0 ml) at 0 °C. The resulting mixture was stirred for 5 h, gradually increasing the temperature to rt. The reaction was quenched with acetic acid (178 µl, 3.11 mmol), and the resulting mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate and then washed with 1 N citric acid. The organic layer was dried over Na₂SO₄ and the organic solvent was removed under reduced pressure. The resulting crude material was purified by flash column chromatography (EtOH : ethyl acetate (1:3) in hexanes, 5 to 100%) to yield (2S,3S,4R,5R)-2-(2-(tert-butoxycarbonyl)-6-(difluoromethyl)-5-fluoro-1,2,3,4- tetrahydroisoquinoline-8-carbonyl)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3- methyltetrahydrofuran-3,4-diyl diacetate as an amorphous solid. MS: 681 [M+H]⁺. Step 2: To a solution of (2S,3S,4R,5R)-2-(2-(tert-butoxycarbonyl)-6-(difluoromethyl)-5-fluoro- 1,2,3,4-tetrahydroisoquinoline-8-carbonyl)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3- methyltetrahydrofuran-3,4-diyl diacetate (95.2 mg, 0.140 mmol) in THF (6.0 ml) was added methylmagnesium bromide (93 µl, 0.280 mmol, 3.0 M in diethyl ether) at 0 °C. The reaction mixture was stirred at 0 °C for 15 min and then quenched with acetic acid (48.0 µl, 0.839 mmol). The resulting mixture was concentrated under reduced pressure and the resulting residue was dissolved in ethyl acetate. The organic solution was washed with 1 N citric acid and the organic layer was dried over Na₂SO₄. The organic solvent was removed under reduced pressure and the crude material was purified by flash column chromatography (EtOH /ethyl acetate (1:3) in hexane, 5 to 100%) to yield (2R,3R,4R,5R)-2-((R)-1-(2-(tert-butoxycarbonyl)-6- (difluoromethyl)-5-fluoro-1,2,3,4-tetrahydroisoquinolin-8-yl)-1-hydroxyethyl)-5-(4-chloro-7H- pyrrolo[2,3-d]pyrimidin-7-yl)-3-methyltetrahydrofuran-3,4-diyl diacetate as an amorphous solid. MS: 697 [M+H]⁺.

Step 3: A solution of (2R,3R,4R,5R)-2-(1-(2-(tert-butoxycarbonyl)-6-(difluoromethyl)-5-fluoro- 1,2,3,4-tetrahydroisoquinolin-8-yl)-1-hydroxyethyl)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7- yl)-3-methyltetrahydrofuran-3,4-diyl diacetate (35.2 mg, 0.050 mmol) in 7 N ammonia in methanol (4.0 ml) was stirred at 130 °C for 4 h in a microwave reactor. The reaction mixture was concentrated under reduced pressure to yield tert-butyl 8-(1-((2R,3S,4R,5R)-5-(4-amino-7H- pyrrolo[2,3-d]pyrimidin-7-yl)-3,4-dihydroxy-3-methyltetrahydrofuran-2-yl)-1-hydroxyethyl)-6- (difluoromethyl)-5-fluoro-3,4-dihydroisoquinoline-2(1H)-carboxylate, which was used for the next step without further purification. MS: 594 [M+H]⁺.

Step 4: To a solution of tert-butyl 8-(1-((2R,3S,4R,5R)-5-(4-amino-7H-pyrrolo[2,3-d]pyrimidin- 7-yl)-3,4-dihydroxy-3-methyltetrahydrofuran-2-yl)-1-hydroxyethyl)-6-(difluoromethyl)-5- fluoro-3,4-dihydroisoquinoline-2(1H)-carboxylate (29.7 mg, 0.05 mmol) in DCM (2.0 ml) was added TFA (1.0 ml) at 0 °C, and the resulting mixture was stirred at 0 °C for 30 min. The reaction mixture was concentrated under reduced pressure, and the residue was purified by reverse-phase HPLC (ACN/water with 0.1% TFA modifier) to yield (2R,3S,4R,5R)-5-(4-amino- 7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((R)-1-(6-(difluoromethyl)-5-fluoro-1,2,3,4- tetrahydroisoquinolin-8-yl)-1-hydroxyethyl)-3-methyltetrahydrofuran-3,4-diol, bis TFA salt as an amorphous solid. MS: 494 [M+H]⁺.¹H-NMR (500 MHz, CD₃OD) d 8.28 (s, 1H), 7.62 (brs, 1H), 7.60 (d, J = 3.7 Hz, 1H), 7.00 (t, J = 54.4 Hz, 1H), 6.89 (d, J = 3.7 Hz, 1H), 6.01 (d, J = 8.1 Hz, 1H), 4.95– 4.88 (m, 2H), 4.70 (s, 1H), 4.42 (d, J = 8.1 Hz, 1H), 3.64– 3.56 (m, 1H), 3.50– 3.41 (m, 1H), 3.21– 3.13 (m, 2H), 1.57 (s, 3H), 0.95 (s, 3H). Table 1: The compounds in the following table were prepared using the methodology herein and the general procedures described in Examples 1-2 and 4-7. Compounds at every step were purified by standard techniques such as chromatography, crystallization, reverse phase HPLC or SFC.

Table 2: The compounds in the following table were prepared using the methodology herein and the general procedures described in Example 3. Compounds at every step were purified by standard techniques such as chromatography, crystallization, reverse phase HPLC or SFC.

Table 3: The compounds in the following table were prepared using the methodology herein and the general procedures described in Examples 8 and 9. Compounds at every step were purified by standard techniques such as chromatography, crystallization, reverse phase HPLC or SFC.

Table 4: The compounds in the following table were prepared using the methodology herein and the general procedures described in Example 10. Compounds at every step were purified by standard techniques such as chromatography, crystallization, reverse phase HPLC or SFC.

Example 82

(2R,3S,4R,5R)-5-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-(1-(3-(difluoromethyl)-5- fluorophenyl)vinyl)-3-methyltetrahydrofuran-3,4-diol

Step 1: To a solution of magnesium (165 mg, 6.81 mmol) and lithium chloride (144 mg, 3.40 mmol) in THF (6.0 mL) was added DIBAL-H (27.2 µL, 0.027 mmol). After 5 minutes at room temperature, the reaction mixture was cooled to 0 °C, and 1-bromo-3-(difluoromethyl)-5- fluorobenzene (612 mg, 2.72 mmol) was added at 0 °C. The resulting mixture was stirred at 0 °C for 1 hour, and then the mixture was added to a solution of (2S,3S,4R,5R)-5-(4-chloro-7H- pyrrolo[2,3-d]pyrimidin-7-yl)-2-(methoxy(methyl)carbamoyl)-3-methyltetrahydrofuran-3,4-diyl diacetate (200 mg, 0.454 mmol) in THF (3.0 mL) at 0 °C. The resulting mixture was stirred for 10 minutes at 0 °C. The reaction was quenched with acetic acid (390 µL, 6.81 mmol) and the resulting mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate and then washed with 1 N citric acid. The organic layer was dried over Na₂SO₄, and the organic solvent was removed under reduced pressure. The crude material was purified by flash column chromatography (EtOAc/EtOH 3:1 in hexanes, 5 to 100%) to yield (2S,3S,4R,5R)-5-(4- chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-(3-(difluoromethyl)-5-fluorobenzoyl)-3- methyltetrahydrofuran-3,4-diyl diacetate as a solid. MS: 526 [M+H]⁺.

Step 2: To a solution of (2S,3S,4R,5R)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-(3- (difluoromethyl)-5-fluorobenzoyl)-3-methyltetrahydrofuran-3,4-diyl diacetate (227 mg, 0.431 mmol) in THF (6.0 ml) was added methylmagnesium bromide (287 µl, 0.862 mmol, 3.0 M in diethyl ether) at 0 °C. The reaction mixture was stirred at 0 °C for 15 min and then quenched with acetic acid (173 µl, 3.02 mmol). The resulting mixture was concentrated under reduced pressure and the residue was dissolved in ethyl acetate. The organic solution was washed with 1 N citric acid and the organic layer was dried over Na₂SO₄. The organic solvent was removed under reduced pressure and the crude material was purified by flash column chromatography (EtOH /ethyl acetate (1:3) in hexane, 5 to 100%) to yield (2R,3R,4R,5R)-5-(4-chloro-7H- pyrrolo[2,3-d]pyrimidin-7-yl)-2-((R)-1-(3-(difluoromethyl)-5-fluorophenyl)-1-hydroxyethyl)-3- methyltetrahydrofuran-3,4-diyl diacetate as an amorphous solid. MS: 542 [M+H]⁺.

Step 3: To a solution of (2R,3R,4R,5R)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((R)-1- (3-(difluoromethyl)-5-fluorophenyl)-1-hydroxyethyl)-3-methyltetrahydrofuran-3,4-diyl diacetate (191 mg, 0.353 mmol) in DCM (4.0 ml) was added Martin's Sulfurane (712 mg, 1.06 mmol) at - 78 °C. After 15 min at -78 °C, the reaction mixture was warmed to -20 °C. The reaction mixture was then gradually warmed up to rt while being stirred for 2 h. The reaction was quenched with methanol and then the resulting mixture was directly purified by flash column chromatography (EtOH/ethyl acetate (1:3) in hexane, 5 to 100%) to yield (2R,3R,4R,5R)-5-(4-chloro-7H- pyrrolo[2,3-d]pyrimidin-7-yl)-2-(1-(3-(difluoromethyl)-5-fluorophenyl)vinyl)-3- methyltetrahydrofuran-3,4-diyl diacetate as an oil. MS: 524 [M+H]⁺.

Step 4: A solution of (2R,3R,4R,5R)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-(1-(3- (difluoromethyl)-5-fluorophenyl)vinyl)-3-methyltetrahydrofuran-3,4-diyl diacetate (23 mg, 0.044 mmol) in 7 N ammonia in methanol (3.0 ml) was stirred at 130 °C for 4 h in a microwave reactor. The reaction mixture was concentrated in vacuo and then purified by reverse-phase HPLC (ACN/water with 0.1% TFA modifier) to yield (2R,3S,4R,5R)-5-(4-amino-7H- pyrrolo[2,3-d]pyrimidin-7-yl)-2-(1-(3-(difluoromethyl)-5-fluorophenyl)vinyl)-3- methyltetrahydrofuran-3,4-diol, TFA salt as an amorphous solid. MS: 421 [M+H]⁺.¹H-NMR (500 MHz, CD₃OD) d 8.30 (s, 1H), 7.63 (d, J = 3.8 Hz, 1H), 7.53 (s, 1H), 7.44 (d, J = 9.9 Hz, 1H), 7.28 (d, J = 8.6 Hz, 1H), 6.99 (d, J = 3.8 Hz, 1H), 6.81 (t, J = 55.9 Hz, 1H), 6.29 (d, J = 7.7 Hz, 1H), 5.65 (s, 1H), 5.63 (s, 1H), 5.10 (s, 1H), 4.26 (d, J = 7.7 Hz, 1H), 1.13 (s, 3H). Examples 83 and 84

(2R,3S,4R,5R)-5-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2- -1-(3-(difluoromethyl)-5-

fluorophenyl)ethyl)-3-methyltetrahydrofuran-3,4-diol and (2R,3S,4R,5R)-5-(4-amino-7H- pyrrolo[2,3-d]pyrimidin-7-yl)-2- -1-(3-(difluoromethyl)-5-fluorophenyl)ethyl)-3-

methyltetrahydrofuran-3,4-diol

To a solution of (2R,3S,4R,5R)-5-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-(1-(3- (difluoromethyl)-5-fluorophenyl)vinyl)-3-methyltetrahydrofuran-3,4-diol (151 mg, 0.359 mmol) in methanol (3.0 ml) was added palladium on carbon (382 mg, 0.359 mmol, 10 wt.%). The reaction vessel was purged with hydrogen gas and then the reaction mixture was stirred for 2 h under an atmosphere of hydrogen. The resulting mixture was filtered through a pad of Celite and the filtrate was concentrated under reduced pressure. The crude material was purified by reverse- phase HPLC (ACN/water with 0.1% NH₄OH modifier) to yield (2R,3S,4R,5R)-5-(4-amino-7H- pyrrolo[2,3-d]pyrimidin-7-yl)-2-(1-(3-(difluoromethyl)-5-fluorophenyl)ethyl)-3- methyltetrahydrofuran-3,4-diol (33.9 mg, 0.080 mmol) and (2R,3S,4R,5R)-5-(4-amino-7H- pyrrolo[2,3-d]pyrimidin-7-yl)-2-(1-(3-(difluoromethyl)-5-fluorophenyl)ethyl)-3- methyltetrahydrofuran-3,4-diol (10.9 mg, 0.026 mmol) as solids.

Example 83 (faster eluting isomer): MS: 423 [M+H]⁺.¹H-NMR (500 MHz, CD₃OD) d 8.05 (s, 1H), 7.28 (s, 1H), 7.19 (d, J = 9.8 Hz, 1H), 7.12 (d, J = 3.7 Hz, 1H), 7.07 (d, J = 8.8 Hz, 1H), 6.69 (t, J = 56.1 Hz, 1H), 6.61 (d, J = 3.7 Hz, 1H), 5.90 (d, J = 5.1 Hz, 1H), 4.03 (d, J = 8.8 Hz, 1H), 3.95 (d, J = 5.1 Hz, 1H), 3.21– 3.12 (m, 1H), 1.43 (s, 3H), 1.38 (d, J = 7.0 Hz, 3H).

Example 84 (slower eluting isomer): MS: 423 [M+H]⁺.¹H-NMR (500 MHz, CD₃OD) d 8.12 (s, 1H), 7.38 (d, J = 3.7 Hz, 1H), 7.33 (s, 1H), 7.24 (d, J = 10.0 Hz, 1H), 7.13 (d, J = 8.7 Hz, 1H), 6.76 (t, J = 56.1 Hz, 1H), 6.71 (d, J = 3.7 Hz, 1H), 6.13 (d, J = 7.1 Hz, 1H), 4.26 (d, J = 7.1 Hz, 1H), 4.25 (d, J = 8.5 Hz, 1H), 3.26– 3.18 (m, 1H), 1.30 (d, J = 6.9 Hz, 3H), 1.15 (s, 3H). Example 85

(2R,3S,4R,5R)-3-methyl-5-(4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-(1-(1,2,3,4- tetrahydroisoquinolin-8-yl)vinyl)tetrahydrofuran-3,4-diol

Step 1: To a solution of tert-butyl 8-iodo-3,4-dihydroisoquinoline-2(1H)-carboxylate (1.08 g, 2.99 mmol) in THF (6 ml), was added isopropylmagnesium chloride lithium chloride (2.58 ml, 3.36 mmol, 1.3 M in THF) at -78 °C. The reaction mixture was stirred at 0 °C for 30 min and then at room temperature for another 30 min. This reaction mixture was added into a solution of (2S,3S,4R,5R)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-(methoxy(methyl)carbamoyl)-3- methyltetrahydrofuran-3,4-diyl diacetate (200 mg, 0.454 mmol) in THF (3 ml) at 0 °C. The reaction was stirred at 0 °C for 3 h and then at rt for 16 h. The reaction was quenched with saturated aqueous NH₄Cl and then extracted with EtOAc (3x). The organic layer was dried over sodium sulfate and then filtered. The residue was purified by flash column chromatography (EtOAc/EtOH 3:1 in hexanes, 0 to 50%) to afford tert-butyl 8-((2S,3S,4R,5R)-5-(4-chloro-7H- pyrrolo[2,3-d]pyrimidin-7-yl)-3,4-dihydroxy-3-methyltetrahydrofuran-2-carbonyl)-3,4- dihydroisoquinoline-2(1H)-carboxylate as an amorphous solid. MS: 529 [M+H]⁺.

Step 2: To a solution of tert-butyl 8-((2S,3S,4R,5R)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7- yl)-3,4-dihydroxy-3-methyltetrahydrofuran-2-carbonyl)-3,4-dihydroisoquinoline-2(1H)- carboxylate (196 mg, 0.371 mmol) in dry acetone (3.71 ml) were added 2,2-dimethoxypropane (1453 µl, 11.9 mmol) and p-toluenesulfonic acid monohydrate (7.05 mg, 0.037 mmol). The reaction vial was purged with nitrogen gas for 1 min. The reaction was stirred at rt for 16 h. The reaction was poured into saturated aqueous sodium bicarbonate and then extracted with EtOAc (3x). The organic layers were combined, dried over sodium sulfate, and then filtered. The filtrate was concentrated under reduced pressure and purified by flash column chromatography

(EtOAc/EtOH 3:1 in hexanes, 0 to 40%) to afford tert-butyl 8-((3aS,4S,6R,6aR)-6-(4-chloro-7H- pyrrolo[2,3-d]pyrimidin-7-yl)-2,2,3a-trimethyltetrahydrofuro[3,4-d][1,3]dioxole-4-carbonyl)- 3,4-dihydroisoquinoline-2(1H)-carboxylate as an oil. MS: 569 [M+H]⁺.

Step 3: To a solution of tert-butyl 8-((3aS,4S,6R,6aR)-6-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin- 7-yl)-2,2,3a-trimethyltetrahydrofuro[3,4-d][1,3]dioxole-4-carbonyl)-3,4-dihydroisoquinoline- 2(1H)-carboxylate (140 mg, 0.246 mmol) in THF (820 µl) was added Tebbe reagent (2.46 ml, 1.23 mmol, 0.5 M in toluene) dropwise under an atmosphere of argon at rt. The reaction was stirred at rt for 2 h and then quenched with saturated aqueous sodium bicarbonate at 0 °C. The mixture was poured into a separatory funnel with saturated aqueous sodium bicarbonate and extracted with EtOAc (3x). The combined organic layers were dried over sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and purified by reverse-phase HPLC (ACN/water with 0.1% NH₄OH modifier) to afford tert-butyl 8-(1-((3aR,4R,6R,6aR)-6- (4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,2,3a-trimethyltetrahydrofuro[3,4-d][1,3]dioxol-4- yl)vinyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate as a solid. MS: 567 [M+H]⁺.

Step 4: To a microwave vial were added tert-butyl 8-(1-((3aR,4R,6R,6aR)-6-(4-chloro-7H- pyrrolo[2,3-d]pyrimidin-7-yl)-2,2,3a-trimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)vinyl)-3,4- dihydroisoquinoline-2(1H)-carboxylate (39 mg, 0.069 mmol) and

tetrakis(triphenylphosphine)palladium(0) (3.97 mg, 3.44 µmol), at which point the vial was sealed and purged with argon gas. THF (688 µl) was added, and dimethylzinc (2 M in toluene, 172 µl, 0.344 mmol) was added while stirring. The reaction was heated to 65 °C for 4 h. The reaction mixture was then poured into saturated aqueous NH₄Cl at 0 °C and extracted with EtOAc (3x). The organic layer was dried over sodium sulfate and then filtered. The crude material was purified by flash column chromatography (EtOAc/EtOH 3:1 in hexanes, 0 to 40%) to afford tert-butyl 8-(1-((3aR,4R,6R,6aR)-2,2,3a-trimethyl-6-(4-methyl-7H-pyrrolo[2,3- d]pyrimidin-7-yl)tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)vinyl)-3,4-dihydroisoquinoline-2(1H)- carboxylate as an oil. MS: 547 [M+H]⁺.

Step 5: A solution of tert-butyl 8-(1-((3aR,4R,6R,6aR)-2,2,3a-trimethyl-6-(4-methyl-7H- pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)vinyl)-3,4- dihydroisoquinoline-2(1H)-carboxylate (56 mg, 0.102 mmol) in 2:1 DCM:TFA (3 ml) was stirred at rt for 15 min. Water (0.1 ml) was added to the reaction mixture. After 16 h at rt the mixture was concentrated under reduced pressure and purified by reverse-phase HPLC

(ACN/water with 0.1% NH₄OH modifier) to afford (2R,3S,4R,5R)-3-methyl-5-(4-methyl-7H- pyrrolo[2,3-d]pyrimidin-7-yl)-2-(1-(1,2,3,4-tetrahydroisoquinolin-8-yl)vinyl)tetrahydrofuran- 3,4-diol as a solid. MS: 407 [M+H]⁺.1H NMR (500 MHz, methanol-d₄) d 8.64 (s, 1H), 7.26 (dd, J = 7.6 Hz, 7.6 Hz, 1H), 7.18 (d, J = 7.6 Hz, 1H), 7.13 (d, J = 7.6 Hz, 1H), 6.61 (d, J = 3.8 Hz, 1H), 6.43 (d, J = 3.8 Hz, 1H), 6.37 (d, J = 7.8 Hz, 1H), 5.60 (s, 1H), 5.17 (s, 1H), 4.84 (s, 1H), 4.13 (d, J = 7.8 Hz, 1H), 3.89 (d, J = 16.3 Hz, 1H), 3.79 (d, J = 16.3 Hz, 1H), 3.03– 2.95 (m, 1H), 2.92– 2.80 (m, 2H), 2.78– 2.73 (m, 1H), 2.72 (s, 3H), 1.43 (s, 3H). Examples 86 and 87

(2R,3S,4R,5R)-3-methyl-5-(4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2- -1-(1,2,3,4-

tetrahydroisoquinolin-8-yl)ethyl)tetrahydrofuran-3,4-diol (Example 86)

(2R,3S,4R,5R)-3-methyl-5-(4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2- -1-(1,2,3,4-

tetrahydroisoquinolin-8-yl)ethyl)tetrahydrofuran-3,4-diol (Example 87)

E^{xample 86 Example 87}

To a solution of (2R,3S,4R,5R)-3-methyl-5-(4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-(1- (1,2,3,4-tetrahydroisoquinolin-8-yl)vinyl)tetrahydrofuran-3,4-diol (20 mg, 0.049 mmol) in MeOH (491 µl) was added palladium on carbon (2.61 mg, 2.454 µmol, 10 wt.%) under an atmosphere of nitrogen. The reaction vial was purged with hydrogen gas, via balloon, for 30 seconds. MeOH (491 µl) was added to the reaction mixture. After 1 h at room temperature the 30 mixture was filtered over Celite, washing with methanol. The filtrate was concentrated and purified by mass triggered reverse phase HPLC (ACN/water with 0.1% NH₄OH modifier) to afford (2R,3S,4R,5R)-3-methyl-5-(4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((R)-1-(1,2,3,4- tetrahydroisoquinolin-8-yl)ethyl)tetrahydrofuran-3,4-diol and (2R,3S,4R,5R)-3-methyl-5-(4- methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((S)-1-(1,2,3,4-tetrahydroisoquinolin-8- yl)ethyl)tetrahydrofuran-3,4-diol, both as solids.

Example 86: MS: 409 [M+H]⁺.¹H NMR (600 MHz, methanol-d₄) d 8.61 (s, 1H), 7.48 (d, J = 3.8 Hz, 1H), 7.23 (d, J = 7.7 Hz, 1H), 7.13 (dd, J = 7.7 Hz, 7.7 Hz, 1H), 6.91 (d, J = 7.7 Hz, 1H), 6.76 (d, J = 3.8 Hz, 1H), 5.92 (d, J = 4.7 Hz, 1H), 4.12– 4.05 (m, 2H), 4.05– 3.97 (m, 2H), 3.26 – 3.18 (m, 1H), 3.01– 2.92 (m, 2H), 2.84– 2.78 (m, 2H), 2.71 (s, 3H), 1.55 (s, 3H), 1.34 (d, J = 6.8 Hz, 3H).

Example 87: MS: 409 [M+H]⁺.¹H NMR (600 MHz, methanol-d₄) d 8.71 (s, 1H), 7.70 (d, J = 3.8 Hz, 1H), 7.19 (d, J = 7.6 Hz, 1H), 7.15 (dd, J = 7.6 Hz, 7.6 Hz, 1H), 7.01 (d, J = 7.6 Hz, 1H), 6.83 (d, J = 3.8 Hz, 1H), 6.18 (d, J = 7.6 Hz, 1H), 4.62 (d, J = 7.6 Hz, 1H), 4.41 (d, J = 9.0 Hz, 1H), 4.21 (d, J = 16.0 Hz, 1H), 4.11 (d, J = 16.0 Hz, 1H), 3.24– 3.18 (m, 1H), 3.18– 3.13 (m, 1H), 3.12– 3.06 (m, 1H), 2.96– 2.88 (m, 2H), 2.77 (s, 3H), 1.18 (d, J = 6.7 Hz, 3H), 1.03 (s, 3H). Example 88

(2R,3S,4R,5R)-2-((R)-1-hydroxy-1-(1,2,3,4-tetrahydroisoquinolin-8-yl)ethyl)-3-methyl-5-(4- methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-3,4-diol

Step 1: To a solution of tert-butyl 8-iodo-3,4-dihydroisoquinoline-2(1H)-carboxylate (1.08 g, 2.99 mmol) in THF (6 ml), was added isopropylmagnesium chloride lithium chloride (2.58 ml, 3.36 mmol, 1.3 M in THF) at -78 °C. The reaction mixture was stirred at 0 °C for 30 min and then at rt for another 30 min. This reaction mixture was added into a solution of (2S,3S,4R,5R)- 5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-(methoxy(methyl)carbamoyl)-3- methyltetrahydrofuran-3,4-diyl diacetate (200 mg, 0.454 mmol) in THF (3 ml) at 0 °C. The reaction was stirred at 0 °C for 3 h and then at rt for 16 h. The reaction was quenched with saturated aqueous NH₄Cl and then extracted with EtOAc (3x). The organic layer was dried over sodium sulfate and then filtered. The residue was purified by flash column chromatography (EtOAc/EtOH 3:1 in hexanes, 0 to 50%) to afford tert-butyl 8-((2S,3S,4R,5R)-5-(4-chloro-7H- pyrrolo[2,3-d]pyrimidin-7-yl)-3,4-dihydroxy-3-methyltetrahydrofuran-2-carbonyl)-3,4- dihydroisoquinoline-2(1H)-carboxylate as an amorphous solid. MS: 529 [M+H]⁺.

Step 2: To a solution of tert-butyl 8-((2S,3S,4R,5R)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7- yl)-3,4-dihydroxy-3-methyltetrahydrofuran-2-carbonyl)-3,4-dihydroisoquinoline-2(1H)- carboxylate (200 mg, 0.378 mmol) in THF (3.78 ml) was added methylmagnesium bromide (3 M in ether, 3.78 ml, 11.3 mmol) at 0 °C. The reaction was stirred for 1 h at 0 °C. The reaction was quenched with saturated aqueous NH₄Cl and then extracted with EtOAc (3x). The organic layer was dried over sodium sulfate then filtered. The crude material was concentrated and purified by flash column chromatography (EtOAc/EtOH 3:1 in hexanes, 0 to 100%) to afford tert-butyl 8-(1- ((2R,3S,4R,5R)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3,4-dihydroxy-3- methyltetrahydrofuran-2-yl)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate as an oil. MS: 545 [M+H]⁺.

Step 3: To a solution of tert-butyl 8-(1-((2R,3S,4R,5R)-5-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin- 7-yl)-3,4-dihydroxy-3-methyltetrahydrofuran-2-yl)-1-hydroxyethyl)-3,4-dihydroisoquinoline- 2(1H)-carboxylate (27 mg, 0.050 mmol) in THF (248 µl) was added

tetrakis(triphenylphosphine)palladium(0) (2.86 mg, 2.48 µmol). The reaction vial was degassed with argon gas for 5 min, and then dimethylzinc (2 M in toluene, 124 µl, 0.248 mmol) was added to the reaction mixture. The reaction vial was degassed with argon again for another 2 min. The reaction mixture was heated to 65 °C for 4 h. The mixture was poured into saturated aqueous NH₄Cl at 0 °C and then extracted with EtOAc (3x). The organic layers were dried over sodium sulfate and then filtered. The filtrate was concentrated under reduced pressure to afford tert-butyl 8-((R)-1-((2R,3S,4R,5R)-3,4-dihydroxy-3-methyl-5-(4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7- yl)tetrahydrofuran-2-yl)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate as a crude oil. The product was used for the next step without further purification. MS: 525 [M+H]⁺.

Step 4: A solution of tert-butyl 8-((R)-1-((2R,3S,4R,5R)-3,4-dihydroxy-3-methyl-5-(4-methyl- 7H-pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-2-yl)-1-hydroxyethyl)-3,4- dihydroisoquinoline-2(1H)-carboxylate in TFA (2 ml) was stirred for 15 min. The reaction mixture was concentrated under reduced pressure and purified by reverse-phase HPLC

(ACN/water with 0.1% NH₄OH modifier) to afford (2R,3S,4R,5R)-2-((R)-1-hydroxy-1-(1,2,3,4- tetrahydroisoquinolin-8-yl)ethyl)-3-methyl-5-(4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7- yl)tetrahydrofuran-3,4-diol as a solid. MS: 425 [M+H]⁺.

1H NMR (500 MHz, methanol-d4) d 8.69 (s, 1H), 7.68 (d, J = 3.7 Hz, 1H), 7.50 - 7.28 (m, 1H), 7.17 (dd, J = 7.7 Hz, 7.7 Hz, 1H), 7.06 (d, J = 7.7 Hz, 1H), 6.79 (d, J = 3.7 Hz, 1H), 5.96 (d, J = 8.2 Hz, 1H), 4.79 (s, 1H), 4.62 (d, J = 8.2 Hz, 1H), 4.59– 4.41 (m, 2H), 3.24– 3.14 (m, 1H), 3.12– 3.03 (m, 1H), 3.03– 2.89 (m, 2H), 2.78 (s, 3H), 1.54 (s, 3H), 0.95 (s, 3H). Examples 89 and 90

(2R,3S,4R,5R)-2-((R)-(6-(difluoromethyl)-5-fluoro-1,2,3,4-tetrahydroisoquinolin-8- yl)(hydroxy)methyl)-3-methyl-5-(4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran- 3,4-diol and (2R,3S,4R,5R)-2-((S)-(6-(difluoromethyl)-5-fluoro-1,2,3,4-tetrahydroisoquinolin-8- yl)(hydroxy)methyl)-3-methyl-5-(4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran- 3,4-diol

Step 1: To a solution of tert-butyl 6-(difluoromethyl)-8-((2S,3S,4R,5R)-3,4-dihydroxy-3-methyl- 5-(4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-2-carbonyl)-5-fluoro-3,4- dihydroisoquinoline-2(1H)-carboxylate (300 mg, 0.156 mmol) in methanol (1.6 ml) was added sodium borohydride (8.86 mg, 0.234 mmol) at 0 °C. After 15 min of stirring at 0 °C the reaction was quenched with acetic acid (35.7 µl, 0.624 mmol). The mixture was concentrated under reduced pressure and purified by flash column chromatography (EtOAc/EtOH 3:1 in hexanes, 0 to 30%) to afford tert-butyl 6-(difluoromethyl)-8-(((2R,3S,4R,5R)-3,4-dihydroxy-3-methyl-5-(4- methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-2-yl)(hydroxy)methyl)-5-fluoro-3,4- dihydroisoquinoline-2(1H)-carboxylate as an oil. MS: 579 [M+H]⁺.

Step 2: A solution of tert-butyl 6-(difluoromethyl)-8-(((2R,3S,4R,5R)-3,4-dihydroxy-3-methyl- 5-(4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-2-yl)(hydroxy)methyl)-5-fluoro- 3,4-dihydroisoquinoline-2(1H)-carboxylate (30 mg, 0.052 mmol) in a 2:1 DCM:TFA solution (2 ml) was stirred at rt for 1 h. The mixture was concentrated under reduced pressure and purified by reverse-phase HPLC (ACN/water with 0.1% NH4OH modifier) to afford (2R,3S,4R,5R)-2- ((R)-(6-(difluoromethyl)-5-fluoro-1,2,3,4-tetrahydroisoquinolin-8-yl)(hydroxy)methyl)-3- methyl-5-(4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-3,4-diol and

(2R,3S,4R,5R)-2-((S)-(6-(difluoromethyl)-5-fluoro-1,2,3,4-tetrahydroisoquinolin-8- yl)(hydroxy)methyl)-3-methyl-5-(4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran- 3,4-diol both as solids. Example 89 (faster eluting isomer): MS: 479 [M+H]⁺. ¹H NMR (500 MHz, methanol-d₄) d 8.65 (s, 1H), 7.69 (d, J = 7.1 Hz, 1H), 7.63 (d, J = 3.8 Hz, 1H), 6.93 (t, J = 55.0 Hz, 1H), 6.76 (d, J = 3.8 Hz, 1H), 6.06 (d, J = 7.6 Hz, 1H), 5.00 (d, J = 7.0 Hz, 1H), 4.64 (d, J = 7.6 Hz, 1H), 4.16 (d, J = 7.0 Hz, 1H), 4.09 (s, 2H), 3.11– 3.04 (m, 1H), 2.97– 2.89 (m, 1H), 2.82– 2.75 (m, 2H), 2.73 (s, 3H), 1.54 (s, 3H).

Example 90 (slower eluting isomer): MS: 479 [M+H]⁺.¹H NMR (500 MHz, methanol-d₄) d 8.68 (s, 1H), 7.98 (d, J = 3.8 Hz, 1H), 7.59 (d, J = 7.2 Hz, 1H), 6.83 (d, J = 3.8 Hz, 1H), 6.77 (t, J = 55.2 Hz, 1H), 6.17 (d, J = 8.0 Hz, 1H), 5.04 (s, 1H), 4.71 (d, J = 8.0 Hz, 1H), 4.17 (d, J = 16.6 Hz, 1H), 4.07 (s, 1H), 3.95 (d, J = 16.6 Hz, 1H), 3.17– 3.10 (m, 1H), 3.10– 3.03 (m, 1H), 2.85– 2.79 (m, 2H), 2.79 (s, 3H), 1.64 (s, 3H). Example 91

(2R,3S,4R,5R)-2- -1-(6-(difluoromethyl)-5-fluoro-1,2,3,4-tetrahydroisoquinolin-8-yl)-1-

hydroxyethyl)-3-methyl-5-(4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-3,4-diol

Step 1: To a solution of (2S,3S,4R,5R)-2-(2-(tert-butoxycarbonyl)-6-(difluoromethyl)-5-fluoro- 1,2,3,4-tetrahydroisoquinoline-8-carbonyl)-3-methyl-5-(4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7- yl)tetrahydrofuran-3,4-diyl diacetate (350 mg, 0.530 mmol) in THF (3.5 ml) was added methylmagnesium bromide (265 µl, 0.795 mmol, 3.0 M in diethyl ether) at 0 °C. After stirring for 1 h at 0 °C the reaction was quenched with acetic acid (121 µl, 2.12 mmol). Saturated aqueous NH₄Cl was added and the mixture and then extracted with EtOAc (3x). The organic layers were combined, dried over sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and purified by flash column chromatography (EtOAc/EtOH 3:1 in hexanes, 0 to 50%) to afford (2R,3R,4R,5R)-2-(1-(2-(tert-butoxycarbonyl)-6-(difluoromethyl)-5-fluoro- 1,2,3,4-tetrahydroisoquinolin-8-yl)-1-hydroxyethyl)-3-methyl-5-(4-methyl-7H-pyrrolo[2,3- d]pyrimidin-7-yl)tetrahydrofuran-3,4-diyl diacetate as a solid. MS: 677 [M+H]⁺.

Step 2: A solution of (2R,3R,4R,5R)-2-(1-(2-(tert-butoxycarbonyl)-6-(difluoromethyl)-5-fluoro- 1,2,3,4-tetrahydroisoquinolin-8-yl)-1-hydroxyethyl)-3-methyl-5-(4-methyl-7H-pyrrolo[2,3- d]pyrimidin-7-yl)tetrahydrofuran-3,4-diyl diacetate (65 mg, 0.096 mmol) in a 2:1 DCM:TFA solution (3 ml) was stirred for 15 min at rt. The crude mixture was then concentrated under reduced pressure to afford (2R,3R,4R,5R)-2-((R)-1-(6-(difluoromethyl)-5-fluoro-1,2,3,4- tetrahydroisoquinolin-8-yl)-1-hydroxyethyl)-3-methyl-5-(4-methyl-7H-pyrrolo[2,3-d]pyrimidin- 7-yl)tetrahydrofuran-3,4-diyl diacetate as an oil. MS: 577 [M+H]⁺.

Step 3: A solution of (2R,3R,4R,5R)-2-((R)-1-(6-(difluoromethyl)-5-fluoro-1,2,3,4- tetrahydroisoquinolin-8-yl)-1-hydroxyethyl)-3-methyl-5-(4-methyl-7H-pyrrolo[2,3-d]pyrimidin- 7-yl)tetrahydrofuran-3,4-diyl diacetate (55 mg, 0.096 mmol) in 7 N ammonia in methanol (5 ml) was stirred for 20 h at rt. The reaction mixture was concentrated under reduced pressure and purified by reverse-phase HPLC (ACN/water with 0.1% NH₄OH modifier) to afford

(2R,3S,4R,5R)-2-((R)-1-(6-(difluoromethyl)-5-fluoro-1,2,3,4-tetrahydroisoquinolin-8-yl)-1- hydroxyethyl)-3-methyl-5-(4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-3,4-diol as a solid. MS: 493 [M+H]⁺.¹H NMR (500 MHz, methanol-d₄) d 8.70 (s, 1H), 7.71 (s, 1H), 7.68 (d, J = 3.7 Hz, 1H), 6.97 (t, J = 54.9 Hz, 1H), 6.79 (d, J = 3.7 Hz, 1H), 5.97 (d, J = 8.2 Hz, 1H), 4.74 (s, 1H), 4.62 (d, J = 8.2 Hz, 1H), 4.42 (s, 2H), 3.22– 3.04 (m, 2H), 2.96– 2.86 (m, 2H), 2.79 (s, 3H), 1.56 (s, 3H), 0.93 (s, 3H). Examples 92 and 93

(2R,3S,4R,5R)-2-((R)-1-(6-(difluoromethyl)-5-fluoro-1,2,3,4-tetrahydroisoquinolin-8-yl)ethyl)- 3-methyl-5-(4-methyl-7H-pyrrol midin-7-yl)tetrahydrofuran-3,4-diol and

(2R,3S,4R,5R)-2-((S)-1-(6-(difluoromethyl)-5-fluoro-1,2,3,4-tetrahydroisoquinolin-8-yl)ethyl)- 3-methyl-5-(4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-3,4-diol

Step 1: To a solution of (2S,3S,4R,5R)-2-(2-(tert-butoxycarbonyl)-6-(difluoromethyl)-5-fluoro- 1,2,3,4-tetrahydroisoquinoline-8-carbonyl)-3-methyl-5-(4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7- yl)tetrahydrofuran-3,4-diyl diacetate (350 mg, 0.530 mmol) in THF (3.5 ml) was added methylmagnesium bromide (265 µl, 0.795 mmol, 3.0 M in diethyl ether) at 0 °C. After stirring for 1 h at 0 °C the reaction was quenched with acetic acid (121 µl, 2.12 mmol). Saturated aqueous NH₄Cl was added and the mixture and then extracted with EtOAc (3x). The organic layers were combined, dried over sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and purified by flash column chromatography (EtOAc/EtOH 3:1 in hexanes, 0 to 50%) to afford (2R,3R,4R,5R)-2-(1-(2-(tert-butoxycarbonyl)-6-(difluoromethyl)-5-fluoro- 1,2,3,4-tetrahydroisoquinolin-8-yl)-1-hydroxyethyl)-3-methyl-5-(4-methyl-7H-pyrrolo[2,3- d]pyrimidin-7-yl)tetrahydrofuran-3,4-diyl diacetate as a solid. MS: 677 [M+H]⁺.

Step 2: To a solution of (2R,3R,4R,5R)-2-(1-(2-(tert-butoxycarbonyl)-6-(difluoromethyl)-5- fluoro-1,2,3,4-tetrahydroisoquinolin-8-yl)-1-hydroxyethyl)-3-methyl-5-(4-methyl-7H- pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-3,4-diyl diacetate (251 mg, 0.371 mmol) in DCM (3.7 ml) a solution of Martin's Sulfurane (998 mg, 1.48 mmol) in DCM (1 ml) was added at 0 °C. The reaction was stirred for 15 min at 0 °C, then warmed to rt for 16 h. The reaction mixture was concentrated under reduced pressure and purified by flash column chromatography

(EtOAc/EtOH 3:1 in hexanes, 0 to 100%) to afford (2R,3R,4R,5R)-2-(1-(2-(tert- butoxycarbonyl)-6-(difluoromethyl)-5-fluoro-1,2,3,4-tetrahydroisoquinolin-8-yl)vinyl)-3- methyl-5-(4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-3,4-diyl diacetate as a solid. MS: 659 [M+H]⁺.

Step 3: A solution of (2R,3R,4R,5R)-2-(1-(2-(tert-butoxycarbonyl)-6-(difluoromethyl)-5-fluoro- 1,2,3,4-tetrahydroisoquinolin-8-yl)vinyl)-3-methyl-5-(4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7- yl)tetrahydrofuran-3,4-diyl diacetate (125 mg, 0.190 mmol) in 7 N ammonia in methanol (3 ml) was stirred at rt for 2 h. The reaction mixture was concentrated under reduced pressure and purified by reverse-phase HPLC (ACN/water with 0.1% NH₄OH modifier) to afford tert-butyl 6- (difluoromethyl)-8-(1-((2R,3S,4R,5R)-3,4-dihydroxy-3-methyl-5-(4-methyl-7H-pyrrolo[2,3- d]pyrimidin-7-yl)tetrahydrofuran-2-yl)vinyl)-5-fluoro-3,4-dihydroisoquinoline-2(1H)- carboxylate as a solid. MS: 575 [M+H]⁺.

Step 4: To a solution of tert-butyl 6-(difluoromethyl)-8-(1-((2R,3S,4R,5R)-3,4-dihydroxy-3- methyl-5-(4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-2-yl)vinyl)-5-fluoro-3,4- dihydroisoquinoline-2(1H)-carboxylate (36 mg, 0.063 mmol) in methanol (2.1 ml) was added Pd-C (6.67 mg, 0.063 mmol, 10 wt.%) at rt and the system was purged with H₂ for 1 min. The vial was stirred for 4 days at rt. The reaction mixture was filtered through a Celite pad, washing with methanol. The filtrate was concentrated under reduced pressure to afford tert-butyl 6- (difluoromethyl)-8-(1-((2R,3S,4R,5R)-3,4-dihydroxy-3-methyl-5-(4-methyl-5,6-dihydro-7H- pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-2-yl)ethyl)-5-fluoro-3,4-dihydroisoquinoline- 2(1H)-carboxylate. MS: 577 [M+H]⁺.

Step 5: A solution of tert-butyl 6-(difluoromethyl)-8-(1-((2R,3S,4R,5R)-3,4-dihydroxy-3- methyl-5-(4-methyl-5,6-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-2-yl)ethyl)-5- fluoro-3,4-dihydroisoquinoline-2(1H)-carboxylate (80 mg, 0.138 mmol) in a 2:1 TFA:DCM solution (1.5 ml) was stirred at rt for 30 min. The reaction mixture was concentrated under reduced pressure and purified by reverse-phase HPLC (ACN/water with 0.1% NH₄OH modifier) to afford (2R,3S,4R,5R)-2-((R)-1-(6-(difluoromethyl)-5-fluoro-1,2,3,4-tetrahydroisoquinolin-8- yl)ethyl)-3-methyl-5-(4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-3,4-diol and (2R,3S,4R,5R)-2-((S)-1-(6-(difluoromethyl)-5-fluoro-1,2,3,4-tetrahydroisoquinolin-8-yl)ethyl)- 3-methyl-5-(4-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-3,4-diol, both as solids. Example 92 (faster eluting isomer): MS: 477 [M+H]⁺.¹H NMR (500 MHz, DMSO-d₆) d 8.63 (s, 1H), 7.54 (d, J = 3.8 Hz, 1H), 7.34 (d, J = 6.9 Hz, 1H), 7.11 (t, J = 54.7 Hz, 1H), 6.76 (d, J = 3.8 Hz, 1H), 5.87 (d, J = 5.2 Hz, 1H), 5.69 (d, J = 5.8 Hz, 1H), 4.91 (s, 1H), 4.00– 3.87 (m, 4H), 3.19– 3.11 (m, 1H), 2.89– 2.82 (m, 1H), 2.82– 2.74 (m, 1H), 2.63 (s, 3H), 2.60– 2.53 (m, 2H), 1.42 (s, 3H), 1.23 (d, J = 6.8 Hz, 3H).

Example 93 (slower eluting isomer): MS: 477 [M+H]⁺.¹H NMR (500 MHz, DMSO-d₆) d 8.70 (s, 1H), 7.87 (d, J = 3.8 Hz, 1H), 7.31 (d, J = 6.9 Hz, 1H), 7.10 (t, J = 54.5 Hz, 1H), 6.82 (d, J = 3.8 Hz, 1H), 6.14 (d, J = 7.8 Hz, 1H), 5.38 (d, J = 7.1 Hz, 1H), 4.83 (s, 1H), 4.44– 4.39 (m, 1H), 4.21 (d, J = 8.3 Hz, 1H), 4.09 (d, J = 16.8 Hz, 1H), 3.94 (d, J = 16.8 Hz, 1H), 3.24– 3.19 (m, 1H), 3.00– 2.90 (m, 2H), 2.68 (s, 3H), 2.67– 2.62 (m, 2H), 1.07 (d, J = 6.7 Hz, 3H), 0.92 (s, 3H). PRMT5-MEP50 Enzyme Methylation Assay

PRMT5-MEP50 biochemical assay is a direct measurement of the methylation activity of the enzyme complex on a short peptide substrate derived from the N-terminus of H4 histone. Methylation experiment is performed with recombinant PRMT5-MEP50 protein complex. The assessment of inhibitory effect of small molecules is measured by the effectiveness of the compounds to inhibit this reaction (EC₅₀).

In this assay, the potency (EC₅₀) of each compound was determined from a twenty-point (1:2 serial dilution; top compound concentration of 100000 nM) titration curve using the following outlined procedure. To each well of a white ProxiPlus 384 well-plate, 100 nL of compound (1% DMSO in final assay volume of 10 µL) was dispensed, followed by the addition of 8 µL of 1x assay buffer (50 mM Bicine pH 8.0, 1 mM DTT, 0.004% Tween20, 0.01% BSA) containing 1.25 nM of Full-length (FL)-PRMT5-MEP50 enzyme complex (recombinant proteins from baculovirus-transfected Sf21 cells: FL-PRMT5; MW = 73837 kDa and FL-MEP50; MW = 38614) and 1 µL of 150 µM S-(5’-Adenosyl)-L-Methionine Chloride (SAM). Plates were sealed and placed in a 37 ^oC humidified chamber for a 60 minutes pre-incubation with compound.

Subsequently, each reaction was initiated by the addition of 1 µL 1x assay buffer containing 750 nM biotinylated H4R3(Me1) peptide. The final reaction in each well of 10 mL consists of 1.0 nM PRMT5-MEP50, 75 nM biotinylated-peptide, and 15 µM SAM. Methylation reactions were allowed to proceed for 150 minutes in a sealed plate at 37 ^oC. Reactions were immediately quenched by the addition of 1 µL of 5% formic acid. Plates were then frozen and shipped to SAMDITM Tech Inc. to determine the percent conversion from H4R3(Me1) to H4R3(Me2). Dose-response curves were generated by plotting percent effect (% product conversion; Y-axis) vs. Log10 compound concentrations (X-axis). EC₅₀ values were determined by non-linear regression according to models for either sigmoidal (4 parameters) or biphasic (7 parameters) dose-response curves.

PRMT5 Cell Target Engagement (TE) Assay

The PRMT5 TE assay is a biomarker assay for identifying compounds that inhibit symmetric dimethylation of arginine (SDMA) of PRMT5 substrates. The following substrates have been reported for PRMT5: Histone H2A and H4 R3, Histone H3 R2, Histone H3 R8, spliceosome Sm proteins, ribosomal protein RPS10, p53, FEN1, nucleoplasmin, nucleolin, EGFR and EBNA. The assay will focus on detecting symmetrically dimethylated nuclear proteins using high content imaging technology. Detection of the expression of symmetrically dimethylated nuclear proteins is through a mixture of primary rabbit monoclonal antibodies to SDMA (CST 13222), which in turn recognized by an Alexafluor 488 dye-conjugated anti-rabbit IgG secondary antibody. The IN Cell Analyzer 2200 or Opera-Phenix measures nuclear Alexafluor 488 fluorescent dye intensity that is directly related to the level of expression of symmetrically dimethylated nuclear proteins at the single cell level. Nuclear AF488 dye intensities are compared to the mean value for DMSO treated cells (MIN) to report percent of inhibition for each compound-treated well.

In this assay, the cell potency (EC₅₀) of each compound was determined from a ten point (1:3 serial dilution; top compound concentration of 10000 nM) titration curve using the following outlined procedure. Each well of a BD falcon collagen coated black/clear bottom 384-well plate was seeded with 4000 MCF-7 cells in 30 µl media and allowed to attach for 5 hours. Media is ATCC-formulated Eagle's Minimum Essential Medium, Catalog No.30-2003. The following components were added to the base medium: 0.01 mg/mL human recombinant insulin; fetal bovine serum to a final concentration of 10%. Additional 30 µl of media containing 2x compounds were added to each well. Cells were treated for 3 days in 37°C CO₂ incubator. On day 3, cells were fixed with Cytofix, permeabilized with 0.4% Triton-X-100/Cytofix, and washed with D-PBS without Ca/Mg. Cells were blocked with Licor Odessey blocking reagent for 1 hour at room temperature, followed by incubation with anti-SDMA (1:1000) antibody at 4°C overnight.1° antibody was removed, followed by three washings with DPBS without Ca/Mg and 0.05% Tween20. Hoechst (5µg/mL), Cell Mask deep stain (1:2000) and Alexa488- conjugated goat anti-rabbit IgG (2 µg/mL) was added for 1 hour at room temperature. A final washing step (three washes) was performed before sealing plate for imaging on In Cell Analyzer 2200 or Opera-Phenix. Images from analyzer were uploaded to Columbus for image analysis. IC₅₀ values were determined by 4 parameters robust fit of percent fluorescence units vs. (Log10) compound concentrations.

Representative compounds of the present invention were tested using the assay protocol described in this example. Results are provided in Table 5 below. Table 5: When only one EC₅₀ is shown, the data was fit to a 4 parameters single site sigmodal model. When two EC50s are shown, the data was fit to a 7 parameters biphasic model

Claims

WHAT IS CLAIMED IS:

1. A compound of Formula I,

or a pharmaceutically acceptable salt thereof, wherein

X is CH₂ or O;

Y is CR⁹R⁹, CR⁹OR⁹, C(CFH₂)(R⁹), C(CF₂H)(R⁹), C(CF₃)(R⁹), C(CFH₂)(OR⁹), C(CF₂H)(OR⁹), C(CF₃)(OR⁹), CHF, or CF₂;

A¹ is CH, CR⁹, or CNH₂;

A² is CH, N, or CF;

Z¹ is N, or CR⁴;

Z² is N, or CR⁵;

Z³ is N, or CR⁶;

Z⁴ is N, or CR⁷;

Z⁵ is N, or CR⁸;

R¹ is H, C_1-6alkyl optionally substituted with one to four halogens, or CH=CH₂;

R² is H, C_1-6alkyl optionally substituted with one to four halogens, or CH=CH₂;

provided that if R¹ is H, then R² cannot be H; and if R² is H, then R¹ cannot be H;

R³ is H, CH₃, NH₂, or NHCH₃;

R⁴ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, OC_1-6alkyl, or taken together with R⁵ to form a 5 or 6 membered ring comprising one N atom;

R⁵ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, OC_1-6alkyl, or taken together with R⁴ or R⁶ to form a 5 or 6 membered ring comprising one N atom;

R⁶ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, OC_1-6alkyl, or taken together with R⁵ or R⁷ to form a 5 or 6 membered ring comprising one N atom;

R⁷ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, OC_1-6alkyl, or taken together with R⁸ to form a 5 or 6 membered ring comprising one N atom;

R⁸ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, OC_1-6alkyl, or taken together with R⁷ to form a 5 or 6 membered ring comprising one N atom; and

each R⁹, when present, is independently H or C1-6alkyl.

2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein Formula I is Formula Ia

or a pharmaceutically acceptable salt thereof, wherein

X is CH₂ or O;

Y is CR⁹R⁹, CR⁹OR⁹, C(CFH₂)(R⁹), C(CF₂H)(R⁹), C(CF₃)(R⁹), C(CFH₂)(OR⁹), C(CF₂H)(OR⁹), C(CF₃)(OR⁹), CHF, or CF₂,

Z¹ is N, or CR⁴;

Z² is N, or CR⁵;

Z³ is N, or CR⁶;

Z⁴ is N, or CR⁷;

Z⁵ is N, or CR⁸;

R¹ is H, C_1-6alkyl optionally substituted with one to four halogens, or CH=CH₂;

R² is H, C_1-6alkyl optionally substituted with one to four halogens, or CH=CH₂;

provided that if R¹ is H, then R² cannot be H; and if R² is H, then R¹ cannot be H;

R³ is H, CH3, NH2, or NHCH3;

R⁴ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, OC_1-6alkyl, or taken together with R⁵ to form a 5 or 6 membered ring comprising one N atom;

R⁵ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, OC_1-6alkyl, or taken together with R⁴ or R⁶ to form a 5 or 6 membered ring comprising one N atom;

R⁶ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, OC_1-6alkyl, or taken together with R⁵ or R⁷ to form a 5 or 6 membered ring comprising one N atom;

R⁷ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, OC_1-6alkyl, or taken together with R⁸ to form a 5 or 6 membered ring comprising one N atom;

R⁸ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, OC_1-6alkyl, or taken together with R⁷ to form a 5 or 6 membered ring comprising one N atom; and

each R⁹, when present, is independently H or C1-6alkyl.

3. The compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein Y is CH₂, CH(CH₃), CH(OH), or C(CH₃)(OH).

4. The compound of claim 2 or 3, or a pharmaceutically acceptable salt thereof, wherein R¹ is CH₃.

5. The compound of claim 2, 3, or 4, or a pharmaceutically acceptable salt thereof, wherein R² is H.

6. The compound of claim 2, 3, 4, or 5, or a pharmaceutically acceptable salt thereof, wherein R³ is NH₂.

7. The compound of claim 2, 3, 4, 5, or 6, or a pharmaceutically acceptable salt thereof, wherein R⁵ is F or CHF₂.

8. The compound of claim 2, 3, 4, 5, 6, or 7, or a pharmaceutically acceptable salt thereof, wherein R⁷ is F or CHF₂.

9. The compound of claim 2, 3, 4, 5, 6, 7, or 8, or a pharmaceutically acceptable salt thereof, wherein

R⁴ is H, CH₃, or halogen;

R⁵ is H, halogen, CH₃, OCH₃, CF₃, or CF₂H;

R⁶ is H, halogen, CH₃, OCH_3, CF₃, or CF₂H;

R⁷ is H, halogen, CH₃, OCH_3, CF₃, or CF₂H; and

R⁸ is H, CH₃, or halogen.

10. The compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein R⁹ is H.

11. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein formula I is formula Ib

or a pharmaceutically acceptable salt thereof, wherein

Z¹ is N, or CR⁴;

Z² is N, or CR⁵;

Z³ is N, or CR⁶;

Z⁴ is N, or CR⁷;

Z⁵ is N, or CR⁸;

R¹ is H, C_1-6alkyl optionally substituted with one to four halogens, or CH=CH₂;

R² is H, C_1-6alkyl optionally substituted with one to four halogens, or CH=CH₂;

provided that if R¹ is H, then R² cannot be H; and if R² is H, then R¹ cannot be H;

R³ is H, CH₃, NH₂, or NHCH₃;

R⁴ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, OC_1-6alkyl, or taken together with R⁵ to form a 5 or 6 membered ring comprising one N atom;

R⁵ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, OC_1-6alkyl, or taken together with R⁴ or R⁶ to form a 5 or 6 membered ring comprising one N atom;

R⁶ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, OC_1-6alkyl, or taken together with R⁵ or R⁷ to form a 5 or 6 membered ring comprising one N atom;

R⁷ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, OC_1-6alkyl, or taken together with R⁸ to form a 5 or 6 membered ring comprising one N atom; and

R⁸ is H, halogen, C1-6alkyl optionally substituted with one to four halogens, OC1-6alkyl, or taken together with R⁷ to form a 5 or 6 membered ring comprising one N atom.

12. The compound of claim 11, or a pharmaceutically acceptable salt thereof, wherein R¹ is CH₃ and R² is H.

13. The compound of claim 11 or 12, or a pharmaceutically acceptable salt thereof, wherein R³ is NH₂.

14. The compound of claim 11, 12, or 13, or a pharmaceutically acceptable salt thereof, wherein

R⁴ is H, CH₃, or halogen;

R⁵ is H, halogen, CH₃, OCH₃, CF₃, or CF₂H;

R⁶ is H, halogen, CH₃, OCH₃, CF₃, or CF₂H;

R⁷ is H, halogen, CH₃, OCH_3, CF₃, or CF₂H; and

R⁸ is H, CH₃, or halogen.

15. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein formula I is formula Ic

or a pharmaceutically acceptable salt thereof, wherein

R³ is H, CH₃, NH₂, or NHCH₃;

R⁴ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, OC_1-6alkyl, or taken together with R⁵ to form a 5 or 6 membered ring comprising one N atom;

R⁵ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, OC_1-6alkyl, or taken together with R⁴ or R⁶ to form a 5 or 6 membered ring comprising one N atom;

R⁶ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, OC_1-6alkyl, or taken together with R⁵ or R⁷ to form a 5 or 6 membered ring comprising one N atom;

R⁷ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, OC_1-6alkyl, or taken together with R⁸ to form a 5 or 6 membered ring comprising one N atom; and

R⁸ is H, halogen, C_1-6alkyl optionally substituted with one to four halogens, OC_1-6alkyl, or taken together with R⁷ to form a 5 or 6 membered ring comprising one N atom.

16. The compound of claim 15, or a pharmaceutically acceptable salt thereof, wherein R³ is NH₂.

17. The compound of claim 15 or 16, or a pharmaceutically acceptable salt thereof, wherein

R⁴ is H, CH₃, or halogen;

R⁵ is H, halogen, CH₃, OCH₃, CF₃, or CF₂H;

R⁶ is H, halogen, CH₃, OCH₃, CF₃, or CF₂H;

R⁷ is H, halogen, CH₃, OCH_3, CF₃, or CF₂H; and

R⁸ is H, CH₃, or halogen.

18. The compound of claim 1, or a pharmaceutically acceptable salt thereof, which is,

, , , or a pharmaceutically acceptable salt thereof.

19. A composition for treating cancer comprising a compound of any of claims 1-18, or a pharmaceutically acceptable salt thereof, and a pharmaceutically carrier.

20. A method for treating cancer comprising administering to a patient in need thereof a compound of any of claims 1-18, or a pharmaceutically acceptable salt thereof.

21. The use of a compound of any of claims 1-18, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating cancer.