OA20817A - PRMT5 inhibitors - Google Patents

Abstract

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

Description

PRMT5 INHIBITORS

BACKGROUND OF THE INVENTION

PRMT5 (aka JBPl, SKBl, 1BP72, SKBlhis 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 rôle în control and modulation of gene transcription. Inter alia, PRMT5 îs known to symmetrically 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). PRMT5 has been reported to perform diverse rôles including but not limited to împacting cell viabîlity, 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 négative regulator of p53, MDM4 resuiting in increased expression of the short isoform of MDM4 (MDM4-S), decreased expression of the full-length isoform (MDM4-FL) and încreased p53 activity (Gerhart et al Sci Rep (2018)). Most of the physiological funclions of p53 are attributable to its rôle 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 malîgnancies, 85% in bones and endocrine glands, and 75% of primary breast cancer. Restoration of p53 in cancer cells harboring 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.

In response to DNA damage cause d b y 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-Gl population and concomitant réduction in G1 cells and, in the presence of p53, a significant increase in apoptosis. Knockdown of PRMT5 also resulted in an increased level of p21, a key p53 target gene that régulâtes cell cycle arrest during the p53 response and MDM2, a p53 E3 ubiquitin ligase, but not PUMA, NOXA, A1P1 & APAF1, p53 target genes linked to apoptosis.

Knockdown of PRMT5 (but not PRMT1 or CARM1/PRMT4) results in decreased p53 stabilization, decreased basal p53 levels, decreased p53 oligomerisation, and also decreased expression of elF4E a major comportent of translation al machinery invol ved in ribosome binding to mRNA. Indeed, e!F4E is a potent oncogene, which has been shown to promote malignant transformation in vitro and human cancer formation.

The rôle of PRMT5 in the DNA damage response has been explored with groups reporting a rôle 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 lînking this mecbanism to cancers. PRMT5 overexpressîon 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-Ce il lymphomas and leukemia (Wang, 2008). Moreover, elevated expression of P RM T 5 in Melanoma, Breast and Colorectal cancers has been demonstrated to correlate with a poor prognosis.

Lympboid malignancies including chronic iymphocytic leukemia (CLL) are associated with over-expression of PRMT5. PRMT5 is over-expressed (at the protein level) in the nucléus and cytosol in a number of patient derived Burkitt's lymphoma; niant le 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, resuiting in increased levels of PRMT5 (Pal et al., 2007; Wang et al., 2008).

In addition to genomic changes, CLL, like almost ail cancers, has aberrant epigenetic abnormalities characterised by global hypomethylation and hot-spots of répressive hyperméthylation of promoters including tumor suppressor genes. While the rôle of epigenetics in the origin and progression of CLL remains unclear, epigenetic changes appear to occur early in the disease and spécifie patterns of DNA méthylation are associated with worse prognosis (Chen et al., 2009; Kanduri et al., 2010). Global symmetrîc méthylation of histones H3R8 and H4R3 îs increased in transfonned iymphoid cell lines and MCL clinical samples (Pal et al., 2007), correlating with the overexpressîon of PRMT5 observed in a wide variety of iymphoid 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 ïs es senti al 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 β subunits. In human infants, the hemoglobin molécule is made up of two a and two y chains. The gamma chains are gradually replaced by β subunits as the infant grows. The developmental switch in human β-like giobin gene subtype from foetal (y) to adult (β) that begins at birth heralds the onset of the hemoglobinopathies β-thalassemia or sickle cell disease (SCD). In β-thalassemia the adult chains are not produced. In SCD, a point mutation in the coding sequence in the β giobin gene leads to the production of a protein with altered polymérisation properties. The observation that increased adult γ-globin gene expression (in the setting of bereditary persistence of foetal hemoglobin (HPFH) mutations) signifïcantly améliorâtes the clinical severity of β-thalassemia and SCD has prompted the search for therapeutic strategies to reverse γ-globin gene silencing. To date, this has been achieved through pharmacological induction, using compounds that broadly influence epigenetîc modifications, including DNA méthylation and histone deacetylation. The development of more targeted thérapies is dépendent on the identification of the molecular mechanisms underpinning foetal globin gene silencing. These mechanisms hâve remained elusîve, despite exhaustive study of the HPFH mutations, and considérable progress m many other aspects of globin gene régulation.

PRMT5 plays a critical rôle in triggering coordinated répressive epigenetîc events that initiale with dîmethylatîon of histone H4 Arginine 3 (H4R3me2s), and culminate in DNA méthylation and transcriptional silencing of the y-genes (Rank et al., 2010). Intégral to the synchronous establishment of the répressive mark ers is the assembly of a PRMT5-dépendent complex containing the DNA methyltransferase DNMT3A, and other repressor proteins (Rank et al., 2010). DNMT3A is directly recruîted 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 lackîng methyltransferase activity leads to marked upregulation of γ-gene expression, and complété abrogation of DNA méthylation at the γ-promoter. Treatment of human erythroid progenitors with non-specific methyltransferase inhibitors (Adox and MTA) also resulted in upregulation of γ-gene expression (He Y, 2013). Inhibitors of PRMT5 thus hâve potential as therapeutics for hemoglobinopathies such as β-thalassemia or Sickle Cell Disease (SCD).

The present inventors hâve 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

The present invention provides a compound selected from:

and the pharmaceutically acceptable salts, esters, and prodrugs thereof, which are PRMT5 inhibitors. Also provided are methods of making compounds disclosed herein, pharmaceutical compositions comprising compounds disclosed herein, and methods of using these compounds to treat cancer, sickle cell, and hereditary persistenee of foetal hemoglobin (HPFH) mutations.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a compound selected from:

or a pharmaceutically acceptable sait thereof.

In one embodiment, the present invention provides a compound selected from:

or a pharmaceutically acceptable sait thereof.

In an embodiment of the invention, the compound is, ( ( 3 R, 3 'R) - 3 ' -hy droxy -1,4-dihy dro- VH, 2 //-sp iro [i so q uin o l ine- 3,4 ' -p i péri di η] - Γ-y l ) [ 8 (methoxymethyl)-6-(trifluoromethyl)imidazo[ l ,2-u]pyridin-2-yI] methanone, (6-bromo-8-((S)-l-methoxyetbyl)imidazo[J,2M|pyridin-2-yl)((3fo3’7î)-3'-hydiOxy-],4-dihydro2/7-spîro[isoquinoline-3,4'-piperidin]-r-yl)methanone, (6-bromo-8~((A)-1 -methoxyethyl)imidazo[ l ,2-a]pyridin-2-yl)((3Æ,3 ’/Q-S'-hydroxy-l ,4-dihydro2f7-spîro[isoquinoline-3,4'-piperidin]-r-yl)methanone, (6-bromo-7-methylimidazo[l,2-i/]pyrimidin-2-yl)[(37ï,3'A)-3'-hydroxy-l,4-dihydro-lW,2//spiro[isoquinoIine-3,4'-piperidin]-r-yl]methanone, (6-cyclopropylimidazo[ l ,2-u]pyriimdin-2-yl)[(37ï,37ï)-3’-hydroxy-l ,4-dihydro-l77,2/7spiro[isoqumoIine-3,4’-piperidm]-l'-yl]methanone, or a pharmaceutically acceptable sait thereof.

In an embodiment of the invention, the compound is,

, or a pharmaceutically acceptable sait thereof.

In an embodiment ofthe invention, the compound is,

In an embodiment of the invention, the compound is,

or a pharmaceutically acceptable sait thereof.

In an embodiment of the invention, the compound îs,

, or a pharmaceutically acceptable sait thereof.

In an embodiment of the invention, the compound is, ((32ï,3’/î)-3'-hydroxy-l,4-dihydror/f,2/7-spiro[isoquinoline-3_J4^,-piperidin]-l'-yl)[8-(methoxymethyl)-6(trifluoromethyl)imidazo[l,2-o]pyridin-2-yl]methanone, or a pharmaceutically acceptable sait thereof.

In an embodiment of the invention, the compound is, (6-bTOmo-8-((5)-lme&oxyethyl)imidazo[l,2-o]pyridin-2-yl)((3_Jfi,3’Â)-3’-hydroxy-l,4-dihydro-2/7spiro[isoqumoline-3,4'-piperidm]-r-yl)methanone, or a pharmaceutically acceptable sait thereof.

In an embodiment of the invention, the compound is, (6-bromo-8-((R)-lmethoxyethyl)imidazo[ 1,2-a]pyridm-2-yl)((3A,3’Æ)-3'-hydroxy-l ,4-dihydro-2//spiro[isoqumoline-3,4'-piperidin]-r-yl)methanone, or a pharmaceutically acceptable sait thereof.

In an embodiment of the invention, the compound is, (6-bromo-7-methylimidazo[l,2û]pyrimidin-2-yl)[(3A,3'A)-3'-hydroxy-l,4-dihydro-]'/7,2ff-spiro[isoqumoline-3,4'-piperidin]-ryl]methanone, or a pharmaceutically acceptable sait thereof.

In an embodiment of the invention, the compound is, (6-cyclopropylimidazo[l,2u]pyrmiidm-2-yl)[(3A3'R)-3’-hydroxy-l ,4-dihydro-r/f,2H-spiro[isoqumoline-3,4'-piperidîn]-ryl]methanone, or a pharmaceutically acceptable sait thereof.

In one embodiment, the présent invention is a composition for treating cancer comprising an effective amount of at least one compound disclosed herein, or a pharmaceutically acceptable sait thereof, and a pharmaceutically acceptable carrier.

The invention also provides a pharmaceutical composition comprising an effective amount of at least one compound disclosed herein, or a pharmaceutically acceptable sait thereof, and an effective amount of at least one other pharmaceutically active ingrédient (such as, for example, a chemotherapeutic agent), and a pharmaceutically acceptable carrier.

In one embodiment, the présent invention is a composition for treating hemoglobinopathies such as β-thalassemia or Sickle Cell Disease (SCD), comprising a compound disclosed herein, or a pharmaceutically acceptable sait thereof.

In one embodiment, the présent invention is a composition for treating hemoglobinopathies such as β-thalassemia or Sickle Cell Disease (SCD), comprising a compound disclosed herein, or a pharmaceutically acceptable sait thereof, and a pharmaceutically acceptable c a nier.

In one embodiment, the présent invention is a method of inhibiting P RM T 5 in a patient in need thereof comprising administering to said patient an effective amount of at least one compound disclosed herein, or a pharmaceutically acceptable sait thereof.

In another embodiment, the présent invention is a method of treating cancer comprising administering to a patient in need thereof a an effective amount of at least one compound disclosed herein, or a pharmaceutically acceptable sait thereof.

In another embodiment, the présent invention provides a method for treating cancer in a patient in need thereof comprising administering to said patient an effective amount of at least one compound disclosed herein, or a pharmaceutically acceptable sait 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 disclosed herein and a pharmaceutically acceptable carrier.

In another embodiment, the present invention includes a method of treating hemoglobinopathies such as β-thalassemia or Sickle Cell Disease (SCD), comprising administering to a patient in need thereof a compound disclosed herein, or a pharmaceutically acceptable sait thereof.

in another embodiment, the present invention is a method of treating cancer comprising administering to a patient in need thereof a compound disclosed herein, or a pharmaceutically acceptable sait thereof.

In another embodiment, the present invention is a method of treating cancer comprising administering to a patient in need thereof a compound disclosed herein, or a pharmaceutically acceptable sait thereof, and a pharmaceutically acceptable carrier.

In another embodiment, the present invention is a method of treating hemoglobinopathies such as β-thalassemia or Sickle Cell Disease (SCD), comprising administering to a patient in need thereof a compound disclosed herein, or a pharmaceutically acceptable sait thereof.

In another embodiment, the present invention is a method of treating hemoglobinopathies such as β-thalassemia or Sickle Cell Disease (SCD), comprising administering to a patient in need thereof a compound disclosed herein, or a pharmaceutically acceptable sait 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 disclosed herein, or a pharmaceutically acceptable sait thereof, and a pharmaceutically acceptable carrier.

In another embodiment, the present invention is a method of treating hemoglobinopathies such as β-thalassemia or Sickle Cell Disease (SCD), comprising administering to a patient in need thereof, a composition comprising a compound disclosed herein, or a pharmaceutically acceptable sait thereof, and a pharmaceutically acceptable carrier.

In another embodiment, the present invention is the use of a compound disclosed herein, or a pharmaceutically acceptable sait thereof, in the manufacture of a médicament for treating cancer.

In another embodiment of the present invention îs the use of a compound disclosed herein, or a pharmaceutically acceptable sait thereof, in the manufacture of a médicament for treating hemoglobinopathies such as β-thalassemia or Sickle Cell Disease (SCD).

In another embodiment, the present invention includes the use of compound disclosed herein, or a pharmaceutically acceptable sait thereof, for the préparation of a médicament for the treatment of cancer, or hemoglobinopathies such as β-thalassemia or Sickle Cell Disease (SCD).

Another embodiment îs the use of compound disclosed herein, or a pharmaceutically acceptable sait thereof, for the préparation of a médicament for the treatment of cancer. In a subembodiment, the cancer is i) cardiac cancer, ii) long cancer, iii) gastrointestinal cancer, iv) genitourinary tract cancer, v) liver cancer, vi) bone cancer, vii) nervous System cancer, vîîi) gynecological cancer, ix) hematological cancer, x) skrn cancer, or xi) adienal cancer.

Another embodiment is the use of a compound described herein, or a pharmaceutically acceptable sait thereof, for the préparation of a médicament for the treatment of hemoglobinopathîes such as β-thalassemia or Sickle Cell Disease (SCD).

In another embodiment, the present invention includes compounds disclosed herein, for use in the treatment of cancer or hemoglobinopathîes such as B-thalassemia or Sickle Cell Disease (SCD). In another embodiment, the present invention includes compounds disclosed herein, or a pharmaceutically acceptable sait thereof, 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 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 disclosed herein, or a pharmaceutically acceptable sait 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 disclosed herein, or a pharmaceutically acceptable sait thereof, and an effective amount of at least one chemotherapeutic agent.

The invention also provides any of the above methods of treating cancer wherein the cancer 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 disclosed herein, or a pharmaceutically acceptable sait 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 disclosed herein, or a pharmaceutically acceptable sait thereof, and an effective amount of at least one chemotherapeutic agent.

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 therapeutîcally effective amount of a compound of the instant invention, or a pharmaceutically acceptable sait thereof, 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 antiproliférative agent, a prenyl-protein transferase inhibitor, an HMG-CoA reductase inhibitor, an HIV protease inhibitor, a reverse transcriptase inhibitor, an angiogenesis inhibitor, PPAR-γ agonists, PPAR-ô agonists, an inhibitor of inhérent multidrug résistance, an anti-emetic agent, an agent useful in the treatment of anémia, an agent useful in the treatment of neutropenia, an immunologic-enhancing drug, an inhibitor of cell prolifération and survival signaling, a bisphosphonate, an aromatase inhibitor, an siRNA therapeutic, γ-secretase and/or NOTCH mhibitors, agents that interfère with receptor tyrosine kinases (RTKs), an agent that interfères with a cell cycle checkpoint, and any of the therapeutic agents listed herein, or a pharmaceutically acceptable sait thereof.

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

In one embodiment, the compound disclosed herein is selected from the group consisting of the compounds exemplified herein, for example, in Examples l — 5, or a pharmaceutically acceptable sait thereof.

The tenu composition is intended to encompass a product comprising the specified ingrédients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingrédients in the specified amounts. The term “anti-cancer agent” means a drug (médicament or pharmaceutically active ingrédient), or antibody for treating cancer. 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 (médicament or pharmaceutically active ingrédient) for treating cancer (i.e., an antineoplastic agent). The term “effective amount” means a “therapeutic ail y effective amount”. The term therapeutically effective amount means ihat amount of active compound or pharmaceutical agent that elicits the biological or médicinal response in a tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician. Thus, for example, în 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 réduction, alleviation or disappearance of one or more symptoms caused by the cancer, (b) the réduction of tumor size, (c) the élimination 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) îs that amount which results in the réduction 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 allevîates 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.

Methods for the safe and effective administration of most of these chemotherapeutîc 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 chemotherapeutîc agents is described in the “Physicians’ Desk Reference” (PDR), e.g., the Physicians’ Desk Référencé, 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 îs 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 dependîng upon the requirements of the patient and the severity of the condition being treated. Détermination of the proper dosage for a particular situation is within the skiH of the art. A détermination to vary the dosages and protocols for administration may be made after the skilled clinician takes into account such factors as the patient’s âge, 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 disclosed herein and the chemotherapeutîc agents will be regulated according to the judgment of the attending clinician (physician) considering such factors as âge, condition and size of the patient as well as severity of the cancer being treated,

The compounds of the invention are also usefui in preparing a médicament that is usefui in treating cancer.

The instant compounds are also usefui in combination with therapeutic, chemotherapeutîc and anti-cancer agents. Combinations of the presently disclosed compounds with therapeutic, chemotherapeutîc 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^lh édition (May 16, 20ll), Lippincott Williams & Wilkins Publishers. A person of ordinary skill in the art would be able to discern which combinations of agents would be usefui based on the partîcular characteristics of the drugs and the cancer involved. Such agents include the following: estrogen receptor modulators, prograimned cell death protein l (PD-l) inhibitors, programmed death-ligand l (PD-Ll) inhibitors, androgcn receptor modulators, retinoid receptor modulators, cytotoxic/cytostatic agents, antiproliférative agents, prenyl-protein transferase inhibitors, HMG-CoA reductase inhibitors and other angiogenesis inhibitors, HIV protease inhibitors, reverse transcriptase inhibitors, inhibitors of cell prolifération and survival signaling, bisphosphonates, aromatase inhibitors, siRNA therapeutics, γ-secretase inhibitors, agents that interfère with receptor tyrosine kinases (RTK.s) and agents that interfère with cell cycle checkpoints. The instant compounds are particularly useful when co-administered with radiation therapy.

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 chcmotherapeutic agent can be varîed 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 modifîed by the skilled clinician.

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

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

Thus, in accordance with expérience 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. Ail such modifications are within the scope of the present invention.

The anti-cancer 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 anti-cancer agent can be varied depending on the cancer being treated and the known effects of the anti-cancer 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

H 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 pro toc ois 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 clinicîan.

The particular choice of anti-cancer agent will dépend upon the diagnosis of the attending physicians and their judgement of the condition of the patient and the appropriate treatment protocol.

The détermination of the order of administration, and the number of répétitions of administration of the anti-cancer agent during a treatment protocol, is well within the knowledge of the skilled physician after évaluation of the cancer being treated and the condition of the patient.

Thus, in accordance with expérience and knowledge, the practicing physician can modify each protocol for the administration of an anti-cancer agent according to the individu al patient’s needs, as the treatment proceeds. AU such modifications are within the scope of the present invention.

The attending clinician, in judging whether treatment is effective at the dosage administered, will consi der the general well-being of the patient as well as more defini te 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 diseaserelated 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, fïbrosarcoma, rhabdomyosarcoma, lip os arc orna), myxoma, rhabdomyoma, fibroma, lipoma and teratoma; (2) Lung: bronchogenîc 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), pancréas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofïbroma, fïbroma), 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, hemangiorna; (6) Bone: ostéogénie sarcoma (osteos arc orna), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (réticulum 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, xantboma, osteitis deformans), méningés (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congénital tumors), spinal cord neuroftbroma, meningioma, glioma, sarcoma); (8) Gynécologie al: utérus (endométrial carcinoma), cervix (cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadcnocarcinoma, unclassifted carcinoma], granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithélial carcinoma, adenocarcinoma, fïbro sarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdoinyos arc orna), fallopian tubes (carcinoma), breast; (9) Hématologie: blood (myeloid leukemia [acute and chronic], acute lymphoblastîc leukemia, chronic lymphocytic leukemia, chronic myelomonocytic (CMML), myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkiris 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: ncuroblastoma. 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 aboveidentified conditions.

In the treatment of breast cancer (e.g., postmenopausal and premenopausal breast cancer, e.g., hormone-dépendent breast cancer) the compounds disclosed herein 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 inciude but are not limited to: Tamoxifen (e.g., Nolvadex), Fulvestrant (e.g., Faslodex), Raloxifene (e.g., Evîsta), and Acolbifene. Examples of LHRH analogues inciude 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 inciude 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).

“Estrogen receptor modulât ors” refers to compounds that interfère with or inhibit the binding of estrogen to the receptor, regardless of mechanism. Examples of estrogen receptor modulators inciude, but are not limited to, tamoxifen, raloxifene, idoxifene, LY353381, LYH7081, toremifene, fulvestrant, 4-[7 -(2,2-dimethyl-1 -oxopropoxy-4-methyl-2-[4-[2-( l piperidinyl)ethoxy]phenyl]-2H-l-benzopyran-3-yl]-phenyl-2,2-dimethylpropanoate, 4,4’dihydroxybenzophenone-2,4-dmîtrophenyl-hydrazone, and SH646.

PD-l inhibitors inciude pembrolizumab (lambrolizumab), nivolumab and MPDL328ÛA. PDL- inhibitors inciude atezolizumab, avelumab, and durvalumab^ “Androgen receptor modulators” refers to compounds which interfère or inhibit the binding of androgens to the receptor, regardless of mechanism. Examples of androgen receptor modulators inciude finasteride and other 5a-reductase inhibitors, niiutamide, fltitamidc, bicalutamide, lîarozole, and abiraterone acetate.

“Retinoid receptor modulators” refers to compounds which interfère or inhibit the binding of retinoid s to the receptor, regardless of mechanism. Ex amp le s of such retinoid receptor modulators inciude bexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoic acid, adifluoromethylomithine, 1LX23-7553, trans-N-(4’-hydroxyphenyl) retinamide, and N-4carboxyphenyl retinamide.

“Cytotoxic/cytostatic agents” refers to compounds which cause cell death or inhibit cell prolifération primarily by interfering dîrectly with the cell’s functioning or inhibit or interfère with cell myosis, including alkylating agents, tumor necrosis factors, intercalators, hypoxîa activatablc compounds, microtubule inhibitors/miciOtubule-stabilizing agents, inhibitors of mitotic kînesins, 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 modifîers, 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 inciude, but are not limited to, sertenef, cachectin, ifosfamide, tasonermin, lonidamîne, carboplatin, altretamine, prednimustine, dibromodulcitol, ranimustine, fotemustine, nedaplatin, oxaliplatin, temozolomide, heptaplatin, estramustine, improsulfan tosilate, trofosfamide, nimustine, dibrospidium chloride, pumitepa, lobapiatin, satraplatin, profiromycin, cisplatin, irofulven, dexifosfamide, cis-aminedîchloro(2-mcthylpyridîne)platinum, benzylguanine, glufosfamide, GPX100, (trans, trans, trans)-bis-mu-(hexanel,6-diamine)-mu-[diamine-platinum(ll)]bis[diamïne(chioro)platinum (II)]tetrachloride, diarizidinylspermine, arsenic trioxide, 1-( 1 l-dodecy]amino-l O-hydroxyundecy 1)-3,7 dimethylxanthine, zorubicin, idarubicin, daunorubicin, bisantrene, mitoxantrone, pirarubicin, pînafide, valrubicin, amrubicin, antineoplaston, 3’-deamino-3 ’-morpholino-13-deoxo-10hydroxycarminomycin, annamycin, galarubicin, elinafide, MEN10755, 4-demethoxy-3-deammo3-aziridinyI-4-methylsulphonyf 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, RPR 109881, 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 topoîsomerase inhibitors are topotecan, hyeaptamine, irinotecan, rubitecan, 6-ethoxypropionyl-3’,4’-0-exo-benzylidene-chartreusin, 9-methoxy-N,N-dimethyl-5nitropyrazolo[3,4,5-kl]acridine-2-(6H) propanamine, l-ammo-9-ethyl-5-fluoro-2,3-dihydro-9hy droxy-4-methyl-1 H, 12H-benzo[de]pyrano [3 ’ ,4’ : b,7]-indolizinof 1,2b]quinoline10,13(9H,15H)dione, lurtotecan, 7-[2-(N-isopropylamino)ethyl]-(20S)camptothecin, BNP1350, BNPI1100, BN80915, BN80942, etoposide phosphate, teniposide, sobuzoxane, 2’-dimethylamino2’-deoxy-etoposide, GL331, N-[2-(dimethylaminü)ethyl]-9-hydroxy-5,6-dimethyl-6H-pyrido[4,3b]carbazole-l-carboxamide, asulacrine, (5a, 5aB, 8aa,9b)-9-[2-[N-[2-(dimethylamino)ethyl]-Nmethy lamino]ethy 1]-5 “[4-hydro0xy-3,5-dimethoxypheny 1J-5,5a,6,8,8a,9hexohydrofuro(3’,4’:6,7)naphtho(2,3-d)-l,3-dioxol-6-one, 2,3-(methylenedioxy)-5-methyl-7hydroxy-8-methoxybenzo[c]-phenanthridmium, 6,9-bîs[(2aminoethyl)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)ethylammo]-7-inethoxy-9-oxo-9H-thioxanÎhen-4-ylmethyl]formamide, N-(2(dimethylamîno)ethyl)acridine-4-carboxamide, 6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy7H-indeno[2,l-c] quinolin-7-one, and dimesna.

Examples of inhibitors of mitotic kinesins, and in particular the human mitotic kîncsin KSP, are described in Publications WO03/039460, WO03/050064, WO03/050122, WO03/049527, WO03/049679, WO03/049678, WO04/039774, WO03/079973, WO03/0992U, WO03/105855, WO03/106417, WO04/037171, WO04/058148, WO04/058700, WO04/126699, WO05/018638, WOÛ5/019206, 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 MKLPl, 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-lîke kinases (PLK; in particular inhibitors of PLK-1), inhibitors of bub-1 and inhibitors of bub-Rl. An example of an “aurora kmase inhibitor” is VX680 (tozasertib).

“Antiproliférative agents” include antisense RNA and DNA oligonucleotides such as G3139, ODN698, GEM23Î, and INX3Û01, 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-dibydro-benzofuryl)sulfonyl]-N’-(3,4-dichlorophenyl)urea, N6-[4deoxy-4-[N2-[2(E),4(E)-tetradecadienoyl]glycylamino]-L-glycero-B-L-mannoheptopyranosyljadenine, aplidine, ecteinascidin, troxacitabine, 4-[2-amino-4-oxo-4,6,7,8teÎrahydro-3H-pyrimidino[5,4-b][ 1,4]thiazin-6-yl-(S)-ethyl]-2,5-thienoyl-L-glutamic acid, aminopterin, 5-flurouracil, alanosine, ll-acetyl-8-(carbamoyloxymethyl)-4-formyl-6-methoxy-14oxa-1,1 l-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-l-B-D-arabino furanosyl cytosine, 3-aminopyridinc-2-carboxaldehyde thiosemicarbazone and trastuzumab.

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

“HMG-CoA reductase inhibitor” 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) 5 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, Cholestérol Lowering Drugs, Chemistry Æ 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 ail pharmaceutically acceptable lactone and 10 open-acid forms (i.e., where the lactone ring is opened to form the free acid) as well as sait and ester forms of compounds which hâve HMG-CoA reductase inhibitory activity, and therefore the use of such salts, esters, open-acid and lactone forms is încluded 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 famesyl-protein transferase 15 (FPTase), geranylgeranyl-protein transferase type I (GGPTase-1), and geranylgeranyl-protein transferase type-II (GGPTase-Π, also called Rab GGPTase). For an example of the rôle of a prenyl-protein transferase inhibitor on angiogenesis see European J. of Cancer, Vol. 35, No. 9, pp.1394-1401 (1999).

“Angiogenesis inhibitor” refers to compounds that inhibât the formation of new blood 20 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-l/KDR (VEGFR2), inhibitors of epidermal-derived, fibroblast-derived, or platelet derived growth factors, MMP (matrix métalloprotéase) inhibitors, integrin blockers, interferon-a, interleukin-12, pentosan polysulfate, cyclooxygênase inhibitors, including nonsteroîdal anti25 inflammatories (NSAIDs) like aspirin and ibuprofen as well as sélective cyclooxy-genase-2 inhibitors like celecoxib and rofocoxib (PNAS, Vol. 89, p. 7384 (1992); JNC1, Vol. 69, p. 475 (1982); Arch. Opthalmol., Vol. 108, p.573 (1990); Anat. Rec., Vol. 238, p. 68 FEBSLetters, 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, minerafocorticoids, dexaméthasone, prednisone, prednisolone, methylpred, betamethasone), carboxyamidotriazole, combretastatin A-4, squalamine, 6-O-chloroacetyl-carbonyl)-fumagilfol, thalidomide, angiostatin, troponin-1, angiotensin II antagoniste (see Fernandez et al., J. Lab. Clin. Med. 105:141-145 (1985)), and antibodies to VEGF (see, Nature Biatechnology, 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 angiogenesîs and may also be used in combination with the compounds of the instant invention include agents that modulate or inhibit the coagulation and fïbrinolysis Systems (see review in Clin. Chem. La. Med. 38:679-692 (2000)). Examples of such agents that modulate or inhibit the coagulation and fïbrinolysis 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 fïbrinolysis inhibitor [TAFIa]) (see Thrombosis Res. 101:329-354 (2001)). TAFla inhibitors hâve been described in U.S. Ser. Nos. 60/310,927 (filed August 8, 2001) and 60/349,925 (fded January 18, 2002).

“Agents that interfère with cell cycle checkpoints” refer to compounds that inhibit protein kinases that transduce cell cycle checkpoint signais, 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 specifïcally exemplîfied by 7-hydroxystaurosporin, flavopiridol, CYC202 (Cyclacel) and BMS-387032.

“Agents that interféré with receptor tyrosine kinases (RTKs)” refer to compounds that inhibit RTKs and therefore mechamsms 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 Buine-Jensen and Hunter, Nature, 411:355-365, 2001.

“Inhibitors of cell prolifération and survival signalling pathway” refers 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 7,454,431, US 7,589,068), inhibitors of Raf kinase (for example BAY-43-9006 ), inhibitors of MEK (for example CI-1040 and PD098059), inhibitors of mTOR (for example Wyeth CCI-779), and inhibitors of PI3K (for example LY294002).

As described above, the combinations with NSAIDs are directed to the use of NSAIDs which are potent COX-2 inhibiting agents. For purposes of the spécification an NSA1D is potent if it possesses an ICso for the inhibition of COX-2 of ΙμΜ or less as measured by cell or microsomal assay s.

The invention also encompasses combinations with NSAIDs which are sélective COX-2 inhibitors. For purposes of the spécification NSAIDs which are sélective inhibitors of COX-2 are defined as those which possess a specificity for inhibiting COX-2 over COX-l of at least 100 fold as measured by the ratio of IC5Q 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, ail of which are hereby incorporated by reference.

Inhibitors of COX-2 that are particularly usefui in the instant method of treatment are: 310 phenyl-4-(4-(methylsulfonyl)phenyl)-2-(577)-furanone; and 5-chloro-3-(4-methylsulfonyl)-phcnyl2-(2-methyl-5-pyridinyl)pyridine; or a pharmaceutical ly acceptable sait thereof.

Compounds that hâve been described as spécifie inhibitors of COX-2 and are therefore usefui in the présent invention include, but are not limited to, the following: rofecoxib, etoricoxib, parecoxib, BEXTRA® and CELEBREX® or a phannaceutically acceptable sait thereof.

Other examples of angiogenesis inhibitors include, but are not limited to, endostatin, ukrain, ranpirnase, 1M862, 5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]-loxaspiro[2,5]oct-6-yl(chloroacetyl)carbamate, acetyldinanaline, 5-amino-l-[[3,5-dichloro-4-(4chlorobenzoyl)phenyl]methyl]-1 H-1,2,3-triazole-4-carboxamîde,CM 101, squalamine, combretastatin, RPI4610, NX31838, sulfated mannopentaose phosphate, 7,7-(carbonyl-bis[imino20 N-methyl-4,2-pyrrolocarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino]-bis-(l,3-naphtbalene disulfonate), and 3-[(2,4-dimethylpyiTol-5-yl)methylene]-2-indolinoiie (SU5416), or a phannaceutically acceptable sait thereof.

As used above, “integrin blockers” refers to compounds which selectively antagonize, inhibit or counteract binding of a physiological ligand to the α_νβ3 integrin, to compounds which 25 selectively antagonize, inhibit or counteract binding of a physiological ligand to the ανβ5 integrin, to compounds which antagonize, inhibit or counteract binding of a physiological ligand to both the (X_vp3 integrin and the a_vp5 integrin, and to compounds which antagonize, inhibit or counteract the activity of the particular integrin(s) expressed on capillary endothélial cells. The term also refers to antagonists of the oc_vp6> «vP8- ^α1βυ ^a2pb ^α5β] > &6pi and «6p4 integrins. The term also refers to antagonists of any combination of α_νβ3, ^ανΡό· ct_vp6> ^avp8> ^alPb ^α2β1> ^a5pb «όβ 1 ^and ^K6p4 integrins.

Some spécifie examples of tyrosine kinase inhibitors include N-(trifluoiOmethylphenyl)-5methylisoxazol-4-carboxamide, 3 - [ (2,4-d imethy Ipy n ol - 5 - y l)me th y ltd eny 1 ) indo 1 i n- 2 -o ne, 17(allylamino)-17-demethoxygeldanamycin,4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-[3-(43 5 morpb 01 îny 1 )propoxy 1] quinazo li ne, N - ( 3 -eth y ny Iphe ny 1) - 6,7 - bis ( 2 -me thoxy ethoxy ) -4 qumazol inami ne, B1BX1382, 2,3,9,10,11,12-hexahydro- 10-(hydroxymethyl)-10-hydroxy-9methy 1-9,12-epoxy-1 H-diindolo[ 1,2,3-fg: 3 ’ ,2 ’, Γ -kl]py rrolo [3,4-i] [ 1,6] benzodiazocin-1 -one, SH268, genistein, ST1571, CEP2563, 4-(3-chlorophenylamino)-5,6-dimethyl-7H-pyrrolo[2,3d] py ri m idmemeth ane sul fon ate, 4 - ( 3-brom o -4-hy droxyphe nyl)amino-6,7-d imetho xyquinazoline, 4-(4’-hydroxyphenyl)amino-6,7-dîmethoxyquinazoline, SU6668, STI571A, N-4-chlorophenyl-4(4-pyridylmethyl)-l-phthalazinamine, and EMD121974, or a pharmaceutically acceptable sait thereof.

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-γ (i.e., PPAR-gamma) agonists and PPAR-Ô (i.e., PPAR-delta) agonists are useful in the treatment of certain malignancies. PPAR-γ and PPAR-δ are the nuclear peroxisome proliferator-activated receptors γ and δ. The expression of PPAR-γ on endothélial 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-γ agonists hâve been shown to inhibit the angiogenic response to VEGF in vitro; both troglitazone and rosiglitazone maleate inhibit the development of retînal neovascularizatîon in mice (Arch. Ophthamol. 2001; 119:709-717). Examples of PPAR-γ agonists and PPAR- γ/α agonists include, but are not limited to, thiazolidinediones (such as DRF2725, CS-011, troglitazone, rosiglitazone, and pioglitazone), fenofibrate, gemfibrozil, clofibrate, GW2570, SB219994, ARH039242, JTT-501, MCC-555, GW2331, GW409544, NN2344, KRP297, NP0110, DRF4158, NN622, GI262570, PNU182716, DRF552926, 2-[(5,7-dipropyl-3-trifluoromethyl-l,2benzîsoxazol-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), or a pharmaceutically acceptable sait thereof.

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 Dissémination 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 inhérent multidrug résistance (MDR), in partîcular MDR associated with high levels of expression of transporter proteins. Such MDR inhibitors include inhibitors of p-glycoproteîn (Pgp), such as LY335979, XR9576, OC144-093, R101922, VX853 and PSC833 (valspodar), or a pharmaceutically acceptable sait thereof.

A compound of the présent invention may be employed in conjunction with anti-emetic 5 agents to treat nausea or emesis, including acute, deiayed, late-phase, and anticipatory emesis, which may resuit from the use of a compound of the présent invention, alone or with radiation therapy. For the prévention or treatment of emesis, a compound of the présent invention may be used in conjunction with other anti-emetic agents, especially neurokînin-1 receptor antagonists, 5FIT3 receptor antagonists, such as ondansetron, granisetron, tropisetron, and zatisetron, G AB AB 10 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 prévention of emesis that may resuit upon administration of the instant compounds.

A compound of the instant invention, or a pharmaceutically acceptable sait thereof, may also be admînistered with an agent useful in the treatment of anémia. Such an anémia treatment 20 agent îs, for example, a continuous erythropoiesis receptor activator (such as epoetin alfa).

A compound of the instant invention, or a pharmaceutically acceptable sait thereof, may also be admînistered with an agent useful in the treatment of neutropenia. Such a neutropenia treatment agent is, for example, a hematopoietic growth factor which régulâtes the production and function of neutrophile such as a human granulocyte colony stimulating factor, (G-CSF). Examples 25 of a G-CSF include filgrastim.

A compound of the instant invention, or a pharmaceutically acceptable sait thereof, may also be admînistered with an immunologic-enhancing drug, such as levamisole, isoprinosîne and Zadaxin, or a pharmaceutically acceptable sait thereof.

A compound of the instant invention, or a pharmaceutically acceptable sait thereof, may 30 also be useful for treating or preventing cancer in combination with P450 inhibitors including: xenobiotics, quinidine, tyramine, kétoconazole, testosterone, quinine, methyrapone, caffeîne, phenelzine, doxorubicin, troleandomycin, cyclobenzaprine, erythromycîn, cocaine, furafyline, cimetidine, dextromethorphan, ritonavir, indinavir, amprenavir, diltiazem, terfcnadine, verapamil, cortisol, itraconazole, mibefradil, nefazodone and nelfinavir, or a pharmaceutically acceptable sait 35 thereof.

A compound of the instant invention, or a pharmaceutically acceptable sait thereof, 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, Kol32, Kol34, Iressa, Imatnib mesylate, EKI-785, Cl 1033, novobiocin, diethylstilbestrol, tamoxifen, respeipine, VX-710, tryprostatin A, flavonoids, ritonavir, saquînavir, nelfinavir, omeprazole, quinidine, verapamil, terfenadine, kétoconazole, nifidepine, FK5O6, amiodarone, XR9576, indinavir, ainprenavir, cortisol, testosterone, LY335979, OC 144-093, erythromycin, vincristine, digoxin and talinolol, or a pharmaceutically acceptable sait thereof.

A compound of the instant invention, or a pharmaceutically acceptable sait thereof, 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: étidronate (Drdronel), pamidronate (Aredia), alendronate (Fosamax), risedronate (Actonel), zolédronate (Zometa), ibandronate (Boniva), incadronate or cimadronate, clodronate, EB-1053, minodronate. neridronate, piridronate and tiludronate including any and ail pharmaceutically acceptable salts, dérivatives, hydrates and mixtures thereof.

A compound of the instant invention, or a pharmaceutically acceptable sait thereof, 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, or a pharmaceutically acceptable sait thereof.

A compound of the instant invention, or a pharmaceutically acceptable sait thereof, 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 ysecretase 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), or a pharmaceutically acceptable sait thereof.

A compound of the instant invention, or a pharmaceutically acceptable sait thereof, may also be useful for treating or preventing cancer in combination with PA RP inhibitors.

A compound of the instant invention, or a pharmaceutically acceptable sait thereof, 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®); carbop latin (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®); daunorubicin, daunomycin (Daunorubicin®); daunorubicin, daunomycin (Cerubidine®); Denileukin 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®); epirnbicin (Ellence®); Epoetin alfa (epogen®); erlotinib (Tarceva®); estramustine (Emcyt®); etoposide phosphate (Etopophos®); etoposide, VP-16 (Vepesid®); exemestane (Aromasin®); Fîlgrastim (Neupogen®); iloxuridîne (intraarterial) (FUDR®); fludarabine (Fludara®); fluorouracil, 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®); idarubîcin (Idamycin®); ifosfamide (1FEX®); 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®); melphaian, L-PAM (Alkeran®); mercaptopurine, 6-MP (Purinethol®); mesna (Mesnex®); mesna (Mesnex tabs®); methotrexate (Methotrexate®); methoxsalen (Uvadex®); mitomycin C (Mutamycin®); mîtotane (Lysodren®); mitoxantrone (Novantrone®); nandrolone phenpropionate (Durabolin-50®); nelarabine (Arranon®); Nofetumomab (Verluma®); Oprelvekin (Nemnega®); oxaliplatin (Eloxatin®); paclitaxel (Paxene®); paclitaxel (Taxol®); paclitaxel protein-bound particles (Abraxane®); palîfermîn (Kepivance®); pamidronate (Aredia®); pegademase (Adagen (Pegademase Bovine)®); pegaspargase (Oncaspar®); Pegfïlgrastim (Neulasta®); pemetrexed disodium (Alimta®); pentostatin (Nipent®); pipobroman (Vercyte®); plicamycin, mithramycin (Mithracin®); porfimer sodium (Photofrin®); procarbazine (Matulane®); quinacrine (Atabrine®); Rasburicase (Elitek®); Rituximab (Rîtuxan®); Rîdaforolimus; sargramostim (Leukine®); Sargramostim (Prokine®); sorafenib (Nexavar®); streptozocin (Zanosar®); sunitinib maleate (Sutent®); talc (Sclerosol®); tamoxifcn (Nolvadex®); temozolomide (Temodar®); teniposide, VM-26 (Vumon®); testolactone (Teslac®); thioguanine, 6-TG (Thioguanine®); thiotepa (Thioplex®); topotecan (Hycamtin®); toremifene (Fareston®); Tosîtumomab (Bexxar®); Tositumomab/1-131 tositumomab (Bexxai®); Trastuzumab (Herceptin®); tretinoin, ATRA (Vesanoid®); Uracil Mustard (Uracil Mustard Capsules®); valrubicin (Valstar®); vînblastine (Velban®); vincristine (Oncovin®); vinorelbine (Navelbine®); vorinostat (Zolinza®) and zolédronate (Zometa®), or a pharmaceutically acceptable sait thereof.

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 integiin blocker, interferon-a, interleukin-12, pentosan polysulfate, a cyclooxygenase inhibitor, carboxyamidotriazole, combretastatin A-4, squalamine, 6-0chloroacetyl-carbonyl)-fumagillol, thalidomide, angiostatin, troponin-1, or an antibody to VEGF. In an example, the estrogen receptor modulator is tamoxifen or raloxifene, or a pharmaceutically acceptable sait thereof.

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 antiproliférative agent, a prenyl-protein transferase inhibitor, an HMG-CoA reductase inhibitor, an HIV protease inhibitor, a reverse transcriptase inhibitor, an angiogenesis inhibitor, PPAR-γ agonists, PPAR-δ agonists, an inhibitor of inhérent multidrug résistance, an anti-emetic agent, an agent useful in the treatment of anémia, an agent useful in the treatment of neutropenia, an immunologic-enhancing drug, an inhibitor of cell prolifération and survival signalîng, a bisphosphonate, an aromatase inhibitor, an siRNA therapeutic, γ-sécrétase and/or NOTCH inhibitors, agents that interfère with receptor tyrosine kinases (RTKs), an agent that interfères with a cell cycle checkpoint, and any of the therapeutic agents listed above.

Also included in the scope of the daims îs a method of treating cancer that comprises administering a therapeutically effective amount of a compound of the instant invention, or a pharmaceutîcally acceptable sait thereof, 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 antiproliférative agent, a prenyl-protein transferase inhibitor, an HMG-CoA reductase inhibitor, an HIV protease inhibitor, a reverse transcriptase inhibitor, an angiogenesis inhibitor, PPAR-γ agonists, PPAR-δ agonists, an inhibitor of inhérent multidrug résistance, an anti-emetic agent, an agent use fui in the treatment of anémia, an agent useful in the treatment of neutropenia, an immunologic-enhancing drug, an inhibitor of cell prolifération and survival signalîng, a bisphosphonate, an aromatase inhibitor, an siRNA therapeutic, γ-secretase and/or NOTCH inhibitors, agents that interfère with receptor tyrosine kinases (RTKs), an agent that interfères with a cell cycle chcckpoint, 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, or a pharmaceutîcally acceptable sait thereof, 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, or a pharmaceutîcally acceptable sait thereof, in combination with a COX-2 inhibitor, or a pharmaceutîcally acceptable sait thereof.

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 prévention, treatment, control, amelioration, or réduction of risk of a particular disease or condition (e.g., cell-prolifération disorders). In one embodiment, a compound disclosed herein is combined with one or more other anti-cancer agents for use in the prévention, treatment, control amelioration, or réduction 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 sequentîally with a compound of the présent 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, or a pharmaceutîcally acceptable sait thereof, and a second compound selected from: an estrogen receptor modulator, an androgen receptor modulator, a retinoid receptor modulator, a cytotoxic/cytostatic agent, an antiproliférative agent, a prenyl-protein transferase inhibitor, an HMG-CoA reductase inhibitor, an HIV protease inhibitor, a reverse transcriptase inhibitor, an angiogenesis inhibitor, a PPAR-γ agonist, a PPAR-δ agonîst, an inhibitor of cell prolifération and survival signaling, a bisphosphonate, an aromatase inhibitor, an siRNA therapeutic, γ-secretase and/or NOTCH inhibitors, agents that interféré with receptor tyrosine kinases (RTKs), an agent that interfères with a cell cycle checkpoint, and any of the therapeutic agents listed above.

The present invention includes compounds disclosed herein, as well as the pharmaceutically acceptable salts, and also salts that are not pharmaceutically acceptable when they are used as precursors to the free compounds or theîr pharmaceutically acceptable salts or in other synthetic manipulations.

The compounds of the present invention may be administered in the form of a pharmaceutically acceptable sait. The term pharmaceutically acceptable sait 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 sait 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. Représentative salts of basic compounds of the present invention inciude, but are not limited to, the following: acetate, ascorbate, adipate, alginate, aspîrate, benzenesulfonatc, 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, glycérophosphate, glycollylarsanilate, hemisulfate, heptanoate, hexanoate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, 2hydrox y ethanesulfonate, hydroxynaphthoate, iodide, isonicotinic, isothionate, lactate, lactobîonate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, methanesulfonate, mucate, 2-naphthalenesulfonate, napsylate, nicotinate, nitrate, Nmethylglucamine ammonium sait, oleate, oxalate, pamoate (embonate), palmitate, pantothenate, pectinate, persulfate, phosphate/diphosphate, pimelic, phenylproptontc, polygalacturonate, propionate, salicylate, stéarate, 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 phannaceutically acceptable salts thereof inciude, but are not limited to, salts derived from inorganic bases including aluminum, ammonium, calcium, copper, ferrie, ferrous, lithium, magnésium, manganic, mangamous, potassium, sodium, zinc, and the like. Particularly preferred are the ammonium, calcium, magnésium, potassium, and sodium salts.

With basic reagents such as hydroxides, carbonates, hydrogencarbonates, alkoxides and ammonia, organic bases or altematively basic amino acids the compounds disclosed herein form stable alkali métal, alkaline earth métal or optionally substituted ammonium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, cyclîc amines, dicyclohexyl amines and basic ion-exchange resins, such as arginine, betaine, caffcine, choline, Ν,Ν-dibenzylethylenediamine, di ethanolamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylamine, ethylenediamine, Nethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropy lamine, 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 nitrogencontaining groups may be quatemized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides and iodidcs; dialkyl sulfates like dimethyl, di ethyl, dibutyl; and diamyl sulfates, long chain halides such as decyl, lauryl, myristyi and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides and others.

The préparation of pharmacologically acceptable salts from compounds disclosed herein capable of sait formation, including their stereoisomeric forms is carried out known methods, for ex ample, by mixing a compound of the present invention with an équivalent amount and a solution containing a desired acid, base, or the like, and then collecting the desired sait by filtering the sait or distilling off the solvent. The compounds of the present invention and salts thereof may form solvatés with a solvent such as water, éthanol, or glycerol. The compounds of the present invention may form an acid addition sait and a sait with a base at the same time according to the type of substituent of the side chain.

The present invention encompasses ail stereoisomeric forms of the compounds disclosed herein. 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 compounds. Similarly, when a compound name is récited without a chiral désignation 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. Absolute stereochemistry may be determined by Xray 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, ail individual tautomers as well as mixtures thereof are included in the scope of the invention. The present invention includes ail such isomers, as well as salts, solvatés (including hydrates) and solvated salts of such isomers 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 partîcular isotope having the same atomtc 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 ail suitable isotopic variations of the specifically and generically described compounds. For example, different isotopic forms of hydrogen (H) include protium (^H) and deuterium (^H). Protium is the prédominant 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 usefui as a standard for characterization of biological samples. Isotopically-enriched compounds can be prepared without undue expérimentation 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.

Furthennore, compounds of the present invention may exist in amorphous form and/or one or more crystalline forms, and as such ail amorphous and crystalline forms and mixtures thereof of the compounds disclosed herein are intended to be included within the scopc of the present invention. In addition, some of the compounds of the instant invention may form solvatés with water (i.e., a hydrate) or common organic solvents. Such solvatés and hydrates, particularly the pharmaceutically acceptable solvatés 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 disclosed herein as well as salts thereof, particularly pharmaceutically acceptable salts, solvatés of such compounds and solvated sait forms thereof, where such forms are possible unless specifted otherwise.

Commonly used abbreviations for aîkyl groups are used throughout the spécification, e.g. methyl may be represented by conventional abbreviations including “Me” or CHj 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. “Cm alkyl” (or “C1-C4 alkyl”) for example, means linear or branched Chain alkyl groups, including ail isomers, having the specifîed number of carbon atoms. For example, the structures mm ,-CH₃ m

HN and 'k'' hâve équivalent meanings. Cm alkyl includes n-, iso-, sec- and t-butyl, n- and isopropyl, ethyl and methyl. If no number is specifîed, 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 dérivatives, such as methyl, ethyl, or pivaloyloxy methyl, or acyl dérivatives of alcohols, such as O-acetyl, O-pivaloyl, O-benzoyl, and Oaminoacyl, 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 orprodrug formulations.

If the compounds disclosed herein simultaneously contain acidic and basic groups in the molécule the invention also includes, in addition to the sait forms mentioned, inncr salts or betaines (zwi Itérions). Salts can be obtained from the compounds disclosed herein 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 ail salts of the compounds disclosed herein which, owing to low physiologtcal compatibility, are not directly suitable for use in phannaceuticals but which can be used, for example, as intermediates for Chemical reactions or for the préparation of physiologicaliy acceptable salts.

The invention also includes dérivatives of the compounds disclosed herein, acting as prodrugs and solvatés. 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 compounds disclosed herein. Such prodrugs include those that demonstrate enhanced bioavailability, tissue specifïcity, and/or cellular delivery, to improve drug absorption of the compounds disclosed herein. The effect of such prodrugs may resuit from modification of physicochemical properties such as lipophilicity, molecular weight, charge, and other physicochemical properties that détermine the perméation properties of the drug. For example, esters can optionally be made by estérification of an available carboxylic acid group or by formation of an ester on an available hydroxy group in a compound. Similarly, labile ami de s 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 back 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, -Ci-salkyl esters and -Ci.salkyl substituted with phenyl esters.

When any variable occurs more than one time in any constituent or in the schemes disclosed herein, its définition on each occurrence is independent of its définition at every other occurrence. Also, combinations of substituents and/or variables are permissible only if such combinations resuit in stable compounds.

Except where noted, the term halogen means fluorine, chlonne, biomine or lodme.

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

the variables are defïned 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 définition will show “N” and will not dcpict the bonds associated with it, e.g., will not show “=N-“. Likewise, when X is a carbon atom that is substîtuted with bromide, the définition will show “C-Br” and will not depict the bonds associated with it, e.g., will not show

C-Br

II

The invention also relates to médicaments containing at least one compound of those disclosed herein and/or of a pharmaceutically acceptable sait of the compound and an optionally stereoisomeric form of the compound or a pharmaceutically acceptable sait of the stereoisomeric form of the compound, together with a pharmaceutically acceptable vehicle, carrier, additive and/or other active substances and auxiliaries.

The médicaments according to the invention can be administered by oral, inhalative, rectal or transdermal administration or by subeutaneous, intraarticular, intraperitoneal or intravenous injection. Oral administration ts preferred. Coating of stents with compounds disclosed herein and other surfaces which corne into contact with blood in the body is possible.

The invention also relates to a process for the production of a médicament, which comprises brînging at least one compound disclosed herein into a suitable administration form using a pharmaceutically acceptable carrier and optionally further suitable active substances, additives or auxiliaries.

Suitable solid or galenical préparation forms are, for example, granules, powders, coated tablets, tablets, (micro)capsules, suppositories, syrups, juices, suspensions, émulsions, drops or injectable solutions and préparations having prolonged release of active substance, in whose préparation 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 magnésium carbonate, titanium dioxide, lactose, mannitoi and other sugars, talc, lactose, gelât in, starch, cellulose and its dérivatives, animal and plant oils such as cod liver oil, sunflower, peanut or sesame oil, polyethylene glycol and solvents such as, for example, stérile 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, âge, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the rénal and hepatic fonction of the patient; and the particular compound or sait thereof employed. An ordînarily skilled physician or veterinarian can readily détermine and prescribe the effective amount of the drug required to prevent, counter, or arrest the progrès s 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 ingrédients are on free base basis). For example, an 80 kg patient wouïd 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 médicament 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 dîvîded doses of two, three, or four times daily. For administration twîce a day, a suitably prepared médicament would contain between 0.4 mg and 4 g, preferably between l 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 ingrédient in quantifies 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 quantifies may be administered in a number of suitable ways, e.g. large volumes of low concentrations of active ingrédient during one extended period of time or several times a day, low volumes of high concentrations of active ingrédient during a short period of time, e.g. once a day. Typîcally, a conventional intravenous formulation may be prepared which contaîns a concentration of active ingrédient 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, reccîving 8 ml twîce a day of an intravenous formulation having a concentration of active ingrédient of 0.5 mg/ml, receives 8 mg of active ingrédient per day. Glucuronic acid, L-lactic acid, acetic acid, citric acid or any pharmaceutîcally 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 lîtcrature or exemplified in the experimental procedures. The illustrative Reaction Schemes below, therefore, are not limited by the compounds listed or by any partîcular substîtuents employed for illustrative purposes.

Methods for Making the Compounds of Présent Invention

General Methods

The compounds of the présent 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 présent invention is not limited to the production processes described below. The invention also includes processes for the préparation of compounds of the invention.

It should be noted that, when a compound disclosed herein 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 subséquent 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, R, 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 présent invention is not limited in scope by the spécifie embodiments disclosed in the examples which are intended as illustrations of a few aspects of the invention and any embodiments that are functionally équivalent aie within the scope of this invention. Indced, 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.

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

Starting materials used were either available from commercial sources or prepared according to lîterature procedures and had experimental data in accordance with those reported.

Abbreviations used aie those conventional in the art of the following.

ACN	acetonitrile
AcOH	acetic acid
Ar	Aryl
Aq. BSA	Aqueous bovine sérum albumin
Boc	tert-Butyloxycarbonyl protecting group
BrettPhos G3	[(2-Di-cyclohexylphosphmo-3,6-dimethoxy-2',4',6'- triisopropyi-l,l'-biphenylF2(2'-amino-l,l' -biphenyl)Jpalladium(II) methanesulfonate methanesulfonate
C	degree Celsius
CDCb	deuterated chloroform
CDjOD	deuterated methanol
CHCb	chloroform
CS2CO3	césium carbonate
CO	carbon monoxide
DCM	dichloromethane
DIEA	N, N -d i i s op ropy lethy lami ne
DMA	N,N-dimethylacetamide
DME	l ,2-dimethoxyethane
DMF	N,N-dimethylformamide
DMSO	dimethylsulfoxide
DTT	dithiothreitol
DPPA	diphenylphosphoryl azide
EtOAc	ethyl acetate
EtOH	éthanol
g h	gram hour(s)
H?	Hydrogen
H2O	Water
HATU	A-[(Dimethylamino)-l//-l,2,3-triazolo-[4,5-Z>]pyridin-l-ylmethylene]-A'methylmethanaminium hexafluorophosphate A-oxide
HCl	hydrochloric acid
HPLC	High Performance Liquid Chromatography
K2CO3	potassium carbonate

KOH	potassium hydroxide
L	Liter
LCMS	liquid chromatography and mass spectrometry
LiBr	lithium bromide
5 M	molar
MHz	Mégahertz
MeCN	Acetonitrile
MeOH	methanol
MS	mass spectrometry
10 MsCl	methanesulfonyl chloride
MTBE	methyl terZ-butyl ether
mmol	millimole
mg	milligram
min	minutes
15 mL	milliliter(s)
n2	nitrogen
NaBH4	sodium borohydride
NaH	sodium hydride
NaHCO3	Sodium Bicarbonate
20 Nal	sodium iodide
NaOH	Sodium Hydroxide
NBS	N-bromosuccinimide
nM	nanomolar
NMP	N-methyl -2-pyrrol i donc
25 N	normal
NH3H2O	ammonia in water
NH4HCO3	ammonium bicarbonate
NH4OH	ammonium hydroxide
NMR	nuclear magnetic résonance
30 Pd/C or Pd-C	palladium on carbon
Pd2(dba)î	tris(dibenzy]ideneacetone)dipalladium(0)
Pd(PPh3)2Cb	bis(triphenylphosphine)palladium(ll) dichloride
PdCbidppf)	[1,1 -bis(diphenylphosphme)fenOcene]dichloropalladium(II)
Pet. Ether	Petroleum ether
35 psi	pound per square inch
PPh3	triphenylphosphine

rt	room température
sat.	saturated
SM	starting material
SFC	Supercritical fluid chromatography
tBuOK	potassium tert-b ut oxide (or t-BuOK)
T3P	propylphosphonîc anhydride
TBAB	tetrabutylammonium bromide
TEA	tri ethy lamine
TFA	trifluoroacetic acid
TfOH	trifluromethane sulfonic acid
THF	tetrahydrofuran
TLC	thin layer chromatography
Prep. TLC TMSCBrF2	préparative TLC (bromodifluoromethyl)trimethylsilane
pL	micro liter
vol	volume
XantPhos	4,5 -bi s (dipheny lp h osphi no )- 9,9 -dî methy Ix ant he ne

General Synthetic Schemes

While the présent invention has been described in conj miction with the spécifie ex amp les set forth above, many alternatives, modifications and variations thereof will be apparent to those of ordinary skili in the art. In some cases, the order of carrying ont the steps of the reaction schemes may be varied to facilitate the reaction or to avoid unwanted reaction products. Ail such alternatives, modifications and variations are intended to fall within the spirit and scope of the présent invention. Starting materials and intermediates are purchased from commercial sources, made from known procedures, or are otherwise illustrated.

Several methods for preparing the compounds of this invention are described in the following Schemes and Examples. Unless otherwise indicated, ail variables are as previously defined. In ail general schemes Ar implies an optionally substituted aryl or heteroaryl moiety.

In scheme 1, R represents H, halogen, C|.j alkyl or Ci.jalkoxy.

In scheme 1, Y¹ represents H or OH.

Scheme 1:

ArCO₂H amine base, HATU or T3P

In Scheme 1, an optionally substituted spiroamîne 1 can be coupled to an approprîately substituted carboxylîc acid using standard amide couplîng conditions to provide amide 2.

Scheme 2:

Ri⁼ alkyl. H

Y = CH, N

Z = CH, N

R = alkyl, H, halogen

R₂ = alkyl, H, halogen

In scheme 2, an optionally substituted heteroaromatic amine 3 can be condensed with an alkyl 3bromo-2-oxopropanoate (4) to form bicyclic products 5 and 6.

SYNTHESIS OF INTERMEDIATES

Intermediate 1: (3A,3’Æ)-l,4-dihydro-2Z/-spiro[isoquinoline-3,4'-piperidin]-3'-ol

1) KOH

2) DPPA.TEA

3) NaOH

PPh₃, CO

PdiPPh^CI^

DMF

1)SFC

2) BHjTHF

3) Hj, Pd/C

Step 1: Ethyl l-benzyi-3-oxopiperidine-4-carboxylate (800 g, 2,69 mol) was added dropwise at 0 °C to a solution of t-BuOK (633 g, 5.64 mol) in THF (2 L) at 0 °C. The mixture was stined at 25 °C for 1 h. The mixture was cooled to 0 °C and a solution of l-bromo-2-(bromomethyl)benzene (705 g, 2.82 mol) in THF (500 mL) was added dropwise over 0.5 h. The mixture was stined at °C for 5 h to afford a solution of ethyl l-benzyl-4-(2-bromobenzyl)-3-oxopiperidme-4carboxylate which was used directly without work-up or purification, MS; 430 and 432 (M + 1), Step 2: Ethyl l-benzyl-4-(2-bromobenzyl)-3-oxopiperidme-4-carboxylate (1.00 kg, 2.32 mol) was added to EtOH ( 1 L) and the solution was purged and degassed with N: (3x). The résultant mixture was cooled to 0°C and NaBH4 (87.9 g, 2.32 mol) was added portionwise over Ih. The mixture was then stirred at 25 °C for 2 h. The reaction mixture was concentrated under reduced pressure, diluted with H₂O (200 mL), and extracted with ethyl acetate (200 mL x 3). The combined organic layers were dried over anhydrous magnésium sulfate, fîltered, and concentrated under reduced pressure to afford ethyl l-benzyl-4-(2-bromobenzyl)-3-hydroxypiperidine-4-carboxylate. MS: 432 and 434 (M + 1).

Step 3î A solution of ethyl l-benzyl-4-(2-bromobenzyl)-3-hydroxypiperidine-4-carboxylate (622 g, 1.44 mol) in DMF (2.5 L) was purged and degassed with N; (3x) and then cooled to 0 °C. To the résultant mixture was added NaH (69.1 g, 1.73 mol, 60% w/w) portionwise at 0 °C over 1 h. The résultant mixture was stirred at 25 °C for 0.5 h. Benzyl bromide (197 g, 1.15 mol, 137 mL) was added dropwise to the mixture at 0 °C over Ih and the résultant mixture was stirred at 25 °C for 5 h. The reaction was quenched with saturated aq. NH4CI (1 L) at 0 °C, and extracted with MTBE (300 mL x 3). The combined organic layers were washed with sat. aqueous NaCI (200 mL x 2), dried over anhydrous sodium sulfate, fîltered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica (50:1 to 1:1 petroleum ether :ethyl acetate) to provide ethyl l-benzyl-3-(benzyloxy)-4-(2-bromobenzyl)piperidine-4-carboxylate. MS; 522 and 524 (M + 1)

Step 4: Ethyl l-benzyl-3-(benzyloxy)-4-(2-bromobenzyl)piperidine-4-carboxyîate (500 g, 957 mmol) and KOH (805 g, 14,4 mol) were added to EtOH (4 L) and the résultant mixture was purged and degassed with Ni (3x). The mixture was stirred at 100 °C for 12 h. The reaction mixture was concentrated under reduced pressure and the residue was diluted with H2O (200 mL). The pH of the mixture was adjusted to pH 6 with 6 N aqueous HCl. The solid was fîltered, washed with H2O (1 L), and concentrated to afford l-benzyl-3-(benzyloxy)-4-(2-bromobenzyl)piperidine4-carboxylic acid, which was used directly without further purification. MS: 494 and 496 (M + 1) Step 5: A solution of 1 -benzyl-3-(benzyloxy)-4~(2-bromobenzyl)piperidine-4-carboxylic acid (200 g, 405 mmol), DPPA (134 g, 486 mmol, 105 mL) and TEA (123 g, 1.21 mol, 169 mL) in dioxane (1 L) was purged and degassed with Ni (3x). Tbe mixture was stirred at 25 °C for 3 h. To the mixture was added MeOH (600 mL) at 25 °C over 0.5 h and then the mixture was stirred at 100 °C for 12 h. The reaction was quenched with saturated aq. NaHCOj (2 L) at 0 °C, and then concentrated under reduced pressure. The residue was diluted with ethyl acetate (750 mL), washed wîth brine (150 mL), dried over anhydrous sodium sulfate, fîltered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica (3:1 petroleum etherethyl acetate) to provide methyl ( 1 -benzyl-3-(benzyloxy)-4-(2-bromobenzyl)piperidin-4yl)carbamate. MS: 523 and 525 (M + 1).

Step 6: To a solution of methyl (l-benzyl-3-(benzyIoxy)-4-(2-bromobenzyl)piperidin-4yl)carbamate (130 g, 248 mmol) in DMSO (700 mL) that was purged and degassed with Ni (3x) was added a solution of NaOH (89.4 g, 2.24 mol) in H2O (400 mL) at 25 °C. The résultant mixture was heated to 100 °C and stirred for 1 h. The reaction was quenched with HiO (1 L) at 0 °C. The mixture was extractcd with ethyl acetate (300 mL x 3) and the combined organic layers were concentrated under reduced pressure. The residue was purificd by column chromatography on silica (petroleum ethenethyl acetate) to provide l-benzyl-3-(benzyloxy)-4-(2bromobenzyl)piperidin-4-amine. MS: 465 and 467 (M + 1).

Step 7: A mixture of l-benzyL3-(benzyloxy)-4-(2-bromobenzyl)pîperidin-4-amine (270 g, 580 mmol), PPL (10.6 g, 40.6 mmol) and PdfPPhjjïCL (40.7 g, 58.0 mmol) in DMF (3 L) was stirred under CO (50 psi) at 120 °C for 12 h. The reaction mixture was cooled to 0 °C and saturated aq. NaHCOi (6 L) was added. The mixture was extracted with ethyl acetate (2 L x 3). The combined organic layers were washed with brine (1 L x 2), dried over anhydrous sodium sulfate, fîltered, and concentrated under reduced pressure to yield a solid. The solid was washed with MTBE (1 L x 3) to afford r-benzyl-3'-(benzyloxy)-2W-spiro[isoquinoline-3,4'-piperidin]-l(4F/)-one. MS: 413 (M + 1).

This solid, ]'-benzyl-3’-(benzyloxy)-2F/-spiro[isoqumoline-3,4'-piperidin]-l(4E/)-one (208 g, 504 mmol), was further purified by SFC on a chiral column (Chiral Pak AD; Mobile phase: A for CO2 and B for EtOH) to obtain two isomers:

(First Eluting) (3A,3’Æ)-T-benzyl-3'-(benzyloxy)-2/7-spiro[isoquinoline-3,4'-piperidin]-I(4f7)-one. MS: 413 (M + J).

(Second Eluting) (35,3'5)-Γ-benzyl-3'-(benzyloxy)-2H-spiro[isoquinoline-3,4’-piperidin]-1(4//)one. MS: 413 (M + 1).

Step 8: A mixture of (3Æ,3’7ï)-r-benzyl-3^,-(benzyloxy)-2//-spiiO[isoquinoline-3,4'-piperidin]l(4//)-one (32.0 g, 77.6 mmol) in THF (150 mL) that was purged and degassed with N2 (3x) was stirred at 15 °C for 4 h. To the mixture was added BH3.THF (1 M in THF, 698 mL, 698 mmol) dropwise at 0 °C over 0.5 h. The résultant mixture was heated to 80 °C and stirred for 48 h. The mixture was cooled to 0 °C and quenched with dropwise addition of MeOH (300 mL). The mixture was then heated to 80 °C and stirred for 20 h. The mixture was cooled to room température and concentrated under reduced pressure. The crude product was washed with MTBE (500 mL) to afford (3Â,3’A)-r-benzyl-3^,-(benzyloxy)-l,4-dihydro-2Zf-spiro[isoquinoiine-3,4'piperidine]. MS: 399 (M + 1).

Step 9: A solution of (3R,3’R)-T-benzyl-3’-(benzyloxy)-l,4-dihydro-217-spiiOrisoquinoIii)e-3,4’piperidine] (20.0 g, 50.2 mmol), HCl (2 M in MeOH, 100 mL, 200 mmol) in MeOH (200 mL) and Pd/C (10.0 g, 10 wt.%) at 25 C was purged and degassed with H? (3x). The mixture was stirred under Hs (50 psi) at 50 °C for 12 h. The reaction mixture was filtered and concentrated under reduced pressure. The resulting residue was washed with EtOAc (30 mL x 3) to afford (3R,3’R)-l,4-dihydro-2//-spiro[isoquinoline-3,4’-piperidin]-3'-ol. MS: 219 (M + 1), 'HNMR: (500 MHz, D₂O) δ 7.44-7.33 (m, 3H), 7.33-7.26 (m, 1H), 4.54 (d, J= 16.5 Hz, 1H), 4.49 (d,J= 16.6 Hz, 1H), 4.33 -4.24 (m, 1H), 3.61 (dd, 7 = 12.9,4.1 Hz, 1H), 3.52-3.39 (m, 2H), 3.37-3.19 (m, 3H), 2.32 (d, J= 14.5 Hz, 1H), 2.11-2.01 (m, 1H).

Intermcdiate 2: 8 -finetho xymeth vl) -6- ( tri fluorometh yllimidazo Γ L 2 -ctl p y ri di ne -2 -carbox v lî c ac id

Step 1: To a solution of 3-bromo-5-(trifluoromethyi)pyridin-2-amine (5.0 g, 21 mmol) in DME (150 mL) was added ethyl 3-bromo-2-oxopropanoate (4.94 g, 25.3 mmol) dropwîse. The reaction mixture was heated to 90 °C for 18 h. The mixture was concentrated under reduced pressure to give crude residue, which was purified by flash silica gel chromatography (ethyl acetate/petroleum ether gradient) to give ethyl 8-bromo-6-(trifluoromethyl)imidazo[L2-Li]pyridine-2-carboxylate. MS: 337 and 338 (M + 1). ’H NMR(400 MHz, CDjOD) δ 9.21 - 9.07 (m, 1H), 8.64 (s, 1H), 7.98 7.87 (m, 1H), 4.42 (q, 7 = 7.1 Hz, 2H), 1.49 - 1.32 (m, 3H).

Step 2: To a solution of ethyl 8-bromo-6-(trifluoromethyl)imidazo[l,2-«]pyridine-2-carboxylate (500 mg, 1.48 mmol) in tert-amyl alcohol (10 mL) was added chloro(2-dicyclo hexylphosphino2',6’-diisopiOpoxy-l,l'-biphenyl)[2-(2'-amino-l,l'-biphenyl)]palladium(lI) (115 mg, 0.148 mmol), potassium methoxymethyltrifluoroborate (451 mg, 2.97 mmol) and Cs₂CCh (1.45 g, 4.45 mmol) in a glove box under an argon atmosphère at 25 °C. The mixture was stirred at 100 °C for 18 h. The mixture was concentrated under reduced pressure. The residue was purified by préparative TLC (petroleum ether/EtOAc) to give ethyl 8-(methoxymethyl)-6-(trifluoromethyl)imidazo[L2iïjpyridine-2-carboxylate. MS: 303 (M + 1)

Step 3: To a solution of ethyl 8-(methoxymethyl)-6-(trifluoiOmethyl)imidazo[l,2-<î]pyridine-2carboxylate (230 mg, 0.76 mmol) in EtOH (6 mL) and water (1 mL) was added LiOH HiO (38.3 mg, 0.91 mmol) at 25 °C. The mixture was stirred at 25 °C for 2 h. The mixture was acidified with 1 M HCl (in water) to pH~3. The mixture was concentrated under reduced pressure to give the crude product 8-(methoxymethyl)-6-(trifluoromethyl)imidazo[l,2-6']pyridine-2-carboxylic acid.

MS: 275 (M + 1).

Intermediates 3 and 4: (Æ)-6-bromo-8-( 1 -methoxyethyliimidazol 1,2-71pvridine-2-carboxylic acid and ('5j - 6-broino-8 - ( 1 -m ethoxy ethyl ) imi dazo 11,2-rd p yrid i ne-2-c arboxv 1 i c ac i d </ O

1) NaH, Mel______________ }--- Br₂ \___

2) benzophenone imine, /=( AcOH 25 “C 2 h* /=\

Pd₂(dba)₃, XantPhos, <\ // NH₂ ^{1 1} Br—G /)— ΝΗ_Ξ

Cs₂CO₃ N

—O

O

Step 1: To a mixture of 2-chloiomcütinaldehyde (4.0 g, 28 mmol) in THF ( 100 mL) was added méthylmagnésium bromide (3M in diethyl ether) (14.1 mL, 42.4 mmol) slowly dropwise at -78 °C. The mixture was stirred at -78 °C for 0.5 h. To the reaction was added sat. NH4CI (50 mL) and water (80 mL). The aqucous layer was extracted with ethyl acetate (70 mL x 3). The combined organic layers were washed with brine (100 mL), dried over anhydrous Nas S Ch, filtered, and concentrated under reduced pressure to give l-(2-chloropyridin-3-yl)ethanol, which was used in the next step directly. MS: 158 and 160 (M+l). Ή NMR (400 MHz, CDCb) δ 8.23-8.28 (m, 1H), 7.96 (dd, 7= 1.3, 7.9 Hz, 1H), 7.24-7.30 (m, JH), 5.18-5.26 (m, 1H),2.6O (d,7=3.5 Hz, 1H), 1.49 (d, 7 = 6.6 Hz, 3H).

Step 2: To a mixture of l-(2-chloropyridin-3-yl)ethanol (4.4 g, 27.9 mmol) and Mel (2.22 mL, 35,4 mmol) in DMF (60 mL) was added NaH (1.34 g, 33.5 mmol) at 0 °C. The mixture was stirred at 0 °C for 0.5 h. To the reaction was added water (500 mL). The aqueous layer was re-extracted with ethyl acetate (200 mL x 3). The combined organic layers were washed with brine (300 mL), dried over anhydrous NasSCh, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ethyl acetate/pet. ether gradient) to give 2-chloro-3-(lmethoxyethyl)pyridine. MS: 172 (M+l). ‘H NMR (400 MHz, CDCh) S 8.34 (dd,7 = 2.0, 4.9 Hz, 1H), 7.85 (dd, 7 = 2.0, 7.8 Hz, 1H), 7.31 (dd, 7 = 4.9, 7.8 Hz, 1H),4.71 (q, 7 = 6.4 Hz, 1H), 3.30 (s, 3H), 1.45 (d, 7 = 6.4 Hz, 3H).

Step 3: To a solution of 2-chloro-3-( l -methoxyethy l)py ridine (4.3 g, 25 mmol), diphenylmethanimine (5.45 g, 30.1 mmol), Xantphos (1.45 g, 2.51 mmol) in l,4-dioxane (60 mL) was added Cs₂COj (19.6 g, 60.1 mmol) and Pd₂(dba)₃ (1.15 g, 1.25 mmol) at 25 °C. The résultant mixture was degassed and backfîlled with N₂ (three times) and stirred at 90 °C for 12 h. The mixture was concentrated under reduced pressure to remove dioxane and H₂O (60 mL) was added. The mixture was extracted with Ethyl acetate (40 mL x 3). The organic layers were dried over NaiSCU, filtered and the filtrate was concentrated under reduced pressure. The residue was dissolved in MeOH (30 mL) and the mixture was adjusted to pH 5 with HCl solution (aq. 1 M) and stirred for 20 min. The mixture was basified with ammonium hydroxide (1 M) to pH 8 and the mixture was concentrated under reduced pressure. The residue was purified by flash sîlica gel chromatography (Ethyl acetate/pet. ether gradient) to give 3-(l-methoxyethyI)pyridin-2-amine. MS: 153 (M+l). Ή NMR (400 MHz, CDCh) δ 8.02 (dd, J = 1.7, 5.1 Hz, 1H), 7.26 (dd, J = 2.0, 7.3 Hz, 1H), 6.63 (dd, J = 4.9, 7.3 Hz, 1H), 5.10 (br s, 2H), 4.34 (q, J = 6.9 Hz, 1H), 3.26-3.31 (m, 3H), 1.53 (d, J = 6.9 Hz, 3H).

Step 4: To a mixture of 3-(l-methoxyethyl)pyridm-2-amine (500 mg, 3.29 mmol) in AcOH (5 mL) was added Br₂ (0.25 mL, 4.93 mmol) at 25 °C. The mixture was stirred at 25 °C for 2 h. The mixture was concentrated under reduced pressure. The residue was dissolved in water (5 mL) and the mixture was adjusted to pH 8 with sat. NaHCOj. The aqueous layer was extracted with Ethyl acetate (10 mL x 5) and the combined organic layers were washed with brine (20 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Ethyl acetate/pet. ether gradient) to give 5-bromo-3-(lmethoxyethyl)pyridin-2-amine. MS: 231 and 233 (M+l). Ή NMR (400 MHz, CDCh) 5 8.05 (d, J = 2.5 Hz, 1H), 7.37 (d, J = 2.5 Hz, 1H), 5.14 (br s, 2H), 4.23-4.33 (m, 1H), 3.29 (s, 3H), 1.51 (d, J = 6.9 Hz, 3H).

Step 5: To a mixture of 5-bromo-3-(l-methoxyethyl)pyridin-2-ainme (270 mg, 1.17 mmol) in 1,4dioxane (15 mL) was added MgSCU (422 mg, 3.51 mmol) and ethyl 3-bromo-2-oxopropanoate (349 mg, 1.75 mmol) at 25 °C. The mixture was stirred at 80 °C for 12 h. After cooling to 25 °C, TE A (0.20 mL, 1.4 mmol) was added. The mixture was stirred at 25 °C for 1 h and the precipitate was filtered off. The filtrate was concentrated under reduced pressure. The residue was purified by flash sîlica gel chromatography (Ethyl acetate/pet. ether gradient) to give ethyl 6-bromo-8-(lmethoxyethyl)imidazo[l,2-cï]pyridine-2-carboxylate. MS: 327 and 329 (M+l). ^lH NMR (400 MHz, CDC1₃)5 8.20 (d, J = 1.8 Hz, 1 H), 8.13 (s, 1H), 7.37-7.42 (m, 1H), 5.20 (q, J = 6.6 Hz, 1H), 4.42-4.50 (m, 2H), 3.38 (s, 3H), 1.54 (d, J= 6.6 Hz, 3H), 1.42 (t, J = 7.0 Hz, 3H).

Step 6: A mixture of isomers of ethyl 6Tromo-8-(l-methoxyethyl)imidazo[l,2-a]pyndine-2carboxylate (210 mg, 0.64 mmol) was separated by SFC (AD Column, NHj H₂O/EtOH/CO₂) to afford ethyl (5 or A)-6-bromo-8-(l -methoxyethyl)imidazo[ 1,2-ü]pyridine-2-carboxylate (Peak 1) and ethyl (S or /<)-6-bromo-8-(l-methoxyethyl)imidazo[l,2-c?]pyridine-2-carboxylate (Peak 2). Peak 1: MS: 327 and 329 (M+l). Ή NMR (400 MHz, CDCh) δ 8.20 (d, J =1.8 Hz, 1 H), 8.13 (s, 1H), 7.40 (s, 1H), 5.20 (q, J = 6.6 Hz, 1H), 4.42-4.49 (m, 2H), 3.38 (s, 3H), 1.54 (d, J = 6.6 Hz, 3 H), 1.42 (t, J = 7.2 Hz, 3H).

Peak 2: MS: 327 and 329 (M+l). Ή NMR (400 MHz, CDCh) S 8.20 (d, J = 1.8 Hz, IH), 8.13 (s, IH), 7.37-7.43 (m, 1H), 5.14-5.25 (m, 1H), 4.40-4.49 (m, 2H), 3.38 (s, 3H), 1.55 (d, J = 6.1 Hz, 3H), 1.42 (t,J = 7.2 Hz, 3H).

Step 7: To a mixture of ethyl (R or S)-6-bromo-8-(l-methoxyethyl)imidazo[l,2-rï]pyridine-2carboxylate (70 mg, 0.21 mmol) in EtOH (2 mL) and water (0.5 mL) was added LiOH HiO (178 mg, 0.43 mmol) at 25 °C. The mixture was stirred at 25 °C for 2 h. The mixture was concentrated under reduced pressure to give crude product (R or S)-6-bromo-8-(l-methoxyethyl)imidazo[l,2iî]pyridine-2-carboxylic acid, which was used in next step without purification. MS: 299 and 301 (M+l). The same was carried out with ethyl (S or A)-6-bromo-8-(l-methoxyethyl)imidazo[l,2cï]pyridine-2-carboxylate isorner.

Intermediate 5: 6-bromo-7-methvlimidazoi 1.2-7lnvrimidine-2-carboxvlic acid

Step 1: To a mixture of 5-bromo-4-methylpyrimidin-2-amine (5.0 g, 27 mmol) in EtOH (50 mL) was added ethyl 3-bromo-2-oxopropanoate (10,37 g, 39,9 mmol) at 20 °C. The mixture was heated to 80 °C for 12 h. The mixture was concentrated under reduced pressure and purified by reverse phase HPLC (ACN/water with 0.1% TFA modifier) to give ethyl 6-bromo-7-methylimidazo[l,2ô/]pyrimidme-2-carboxylate (peak 1) and ethyl 6-bromo-5-methylimidazo[l,2-ii]pyrimidine-2carboxylate (peak 2). MS: 284 and 286 (M + 1). For ethyl 6-bromo-7-methylimidazo[l,2«]pyrimidine-2~carboxylate: Ή NMR (400 MHz, CDCfi) δ 8.72 (s, IH), 8.09 (s, 1H), 4.41 (q, 7 = 7,2 Hz, 2H), 2,79 (s, 3H), 1.39 (t, J = 7.0 Hz, 3H). For ethyl 6-bromo-5-methylimidazo[l,2£i]pyrimidine-2-carboxylate: ^lH NMR (400 MHz, CDC1₃) δ 8.71 (s, IH), 8.13 (s, IH), 4.44 (q, 7 = 7,2 Hz, 2H), 2.85 (s, 3H), 1.41 (t, 7= 7.2 Hz, 3H).

Step 2: A mixture of ethyl 6-bromo-7-methylimidazo[l,2-ri]pyrimidine-2-carboxylate (1,0 g, 3.5 mmol) in HCl (12 M, 8 mL) was heated to 75 °C for 2 h. The mixture was concentrated under reduced pressure to give 6-bromo-7-methyîimidazo[l,2-7jpyrimidine-2-carboxylic acid. MS: 256 and 258 (M+l).

Intermediate 6: 6-cyclopropylimidazor 1.2-g ]pvrimidme-2-carboxylic acid /~^{N N}\^k.OH O

Step 1: To a solution of 5-cyclopropylpyrimidm-2-amine (3.5g, 26 mmol)) in EtOH (50 mL) was added ethyl 3-bromo-2-oxopropanoate (6.06 g, 31.1 mmol) and heated to 80 °C for 16 h. The mixture was cooled to room température, treated with TEA (7.22 ml, 51.8 mmol), and left to stir for 30 min. The mixture was concentrated and purified by column chromatography on silica (60% ethyl acetate in pet. ether) to give ethyl 6-cyclopropylimidazo[ 1,2-a]pyrimidinc-2-carboxylate. MS: 232 (M+l). Ή NMR (500 MHz, CDCh) 5 8.50 (d, J=2.4 Hz, 1H), 8.16 (d, J = 2.0 Hz, 1H), 8.04 (s, 1H), 4.44 (q, J = 7.2 Hz, 2H), 1.87-2.03 (m, 1H), 1.42 (t, 7.2 Hz, 3H), 1.04-1.12 (m,

2H), 0.72-0.80 (m, 2H).

Step 2: A mixture of ethyl 6-cyclopropylimidazo[l,2-a]pyrimidine-2-carboxylate (100 mg, 0.432 mmol) in HCl (4 M, 2 mL) was heated to 80 °C for 3 h. The mixture was concentraed under reduced pressure to give 6-cyclopropylimidazo[l,2-a]pyrimîdine-2-carboxylic acid. MS: 204 (M+l).

EXAMPLES

The following experimental procedures detail the préparation of spécifie examples of the instant disclosure.

Note: Many of the compounds claimed exist as a mixture of rotamers in solution at room température, which compilâtes their analyses by 1H-NMR spectroscopy. In these cases, the peak shifts are listed as ranges of multiplets that encompass the signais from both rotamers, rather than describing individual rotamer peaks.

Example 1 : ((3Æ.3’A)-3'-hvdroxv-L4-dihvdro-rZ/,2//-spirorisoquinoline-3.4'-niperidin1-r-yl')r8(mcthoxvmethvl)-6-itrifluoromethvÎ')imidazoÎL2-alpvridin-2-yl1methanone

To a solution of 8-(methoxymethyl)-6-(trifluoromethyl)îmidazo[l,2-g]pyridine-2carboxylic acid (22 mg, 0.080 mmol), (3Æ,3’Λ)-1,4-dihydro-277-spiro[isoquinoline-3,4'-piperidin] s

3'-ol ( 19.3 mg, 0.088 mmol), and DIEA (0.070 mL, 0.40 mmol) in DMF (l mL) was added T3P (0. lû mL, 0.16 mmol) (48% Wt, in DMF) at 25 °C. The mixture was stirred at 25 °C for 5 min and purified by prep-HPLC (ACN/water, 10 mM NH4HCO3 modifier) to afford ((3A,3’Æ)-3'-hydroxyl,4-dihydro-lW,2/7-spiro[isoquinoline-3,4'-piperidin]-l'-yl)[8-(methoxymethyl)-6(trifluoromethyl)îmidazo[l,2-<a]pyridin-2-yl]methanone. MS: 475 (M+l). ’H NMR (400 MHz, CDjOD) 5 8.95-9.06 (m, IH), 8.28- 8.38 (m, 1H), 7.47-7.58 (m, 1H), 7.14 (d, J = 2.5 Hz, 3H), 7.07 (br s, 1H), 4.61 (br s, 1H), 4.06 - 4.27 (m, 3H), 4.02 (br s, 2H), 3.75 - 3.96 (m, 1H), 3.51 3.71 (m, 5H), 2.92-3.10 (m, 1H), 2.70-2.87 (m, 1H), 1.84-2.01 (m, 1H), 1.60 (br d, J = 15.0 Hz, 1H).

Example 2: ( 6-bromo-8-{(SU 1 -me thoxy eth yi) im i dazo IL 2-al py ri dîn-2-y Γ) 1(3 /6 3 ’ 3?)-3 '-h vdro x vLd-dilivdroMZf-spirorisoquinoÎine-SA'-pmeridinl-Î'-vDmethanone

Example 3: ( 6 -bromo - 8 -(Ï3?) -1 -me thoxy ethy D imi dazo 11.2 -al D vri di n-2 - vl 1 ( ( 3 7?, 3 ’ 3? ) -3 ' -h ydroxy L4-dihvdrO2/7-spiiOrisoquinoline-3,4'-proeridin1-I^l-vl)methanone

To a mixture of 6-bromo-8-(l-inethoxyethyl)imidazo[l,2-«]pyridine-2-carboxylic acid (70 mg, 0.23 mmol) in DMF (3 mL) were added HATU (107 mg, 0.281 mmol), DIEA (90.7 mg, 0.702 mmol) and (3A,3’7ï)-l,4-dihydro-2//-spiro[isoquinoline-3,4’-pipendin]-3’-ol (51.1 mg, 0.234 mmol) at 25 °C. The mixture was stirred at 25 °C for 1 h. The mixture was purified by préparative HPLC (water/MeCN, lOmM NH4HCO3) to give (6-biOmo-8-((S)-l-methoxyethyI)imidazo[l,2a]pyridin-2-yl)((3R,3’JÎ)-3'-bydroxy-l,4-dihydro-227-spiro[isoquinolme-3,4'-piperidm]-ryl)methanone. Peak 1: MS: 499 and 501 (M+l). Ή NMR (400 MHz, CD₃OD) δ 8.62-8.71 (m, 1H), 8.13-8.22 (m, 1H), 7.35-7.44 (m, 1H), 7.12 (d, J = 2.2 Hz, 3H), 7.05 (br s, 1H), 4.95 (br t, J = 6.4 Hz, 1H), 3.83-4.26 (m, 5H), 3.49-3.80 (m, 2H), 3.33-3.39 (m, 3H), 2.90-3.10 (m, 1H), 2.64-2.85 (m, 1H), 1.84-1.96 (m, 1H), 1.46-1.62 (m, 4H).

( 6-Bromo-8-((J?)-1 -methoxy ethyl )imidazo[ 1,2-a] pyridin-2-y 1)((3Æ,3 ^)-3 '-hy droxy-1,4-dihydro2/7-spiro[isoquinoline-3,4’-piperidm]-l'-yl)methanone. Peak 2: MS: 499 and 501 (M+l). Ή NMR (400 MHz, CDjOD) δ 8.67 (br s, 1H), 8.10-8.23 (m, 1H), 7.32-7.47 (m, 1H), 7.11 (br s, 3H), 7.05 (br s, 1H), 4.92-4.99 (m, 1H), 3.82-4.24 (m, 5H), 3.49-3.81 (m, 2H), 3.32-3.39 (m, 3H), 2.90-3.08 (m, 1H), 2.66-2.84 (m, 1 H), 1.83-1.99 (m, 1H), 1.43-1.62 (m, 4H).

Example 4: (6-bromo-7-methvlimidazori,2-alnvnmidin-2-yl)r(3/?.3 K)-3 -hvdroxv-1,4-diliydro r/7.2/f-spirorisoquinoline-3,4'-piperidinl-T-yllmethanone

To a mixture of 6-bromo-7-meÎhylimidazo[l,2-n]pyrimidine-2-carboxylic acid (70 mg, 0.27 mmol) in DMF (2 mL) was added HATU (125 mg, 0.328 mmol), DIEA (0.143 ml, 0.820 mmol) and (3A3’A)-l,4-dihydro-2H-sp!ro[isoquinoline-3,4'-piperidin]-3'-ol (60 mg, 0.27 mmol) at 15 °C. The mixture was stirred at 15 °C for 10 min and purified by préparative HPLC (water/MeCN, ΙΟιηΜ NH4HCO3) to give (6Tromo-7-methylimidazo[l_:2-«]pynmidm-2yl)[(33i,3’JÏ)-3'-hydiOxy-l,4-dihydro-r/f,2ff-spiro[isoquinoline-3,4'-piperidin]-r-yl]methanone. MS: 456 and 458 (M+l). Ή NMR (400 MHz, CD₃OD) δ 9.13 (br s, 1H), 7.99-8.08 (m, 1H), 7.11 (br d, 7= 2.6 Hz, 3H), 7.05 (br s, 1H), 4.04-4.23 (m, 2H), 3.53-4.03 (m, 5H), 2.90-3.05 (m, 1H), 2.67-2,84 (m, 4H), 1.77-1.96 (m, 1H), 1.55 (brd,J = 16.2 Hz, 1H).

Example 5: (6-cvclopropvlimidazorL2-_lj1pyrimidin-2-¥l)r(3A.3'/f')-3'-hYdroxv-l.4-dihvdro1 W,2//-spirorisoqumoliiie-3.4^,-pÎperidm]-r-yl1methaiÎone

O

To a solution of 6-cyclopropylîmidazo[l,2-a]pyrimidine-2-carboxylic acid (250 mg, 1.23 mmol) in DMF (5 mL) were added HATU (468 mg, 1.23 mmol), DIEA (0.645 ml, 3.69 mmol) and (3/î,3’Æ)-l,4-dihydro-2f/-spiro[isoquinoline-3,4'-piperidin]-3'-ol (215 mg, 0.984 mmol) .The mixture was stirred at 15 °C for 0.5 h, concentrated, and purifïed by préparative HPLC (water/MeCN, lOmM NH4HCO3) to give (6-cyclopropylimidazo[l,2-iï]pyrimidm-2-yl)[(3JÎ,3'.ff)3'-hydroxy-l,4-dihydro-17f,2/TspÎiO[isoquinoline-3,4'-piperidin]-r-yl]methanone. MS 404 (M+l). Ή NMR (400 MHz, CDCL) 5 8.41 (d, J= 2.4 Hz, 1H), 8.15 (s, 1H), 7.96-7.94 (m, 1H), 7.11 (m, 3H), 7.01-6.99 (m, 1H), 4.58-4.54 (m, 1 H), 4.34-4.31 (m, 1H), 4.05-3.92 (m, 3H), 3.593.58 (m, 1H), 3,32-3.29 (m, 1H), 2.92-2.72 (m, 2H), 1.98-1.90 (m, 2H), 1.57-1.53 (m, 1H),1.ΟΤΙ .05 (m, 2H), 0.74-0.73 (m, 2H).

PRMT5-MEP50 Enzyme Méthylation Assay

PRMT5-MEP50 biochemical assay is a direct measurement of the méthylation activity of the enzyme complex on a short peptide substrate derived from the N-terminus of H4 histone. Méthylation experiment was performed with recombinant PRMT5-MEP5O protein complex. The assessment of inhibitory effect of smali molécules was measured by the effective ne ss of the compounds to inhibit this réaction (ECso).

In this assay, the potency (ECso) 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 pL) was dispensed, followed by the addition of 8 pL of Ix 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-MEP5Û enzyme compte x (recombinant p rote ins from baculovirus-transfected Sf21 cells: FL-PRMT5; MW = 73837 kDa and FL-MEP50; MW = 38614) and 1 pL of 150 pM S-(5’-Adenosyl)-L-Methionine Chloride (SAM). Plates were sealed and placed in a 37 ⁿC humidifïed chamber for a 60 minutes pre-incubât ion with compound. Subsequently, each reaction was initiated by the addition of 1 pL Ix assay buffer containing 750 nM biotinylated H4R3(Mel) peptide. The final réaction in each well of 10 pL consists of 1.0 nM PRMT5-MEP5Û, 75 nM biotinylated-peptide, and 15 pM SAM. Méthylation reactions were allowed to proceed for 150 minutes in a sealed plate at 37 “C. Reactions were îmmediately quenched by the addition of 1 pL of 5% fonnic acid. Plates were then frozen and shipped to SAMDITM Tech Inc. to déterminé the percent conversion from H4R3(Mel) to H4R3(Me2). Doseresponse curves were generated by plotting percent effect (% product conversion; Y-axis) vs. LoglO compound concentrations (X-axis). ECso values were determined by non-linear régression according to models for sigmoidal (4 parameters) dose-response curves.

PRMT5 Cell Target Engagement (TE) Assay

The PRMT5 TE assay is a biomarker assay for identifying compounds that inhibit symmetric dimethylatîon of arginine (SDMA) of PRMT5 substrates. The following substrates hâve been reported for PRMT5: histone H2Aand H4 R3, Histone H3 R2, Histone H3 R8, spliceosome Sm proteins, ribosomal protein RPS10, p53, FENi, nucleoplasmin, nucleolin, EGFR and EBNA. The assay focuses on detecting symmetric ail y dimethylated nuclear proteins using high content imagîng technology. Détection of the expression of symmetric ally dimethylated nuclear proteins is through a mixture of primary rabbii monoclonal antibodies to SDMA (CST 13222), which in tum 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 mcan value for DMSO treated cells (MIN) to report percent of inhibition for each compound-treated well.

In this assay, the cell potency (ECso) of each compound was determined from a ten point (1:3 serial dilution; top compound concentration of 10000 nM) titration curve using the following outhned procedure, Each weil of a BD falcon collagen coated black/clear bottom 384-well plate was seeded with 4000 MCF-7 cells in 30 μΐ media and allowed to attach for 5 h. Media is ATCCformulated Eagle's Minimum Essential Medium, Catalog No. 30-2003, To make the complété growth medium, the following components were added to the base medium: 0.01 mg/mL human recom binant insu lin; fêtai bovine sérum to a final concentration of 10%. Additional 30 μΐ of media containing 2x compounds were added to each well. Cells were treated for 3 days in 37°C CO: incubator. On day 3, cells were fixed with CytofÎx, permeablized with 0,4% Triton-X-100/Cytofix, and washed with D-PBS without Ca/Mg. Cells were blocked with Licor Odessey blocking reagent for l h at room température, followed by incubation with anti-SDMA ( 1:1000) antibody at 4°C ovemight. 1 ° antibody was removed, followed by three washings with DPBS without Ca/Mg and 0.05% Twecn20. Hoechst (5pg/mL), Cell Mask deep stain (1:2000) and Alexa488-conj ugated goat anti-rabbit IgG (2 pg/mL) was added for 1 h at room température. A final washing step (three washes) was performed before sealing plate for imaging on In Cell Analyzer 2200 or OperaPhenix. Images from analyzer were uploaded to Columbus (at WP or BOS) for image analysis. ICso values were determined by 4 parameters robust fit of percent fluorescence units vs. (Logio) compound concentrations.

Représentative compounds of the présent invention were tested using the assay protocol described in this example. Results are provided in Table 3 below.

Table 3:

Ex. No.	Enzyme Méthylation Assay (ECso, nM)	TE Assay (ECso, nM)
1	0.525,223.9	6.226
2	0.3693, 113.9	2.11
3	0.5807	1.572
4	0.6523, 280	13.99
5	0.2164, 72.44	4.425

When only one ECso 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 (13)

1. A compound selected from:

2. The compound of claim 1 selected from:

, or a pharmaceutically acceptable sait thereof.

3. The compound of claim 1, or a pharmaceutically acceptable sait thereof,

15 which is:

((3A,3’Æ)-3'-hydroxy-l,4-dihydro-l W,2//-spiiO[isoqumoline-3,4'-piperidm]-r-yl)[8( methoxymethyl)-6-(tri fluoromethy l)imidazo [ 1,2-a]pyrîdin-2-y IJmethanone, (6-bromo-8-((S)-l-methoxyethyl)imidazo[l,2-n]pYridin-2-y 1)((3//,3 ’//)-3'-hydroxy-l,4-dihYdro2/7-spiro[ i soquinoline-3,4’-pip eri di n] -1 '-yl)methanone, (6-bromo-8-((A)-1 -methoxyethyl)imidazo[ l ,2-<7]pyridin-2-y l)((3Æ,3 '//)-3'-hydroxy-1,4-dihydro2/7-spiro[isoquinoline-3,4'-piperidin]-r-yl)methanoue, (6-bromo-7-methylimidazo[l,2-iï]pyrimidin-2-yl)[(37î,3^,^)-3'-hydroxy-l,4-dihydro-r//,2Zfspiro[isoquinoline-3,4^,-piperidin]-r-yl]methanone, or (6-cyclopropylimidazo[ 1,2-â]pyiïmidin-2-yl)[(3Æ,3'Æ)-3'-hydroxy-1,4-dihydro-1 spîrotisoqumoline-SA'-pipcridinj-l'-ylJmethanone.

The compound of claim 1, which is:

, or a pharmaceutîcally acceptable sait thereof.

5. The compound of claim 1, which is:

, or a pharmaceutîcally acceptable sait thereof.

6. The compound of claim 1, which is:

Br

, or a pharmaceutîcally acceptable sait thereof.

7. The compound of claim 1, which is:

, or a pharmaceutîcally acceptable sait thereof.

8. The compound of claim l, which is: ((3R,3 R)-3 -hydroxy-l,4-dihydro-l W,2H- spi ro [i s oqui η ο I i n e - 3,4'-p iperi di η] - Γ-y 1) [ 8 - ( methoxy methy l) - 6 - (tri fki oromethy l) i m idazo [ 1,2 «]pyridin-2-yl]methanone, or a pharmaceutically acceptable sait thereof.

9. The compound of claim 1, which is: (6-bromo-8-((5)-l- methoxyethyl)imidazo[l,2-<i|pyridin-2-yl)((32ï,3’A)-3-hydroxy-l,4-dihydro-2/7spiro[isoquinolme-3,44piperidinJ-T-yOmethanonc, or a pharmaceutically acceptable sait thereof.

10. The compound of claim 1, which is: (6-bromo-8-((/ï)-l- methoxyethyl)imidazo[l ,2-a]pyridin-2-yl)((3/?,3’A')-3^,-hydroxy-l_:4-dihydiO-2iyspiro[isoquinoline-3,4'-piperidin]-r-yl)methanone, or a pharmaceutically acceptable sait thereof.

H. The compound of claim 1, which is: (64>romo-7-methylimidazo[l,2-a]pyrimidin2-yl)[(37L37î)-3^,4aydroxy-l,4-dihydro-1742/7-spiro[isoquinolme-3,4'-piperidin]-ryljmethanone, or a pharmaceutically acceptable sait thereof.

12. The compound of claim 1, which is: (6-cyclopropylimidazo[l,2-«]pyrimidm-2- yl)[(3R,37?)-3'4iydroxy-l,4-dihydro-17/,2Z/-spiro[isoquinoline-3,4'-piperidin]-ryl]methanone, or a pharmaceutically acceptable sait thereof.

13. A pharmaceutical composition comprising a compound of any one of daims 1-12, or a pharmaceutically acceptable sait thereof, and a pharmaceutically acceptable carrier.

14. A pharmaceutical composition comprising a compound of any one of daims 1-12, or a pharmaceutically acceptable sait thereof, for treating cancer, sickle cell disease or hereditary persistence of foetal hemoglobin (HPFH) mutations.

15. The use of a compound of any of daims 1-12, or a pharmaceutically acceptable sait thereof, for the manufacture of a médicament for treating cancer, sickle cell disease or hereditary persistence of foetal hemoglobin (HPFH) mutations.