WO2025039073A1 - Rna helicase inhibitor - Google Patents

Abstract

There is provided A compound of formula I, a salt or solvate thereof as described herein, for treating MYC positive cancers including lung cancer, leukemia, breast cancer, myeloproliferative disorders, colorectal cancer, medulloblastoma, renal, hepatocellular cancer, melanoma, ovarian cancer, prostate cancer, esophageal adenocarcinoma, liposarcoma, esophageal squamous cancer, gastrointestinal stromal tumor, glioma, myxofibrosarcoma, leiomyosarcoma, neuroblastoma, synovial sarcoma, mesothelioma, gastric cancer, thyroid cancer, lymphoma, osteosarcoma, rhabdomyosarcoma, fibrosarcoma, epithelial cancer, and neural cancer.

Description

RNA HELICASE INHIBITOR

CROSS REFERENCE TO A RELATED APPLICATION

[0001] This disclosure claims priority from U.S. provisional application number 63/520,384 filed on August 18, 2023 and is incorporated herein by reference in its entirety.

TECHNICAL FIELD

[0002] This disclosure relates to the field of inhibitors of RNA helicases, methods of making same and uses thereof in the treatment of cancer.

BACKGROUND OF THE ART

[0003] Breast cancer is a heterogeneous disease that is classified into three main histological subtypes. The histological subtypes inform on patient outcome and treatment options for clinical management of the disease. The treatments include estrogen/progesterone receptor (ER/PR)- positive (65% incidence), human epidermal growth factor receptor 2 (HER2)-positive (~20% incidence) and triple negative breast cancers (TNBC: ER-/PR-/HER2-) (15% incidence). Notably, TNBC tumors tend to be more aggressive and are more likely to be diagnosed in younger (premenopausal) women. Standard of care therapy for women with ER+ disease includes endocrine therapies that disrupt ER signaling and/or reduce ER levels within breast cancer cells (tamoxifen, fulvestrant) or alternatively impair estrogen production (aromatase inhibitors such as letrozole). HER2+ patients are treated with trastuzumab, a HER2+ monoclonal antibody, which is highly effective for women diagnosed with early stage disease. In contrast, for women with TNBC, effective targeted therapies remain elusive, and chemotherapy is the standard of care.

[0004] Unfortunately, chemotherapeutic regimens minimally impact disease-free and overall survival rates in non-responders or in women who experience relapse. A high degree of intra- tumoral heterogeneity was thought to, at least in part, underpin intrinsic or acquired chemoresistance for many TNBC tumors. Indeed, following neo-adjuvant chemotherapy, patients diagnosed with TNBC tumors showed decreased 3-year survival rates, and increased rate of recurrence and progression to metastatic disease following treatment compared to women with non-TNBC disease. The high degree of genomic instability for many TNBCs has informed pre- clinical studies to explore whether poly ADP ribose polymerase (PARP) inhibitors and/or immune checkpoint inhibitors represent promising classes of compounds for this subtype. Unfortunately, clinical trials have shown limited efficacy for either class of drugs regarding survival outcomes for women diagnosed with TNBC. There is therefore a need to identify targetable essential vulnerabilities that are not affected by inherent heterogeneity of TNBC malignancies and to thus develop a therapeutic agent that can target the vulnerability to treat the cancer.

SUMMARY

[0005] In one aspect, there is provided a compound of formula lb:

salt or solvate thereof.

[0006] In another aspect, there is provided an enantiomeric mixture comprising the compounds of formula la and lb:

salts or solvates thereof.

[0007] In a further aspect, there is provided a full racemate of the compound of formula I:

salt or solvate thereof.

[0008] In still a further aspect, there is provided a composition comprising the compound of formula, the enantiomeric mixture or the full racemate as defined herein. There is also provided a composition comprising a full racemate of the compound of formula I and a pharmaceutically acceptable excipient. There is further provided a composition comprising a racemate of the compounds of formula la and lb and a pharmaceutically acceptable excipient.

[0009] The compounds and compositions of the present disclosure are useful in the treatment of MYC positive cancers including lung cancer, leukemia, breast cancer, myeloproliferative disorders, colorectal cancer, medulloblastoma, renal, hepatocellular cancer, melanoma, ovarian cancer, prostate cancer, esophageal adenocarcinoma, liposarcoma, esophageal squamous cancer, gastrointestinal stromal tumor, glioma, myxofibrosarcoma, leiomyosarcoma, neuroblastoma, synovial sarcoma, mesothelioma, gastric cancer, thyroid cancer, lymphoma, osteosarcoma, rhabdomyosarcoma, fibrosarcoma, epithelial cancer, and neural cancer. In particular, the MYC positive cancer can be breast cancer. Many further features and combinations thereof concerning the present improvements will appear to those skilled in the art following a reading of the instant disclosure.

DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1 A is a graph assessing the compound-induced clamping of elF4A1 to fluorescein amidities (FAM) - labelled RNA of the indicated nucleotide composition. The AmP obtained with elF4A1 «ATP «poly (NN)s RNA was measured for the indicated compounds at 10 pM. The AmP obtained relative to dimethyl sulfoxide (DMSO) is shown. n=3 ± standard deviation (SD).

[0011] FIG. 1 B is a graph assessing compound-induced clamping of DDX3X to FAM-labelled poly(AG)8 RNA. The AmP was measured for 2 pM protein and 2 pM compound concentration. The AmP obtained relative to DMSO is shown. n=3 ± SD

[0012] FIG. 1C is a graph showing the relative dissociation of pre-formed elF4A1 «ATP«Cmpd*FAM-poly (AG)a complexes measured as a function of time in the presence of 1000-fold molar excess poly (AG)s RNA. DMSO, ti/2 ~4.1 ± 1 min; CR-1-31 B (10 pM), ti/2 ~59 ± 6.5 min; MG-002 (10 pM), ti/2 ~68 ± 2.8 min; eFT226 (10 pM), t-1/2 ~ 27 ± 5.8 min. Error values were calculated from the 95% confidence intervals.

[0013] FIG. 1 D is a graph of the differential scanning fluorimetry (DSF) analysis of elF4A1 (8 pM) in the presence of the indicated compounds (15 pM), 15 pM poly(AG)s, and 1 mM AMPPNP. The transition midpoint temperature shifts (AT50) are: CR-1-31 B, 7.3°C; eFT226, 7°C; MG-002, 7.2°C; PatA, 10°C. n=3 ± SD.

[0014] FIG. 1 E is a graph showing the concentration of MG-002, a potent inhibitor of capdependent translation. Inhibition of cap-dependent (FLuc) and independent (RLuc) translation was measured in response to the indicated compounds in Krebs-2 translation extracts programmed with the noted bicistronic mRNA. IC50s towards inhibition of FLuc synthesis from (CAG)-FF/HCV-IRES/Ren mRNA were: CR-1 -31 B, 54 ± 4 nM; eFT226, 813 ± 91 nM; MG-002, 43 ± 4 nM. n=2 ± SD.

[0015] FIG. 1 F is a schematic of bicistronic mRNA reporters with (AG)- or (UC)- enriched 5’ leader regions. [0016] FIG. 1G is a graph showing the translation response (AG)io of indicated reporter mRNAs used to program Krebs-2 translation extracts. FLuc on the left for each condition and RLuc on the right for each condition. n=3 ± SD.

[0017] FIG. 1 H is a graph showing the translation response (UC)io of indicated reporter mRNAs used to program Krebs-2 translation extracts. FLuc on the left for each condition and RLuc on the right for each condition. n=3 ± SD.

[0018] FIG. 11 is a bar graph showing that MG-002 is a potent inhibitor of cellular translation. eHAP1 cells were incubated in the presence of the indicated concentrations of compound for 1 h. During the last 15 min of incubation, ³⁵S-Met was added to the cells before harvesting, followed by trichloroacetic acid (TCA) precipitation and quantitation of ³⁵S-Met incorporation into protein. n=3 ± SD.

[0019] FIG. 1J is a graph showing the sucrose density gradient for polysomes isolated from eHAP1 cells exposed to vehicle or 15 nM MG-002 for 1 h.

[0020] FIG. 2A is a bar graph showing the time for MDA-MB-231 cells to enter mitosis following release from S-phase DNA synthesis block. Release was performed in the presence of DMSO or 10 nM of the indicated compounds. n=2 ± SD.

[0021] FIG. 2B is a bar graph showing the activity of CR-1 -31 B, eFT226, and MG-002 towards the TNBC cell line 4T1. Cells were exposed to the indicated concentrations of compound for 2 days and viability measured using the SRB assay. n=3 ± SD.

[0022] FIG. 2C is a bar graph showing the activity of CR-1 -31 B, eFT226, and MG-002 towards the TNBC cell line BT474. Cells were exposed to the indicated concentrations of compound for 2 days and viability measured using the SRB assay. n=3 ± SD.

[0023] FIG. 2D is a bar graph showing the activity of CR-1 -31 B, eFT226, and MG-002 towards the TNBC cell line MDA_MB-31 . Cells were exposed to the indicated concentrations of compound for 2 days and viability measured using the SRB assay. n=3 ± SD.

[0024] FIG. 2E is a graph showing the activity of CR-1 -31 B, eFT226, and MG-002 towards non-transformed, immortalized IMR-90 cells. Cells were exposed to the indicated concentrations of compound for 2 days and stained with SRB. n=3 ± SD. [0025] FIG. 2F is a graph showing the activity of CR-1-31 B, eFT226, and MG-002 towards non-transformed, immortalized MRC-5 cells. Cells were exposed to the indicated concentrations of compound for 2 days and stained with SRB. n=3 ± SD.

[0026] FIG. 2G is a graph showing the activity of CR-1-31 B, eFT226, and MG-002 towards non-transformed, immortalized HUVECs. Cells were exposed to the indicated concentrations of compound for 2 days and then stained with SRB. n=3 ± SD.

[0027] FIG. 2H is a Western blot analysis of the indicated proteins in cells exposed to MG- 002 (100 nM), CR-1-31 B (100 nM), or doxorubicin (Dox, 0.5 pg/ml) for 24 h.

[0028] FIG. 3A is a graph showing the activity of MG-002 cells towards parental eHAP1 or elF4A1^F163L/elF4A2- cells.

[0029] FIG. 3B is a schematic showing that in eHAP1 cells, rocaglate-induced clamping of elF4A1 to mRNAs leads to inhibition of translation and cell death.

[0030] FIG. 3C is a schematic showing that elF4A1 ^F163L/elF4A2^_ eHAP1 cells are significantly resistant to rocaglates as clamping of elF4A1^F163L to RNA is impaired.

[0031] FIG. 3D is a schematic showing an overexpression of wild type (wt) elF4A1 in elF4A1^F163L/elF4A2^_ cells is expected to re-sensitize these to rocaglate-induced translation inhibition and cell death.

[0032] FIG. 3E is a Western blot of proteins from stable cell lines ectopically expression elF4A1 , elF4A2, or DDX3X. Blots were probed with antibodies targeting the V5 tag or proteins indicated to the right.

[0033] FIG. 3F is a graph showing the cytotoxicity of elF4A1 ^F163L/elF4A2^_ eHAP1 cells ectopically expressing GFP, elF4A1 , elF4A2, or DDX3X following a two-day exposure to MG-002. n=3 ± SD.

[0034] FIG. 3G is a graph showing the response of wt eHAP1 cells ectopically expressing GFP, elF4A1 , elF4A2, or DDX3X following a two-day exposure to the indicated compound concentration. n=3 ± SD.

[0035] FIG. 3H is a Western blot of elF4A1^F163L/elF4A2^_ eHAP1 cells ectopically expressing GFP, elF4A1 , or elF4A3. Blots were probed with antibodies targeting the V5 tag or elF4A3. [0036] FIG. 31 is a graph showing the cytotoxicity of elF4A1 ^F163L/elF4A2' eHAP1 cells ectopically expressing GFP, elF4A1 , or elF4A3 following a two-day exposure to the indicated compound concentration. n=3 ± SD.

[0037] FIG. 3J is a graph showing the cytotoxicity of eHAP1 cells ectopically expressing elF4A1^F163L following a two-day exposure to MG-002. n=3 ± SD.

[0038] FIG. 3K is a graph showing the cytotoxicity of elF4A1^F163L/elF4A2^_ cells ectopically expressing elF4A1 ^F163L following a two-day exposure to MG-002. n=3 ± SD.

[0039] FIG. 4A is a graph showing the average plasma concentration of MG-002 at the indicated time points following delivery of 0.5 mg/kg MG-002 by oral gavage (PO).

[0040] FIG. 4B is a graph showing polysomes isolated from livers of mice treated with vehicle (control).

[0041] FIG. 4C is a graph showing polysomes isolated from livers of mice treated at the indicated time points following delivery of 0.5 mg/kg MG-002 PO (time point 7 h).

[0042] FIG. 4D is a graph showing polysomes isolated from livers of mice treated at the indicated time points following delivery of 0.5 mg/kg MG-002 PO (time point 24 h).

[0043] FIG. 4E is a bar graph showing the concentration of MG-002 in the indicated tissues 4 h after PO delivery of MG-002 (5 mg/kg). n=3 ± SD.

[0044] FIG. 4F shows blood cell counts from mice treated with vehicle or 0.5 mg/kg MG-002 PO on Mon/Wed/Fri for two consecutive weeks. n= 3 ± SD.

[0045] FIG. 4G is a complete blood count (CBC) analysis for the erythrocyte count from mice treated with vehicle or 0.5 mg/kg MG-002 PO Mon/Wed/Fri for two consecutive weeks. n=4-5 ± SD.

[0046] FIG. 4H is a complete blood count (CBC) analysis for the hemoglobin from mice treated with vehicle or 0.5 mg/kg MG-002 PO Mon/Wed/Fri for two consecutive weeks. n=4-5 ± SD.

[0047] FIG. 4I is a complete blood count (CBC) analysis for the hematocrit from mice treated with vehicle or 0.5 mg/kg MG-002 PO Mon/Wed/Fri for two consecutive weeks. n=4-5 ± SD. [0048] FIG. 4J is a complete blood count (CBC) analysis for the platelet count from mice treated with vehicle or 0.5 mg/kg MG-002 PO Mon/Wed/Fri for two consecutive weeks. n=4-5 ± SD.

[0049] FIG. 4K is a complete blood count (CBC) analysis for the reticulocytes count from mice treated with vehicle or 0.5 mg/kg MG-002 PO Mon/Wed/Fri for two consecutive weeks. n=4-5 ± SD.

[0050] FIG. 4L shows tissue weights from mice treated with vehicle or 0.5 mg/kg MG-002 PO Mon/Wed/Fri for two consecutive weeks, after which time organs were harvested. n=3 ± SD.

[0051] FIG. 4M here should we add FIG. 4C of the paper?!

[0052] FIG. 5A shows the gel of the puromycin assays assessing de novo protein synthesis in 4T1-526 cells treated with increasing concentration of MG-002 (1-50 nM) compared to vehicle control. Puromycin incorporation was visualized by immunoblot analysis using puromycin-specific antibodies and Ponceau S staining verified similar protein content between the various conditions.

[0053] FIG. 5B is a quantification of the data of Fig. 5A to determine the IC50 for the ability of MG-002 to inhibit protein synthesis in 4T1-526 cells.

[0054] FIG. 5C is a comparative to Fig. 5B with eFT226 instead of MG-002 in 4T1-526 cells.

[0055] FIG. 5D is a graph of the live cell counts using the IC50 concentrations calculated from

Figs. 5B-5C, MG-002 was tested for its ability to inhibit the growth of 4T1-526 cells after 72 hours in culture, compared to the vehicle control. The live cell count was determined by trypan blue exclusion. Representative of three independent biological replicates (n=3) is shown. Technical duplicate mean ± SD was plotted, and statistical analysis performed with a One-way ANOVA (Dunnett’s multiple comparisons test). *, p<0.05; **, p<0.01 .

[0056] FIG. 6A is a graph showing the tumour volume of primary tumor growth of lung metastatic 4T1-526 TNBC over time up to reaching ~100 mm³ in mouse.

[0057] FIG. 6B is a bar graph showing the change in mouse weight of the mice of Fig. 6A.

[0058] FIG. 6C is a bar graph showing the change in liver weight of the mice of Fig. 6A. [0059] FIG. 6D is a bar graph showing the change in % Ki67 positive cells in the mice of Fig. 6A.

[0060] FIG. 6E is a bar graph showing the change in % cleaved Casp3 positive cells in the mice of Fig. 6A.

[0061] FIG. 6F is a bar graph showing the change in % Myc positive pixels for the mice of Fig. 6A.

[0062] FIG. 6G is a graph showing the Pearson correlation examining the relationship between Myc positivity and the percentage Ki67 positive cells (individual vehicle-treated tumors are represented by circles; individual MG-002 treated tumors are represented by squares).

[0063] FIG. 6H is a graph showing the Pearson correlation examining the relationship between Myc positivity and the percentage of cleaved Casp3 positive cells (individual vehicle- treated tumors are represented by circles; individual MG-002 treated tumors are represented by square.

[0064] FIG. 7A is a graph showing the tumor volume overtime for a 4T1-526 mammary tumor that was allowed to develop in the mammary fat pads of BALB/c mice and when the tumors reached ~100 mm³ (arrow), the animals were randomized into three groups: 0.5 mg/kg MG-002, 0.5 mg/kg eFT226, or vehicle control with intraperitoneal administration.

[0065] FIG. 7B is a bar graph showing the change in weight for the mice of Fig. 7A.

[0066] FIG. 7C is a bar graph showing the change in liver weight at the end point of the mice of Fig. 7A.

[0067] FIG. 7D is a graph showing the tumor volume overtime for a 4T1-526 mammary tumor that was allowed to develop in the mammary fat pads of BALB/c mice and when the tumors reached ~100 mm³ (arrow), the animals were randomized into three groups: 0.5 mg/kg MG-002, 0.5 mg/kg eFT226, or vehicle control with intravenous administration.

[0068] FIG. 7E is a bar graph showing the change in weight for the mice of Fig. 7D.

[0069] FIG. 7F is a bar graph showing the change in liver weight at the end point of the mice of Fig. 7D. [0070] FIG. 7G is a graph showing the tumor volume overtime for a MDA-MB-231 mammary tumor that was allowed to develop in the mammary fat pads of BALB/c mice and when the tumors reached ~100 mm³ (arrow), the animals were randomized into two groups: 0.5 mg/kg MG-002 or vehicle control with intraperitoneal administration.

[0071] FIG. 7H is a bar graph showing the change in weight for the mice of Fig. 7G.

[0072] FIG. 7I is a bar graph showing the change in liver weight at the end point of the mice of Fig. 7G.

[0073] FIG. 8A is a schematic showing diagram outlining the experimental schedule to assess the ability of MG-002 to prevent the formation of spontaneous lung metastases from the primary tumor (MFP, mammary fat pad).

[0074] FIG. 8B is a bar graph showing the experimental results of 4T1- 526 following the schedule of Fig. 8A where mice were either treated by oral gavage with MG-002 (0.5 mg/kg), or vehicle control every three days until surgical resection. Lung (4T1-526) metastatic area was quantified by histology (15-16 mice/group) and the average metastatic burden is shown ± SD.

[0075] FIG. 8C is a bar graph showing the experimental results of MDA-MB-231 following the schedule of Fig. 8A where mice were either treated by oral gavage with MG-002 (0.5 mg/kg), eFT226 (0.5 mg/kg), or vehicle control every three days until surgical resection. Liver (MDA-MB- 231) metastatic area was quantified by histology (15-16 mice/group) and the average metastatic burden is shown ± SD.

[0076] FIG. 8D is a schematic diagram outlining the experimental schedule to assess the ability of MG-002 to treat established lung metastases following lung colonization.

[0077] FIG. 8E is a bar graph showing the experimental results of the schedule of Fig. 8D where mice were either treated by oral gavage with MG-002 (0.5 mg/kg) or vehicle control every three days until the experimental endpoint. The metastatic area in the lungs was quantified by histology and the average metastatic burden is presented ± SD.

[0078] FIG. 9A is a graph of tumor volume overtime. 4T1-526 mammary tumors were allowed to develop in the mammary fat pads of Balb/c mice and when tumors reached ~100 mm3, animals were randomized into four groups: (1) 0.5 mg/kg MG-002 PO; (2) 2.5 mg/kg doxorubicin IP; (3) MG-002/doxorubicin combination treatment using the same concentrations and (4) vehicle control. Animals were treated every three days until the experimental endpoint (n=10-14 tumors/group).

[0079] FIG. 9B is a bar graph showing the change in mouse weight of the mice of Fig. 9A.

[0080] FIG. 9C is a graph of tumour volume over time for the mice of Fig. 9A sacrificed four days after the start of drug treatment.

[0081] FIG. 9D is a bar graph showing the histology analysis results with Ki67 specific antibodies on the mice harvested in Fig. 9C.

[0082] FIG. 9E is a bar graph showing the histology analysis results with cleaved caspase-3 specific antibodies on the mice harvested in Fig. 9C.

[0083] FIG. 9F is a bar graph showing mice with established metastases that were randomized into four groups and treated as outlined in Fig. 9A. The metastatic area in the lungs was quantified by histology (n=16 mice/group) and the average metastatic burden is presented ± SD. Statistical analysis was performed with a Two-way ANOVA (Tukey’s multiple comparisons test).

[0084] FIG. 10A is a graph showing the activity of MG-002 towards primary human urothelial carcinoma cell line JP-C01. Cells were exposed to the indicated concentrations of compound for 2 days and stained with SRB. n=3 ± SD.

[0085] FIG. 10B is a graph of tumor volume over time of a patient-derived xenograft (PDX) model of human urothelial carcinoma JP-PDX01 grafted in the subcutaneous space of NSG mice, which were treated with either vehicle control or 0.0625 mg/kg MG-002 by oral gavage (n=3/group) every three days until the experimental endpoint. Two-way ANOVA; ***, p<0.001 .

[0086] FIG. 11 is an image showing the three dimensional structure determined for the compound of formula lb.

[0087] FIG. 12 is a graph showing the activity of MG-002 cells towards wild type eHAP1 or elF4A1^F163L/elF4A2- cells.

[0088] FIG. 13 is a graph showing the activity of a full racemate of compound I as assessed by luciferase activity (FF = firefly and Ren = Ren ilia) . [0089] FIG. 14 is a titration curve for MG-002 synthesized by Wuxi or Nuchem and for compound lb.

DETAILED DESCRIPTION

[0090] Dysregulation of mRNA translation is associated with tumor initiation, maintenance, and drug resistance. Unchecked translation in tumor cells can arise as a consequence of hyperactivation of MAPK or PI3K/Akt/mTOR pathways - both of which converge on eukaryotic initiation factor (elF) 4F to regulate its activity. Attempts to target the PI3K/Akt/mTOR pathway for the treatment of women diagnosed with TNBC have thus far failed in the clinic. The present disclosure instead focuses on the downstream effector, eukaryotic initiation factor (elF4F), which is responsible for pro-survival and proliferative outputs upon pathway activation. The elF4F complex contains the elF4E cap binding protein, the DEAD-box RNA helicase elF4A, and a large scaffolding protein, elF4G. elF4F catalyzes the recruitment of 40S ribosomes (and associated initiation factors) to mRNA 5’ cap structures. Mammalian cells encode three related elF4A proteins: (i) elF4A1 [DDX2A], (ii) elF4A2 [DDX2B], and (iii) elF4A3 [DDX48], elF4A1 and elF4A2 share 90% amino acid identity, can recycle through the elF4F complex, and are implicated in translation. DDX48 is a component of the exon junction complex (EJC) and shares 66% amino acid identity with elF4A1. elF4A1 is the more abundant and best studied family member. The requirement for elF4A in translation is directly proportional to cap-proximal secondary structure, which imparts unequal responses to different mRNAs following changes in elF4F levels/activity. For example, increased elF4F activity preferentially leads to the production of proteins regulating cell proliferation, survival, migration, and differentiation, as well as chemoresistance. This, in combination with high reliance of TNBC cells on elF4F activity, positions elF4F complex as a target to overcome intra-tumor heterogeneity. The compound of formula I has been identified and characterized as an inhibitor targeting the elF4A helicase subunit of elF4F. The compound for formula I has a plurality of possible stereoisomers. Two enantiomers were identified and are shown in formulas la and lb. It was found that formula lb and enantiomeric and stereoisomeric mixtures containing same are effective inhibitors targeting the elF4A helicase subunit of elF4F. Accordingly, in some embodiments, the compound of formula I refers to a stereoisomeric mixture that contains two or more stereoisomers, where at least one of the stereoisomers is the compound of formula lb. In some embodiments, the enantiomer mixture has four stereoisomers and includes both the compounds of formula la and lb. In other embodiments, there is provided an enantiomeric mixture containing both formula la and lb. In still further embodiments, there is provided a full racemate of the compound of formula I (which comprises formulas la and lb) as well as other possible stereoisomers. The full racemate can refer to a mixture containing all possible stereoisomers. In yet further embodiments, there is provided a racemic mixture of compounds la and lb. This racemic mixture is referred to herein as MG-002. The present compounds can be formulated as salts or solvates thereof and can be prepared as crystals suitable for pharmaceutical use

[0091] The compound of formula I is derived from rocaglates. Rocaglates (Roc) are cyclopenta[b]benzofuran natural products produced by plants of the Aglaia genus that inhibit eukaryotic protein synthesis. To date, several hundred natural and synthetic rocaglates have been isolated, synthesized, and studied. Rocaglates, such as Roc, target the translation process by acting as molecular staples to induce clamping of elF4A (and elF4F) to polypurine-rich RNA sequences.

[0092] Unscheduled rocaglate-induced clamping of elF4A1 to RNA leads to gain-of-function complexes that exert multiple effects on translation initiation. When elF4A«Roc«RNA complexes form in mRNA 5’ leader regions they can block ribosome scanning. In addition, elF4F«Roc«RNA complexes are not competent for ribosome recruitment. Blocking of scanning can also lead to 40S ribosome collisions, activating initiation ribosome-associated quality control (iRQC) and 40S ribosome degradation. It was presently found that rocaglates have an ability to induce RNA clamping which correlates well with inhibition of translation; however, there are notable exceptions. For example, a functional distinction is that CR-1-31 B exhibits clear selectivity for blocking translation of mRNAs with polypurine-rich 5’ leader regions (over those with polypyrimidine rich 5’ leaders); whereas, the same does not hold true for silvestrol which inhibits both class of reporters similarly. Moreover, RocA has been shown to also induce clamping of DDX3X to poly (AG) RNA, albeit with significantly lower affinity compared to elF4A1 .

[0093] Silvestrol and RocA are well studied rocaglates. They have been very useful in unraveling the mechanism of action for this class of compounds, they demonstrate cytotoxic selectivity towards transformed cells, and this class of compounds have anti-tumor activity. The complexity associated with silvestrol synthesis, as well as its in vivo conversion to inactive silvestric acid following intravenous administration, prompted synthetic exploration of the rocaglamide core for the generation of simpler compounds. Introduction of a hydroxamate group at the C-2 position yielded a series of bioactive molecules with improved activity, among which CR-1-31 B is the best characterized. The improved potency of CR-1 -31 B compared to RocA is likely related to the ability of the hydroxamate moiety to act as a hydrogen bond acceptor and/or as a bidentate chelating group, possibly stabilizing interactions with elF4A1 Q195 and D194. A second modification introduced to the rocaglate core that improved activity was addition of a nitrile group on one of the ring, which can form hydrogen bonds with N167 of elF4A1. The substitution of one of the aryl ring for a pyridine ring was found to lower lipophilicity of the core while maintaining n-stacking with RNA, leading to the development of a clinical candidate, eFT226 (Zotatifin). The compound I of the present disclosure incorporates the advantages including having the pyridine ring, the hydroxamate moiety and the nitrile group to arrive at an improved inhibitory activity.

[0094] There is provided a method of preventing, treating and/or alleviating the symptoms of a cancer in a subject in need thereof by administering the compound of formula lb or an stereoisomeric mixture comprising the same. In some embodiments, the cancer is characterized by cell proliferation dependent, driven or promoted by elF4A1 or elF4F. The compound of formula lb is an inhibitor of elF4A1 and elF4F, and therefore limits cell proliferation and tumor growth. The compound of formula lb or stereoisomeric mixtures comprising the same cause unscheduled and non-productive RNA clamping by the RNA helicase eukaryotic initiation factor (elF) 4A. Compound lb or stereoisomeric mixtures comprising the same potently inhibit mRNA translation initiation by preventing ribosome recruitment to mRNAs. Moreover, compound lb or stereoisomeric mixtures comprising the same have a potent efficacy in limiting primary TNBC tumor growth. Even more importantly, considering that metastatic spread is a major cause of mortality in TNBC, compound lb or stereoisomeric mixtures comprising the same attenuate metastatic spread. Compound lb or stereoisomeric mixtures comprising the same showed a superior translation inhibition activity compared to the compounds of the prior art, is orally bioavailable, and shows single agent efficacy toward primary and metastatic TNBC pre-clinical models. Moreover, compound lb or stereoisomeric mixtures comprising the same are particularly useful for the treatment of MYC positive cancers. The MYC oncogene has a central role in almost every aspect of the oncogenic process, orchestrating proliferation, apoptosis, differentiation, and metabolism. MYC positive cancers include lung cancer (small cell or non-small cell), leukemia (acute or chronic), breast cancer, myeloproliferative disorders (e.g. myelodysplasia and multiple myeloma), colorectal cancer, medulloblastoma, renal, hepatocellular cancer, melanoma, ovarian cancer, prostate cancer, esophageal adenocarcinoma, liposarcoma (pleomorphic or dedifferentiated), esophageal squamous cancer, gastrointestinal stromal tumor, glioma, myxofibrosarcoma, leiomyosarcoma, neuroblastoma, synovial sarcoma, mesothelioma, gastric cancer, thyroid cancer, lymphoma (e.g. non-Hodgkin’s lymphoma), osteosarcoma, rhabdomyosarcoma, fibrosarcoma, epithelial cancer (e.g. bladder, pancreatic, cervical, esophageal, endometrial, and head and neck), and neural cancer (e.g. meningioma, schwannoma, and retinoblastoma) (see Beroukhim, Rameen, et al. "The landscape of somatic copy-number alteration across human cancers." Nature 463.7283 (2010): 899-905). In some embodiments, the cancer is a breast cancer such as a triple negative breast cancer or is a metastatic cancer for example a cancer that metastasized into the lungs. More generally, the present disclosure provides a method for the prevention, treatment and/or alleviation of a cancer comprising administering to a subject in need thereof a therapeutically effective amount of the compound of formula lb or an stereoisomeric mixture comprising the same. The prevention of a metastatic cancer is particularly advantageous as metastasis is linked with poor prognosis. The prevention is only contemplated for breast cancer types having a known genetic factor that increases the likelihood of developing cancer. For example, the BRCA1 and BRCA2 genes are known to have an impact on an individual’s chances of developing breast cancer. The compound of the present disclosure can be used to prevent the cancer. The prevention of cancer as contemplated herein refers to patients in remission that are receiving treatments to maintain remission, in the context of a relapse following a remission or a partial remission.

[0095] Administration is by any of the routes normally used for introducing an agent into ultimate contact with blood. The agent described herein can be administered in any suitable manner, preferably with pharmaceutically acceptable carriers or excipients. An advantage of the compound of formula lb or stereoisomeric mixtures comprising the same is that they have an excellent oral bioavailability which allows the non-invasive and most convenient form of administration. The terms “pharmaceutically acceptable carrier”, “excipients”, “physiologically acceptable vehicle” and the like are to be understood as referring to an acceptable carrier that may be administered to a subject, together with the compound, and which does not impair the pharmacological activity thereof. Further, as used herein "pharmaceutically acceptable carrier" or "pharmaceutical carrier" are known in the art and include, but are not limited to, 0.01-0.1 M and preferably 0.05 M phosphate buffer or 0.9% saline. Additionally, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, collating agents, inert gases and the like.

[0096] In some embodiments, there is provided a pharmaceutical composition comprising a compound of formula lb and a pharmaceutically acceptable carrier. The pharmaceutical composition can be used in the prevention, alleviation or treatment methods described herein. As used herein, “pharmaceutical composition” means therapeutically effective amounts (dose) of the compound together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, and/or carriers. A “therapeutically effective amount” as used herein in the context of the pharmaceutical composition refers to that amount which provides a therapeutic effect for a given condition and administration regimen. Such compositions are liquids or lyophilized or otherwise dried formulations and include diluents of various buffer content (e.g., Tris-HCI, acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, and detergents (e.g., Tween 20™, Tween 80™, Pluronic F68™, bile acid salts). The pharmaceutical composition can comprise pharmaceutically acceptable solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol, parabens), bulking substances or tonicity modifiers (e.g., lactose, mannitol), covalent attachment of polymers such as polyethylene glycol, complexation with metal ions, or incorporation of the material into or onto particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance. Controlled or sustained release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils). Also contemplated by the present disclosure are particulate compositions coated with polymers (e.g., poloxamers or poloxamines).

[0097] The pharmaceutical composition further comprises or be administered in conjunction with one or more other therapeutic agents particularly in the case of metastatic cancer. Examples of other therapeutic agents include but are not limited to doxorubicin, dexamethasone, and cisplatinum. [0098] Suitable methods of administering the agent are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route. The preventive or therapeutic compounds of the present invention may be administered, either orally or parenterally, systemically or locally. In preferred embodiments, the compound of formula lb is administered orally for example in the form of a tablet ordroplets. Other examples include, intravenous injection such as drip infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection, suppositories, intestinal lavage, and the like.

[0099] The excipient(s) or carrier(s) must be “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of the formulation and not being deleterious to the recipient thereof. Standard accepted excipient(s) or carrier(s) are well known to skilled practitioners and described in numerous textbooks.

[0100] It will be clearto a person skilled in the art that the amount of the compound described herein and used in accordance with the disclosure (or if a further additional therapeutic agent is required or desired) can be determined by the attending physician or pharmacist. It will be appreciated that the amount of a compound required will vary not only with the particular compound selected but also with the route of administration, the extent of the condition for which treatment is required (e.g. stage I, II, III or IV of cancer) and the age and condition of the patient. It will be understood that the scope of the method of treatment or uses described herein is not particularly limited, but includes in principle any therapeutically useful outcome including preventing, treating or slowing the progression of conditions defined herein.

EXAMPLE 1

Materials

[0101] Unlabelled and 5’-end fluorescein amidities (FAM) labelled poly (AG)s and (UC)s RNAs were obtained from Integrated DNA Technologies (IDT). MG-002 was custom synthesized by WuXi Chemistry Services (China) and NuChem (Sygnature Discovery - Canada). eFT226 was also purchased from GLPBIO (Montclair, CA) or was synthesized by WuXi. CR-1-31 B was purchased from MedChem Express.

[0102] eHAP1 cells were obtained from Horizon Discovery and maintained in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 10% fetal bovine serum, 1 % penicillin- streptomycin antibiotics (Pen-Strep), and 2 mM L-glutamine at 37 °C and 5% CO2. 293T/17 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% bovine growth serum, 1 % penicillin-streptomycin (Pen-Strep), and 2 mM L-glutamine. Tests for mycoplasma contamination were routinely performed. MDA-MB-231 cells were obtained from the American Type Culture Collection (ATCC) and were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% bovine growth serum, 1 % Pen-Strep, and 2 mM L-glutamine. IMR-90 cells were obtained from ATCC and were grown in Eagles Minimum Essential Medium (EMEM) supplemented with 10% bovine growth serum, 1 % Pen-Strep, and 2 mM L-glutamine. MRC-5 were obtained from ATCC and were grown in Eagles Minimum Essential Medium (EMEM) supplemented with 10% bovine growth serum, 1 % Pen-Strep, and 2 mM L-glutamine. HUVEC cells were obtained from Lonza and propagated in Endothelial Basal Medium (EBM) supplemented with Endothelial Supplemental Mix.

Synthesis of the compounds

[0103] As previously stated, the compounds were synthesized by WuXi or NuChem. Many synthesis pathways are possible and the scheme below presents a non-limitative exemplary synthesis path. Specifically, the scheme below details the synthesis steps by WuXi to obtain a stereoisomeric mixture from which a racemate of the compounds of formula la and lb was extracted. As used in the present example, unless stated otherwise, the term MG-002 refers to the racemate of the compounds of formula la and lb. MG-002 racemate was used in the experiments of the present example unless stated otherwise.

Purification of recombinant elF4A1 Protein

[0104] BL21 (DE3) codon⁺ E. coli cells were transformed with pET15b-HiS6-elF4A1 , cultured at 37°C until the optical density (ODeoo) reached 0.6, at which point induction was undertaken by the addition of 1 mM (isopropyl-p-D-thiogalactopyranoside) IPTG at 37°C for 3 h. Recombinant HiS6-elF4A1 was purified on a Ni(2⁺)-NTA agarose column and the eluent applied to a Q- Sepharose fast flow matrix. The protein was eluted with a linear salt gradient (100-500 mM KCI), dialyzed against Buffer A (20 mM Tris-CI [pH 7.5], 10% glycerol, 0.1 mM ethylenediaminetetraacetic acid (EDTA)) overnight at 4°C, and stored in aliquots at -80°C.

Fluorescence polarization (FP) assay

[0105] FP assays were performed as per Chu J, Zhang W, Cencic R, O'Connor PBF, Robert F, Devine WG, et al. Rocaglates Induce Gain-of-Function Alterations to elF4A and elF4F. Cell Rep. 2020;30(8):2481-8 e5. Briefly, 1.5 pM recombinant elF4A1 protein was added to 10 nM FAM-labeled RNA in binding buffer (14.4 mM 4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid (HEPES) - NaOH [pH 8], 108 mM NaCI, 1 mM MgCI₂, 14.4% glycerol, 0.1 % dimethyl sulfoxide (DMSO), 2 mM dithiothreitol (DTT)) and 1 mM adenosine triphosphate (ATP) in the presence or absence of indicated compound in black, low volume 384 well plates (Corning 3820). Following assembly, binding reactions were incubated 30 min at room temperature (RT) in the dark, after which polarization values were determined on a Pherastar™ FS microplate reader (BMG Labtech). In experiments monitoring the dissociation of the elF4A1 «Roc«FAM-poly (AG)a complexes, after the initial 30 min incubation, reactions were supplemented with 1000-fold molar excess unlabelled poly (AG)s RNA and polarization measurements performed. The relative dissociation was measured as a function of time. The half-lives of complexes were calculated using the “one phase decay” method on Graph Pad.

Differential scanning fluorimetry (DSF)

[0106] Experiments were performed as described in Liu T, Nair SJ, Lescarbeau A, Belani J, Peluso S, Conley J, et al. Synthetic silvestrol analogues as potent and selective protein synthesis inhibitors. J Med Chem. 2012;55(20):8859-78. Briefly, 8 pM of recombinant elF4A1 was incubated with compound (15 pM) or DMSO in DSF buffer (20 mM HEPES [pH 7.5], 70 mM KCI, 2 mM DTT, 1 mM Mg(OAc)₂, 1 mM AMPPNP, 7.5X Sypro Orange (S-6650, Thermo Fisher), and 15 pM poly (AG)s RNA. The samples were heated and read from 37°C to 75°C using 1 °C/min ramp rate in the CFX96 Touch™ Real-Time PCR Detection System (BioRad).

Cell viability assays and EC50 determination

[0107] Cell viability assays were performed using sulforhodamine B reagent (SRB). Cells (5000/well) were seeded into 96 well plates and treated with compound at the indicated concentrations. Cells were then washed with phosphate buffered saline (PBS) and fixed with 50% trichloroacetic acid (TCA) for 1 h at 4°C, rinsed with water and air-dried. Fixed cells were stained with 0.5% SRB/1 % acetic acid for 1 h, plates were washed with 1% acetic acid four times, and dried. The bound SRB was resuspended in 100 pl 20mM Tris base (pH adjusted to 9) and the OD550nm was measured using a SpectraMax™ M5 microplate reader (Molecular Devices). Relative viability was calculated by normalizing to the DMSO control.

In vitro translations

[0108] In vitro translations were performed using 5 ng/pL of reporter mRNA and the indicated compound concentrations in Krebs-2 extracts at 30°C for 1 h, as described in Novac O, Guenier AS, and Pelletier J. Inhibitors of protein synthesis identified by a high throughput multiplexed translation screen. Nucleic Acids Res. 2004;32(3):902-15. FLuc and RLuc luciferase activities were assessed on a Berthold Lumat LB 9507 luminometer (Berthold Technologies). Half maximal inhibitory concentrations (IC50) were determined using a non-linear regression model on GraphPad Prism 8.4.0.

Immunoblotting

[0109] Cells were pelleted, washed in PBS and lysed with radioimmunoprecipitation assay buffer (RIPA) buffer (20 mM Tris-HCI [pH 7.6], 100 mM NaCI, 1 mM EDTA,1 mM ethylene glycol- bis(p-aminoethyl ether (EGTA), 1% Thermo Scientific Surfact-Amps NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 20 mM B-glycerophosphate, 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride (PMSF), 4 pg/mL aprotinin, 2 pg/mL leupeptin, 2 pg/mL pepstatin). Cell lysates were collected after centrifuging the samples at 16000 xg for 10 mins, resolved on a 10% SDS-polyacrylamide gel, and transferred to polyvinylidene fluoride (PVDF) membrane (Bio-Rad). Antibodies against elF4A1 (ab31217) and elF4A2 (ab31218) were obtained from Abeam, anti-eEF2 antibodies (#2332), anti-V5 tag (#13202), anti-MYC (#5605), and anti-PARP (#9542) antibodies were obtained from Cell Signaling Technologies. Anti-elF4A3 (HPA021878) antibody was obtained from Atlas Antibodies. Anti-DDX3X antibody was obtained from Novus Biologicals.

Time-lapse microscopy for cell cycle analysis

[0110] Eighty thousand MDA-MB231 cells were seeded in 12-well plates in DMEM supplemented with 10% FBS and antibiotics. Twenty-four hours later, cells were treated with 2.5 mM thymidine dissolved in media to synchronize them in S phase, as described in Whitfield ML, Sherlock G, Saldanha AJ, Murray JI, Ball CA, Alexander KE, et al. Identification of genes periodically expressed in the human cell cycle and their expression in tumors. Mol Biol Cell. 2002;13(6):1977-2000. Twenty hours later, cells were washed twice with warm PBS and released into fresh media that contained either DMSO or 10 nM of CR-1-31-B, MG-002, or eFT226. Following drug addition, cells were placed in an incubation chamber of a Zeiss Axiovert 200M microscope to maintain temperature and CO2 levels. Bright-field imaging was performed. Images were taken every 10 minutes at 10x total magnification for a total of 40 hours. For each condition, 50 cells were followed from the time of starting the experiment to the time they entered mitosis (minutes to mitotic entry), which roughly represent the time they spent in late S and G2 phase of the cell cycle. A one-way ANOVA with Tukey multiple comparisons test was used to compare the average minutes to mitotic entry for each treatment to the vehicle control.

Sensitization assay

[0111] Lentivirus (pLX-TRC317) harboring cDNAs to elF4A1 (TRCN0000491404) and elF4A3 (TRCN0000471580) were obtained from the Genetic Perturbation Service (GPS) of the Goodman Cancer Research Center and McGill Biochemistry Dept. The elF4A1 cDNA was cloned into pLX-TRC317 and DDX3X was cloned into pPRIME-CMV-GFP-PGK-Puro-recipient. Virus was produced in 293T cells and used to infect parental or elF4A1^F163L/elF4A2- eHAP1 cells. Following puromycin selection (2 pg/pL for 2 days), cells were expanded for experiments.

Bioanalytic analysis

[0112] Analytical methodology development, plasma protein binding, simulated gastric fluid stability, and pharmacokinetic studies were performed by the Platform of Biopharmacy at the Universite de Montreal. CBC analyses were performed by the Comparative Medicine and Animal Resources Centre (CMARC) at McGill University.

Polysome profiles

[0113] Polysomes from livers were isolated from mice that had received 0.5 mg/kg MG-002 PO. At 7h and 24h, animals were sacrificed, and the livers excised and washed in cold PBS containing 100 pg/mL cycloheximide. Tissue was homogenized in an Eurostar Power-b homogenizer (IKA Liver Labortechnik, Staufen, Germany) in 3 volumes of lysis buffer (40 mM HEPES [7.5], 100 mM KCI, 5 mM MgCh, 100 pg/mL cycloheximide). Homogenates were centrifuged for 10 min at 1 ,200xg at 4°C. Triton X-100 and sodium deoxycholate were added to the supernatant to a final concentration of 0.5%. Samples were loaded on 10-50% sucrose gradients and centrifuged in an SW40 rotor at 35,000 rpm for 2 h. Gradients were collected using a Brandel tube piercer and delivering 60% sucrose through the bottom of the centrifuge tube. Recording of the data was performed using InstaCal™ Version 5.70 and TracerDaq™ Version 1 .9.0.0 (Measurement Computing Corporation, Norton, MA).

³⁵S-Met labelling

[0114] To measure protein synthesis, 60,000 cells/well were seeded in a 24-well plate. The following day, the medium was removed, cells washed with PBS and incubated in methionine- free DMEM supplemented with 10% dialyzed serum for 1 hr. For the last 15 min, cells were labeled with ³⁵S-methionine. Medium was removed, cells washed in PBS and lysed in RIPA buffer (20 mM Tris [pH 7.5], 100 mM NaCI, 1 mM EDTA, 1 mM EGTA, 0.1 % NP-40, 0.5% sodium desoxycholate, 0.1 % SDS, 20 mM B-glycerophosphate, 10 mM NaF, 1 mM PMSF, 4 pg/mL aprotinin, 2 pg/mL leupeptin, 2 pg/mL pepstatin) for 20 min with shaking at 4°C. The protein was applied to Whatman 3M paper, precipitated with TCA, washed, and radioactivity quantitated by scintillation counting. Protein content in the RIPA extracts was determined using the Bio-Rad DC ProteinAssay (Bio-Rad Laboratories) and used to standardize the TCA precipitable counts.

Animal studies

[0115] Balb/c and SCID-beige female mice (8-10 weeks old) were purchased from Charles River Laboratories (Quebec, Canada). 4T1-526 (50,000 cells) or MDA-MB-231 (1x10⁶ cells) were injected into the mammary fat pads of Balb/c or SCID-Beige mice, respectively as described. For the experimental metastasis assays, 1x10⁵ 4T1-526 cells were injected into the tail vein of animals. All mice had ad libitum access to food and water and were housed within the animal facilities of the Lady Davis Institute, on a 12h light day cycle, mean temperature 22.5 C ± 1 .5 °C. As indicated, MG-002 and eFT226 were injected either intraperitoneally, intravenously or by oral gavage (0.5 mg/kg every three days). Urothelial carcinoma patient-derived xenograft (PDX) JP- PDX01 were established and viably preserved at McGill University, were cut into pieces and then inserted into a pocket in the subcutaneous space of NSG mice. Mice were randomized to treatment arms once the average tumor volume reached at least 100 mm³. MG-002 was resuspended in carrier (PBS) and administered by PO at 0.0625 mg/kg (every three days). Tumor volume and mouse weight were measured twice to thrice weekly. Tumor volume was calculated as length*(width)²*0.52. Animal studies were approved by the Animal Resource Centre at McGill University in accordance with guidelines from the Canadian Council of Animal Care. MG-002 induces elF4A1'RNA clamping to potently inhibit translation

[0116] The RNA clamping activity of rocaglates can be conveniently measured using the fluorescence polarization (FP) assay. It was found that like CR-1-31 B, both eFT226 and MG-002 discriminated between poly (AG)s and poly (UC)8 RNA templates (Fig. 1A). As previously noted, the unrelated natural product, pateamine A (PatA), stabilized binding of elF4A1 to both polypurine and polypyrimidine RNA templates. Relative to elF4A, the tested rocaglates only minimally influenced DDX3X:RNA clamping (Fig. 1 B). The stabilizing effects of these compounds on elF4A1 «poly(AG)8’ATP complexes were then assessed. The dissociation of pre-formed complexes was monitored following addition of excess unlabelled RNA. Complexes formed with CR-1-31 B or MG-002, in the presence of ATP, had a ~2 - fold longer half-life than those containing eFT226 (Fig. 1 C). Stabilization of elF4A1 by CR-1 -31 B, eFT226 and MG-002 was independently confirmed by differential scanning fluorimetry (DSF) (Fig. 1 D). This approach monitors temperature-dependent protein unfolding, an event that is attenuated by target/ligand complex formation. A transition midpoint temperature shift (AT50) of ~7°C was noted, consistent with the ability of CR-1 -31 B, eFT226 and MG-002 to induce RNA clamping to elF4A1 .

[0117] In in vitro translation assays, CR-1 -31 B and MG-002 were similarly effective at inhibiting cap-dependent translation from a reporter harboring a 37% (AG)-rich 5’ leader region (Fig. 1 E). eFT226 was the least active of the three tested compounds (Fig. 1 E). MG-002, like CR- 1 -31 B, also preferentially inhibited translation of an mRNA reporter enriched for (AG)-repeats, relative to a reporter with a (UC)-rich 5’ leader (Figs. 1 F-1 H). As expected, all three compounds preferentially targeted cap-dependent translation versus hepatits C virus (HCV) internal ribosome entry site (IRES) driven translation. After 1 h of exposure of eHAP1 cells to MG-002 a robust inhibition of protein synthesis was observed, as assessed by ³⁵S-Met metabolic labelling (Fig. 11) and polysome profiling (Fig. 1J). Taken together, these results indicate that MG-002 stabilizes elF4A1-RNA complexes, preferentially inhibits mRNAs with polypurine leader regions, and is a potent inhibitor of translation in vitro and in cellula.

Biological activity of MG-002

[0118] The anti-tumorigenic properties of MG-002 towards TNBCs were assessed. Exposure of MDA-MB-231 cells to MG-002 or CR-1 -31 B caused a profound delay in G2/M progression (Fig 2A), consistent with the previously reported activity of rocaglates against other cell lines. The consequences of prolonged exposure (2 days) to MG-002 was then assessed against two TNBC lines (4T1 , MDA-MB-231) and a model of ER+/HER2+ breast cancer (BT-474) to evaluate the generalizability of our findings (Figs. 2B-2D). In all instances, cell viability was compromised with half maximal effective concentration (EC50) values for MG-002 ranging from ~1 -10 nM, indicating potent activity. When these same compounds were tested against non-transformed, immortalized cells (IMR90, MRC5) (Figs. 2E-2F) or primary endothelial cells (HUVEC) (Fig. 2G), only a modest cytostatic effect was observed as the number of viable cells never fell below 50%, even at high 3 pM concentrations. Reduction in MYC protein which is encoded by elF4F-sensitive mRNA was observed in MDA-MB-231 , 4T1 and MRC5 cells exposed to MG-002 or CR-1-31 B (but not if exposed to doxorubicin [Dox]) (Fig. 2H). Cleavage of PARP was observed when MDA-MB-231 or 4T1 cancer cells were exposed to MG-002, CR-1-31 B, or DXR but not in MRC5 cells treated with MG-002 or CR-1 -31 B, consistent with the cell viability studies (Fig. 2H). In summary all three compounds are comparable in their ability to preferentially inhibit the growth of breast cancer cells, relative to non-transformed cells, in cellula.

MG-002 cytotoxicity is elF4A1 and elF4A2 dependent

[0119] Rocaglates primarily exert their biological effects through elF4A1 engagement. However, RocA can induce RNA clamping of DDX3X, although the binding is at least 30-fold lower than for elF4A1. To determine if MG-002 exerted its cytotoxic effects through elF4A1/2 engagement, HAP1 cells expressing either wild-type elF4A or an elF4A1 ^F163L mutant were tested to determine which is unable to bind the rocaglate compound. It was found that elF4A1 ^F163L/elF4A2^_ Hap1 cells were significantly more resistant to MG-002 when compared to those expressing the wild-type protein (Fig. 3A). It was rationalized that it is possible to further engineer elF4A1 ^F163L/elF4A2^_ cells to ectopically express wild-type elF4A1 , elF4A2, elF4A3, or DDX3X; which should re-sensitize them to MG-002 induced cytotoxic activity due to compound- induced clamping of wild-type proteins (Figs. 3B-3D). elF4A1 ^F163L/elF4A2^_ cells ectopically expressing elF4A1 or elF4A2 indeed showed increased sensitivity to MG-002 (Figs. 3E and 3F). Sensitization to MG-002 was not observed with parental eHAP1 cells ectopically expressing elF4A1 or elF4A2 (Fig. 3G), suggesting that elF4A1 is not limiting for target engagement in these cells. Sensitization was also not observed in elF4A1 ^F163L/elF4A2^_ cells ectopically expressing DDX3X (Fig. 3F) or elF4A3 (Figs. 3H and 3I). Sensitization was dependent on the presence of wild-type elF4A1 in this assay since the ectopic expression of elF4A1 ^F163L failed to sensitize elF4A1 ^F163L/elF4A2^_ cells to MG-002 (Figs. 3J and 3K). Taken together, these results indicate that MG-002 is primarily exerting its cytotoxic effects through elF4A1/2 engagement. MG-002 is an orally bioavailable inhibitor of translation

[0120] MG-002 shows similar mouse and human plasma protein binding as eFT226 (Table

1). MG-002 was also found to be stable in simulated gastric fluid (pH 1.2) (Table 1), prompting the investigation of its plasma concentration following drug delivery by oral gavage (PO). Surprisingly, it was found that PO delivery resulted in sustained presence of MG-002 in the plasma, with a terminal half-life of ~ 10 h (Fig. 4A, Table 1). The consequences of PO delivery on translation in vivo was assessed by analyzing liver polysomes following compound delivery by gavage (Figs. 4B-4D). The results indicate strong suppression of translation in the liver following PO delivery of MG-002 as evidenced by >3 fold decrease in polysome-to-monosome ratio 7h post-delivery, followed by partial translational recovery 24h post-treatment. MG-002 distribution to plasma, spleen, lungs, and liver was also quite extensive 4h post-delivery PO (Fig. 4E). MG- 002 administration was also well tolerated, with no observable impact on blood cells (Figs. 4F- 4K), organ weights (Fig. 4L), total body weights. Histopathological analysis of heart, lung, spleen, kidney, and bone marrow following six treatments with MG-002 did not uncover any overt tissue damage (Table 2). Based on these encouraging results, it was decided to undertake studies to assess the anti-cancer activity of MG-002 in vivo towards TNBC.

Table 1 . Plasma protein binding, acid stability, and plasma half-life for MG-002

a Cmpd (50 pM) was incubated in Simulated Gastric Fluid (SGF) containing pepsin at pH 1 .2 for 1 h at 37°C. b Delivered PO 5 mg/kg. n=3 ± standard deviation (SD). c Delivered IV 0.5 mg/kg. n=3 ± SD.

Table 2. Histopathological Analysis following PO Delivery of MG-002 (0.5 mg/kg) for 6 treatments (M/W/F). Tissues were harvested 12 days after the first injection

N: Normal

F: Finding

Grade 1 = modest, rare

Grade 2 = mild, infrequent

Grade 3 = moderate, frequent

Grade 4 = severe, diffuse

MG-002 inhibits tumor cell growth in vivo following PO delivery

[0121] The results described above indicate a potent activity of MG-002 towards TNBC lines in vitro, which was then confirmed in vivo. 4T1-526 lung metastatic breast cancer cells were selected as they represent an immunocompetent model of TNBC disease. They also represent a 4T1 subline that was selected in vivo for its propensity to metastasize to the lungs. Respectively, MG-002 and eFT226 showed IC50 values of ~ 7 and 8 nM for inhibition of translation towards 4T1 -526 cells, as assessed by puromycin incorporation (Figs. 5A-5C). Significant cell death was observed following chronic exposure of 4T1-526 cells to MG-002 and eFT226; with MG-002 being the more potent molecule (Fig. 5D). [0122] Following mammary fat pad injection, tumor-bearing mice were randomized into three groups when 4T1 -526 tumors reached a volume of ~100 mm³. Cohorts were then treated by oral gavage (PO) with MG-002, eFT226, or vehicle control until the experimental endpoint (Fig. 6A). The results indicated that MG-002 more effectively impaired mammary tumor growth when compared to eFT226 (Fig. 6A) without adversely affecting total body (Fig. 6B) or liver weights (Fig. 6C) in tumor-bearing animals. Immunohistochemical analysis further revealed that MG-002 treatment significantly reduced proliferation (Fig. 6D), increased apoptotic cell death (Fig. 6E), and diminished Myc protein levels (Fig. 6F), which were used as a surrogate marker for “elF4F- sensitive” translation. Despite some variability in Myc protein levels between individual tumors, elevated Myc levels positively correlated with increasing Ki67 positivity and inversely correlated with the percentage of cleaved Caspase 3 positive cells (Figs. 6G-6H).

[0123] The differences in response to oral delivery of MG-002 and eFT226 prompted us to compare the in vivo response of both drugs following intraperitoneal or intravenous delivery (Figs. 7A-7I). Here, it was found that both MG-002 and eFT226 similarly delayed tumor growth (Figs. 8A-8C). An anti-neoplastic effects of intraperitoneal delivery of MG-002 against MDA-MB-231 tumors was observed, representing a model of human TNBC (Fig. 8D). Hence, the inferior response of eFT226 following PO delivery in vivo is not due to inherent differences in potency compared to MG-002, which were additionally confirmed in cellula (Figs. 2A-2H and 8E). The results demonstrate that MG-002 is effective when administered by oral gavage and appears well tolerated.

Activity of MG-002 towards metastatic TNBC

[0124] The ability of MG-002 to accumulate in lung tissue following PO delivery (Fig. 4E), a frequent site of breast cancer metastasis, prompted us to assess the efficacy of MG-002 towards metastatic lesions. It was first assessed whether neo-adjuvant MG-002 treatment could prevent the formation of spontaneous lung and liver metastases (Figs. 8A-8C). 4T1-526 or MDA-MB-231 tumor bearing mice were treated with MG-002, eFT226 or vehicle control (0.5 mg/kg every 3 days) and primary tumors were subsequently surgically resected at a volume of 500 mm³. Drug treatment was terminated following tumor resection and the animals were followed for the development of lung (4T1-526) and liver (MDA-MB-231) metastases. It was found that MG-002 was effective as a monotherapy at reducing the formation of the murine spontaneous 4T1 -526 lung metastases (Fig. 8B). Furthermore, in the human metastatic MDA-MB-231 TNBC xenograft model, MG-002, but not eFT226, significantly decreased the formation of spontaneous liver metastases (Fig. 8C). Next, the ability of MG-002 to shrink breast cancer metastases that had already colonized the lungs was assessed (Fig. 8D). 4T1-526 breast cancer cells were injected into the tail vein to directly seed the lung. Following ten days post-injection to ensure lung colonization, treatment with MG-002 (0.5 mg/kg every 3 days) or vehicle was initiated over a 21 day period. It was thus found that MG-002 is associated with reduced lung metastatic burden, compared to vehicle control treated mice, although these differences were not statistically significant (Fig. 8E). Thus, MG-002 is likely not sufficient as a monotherapy to treat individuals who present with distant metastases but metastatic cancer are considerably harder to treat and will require are known to require a multi-faced approach including more than one treatment agent. Combined, the present data suggest that MG-002 monotherapy demonstrates anti-metastatic properties, primarily in preventing the likelihood that a patient will progress to metastatic disease.

[0125] In addition to breast cancers, MG-002 was also tested as a monotherapy to target urothelial carcinoma. The consequences of prolonged exposure (2 days) to MG-002 was first assessed against one primary urothelial carcinoma cell line established from a metastatic tumor and cell viability was compromised with half maximal effective concentration (EC50) value for MG- 002 ~10 nM, indicating potent activity similar to other cancer cells tested (Fig. 2, 3). Furthermore, in a patient-derived xenograft (PDX) model of urothelial carcinoma stablished from a metastatic tumor, treatment of mice with MG-002 as a monotherapy at a reduced dosage (0.0625 mg/kg, every 3 days) strongly suppressed tumor growth (Fig. 10B). This was also confirmed in vitro with the compound of formula lb only rather than MG-002 (Fig. 10A).

MG-002/doxorubicin combination therapy reduces primary and metastatic disease burden

[0126] Aggressive breast cancers are likely to require combination therapies to exploit the therapeutic benefit of MG-002. This was assessed by combining MG-002 with doxorubicin (Dox); a front-line chemotherapy that is already standard-of-care for TNBC. It was therefore investigated whether a combination of MG-002/Dox might be more effective than either single compound as monotherapy. Following mammary fat pad injection of 4T1-526, either the monotherapy or the combination treatment was started when tumors were ~ 100 mm³. Tumors were equally responsive to Dox or MG-002, but significantly more sensitive to combination treatment (Fig. 9A). Combination treatment did not lead to significant weight loss in treated animals, although animals failed to gain weight during treatment (Fig. 9B). To better understand the basis for the improved anti-neoplastic activity of MG-002/Dox combination treatment, breast tumors were harvested early during their treatment schedule, at a timepoint when reduced tumor volumes was first observed with MG-002 as a monotherapy, and an even further reduction with MG-002/Dox combination treatment (Fig. 9C). Immunohistochemistry (IHC) analysis at these time points revealed no significant differences in the rates of cancer cell proliferation, with either MG-002 or doxorubicin single or combination treatments (Fig. 9D). However, it was observed that MG-002 treatment increased the rate of apoptosis in breast tumors, which was even more pronounced following MG-002/Dox treatment (Fig. 9E). Finally, treatment of mice already bearing lung metastases with MG-002/Dox combination therapy significantly reduced the lung tumor burden compared to animals treated with either drug as a monotherapy (Fig. 9F). Combined, these results support that more effective cytotoxic responses towards primary TNBC tumors and TNBC metastatic lesions are obtained with a combination of MG-002/Dox treatment.

Discussion

[0127] Cancer cells universally activate signal transduction pathways that upregulate the activity of protein synthesis machinery and/or perturb the expression of genes encoding the components of the translational apparatus to reprogram and augment translation. As a result, pharmacological inhibition of mRNA translation initiation has great therapeutic potential. To date, most of the clinical trial studies have focused on mTOR inhibitors, which function upstream to prevent elF4F complex formation. In breast cancer, mTORCI inhibitors have been extensively studied, at least in hormone receptor (HR) positive breast tumors, starting with the BOLERO-2 trial. This phase III randomized study that compared the effects of everolimus and exemestane (an aromatase inhibitor) with exemestane alone in women with advance HR+ disease. Study results revealed a transient and marginal (4 month) improvement in progression-free survival in these women, which was further substantiated with subsequent phase II studies combining everolimus with other estrogen receptor-targeted therapies. Although few studies have examined the efficacy of mTOR inhibitors in treating triple negative breast cancers, these clinical trials suggest that inhibitors of elF4F activity would be superior in sustaining prolonged anti-neoplastic functions in this already more heterogenous and poor outcome subtype. In this regard, elF4A is an attractive therapeutic target for treating TNBC tumors given that human ER negative primary breast cancers overexpress elF4A compared to other breast cancer subtypes. Herein it is reported an orally bioavailable, rocaglate-inspired molecule targeting elF4A1 and elF4A2 that inhibits TNBC cell proliferation, induces cell death, and sensitizes to chemotherapy. Importantly, MG-002 also significantly delayed the growth of primary TNBC tumors and impaired the formation of spontaneous lung metastases. Furthermore, MG-002 inhibited urothelial carcinoma cell proliferation in vitro and as a monotherapy, at a reduced dosage, strongly suppressed the growth of primary urothelial carcinoma in vivo. Moreover, MG-002 is likely to exert cytotoxic effects in other malignancies given promising pre-clinical studies showing that other elF4A inhibitors elicit strong anti-neoplastic effects in models of leukemia and pancreatic cancer.

[0128] As has been noted for other rocaglates, MYC expression is quite sensitive to MG-002 treatment. Since MYC protein has a short half-life, levels are rapidly depleted from cells when exposed to MG-002. MYC is a well-known cancer driver, promoting tumorigenesis through a variety of mechanisms including stimulating cell proliferation, altering metabolism, inhibiting cell death, and impacting the tumor microenvironment. Genetic modelling of both the therapeutic impact and side effects of systemic MYC suppression in the mouse has shown profound anticancer activity, as well as the tolerability of such an approach. Indeed, exogenous administration of a MYC dominant-negative (Omomyc) is efficacious against TNBC and displays antimetastatic properties. However, targeting MYC for cancer treatment had proven quite challenging. The sensitivity of c-MYC translation to MG-002 offers a powerful approach by which to rapidly block MYC expression.

[0129] It is presently shown that MG-002, functions as a molecular staple and clamps elF4A onto RNA with poly(AG)-rich tracts. Cells encode two elF4A homologs, elF4A1 and elF4A2, both of which are targets for rocaglates. In general, elF4A1 is the more abundant isoform (58, 59) and overall levels of elF4A1 and elF4A2 in a tumor cell may contribute to MG-002 sensitivity. This was modeled by re-expressing either elF4A1 or elF4A2 wild-type proteins in elF4A1 F163L/elF4A2- eHAP1 cells (Fig. 3). Since elF4A1 is essential (60), loss of elF4A1 can never be a mechanism of rocaglate resistance.

[0130] Clear differences in activity between MG-002 and eFT226 were noted. The eFT226 compound is not as potent as MG-002 at inhibiting translation when compared to MG-002 (Figs. 1 B, and 1 E-1 G). Although eFT226 is slightly better at clamping elF4A to poly(AG) RNA sequences (Fig. 1 A), the data shows that the complexes formed are not as long lived and show a half-life that is approximately 2.5-fold less than those formed in the presence MG-002 (Fig. 1 C). This latter point may explain the lower potency at inhibiting protein synthesis on the part of eFT226. M-002 was also more effective than eFT226 towards TNBCs in general in vitro (Figs. 2A-2H). Lastly, when delivered by gavage, eFT226 was less potent that MG-002. In particular as shown in Fig. 2A, MG-002 outperformed CR-1-31 B and eFT226 when evaluating the effect on cell division and time to mitotic entry. [0131] Identified in a yeast-based screen for rocaglate-resistant alleles, there are missense mutations in six amino acids that could confer resistance to two rocaglates. These are: T158, P159, F163, F192, Q195, and 1199 (human amino acid numbering used). From the crystal structure of elF4A1 bound to RocA, two of these amino acids, F163 and Q195, make direct contacts with RocA. A search of COSMIC (Catalogue of Somatic Mutations in Cancer) revealed only a single missense mutation (F192C) arising in one tumor, a skin adenocarcinoma. Hence mutations in elF4A1 that would confer resistance to MG-002 are unlikely to be pre-existing in a tumor cell population. Should such mutations be selected for following exposure to drug, the present data suggests that all alleles of elF4A1 and elF4A2 would have to be compromised in order to confer MG-002 resistance since the presence of the wild-type protein should still be sufficient to maintain sensitivity to MG-002. Hence, it is unlikely that missense mutations simultaneously targeting all alleles of elF4A1 and elF4A2 could easily arise to confer drug resistance. DDX3X has also been identified as a target of RocA, although the affinity is at least 30-fold lower. It was found that MG-002 did not induce significant clamping to DDX3X (Fig. 1 B). In fact, it was shown that DDX3X cannot re-sensitize cells to the cytotoxic effects of MG-002 (Figs. 3A-3K), suggesting that DDX3X does not play a significant role (if any) in the anti-tumor activity of MG-002.

[0132] Suppression of the elF4E cap-binding protein is effective at reducing metastatic breast cancer cell migration and invasion in vitro, decreasing both pulmonary colonization and metastasis growth by breast cancer cells. This correlates with a reduction in the translation of mRNAs encoding proteins of the metastatic cascade. Silvestrol was shown to inhibit primary MDA-MB-231 tumor outgrowth in a xenograft setting and in vitro could also inhibit breast cancer cell migration and invasion. The present results highlight the value of elF4A as a therapeutic target in vivo, as well as offer a strategy to directly inhibit mRNA translation by targeting elF4A activity. Studies testing other rocaglates have shown that elF4A inhibition increases the radiosensitivity of tumors and also improves the response to targeted therapies, including MEK and CDK4/6 inhibitors. Herein, it is shown that elF4A inhibitors, and MG-002 specifically, increase responsiveness to standard-of-care chemotherapies, including doxorubicin. In conclusion, MG- 002 is an orally available rocaglate capable of reducing primary tumor growth and blocking metastatic seeding.

EXAMPLE 2 [0133] Example 1 demonstrated the activity of MG-002 which is a racemic mixture of the compounds of formula la and lb. Example 1 also demonstrated the activity of the compound of formula lb alone (Fig. 10A). The specific structure of the compounds and enantiomeric mixtures were investigated to confirm chirality, particularly for the active compound lb.

[0134] The compound of formula lb, crystallized from DMF/H2O, was collected from a shock- cooled single crystal at 150 K on a Bruker™ Venture Metaljet K-geometry diffractometer with a Metal Jet using a Helios™ MX Mirror Optics as monochromator and a Bruker™ CMOS Photon III detector. The diffractometer was equipped with an Oxford Cryostream™ 700 low temperature device and used Ga K_a radiation (A = 1 .34139 A). All data were integrated with SAINT and a multiscan absorption correction using SADABS was applied. The structure was solved by intrinsic phasing methods with XT and refined by full-matrix least-squares methods against F² using XL within the graphical user interface of OLEX2. All non-hydrogen atoms were refined with anisotropic displacement parameters. The hydrogen atoms were refined isotropically on calculated positions using a riding model with their U_ISO values constrained to 1 .5 times the L/_eq of their pivot atoms for terminal sp³ carbon atoms and 1 .2 times for all other carbon atoms. The Crystallographic Information Framework (CIF) file was generated using FinalCif™. The three dimensional structure identified is shown in Fig. 11 . Ellipsoids were drawn at the 50% probability level and hydrogen atoms are shown as sphere of arbitrary size.

[0135] The compound of formula lb was confirmed to contain the correct structure by nuclear magnetic resonance (NMR) and liquid chromatography mass spectroscopy (LCMS). The ¹H NMR spectra obtained was as follows: ¹H NMR (CHCls-d, 400 MHz): 5H 8.55 (1 H, br s), 7.39-7.41 (2H, m), 7.29-7.31 (2H, m), 7.07-7.09 (3H, m), 6.93 (2H, br s), 6.08 (1 H, s), 4.96 (1 H, br s), 4.32 (1 H, br s), 4.02 (3H, s), 3.94 (3H, s), 3.50-3.84 (5H, br m), 1.97 (1 H, br s).

[0136] The experiment performed in Fig. 3A of Example 1 above was repeated with the racemate MG-002 that was confirmed by crystallography as explained above. Similar results were obtained (see Fig. 12) indicating that the racemate used in Example 1 is the same as the structure confirmed in present Example 2.

[0137] A FLuc and Rluc luciferase assay was performed as described in Example 1 a full racemate of the compound of formula I containing four stereoisomers, two of which were compounds la and lb and two had an unconfirmed structure (Fig. 13). Firefly (FF) and Renilla (Ren) luciferase reporter genes were used to determine expression. As can be seen from Fig. 13, the full racemate was active.

[0138] Half maximal inhibitory concentrations (IC50) were determined using a non-linear regression model as explained in Example 1. The titration graph is shown in Fig. 14 and was produced following the experimental protocol detailed in Example 1 . The IC50 for the MG-002 racemate as obtained by Wuxi was 8.8 nM, for the MG-002 racemate as obtained by NuChem it was 11.8 nM and for the purified compound lb it was 7.9 nM.

Claims

WHAT IS CLAIMED IS:

1 . A compound of formula lb:

salt or solvate thereof.

2. An enantiomeric mixture comprising the compounds of formula la and lb:

salts or solvates thereof.

3. A full racemate of the compound of formula I:

salt or solvate thereof.

4. A composition comprising the compound of formula lb as defined in claim 1 , the enantiomeric mixture as defined in claim 2, or the full racemate as defined in claim 3 and a pharmaceutically acceptable excipient.

5. A composition comprising a full racemate of the compound of formula I:

and a pharmaceutically acceptable excipient.

6. A composition comprising a racemate of the compounds of formula la and lb:

a pharmaceutically acceptable excipient.

7. The compound as defined in claim 1 , for use in the treatment of a MYC positive cancer.

8. The compound as defined in claim 1 , for use in the manufacture of a medicament for treating a MYC positive cancer.

9. The compound of claim 7 or 8, wherein the MYC positive cancer is selected from lung cancer, leukemia, breast cancer, myeloproliferative disorders, colorectal cancer, medulloblastoma, renal, hepatocellular cancer, melanoma, ovarian cancer, prostate cancer, esophageal adenocarcinoma, liposarcoma, esophageal squamous cancer, gastrointestinal stromal tumor, glioma, myxofibrosarcoma, leiomyosarcoma, neuroblastoma, synovial sarcoma, mesothelioma, gastric cancer, thyroid cancer, lymphoma, osteosarcoma, rhabdomyosarcoma, fibrosarcoma, epithelial cancer, and neural cancer.

10. The compound of claim 9, wherein the MYC positive cancer is breast cancer or pancreatic cancer.

11 . The composition as defined in any one of claims 4 to 6, for use in the treatment of a MYC positive cancer.

12. The composition as defined in claim 11 , wherein the MYC positive cancer is selected from lung cancer, leukemia, breast cancer, myeloproliferative disorders, colorectal cancer, medulloblastoma, renal, hepatocellular cancer, melanoma, ovarian cancer, prostate cancer, esophageal adenocarcinoma, liposarcoma, esophageal squamous cancer, gastrointestinal stromal tumor, glioma, myxofibrosarcoma, leiomyosarcoma, neuroblastoma, synovial sarcoma, mesothelioma, gastric cancer, thyroid cancer, lymphoma, osteosarcoma, rhabdomyosarcoma, fibrosarcoma, epithelial cancer, and neural cancer.

13. The composition of claim 12, wherein the MYC positive cancer is breast cancer.

14. Use of a compound as defined in claim 1 for treating a MYC positive cancer.

15. Use of a compound as defined in claim 1 for the manufacture of a medicament for treating a MYC positive cancer.

16. Use of a composition as defined in any one of claims 4 to 6 for treating a MYC positive cancer.

17. Use of a composition as defined in any one of claims 4 to 6 for the manufacture of a medicament for treating a MYC positive cancer.

18. The use of any one of claims 14 to 17, wherein the MYC positive cancer is selected from lung cancer, leukemia, breast cancer, myeloproliferative disorders, colorectal cancer, medulloblastoma, renal, hepatocellular cancer, melanoma, ovarian cancer, prostate cancer, esophageal adenocarcinoma, liposarcoma, esophageal squamous cancer, gastrointestinal stromal tumor, glioma, myxofibrosarcoma, leiomyosarcoma, neuroblastoma, synovial sarcoma, mesothelioma, gastric cancer, thyroid cancer, lymphoma, osteosarcoma, rhabdomyosarcoma, fibrosarcoma, epithelial cancer, and neural cancer.

19. The use of claim 18, wherein the MYC positive cancer is breast cancer or pancreatic cancer.

20. A method of treating a MYC positive cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the compound as defined in claim 1.

21. A method of treating a MYC positive cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition as defined in any one of claims 4 to 6.

22. The method of claim 20 or 21 , wherein the MYC positive cancer is selected from lung cancer, leukemia, breast cancer, myeloproliferative disorders, colorectal cancer, medulloblastoma, renal, hepatocellular cancer, melanoma, ovarian cancer, prostate cancer, esophageal adenocarcinoma, liposarcoma, esophageal squamous cancer, gastrointestinal stromal tumor, glioma, myxofibrosarcoma, leiomyosarcoma, neuroblastoma, synovial sarcoma, mesothelioma, gastric cancer, thyroid cancer, lymphoma, osteosarcoma, rhabdomyosarcoma, fibrosarcoma, epithelial cancer, and neural cancer.

23. The method of claim 22, wherein the MYC positive cancer is breast cancer or pancreatic cancer.