Methods of treating ovarian and pancreatic cancer

Abstract

The present application relates to small molecule compounds useful for treating cancer, pharmaceutical compositions containing such compounds. The invention also describes methods for treating cancer in a subject in need thereof, comprising administering to the subject, a therapeutically effective amount of a compound of the present invention to a subject in need thereof. The cancers to be treated include ovarian cancers and pancreatic cancers.

Description

METHODS OF TREATING OVARIAN AND PANCREATIC CANCERCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority under 35 U. S. C. § 119(e) to U. S.Provisional Application No. 63 / 717,728, filed November 7, 2024. The disclosure of the prior application is considered part of and is herein incorporated by reference in the disclosure of this application in its entirety.INCORPORATION OF SEQUENCE LISTING

[0002] The material in the accompanying sequence listing is hereby incorporated by reference into this application. The accompanying sequence listing xml file, name 395334-000033_SL.xml, was created on November 7, 2024, and is 5,077 bytes.FIELD OF THE INVENTION

[0003] The present invention relates generally to small molecules and more specifically to the use thereof to treat cancer.BACKGROUND INFORMATION

[0004] The Gl-S phase cell cycle transition is governed by two cyclin- CDK complexes, cyclin D-CDK4 / 6 and cyclin E- CDK2. Cyclin D-CDK4 / 6 (hereafter CDK4 / 6) phosphorylates the G1 gatekeeper Rb, causing the release of S-phase specific transcription factors, such as E2F. E2F causes the transcriptional induction of Cyclin E, which in turn partners with CDK2 to further phosphorylate Rb and irreversibly cause the transition into S-phase. Cyclin DI and CDK4 / 6 are overexpressed in a variety of human cancers, and, in mouse models, loss of either prevents the development of certain oncogene-driven tumors. Targeting CDK4 / 6 activity has been a longstanding goal in the oncology field and because CDK4 / 6 is downstream of most oncogenic signaling pathways, targeting this kinase might prevent the resistance that frequently occurs when cell surface or upstream signal transducers are inhibited. The advent of CDK4 / 6 specific inhibitors (CDK4i), such as Palbociclib (PD 0332991, hereafter PD), Abemaciclib, or Ribociclib has demonstrated that CDK4 / 6 is a promising target. In combination with Letrozole, PD extended median Progression Free Survival (PFS) for metastatic breast cancer patients from 10.2 to 20.2 months (PALOMA trial). However, the overall survival (OS) of patients treated with PD mirrored that seen in patients treated with Letrozole alone, suggesting that resistance to this combinationtherapy occurs. In tissue culture lines, PD- or Ribociclib-mediated arrest did not appear durable either. While loss of Rb appears to distinguish primary PD-non-responsiveness in cell lines, differences in Ki67, cyclin D, CDK4 / 6, and pl6 do not appear able to stratify responsive and non-responsive subgroups.

[0005] Cyclin D is a transcriptional target of the MAPK pathway, but after cyclin D partners with CDK4 / 6, the dimer is unstable, and rapidly dissociates back into the monomeric forms, unless a third protein, p27Kipl (hereafter p27) or p21Cipl, holds the complex together. However, p27 binds to D-K4 in two different conformations: a closed and inactivating conformation or alternatively, an open and activating form. This transition is mediated by the tyrosine (Y) phosphorylation of p27 on residues Y88 (or Y89) and also on residue Y74 which causes a conformational change, opening the D-K4-p27 ternary complex, thus rendering it able to phosphorylate substrates such as Rb. Non-phosphorylated p27-D-K4 complexes are catalytically inactive because the associated p27 both blocks the ATP binding site on CDK4 / 6 and prevents the required CDK Activating Kinase (CAK) phosphorylation of the CDK4 / 6 domain itself. p27 Y88 is phosphorylated by the Y kinase Brk (Breast tumor Related Kinase or PTK6, Protein Tyrosine Kinase 6) and can be phosphorylated by other non-receptor bound tyrosine kinases including Src and Abl, and interaction between Brk and p27 is mediated though Brk’s SH3 domain and a proline-rich binding site in p27. Addition of Brk SH3 -containing peptides in vitro blocks this interaction, preventing p27 Y88 phosphorylation, which in turn causes inhibition of CDK4 / 6. Overexpression of a naturally occurring ALTternatively-spliced form of Brk (ALT), which contains Brk’s SH3 domain, but lacks the SHI kinase domain, also inhibits Brk’s phosphorylation of p27, inhibits CDK4 / 6, and causes growth arrest, suggesting that inhibition of p27 Y88 phosphorylation might be an alternative way to target CDK4-dependent tumors.

[0006] Data suggests that the CDK4i palbociclib associates with the CDK4 / 6 free monomer. While it might seem unusual that a kinase inhibitor does not associate with the active form of the kinase, association with monomeric CDK4 / 6 would reduce the amount of the ternary complex as well, freeing p27 to be able to associate with and inhibit CDK2. In contrast to CDK4 / 6, CDK2 does not require p27 to stabilize the interaction with its cyclin; actually CDK2’s phosphorylation of RB is inhibited whenever p27, phosphorylated or not phosphorylated, is associated with the complex. But, even when unable to phosphorylate RB, Y-phosphorylated p27-cyclin E- CDK2 complexes are able to phosphorylate p27 on residue T187, which in vivo, results in decreased p27 stability as it becomes a target for ubiquitin-mediated degradation, reducing p27 association with CDK2, and indirectly activating the cyclin E- CDK2 complex. Thus, blocking pY88 might havethe added benefit of preventing p27 degradation and stabilizing p27 in the non-phosphorylated form, permitting it to inhibit CDK2 as well as CDK4 / 6. The root of resistance to CDK4 / 6 inhibiting therapies, such as PD, is unknown, but one candidate that could compensate for loss of CDK4 / 6 activity is CDK2, so a therapy that inhibits both kinases at the outset might offer therapeutic advantages.

[0007] Ovarian cancer is a cancerous tumor of an ovary which may originate from the ovary itself or more commonly from communicating nearby structures such as fallopian tubes or the inner lining of the abdomen. In 2020, new cases occurred in approximately 313,000 women. In 2019 it resulted in 13,445 deaths in the United States. Ovarian cancer is the second-most common gynecologic cancer in the United States. It causes more deaths than any other cancer of the female reproductive system. Among women it ranks fifth in cancer-related deaths. The typical age of diagnosis is 63. Pancreatic cancer is among the deadliest forms of cancer globally, with one of the lowest survival rates. In 2015, pancreatic cancers of all types resulted in 411,600 deaths globally. Pancreatic cancer is the fifth-most-common cause of death from cancer in the United Kingdom, and the third most-common in the United States. The disease occurs most often in the developed world, where about 70% of the new cases in 2012 originated. Pancreatic adenocarcinoma typically has a very poor prognosis; after diagnosis, 25% of people survive one year and 12% live for five years. For cancers diagnosed early, the five-year survival rate rises to about 20%. Neuroendocrine cancers have better outcomes; at five years from diagnosis, 65% of those diagnosed are living, though survival considerably varies depending on the type of tumor.

[0008] Growing knowledge of the molecular underpinnings comprising the etiology of cancer has driven the field of personalized or “precision” medicine to identify specific tumor characteristics and exploit these features by developing targeted therapies against these entities. However, there is still an unmet need for personalized medicine that specifically targets cancer cells and their ability to circumvent cell cycle regulation.SUMMARY

[0009] The present invention relates to novel compounds and pharmaceutical compositions used for the treatment of cancer in a subject in need thereof. Additional information relevant to the present disclosure can be found in International Patent Application No. PCT / US2024 / 29543, which is hereby incorporated by reference in its entirety.

[0010] In one embodiment, the invention provides a method of treating ovarian cancer in a subject including administering to the subject a therapeutically effective amount of apharmaceutical composition including a compound selected fromthereby treating ovarian cancer in the subject.

[0011] In one aspect, the compound inhibits phosphorylation of p27. In another aspect, the compound inhibits CDK2 and CDK4 / 6. In one aspect the compound inhibits ovarian cancer cell proliferation and / or decreases ovarian cancer cell viability. In another aspect, the compound increases ovarian cancer cell death.

[0012] In another embodiment, the invention provides method of treating pancreatic cancer in a subject including administering to the subject a therapeutically effective amount of a pharmaceutical composition including a compound selected fromthereby treating pancreatic cancer in the subject.

[0013] In one aspect, the compound inhibits phosphorylation of p27. In another aspect, the compound inhibits CDK2 and CDK4 / 6. In one aspect the compound inhibits pancreatic cancer cell proliferation and / or decreases pancreatic cancer cell viability. In another aspect, the compound increases pancreatic cancer cell death.

[0014] In various aspects, the methods described herein further include administering to the subject an anti-cancer treatment. In one aspect, the anti-cancer treatment is selected from the group consisting of chemotherapy, radiation treatment, immunotherapy, resection of a tumor, and any combination thereof. In another aspect, the anti-cancer treatment is administered prior to, simultaneously with, or after administration of the pharmaceutical composition. In one aspect, the anti-cancer treatment is an anti-cancer agent selected from the group consisting of palbociclib, ribociclib, abemaciclib, osirmetinib, gefitinib, lapatinib, pantitumumab, vandetanib, necitumumab, vemurafenib, sorafenib tosylate, PLX-4720, dabrafenib, paclitaxel, cisplatin,docetaxol, carboplatin, vincristine, vinblastine, methotrexate, cyclophosphamide, CPT-11, 5-fluorouracil, gemcitabine, estramustine, carmustine, adriamycin, etoposide, arsenic trioxide, irinotecan, Herceptin, vemurafenib, erlotinib, cetuximab, letrozole, fulvestrant, epolhilone derivatives, elacestrant, lasofoxifene, anastrozole, sotorasib, and adagrasib. trametinib, cobmetinib, Bay-293, everolimus, erolintib, cetaxumab, panitumumab, and afatinib, imatinib, sunitinib, panatinib, axitinib, foretinib, nintedanib, and amuvatinib, adavorsertib, pertuzumab, am-trastuzumab-deruxtecan, T-DM1 or ado-trastuzumab emtansine, pertuzumab / trastuzumab / hyaluronidase, neratinib), and tucatinib.

[0015] In one aspect, the methods described herein further include administering to the subject a second CDK2-selective inhibitor. In some aspects, the CDK2-selective inhibitor is PF-07104091.

[0016] In one aspect, the subject is a human. In another aspect, administration of the pharmaceutical composition is intracutaneous, subcutaneous, intravenous, intraperitoneal, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transdermal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoidal, intraspinal, intrasternal, oral, sublingual, buccal, rectal, vaginal, nasal or ocular, or by infusion, inhalation, or nebulization.BRIEF DESCRIPTION OF DRAWINGS

[0017] FIG. 1 shows the dose dependent binding of His-p27 binding to CDK4 / 6 / CyclinDl in a TR-FRET deconvolution assay.

[0018] FIGs 2A-2B show the effect of altering pH and buffer on binding and kinetics His-p27 to CDK4 / 6 / CyclinDl in a TR-FRET deconvolution assay. FIG. 2Aillustrates the effect of change in pH on assay signal. FIG. 2Billustrates the effect of Buffer change on assay kinetics.

[0019] FIGs 3A-3C; FIG. 3A shows Kd Assessment His-p27 binding to GST-tagged CDK4 / 6 / CyclinDl. FIG. 3B shows IC50 determination of no-tag p27 competing with his-p27 in binding to GST-CDK4 / 6 / CyclinDl. FIG. 3C shows IC50 assessment of no-tag CDK4 / 6 / Cyclin DI competing with GST variant to bind his-p27.

[0020] FIG. 4 shows TR-FRET analysis of ALT-BRK binding to free p27 (square) or p27 in complex with CDK4 / 6 / CyclinDl (dots). ALT bound to free p27 and p27 / CDK4 / 6 / Cyclin DI ternary complex with Kd of 27.16 nM and 32.65 nM, respectively.

[0021] FIG. 5 shows signal specificity in ALT-b inding assay. Addition of no-tagged p27 abolished ALT binding interaction with the ternary complex.

[0022] FIGs 6A-6C; FIG. 6 A shows assessment of assay activity in increasing concentrations of NP40. FIG. 6B shows assay activity levels in the presence of increasing concentrations of Tween20. FIG. 6C shows assay sensitivity to increasing concentrations of DMSO.

[0023] FIG. 7 shows peptide inhibition for ALT:p27 / CDK4 / 6 / Cyclin DI in an interaction assay.

[0024] FIG. 8 shows the Brk phosphorylation assay in the presence and absence of the CDK4 / 6 / Cyclin DI complex. p27 is phosphorylated by BRK in the absence of CDK4 / 6 / Cyclin DI in a dose-dependent manner as detected by the phospho pY88 antibody in a TR-FRET format. p27 phosphorylation is much slower in the presence of CDK4 / 6 / Cyclin DI ternary complex.

[0025] FIG. 9 shows CDK4 / 6 / Cyclin DI inhibition of p27 phosphorylation by BRK. p27 phosphorylation by BRK is inhibited by increasing concentrations of CDK4 / 6 / Cyclin DI with an IC50of 164.6 nM.

[0026] FIG. 10 shows p27 phosphorylation under different conditions.

[0027] FIG. 11 shows Tilfrinib inhibition (a potent BRK kinase inhibitor) in BRK TR-FRET kinase assay using p27 as substrate.

[0028] FIGs 12A-12C show isolation of BRK-phosphorylated p27. FIG. 12A shows a Coomassie blue stain of BRK phosphorylated p27 on phosphoprotein column. FIG. 12Bshows Western blot using anti-pTyr Ab; detecting p27 and pBrk. FIG. 12C shows non phosphorylated p27 loaded onto Pro-Q column does not bind.

[0029] FIGs 13A-13C depict: FIG. 13A which illustrates a screening assay: TR-FRET Assay for ALT-BRK Binding to p27 / Cyclin D1 / CDK4 / 6 Ternary Complex (ALT:p27 / CDK4 / 6 / Cyclin DI). FIG. 13B shows a deconvolution assay: TR-FRET Assay for p27 Binding to Cyclin D1 / CDK4 / 6 (p27: CDK4 / 6 / Cyclin DI). FIG. 13C shows a validation assay: TR-FRET assay for BRK phosphorylation of p27 on residue Y88 (Brk Kinase Assay).

[0030] FIGs 14A-14C show GST-PreScission-BRK (1-451) is highly expressed. FIG. 14A shows expression 48h after infection. FIG. 14B shows expression 72h after infection. FIG. 14C shows pull-down after 48h.

[0031] FIG. 15 shows a GST-PreScission-Rbc(773-928) is highly expressed.

[0032] FIGs 16A-16D show GST-PreScission-CDK4 / 6 / GST-PreScission-Cyclin DI Complex Production. FIG. 16A shows the results 48h post infection. FIG. 16B shows a comparison between the pull-down and the pull-down + cleavage. FIG. 16C shows the GST-CDK4 pull-down. FIG. 16D shows the GST-cyclin DI pull-down.

[0033] FIGs 17A-17C: FIG. 17A shows CDK4 / 6 / Cyclin DI Complex Production [tag cleaved]. FIG. 17B shows p27Kipl (1-198) Production [tag cleaved]. FIG. 17C shows GST-PreScission-p27Kipl(l-198) is highly expressed.

[0034] FIG. 18 shows a graph of the results of the TR-FRET Assay for ALT BRK Binding to p27 / CDK4 / 6 / Cyclin D Ternary Complex (Screening Assay) showing the IC50 of compounds L3, 14, 39, 40, 35A, 35, and 34, CCL20 peptide, CCL28 peptide variant and endogenous ALT.

[0035] FIG. 19 is a graph of the results of BRK phosphorylation of p27 on residue Y88 (Brk Kinase Assay) showing the IC50 of compounds L3, 14, 39, 40, 35A, 35, and 34, CCL20 peptide, CCL28 peptide (negative control) and endogenous ALT.

[0036] FIG. 20 shows a graph of % Brk kinase activity (polyE4Y substrate, Y axis) to concentration of compound (x-axis). Tilfrinib, a potent Brk inhibitor, was used as a positive control in this assay

[0037] FIGs 21A-21D shows graphs from the Thermal Shift Assay: FIG. 21A shows the starting temperature of p27, CDK4 / 6 / Cyclin D and p27 / CDK4 / 6 / Cyclin D. FIG. 21B is a graph of the maximum temperature increase of p27, CDK4 / 6 / Cyclin D and p27 / CDK4 / 6 / Cyclin D when compounds Palbociclib (CDK4 / 6 inhibitor, CDK4i), L-9 (+ve control) and L-8 (-ve control) were added, wherein an increase in temperature is indicative of binding. FIG. 21C is a graph of the maximum temperature increase of p27 / CDK4 / 6 / Cyclin D Trimer when compounds Palbociclib, L3, 14, 39, 40, 35A, 35, and 34 were added in a cell-free thermal shift assay, wherein an increase in temperature is indicative of binding. FIG.21D is a graph of the maximum temperature increase of CDK4 / 6 / Cyclin D Dimer when compounds Palbociclib, L3, 14, 39, 40, 35A, 35, and 34 were added in a cell-free thermal shift assay, wherein an increase in temperature is indicative of binding.

[0038] FIGs 22A-22W shows graphs from the BrdU Incorporation Assay of ER+ human breast tumor cell, T47D (n=3), where the y- axis is the BrdU incorporation (% to DMSO control) vs. concentrations of various compounds. In FIG. 22A the compound is the vehicle control DMSO, and palbociclib. In FIG. 22B the compound is DMSO. In FIG. 22C the compound is compound L2. In FIG. 22D the compound is compound LI. In FIG. 22E the compound is compound L4. In FIG. 22F the compound is compound L6. In FIG. 22G the compound is compound L3. In FIG. 22H the compound is compound L5. In FIG. 221 the compound is compound 3. In FIG. 22 J the compound is compound 14. In FIG. 22K the compound is compound 13. In FIG. 22L the compound is compound 39. In FIG. 22M the compound is compound 16. In FIG. 22N the compound is compound 40. In FIG. 220 the compound is compound 20. In FIG. 22P the compound is compound 17A. In FIG. 22Q the compound iscompound 17B. In FIG. 22R the compound is compound 26. In FIG. 22S the compound is compound 27. In FIG. 22T the compound is compound 35 A. In FIG. 22U the compound is compound 35. In FIG. 22V the compound is compound 34. In FIG. 22W the compound is compound L7.

[0039] FIGs 23A-23I show graphs from the BrdU Incorporation Assay of ER+ human breast tumor cells that have been conditioned to become resistant to palbociclib, T47D PalboR (n=3), where the y- axis is the BrdU incorporation (% to DMSO control) vs. concentration of various compounds. In FIG.23A the compound is palbociclib tested in T47D cells (Palbociclib sensitive) and T47D PalboR cells (palbociclib resistant). In FIG.23B the compound is DMSO. In FIG.23C the compound is compound L3. In FIG. 23D the compound is compound 14. In FIG. 23E the compound is compound 39. In FIG. 23F the compound is compound 40. In FIG. 23G the compound is compound 35 A. In FIG. 23H the compound is compound 35. In FIG. 231 the compound is compound 34.

[0040] FIGs 24A-24I show graphs from the BrdU Incorporation Assay of ER- human normal breast cells, MCF10A(n=3), where the y- axis is the BrdU incorporation (% to DMSO control) vs. concentration of various compounds. In FIG. 24A the compound is palbociclib. In FIG. 24B the compound is DMSO. In FIG. 24C the compound is compound L3. In FIG. 24D the compound is compound 14. In FIG. 24E the compound is compound 39. In FIG. 24F the compound is compound 40. In FIG. 24G the compound is compound 35A. In FIG. 24H the compound is compound 35. In FIG. 241 the compound is compound 34.

[0041] FIGs 25A-25I show graphs from the BrdU Incorporation Assay of ER+ mouse normal breast cells, HC1 l(n=2), where the y- axis is the BrdU incorporation (% to DMSO control) vs. concentration of various compounds. In FIG. 25A the compound is palbociclib. In FIG. 25B the compound is DMSO. In FIG.25C the compound is compound L3. In FIG.25D the compound is compound 14. In FIG. 25E the compound is compound 39. In FIG. 25F the compound is compound 40. In FIG. 25G the compound is compound 35A. In FIG. 25H the compound is compound 35. In FIG. 251 the compound is compound 34.

[0042] FIGs 26A-26B show graphs of the BrdU Incorporation Assay. FIG. 26A shows that addition of 10 pM compounds L3, 14, 39, 40, 35A, 35 and 34 enhance response of palbociclib at 50nM concentration in T47D cells. FIG 26B shows that addition of 10 pM compounds L3, 14, 39, 40, 35 A, 35 and 34 enhance response of ribociclib at lOOnM concentration in T47D cells.

[0043] FIGs 27A-27B: FIG 27A shows a graph of the cell cycle phases 48 hours posttreatment of T47D cells with 15 pM compounds L3, 14, 39, 40, 35A, 35 and 34 or 400nM ofpalbociclib (PD). Gap 1 stage (Gl), synthesis stage (S), gap 2 and mitosis stages (G2 / M) and Sub-G1 which represents apoptotic cells are illustrated. FIG. 27B shows a graph of the cell cycle phases 48 hours post-treatment of T47D cells with 30 pM compounds L3, 14, 39, 40, 35A, 35 and 34 or 400nM of palbociclib (PD). Gap 1 stage (Gl), synthesis stage (S), gap 2 and mitosis stage (G2 / M) and Sub-Gl which represents apoptotic cells are illustrated.

[0044] FIGs 28A-28I show graphs from the cell viability assay of ER+ human breast tumor cells, T47D (n=2) at 48 hr or 72 hr where the Y-axis represents the percent viability (ATP) relative to control (DMSO treated cells) and the X axis represents concentration of compounds. In FIG.28A the compound is cisplatin. In FIG.28B the compound is DMSO. In FIG.28C the compound is compound L3. In FIG. 28D the compound is compound 14. In FIG. 28E the compound is compound 39. In FIG. 28F the compound is compound 40. In FIG. 28G the compound is compound 35 A. In FIG. 28H the compound is compound 35. In FIG. 281 the compound is compound 34.

[0045] FIGs 29A-29I show graphs from the cell viability assay of ER+ mouse normal breast cells, HC 11 (n=2) at 48 hr or 72 hr where the Y -axis represents the percent viability (ATP) relative to control (DMSO treated cells) and the X axis represents concentration of compounds. In FIG.29A the compound is cisplatin. In FIG.29B the compound is DMSO. In FIG.29C the compound is compound L3. In FIG. 29D the compound is compound 14. In FIG. 29E the compound is compound 39. In FIG. 29F the compound is compound 40. In FIG. 29G the compound is compound 35 A. In FIG. 29H the compound is compound 35. In FIG. 291 the compound is compound 34.

[0046] FIGs 30A-30I show graphs from the cell viability assay of ER- human normal breast cells MCF10A(n=l) at 48 hr or 72 hr where the Y-axis represents the percent viability (ATP) relative to control (DMSO treated cells) and the X axis represents concentration of compounds. In FIG. 30A the compound is cisplatin. In FIG. 30B the compound is DMSO. In FIG. 30C the compound is compound L3. In FIG. 30D the compound is compound 14. In FIG. 30E the compound is compound 39. In FIG. 30F the compound is compound 40. In FIG. 30G the compound is compound 35 A. In FIG. 30H the compound is compound 35. In FIG. 301 the compound is compound 34.

[0047] FIG. 31 shows Western blots of (A) phospho-retinoblastoma tumor suppressor protein (Rb) (Ser807 / 811), (B) Rb, (C) phospho-CDK2 (Thrl60), (D) CDK2 and (E) beta-actin from T47D cell lysates treated with vehicle DMSO and 30uM compounds 14, 40, 35A and 34 for 3, 7, 24 and 48 hrs.

[0048] FIGs 32A-32F illustrate responses of OVCAR3 and SK-OV-3 cells to CDK2 inhibitor (PF-07104091, PF4091) monotherapy and combination therapy with compounds 35 A and 34. FIG. 32A is a graph showing PF4091 dose-response curve in OVCAR3 at 48hrs post treatment. Data shown as mean±SD (n=3). FIG. 32B is a graph showing BrdU incorporation in OVCAR3 cells treated with 10 and 30uM of 35 A or 34. Data shown as mean±SD (n=2). FIG.32C shows graph illustrating BrdU incorporation in OVCAR3 cells treated with 0, 150, 350 and 750 nM of PF-07104091 alone or in combination with lOuM or 30uM of compounds 35A (left panel) and 34 (right panel) for 48hr. Data shown as mean±SD (n=2). FIG.32D is a graph showing PF4091 dose-response curve in SK-OV-3 at 48hrs post treatment. Data shown as mean±SD (triplicates, n=l). FIG.32E is a graph showing BrdU incorporation in SK-OV-3 cells treated with 10 and 30uM of 35 A or 34. Data shown as mean±SD (n=2). FIG. 32F is a graph showing BrdU incorporation in SK-OV-3 cells treated with 0, 150, 350 and 750 nM of PF-07104091 alone or in combination with lOuM or 30uM of compounds 35 A (left panel) and 34 (right panel) for 48hr. Data shown as mean±SD (n=2).

[0049] FIGs 33A-33B illustrate response of OVCAR3 to CDK2 / 4 / 6 inhibitor (PF-06873600, PF3600) monotherapy and combination therapy with compounds 35A and 34. FIG.33A is a graph showing PF3600 dose-response curve in OVCAR3 at 48hrs post treatment. Data shown as mean±SD (triplicates, n=l). FIG.33B shows graphs illustrating BrdU incorporation in OVCAR3 cells treated with 0, 50, and 150 nM of PF3600 alone or in combination with lOuM or 30uM of compounds 35A (left panel) and 34 (right panel) for 48hr. Data shown as mean±SD (n=2).

[0050] FIG. 34 illustrates pharmacodynamic (PD) of biomarkers, p27, p21, pCdc6 (S54) in response to compound 34 and PF4091 monotherapies, and combination therapy of 34+PF4091 in OVCAR3 cells. Cells were treated with vehicle (DMSO), 30uM 34, 150, 350, 750nM PF4091, and 30uM 34+150nM, 30uM 34+350nM PF4091 for 18 and 24hr. pCdc6(S54) is the lower band, * indicates the nonspecific band.

[0051] FIGs 35A-35B illustrate response of MIA PaCa-2 and PL45 cells to gemcitabine, RMC-9805 (KRAS G12D inhibitor) and compound 34. FIG. 35A shows graph illustrating gemcitabine dose-response curve in MIA PaCa-2 cells at 72hr post treatment (left panel) (data shown as mean±SD (n=3)) and compounds 34 dose-response curve in MIA PaCa-2 cells at 72hr post treatment (right panel). MIA Pa Ca-2 cells were treated with 0, 2.5, 5, 10, 30, 60 and 120uM 34 and cell viability was determined at 72hr post treatment. Data shown as mean±SD (n=2). FIG.35B shows graph illustrating gemcitabine dose-response curve in PL45 cells at 72hr post treatment (left panel) (data shown as mean±SD (n=2)); RMC-9805 dose-response curve in PL45 cells(middle panel) (data shown as mean±SD (n=2)); and compounds 34 dose-response curve in PL45 cells (right panel). PL45 cells were treated with compound 34 and cell viability was determined at 72hr post treatment. Data shown as mean±SD (n=2).DETAILED DESCRIPTION

[0052] This invention is not limited to the particular processes, compositions, or methodologies described, as these may vary. The terminology used in the description is for the purpose of describing the particular versions or embodiments only and is not intended to limit the scope of the present invention. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. All publications mentioned herein are incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.Definitions

[0053] Where a range of values is provided, it is intended that each intervening value between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure. For example, if a range of 1 mg to 8 mg is stated, it is intended that 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, and 7 mg, are also explicitly disclosed.

[0054] At various places in the present specification, substituents of compounds of the disclosure are disclosed in groups or in ranges. It is specifically intended that embodiments of the disclosure include each and every individual subcombination of the members of such groups and ranges. For example, the term “Ci-6 alkyl” is specifically intended to individually disclose, e.g., methyl (Ci alkyl), ethyl (C2 alkyl), propyl (C3 alkyl), butyl (C4 alkyl), pentyl (C5 alkyl), and hexyl (Ce alkyl) as well as, e.g., C1-C2 alkyl, C1-C3 alkyl, C1-C4 alkyl, C2-C3 alkyl, C2-C4 alkyl, C3-C6 alkyl, C4-C5 alkyl, and Cs-Ce alkyl.

[0055] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” includes one or more methods, and / or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0056] As used herein, the term “about” means plus or minus 10% of the numerical value of the number with which it is being used. Therefore, about 50 mL means in the range of 45 mL-55 mL

[0057] The terms “active ingredient”, “active compound” “active pharmaceutical ingredient”, “effective ingredient” or “API” are interchangeable and is meant to refer to any agent that is capable of inducing a sought-after effect upon administration. In one embodiment, the active ingredient includes a biologically active molecule. As used herein, the phrase "biologically active molecule" refers to a molecule that has a biological effect in a cell. In one embodiment, the active ingredient is a compound of the present invention.

[0058] “Administering,” or "administration" and the like, when used in conjunction with the compounds of the disclosure refers to providing the compounds or pharmaceutical compositions according to any of the embodiments described herein, to a subject in need of treatment. Preferably the subject is a mammal, more preferably a human. Administration routes can be enteral, topical or parenteral. As such, administration routes include but are not limited to intracutaneous, subcutaneous, intravenous, intraperitoneal, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transdermal, transtracheal, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal, oral, sublingual buccal, rectal, vaginal, nasal ocular administrations, as well infusion, inhalation, and nebulization. The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration. In some aspects, the administration of the pharmaceutical composition is intracutaneous, subcutaneous, intravenous, intraperitoneal, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transdermal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoidal, intraspinal, intrasternal, oral, sublingual, buccal, rectal, vaginal, nasal or ocular, or by infusion, inhalation, or nebulization. In other aspects, the pharmaceutical compositions can be delivered in a controlled release system, such as using an intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one aspect, the pharmaceutical composition is administered intravenously. The present invention comprises administering the pharmaceutical composition of the invention alone or in conjunction with another therapeutic agent. When a pharmaceutical composition of the invention is administered in conjunction with another therapeutic agent, the pharmaceutical composition of the invention and the other therapeutic agent, can be administered at the same time or different times.

[0059] As used herein, the term “alkyl” is meant to refer to a saturated hydrocarbon group which is straight-chained or branched. Example alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, t-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), and the like. An alkyl group can contain from1 to about 20, from 2 to about 20, from 1 to about 10, from 1 to about 8, from 1 to about 6, from 1 to about 4, or from 1 to about 3 carbon atoms. "Ci-Ce alkyl" or "Ci-6 alkyl", is intended to include Ci, C2, C3, C4, C5, Ce, C7, Cs, C9, and C10 alkyl groups. Additionally, for example, "Ci-Ce alkyl" or "C1-6 alkyl" denotes alkyl having 1 to 6 carbon atoms. The term “alkylene” refers to a divalent alkyl linking group. An example of alkylene is methylene (CH2).

[0060] “7V-(alkylsulfonyl)aminocarbonyl” as used herein, denotes a radical of the formula:An example of an / V-(alkylsul fonyl (aminocarbony I is / V-(mcthylsul fonyl)aminocarbonyl.

[0061] As used herein, the term “aminocarbonyl” denotes a carbonyl radical adjacent to anamino group, also depicted

[0062] As used herein, “aryl” refers to monocyclic or polycyclic (e.g., having 2, 3 or 4 fused rings) aromatic hydrocarbons such as, for example, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, and the like. In some embodiments, aryl groups have from 6 to about 20 carbon atoms. In some embodiments, aryl groups have from 6 to about 10 carbon atoms. In some embodiments, aryl group has 6 carbon atoms.

[0063] The term “cancer” refers to a group of diseases characterized by abnormal and uncontrolled cell proliferation starting at one site (primary site) with the potential to invade and to spread to other sites (secondary sites, metastases) which differentiate cancer (malignant tumor) from benign tumor. Virtually all the organs can be affected, leading to more than 200 types of cancer that can affect humans. Cancers can result from many causes including genetic predisposition, viral infection, exposure to ionizing radiation, exposure environmental pollutant, tobacco and or alcohol use, obesity, poor diet, lack of physical activity or any combination thereof.

[0064] As used herein, “cycloalkyl” refers to non-aromatic cyclic hydrocarbons including cyclized alkyl, alkenyl, and alkynyl groups that contain up to 20 ring-forming carbon atoms. Cycloalkyl groups can include mono- or polycyclic (e.g., having 2, 3 or 4 fused rings) ring systems as well as spiro ring systems. A cycloalkyl group can contain from 3 to about 15, from 3 to about 10, from 3 to about 8, from 3 to about 6, from 4 to about 6, from 3 to about 5, or from 5 to about 6 ring-forming carbon atoms. Ring-forming carbon atoms of a cycloalkyl group can be optionallysubstituted by oxo or sulfido. Example of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbornyl, norpinyl, norcarnyl, adamantyl, and the like. Also included in the definition of cycloalkyl are moieties that have one or more aromatic rings fused (i.e., having a bond in common with) to the cycloalkyl ring, for example, benzo or thienyl derivatives of cyclopentane, cyclopentene, cyclohexane, and the like (e.g., 2,3-dihydro-lH-indene-l-yl, or lH-inden-2(3H)-one-l-yl). Preferably, “cycloalkyl” refers to cyclized alkyl groups that contain up to 20 ring-forming carbon atoms. Examples of cycloalkyl preferably include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, and the like.

[0065] As used herein, “halo” or “halogen” includes fluorine, chlorine, bromine, and iodine.

[0066] As used herein, “heteroaryl” groups refer to an aromatic heterocycle having up to 20 ring-forming atoms and having at least one heteroatom ring member (ring-forming atom) such as sulfur, oxygen, or nitrogen. In some embodiments, the heteroaryl group has at least one or more heteroatom ring-forming atoms each independently selected from sulfur, oxygen, and nitrogen. Heteroaryl groups include monocyclic and polycyclic (e.g., having 2, 3 or 4 fused rings) systems. Examples of heteroaryl groups include without limitation, pyridyl (a.k.a. pyridinyl), pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furyl, quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrrolyl, oxazolyl, benzofuryl, benzothienyl, benzthiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1,2,4-thiadiazolyl, isothiazolyl, benzothienyl, purinyl, carbazolyl, benzimidazolyl, indolinyl, and the like. In some embodiments, the heteroaryl group has from 1 to about 20 carbon atoms, and in further embodiments from about 1 to about 5, from about 1 to about 4, from about 1 to about 3, from about 1 to about 2, carbon atoms as ring-forming atoms. In some embodiments, the heteroaryl group contains 3 to about 14, 3 to about 7, or 5 to 6 ring-forming atoms. In some embodiments, the heteroaryl group has 1 to about 4, 1 to about 3, or 1 to 2 heteroatoms. Unless otherwise specified, heteroaryl groups may be unsubstituted or substituted with at least one groups selected from oxo, cyano, hydroxyl, alkoxy, -acylamino, carboxyamido, -SO2CH3, -CF3, Ci-Ce alkyl, halo, and acyl. Examples of optionally substituted heteroaryl include, without limitation, l,2,4-oxadiazol-5(4H)-one, l,2,4-thiadiazol-5(4H)-one, thiazolidine-2,4-dione, hydroxyisoxazole, hydroxythiazole.

[0067] The term “heterocyclyl,” “heterocyclic” or “heterocyclyl ring” is defined as a saturated or partially unsaturated ring containing one to four hetero atoms or hetero groups selected from O, N, NH, -N(RZ)-, -S(O)- or -S(O)2-, wherein Rzis selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, optionally substituted heterocyclyl, in a single or fusedheterocyclic ring system having from three to twelve ring members. In a preferred embodiment, a heterocyclyl is a ring system having three to seven ring members. Examples of a heterocyclyl group include, without limitation, azetindinyl, pyrrolidinyl, piperidinyl, piperazinyl, oxopiperazinyl, morpholinyl, dioxothiomorphylinyl, tetrahydrofuranyl and azabicyclo[3.2.1]octanyl. Unless otherwise specified, hetercyclyl groups may be unsubstituted or substituted with at least one group selected from oxo, cyano, hydroxyl, alkoxy, -acylamino, carboxyamido, -SO2CH3, -CF3, Ci-Ce alkyl, halo, and acyl. Examples of optionally substituted heterocycles include, without limitation, oxazolidine-2, 4-dione, thiazolidine-2, 4-dione,

[0068] The terms “hydroxyl” and “hydroxy” are used interchangeably to mean an OH group.

[0069] The term “improves” is used to convey that the disclosure changes either the characteristics and / or the physical attributes of the tissue to which it is being provided, applied or administered. The term “improves” may also be used in conjunction with a disease state such that when a disease state is “improved” the symptoms or physical characteristics associated with the disease state, are diminished, reduced, eliminated, delayed or averted.

[0070] The term “inhibiting” includes the blockade, aversion of a certain result or process, or the restoration of the converse result or process. In terms of prophylaxis or treatment by administration of a compound of the disclosure, “inhibiting” includes protecting against (partially or wholly) or delaying the onset of symptoms, alleviating symptoms, or protecting against, diminishing or eliminating a disease, condition or disorder.

[0071] As used herein, “neoplasm” or “tumor” including grammatical variations thereof, means new and abnormal growth of tissue, which may be benign or cancerous. In a related aspect, the neoplasm is indicative of a neoplastic disease or disorder, including but not limited, to various cancers. For example, such cancers can include prostate, pancreatic, biliary, colon, rectal, liver, kidney, lung, testicular, breast, ovarian, pancreatic, brain, and head and neck cancers, melanoma, sarcoma, multiple myeloma, leukemia, lymphoma, and the like.

[0072] As used herein, the term “optionally substituted” means that substitution is optional and therefore includes both unsubstituted and substituted atoms and moieties. A “substituted” atom or moiety indicates that any hydrogen on the designated atom or moiety can be replaced with a selection from the indicated substituent group, provided that the normal valence of the designated atom or moiety is not exceeded, and that the substitution results in a stable compound. For example, if a methyl group (i.e., CH3) is optionally substituted, then up to 3 hydrogen atoms on the carbon atom can be replaced with substituent groups.

[0073] The phrase “pharmaceutically acceptable” refers to molecular entities and compositions that are generally regarded as safe and nontoxic. In particular, pharmaceutically acceptable carriers, diluents or other excipients used in the pharmaceutical compositions of this disclosure are physiologically tolerable, compatible with other ingredients, and do not typically produce an allergic or similar untoward reaction (for example, gastric upset, dizziness and the like) when administered to a patient. Preferably, as used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.

[0074] The phrase "pharmaceutically acceptable salt(s)", as used herein, includes those salts of compounds of the disclosure that are safe and effective for use in mammals and that possess the desired biological activity. Pharmaceutically acceptable salts include salts of acidic or basic groups present in compounds of the disclosure or in compounds identified pursuant to the methods of the disclosure. Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate, p-toluenesulfonate and pamoate (i.e., l,l'-methylene-bis-(2 -hydroxy-3 -naphthoate)) salts. Certain compounds of the disclosure can form pharmaceutically acceptable salts with various amino acids. Suitable base salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, iron and diethanolamine salts. Pharmaceutically acceptable base addition salts are also formed with amines, such as organic amines. Examples of suitable amines are N, N’ -dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine.

[0075] The terms "subject," “individual” or “patient” are used interchangeably. Generally, the subject is human, although as will be appreciated by those in the art, the subject may be an animal. Thus, other animals, including vertebrate such as rodents (including mice, rats, hamsters, and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, chickens, etc., and primates (including monkeys, chimpanzees, orangutans, and gorillas) are included within the definition of subject.

[0076] By “therapeutically effective amount”, “effective dose”, “therapeutically effective dose”, “effective amount,” or the like it is meant an amount a compound, or compound of thepharmaceutical composition of the invention, that will elicit the biological or medical response of a tissue, system, animal, or human that is being sought by the researcher, veterinarian, medical doctor or other clinician. Generally, the response is either amelioration of symptoms in a patient or a desired biological outcome (e.g., reduction of tumor volume, or increased survival of the subject). The therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect or physician observes a change). A therapeutically effective amount of a compound, according to any embodiment described herein, may broadly range from 0.01 mg / kg to about 500 mg / kg, about 0.01 to about 250 mg / kg, about 0.01 to about 25 mg / kg, about 0.05 mg / kg to about 20 mg / kg, about 0.1 mg / kg to about 400 mg / kg, about 0.1 mg / kg to about 200 mg / kg, about 0.1 mg / kg to about 25 mg / kg, about 0.1 to about 10 mg / kg, about 0.2 to about 5 mg / kg, about 1 mg / kg to about 300 mg / kg, about 10 mg / kg to about 100 mg / kg, body weight. The effect contemplated herein includes both medical therapeutic and / or prophylactic treatment, as appropriate. The specific dose of a compound administered according to this disclosure to obtain therapeutic and / or prophylactic effects is determined by the particular circumstances surrounding the case, including, for example, the compound administered, the route of administration, the co-administration of other active ingredients, the condition being treated, the activity of the specific compound employed, the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed and the duration of the treatment;. The therapeutically effective amount administered will be determined by the physician in the light of the foregoing relevant circumstances and the exercise of sound medical judgment. A therapeutically effective amount of a compound, according to any embodiment described herein, is typically an amount such that when it is administered in a physiologically tolerable carrier / excipient composition, it is sufficient to achieve an effective systemic concentration or local concentration in the tissue. The total daily dose of the compounds according to any embodiment described herein administered to a human or other animal in single or in divided doses can be in amounts, for example, from about 0.01 mg / kg to about 500 mg / kg, about 0.01 to about 250 mg / kg, about 0.01 to about 25 mg / kg, about 0.05 mg / kg to about 20 mg / kg, about 0.1 mg / kg to about 400 mg / kg, about 0.1 mg / kg to about 200 mg / kg, about 0.1 mg / kg to about 25 mg / kg, about 0.1 to about 10 mg / kg, about 0.2 to about 5 mg / kg, about 1 mg / kg to about 300 mg / kg, about 10 mg / kg to about 100 mg / kg, body weight per day. Single dose pharmaceutical compositions of any embodiment described herein may contain such amounts or submultiples thereof to make up the daily dose. For example, the compounds according to anyembodiment described herein, may be administered on a regimen of 1 to 4 times per day, such as once, twice, three times or four times per day. In some embodiments, the therapeutically effective amount of a compound according to any embodiment disclosed herein, can range between about 0.01 and about 25 mg / kg / day. In some embodiments the therapeutically effective amount is between a lower limit of about 0.01 mg / kg of body weight, about 0.1 mg / kg of body weight, about 0.2 mg / kg of body weight, about 0.3 mg / kg of body weight, about 0.4 mg / kg of body weight, about 0.5 mg / kg of body weight, about 0.60 mg / kg of body weight, about 0.70 mg / kg of body weight, about 0.80 mg / kg of body weight, about 0.90 mg / kg of body weight, about 1 mg / kg of body weight, about 2.5 mg / kg of body weight, about 5 mg / kg of body weight, about 7.5 mg / kg of body weight, about 10 mg / kg of body weight, about 12.5 mg / kg of body weight, about 15 mg / kg of body weight, about 17.5 mg / kg of body weight, about 20 mg / kg of body weight, about 22.5 mg / kg of body weight, and about 25 mg / kg of body weight; and an upper limit of 25 mg / kg of body weight, about 22.5 mg / kg of body weight, about 20 mg / kg of body weight, about 17.5 mg / kg of body weight, about 15 mg / kg of body weight, about 12.5 mg / kg of body weight, about 10 mg / kg of body weight, about 7.5 mg / kg of body weight, about 5 mg / kg of body weight, about 2.5 mg / kg of body weight, about 1 mg / kg of body weight, about 0.9 mg / kg of body weight, about 0.8 mg / kg of body weight, about 0.7 mg / kg of body weight, about 0.6 mg / kg of body weight, about 0.5 mg / kg of body weight, about 0.4 mg / kg of body weight, about 0.3 mg / kg of body weight, about 0.2 mg / kg of body weight, about 0.1 mg / kg of body weight, and about 0.01 mg / kg of body weight. In some embodiments, the therapeutically effective amount is about 0.1 mg / kg / day to about 10 mg / kg / day; in some embodiments the therapeutically effective amount is about 0.2 and about 5 mg / kg / day. In some embodiments, treatment regimens according to the disclosure comprise administration to a patient in need of such treatment will usually include from about 1 mg to about 5000 mg, about 10 mg to about 2000 mg, about 10 mg to about 200 mg, about 20 to about 1000 mg, about 20 to about 500 mg, about 20 to about 400 mg, about 40 to about 800 mg, about 50 mg to about 500 mg, about 80 to about 1600 mg and about 50 mg, of a compound according to any embodiment disclosed herein, or a pharmaceutically acceptable salt thereof, per day in single or multiple doses. In some embodiments the therapeutically effective amount is a total daily dose of 50 mg to 500 mg. In some embodiments, the daily dose is between a lower limit of about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg; about 120 mg, about 125 mg, about 130 mg, about 135 mg, about 140 mg, about 145 mg, about 150 mg, about 155 mg, about 160 mg, about 165 mg, about 170 mg, about 175 mg, about 180 mg, about 185 mg, about190 mg, about 195 mg, about 200 mg, about 205 mg, about 210 mg, about 215 mg; about 220 mg, about 225 mg, about 230 mg, about 235 mg, about 240 mg, about 245 mg, about 250 mg, about 255 mg, about 260 mg, about 265 mg, about 270 mg, about 275 mg, about 280 mg, about 285 mg, about 290 mg, about 295 mg, 300 mg, about 305 mg, about 310 mg, about 315 mg; about 320 mg, about 325 mg, about 330 mg, about 335 mg, about 340 mg, about 345 mg, about 350 mg, about 355 mg, about 360 mg, about 365 mg, about 370 mg, about 375 mg, about 380 mg, about 385 mg, about 390 mg, about 395, about 400 mg, about 405 mg, about 410 mg, about 415 mg; about 420 mg, about 425 mg, about 430 mg, about 435 mg, about 440 mg, about 445 mg, about 450 mg, about 455 mg, about 460 mg, about 465 mg, about 470 mg, about 475 mg, about 480 mg, about 485 mg, about 490 mg, about 495 mg, and about 500 mg and an upper limit of about 500 mg, about 495 mg, about 490 mg, about 485 mg, about 480 mg, about 475 mg, about 470 mg, about 465 mg, about 460 mg, about 455 mg, about 450 mg, about 445 mg, about 440 mg, about 435 mg, about 430 mg, about 425 mg, about 420 mg, about 415 mg, about 410 mg, about 405 mg, about 400 mg, about 395 mg, about 390 mg, about 385 mg, about 380 mg, about 375 mg, about 370 mg, about 365 mg, about 360 mg, about 355 mg, about 350 mg, about 345 mg, about 340 mg, about 335 mg, about 330 mg, about 325 mg, about 320 mg, about 315 mg, about 310 mg, about 305 mg about 300 mg, about 295 mg, about 290 mg, about 285 mg, about 280 mg, about 275 mg, about 270 mg, about 265 mg, about 260 mg, about 255 mg, about 250 mg, about 245 mg, about 240 mg, about 235 mg, about 230 mg, about 225 mg, about 220 mg, about 215 mg, about 210 mg, about 205 mg 200 mg, about 195 mg, about 190 mg, about 185 mg, about 180 mg, about 175 mg, about 170 mg, about 165 mg, about 160 mg, about 155 mg, about 150 mg, about 145 mg, about 140 mg, about 135 mg, about 130 mg, about 125 mg, about 120 mg, about 115 mg, about 110 mg, about 105 mg, about 100 mg, about 95 mg, about 90 mg; about 85 mg, about 80 mg, about 75 mg, about 70 mg, about 65 mg, about 60 mg, about 55 mg, and about 50 mg of a compound according to any embodiment herein. In some embodiments, the total daily dose is about 50 mg to 150 mg. In some embodiments, the total daily dose is about 50 mg to 250 mg. In some embodiments, the total daily dose is about 50 mg to 350 mg. In some embodiments, the total daily dose is about 50 mg to 450 mg. It will be understood that the pharmaceutical formulations of the disclosure need not necessarily contain the entire amount of the compound that is effective in treating the disorder, as such effective amounts can be reached by administration of a plurality of divided doses of such pharmaceutical formulations. The compounds may be administered on a regimen of 1 to 4 times per day, such as once, twice, three times or four times per day.

[0077] The terms “treat,” “treated,” or “treating” as used herein,

[0078] refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to protect against (partially or wholly) or slow down (e.g., lessen or postpone the onset of) an undesired physiological condition, disorder or disease, or to obtain beneficial or desired clinical results such as partial or total restoration or inhibition in decline of a parameter, value, function or result that had or would become abnormal. For the purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent or vigor or rate of development of the condition, disorder or disease; stabilization (i.e., not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of the condition, disorder or disease state; and remission (whether partial or total), whether or not it translates to immediate lessening of actual clinical symptoms, or enhancement or improvement of the condition, disorder or disease. Treatment seeks to elicit a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.

[0079] Embodiments of the invention are directed to compounds according to any embodiment described herein, useful for treating cancer, pharmaceutical compositions containing such compounds and pharmaceutically acceptable carriers, and methods for treating cancer by administering such compounds and pharmaceutical compositions in a pharmaceutically acceptable amount.Compounds of the Invention

[0080] Various embodiments are directed to a compound of Formula (I):or a pharmaceutically acceptable salt thereof.

[0081] Substituent R of Formula (I) is selected from the group consisting of H, aminocarbonyl, cyano, -(CH2)nC(O)OH, N-(C i -Ce-al kylsulfony 1 )ami nocarbonyl, 5 to 6-membered optionally substituted heteroaryl and 5 to 6-membered optionally substituted heterocyclyl; and n is an integer selected from 1-6.

[0082] Each of substituents R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10of Formula (I) is independently selected from the group consisting of H and Ci-Ce-alkyl.

[0083] In some embodiments, R of Formula (I) is selected from the group consisting of H, aminocarbonyl, cyano, -(CH2)nC(O)OH,;'V-(mcthylsulfonyl)aminocarbonyl, tetrazolyl, oxadiazolonyl, thiadiazolonyl, hydroxyisoxazolyl, hydroxythiazolyl, oxazolidinedionyl, and thiazolidinedionyl.

[0084] In some embodiments, R of Formula (I) is selected from the group consisting of H,, aminocarbonyl, cyano, -(CH2)nC(O)OH,;'V-(mcthylsulfonyl)aminocarbonyl,

[0085] In some embodiments, each of R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10, is independently selected from the group consisting of H and methyl.

[0086] In some embodiments, the compound of Formula (I) is a compound of Formula (I-A):or a pharmaceutically acceptable salt thereof.

[0087] Some embodiments describe a compound of Formula (I), selected from the group consisting of:or a pharmaceutically acceptable salt thereof.

[0088] Various embodiments are directed to a compound of Formula (II):or a pharmaceutically acceptable salt thereof.

[0089] Each of R11, R12, R13, R14, R15, R16, R17, R18, R19, and R20of Formula II is independently selected from the group consisting of H and Ci-Ce-alkyl.

[0090] Substituent Z of Formula (II) is selected from the group consisting ofm is an integer selected from 1, 2, 3, 4, 5, or 6; andp is an integer selected from 1, 2, or 3.

[0091] In some embodiments, each of R11, R12, R13, R14, R15, R16, R17, R18, R19, and R20of Formula (II) is independently selected from the group consisting of H and methyl.

[0092] In some embodiments, m is 1.

[0093] In some embodiments, the compound of Formula (II) is a compound of Formula (II- A):or a pharmaceutically acceptable salt thereof.

[0094] Some embodiments describe a compound of Formula (II), selected from the group consisting of:or a pharmaceutically acceptable salt thereof.

[0095] Various embodiments are directed to a compound of Formula (III):or a pharmaceutically acceptable salt thereof.

[0096] Each of R21, R22, 223, R24, R25, R26, R27, R28, R29, and R30of Formula (III) is independently selected from the group consisting of H and Ci-Ce-alkyl.

[0097] Substituent Y of Formula (III) is selected from the group consisting of oxopiperazinyl, piperidinyl, pyrrolidinyl and azetidinyl.

[0098] In some embodiments, Y of Formula (III) is Y is selected from the group consisting of

[0099] In some embodiments, each of R21, R22, 223, R24, R25, R26, R27, R28, R29, and R30, is independently selected from the group consisting of H and methyl.

[0100] In some embodiments the compound of Formula (III) is a compound of Formula (III- A):or a pharmaceutically acceptable salt thereof.

[0101] Some embodiments describe a compound of Formula (III), selected from the group consisting of:or a pharmaceutically acceptable salt thereof.

[0102] Various embodiments are directed to a compound of Formula (IV):or a pharmaceutically acceptable salt thereof.

[0103] Each of R32, R33, 234, R35, and R36, is independently selected from the group consisting of H and Ci-Ce-alkyl.

[0104] Substituent Q is selected from H, -NHSO2-(Ci-C6)-alkyl and optionally substituted -NHSCh-cycloalkyl.

[0105] In some embodiments, Q is selected from H, NHSCh-cyclohexyl, NHSO2-4-methylcyclohexyl, -NHSO2CH3, -NHSCh-n-butyl and -NHSCh-zso-pentyl.

[0106] In some embodiments, each of R32, R33, 234, R35, and R36, is independently selected from the group consisting of H and methyl.

[0107] In some embodiments the compound of Formula (IV), is a compound of Formula (IV-A):or a pharmaceutically acceptable salt thereof.

[0108] Some embodiments describe a compound of Formula (IV), selected from the group consisting of:or a pharmaceutically acceptable salt thereof.

[0109] Various embodiments are directed to a compound of Formula (V):or a pharmaceutically acceptable salt thereof.

[0110] Each of R37, R38, R39, R40, and R41, is independently selected from the group consisting of H and Ci-Ce-alkyl.

[0111] Substituent T is selected from -NRARB, and

[0112] Each of RAand RBis independently selected from Ci-Ce-alkyl.

[0113] Substituent R42is selected fromO-(Ci-C6)-alkyl, -(C3-C?)-cycloalkyl, -CH2-aryl, -CH2CH2-aryl0^^(C’3-(.’7)cycloalkylaryl

[0114] In some embodiments, T is selected from the group consisting of -N(CH3)2,

[0115] In some embodiments, each of R37, R38, R39, R40, and R41, is independently selected from the group consisting of H and methyl.

[0116] In some embodiments the compound of Formula (V) is a compound of Formula (V-A) or Formula (V-B):or a pharmaceutically acceptable salt thereof.

[0117] Some embodiments describe a compound of Formula (V), selected from the group consisting of:or a pharmaceutically acceptable salt thereof.

[0118] Various embodiments are directed to a compound of Formula (VI):or a pharmaceutically acceptable salt thereof.

[0119] Each of R43, R44, R45, R46, R47, R48, R49, R50, R51, and R52, is independently selected from the group consisting of H and Ci-Ce-alkyl.

[0120] In some embodiments, the compound of Formula (VI) is not a compound of Formula

[0121] In some embodiments the compound of Formula (VI) is selected from a compound of Formula (VI-A), (VI-B), (VI-C) or (VI-D):or a pharmaceutically acceptable salt thereof.

[0122] In some embodiments, each of R43, R44, R45, R46, R47, R48, R49, R50, R51, and R52, is independently selected from the group consisting of H and methyl.

[0123] Some embodiments describe a compound of Formula (VI), selected from the group consisting of:or a pharmaceutically acceptable salt thereof.

[0124] Various embodiments are directed to a compound of Formula (VIII):or a pharmaceutically acceptable salt thereof.

[0125] Substituent R53of Formula (VIII) is selected from the group consisting of

[0126] Each of R54, R55, R56, R57, R58, R59, R60, R61, R62, and R63of Formula (VIII) is independently selected from the group consisting of H and Ci-Ce-alkyl.

[0127] Substituent X of Formula (VIII) is a halogen.

[0128] In some embodiments, R53of Formula VIII is selected from the group consisting of

[0129] In some embodiments each of R54, R55, R56, R57, R58of Formula (VIII) is independently selected from the group consisting of H and methyl.

[0130] In some embodiments, each of R59, R60, R61, R62, and R63of Formula (VIII) is independently selected from the group consisting of H and methyl.

[0131] In some embodiments X is chloro.

[0132] In some embodiments the compound of Formula (VIII) is a compound of Formula (VIII-A):or a pharmaceutically acceptable salt thereof.

[0133] Some embodiments describe a compound of Formula (VIII), selected from the group consisting of:or a pharmaceutically acceptable salt thereof.

[0134] Various embodiments are directed to a compound of Formula (IX):or a pharmaceutically acceptable salt thereof.

[0135] Substituent R64of Formula (IX) is selected from the group consisting of

[0136] Each of R65, R66, R67, R68, and R69of Formula (IX) is independently selected from the group consisting of H and Ci-Ce-alkyl.

[0137] Substituent X of Formula (IX) is a halogen.

[0138] In some embodiments R64of Formula (IX) is selected from the group consisting of CH3- Q?'and.

[0139] In some embodiments each of R65, R66, R67, R68, and R69of Formula (IX) is independently selected from the group consisting of H and methyl.

[0140] In some embodiment, X of Formula (IX) is chloro.

[0141] Some embodiments describe a compound of Formula (IX), selected from the group consisting of:or a pharmaceutically acceptable salt thereof.

[0142] In some embodiments, a compound of the present invention is a compound selected from a compound in Table 1.or a pharmaceutically acceptable salt thereof.

[0143] In some embodiments, a compound of the present invention is a compound selected from a compound in Table 1 A.

[0144] Table 1A:

[0145] Additional embodiments include salts, solvates, stereoisomers, or polymorphs of the compounds according to any embodiment described herein.

[0146] Some embodiments are directed to free base forms of the compounds according to any embodiment described herein. Other embodiments include salts of such compounds including, for example, pharmaceutically acceptable acid addition salts or pharmaceutically acceptable addition salts of free bases. Examples of pharmaceutically acceptable acid addition salts include, but are not limited to, salts derived from nitric, phosphoric, sulfuric, or hydrobromic, hydroiodic, hydrofluoric, phosphorous, as well as salts derived from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyl alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and acetic, maleic, succinic, or citric acids. Non-limiting examples of such salts include napadisylate, besylate, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, trifluoroacetate, propionate, caprylate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate, methanesulfonate, and the like. Additional salt forms of the compounds described above include salts of amino acids such as arginate and the like and gluconate, and galacturonate (see e.g., Berge, et al. “Pharmaceutical Salts,” J. Pharma. Sci. 1977;66: 1).

[0147] Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines include N, N’~ dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine. The base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desiredbase to produce the salt in the conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid.

[0148] Various embodiments include total and partial salts, i.e., salts with 1, 2 or 3, preferably 2, equivalents of base per mole of acid of a compound or salt described above, with 1, 2 or 3 equivalents, preferably 1 equivalent, of acid per mole of base of a compound of according to any embodiment described herein. Typically, a pharmaceutically acceptable salt of a compound according to any embodiment described herein, may be readily prepared by using a desired acid or base as appropriate. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent. For example, an aqueous solution of an acid such as hydrochloric acid may be added to an aqueous suspension of a compound according to any embodiment described herein, and the resulting mixture evaporated to dryness (lyophilized) to obtain the acid addition salt as a solid. Alternatively, a compound according to any embodiment described herein, may be dissolved in a suitable solvent, for example an alcohol such as isopropanol, and the acid may be added in the same solvent or another suitable solvent. The resulting acid addition salt may then be precipitated directly, or by addition of a less polar solvent such as diisopropyl ether or hexane, and isolated by filtration.

[0149] Further embodiments include N-oxides of the compounds according to any embodiment described herein. N-oxides include heterocycles containing an otherwise unsubstituted sp2N atom. Examples of such N-oxides include pyridyl N-oxides, pyrimidyl N-oxides, pyrazinyl N-oxides and pyrazolyl N-oxides.

[0150] Compounds, according to any embodiment described herein, may have one or more chiral centers and, depending on the nature of individual substituents, they can also have geometrical isomers. Thus, embodiments include stereoisomers, diastereomers, and enantiomers of the compounds according to any embodiment described herein. A chiral compound can exist as either an individual enantiomer or as a mixture of enantiomers. A mixture containing equal proportions of the enantiomers is called a “racemic mixture.” A mixture containing unequal portions of the enantiomers is described as having an “enantiomeric excess” (ee) of either the R or S compound. The excess of one enantiomer in a mixture is often described with a % enantiomeric excess. The ratio of enantiomers can also be defined by “optical purity” wherein the degree at which the mixture of enantiomers rotates plane polarized light is compared to the individual optically pure R and S compounds. The compounds can also be a substantially pure (+) or (-) enantiomer of the compounds described herein. In some embodiments, a composition can include a substantially pure enantiomer that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% of one enantiomer. In certain embodiments, a composition may include a substantially pure enantiomer that is at least 99.5% one enantiomer.

[0151] The description above encompasses all individual isomers of the compounds according to any embodiment described herein, and the description or naming of a particular compound in the specification and claims is intended to include both individual enantiomers and mixtures thereof. Methods for the determination of stereochemistry and the resolution or stereotactic synthesis of stereoisomers are well-known in the art. Diastereomers differ in both physical properties and chemical reactivity. A mixture of diastereomers can be separated into enantiomeric pairs based on solubility, fractional crystallization, or chromatographic properties, e.g., thin layer chromatography, column chromatography or HPLC. Purification of complex mixtures of diastereomers into enantiomers typically requires two steps. In a first step, the mixture of diastereomers is resolved into enantiomeric pairs, as described above. In a second step, enantiomeric pairs are further purified into compositions enriched for one or the other enantiomer or, more preferably resolved into compositions comprising pure enantiomers. Resolution of enantiomers typically requires reaction or molecular interaction with a chiral agent, e.g., solvent or column matrix. Resolution may be achieved, for example, by converting the mixture of enantiomers, e.g., a racemic mixture, into a mixture of diastereomers by reaction with a pure enantiomer of a second agent, i.e., a resolving agent. The two resulting diastereomeric products can then be separated. The separated diastereomers are then reconverted to the pure enantiomers by reversing the initial chemical transformation.

[0152] Resolution of enantiomers can also be accomplished by differences in their non-covalent binding to a chiral substance, e.g., by chromatography on homochiral adsorbants. The noncovalent binding between enantiomers and the chromatographic adsorbant establishes diastereomeric complexes, leading to differential partitioning in the mobile and bound states in the chromatographic system. The two enantiomers therefore move through the chromatographic system, e.g., column, at different rates, allowing for their separation.

[0153] The invention also embraces isolated compounds. An isolated compound refers to a compound which represents at least 10%, preferably at least 20%, more preferably at least 50% and most preferably at least 80% of the compound present in the mixture.

[0154] In some embodiments of the invention, one or more hydrogen atoms is replaced by a deuterium. It is well established that deuteration of physiologically active compounds offer the advantage of retaining the pharmacological profile of their hydrogen counterparts while positively impacting their metabolic outcome. Selective replacement of one or more hydrogen withdeuterium, in a compound of the present invention, could improve the safety, tolerability and efficacy of the compound when compared to its all-hydrogen counterpart.

[0155] M ethods for incorporation of deuterium into compounds is well established. Usingmetabolic studies established in the art, the compound of the present invention can be tested to identify sites for s ele ctive placement of a deuterium isotope, wherein the isotope will not be metabolized. Moreover, these studies identify sites of metabolism as the location where a deuterium atom would be placed.Pharmaceutical compositions

[0156] Some embodiments describ e a pharmaceutical composition comprising: a compoundaccording to an embodiment described herein, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. In some embodiments the compound is a compound disclosed in Table 1. In some embodiments the compound is a compound disclosed in Table 1A.

[0157] Some embodiments describe a pharmaceutical composition comprising a compound selected of Table 2:Table 2.and a pharmaceutically acceptable carrier.

[0158] Some embodiments describe a pharmaceutical composition comprising a compound selected of Table 2 A:Table 2A.and a pharmaceutical acceptable carrier.

[0159] The pharmaceutical compositions can be prepared in a manner well known in the pharmaceutical arts, and can be administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated.

[0160] While it is possible that a compound as described in any embodiment herein may be administered as the bulk substance, it is preferable to present the compound in a pharmaceutical formulation, e.g., wherein the active agent is in an admixture with a pharmaceutically acceptablecarrier selected with regard to the intended route of administration and standard pharmaceutical practice.

[0161] In particular, the disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of at least one compound according to any embodiment described herein, and optionally, a pharmaceutically acceptable carrier.

[0162] The pharmaceutically compositions of the present invention comprise one or more compounds of the invention in association with one or more non-toxic, pharmaceutically acceptable carriers and / or diluents and / or adjuvants and / or excipients, collectively referred to herein as “carrier” materials, and if desired other active ingredients. The pharmaceutical compositions may contain common carriers and excipients, such as corn starch or gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid. The pharmaceutical compositions may contain croscarmellose sodium, microcrystalline cellulose, corn starch, sodium starch glycolate and alginic acid.

[0163] Preservatives, stabilizers, dyes and flavoring agents may be provided in any pharmaceutical composition described herein. Examples of preservatives include sodium benzoate, ascorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.

[0164] With respect to combinations including biologies such as monoclonal antibodies or fragments, suitable excipients will be employed to prevent aggregation and stabilize the antibody or fragment in solution with low endotoxin, generally for parenteral administration, for example, intravenous, administration. For example, see Formulation and Delivery Issues for Monoclonal Antibody Therapeutics, Daugherty et al., in Current Trends in Monoclonal Antibody Development and Manufacturing, Part 4, 2010, Springer, New York pp 103-129.

[0165] The compounds according to any embodiment described herein, may be milled using known milling procedures such as wet milling to obtain a particle size appropriate for tablet formation and for other formulation types. Finely divided (nanoparticulate) preparations of the compounds may be prepared by processes known in the art, for example see WO 02 / 00196 (SmithKline Beecham).

[0166] The compounds of the invention can be used in antibody drug conjugates, or ADCs. The term “antibody-drug-conjugate” or “ADC” refers to a binding protein, such as an antibody or antigen binding fragment thereof, chemically linked to one or more chemical drug(s) (also referred to herein as agent(s)) that may optionally be therapeutic or cytotoxic agents. In a preferred embodiment, an ADC includes an antibody, a cytotoxic or therapeutic drug, and a linker thatenables attachment or conjugation of the drug to the antibody. An ADC typically has anywhere from 1 to 8 drugs conjugated to the antibody, including drug loaded species of 2, 4, 6, or 8. Nonlimiting examples of drugs that may be included in the ADCs are mitotic inhibitors, antitumor antibiotics, immunomodulating agents, vectors for gene therapy, alkylating agents, antiangiogenic agents, antimetabolites, boron-containing agents, chemoprotective agents, hormones, antihormone agents, corticosteroids, photoactive therapeutic agents, oligonucleotides, radionuclide agents, topoisomerase inhibitors, tyrosine kinase inhibitors, and radiosensitizers. In the context of the present invention, at a minimum the drug includes a compound of the present invention.Combinations

[0167] For the pharmaceutical compositions and methods of the disclosure, a compound according to any embodiment described herein, may be used in combination with other therapies and / or active agents.

[0168] Accordingly, the disclosure provides, in a further aspect, a pharmaceutical composition comprising at least one compound according to any embodiment described herein, or pharmaceutically acceptable derivative thereof; a second active agent; and optionally a pharmaceutically acceptable carrier.

[0169] In various aspects, the pharmaceutical composition further includes at least one anticancer agent.

[0170] The term “anti-cancer agent” can refer to any agent, small molecule, drug and the like that can be used to treat cancer, such as chemotherapy, immunotherapy, targeted therapy, and checkpoint inhibitor therapy.

[0171] Examples of chemotherapy include treatment with a chemotherapeutic, cytotoxic or antineoplastic agents including, but not limited to, (i) anti-microtubules agents comprising vinca alkaloids (vinblastine, vincristine, vinflunine, vindesine, and vinorelbine), taxanes (cabazitaxel, docetaxel, larotaxel, ortataxel, paclitaxel, and tesetaxel), epothilones (ixabepilone), and podophyllotoxin (etoposide and teniposide); (ii) antimetabolite agents comprising anti-folates (aminopterin, methotrexate, pemetrexed, pralatrexate, and raltitrexed), and deoxynucleoside analogues azacitidine, capecitabine, carmofur, cladribine, clofarabine, cytarabine, decitabine, doxifluridine, floxuridine, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, mercaptopurine, nelarabine, pentostatin, tegafur, and thioguanine); (iii) topoisomerase inhibitors comprising Topoisomerase I inhibitors (belotecan, camptothecin, cositecan, gimatecan, exatecan, irinotecan, lurtotecan, silatecan, topotecan, and rubitecan) and Topoisomerase II inhibitors(aclarubicin, amrubicin, daunorubicin, doxorubicin, epirubicin, etoposide, idarubicinm, merbarone, mitoxantrone, novobiocin, pirarubicin, teniposide, valrubicin, and zorubicin); (iv) alkylating agents comprising nitrogen mustards (bendamustine, busulfan, chlorambucil, cyclophosphamide, estramustine phosphate, ifosamide, mechlorethamine, melphalan, prednimustine, trofosfamide, and uramustine), nitrosoureas (carmustine (BCNU), fotemustine, lomustine (CCNU), N-Nitroso-N-methylurea (MNU), nimustine, ranimustine semustine (MeCCNU), and streptozotocin), platinum-based (cisplatin, carboplatin, dicycloplatin, nedaplatin, oxaliplatin and satraplatin), aziridines (carboquone, thiotepa, mytomycin, diaziquone (AZQ), triaziquone and triethylenemelamine), alkyl sulfonates (busulfan, mannosulfan, and treosulfan), non-classical alkylating agents (hydrazines, procarbazine, triazenes, hexamethylmelamine, altretamine, mitobronitol, and pipobroman), tetrazines (dacarbazine, mitozolomide and temozolomide); (v) anthracyclines agents comprising doxorubicin and daunorubicin. Derivatives of these compounds include epirubicin and idarubicin; pirarubicin, aclarubicin, and mitoxantrone, bleomycins, mitomycin C, mitoxantrone, and actinomycin; (vi) enzyme inhibitors agents comprising FI inhibitor (Tipifamib), CDK inhibitors (Abemaciclib, Alvocidib, Palbociclib, Ribociclib, and Seliciclib), PrI inhibitor (Bortezomib, Carfilzomib, and Ixazomib), Phi inhibitor (Anagrelide), IMPDI inhibitor (Tiazofurin), LI inhibitor (Masoprocol), PARP inhibitor (Niraparib, Olaparib, Rucaparib), HDAC inhibitor (Belinostat, Panobinostat, Romidepsin, Vorinostat), and PIKI inhibitor (Idelalisib); (vii) receptor antagonist agent comprising ERA receptor antagonist (Atrasentan), Retinoid X receptor antagonist (Bexarotene), Sex steroid receptor antagonist (Testolactone); (viii) ungrouped agent comprising Amsacrine, Trabectedin, Retinoids (Alitretinoin Tretinoin) Arsenic trioxide, Asparagine depleters (Asparaginase / Pegaspargase), Celecoxib, Demecolcine Elesclomol, Elsamitrucin, Etoglucid, Lonidamine, Lucanthone, Mitoguazone, Mitotane, Oblimersen, Omacetaxine mepesuccinate, and Eribulin.

[0172] Examples of immunotherapy include treatment with antibodies including, but not limited to, alemtuzumab, AVASTIN™ (bevacizumab), BEXXAR™ (tositumomab), CDP 870, and CEA-Scan (arcitumomab), denosumab, ERBITUX™ (cetuximab), HUMIRA™ (adalimumab), IMC-IIF 8, LEUKOSCAN™ (sulesomab), MABCAMPATH™ (alemtuzumab), MABTHERA™ (Rituximab), matuzumab, MYLOTARG™ (gemtuzumab oxogamicin), natalizumab, NEUTROSPEC™ (Technetium (99mTc) fanolesomab), panitumamab, PANOREX™ (Edrecolomab), PROSTASCINT™ (Indium-Ill labeled Capromab Pendetide), RAPTIVA™ (efalizumab), REMICADE™ (infliximab), REOPRO™ (abciximab), rituximab,SIMULECT™ (basiliximab), SYNAGIS™ (palivizumab), THERACIM HR3™, tocilizumab, TYSABRI™ (natalizumab), VERLUMA™ (nofetumomab), XOLAIR™ (omalizumab), ZENAPAX™ (dacliximab), ZEVALIN™ (ibritumomab tiuxetan (IDEC-Y2B8) conjugated to yttrium 90), GILOTRIF™ (afatinib), LYNPARZA™ (olaparib), OPDIVO™ (nivolumab), BOSULIF™ (bosutinib), CABOMETYX™ (cabozantinib), trastuzumab-dkst (OGIVRI™), SUTENT™ (sunitinib malate), ADCETRIS™ (brentuximab vedotin), ALECENSA™ (alectinib), CALQUENCE™ (acalabrutinib), YESCARTA™ (ciloleucel), VERZENIO™ (abemaciclib), KEYTRUDA™ (pembrolizumab), ALIQOPA™ (copanlisib), IMFINZI™ (durvalumab), DARZALEX™ (daratumumab), TECENTRIQ™ (atezolizumab), and TARCEVA™ (erlotinib).

[0173] In some embodiments, the anti-cancer agent is a peptide as described in WO 2022 / 010914, herein incorporated by reference in its entirety.

[0174] In some embodiments the anti-cancer agent is an estrogen modulating therapy including, but not limited to, fulvestrant, elacestrant, or aromatase inhibitors including, but not limited to, as lasofoxifene, anastrozole, and letrozole.

[0175] In some embodiments the anti-cancer agent is an RAS inhibitor including, but not limited to, sotorasib, and adagrasib.

[0176] In some embodiments the anti-cancer agent is an RAF inhibitor including, but not limited to, vemurafenib, and dabrafenib.

[0177] In some embodiments the anti-cancer agent is an MEK inhibitor including, but not limited to trametinib, and cobmetinib.

[0178] In some embodiments the anti-cancer agent is an S0S1 inhibitor including, but not limited to, Bay-293.

[0179] In some embodiments the anti-cancer agent is an mTOR inhibitor including, but not limited to, everolimus.

[0180] In some embodiments the anti-cancer agent is an EGFR inhibitor including, but not limited to, erolintib, cetaxumab, panitumumab, and afatinib.

[0181] In some embodiments the anti-cancer agent is an PDGFR inhibitor including, but not limited to, imatinib, sunitinib, panatinib, axitinib, foretinib, nintedanib, and amuvatinib.

[0182] In some embodiments the anti-cancer agent is a Weel inhibitor including, but not limited to, adavorsertib.

[0183] In some embodiments the anti-cancer agent is a precision oncology targeted therapy including, but not limited to Herceptin® (trastuzumab), Perjeta® (pertuzumab), Enhertu® (am-trastuzumab-deruxtecan-nxki), Kadcyla® (T-DM1 or ado-trastuzumab emtansine), Phesgo®(pertuzumab / trastuzumab / hyaluronidase-zzxf), Nerlynx® (neratinib), Tykerb® (lapatinib) AND Tukysa® (tucatinib).

[0184] When combined in the same formulation it will be appreciated that the two or more compounds must be stable and compatible with each other and the other components of the formulation. When formulated separately they may be provided in any convenient formulation, in such manner as are known for such compounds in the art.

[0185] Compounds according to any embodiment described herein, or pharmaceutically acceptable salts thereof, can be formulated for any route of administration.Routes of Administration and Unit Dosage Forms

[0186] The compounds according to any embodiment described herein, may be formulated for administration in any convenient way for use in human or veterinary medicine and the disclosure therefore includes within its scope pharmaceutical compositions comprising a compound according to any embodiment described herein, adapted for use in human or veterinary medicine. Such pharmaceutical compositions may be presented for use in a conventional manner with the aid of one or more suitable carriers. Acceptable carriers for therapeutic use are well-known in the pharmaceutical art, and are described, for example, in Remington’s Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The choice of pharmaceutical carrier can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise, in addition to the carrier, any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), and / or solubilizing agent(s).

[0187] The routes for administration (delivery) include, but are not limited to, one or more of: gastrointestinal, oral (e.g., as a tablet, capsule, elixers, suspension or as an ingestible solution), topical, mucosal (e.g., as a nasal spray or aerosol for inhalation), buccal (e.g., tablets, lozenges), ovules, parenteral (e.g., by an injectable form), subcutaneously, intraspinal, intraperitoneal, intramuscular, intravenous, intracerebroventricular, or other depot administration etc. Therefore, the pharmaceutical compositions according to any embodiment described herein, include those in a form especially formulated for the mode of administration.

[0188] There may be different pharmaceutical composition / formulation requirements depending on the different delivery systems. It is to be understood that not all of the compounds need to be administered by the same route. Likewise, if the pharmaceutical composition comprises more than one active component, then those components may be administered by different routes. By way of example, the pharmaceutical composition of the disclosure may be formulated to be delivered orally, for example, as a tablet or capsule for ingestible, or parenterally in which thepharmaceutical composition is formulated by an injectable form, for delivery by, for example, an intravenous, intramuscular, or subcutaneous route. Alternatively, the formulation may be designed to be delivered by multiple routes.

[0189] Where the compound according to any embodiment described herein, is to be delivered mucosally through the gastrointestinal mucosa, it should be able to remain stable during transit though the gastrointestinal tract; for example, it should be resistant to proteolytic degradation, stable at acid pH and resistant to the detergent effects of bile. For example, compounds according to any embodiment described herein, prepared for oral administration may be coated with an enteric coating layer. The enteric coating layer material may be dispersed or dissolved in either water or in a suitable organic solvent. As enteric coating layer polymers, one or more, separately or in combination, of the following can be used, e.g., solutions or dispersions of methacrylic acid copolymers, cellulose acetate phthalate, cellulose acetate butyrate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, cellulose acetate trimellitate, carboxymethylethylcellulose, shellac or other suitable enteric coating layer polymer(s). In some embodiments, the aqueous enteric coating layer is a methacrylic acid copolymer.

[0190] The compounds according to any embodiment described herein, may be presented for human or veterinary use in a form suitable for oral or buccal administration, for example in the form of solutions, gels, syrups, or suspensions, or a dry powder for reconstitution with water or other suitable vehicle before use. Solid and liquid pharmaceutical compositions for oral use may be prepared according to methods well-known in the art. The pharmaceutical compositions may be administered orally, in the form of rapid or controlled release tablets, microparticles, mini tablets, capsules, sachets, and oral solutions or suspensions, or powders for the preparation thereof. Oral preparations may optionally include various standard pharmaceutical carriers and excipients, such as binders, fillers, buffers, lubricants, glidants, dyes, disintegrants, odorants, sweeteners, surfactants, mold release agents, antiadhesive agents and coatings. Some carriers / excipients may have multiple roles in the pharmaceutical compositions, e.g., act as both binders and disintegrants.

[0191] Dispersions can be prepared in a liquid carrier or intermediate, such as glycerin, liquid polyethylene glycols, triacetin oils, and mixtures thereof. The liquid carrier or intermediate can be a solvent or liquid dispersive medium that contains, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol or the like), vegetable oils, non-toxic glycerine esters and suitable mixtures thereof. Suitable flowability may be maintained, by generation of liposomes, administration of a suitable particle size in the case of dispersions, or by the addition of surfactants.

[0192] Tablets may contain excipients / carriers such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably com, potato, or tapioca starch), sodium starch glycolate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin, and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

[0193] Examples of pharmaceutically acceptable disintegrants for oral pharmaceutical compositions according to any embodiment described herein include, but are not limited to, starch, pre-gelatinized starch, sodium starch glycolate, sodium carboxymethylcellulose, croscarmellose sodium, microcrystalline cellulose, alginates, resins, surfactants, effervescent compositions, aqueous aluminum silicates and cross-linked polyvinylpyrrolidone.

[0194] Examples of pharmaceutically acceptable binders for oral pharmaceutical compositions according to any embodiment described herein, include, but are not limited to, acacia; cellulose derivatives, such as methylcellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose or hydroxyethylcellulose; gelatin, glucose, dextrose, xylitol, polymethacrylates, polyvinylpyrrolidone, sorbitol, starch, pregelatinized starch, tragacanth, xanthine resin, alginates, magnesium aluminum silicate, polyethylene glycol or bentonite.

[0195] Examples of pharmaceutically acceptable fillers for oral pharmaceutical compositions according to any embodiment described herein include, but are not limited to, lactose, anhydrolactose, lactose monohydrate, sucrose, dextrose, mannitol, sorbitol, starch, cellulose (particularly microcrystalline cellulose), dihydro- or anhydro-calcium phosphate, calcium carbonate and calcium sulphate.

[0196] Examples of pharmaceutically acceptable lubricants useful in the pharmaceutical compositions according to any embodiment described herein, include, but are not limited to, magnesium stearate, talc, polyethylene glycol, polymers of ethylene oxide, sodium lauryl sulphate, magnesium lauryl sulphate, sodium oleate, sodium stearyl fumarate, and colloidal silicon dioxide.

[0197] Examples of suitable pharmaceutically acceptable odorants for the oral pharmaceutical compositions according to any embodiment described herein, include, but are not limited to, synthetic aromas and natural aromatic oils such as extracts of oils, flowers, fruits (e.g., banana, apple, sour cherry, peach) and combinations thereof, and similar aromas. Their usedepends on many factors, the most important being the organoleptic acceptability for the population that will be taking the pharmaceutical compositions.

[0198] Examples of suitable pharmaceutically acceptable dyes for the oral pharmaceutical compositions according to any embodiment described herein, include, but are not limited to, synthetic and natural dyes such as titanium dioxide, beta-carotene, and extracts of grapefruit peel.

[0199] Examples of useful pharmaceutically acceptable coatings for the oral pharmaceutical compositions according to any embodiment described herein, typically used to facilitate swallowing, modify the release properties, improve the appearance, and / or mask the taste of the pharmaceutical compositions include, but are not limited to, hydroxypropylmethylcellulose, hydroxypropylcellulose and acrylate -methacrylate copolymers.

[0200] Suitable examples of pharmaceutically acceptable sweeteners for the oral pharmaceutical compositions according to any embodiment described herein, include, but are not limited to, aspartame, saccharin, saccharin sodium, sodium cyclamate, xylitol, mannitol, sorbitol, lactose, and sucrose.

[0201] Suitable examples of pharmaceutically acceptable buffers include, but are not limited to, citric acid, sodium citrate, sodium bicarbonate, dibasic sodium phosphate, magnesium oxide, calcium carbonate and magnesium hydroxide.

[0202] Suitable examples of pharmaceutically acceptable surfactants include, but are not limited to, sodium lauryl sulphate and polysorbates.

[0203] Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients / carriers in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and / or elixirs, the agent may be combined with various sweetening or flavoring agents, coloring matter or dyes, with emulsifying and / or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

[0204] The compounds according to any embodiment described herein, may be formulated for use in human or veterinary medicine by injection (e.g., by intravenous bolus injection or infusion or via intramuscular, subcutaneous, or intrathecal routes) and may be presented in unit dose form, in ampoules, or other unit-dose containers, or in multi-dose containers, if necessary, with an added preservative. The pharmaceutical compositions for injection may be in the form of suspensions, solutions, or emulsions, in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, solubilizing and / or dispersing agents. Alternatively, theactive ingredient may be in sterile powder form for reconstitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

[0205] Where the pharmaceutical composition according to any embodiment described herein, is to be administered parenterally, such administration includes without limitation: intravenously, intraarterially, intrathecally, intraventricularly, intracranially, intramuscularly, or subcutaneously administering the compound of the disclosure; and / or by using infusion techniques. Antibodies or fragments are typically administered parenterally, for example, intravenously.

[0206] Pharmaceutical compositions according to any embodiment described herein, suitable for injection or infusion may be in the form of a sterile aqueous solution, a dispersion or a sterile powder that contains the active ingredient, adjusted, if necessary, for preparation of such a sterile solution or dispersion suitable for infusion or injection. This preparation may optionally be encapsulated into liposomes. In all cases, the final preparation must be sterile, liquid, and stable under production and storage conditions. To improve storage stability, such preparations may also contain a preservative to prevent the growth of microorganisms. Prevention of the action of microorganisms can be achieved by the addition of various antibacterial and antifungal agents, e.g., paraben, chlorobutanol, or ascorbic acid. In many cases isotonic substances are recommended, e.g., sugars, buffers, and sodium chloride to assure osmotic pressure similar to those of body fluids, particularly blood. Prolonged absorption of such injectable mixtures can be achieved by introduction of absorption-delaying agents, such as aluminum monostearate or gelatin.

[0207] For parenteral administration, the compound, according to any embodiment described herein, is best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.

[0208] Sterile injectable solutions can be prepared by mixing a compound according to any embodiment described herein, with an appropriate solvent and one or more of the aforementioned carriers, followed by sterile filtering. In the case of sterile powders suitable for use in the preparation of sterile injectable solutions, preferable preparation methods include drying in vacuum and lyophilization, which provide powdery mixtures of the compounds and desired excipients / carriers for subsequent preparation of sterile solutions.

[0209] For intramuscular preparations, a sterile formulation of a compound or a suitable soluble salt form of the compound, for example the hydrochloride salt, can be dissolved and administered in a pharmaceutical diluent such as Water- for-Inj ection (WFI), physiological saline or 5% glucose. A suitable insoluble form of the compound may be prepared and administered as a suspension in an aqueous base or a pharmaceutically acceptable oil base, e.g., an ester of a long chain fatty acid such as ethyl oleate.

[0210] A dose of an intravenous, intramuscular or parental formulation of a compound may be administered as a bolus or by slow infusion. A bolus is a dose that is administered in less than 30 minutes. In a preferred embodiment, a bolus is administered in less than 15 or less than 10 minutes. In a more preferred embodiment, a bolus is administered in less than 5 minutes. In an even more preferred embodiment, a bolus is administered in one minute or less. An infusion is a dose that is administered at a rate of 30 minutes or greater. In a preferred embodiment, the infusion is one hour or more. In another embodiment, the infusion is substantially constant.

[0211] For topical use the compounds of the present invention can also be prepared in suitable forms to be applied to the skin, or mucus membranes of the nose and throat, and can take the form of creams, ointments, liquid sprays or inhalants, lozenges, or throat paints. Such topical formulations further can include chemical compounds such as dimethylsulfoxide (DMSO) to facilitate surface penetration of the active ingredient.

[0212] For application to the eyes or ears, the compounds of the present invention can be presented in liquid or semi-liquid form formulated in hydrophobic or hydrophilic bases as ointments, creams, lotions, paints or powders.

[0213] For rectal administration the compounds of the present invention can be administered in the form of suppositories admixed with conventional carriers such as cocoa butter, wax or another glyceride.

[0214] As indicated, a compounds according to any embodiment described herein, can be administered intranasally or by inhalation and is conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurized container, pump, spray or nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichloro fluoromethane, dichlorotetrafluoroethane, a hydro fluoroalkane such as 1, 1,1,2-tetrafluoroethane (HFA 134AT) or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA), carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurized container, pump, spray, or nebulizer may contain a solution or suspension of the active compound, e.g., using amixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g., sorbitan trioleate.

[0215] Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a compound according to any embodiment described herein, and a suitable powder base such as lactose or starch.

[0216] For topical administration by inhalation a compound according to any embodiment described herein may be delivered for use in human or veterinary medicine via a nebulizer.

[0217] The combination of a compound according to any embodiment described herein, and an antibody or antibody fragment molecule can be formulated and administered by any of a number of routes and are administered at a concentration that is therapeutically effective in the indication or for the purpose sought. To accomplish this goal, the antibodies may be formulated using a variety of acceptable excipients known in the art. Typically, the antibodies are administered by injection, for example, intravenous injection. Methods to accomplish this administration are known to those of ordinary skill in the art. For example, Gokarn et al., 2008, J Pharm Sci 97(8): 3051-3066, incorporated herein by reference, describe various high concentration antibody self-buffered formulations. For example, monoclonal antibodies in self-buffered formulation at e.g., 50 mg / mL mAb in 5.25% sorbitol, pH 5.0; or 60 mg / mL mAb in 5% sorbitol, 0.01% polysorbate 20, pH 5.2; or conventional buffered formulations, for example, 50 mg / mL mAbl in 5.25% sorbitol, 25 or 50 mM acetate, glutamate or succinate, at pH 5.0; or 60 mg / mL in 10 mM acetate or glutamate, 5.25% sorbitol, 0.01% polysorbate 20, pH 5.2; other lower concentration formulations can be employed as known in the art.

[0218] The pharmaceutical compositions of the disclosure may contain from 0.01 to 99% weight per volume of the active material. For topical administration, for example, the pharmaceutical composition will generally contain from 0.01-10%, more preferably 0.01-1% of the active material.

[0219] A compound, according to any embodiment described herein, can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.

[0220] The pharmaceutical composition or unit dosage form, according to any embodiment described herein, may be administered according to a dosage and administration regimen defined by routine testing in the light of the guidelines given above in order to obtain optimal activity while minimizing toxicity or side effects for a particular patient. The dosage of the compounds orunit dosage form may vary according to a variety of factors such as underlying disease conditions, the individual’s condition, weight, sex and age, and the mode of administration. The exact amount to be administered to a patient will vary depending on the state and severity of the disorder and the physical condition of the patient. A measurable amelioration of any symptom or parameter can be determined by a person skilled in the art or reported by the patient to the physician. It will be understood that any clinically or statistically significant attenuation or amelioration of any symptom or parameter is within the scope of the disclosure. Clinically significant attenuation or amelioration means perceptible to the patient and / or to the physician.

[0221] In certain embodiments, the pharmaceutical compositions of the disclosure are formulated in a form that is suitable for oral delivery. In some embodiments, the compound is an orally bioavailable compound, suitable for oral delivery. In other embodiments, the pharmaceutical compositions of the disclosure are formulated in a form that is suitable for parenteral delivery.

[0222] In some embodiments, the amount of the compound to be administered can range between about 0.01 and about 100 mg / kg / day. Generally, dosage levels of between 0.01 to 100 mg / kg of body weight daily are administered to the patient, e.g., humans. In some embodiments the therapeutically effective amount is between a lower limit of about 0.01 mg / kg of body weight, about 0.1 mg / kg of body weight, about 0.2 mg / kg of body weight, about 0.3 mg / kg of body weight, about 0.4 mg / kg of body weight, about 0.5 mg / kg of body weight, about 0.60 mg / kg of body weight, about 0.70 mg / kg of body weight, about 0.80 mg / kg of body weight, about 0.90 mg / kg of body weight, about 1 mg / kg of body weight, about 2.5 mg / kg of body weight, about 5 mg / kg of body weight, about 7.5 mg / kg of body weight, about 10 mg / kg of body weight, about 12.5 mg / kg of body weight, about 15 mg / kg of body weight, about 17.5 mg / kg of body weight, about 20 mg / kg of body weight, about 22.5 mg / kg of body weight, about 25 mg / kg of body weight, about 27.5 mg / kg of body weight, about 30 mg / kg of body weight, about 32.5 mg / kg of body weight, about 35 mg / kg of body weight, about 37.5 mg / kg of body weight, about 40 mg / kg of body weight, about 42.5 mg / kg of body weight, about 50 mg / kg of body weight, about 52.5 mg / kg of body weight, about 60 mg / kg of body weight, about 62.5 mg / kg of body weight, about 70 mg / kg of body weight, about 72.5 mg / kg of body weight, about 80 mg / kg of body weight, about 82.5 mg / kg of body weight, about 90 mg / kg of body weight, about 92.5 mg / kg of body weight, about 95 mg / kg of body weight, about 97.5 mg / kg of body weight, and about 100 mg / kg of body weight; and an upper limit of 100 mg / kg of body weight, about 97.5 mg / kg of body weight, 95 mg / kg of body weight, about 92.5 mg / kg of body weight, 90 mg / kg of body weight, about 87.5 mg / kg of bodyweight, 85 mg / kg of body weight, about 82.5 mg / kg of body weight, 80 mg / kg of body weight, about 77.5 mg / kg of body weight, 75 mg / kg of body weight, about 72.5 mg / kg of body weight, 70 mg / kg of body weight, about 67.5 mg / kg of body weight, 65 mg / kg of body weight, about 62.5 mg / kg of body weight, 60 mg / kg of body weight, about 57.5 mg / kg of body weight, 55 mg / kg of body weight, about 52.5 mg / kg of body weight, 50 mg / kg of body weight, about 47.5 mg / kg of body weight, 45 mg / kg of body weight, about 42.5 mg / kg of body weight, 40 mg / kg of body weight, about 37.5 mg / kg of body weight, 35 mg / kg of body weight, about 32.5 mg / kg of body weight, 30 mg / kg of body weight, about 27.5 mg / kg of body weight, 25 mg / kg of body weight, about 22.5 mg / kg of body weight, about 20 mg / kg of body weight, about 17.5 mg / kg of body weight, about 15 mg / kg of body weight, about 12.5 mg / kg of body weight, about 10 mg / kg of body weight, about 7.5 mg / kg of body weight, about 5 mg / kg of body weight, about 2.5 mg / kg of body weight, about 1 mg / kg of body weight, about 0.9 mg / kg of body weight, about 0.8 mg / kg of body weight, about 0.7 mg / kg of body weight, about 0.6 mg / kg of body weight, about 0.5 mg / kg of body weight, about 0.4 mg / kg of body weight, about 0.3 mg / kg of body weight, about 0.2 mg / kg of body weight, about 0.1 mg / kg of body weight, and about 0.01 mg / kg of body weight. In some embodiments, the therapeutically effective amount is about 0.1 mg / kg / day to about 10 mg / kg / day; in some embodiments the therapeutically effective amount is about 0.2 and about 5 mg / kg / day. It will be understood that the pharmaceutical formulations of the disclosure need not necessarily contain the entire amount of the compound that is effective in treating the disorder, as such effective amounts can be reached by administration of a plurality of divided doses of such pharmaceutical formulations. The compounds may be administered on a regimen of 1 to 4 times per day, such as once, twice, three times or four times per day.

[0223] In some embodiments of the disclosure, a compound, according to any embodiment described herein, is formulated in capsules or tablets, usually containing about 10 to about 7000 mg of the compounds. In some embodiments the capsule or tablet contains between a lower limit of about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg; about 120 mg, about 125 mg, about 130 mg, about 135 mg, about 140 mg, about 145 mg, about 150 mg, about 155 mg, about 160 mg, about 165 mg, about 170 mg, about 175 mg, about 180 mg, about 185 mg, about 190 mg, about 195 mg, and about 200 mg about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about900 mg, about 950 mg, about 1000 mg, about 1050 mg, about 1100 mg, about 1150 mg, about 1200 mg, about 1250 mg, about 1300 mg, about 1350 mg, about 1400 mg, about 1450 mg, about 1500 mg, about 1550 mg, about 1600 mg, about 1650 mg, about 1700 mg, about 1750 mg, about 1800 mg, about 1850 mg, about 1900 mg, about 1950 mg, about 2000 mg, 2050 mg, about 2100 mg, about 2150 mg, about 2200 mg, about 2250 mg, about 2300 mg, about 2350 mg, about 2400 mg, about 2450 mg, about 2500 mg, about 2550 mg, about 2600 mg, about 2650 mg, about 2700 mg, about 2750 mg, about 2800 mg, about 2850 mg, about 2900 mg, about 2950 mg, about 3000 mg, about 3050 mg, about 3100 mg, about 3150 mg, about 3200 mg, about 3250 mg, about 3300 mg, about 3350 mg, about 3400 mg, about 3450 mg, about 3500 mg, about 3550 mg, about 3600 mg, about 3650 mg, about 3700 mg, about 3750 mg, about 3800 mg 3850 mg, about 3900 mg, about 3950 mg, about 4000 mg, about 4050 mg, about 4100 mg, about 4150 mg, about 4200 mg, about 4250 mg, about 4300 mg, about 4350 mg, about 4400 mg, about 4450 mg, about 4500 mg, about 4550 mg, about 4600 mg, about 4650 mg, about 4700 mg, about 4750 mg, about 4800 mg 4850 mg, about 4900 mg, about 4950 mg, about 5000 mg5050 mg, about 5100 mg, about 5150 mg, about 5200 mg, about 5250 mg, about 5300 mg, about 5350 mg, about 5400 mg, about 5450 mg, about 5500 mg, about 5550 mg, about 5600 mg, about 5650 mg, about 5700 mg, about 5750 mg, about 5800 mg, about 5850 mg, about 5900 mg, about 5950 mg, about 6000 mg, about 6050 mg, about 6100 mg, about 6150 mg, about 6200 mg, about 6250 mg, about 6300 mg, about 6350 mg, about 6400 mg, about 6450 mg, about 6500 mg, about 6550 mg, about 6600 mg, about 6650 mg, about 6700 mg, about 6750 mg, about 6800 mg, 6850 mg, about 6900 mg, about 7000 mg; and an upper limit of about 7000 mg, about 6950 mg, about 6900 mg, about 6850 mg, about 6800 mg, about 6750 mg, about 6700 mg, about 6650 mg, about 6600 mg, about 6550 mg, about 6500 mg, about 6450 mg, about 6400 mg, about 6350 mg, about 6300 mg, about 6250 mg, about 6200 mg, about 6150 mg, about 6100 mg, about 6050 mg, about 6000 mg, about 5950 mg, about 5900 mg, about 5850 mg, about 5800 mg, about 5750 mg, about 5700 mg, about 5750 mg, about 5700 mg, about 5650 mg, about 5600 mg, about 5550 mg, about 5500 mg, about 5450 mg, about 5400 mg, about 5350 mg, about 5300 mg, 5250 mg, about 5200 mg, about 5150 mg, about 5100 mg, about 5050 mg, about 5000 mg, about 4950 mg, about 4900 mg, about 4850 mg, about 4800 mg, about 4750 mg, about 4700 mg, about 4650 mg, about 4600 mg, about 4550 mg, about 4500 mg, about 4450 mg, about 4400 mg, about 4350 mg, about 4300 mg, about 4250 mg, about 4200 mg, about 4150 mg, about 4100 mg, about 4050 mg, about 4000 mg, about 3950 mg, about 3900 mg, about 3850 mg, about 3800 mg, about 3750 mg, about 3700 mg, about 3650 mg, about 3600 mg, about 3550 mg, about 3500 mg 3450 mg, about 3400 mg, about 3350 mg, about 3300 mg, about3250 mg, about 3200 mg, about 3150 mg, about 3100 mg, about 3050 mg, about 3000 mg, about 2950 mg, about 2900 mg, about 2850 mg, about 2800 mg, about 2750 mg, about 2700 mg, about 2650 mg, about 2600 mg, about 2550 mg, about 2500 mg 2450 mg, about 2400 mg, about 2350 mg, about 2300 mg, 2250 mg, about 2200 mg, about 2150 mg, about 2100 mg, about 2050 mg, about 2000 mg, about 1950 mg, about 1900 mg, about 1850 mg, about 1800 mg, about 1750 mg, about 1700 mg, about 1650 mg, about 1600 mg, about 1550 mg, about 1500 mg, about 1450 mg, about 1400 mg, about 1350 mg, about 1300 mg, about 1250 mg, about 1200 mg, about 1150 mg, about 1100 mg, about 1050 mg, about 1000 mg, about 950 mg, about 900 mg, about 850 mg, about 800 mg, about 750 mg, about 700 mg, about 650 mg, about 600 mg, about 550 mg, about 500 mg, about 450 mg, about 400 mg, about 350 mg, about 300 mg, about 250 mg, about 200 mg, about 195 mg, about 190 mg, about 185 mg, about 180 mg, about 175 mg, about 170 mg, about 165 mg, about 160 mg, about 155 mg, about 150 mg, about 145 mg, about 140 mg, about 135 mg, about 130 mg, about 125 mg, about 120 mg, about 115 mg, about 110 mg, about 105 mg, about 100 mg, about 95 mg, about 90 mg; about 85 mg, about 80 mg, about 75 mg, about 70 mg, about 65 mg, about 60 mg, about 55 mg, about 50 mg, about 45 mg, about 40 mg, about 35 mg, about 30 mg, about 25 mg, about 20 mg, about 15 mg, and about 10 mg of a compound according to any embodiment herein.

[0224] In some embodiments, a compound according to any embodiment herein is administered to a patient at a total daily dose of 50 mg to 500 mg. In some embodiments, the daily dose is between a lower limit of about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg; about 120 mg, about 125 mg, about 130 mg, about 135 mg, about 140 mg, about 145 mg, about 150 mg, about 155 mg, about 160 mg, about 165 mg, about 170 mg, about 175 mg, about 180 mg, about 185 mg, about 190 mg, about 195 mg, about 200 mg, about 205 mg, about 210 mg, about 215 mg; about 220 mg, about 225 mg, about 230 mg, about 235 mg, about 240 mg, about 245 mg, about 250 mg, about 255 mg, about 260 mg, about 265 mg, about 270 mg, about 275 mg, about 280 mg, about 285 mg, about 290 mg, about 295 mg, 300 mg, about 305 mg, about 310 mg, about 315 mg; about 320 mg, about 325 mg, about 330 mg, about 335 mg, about 340 mg, about 345 mg, about 350 mg, about 355 mg, about 360 mg, about 365 mg, about 370 mg, about 375 mg, about 380 mg, about 385 mg, about 390 mg, about 395, about 400 mg, about 405 mg, about 410 mg, about 415 mg; about 420 mg, about 425 mg, about 430 mg, about 435 mg, about 440 mg, about 445 mg, about 450 mg, about 455 mg, about 460 mg, about 465 mg, about 470 mg, about 475 mg, about 480 mg, about 485 mg, about 490 mg, about 495 mg,and about 500 mg and an upper limit of about 500 mg, about 495 mg, about 490 mg, about 485 mg, about 480 mg, about 475 mg, about 470 mg, about 465 mg, about 460 mg, about 455 mg, about 450 mg, about 445 mg, about 440 mg, about 435 mg, about 430 mg, about 425 mg, about 420 mg, about 415 mg, about 410 mg, about 405 mg, about 400 mg, about 395 mg, about 390 mg, about 385 mg, about 380 mg, about 375 mg, about 370 mg, about 365 mg, about 360 mg, about 355 mg, about 350 mg, about 345 mg, about 340 mg, about 335 mg, about 330 mg, about 325 mg, about 320 mg, about 315 mg, about 310 mg, about 305 mg about 300 mg, about 295 mg, about 290 mg, about 285 mg, about 280 mg, about 275 mg, about 270 mg, about 265 mg, about 260 mg, about 255 mg, about 250 mg, about 245 mg, about 240 mg, about 235 mg, about 230 mg, about 225 mg, about 220 mg, about 215 mg, about 210 mg, about 205 mg 200 mg, about 195 mg, about 190 mg, about 185 mg, about 180 mg, about 175 mg, about 170 mg, about 165 mg, about 160 mg, about 155 mg, about 150 mg, about 145 mg, about 140 mg, about 135 mg, about 130 mg, about 125 mg, about 120 mg, about 115 mg, about 110 mg, about 105 mg, about 100 mg, about 95 mg, about 90 mg; about 85 mg, about 80 mg, about 75 mg, about 70 mg, about 65 mg, about 60 mg, about 55 mg, and about 50 mg of a compound according to any embodiment herein. In some embodiments, the total daily dose is about 50 mg to 150 mg. In some embodiments, the total daily dose is about 50 mg to 250 mg. In some embodiments, the total daily dose is about 50 mg to 350 mg. In some embodiments, the total daily dose is about 50 mg to 450 mg. In some embodiments, the total daily dose is about 50 mg.

[0225] A pharmaceutical composition for parenteral administration contains from about 0.01% to about 100% by weight of the active compound according to any embodiment described herein, based upon 100% weight of total pharmaceutical composition.

[0226] Generally, transdermal dosage forms contain from about 0.01% to about 100% by weight of the active compound according to any embodiment described herein, versus 100% total weight of the dosage form.

[0227] The pharmaceutical composition or unit dosage form may be administered in a single daily dose, or the total daily dosage may be administered in divided doses. In addition, co administration or sequential administration of another compound for the treatment of the disorder may be desirable. To this purpose, the combined active principles are formulated into a simple dosage unit.Methods of Use

[0228] Some embodiments describe a method of treating cancer, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound or a pharmaceutical composition according to any embodiment described herein.

[0229] In some embodiments, the invention describes a method of treating cancer, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound according to any embodiment described herein and a pharmaceutically acceptable carrier.

[0230] In some embodiments, the invention describes a method of treating cancer, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a compound as described in Table I, and a pharmaceutically acceptable carrier.

[0231] In some embodiments, the invention describes a method of treating cancer, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a compound as described in Table II, and a pharmaceutically acceptable carrier.

[0232] In some embodiments the cancer is selected from Acute Lymphoblastic Leukemia, Adult; Acute Lymphoblastic Leukemia, Childhood; Acute Myeloid Leukemia, Adult; Adrenocortical Carcinoma; Adrenocortical Carcinoma, Childhood; AIDS-Related Lymphoma; AIDS-Related Malignancies; Anal Cancer; Astrocytoma, Childhood Cerebellar; Astrocytoma, Childhood Cerebral; Bile Duct Cancer, Extrahepatic; Bladder Cancer; Bladder Cancer, Childhood; Bone Cancer, Osteosarcoma / Malignant Fibrous Histiocytoma; Brain Stem Glioma, Childhood; Brain Tumor, Adult; Brain Tumor, Brain Stem Glioma, Childhood; Brain Tumor, Cerebellar Astrocytoma, Childhood; Brain Tumor, Cerebral Astrocytoma / Malignant Glioma, Childhood; Brain Tumor, Ependymoma, Childhood; Brain Tumor, Medulloblastoma, Childhood; Brain Tumor, Supratentorial Primitive Neuroectodermal Tumors, Childhood; Brain Tumor, Visual Pathway and Hypothalamic Glioma, Childhood; Brain Tumor, Childhood (Other); Breast Cancer; Breast Cancer, Pregnancy; Breast Cancer, Childhood; Breast Cancer, Male; Bronchial Adenomas / Carcinoids, Childhood: Carcinoid Tumor, Childhood; Carcinoid Tumor, Gastrointestinal; Carcinoma, Adrenocortical; Carcinoma, Islet Cell; Carcinoma of Unknown Primary; Central Nervous System Lymphoma, Primary; Cerebellar Astrocytoma, Childhood; Cerebral Astrocytoma / Malignant Glioma, Childhood; Cervical Cancer; Childhood Cancers; Chronic Lymphocytic Leukemia; Chronic Myelogenous Leukemia; Chronic MyeloproliferativeDisorders; Clear Cell Sarcoma of Tendon Sheaths; Colon Cancer; Colorectal Cancer, Childhood; Cutaneous T-Cell Lymphoma; Endometrial Cancer; Ependymoma, Childhood; Epithelial Cancer, Ovarian; Esophageal Cancer; Esophageal Cancer, Childhood; Ewing’s Family of Tumors; Extracranial Germ Cell Tumor, Childhood; Extragonadal Germ Cell Tumor; Extrahepatic Bile Duct Cancer; Eye Cancer, Intraocular Melanoma; Eye Cancer, Retinoblastoma; Gallbladder Cancer; Gastric (Stomach) Cancer; Gastric (Stomach) Cancer, Childhood; Gastrointestinal Carcinoid Tumor; Germ Cell Tumor, Extracranial, Childhood; Germ Cell Tumor, Extragonadal; Germ Cell Tumor, Ovarian; Gestational Trophoblastic Tumor; Glioma. Childhood Brain Stem; Glioma. Childhood Visual Pathway and Hypothalamic; Hairy Cell Leukemia; Head and Neck Cancer; Hepatocellular (Liver) Cancer, Adult (Primary); Hepatocellular (Liver) Cancer, Childhood (Primary); Hodgkin’s Lymphoma, Adult; Hodgkin’s Lymphoma, Childhood; Hodgkin’s Lymphoma During Pregnancy; Hypopharyngeal Cancer; Hypothalamic and Visual Pathway Glioma, Childhood; Intraocular Melanoma; Islet Cell Carcinoma (Endocrine Pancreas); Kaposi’s Sarcoma; Kidney Cancer; Laryngeal Cancer; Laryngeal Cancer, Childhood; Leukemia, Acute Lymphoblastic, Adult; Leukemia, Acute Lymphoblastic, Childhood; Leukemia, Acute Myeloid, Adult; Leukemia, Acute Myeloid, Childhood; Leukemia, Chronic Lymphocytic; Leukemia, Chronic Myelogenous; Leukemia, Hairy Cell; Lip and Oral Cavity Cancer; Liver Cancer, Adult (Primary); Liver Cancer, Childhood (Primary); Lung Cancer, Non-Small Cell; Lung Cancer, Small Cell; Lymphoblastic Leukemia, Adult Acute; Lymphoblastic Leukemia, Childhood Acute; Lymphocytic Leukemia, Chronic; Lymphoma, AIDS — Related; Lymphoma, Central Nervous System (Primary); Lymphoma, Cutaneous T-Cell; Lymphoma, Hodgkin’s, Adult; Lymphoma, Hodgkin’s; Childhood; Lymphoma, Hodgkin’s During Pregnancy; Lymphoma, Non-Hodgkin’s, Adult; Lymphoma, Non-Hodgkin’s, Childhood; Lymphoma, NonHodgkin’s During Pregnancy; Lymphoma, Primary Central Nervous System; Macroglobulinemia, Waldenstrom’s; Male Breast Cancer; Malignant Mesothelioma, Adult; Malignant Mesothelioma, Childhood; Malignant Thymoma; Medulloblastoma, Childhood; Melanoma; Melanoma, Intraocular; Merkel Cell Carcinoma; Mesothelioma, Malignant; Metastatic Squamous Neck Cancer with Occult Primary; Multiple Endocrine Neoplasia Syndrome, Childhood; Multiple Myeloma / Plasma Cell Neoplasm; Mycosis Fungoides; Myelodysplasia Syndromes; Myelogenous Leukemia, Chronic; Myeloid Leukemia, Childhood Acute; Myeloma, Multiple; Myeloproliferative Disorders, Chronic; Nasal Cavity and Paranasal Sinus Cancer; Nasopharyngeal Cancer; Nasopharyngeal Cancer, Childhood; Neuroblastoma; Non-Hodgkin’s Lymphoma, Adult; Non-Hodgkin’s Lymphoma, Childhood; Non-Hodgkin’sLymphoma During Pregnancy; Non-Small Cell Lung Cancer; Oral Cancer, Childhood; Oral Cavity and Lip Cancer; Oropharyngeal Cancer; Osteosarcoma / Malignant Fibrous Histiocytoma of Bone; Ovarian Cancer, Childhood; Ovarian Epithelial Cancer; Ovarian Germ Cell Tumor; Ovarian Low Malignant Potential Tumor; Pancreatic Cancer; Pancreatic Cancer, Childhood’, Pancreatic Cancer, Islet Cell; Paranasal Sinus and Nasal Cavity Cancer; Parathyroid Cancer; Penile Cancer; Pheochromocytoma; Pineal and Supratentorial Primitive Neuroectodermal Tumors, Childhood; Pituitary Tumor; Plasma Cell Neoplasm / Multiple Myeloma; Pleuropulmonary Blastoma; Pregnancy and Breast Cancer; Pregnancy and Hodgkin’s Lymphoma; Pregnancy and Non-Hodgkin’s Lymphoma; Primary Central Nervous System Lymphoma; Primary Liver Cancer, Adult; Primary Liver Cancer, Childhood; Prostate Cancer; Rectal Cancer; Renal Cell (Kidney) Cancer; Renal Cell Cancer, Childhood; Renal Pelvis and Ureter, Transitional Cell Cancer; Retinoblastoma; Rhabdomyosarcoma, Childhood; Salivary Gland Cancer; Salivary Gland’s Cancer, Childhood; Sarcoma, Ewing’s Family of Tumors; Sarcoma, Kaposi’s; Sarcoma (Osteosarcoma Malignant Fibrous Histiocytoma of Bone; Sarcoma, Rhabdomyosarcoma, Childhood; Sarcoma, Soft Tissue, Adult; Sarcoma, Soft Tissue, Childhood; Sezary Syndrome; Skin Cancer; Skin Cancer, Childhood; Skin Cancer (Melanoma); Skin Carcinoma, Merkel Cell; Small Cell Lung Cancer; Small Intestine Cancer; Soft Tissue Sarcoma, Adult; Soft Tissue Sarcoma, Childhood; Squamous Neck Cancer with Occult Primary, Metastatic; Stomach (Gastric) Cancer; Stomach (Gastric) Cancer, Childhood; Supratentorial Primitive Neuroectodermal Tumors, Childhood; T-Cell Lymphoma, Cutaneous; Testicular Cancer; Thymoma, Childhood; Thymoma, Malignant; Thyroid Cancer; Thyroid Cancer, Childhood; Transitional Cell Cancer of the Renal Pelvis and Ureter; Trophoblastic Tumor, Gestational; Unknown Primary Site, Cancer of, Childhood; Unusual Cancers of Childhood; Ureter and Renal Pelvis, Transitional Cell Cancer; Urethral Cancer; Uterine Sarcoma; Vaginal Cancer; Visual Pathway and Hypothalamic Glioma, Childhood; Vulvar Cancer; Waldenstrom’s Macro globulinemia; Wilms’ Tumor; and metastases thereof.

[0233] In various aspects, the cancer is selected from the group consisting of breast, brain, thyroid, prostate, colorectal, pancreas, cervix, stomach, endometrium, liver, bladder, ovary, testis, head and neck, skin, mesothelial lining white blood cells, esophagus, muscle, connective tissue, lung, adrenal gland, kidney, bone or testicle cancer, and metastasis thereof.

[0234] In some embodiments, the compound inhibits the phosphorylation of p27. In other embodiments, the compound inhibits CDK2 and CDK4 and CDK6. In some embodiments, the compound inhibits cancer cell proliferation and / or decreases cancer cell viability.

[0235] Without wishing to be bound be any theory, the compounds of the present invention may act as p27 mimetics, and / or may inhibit the phosphorylation of tyrosine of p27, and / or affect a conformational change of the D-K4-p27 ternary complex, and / or affect the protein-protein interaction between CDK4 / 6 and p27, thereby disturbing the kinase activity of CDK4 / 6 and inhibiting cancer cell progression into the cell cycle.

[0236] Alt-Brk is an ALTternatively-spliced form of Brk containing the SH3 domain, which blocks pY88 and acts as an endogenous CDK4 / 6 inhibitor, and this binding area therefore was identified as a targetable regulatory region within p27. Brk is overexpressed in 60% of breast carcinomas, suggesting that it facilitates cell cycle progression by modulating CDK4 / 6 through p27 tyrosine phosphorylation. Phosphorylation of Tyr-88 / Tyr-89 in the 3io helix of p27 and possibly Y74 reduces its cyclin-dependent kinase (CDK) inhibitory activity. This causes a conformational change in the p27-cyclin D- CDK4 / 6 complex, permitting p27 to vacate the catalytic cleft to allow ATP access and further phosphorylation of the active site. Thus, phosphorylation of this site can switch the tumor suppressive CDK inhibitory activity to an oncogenic activity.

[0237] Blocking CDK4 / 6 activity has long been a goal in cancer therapy. However, this has proven difficult due to the conservation between the active sites of serine / threonine kinases. Most inhibitors reacted with too many other essential kinases to provide any therapeutic benefit. Palbociclib is a CDK4 / 6 inhibitor, that appears to be extremely specific for CDK4 / 6 activity. The advantage of targeting p27 tyrosine (Y) phosphorylation as an indirect way to target CDK4 / 6 activity is that p27 has few substrates and as such its targeting should be more specific. Additionally, use of Palbociclib has shown that targeting CDK4 / 6 is a valid approach. The p27 tyrosine phosphorylation mimetic provides an additional approach for targeting this important kinase, which may have additional benefits.

[0238] Small molecule mimetics of peptide domains are known in the art. For example, VENCLEXTA™ is a mimetic that functions as a BH3 domain of Bcl2 which inhibits Bcl2 action. In a similar fashion, without being bound by any theory, the compounds of the present invention may act as p27 mimetics. Thus, a compound of the present invention may be a functional mimetic of the KI -containing peptide of p27 or an SH3 -containing peptide of Brk having a three-dimensional structure that is similar to that of the native peptide(s), that are capable of inhibiting p27 phosphorylation. The inhibition of p27 phosphorylation prevents CDK2 and CDK4 / 6 activity and therefore prevents cancer cell progression into the cell cycle.

[0239] In one embodiment, the invention provides a method of treating ovarian cancer in a subject including administering to the subject a therapeutically effective amount of a pharmaceutical composition including a compound selected fromthereby treating ovarian cancer in the subject.

[0240] Ovarian cancer is a cancerous tumor of an ovary which may originate from the ovary itself or more commonly from communicating nearby structures such as fallopian tubes or the inner lining of the abdomen. The ovary is made up of three different cell types including epithelial cells, germ cells, and stromal cells, ovarian cancer can emerge from any of those cells, once they become abnormal.

[0241] About 10% of cases are related to inherited genetic risk; women with mutations in the genes BRCA1 or BRCA2 have about a 50% risk of developing the disease. Some family cancer syndromes such as hereditary nonpolyposis colon cancer and Peutz-Jeghers syndrome also increase the risk of developing ovarian cancer. Epithelial ovarian carcinoma is the most common type of ovarian cancer, comprising more than 95% of cases. There are five main subtypes of ovarian carcinoma, of which high-grade serous carcinoma (HGSC) is the most common. Less common types of ovarian cancer include germ cell tumors and sex cord stromal tumors. A diagnosis of ovarian cancer is confirmed through a biopsy of tissue, usually removed during surgery. Ovarian cancers are histologically and genetically divided into type I or type II. Type I cancers are of low histological grade and include endometrioid, mucinous, and clear-cell carcinomas. Type II cancers are of higher histological grade and include serous carcinoma and carcinosarcoma.

[0242] As used herein, the term “ovarian cancer” is meant to refer to any of the various ovarian cancers types, including but not limited to epithelial carcinoma, (derived from the malignant transformation of the epithelium of the ovarian surface, peritoneum, or fallopian tube), serous carcinoma (the most common type of epithelial ovarian cancer), serous tubal intraepithelial carcinoma (STIC), small-cell carcinoma, primary peritoneal carcinoma, clear-cell carcinoma, clear-cell adenocarcinoma, endometrioid adenocarcinoma, malignant mixed Mulllerian tumor (carcinosarcoma), mucinous adenocarcinoma and mucinous cystadenocarcinoma,pseudomyxoma peritonei, undifferentiated epithelial tumor, malignant Brenner tumor, transitional cell carcinoma, sex cord-stromal tumor, granulosa cell tumor (adult and juvenile), Sertoli-Leydig cell tumor, sclerosing stromal tumors, germ cell tumor, dysgerminoma, choriocarcinoma, immature (solid) teratoma, mature cystic teratoma of the ovary, mature teratoma (dermoid cyst), yolk sac tumor / endodermal sinus tumor, embryonal carcinoma, polyembryoma, squamous cell carcinoma, mixed tumors, secondary ovarian cancer and borderline tumors.

[0243] The compounds described herein, for use for the treatment of ovarian cancer (andpancreatic cancer as described below) are:also referredherein as compound 34, or compounds 34;also referred herein as compound 35 A or compound 35 A.

[0244] In one aspect, the compound inhibits phosphorylation of p27. In another aspect, the compound inhibits CDK2 and CDK4 / 6. In one aspect the compound inhibits ovarian cancer cell proliferation and / or decreases ovarian cancer cell viability. In another aspect, the compound increases ovarian cancer cell death.

[0245] In another embodiment, the invention provides method of treating pancreatic cancer in a subject including administering to the subject a therapeutically effective amount of a pharmaceutical composition including a compound selected fromthereby treating pancreatic cancer in the subject.

[0246] Pancreatic cancer arises from cells in the pancreas. The most common, pancreatic adenocarcinoma, accounts for about 90% of cases. Pancreatic cancer rarely occurs before the age of 40, and more than half of cases of pancreatic adenocarcinoma occur in those over 70. Risk factors for pancreatic cancer include tobacco smoking, obesity, diabetes, and certain rare genetic conditions. About 25% of cases are linked to smoking, and 5-10% are linked to inherited genes.

[0247] As used herein, the term “pancreatic cancer” is meant to refer to any of the various pancreatic cancers types, including but not limited to exocrine pancreatic cancers (including pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma (PDAC), acinar cell carcinoma of the pancreas, cystadenocarcinomas, pancreatoblastoma, adenosquamous carcinomas, signet ring cell carcinomas, hepatoid carcinomas, colloid carcinomas, undifferentiated carcinomas, undifferentiated carcinomas with osteoclast-like giant cells, and solid pseudopapillary tumor), pancreatic mucinous cystic neoplasms, and neuroendocrine pancreatic tumor (PanNETs, including insulinomas, gastrinomas, and nonfunctioning tumors)

[0248] In one aspect, the compound inhibits phosphorylation of p27. In another aspect, the compound inhibits CDK2 and CDK4 / 6. In one aspect the compound inhibits pancreatic cancer cell proliferation and / or decreases pancreatic cancer cell viability. In another aspect, the compound increases pancreatic cancer cell death.

[0249] In various aspects, the methods described herein further include administering to the subject an anti-cancer treatment.

[0250] In one aspect, the anti-cancer treatment is selected from the group consisting of chemotherapy, radiation treatment, immunotherapy, resection of a tumor, and any combination thereof. In another aspect, the anti-cancer treatment is administered prior to, simultaneously with, or after administration of the pharmaceutical composition. In one aspect, the anti-cancer treatment is an anti-cancer agent selected from the group consisting of palbociclib, ribociclib, abemaciclib, osirmetinib, gefitinib, lapatinib, pantitumumab, vandetanib, necitumumab, vemurafenib, sorafenib tosylate, PLX-4720, dabrafenib, paclitaxel, cisplatin, docetaxol, carboplatin, vincristine, vinblastine, methotrexate, cyclophosphamide, CPT-11, 5 -fluorouracil, gemcitabine, estramustine, carmustine, adriamycin, etoposide, arsenic trioxide, irinotecan, Herceptin, vemurafenib, erlotinib, cetuximab, letrozole, fulvestrant, epolhilone derivatives, elacestrant, lasofoxifene, anastrozole, sotorasib, and adagrasib. trametinib, cobmetinib, Bay-293, everolimus, erolintib, cetaxumab, panitumumab, and afatinib, imatinib, sunitinib, panatinib, axitinib, foretinib, nintedanib, and amuvatinib, adavorsertib, pertuzumab, am-trastuzumab-deruxtecan, T-DM1 or ado-trastuzumab emtansine, pertuzumab / trastuzumab / hyaluronidase, neratinib), and tucatinib.

[0251] In one aspect, the methods described herein further include administering to the subject a second CDK2-selective inhibitor.

[0252] In some aspects, the CDK2-selective inhibitor is PF-07104091.

[0253] In one aspect, the subject is a human.

[0254] In another aspect, administration of the pharmaceutical composition is intracutaneous, subcutaneous, intravenous, intraperitoneal, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transdermal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoidal, intraspinal, intrastemal, oral, sublingual, buccal, rectal, vaginal, nasal or ocular, or by infusion, inhalation, or nebulization.

[0255] The following examples are provided to further illustrate the embodiments of the present invention but are not intended to limit the scope of the invention. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.EXAMPLES

[0256] General Procedures for the Preparation of the Compounds of the Invention

[0257] The compounds of the invention may be prepared according to one of the following general procedures A-F

[0258] General Procedure A:Each of R1and R2is selected from alkyl, or aryl; X’ is selected from CH, or N; and Y’ is selected from C(O), or SO2.

[0259] General Procedure B:Substituent R2is selected from alkyl, or aryl; and Y’ is selected from C(O), or SO2.

[0260] General Procedure C:Substituent R1, is selected from alkyl, or aryl.Each of R1and R2is selected from alkyl, or aryl; X’ is selected from CH, or N; and Y’ is selected from C(O), or SO2.

[0262] General Procedure E:Each of R1and R3is selected from alkyl, or aryl and X’ is selected from CH, or N.

[0263] General Procedure F:Substituent R1is selected from alkyl, or aryl.Example 1: Preparation of 5-(4-(2,3-dimethylphenyl)piperazin-l-yl)-2-((2,5-dimethylphenyl)sulfonamido)benzoic acid (Compound LI)

[0264] Step 1: Synthesis of methyl 5-(4-(2,3-dimethylphenyl) piperazin- l-yl)-2-nitrobenzoate

[0265] To a stirred solution of methyl 5-fluoro-2-nitrobenzoate (0.50 g, 2.5 mmol) in acetonitrile (15 mL) at room temperature was added l-(2,3-dimethylphenyl) piperazine hydrochloride (0.854 g, 3.77 mmol) followed by potassium carbonate (0.694 g, 5.02 mmol). The mixture was heated at 80 °C for 12 h. After this time, the mixture was cooled to room temperature, combined with water (20 mL), and extracted with ethyl acetate (3 x 40 mL). The organic extracts were collected, dried over sodium sulfate, fdtered and concentrated at reduced pressure. The residue obtained was chromatographed (silica, 0-10% ethyl acetate in hexanes) to afford the title compound as a yellow solid (0.40 g, 43%).

[0266] 'H NMR (400 MHz, CDC13) 58.06-8.03 (m, 1H), 7.10 (t, J= 8.0 Hz, 1H), 6.95 (d, J = 8.0 Hz, 1H), 6.92-6.89 (m, 3H), 3.93 (s, 3H), 3.58 (br s, 4H), 3.01 (t, J= 5.2 Hz, 4H), 2.29 (s, 3H), 2.26 (s, 3H). MS (ESI) 370.2.

[0267] Step 2: Synthesis of methyl 2-amino-5-(4-(2,3-dimethylphenyl) piperazin- 1-yl)benzoate

[0268] To a stirred solution of methyl 5-(4-(2,3-dimethylphenyl) piperazin- l-yl)-2-nitrobenzoate (0.40 g, 1.1 mmol) in tetrahydro furan (10 mL) and water (3 mL) was added ammonium chloride (0.579 g, 10.8 mmol). Zinc dust (0.354 g, 5.41 mmol) was charged portionwise over 10 min. The reaction mixture was stirred at 25 °C for 4 h. After this time, the solids were removed by filtration through a pad of diatomaceous earth, and the filter cake was washed with ethyl acetate (2 x 20 mL). The filtrate was washed with water and brine, dried over sodium sulfate, filtered and concentrated at reduced pressure. The residue obtained was chromatographed (silica, 0-15% ethyl acetate in hexanes) to afford the title compound (0.30 g, 82%) as light-brown solid.

[0269] 'H NMR (400 MHz, DMSO-d6) 57.26 (d, J= 2.8 Hz, 1H), 7.15 (dd, J= 8.4, 2.8 Hz, 1H), 7.05 (t, J= 7.6 Hz, 1H), 6.93-6.87 (m, 2H), 6.75 (d, J= 8.8 Hz, 1H), 6.30 (br s, 2H), 3.78 (s, 3H), 3.07 (br s, 4H), 2.93-2.91 (m, 4H), 2.21 (s, 3H), 2.18 (s, 3H). MS (ESI) 340.2.

[0270] Step 3: Synthesis of methyl 5-(4-(2,3-dimethylphenyl) piperazin- l-yl)-2-((2, 5-dimethylphenyl)-sulfonamido)benzoate

[0271] To a stirred solution of methyl 2-amino-5-(4-(2,3-dimethylphenyl)piperazin-l-yl)benzoate (0.30 g, 0.88 mmol) in dichloromethane (10 mL) was added N, N-diisopropylethylamine (0.463 mL, 2.65 mmol) at 0 °C. 2,5-Dimethylbenzenesulfonyl chloride (0.362 g, 1.77 mmol) was added dropwise, and the mixture was allowed to warm to room temperature and stir for 12 h. After this time, water was added, and the mixture was extracted with ethyl acetate (3 x 30 mL). The organic extracts were collected, dried over sodium sulfate, filtrated and concentrated at reduced pressure. The residue obtained was chromatographed (silica, 0-10% ethyl acetate in 95:5 hexanes / dichloromethane) to afford the title compound (0.25 g, 56%) as lightbrown solid.

[0272] 'H NMR (400 MHz, CDC13) 5 10.30 (s, 1H), 7.81 (s, 1H), 7.50 (d, J= 9.2 Hz, 1H), 7.48 (d, J= 2.8 Hz, 1H), 7.19 (d, J= 7.6 Hz, 1H), 7.11-7.07 (m, 3H), 6.94-6.92 (m, 2H), 3.87 (s, 3H), 3.24 (br s, 4H), 3.01 (t, J = 4.8 Hz, 4H), 2.57 (s, 3H), 2.33 (s, 3H), 2.28 (s, 3H), 2.23 (s, 3H). MS (ESI) 508.2.

[0273] Step 4: Synthesis of 5-(4-(2,3-dimethylphenyl)piperazin-l-yl)-2-((2,5-dimethylphenyl)sulfonamido)-benzoic acid

[0274] To a stirred solution of methyl 5-(4-(2,3-dimethylphenyl)piperazin-l-yl)-2-((2,5-dimethylphenyl)sulfon-amido)benzoate (0.25 g, 0.49 mmol) in tetrahydro furan (10 mL) and water(3.0 mL) was added lithium hydroxide (0.059 g, 2.5 mmol), and the mixture was stirred at 25 °C for 12 h. After this time, the volatiles were removed under reduced pressure, and the aqueous residue was diluted with water (5 mL) and extracted with dichloromethane (10 mL). The aqueous layer was acidified with 3 N HC1 to pH 2 and extracted with ethyl acetate (3 x 10 mL). The organic extracts were collected, dried over sodium sulfate, fdtered and concentrated under reduced pressure to afford the title Compound LI (0.100 g, 41%) as an off-white solid.

[0275] 'H NMR (400 MHz, DMSO-<76) 5 13.92 (br s, 1H), 10.79 (br s, 1H), 7.73 (s, 1H), 7.42 (d, J= 2.8 Hz, 1H), 7.36-7.32 (m, 2H), 7.27-7.21 (m, 2H), 7.04 (t, J= 7.6 Hz, 1H), 6.90 (t, J= 12 Hz, 2H), 3.20 (br s, 4H), 2.89-2.86 (m, 4H), 2.48 (s, 3H), 2.30 (s, 3H), 2.20 (s, 3H), 2.16 (s, 3H). MS (ESI) 494.2.Example 2: Preparation of Compounds 14, 17A, 17B, 17C, 24, 25, 26, 27 and 29

[0276] Compounds 14, 17A, 17B, 17C, 24, 25, 26, 27, 29, L2, L4, and L6 were prepared in a similar manner to Example 1:Table 3.Example 3: Preparation of 5-(4-(2,3-dimethylphenyl)piperidin-l-yl)-2-((2,5-dimethylphenyl)sulfonamido)benzoic acid (Compound 20):

[0277] Step 1: Synthesis of te / -butyl 4-(2,3-dimethylDhenyl)-3,6-dihvdroDyridine-l(2H)-carboxylate

[0278] To a stirred solution of l-bromo-2,3-dimethylbenzene (2.0 g, 11 mmol) in 1,4-dioxane (30 mL) was added tert-butyl 4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-l(2H)-carboxylate (5.01 g, 16.2 mmol) and 2 M potassium carbonate (16.2 mL, 32.4 mmol) in a reaction tube. The mixture was purged with argon for 5 min. Dichlorofl, T-bis(diphenylphosphino) ferrocene]palladium dichloromethane adduct (0.441 g, 0.540 mmol) was added, and argon purge was continued for 5 min. The vial was sealed, and the mixture was heated at 100 °C for 4 h. After this time, water was added and the mixture was extracted with ethyl acetate (3 x50 mL). The organic extracts were combined, washed with water and brine, dried over sodium sulfate, filtered and concentrated at reduced pressure. The residue obtained was chromatographed (silica, 0-6% ethyl acetate in hexanes) to afford the title compound (2.0 g, 64%) as an oil.

[0279] ‘HNMR (400 MHz, CDC13) 57.07-7.03 (m, 2H), 6.92 (dd, J= 6.0, 2.4 Hz, 1H), 5.51 (br s, 1H), 4.02 (br s, 2H), 3.61 (t, J= 6.8 Hz, 2H), 2.31 (br s, 2H), 2.28 (s, 3H), 2.16 (s, 3H), 1.50 (s, 9H).

[0280] Step 2: Synthesis of butyl 4-(2,3-dimethylphenyl)piperidine-l -carboxylate

[0281] To a stirred solution of tert-butyl 4-(2,3-dimethylphenyl)-3,6-dihydropyridine-l(2H)-carboxylate (1.8 g, 6.3 mmol) in ethanol (40 mL) was added 10% Pd / C (1.33 g, 6.26 mmol) under nitrogen. The flask was evacuated and backfilled with hydrogen (2x), and the mixture was stirred at room temperature for 3 h. After this time, the catalyst was removed by filtration through a pad of diatomaceous earth, and the filter cake was rinsed with ethyl acetate (3 x 30 mL). The filtrate was concentrated under reduced pressure to afford the title compound (1.8 g, 99%) as an oil.

[0282] 'H NMR (400 MHz, CDC13) 5 7.10-7.01 (m, 3H), 4.25 (br s, 2H), 2.91 (tt, J= 12.0 Hz, 3.2 Hz, 1H), 2.82 (br t, J= 12.0 Hz, 2H), 2.29 (s, 3H), 2.24 (s, 3H), 1.75 (br d, J= 13.2 Hz, 2H), 1.67-1.55 (m, 2H), 1.49 (s, 9H).

[0283] Step 3: Synthesis of 4-(2,3-dimethylphenyl)piperidine hydrochloride

[0284] To a stirred solution of tert-butyl 4-(2,3-dimethylphenyl)piperidine-l-carboxylate (1.8 g, 6.2 mmol) in 1,4-dioxane (10 mL) was added 4 M hydrogen chloride in 1,4-dioxane (15.5 mL, 62.2 mmol), and the mixture was stirred at room temperature for 1 h. After this time, the solvent was removed under reduced pressure. The residue obtained was tritiurated with pentane to afford the title compound (1.3 g, 93%) as a white solid.

[0285] 'H NMR (400 MHz, DMSO-d6) 5 9.08-9.03 (m, 2H), 7.08 (t, J= 7.2 Hz, 1H), 7.01 (t, J= 8.0 Hz, 2H), 3.32 (br d, J= 12.0 Hz, 2H), 3.16-2.98 (m, 3H), 2.24 (s, 3H), 2.20 (s, 3H), 1.92-1.76 (m, 4H).

[0286] Step 4: Synthesis of methyl 5-(4-(2,3-dimethylphenyl)piperidin-l-yl)-2-nitrobenzoate

[0287] To a stirred solution of methyl 5-fluoro-2-nitrobenzoate (1.0 g, 5.0 mmol) in N, N-dimethylformamide (15 mL) was added 4-(2,3-dimethylphenyl)piperidine hydrochloride (1.36 g, 6.03 mmol) followed by potassium carbonate (2.082 g, 15.06 mmol) at room temperature. The reaction mixture was stirred at 90 °C for 8 h. After this time, the mixture was cooled to room temperature, mixed with water (50 mL) and extracted with ethyl acetate (3 x 40 mL). The organic layers were collected, dried over sodium sulfate, filtered and concentrated at reduced pressure. The residue obtained was chromatographed (silica, 0-10% ethyl acetate in hexanes) to afford the title compound (1.7 g, 92%) as yellow solid.

[0288] ’HNMR (400 MHz, CDCI3) 5 8.08-8.02 (m, 1H), 7.11-7.01 (m, 3H), 6.89-6.87 (m, 2H), 4.10 (br d, J= 12.0 Hz, 2H), 3.93 (s, 3H), 3.16-3.04 (m, 3H), 2.31 (s, 3H), 2.28 (s, 3H), 1.91 (br d, J= 13.2 Hz, 2H), 1.89 (qd, J= 12.4 Hz, 4.0 Hz, 2H).

[0289] Step 5: Synthesis of methyl 2-amino-5-(4-(2,3-dimethylphenyl)piperidin-l-yl)benzoate

[0290] To a stirred solution of methyl 5-(4-(2,3-dimethylphenyl)piperidin-l-yl)-2-nitrobenzoate (1.5 g, 4.1 mmol) in tetrahydrofuran (15 mL) and water (5 mL) was added ammonium chloride (2.178 g, 40.72 mmol). Zinc dust (1.331 g, 20.36 mmol) was then charged portion- wise over 10 min. The resulting mixture was stirred at 25 °C for 4 h. After this time, the solids were removed by filtration through diatomaceous earth, and the filter cake was washed with ethyl acetate (2 x 20 mL). The filtrate was washed with water (20 mL) and brine (20 mL), dried over sodium sulfate, filtered and concentrated at reduced pressure. The residue obtained was triturated with hexanes to afford the title compound (1.3 g, 94%) as an off-white solid.

[0291] MS (ESI) 339.3.

[0292] Step 6: Synthesis of methyl 5-(4-(2,3-dimethylphenyl)piperidin-l-yl)-2-((2,5-dimethylphenyl)-sulfonamido)benzoate

[0293] To a stirred solution of methyl 2-amino-5-(4-(2,3-dimethylphenyl)piperidin-l-yl)benzoate (0.3 g, 0.9 mmol) in dichloromethane (20 mL) was added 2,5-dimethylbenzenesulfonyl chloride (0.363 g, 1.77 mmol) and pyridine (0.215 mL, 2.66 mmol), and the mixture was stirred at room temperature for 16 h. After this time, water (15 mL) was added, and the mixture was extracted with di chloromethane (20 mL). The organic layers were combined, washed with brine (10 mL), dried over sodium sulfate, filtered and concentrated at reduced pressure. The residue obtained was chromatographed (silica, 0-10% ethyl acetate in hexanes) to afford the title compound (0.200 g, 44%) as an off-white solid.

[0294] 'H NMR (400 MHz, CDC13) 5 10.30 (s, 1H), 7.81 (s, 1H), 7.50-7.47 (m, 2H), 7.20 (d, J= 6.4 Hz, 1H), 7.19-7.08 (m, 4H), 7.06-7.01 (m, 1H), 3.87 (s, 3H), 3.70 (br d, J= 13.4 Hz, 2H), 2.92-2.85 (m, 1H), 2.79 (td, J= 11.6 Hz, 3.2 Hz, 2H), 2.57 (s, 3H), 2.33 (s, 3H), 2.30 (s, 3H), 2.24 (s, 3H), 1.89-1.78 (m, 4H). MS (ESI) 507.2.

[0295] Step 7: Synthesis of 5-(4-(2,3-dimethylphenyl)piperidin-l-yl)-2-((2,5-dimethylphenyl)sulfonamido)-benzoic acid

[0296] To a stirred solution of methyl 5-(4-(2,3-dimethylphenyl)piperidin-l-yl)-2-((2,5-dimethylphenyl)-sulfonamido)benzoate (0.200 g, 0.395 mmol) in tetrahydro furan (10 mL) and water (5.0 mL) was added lithium hydroxide (0.0473 g, 1.97 mmol). The reaction mixture was stirred at room temperature for 8 h. After this time, the volatiles were removed under reduced pressure. The aqueous residue was diluted with water (5 mL) and extracted with dichloromethane (20 mL). The aqueous layer was acidified with 3 N HC1 to pH 2 and extracted with ethyl acetate(3 x 10 mL). The organic extracts were collected, dried over sodium sulfate, filtered and concentrated under reduced pressure to afford the title compound 20 (0.150 g, 77%) as an off-white solid.

[0297] 'H NMR (400 MHz, DMSO-<76) 5 13.85 (br s, 1H), 10.80 (br s, 1H), 7.73 (s, 1H), 7.45 (br s, 1H), 7.34 (d, J= 7.2 Hz, 2H), 7.26 (d, J= 8.0 Hz, 2H), 7.04-6.97 (m, 3H), 3.68 (br d, J= 12.0 Hz, 2H), 2.94-2.83 (m, 1H), 2.83-2.72 (m, 2H), 2.45 (s, 3H), 2.30 (s, 3H), 2.22 (s, 3H), 2.19 (s, 3H), 1.76-1.68 (m, 4H). MS (ESI) 493.2.Example 4: 2-((2,5-dimethylbenzyl)oxy)-5-(4-(2,3-dimethylphenyl)piperazin-l-yl)benzoic acid (Compound 16):

[0298] Step 1: Synthesis of methyl 5-bromo-2-((2,5-dimethylbenzyl)oxy)benzoate

[0299] A mixture of l-(2,3-dimethylphenyl)piperazine (0.246 g, 1.29 mmol) and methyl 5-bromo-2-((2,5-dimethylbenzyl)oxy)benzoate (0.307 g, 0.879 mmol) in toluene (2 mL) was sparged with argon for 5 min. Cesium carbonate (0.430 g, 1.32 mmol), BINAP (0.055 g, 0.088 mmol) and palladium(II) acetate (0.0099 g, 0.044 mmol) were added, the mixture was sparged with argon for three min and was heated at 100 °C overnight. After this time, second portions of cesium carbonate (0.240 g, 0.737 mmol), BINAP (0.020 g, 0.032 mmol) and palladium (II) acetate (0.0097 g, 0.043 mmol) were added, and heating was continued for 8 h. Third portions of cesium carbonate (0.240 g, 0.737 mmol), BINAP (0.020 g, 0.032 mmol) and palladium(II) acetate (0.0097 g, 0.043 mmol) were added, and heating was continued overnight. After this time, the mixture was cooled to room temperature, diluted with ethyl acetate (30 mL) and filtered through diatomaceousearth. The filtrate was washed with brine (50 mL), dried over sodium sulfate, filtered and concentrated at reduced pressure. The residue obtained was chromatographed (silica, 0-25% ethyl acetate in heptane) to afford the title compound (0.2763 g, 69%) as a white solid.

[0300] 'H NMR (500 MHz, DMSO-<76) 67.79 (d, J= 2.5 Hz, 1H), 7.73 (dd, J= 8.8, 3.0 Hz, 1H), 7.32-7.29 (m, 2H), 7.10 (d, J= 7.5 Hz, 1H), 7.05 (dd, J= 7.8, 1.5 Hz, 1H), 5.12 (s, 2H), 3.79 (s, 3H), 2.28 (s, 3H), 2.27 (s, 3H).

[0301] Step 2: Synthesis of methyl 2-((2,5-dimethylbenzyl)oxy)-5-(4-(2,3-dimethylphenyl)pmerazin-l-yl)-benzoate

[0302] A mixture of l-(2,3-dimethylphenyl)piperazine (0.246 g, 1.29 mmol) and methyl 5-bromo-2-((2,5-dimethylbenzyl)oxy)benzoate (0.307 g, 0.879 mmol) in toluene (2 ml) was sparged with argon for 5 min. Cesium carbonate (0.430 g, 1.32 mmol), BINAP (0.055 g, 0.088 mmol) and palladium(II) acetate (9.87 mg, 0.044 mmol) were added. The mixture was sparged for 3 min and heated under argon at 100 °C overnight. After this time, second portions of cesium carbonate (0.240 g, 0.737 mmol), palladium (II) acetate (0.0097 g, 0.043 mmol) and BINAP (0.020 g, 0.032 mmol), were added and heating continued. After another 8 h, third portions of cesium carbonate (0.240 g, 0.737 mmol), palladium (II) acetate (0.0097 g, 0.043 mmol) and BINAP (0.020 g, 0.032 mmol), were added and heating continued overnight. The mixture was cooled to room temperature, diluted with ethyl acetate (30 mL) and filtered through diatomaceous earth. The filtrate was washed with brine (50 mL), dried over sodium sulfate, filtered and concentrated at reduced pressure. The residue obtained was chromatographed (silica, 0-25% ethyl acetate in heptane) to afford the title compound (0.276 g, 69%) as a white solid.

[0303] 'H NMR (500 MHz, DMSO-<76) 6 7.32 (s, 1H), 7.27 (d, J= 2.5 Hz, 1H), 7.24-7.19 (m, 4H), 7.10-7.03 (m, 4H), 6.94 (d, J= 8.0 Hz, 1H), 6.90 (d, J= 7.5 Hz, 1H), 5.04 (s, 2H), 3.78 (s, 3H), 3.22 (br s, 4H), 2.95 (t, J= 4.5 Hz, 4H), 2.28 (s, 3H), 2.23 (s, 3H), 2.20 (s, 3H). MS (ESI) 459.1.

[0304] Step 3: Synthesis of 2-((2,5-dimethylbenzyl)oxy)-5-(4-(2,3-dimethylphenyl)piperazin- 1 -yl)benzoic acid

[0305] A 1.25 M aqueous sodium hydroxide solution (0.654 mL, 0.818 mmol) was added to a solution of methyl 2-((2,5-dimethylbenzyl)oxy)-5-(4-(2,3-dimethylphenyl)piperazin-l-yl)benzoate (0.125 g, 0.273 mmol) in tetrahydrofuran (3 mL) and methanol (1 mL). The mixture was stirred at room temperature. After 1.5 h, methanol (3 mL) was added to redissolve the formed precipitate. After two more hours, 1.25 M sodium hydroxide (0.654 mL, 0.818 mmol) and methanol (1.5 mL) were added and stirring was continued overnight. The volatiles were thenremoved under reduced pressure, and the aqueous residue was diluted with water (15 mL), acidified to pH 3 with 3 N hydrochloric acid and extracted with ethyl acetate (3 x 30 mL). The product was not fully soluble, so the organic layer as a suspension was concentrated at reduced pressure. The residue obtained was chromatographed (silica, 0-2% methanol in dichloromethane), and the isolated product was recrystallized from acetonitrile to afford the title compound 16 (0.0872 g, 72%) as a white solid.

[0306] 'H NMR (500 MHz, DMSO-<76) 6 12.58 (br s, 1H), 7.34 (s, 1H), 7.26 (d, J= 2.5 Hz, 1H), 7.19-7.15 (m, 2H), 7.09-7.03 (m, 3H), 6.94 (d, J= 8.0 Hz, 1H), 6.90 (d, J= 7.5 Hz, 1H), 5.03 (s, 2H), 3.22 (br s, 4H), 2.95 (t, J= 5.0 Hz, 4H), 2.28 (s, 3H), 2.27 (s, 3H), 2.22 (s, 3H), 2.20 (s, 3H). MS (ESI) 445.2.Example 5: 3-(4-(2,3-dimethylphenyl)piperazin-l-yl)benzoic acid (Compound 23):

[0307] Step 1: Synthesis ofterLbutyl 3-(4-(2,3-dimethylphenyl)piperazin-l-yl)benzoate

[0308] A solution of tert-butyl 3 -bromobenzoate (0.0415 g, 0.161 mmol) and l-(2,3-dimethylphenyl)piperazine (0.038 g, 0.20 mmol) in toluene (3 mL) was sparged with argon for 5 min. Cesium carbonate (0.084 g, 0.26 mmol), BINAP (0.013 g, 0.021 mmol) and palladium(II) acetate (0.007 g, 0.03 mmol) were added, the mixture was sparged for 5 min, and heated at 100 °C for 18 h. After this time, the mixture was allowed to cool to room temperature and was filtered through diatomaceous earth, and the filter cake rinsed with dichloromethane (3 x 5 mL). The filtrate was concentrated at reduced pressure, and the residue obtained was chromatographed (silica, heptane to dichloromethane) to afford the title compound (0.036 g, 61%) as a white solid.

[0309] 'H NMR (500 MHz, CDCh) 57.65 (br s, 1H), 7.51 (br s, 1H), 7.33 (br t, J= 8.5 Hz, 1H), 7.17 (br s, 1H), 7.11 (t, J= 7.5 Hz, 1H), 6.97 (d, J= 7.5 Hz, 1H), 6.94 (d, J= 7.5 Hz, 1H), 3.41 (br s, 4H), 3.08 (br s, 4H), 2.29 (s, 3H), 2.27 (s, 3H), 1.60 (s, 9H). MS (ESI) 367.2.

[0310] Step 2: Synthesis of 3-(4-(2,3-dimethylphenyl)piperazin-l-yl)benzoic acid

[0311] Trifluoroacetic acid (1.0 mL, 13 mmol) was added dropwise to a solution of tert-butyl 3-(4-(2,3-dimethyl-phenyl)piperazin-l-yl)benzoate (0.0350 g, 0.0955 mmol) in dichloromethane (5 mL) at room temperature, and the mixture was stirred for 3 h. After this time, the volatiles were removed at reduced pressure. Saturated sodium bicarbonate solution (8 mL) was added to the residue, and the pH was adjusted to 4-5 by addition of acetic acid. The product was extracted into dichloromethane (4 x 8 mL), and the organic layers were combined, dried over sodium sulfate, fdtered and concentrated at reduced pressure. The crude product was recrystallized from acetonitrile to afford the title compound (0.0086 g, 29%) as an off-white solid.

[0312] 'H NMR (500 MHz, DMSO-d6) 6 12.82 (br s, 1H), 7.52 (br s, 1H), 7.39 (dt, J= 6.0, 1.5 Hz, 1H), 7.35 (t, J= 8.0Hz, 1H), 7.26 (ddd, J= 8.0, 2.5, 1.0 Hz, 1H), 7.06 (t, J= 7.5 Hz, 1H), 6.94 (d, J= 7.5 Hz, 1H), 6.90 (d, J= 7.5 Hz, 1H), 3.37-3.32 (m, 4H), 2.96 (t, J= 5.0 Hz, 4H), 2.23 (s, 3H), 2.20 (s, 3H). MS (ESI) 311.2.Example 6: N-(4-(4-(2,3-dimethylphenyl)piperazin-l-yl)phenyl)-2,5-dimethylbenzenesulfonamide: (Compound 11):

[0313] Step 1: Synthesis of l-(2,3-dimethylphenyl)-4-(4-nitrophenyl)piperazine

[0314] To l-fhioro-4-nitrobenzene (0.2184 g, 1.548 mmol) was added a solution of 1 -(2,3-dimethylphenylj-piperazine (0.292 g, 1.53 mmol) in dimethyl sulfoxide (10 mL) followed bypotassium carbonate (0.318 g, 2.30 mmol), and the mixture was stirred at room temperature for 5.5 h. After this time, water (15 mL), was added, and the precipitate was collected by filtration, washed with water (3 x 5 mL) and dried under vacuum to afford the title compound (0.231 g, 48%) as a yellow solid.

[0315] ‘HNMR (500 MHz, DMSO-<76) 68.10-8.07 (m, 2H), 7.12-7.08 (m, 2H), 7.06 (t, J= 7.5 Hz, 1H), 6.93-6.90 (m, 2H), 3.61 (br s, 4H), 2.93 (t, J= 5.0 Hz, 4H), 2.23 (s, 3H), 2.22 (s, 3H). MS (ESI) 312.1.

[0316] Step 2: Synthesis of 4-(4-(2,3-dimethylphenyl)piperazin-l-yl)aniline

[0317] A suspension of l-(2,3-dimethylphenyl)-4-(4-nitrophenyl)piperazine (0.102 g, 0.326 mmol) in ethanol (30 mL) was purged with nitrogen. Palladium hydroxide on carbon (5% w / w, 0.030 g, 0.014 mmol) was added, and the mixture was purged with nitrogen, then hydrogen and stirred under 1 atm of hydrogen for 5.5 h. After this time, the mixture was purged with nitrogen and filtered through diatomaceous earth. The filter cake was washed with ethanol (3 x 10 mL), and the filtrate was concentrated at reduced pressure to afford the title compound (0.085 g, 93%) as an off-white solid.

[0318] 'H NMR (500 MHz, DMSO-d6) 67.05 (t, J = 8.0 Hz, 1H), 6.93 (d, J = 7.5 Hz, 1H), 6.88 (d, J= 7.0 Hz, 1H), 6.76-6.73 (m, 2H), 6.53-6.50 (m, 2H), 4.58 (br s, 2H), 3.06 (br s, 4H), 2.91 (t, J= 4.5 Hz, 4H), 2.21 (s, 3H), 2.18 (s, 3H). MS (ESI) 282.0.

[0319] Step 3: A-(4-(4-(2,3-dimethylphenyl)piperazin-l-yl)phenyl)-2,5-dimethylbenzenesulfonamide

[0320] To a solution of 4-(4-(2,3-dimethylphenyl)piperazin-l-yl)aniline (0.085 g, 0.30 mmol) in dichloromethane (10 mL) was added pyridine (0.49 mL, 0.60 mmol), followed by a solution of 2,5-dimethylbenzene-sulfonyl chloride (0.062 g, 0.30 mmol) in dichloromethane (5 mL), added dropwise over 2 min at room temperature. The mixture was stirred overnight at room temperature. After this time, the solution was washed with saturated ammonium chloride solution (20 mL) and water (3 x 20 mL), dried over sodium sulfate, filtered and concentrated at reduced pressure. The residue obtained was chromatographed (silica, 0-10% ethyl acetate in dichloromethane), and the product was recrystallized from acetonitrile to afford the title compound (0.0707 g, 52%) as a white solid.

[0321] 'H NMR (500 MHz, DMSO-d6) 69.84 (s, 1H), 7.61 (d, J= 1.0 Hz, 1H), 7.29 (dd, J = 7.8, 1.0 Hz, 1H), 7.23 (d, J= 8.0 Hz, 1H), 7.04 (t, J= 8.0 Hz, 1H), 6.93-6.87 (m, 4H), 6.86-6.84 (m, 2H), 3.18 (br s, 4H), 2.89 (t, J= 5.0 Hz, 4H), 2.28 (s, 3H), 2.50 (s, 3H), 2.21 (s, 3H), 2.17 (s, 3H). MS (ESI) 450.1.Example 7: Synthesis of 5-(4-(2,3-dimethylphenyl)piperazin-l-yl)-2-(N-(2,5-dimethylphenyl)sulfamoyl)benzoic acid (Compound 13):Step 2

[0322] Step 1: Synthesis of 5-bromo-2-(2,5-climcthylphcnyl)-3-imino-2,3-dihydrobenzo[d1isothiazole 1, 1 -dioxide

[0323] Pyridine (0.231 mL, 2.85 mmol) was added to a solution of 2, 5 -dimethylaniline (0.1884 g, 1.555 mmol) in dichloromethane (3 mL). The solution was cooled in an ice-water bath, and a solution of 4-bromo-2-cyanobenzenesulfonyl chloride (0.4000 g, 1.426 mmol) in dichloromethane (3 mL) was added dropwise over 15 min. After complete addition, the mixture was allowed to warm to room temperature and stir for 3 h. After this time, dichloromethane (20 mL) was added, and the solution was washed with saturated ammonium chloride solution (3 mL) and water (3 x 30mL). The layers were separated, and the aqueous layer was extracted with dichloromethane (30 mL). The organic layers were combined, washed with brine (30 mL), dried over sodium sulfate, fdtered, and concentrated under reduced pressure. The residue obtained was chromatographed (silica, 0-5% ethyl acetate in dichloromethane) to afford the title compound (0.264 g, 51%) as a light-orange foam.

[0324] 'H NMR (500 MHz, DMSO-d6, 1:0.4 ratio of E / Z isomers) 59.91 (s, 1H), 8.83 (d, J = 1.5 Hz, 1H), 8.32 (d, J= 1.5 Hz, 0.4H), 8.26 (d, J= 8.5 Hz, 0.4H), 8.24 (d, J= 8.0 Hz, 1H), 8.22 (s, 0.4H), 8.20 (dd, J= 8.5, 1.5 Hz, 0.4H), 8.17 (dd, J= 8.5, 1.5 Hz, 1H), 7.42 (d, J= 7.5 Hz, 0.4H), 7.38 (dd, J= 8.0, 1.0 Hz, 0.4H), 7.33 (d, J= 8.0 Hz, 1H), 7.29 (dd, J= 8.0, 1.5 Hz, 1H), 7.19 (br s, 0.4H), 7.16 (br s, 1H), 2.35 (s, 1.2H), 2.34 (s, 3H), 2.14 (s, 3H), 2.11 (s, 1.2H). MS (ESI) 366.8Example 8: Preparation of Compounds 1, 2, 3, 28, 34, 35, L-3, L-5 and L-7

[0325] Compounds, 2, 3, 28, 34, 35, L-3, L-5 and L-7 were also prepared:Table 3A.Example 9: Preparation of 2-(2,5-dimethylphenyl)-5-(4-(2,3-dimethylphenyl)piperazin-l-yl)benzo[fif]isothiazol-3(2H)-one 1,1-dioxide (Compound 40)

[0326] To a solution of 5-(4-(2,3-dimethylphenyl)piperazin-l-yl)-2-(N-(2,5-dimethylphenyl)sulfamoyl)benzamide (0.083 g, 0.17 mmol) in 1,4-dioxane (13 mL) was added 3 N hydrochloric acid (9.21 ml, 27.6 mmol), and the mixture was stirred at 85 °C for 9 h and at room temperature for 14 h. After this time, water (40 mL) was added, the pH was adjusted to 7 by addition of saturated sodium bicarbonate, and the mixture was extracted with ethyl acetate (3 x 30 mL). The organic extracts were combined, dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue obtained was adsorbed onto diatomaceous earth and chromatographed (silica, heptane to dichloromethane). The product obtained was lyophilized from 1:1 acetonitrile / water to afford the title compound (0.0506 g, 63%) as a white solid.

[0327] 'H NMR (500 MHz, DMSO-<76) 6 8.13 (d, J= 9.0 Hz, 1H), 7.57-7.54 (m, 2H), 7.38 (d, J= 7.5 Hz, 1H), 7.32 (d, J= 8.0 Hz, 1H), 7.20 (s, 1H), 7.07 (t, J= 8.0 Hz, 1H), 6.94 (d, J =8.0 Hz, 1H), 6.91 (d, J= 7.5 Hz, 1H), 3.65 (br s, 4H), 2.95 (t, J= 4.0 Hz, 4H), 2.35 (s, 3H), 2.23 (s, 3H), 2.22 (s, 3H), 2.15 (s, 3H). MS (ESI) 475.9.Example 10

[0328] The purpose of this study was to develop a series of cell free, high throughput screening assays to screen and functionally test potential small molecule candidates that will recapitulate part or all of the functions of an illustrative truncated ALT peptide that inhibits cancer cell proliferation, called CCL20, or IpY.20 herein.SEQ ID NO:1 CCL-2O / IpY.2O 'MVSRDQAHLGPKYVGLWDFKSRTDEELSFRAGDVFHVARKEEQWWWATLLDEAGG AVAQGYVPHNYLAERETVESEPAGHAGSAALQDLAA91

[0329] PURPOSE:

[0330] While IpY.20 harnesses the biologic power of a naturally occurring inhibitor of p27, it might pose several challenges in its manufacturing and delivery in the clinic. A small molecule that would recapitulate some or all the functions of the biologic peptide could provide a different approach to drug p27. A small molecule drug is typically administered orally (vs. IV in the case of the biologic), both patients and large Pharma partners are more familiar with a small molecule drug formulation and the manufacturing of the drug, if a successful candidate is found, can be much cheaper and scalable. To try and find potential small molecule candidates we developed a series of cell free, high-throughput assays that permit both screening and functionally tests for small molecule compounds that bind to the target of interest and recapitulate the effect of IpY.20, our lead biologic drug candidate.

[0331] ASSAY DESIGN:

[0332] To screen and functionally test small molecule candidates 3 different assays were designed. Assay development was comprised of two phases:

[0333] Phase I: Purification of tagged protein components utilized for assay development

[0334] Phase II: Assay assembly and validation

[0335] The assays included:1. Screening Assay: TR-FRET Assay for ALT-BRK Binding to p27 / Cyclin D1 / CDK4 / 6 Ternary Complex (ALT:p27 / CDK4 / 6 / Cyclin DI)

[0336] This assay measures biotin-ALT BRK binding to p27 / CDK4 / 6 / Cyclin D ternary complex. His-p27 was mixed with GST-CDK4 / 6 / cyclin DI first to form ternary complex. Biotin-ALT BRK was then added. The binding was measured by TR-FRET using Eu-streptavidin (Eu-SA, fluorescence donor) and APC-anti His antibody (fluorescence acceptor). This assay was usedas a primary assay for identifying compounds that block ALT BRK binding to p27 / CDK4 / 6 / Cyclin DI ternary complex. Since ALT BRK binding to p27 is dependent on CDK4 / 6 / Cyclin DI, this assay also identified compounds that block p27 binding to CDK4 / 6 / Cyclin DI. See FIG. 13A. Results in Table 4 and FIG. 18.2. Deconvolution Assay: TR-FRET Assay for p27 Binding to Cyclin D1 / CDK4 / 6 (p27: CDK4 / 6 / Cyclin DI)

[0337] ALT BRK binding to p27 is very much dependent on CDK4 / 6 / Cyclin D 1. Therefore, it is important to set up an assay for measuring p27 binding to CDK4 / 6 / Cyclin DI to make sure that purified p27 can form a tight ternary complex with purified CDK4 / 6 / Cyclin DI. This assay measures His-p27 binding to GST-CDK4 / 6 / Cyclin DI. The binding is detected by TR-FRET using Eu-anti GST antibody (fluorescence donor) and APC-anti His antibody (fluorescence acceptor). This assay was used as a deconvolution assay for identifying compounds that block the p27: CDK4 / 6 / Cyclin DI interaction. See FIG. 13B. Results in Table 53. Functional assay: TR-FRET assay for BRK phosphorylation assay of p27 on residue Y88 (Brk Kinase Assay).Brk is able to phosphorylate p27 on reside Y88 as detected by a pY88 phospho-specific antibody. This assay uses the p27 substrate and measures its tyrosine phosphorylation at residue 88 of His-p27 in the presence of the kinase BRK and ATP. The phosphorylation of His-p27 was detected by TR-FRET using anti-pY88 antibody+Eu-anti-rabbit IgG antibody (fluorescence donor) and an APC anti-His antibody (fluorescence acceptor). See FIG. 13C. Results in Table 6 and FIG. 19.

[0338] RESULTS

[0339] Assay Development Phase I: Tagged Protein components purification summary

[0340] Table 3B represents the purified tagged proteins isolated:Table 3B.

[0341] Examples of key purified protein component westerns are shown in FIGs 14A-14C.

[0342] BRK(1-451) Production [tag cleaved]

[0343] GST-PreScission-BRK (1-451) is highly expressed Infect with 1 x virus for 48 h is the best condition for yielding soluble target protein

[0344] FIG. 15

[0345] GST-PreScission-Rbc(773-928) Expression Matrix

[0346] GST-PreScission-Rbc(773-928)-TEV-His is highly expressed 1 mM IPTG @ 23°C is the best condition for yielding soluble target protein

[0347] FIGs 16A-16D

[0348] GST-PreScission-CDK4 / 6 / GST-PreScission-Cyclin DI Complex Production

[0349] Both proteins were expressed Infection with 1 x virus for 48 h is the best condition for yielding soluble target protein GST tag was cleavable by PreScission proteaseFIGs 17A-17C provide schematics for assays used in the Examples. FIG. 17A- CDK4 / 6 / Cyclin DI Complex Production [tag cleaved]. FIG. 17B p27Kipl (1-198) Production [tag cleaved]. FIG.17C GST-PreScission-p27Kipl(l-198) is highly expressed1 mM IPTG @ 23°C is the best condition for yielding soluble target protein PreScission cleavage was efficient.

[0350] Assay Development Results- Phase II: Assay assembly and validation

[0351] TR-FRET Assay for p27 Binding to CDK4 / 6 / Cyclin DI (Deconvolution assay)

[0352] To assemble this assay purified His-tag p27 was mixed with GST-tag CDK4 / 6 / Cyclin DI the p27binding was measured by TR-FRET. His-p27 showed dose-dependent binding to GST-tag CDK4 / 6 / Cyclin DI. These results showed that purified p27 and CDK4 / 6 / Cyclin DI proteins are active in forming p27: CDK4 / 6 / Cyclin DI ternary complex (FIG. 1).

[0353] FIG. 1. Dose dependent binding of His-p27 binding to CDK4 / 6 / CyclinDl in a TR-FRET deconvolution assay.

[0354] The effect of both pH and buffer effect on the binding kinetics of p27 to CDK4 / 6 / Cyclin DI was examined utilizing different assay conditions and varied pH in Hepes buffer.

[0355] FIG. 2. Effect of altering pH and buffer on binding and kinetics His-p27 to CDK4 / 6 / CyclinDl in a TR-FRET deconvolution assay. A, Effect of Change in pH on assay signal. pH 7.0 showed the best overall conditions. B. Effect of Buffer Change on assay kinetics.

[0356] The optimal conditions for this assay were determined to be 50 mM Hepes, pH 7.0, 100 mM NaCl, 0.65 mM DTT and 0.2 mg / ml BSA (FIG. 2B). Under these conditions, the signal / background ratio in the assay was higher than 15 indicating a great ratio. At pH 7.0, the binding is very fast and reaches plateau in 30 min. On the other hand, the binding is slower at pH 7.5 and 8.0 (FIG. 2A).

[0357] Under these optimal conditions, the Kd of his-p27 binding to GST-CDK4 / 6 / cyclinDl was calculated to be 16.6nM reflecting the previously observed tight binding interaction (FIG.3 A). As both p27 and CDK4 / 6 / CyclinDl will be utilized in the no-tagged forms in future assays, it was important to determine if these no-tagged variant function just as well as their tagged counterparts. To test that, the inhibition potential of no-tag p27 and no-tag CDK / CyclinDl was assessed in this TR-FRET binding assay. Increasing concentrations of no-tagged p27 was added to 43nM of his-p27 and 34nM of GST-CDK4 / 6 / CyclinDl and following incubation the TR-FRET signal was measured. Increasing concentrations of no-tagged p27 inhibited the binding of the his-tagged variant and the TR-FRET signal in a dose dependent manner. The IC50 of the no-tag p27 protein was found to be 59.88 nM, which is very close to the His-p27 concentration (43 nM) in the assay. These results indicate that His-tag p27 and no-tag p27 have similar binding activities to GST-CDK4 / 6 / Cyclin DI (FIG.3B). A similar analysis was performed for the non-tagged variantof CDK4 / 6 / CyclinDl where the IC50 of no-tagged protein complex was found to be 66.68 nM, which is very close to the GST-CDK4 / 6 / Cyclin DI concentration (43 nM) in the assay (FIG.3C).These results indicated that GST-tag CDK4 / 6 / Cyclin DI and no-tag CDK4 / 6 / Cyclin DI have similar binding activities to His-p27, confirming the binding activity of both no-tagged variants.

[0358] FIG. 3A. Kd Assessment his-p27 binding to GST-tagged CDK4 / 6: CyclinD 1. B. IC50 determination of no-tag p27 competing with his-p27 in binding to GST-CDK4 / 6 / CyclinDl / C. IC50 assessment of no-tag CDK4 / 6 / Cyclin DI competing with GST variant to bind his-p27.

[0359] TR-FRET Assay for ALT BRK Binding to p27 / CDK4 / 6 / Cyclin D Ternary Complex (Screening Assay) (ALT:p27 / CDK4 / CyclinDl)

[0360] To assemble this assay His-p27 is mixed with GST-CDK4 / 6 / cyclin DI first to form ternary complex. Biotin-ALT BRK is then added. The binding is measured by TR-FRET using Eu-streptavidin (Eu-SA, fluorescence donor) and APC-anti His antibody (fluorescence acceptor). ALT binding to either free p27 or p27 in complex with CDK4 / 6 / CyclinDl was examined under optimized conditions of pH 6.5. At this pH, ALT BRK binding to free p27 and p27 / CDK4 / 6 / Cyclin DI ternary complex is very tight at pH 6.5 with Kd of 27.16 nM and 32.65 nM, respectively. Although ALT BRK was also able bind to free p27 tightly, its binding signal is less than that of p27 / CDK4 / 6 / Cyclin DI complex. These results suggest that free p27 may have different conformation so that it cannot bind ALT BRK as well as p27 / CDK4 / 6 / Cyclin DI complex (FIG. 4).

[0361] FIG. 4. TR-FRET analysis of ALT-BRK binding to free p27 (squares) or p27 in complex with CDK4 / 6 / CyclinDl (dots). ALT bound to free p27 and p27 / CDK4 / 6 / Cyclin DI ternary complex with Kd of 27.16 nM and 32.65 nM, respectively.

[0362] To examine the binding specificity of ALT-BRK to the ternary complex, the inhibition potential of no-tagged p27 added to the tagged ternary complex and ALT mix was assessed. When no tagged p27 was added to the reaction, it was able to completely inhibit biotin-ALT BRK binding to His-p27 / CDK / Cyclin DI complex, indicating that binding is specific (FIG. 5).

[0363] FIG. 5. Signal specificity in ALT-binding assay. Addition of no-tagged p27 abolished ALT binding interaction with the ternary complex.

[0364] To examine assay sensitivity to different detergents and DMSO, the effect of increasing concentrations of NP 40 and Tween 20 on assay activity was assessed. ALT:p27 / CDK4 / 6 / Cyclin DI interaction was found to be very sensitive to detergent (NP40 or Tween 20) as more than 90% activity was lost even in the presence of 0.001 % NP40 or Tween 20 (FIGs 6A-6B). As most small molecule compounds are typically dissolved in DMSO, it is thusimportant to determine the effect of DMSO on assay activity. Low concentrations of DMSO showed slight activation in the ALT:p27 / CDK4 / 6 / Cyclin DI screening assay (1.5 - 6%) while high concertation (>7.7%) showed inhibition. (FIG. 6C).

[0365] FIG. 6A. Assessment of assay activity in increasing concentrations of NP40. B. Assay activity levels in the presence of increasing concentrations of Tween20. C. Assay sensitivity to increasing concentrations of DMSO.

[0366] In order to validate the assay, we have utilized positive and negative controls. As a negative control for binding specificity, we used a mutant form of ALT(ALTMT2), which contains a three Tryptophan (W) substitution to Alanine (A), as shown in SEQ ID NO:3 below. Full length ALT (ALTWT) was able to efficiently block the binding of ALT-His-p27, displacing it from the complex and thus inhibiting the FRET binding interaction signal. The ALTMT2 mutant was much less efficient at blocking the ALT-His-p27 interaction compared to ALTWT (FIG. 7).

[0367] Table 3C-proteins tested in ALT:p27 / CDK4 / 6 / CyclinDl screening assay*See paragraph

[0370]

[0368] Validation Assay: TR-FRET assay for BRK phosphorylation of p27 on residue Y88 (Brk Kinase assay).

[0369] This assay uses the p27 substrate without cyclin D1 / CDK4 / 6 and measures its tyrosine phosphorylation at residue 88 of His-p27 in the presence of the kinase BRK and ATP. The phosphorylation of His-p27 is detected by TR-FRET using anti-pY88 antibody+Eu-anti-rabbit IgG antibody (fluorescence donor) and an APC anti-His antibody (fluorescence acceptor) (FIG.8).

[0370] FIG. 8. Brk phosphorylation assay in the presence and absence of the CDK4 / 6 / Cyclin DI complex. p27 is phosphorylated by BRK in the absence of CDK4 / 6 / Cyclin DI in a dosedependent manner as detected by the phospho pY88 antibody in a TR-FRET format. However, p27 phosphorylation is much slower in the presence of CDK4 / 6 / Cyclin DI ternary complex.

[0371] p27 phosphorylation by BRK is inhibited by increasing concentrations of CDK4 / 6 / Cyclin DI with an IC50 of 164.6 nM.

[0372] FIG. 9. CDK4 / 6 / Cyclin DI inhibition of p27 phosphorylation by BRK. p27 phosphorylation by BRK is inhibited by increasing concentrations of CDK4 / 6 / Cyclin DI with an IC50 of 164.6 nM.

[0373] FIG. 10. p27 phosphorylation under different conditions. If p27 is phosphorylated by BRK first and then CDK4 / 6 / Cyclin DI is added, 89% of p27 phosphorylation is observed comparing with that without CDK4 / 6 (Sample 2). However, if p27 is mixed with CDK4 / 6 / Cyclin DI first and then BRK is added, only 9.8% of p27 phosphorylation is observed. These results suggest that pY88 antibody can recognize phosphorylated p27 well with or without CDK4 / 6 / Cyclin DI.

[0374] FIG. 11. Tilfrinib prevents Brk’s phosphorylation of p27. Tilfrinib (a potent BRK kinase inhibitor) is a very potent inhibitor (IC50 = 3.1 nM) in BRK TR-FRET kinase assay using p27 as substrate, consistent with that of reported by literature.

[0375] FIG. 12. Isolation of BRK-phosphorylated p27. A. Coomassie blue stain of BRK phosphorylated p27 on phosphoprotein column. B. Western blot using anti-pTyr Ab; detecting p27 and pBrk. C. Non phosphorylated p27 loaded onto Pro-Q column does not bind.Table 4. Biological Data for ALT:p27 / CDK4 / 6 / Cyclin DI Assay (Screening Assay)Table 5. Biological Data for p27: CDK4 / 6 / Cyclin DI (Deconvolution assay)Table 6. Biological Data for BRK phosphorylation of p27 on residue Y88 (Brk Kinase assay)Example 11: BRK Kinase Assay (generic peptide substrate)

[0376] BRK kinase inhibitor Tilfrinib was resuspended in DMSO to make 10 mM stock. Dose response curve consists of 4-fold serial dilutions starting with top dose of 10 pM. ADP Gio kinase assays were performed according to the manufacturer's instructions in 96-well format (Promega V4055). Kinase reactions were performed at room temperature in kinase buffer D (40 mM Tris, pH=7.5, 20 mM MgCl2, 0.1 mg / mL BSA, 2.5 mM MnCh, 50 pM DTT) with 250 pM ATP, 25 ng BRK, and 0.2 pg / pL PolyE4Y substrate. Small molecule compounds were prepared as 2-fold serial dilutions with top dose of 100 pM. Following addition of BRK kinase, 20 pL of the kinase reaction mixture was mixed with 5 pL of each inhibitor dilution per well of white multiwell plate (in triplicate wells) and kinase reactions were allowed to proceed for 1 hr at room temperature. Subsequently, 25 pL of ADP Gio reagent was added to wells and incubated at room temperature for 40 min. Kinase detection reagent was added to each well for 40 min (50 pL per well) prior to luminescence detection on luminometer (Promega Glomax instrument). Results are shown in FIG. 20, which shows that except for compound 35A, none of the small molecule compounds tested reduces general Brk kinase activity toward polyE4Y peptide substrate by more than 50% even at lOOuM, the highest concentration tested. Tilfrinib, a Brk kinase inhibitor has an IC50 of 15 nM in this assay. Data shown as mean±SD (triplicates, n=l).Example 12: Thermal Shift Assay

[0377] The thermal shift assay was used to test direct binding of the compounds of the invention to p27 / CDK4 / 6 / Cyclin D Trimer and CDK4 / 6 / Cyclin D dimer. Assay Principle: The melting temperature (Tm) of proteins is changed upon addition of ligand, typically increasing if the ligand has stabilizing effect and decreasing if a ligand has a degrading effect. Since binding affinities are inherently temperature dependent, these changes in Tm are quantitatively linked to the affinity of the interaction. Therefore, if a compound of the invention binds to target (e.g., tothe p27 / CDK4 / 6 / Cyclin D trimer) then an increase in temperature will be observed. Absence of a temperature increase is indicative of a lack of binding, while a temperature decrease is typically indicative of target protein degrading.

[0378] Testing compounds were reconstituted in 100% DMSO at 2.12 mM and serially diluted 3-fold in DMSO to the total of 5 testing concentrations per each compound (100 pM, 33 pM, 11 pM, 3.7 pM and 1.2 pM). p27 and CDK4 / 6 / CyclinDl proteins were purified previously as components in other cell-free assays, TR-FRET assay for ALT BRK binding to p27 / CDK4 / 6 / Cyclin D and BRK kinase assay as described in Table 3B. Stock solution of p27 / CDK4 / 6 / CyclinDl trimer complex (29.97 pM) was generated by mixing of CDK4 / 6 / CyclinDl with p27 and incubation on ice for Ihr. Subsequently, protein working solutions of p27, CDK4 / 6 / CyclinDl and p27 / CDK4 / 6 / CyclinDl complexes at 15.29 pM were prepared by dilution in assay buffer 1 (50mM Tris, pH 7.4, 100 mM NaCl, 1 mM DTT). For thermal shift assay solution 1.3 pL / well of testing compound solutions were transferred to 384-well polypropylene black plate (Thermo Scientific #264576) and mixed with 11 pL / well of assay buffer 1 by centrifugation, followed by addition of 7.2 pL / well of individual protein working solution and 6 pL / well of assay buffer 1. The plate was then centrifuged and incubated at ambient temperature for 15 minutes. Sypro Orange stock solution was prepared by 500 fold dilution of Sigma- Aldrich reagent (cat#S5692-500UL) with assay buffer (50 mM Tris, pH 7.4, 100 mM NaCl). Subsequently, 1.5 pL / well of diluted Sypro Orange stock was then added to a 384-well PCR plate, mixed with 19 pL / well of thermal shift assay solution and centrifuged. Subsequently 10 pL / well of mineral oil (Sigma- Aldrich #M8410) was gently added on top of each testing well and the plate was read by high temperature fluorescence microplate reader at fluorescence intensity (Ex470 / Em590) from 25.5 °C to 70. 5°C at 1.5 °C / min temperature increment. Tm of target protein complex was measured per each test compound concentration and maximim Tm shift was chosen by comparing Tm at each test concentration to DMSO control.

[0379] Results are shown in FIGs 21A-21D.

[0380] FIG. 21A shows p27 binding to CDK4 / 6 / Cyclin D dramatically increases thermal stability (Tm) of tertiary complex p27 / CDK4 / 6 / Cyclin D (consistent with literature).

[0381] FIG. 21B shows the validation of thermal shift assay: Palbociclib binds to cyclinD-cdk4 / 6 dimer and not to p27 / CDK4 / 6 / cyclinD trimer (consistent with literature). Non-specific compound L8which previously failed ALT:p27 / CDK4 / 6 / Cyclin DI Assay (IC50 > 150mM, negative control)) does not bind to any components, while compound L9,(similar to LI (IC50 of 3.78. M in ALT:p27 / CDK4 / 6 / CyclinDl Assay) shows preferential binding to p27 / CDK4 / 6 / CyclinD trimer complex.

[0382] FIG. 21C shows the results of the thermal shift assay of compounds palbociclib (PD) L3, 14, 39, 40, 35A, 35, and 34 binding to the p27 / CDK4 / 6 / Cyclin D trimer. Compounds L3, 14, 35A, 35 and 34 show binding affinity to the trimer.

[0383] FIG. 21D shows the results of the thermal shift assay of compounds L3, 14, 39, 40, 35A, 35, and 34 binding to the CDK4 / 6 / Cyclin D dimer. Palbociclib (PD) binds to the dimer (consistent with literature), compound L3 shows similar binding affinity to the dimer as PD, whereas compounds 14, 39. 40, 35A, 35, 34 show much weaker binding affinity to the dimer than PDExample 13: Cell Culture

[0384] T47D cells were cultured in RPMI1640 media with 10% FBS, 1% nonessential amino acids, 10 pg / mL insulin solution, and Antibiotic-Antimycotic. Palbociclib-resistant T47D (T47D PalboR) cells were cultured in 2 pM palbociclib in the same culture media as T47D cells. Cells were routinely passaged at either 1:2 or 1:3 split ratio every 2-3 days.

[0385] MCF10A were cultured in MEM media supplemented with a Cell Growth Factor Kit (Lonza / Clonetics #CC-3150),10% FBS, 100 ng / mL cholera toxin and antibiotic-antimyotic.

[0386] HC11 cells were cultured in RPMI 1640 media with 10% FBS and antibiotic- antimyotic.Example 14: Generation of palbociclib-resistant T47D cells

[0387] T47D PalboR cells were generated by culturing cells in 400 nM Palbo for 1 month and gradually increasing dose to 2 pM over 2 months. Prior to using these cells for experiments, cells were plated in media without palbociclib and allowed to attach for 24 hr prior to treatment with indicated doses of CDK4 / 6 inhibitors (CDK4i) or small molecule compounds. To test resistance to CDK4i, cells were tested with increasing doses of palbociclib (50, 100, 400 nM, palbociclib from Selleckchem was reconstituted in DMSO at 2 mM stock) and ribociclib (100, 250, 1000 nM, ribociclib from Selleckchem was reconstituted in DMSO at 2 mM stock) in BrdU assay (as described in example 15).Example 15: BrdU Incorporation Assay

[0388] Cell proliferation was measured with BrdU (5 -bro mo-2’ -deoxyuridine) incorporation assay where BrdU is incorporated into de novo-synthesized DNA as a substitute for thymidine and thereby permanently labels proliferating cells. T47D, palbociclib-resistant T47D (T47D PalboR), MCF10A and HC11 cells were plated at a density of 6000 cells / well in 96-well plate the night before the treatment. On the day of the treatment, 1000, 333, 111, 37, 12.3, 4.11,1.37, 0.45, 0 nM palbociclib and / or ribociclib and 10 and 30 M of small molecule compounds were added to the cells. BrdU was added to the culture 24hrs after the treatment. 48hrs after the treatment, media were removed, and cells were fixed for BrdU detection according to the manufacturer’s instructions (Abeam BrdU Cell proliferation ELISA kit abl26556). Plates were read using a spectrophotometric microtiter plate set at 450 nm. (Spectramax setttings: Endpoint; Abs 450nm, Costar 96 well clear (lidded)). Results are shown in FIG.22 (T47D cells), FIG.23 (T47D PalboR cells), FIG. 24 (MCF10A cells), and FIG. 25 (HC11 cells).

[0389] For combination therapies, the cells were treated with lOuM of small molecule compounds plus 0, 50, 100 nM of palbociclib or with lOuM of small molecule compounds plus 0, 100, 250nM of ribociclib. BrdU was added to the culture 24hrs after the treatment. 48hrs after the treatment, media were removed, and cells were fixed for BrdU detection according to the manufacturer’s instructions (Abeam BrdU Cell proliferation ELISA kit ab 126556). Plates were read using a spectrophotometric microtiter plate set at 450 nm. (Spectramax setttings: Endpoint; Abs 450nm, Costar 96 well clear (lidded)). Results are shown in FIG.26. FIG.26 shows that the addition of 10 pM of compounds L3, 14, 39, 40, 35A, 35 and 34 enhance response of palbociclib at 50nM concentration in T47D cells. FIG. 26B shows that addition of 10 pM of compounds L3, 14, 39, 40, 35A, 35 and 34 enhance response of ribociclib at lOOnM concentration in T47D cells.Example 16: Cell Cycle Analysis

[0390] To determine cell cycle profiles, we combined EdU labeling (5-ethynyl-2’-deoxyuridine, another thymidine analogue that will label replicating DNA during DNA synthesis phase (S phase)) and cellular DNA staining with DAPI. DAPI stains DNA stoichiometrically, allowing differentiation of cells in 4 stages of cell cycle: Gap 1 (Gl), synthesis (S), Gap 2 (G2) and mitosis (M) stages. In addition, during apoptosis, genomic DNA is cleaved into smaller fragments and DNA stained less intensely which shows up as a peak (sub-Gl phase) below the Gl peak, representing apoptotic cells. Since CDK4 / 6 / cyclin D and CDK2 / cyclin E are only active in the Gl phase of the cell cycle, an increase in the percentage of cells in Gl phase and a reduction in % of cells in S phase compared to vehicle DMSO, would suggest the compounds affect the CDK4 / 6 / cyclinD and CDK2 / cyclin E activity. An increase in sub Gl phase cells would indicate cell death. Cells were treated with 15 pM or 30 pM of the indicated compounds or DMSO or 400nM palbociclib for 48 hr. Cells were then pulse-labeled with 10 pM EdU for 1 hr prior to trypsinization and fixation in 4% paraformaldehyde. EdU incorporation was detected using the Alexa Fluor 594 Click-iT EdU Flow Cytometry assay kit (Invitrogen #C 10646) according to the manufacturer’s instructions and DNA was counterstained with 1 pg / mL DAPI prior to analysis. Flow cytometry acquisition was performed on a BD FACSymphony Al cell analyzer (NYU cytometry and cell sorting facility) and data was analyzed using Flow Jo software. Compounds 40 and 35 were shown to induce Gl phase cell cycle arrest in T47D cells at 15 pM (FIG. 27A), whereas compounds L3, 14, 35A and 34 were shown to induce Gl phase cell cycle arrest in T47D cells at 30 pM (FIG. 27B).Example 17: Cell Viability Analysis

[0391] Cell viability was measured using Promega CellTiter Gio luminescent cell viability assay which is a method of determining the number of viable cells in culture based on quantitation of the ATP present, an indicator of metabolically active cells. This is a general measure of cell viability, which could be due to a combination of reduced proliferation and death. Cisplatin stock solution was made fresh by dissolving in PBS to a concentration of 10 mM. Top working concentration was 100 pM, with 1 to 3.16 serial dilutions to generate 8-point dose response curve. All compounds were diluted to 10X the desired concentrations (30 pM and 10 pM) in cell culture media and 10 pL of working stock was added to 90 pL of media per well. Cells were incubated with small molecule compounds for 48 hr or 72 hr. Cell viability was assessed using Promega CellTiter Gio viability assay according to the manufacturer instructions.

[0392] Several of the compounds tested do not cause significant cell death in T47D (FIG.28), HC11 (FIG. 29) or MCF10A cells (FIG. 30) at 48 hr and 72 hr.Example 18: Western Blot

[0393] Intracellular CDK4 / 6 and CDK2 kinase activities in T47D cells was determined by examining the phosphorylation of their substrate, retinoblastoma tumor suppressor protein Rb (pRb) and phosphorylation of CDK2 (as a surrogate marker for CDK2 activity).

[0394] T47D cells were seeded in 6-well culture dishes (Corning) at a density of 250,000 cells per well and allowed to attach for 24 hr prior to treatment with 30 pM of the small molecule compounds 14, 40, 35A, and 34 for 3, 7, 24, and 48 hours. Additionally, a control group was treated with 1.5% of dimethyl sulfoxide (DMSO), which is equivalent to the amount of DMSO used to prepare 30 pM of small molecule compounds, for 48 hours to serve as a baseline comparison.

[0395] For Western blot analysis, cells were lysed in denaturing SDS buffer (100 mM Tris, pH 6.8, 2% SDS, and 20 mM P-mercaptoethanol) and heated at 100 °C for 20 minutes in a heat block. Protein concentrations were determined using the RC DC protein quantification assay (BioRad) with a Bovine Serum Albumin (BSA) standard curve in sample buffer according to the manufacturer’s instructions.

[0396] Cell extracts were prepared with sample buffer (Tris 1 M, pH 6.8 (80 mM), SDS (20%), Glycerol (10%), and Bromophenol blue (0.1%)) and separated on 10% Mini-PROTEAN® TGX™ Precast Gels (Bio-Rad) alongside a Protein Ladder (Peacock™ Prestained Protein Marker, Biotium). Electrophoresis was carried out at 115V for 1 hour or until the dye front reached the end of the glass plates. Proteins were then transferred onto 0.45 pm Nitrocellulose membranes (Amersham) or 0.45 pm PVDF membranes (Millipore) in Transfer Buffer (25 mM Tris, 192 mM glycine, 10% methanol) at 100V for 1 hour and 40 minutes.

[0397] Following transfer, membranes were blocked in 5% milk in TBS-T for 1 hour prior to overnight incubation with primary antibodies at the following dilutions: Anti-beta Actin Polyclonal Antibody (1:2000 dilution, Invitrogen), Anti-Phospho-CDK2 (Thrl60) (1:1000 dilution, Cell Signaling), Anti-CDK2 Monoclonal Antibody (1:1000 dilution, Thermo Fisher), Anti-Phospho-Rb (Ser807 / 811) (1:1000 dilution, Cell Signaling), Anti-Rb (4H1) Mouse (1:2000 dilution, Cell Signaling).

[0398] The following day, membranes were incubated with fluorescence-conjugated secondary antibodies [IRDye® 800CW Goat anti-Rabbit IgG Secondary Antibody (1:10,000 dilution, Licor) and IRDye® 680RD Goat anti-Mouse IgG Secondary Antibody (1:10,000dilution, Licor)] or HRP-conjugated secondary antibodies [Goat Anti-Mouse IgG H& L (HRP) (1:10,000 dilution, Abeam) and Goat Anti-Rabbit IgG (HRP) (1:5000 dilution, Sigma)] in 5% milk in TBS-T. Membranes were then developed using ECL Prime Western Blot detection (Amersham).

[0399] Results as shown in FIG. 31 indicate that treatment of compounds 14, 40, 35A and 34 reduce phosphorylation of Rb and CDK2 48 hours post treatment in T47D cells, suggesting these compounds reduce CDK4 / 6 and CDK2 kinase activities.Example 19: Effects of Compounds 35A and 34 on ovarian and pancreatic cancer cells -Material and Methods

[0400] Cell Culture

[0401] OVCAR-3 cells were cultured in RPMI 1640 media with 10% FBS and 1% antibiotic-antimycotic. SK-OV-3 cells were cultured in McCoy’s 5 A medium with 10% FBS and 1% antibiotic-antimycotic. MIA PaCa-2 and PL45 cells were cultured DMEM with 10% FBS and 1% antibiotic-antimycotic

[0402] BrdU incorporation assay

[0403] OVCAR-3 and SK-OV-3 cells were plated at a density of 5000 cells / well and 5500 cells / well, respectively, in 96-well plate the night before the treatment. On the day of the treatment, OVCAR-3 cells were treated with 10, 3.33, 1.11, 0.37, 0.123, 0.0411, 0.0137. 0.005 uM of PF-07104091 (5mM stock in DMSO,). SK-OV-3 cells were treated with 20, 10, 5, 2.5, 1.25, 0.625, 0.313, 0.156 uM of PF-07104091. OVCAR-3 and SK-OV-3 cells were also treated with 10 and 30M of compound 35 A or 34. For combination therapies, cells were treated with lOuM or 30uM 35A and 34 plus 150, 350 and 750nM PF-07104091. Cells were treated for a total of 48hr. Cells were labeled with BrdU 2h prior to the end of treatment. 48hrs after the treatment, media were removed, and cells were fixed for BrdU detection according to the manufacturer’s instructions. Plates were read using a spectrophotometric microtiter plate set at wavelength of 450 nm. OVCAR-3 were plated at a density of 5000 cells / well in 96-well plate the night before the treatment. On the day of the treatment, OVCAR-3 cells were treated with 10, 3.33, 1.11, 0.37, 0.123, 0.0411, 0.0137. 0.005 uM of PF-06873600 (5mM stock in DMSO). For combination therapies, cells were treated with lOuM or 30uM 35A and 34 plus 50 and 150nM of PF-06873600. Cells were treated for a total of 48hr. Cells were labeled with BrdU 2h prior to the end of treatment.48hrs after the treatment, media were removed, and cells were fixed for BrdU detection according to the manufacturer’s instructions. Plates were read using a spectrophotometric microtiter plate set at wavelength of 450 nm.

[0404] Cell Viability Assay

[0405] MIA PaCa-2 and PL45 cells were plated at a density of 1800 cells / well and 3000 cells / well, respectively, in a 96-well plate the night before the treatment. Gemcitabine was reconstituted in water to a concentration of lOmM (stock concertation). RMC-9805 (KRAS G12D inhibitor) was reconstituted in DMSO to a concentration of 5mM (stock concentration) compound 34 was reconstituted in DMSO at a concentration of lOmM (stock concentration). Top working concentration of gemcitabine was lOOOuM, with 1:10 serial dilutions to generate 7-point dose response curve. Top working concentration of RMC-9805 was 3uM, with 1:3 serial dilutions to generate 7-point dose response curve. Compound 34 was reconstituted in DMSO at a concentration of lOmM (stock concentration). Top working concentration of 34 was 120uM, with 1:2 serial dilutions to generate 6-point dose response curve. All compounds were diluted to 10X the desired concentrations in cell culture media and 10 ul of working stock was added to 90 ul of media per well. Cells were incubated with treatments for 72 hr. Cell viability was assessed using Promega CellTiter Gio Luminescent cell viability assay according to the manufacturer instructions.

[0406] Western Blot

[0407] OVCAR3 cells were seeded in 6-cm dishes at a density of 250,000 cells per dish and allowed to attach for 24 hr prior to treatment with (1) 30 pM of compound 34; (2) 150nM, 350nM and 750nM PF-07104091; (3) 30 pM of compound 34+150nM PF-07104091, 30 pM of compound 34+350nM PF-07104091 for 18 and 24 hours. Additionally, a control group was treated with 0.3% of dimethyl sulfoxide (DMSO), which is equivalent to the amount of DMSO used to prepare 30uM of compound 34, for 18 and 24 hours to serve as a baseline comparison.

[0408] For Western blot analysis, cells were lysed in denaturing SDS buffer (100 mM Tris, pH 6.8, 2% SDS, and 20 mM P-mercaptoethanol) and boiled for 15-20 min at 95oC. Protein concentrations were determined using detergent-compatible Bradford protein with a Bovine Serum Albumin (BSA) standard curve in sample buffer according to the manufacturer’s instructions.

[0409] Cell extracts were prepared with sample buffer (Tris 1 M, pH 6.8 (80 mM), SDS (20%), Glycerol (10%), and Bromophenol blue (0.1%)) and separated on 4-20% BioRad TGx gels with lOug of whole cell lysates alongside a protein ladder. Electrophoresis was carried out at 115V for 1 hour or until the dye front reached the end of the glass plates. Proteins were then transferred onto 0.45 pm PVDF membranes in Transfer Buffer (25 mM Tris, 192 mM glycine, 10% methanol) at 100V for 1.5 hour or at 85V for 2.5 hour. Following transfer, membranes were blocked in 5%milk in TBS-T for 1 hour prior to overnight incubation with primary antibodies overnight at 4°C at the following dilutions: anti-p27 (1:5000, BD), anti-p21 (1:1000, Cell Signaling Technology), anti-GAPDH (1:2000, Novus), anti-phopsho-CDC6 (S54) (1:2000, Abeam).

[0410] The following day, membranes were washed and incubated with HRP-conjugated anti-mouse IgG (1:5000, Cytiva) or anti-rabbit IgG (H+L, 1:5000, Southern Biotech), Membranes were then developed using ECL Prime Western Blot detection.Example 20: Effects of 35A and 34 on ovarian and pancreatic cancer cells - Results

[0411] As illustrated in FIGs 32A-32F compounds 35A and 34 were found to enhance the potency of CDK2i (PF4091) in OVCAR3 and SK-OV-3 cells.

[0412] The efficacy of compounds 35A and 34 was tested in two different ovarian cell lines, OVCAR3 and SK-OV-3. Both cell lines have high expression of Cyclin E protein because of CCNE1 gene amplification; therefore, both are CDK2-depenend and susceptible to CDK2 inhibitor. However, OVCAR3 expresses pl6INK4a, a cyclin-dependent kinase inhibitor that suppresses CDK4 / 6 activity, SK-OV-3 is pl6-deficient, making SK-OV-3 more resistant to CDK2i through compensatory activation of CDK4 / 6 than OVCAR3 cells. The efficacy of compounds in both ovarian cell lines was tested. The data demonstrated that while monotherapy of compounds up to 30uM did not significantly impact the cell proliferation, combining compounds to CDK2i enhanced the potency of CDK2i in both cell lines (FIGs 32A-32F).Consistent with published data, it was demonstrated that SK-OV-3 cells were more resistant to CDK2i with an IC50 of ~4uM compared to IC50 of 0.36uM in OVCAR3 cells (FIG. 32A and 32D). In both cell lines, combination therapy with compounds enhanced the CDK2i potency (FIG.32C and 32F).

[0413] As illustrated in FIGs 33A-33B, compounds 35A and 34 were found to enhance the potency of CDK2 / 4 / 6i (PF3600) in OVCAR3 cells.

[0414] The efficacy of compounds 35A and 34 in combination with CDK2 / 4 / 6i PF-06873600 which has been discontinued due to clinical toxicity were also tested. In OVCAR3 cells, combining compounds 35 A and 34 strongly enhanced the potency of PF3600 (FIGs 33A-33B)

[0415] As illustrated in FIG. 34, cellular target engagement following compound 34 and CDK2i treatment was observed in OVCAR3 cells.

[0416] To investigate whether the anti-proliferative activities of combining compound 34 with CDK2i in OVCAR3 cells resulted from modulation of p27 and p21 protein level and inhibition of CDK4 / 6 and CDK2 kinase in these cells, (1) p27 and p21 protein expression and (2) phosphorylation of CDK2 substrate, CDC6, at residue serine 54 (S54) were examined. The proteinlevel and function of p27 is controlled by phosphorylation of p27 on residue tyrosine 88 (pY88). This phosphorylation event keeps CDK4 / 6-cyclin D complex active and allows subsequent phosphorylation of p27 on residue threonine 187 (T187), leading to ubiquitin-mediated degradation of p27 and the release and activation of CDK2-cyclin E. Therefore, inhibiting phosphorylation of Y88 on p27 with compounds can regulate the activities of CDK2 / 4 / 6 simultaneously and block ubiquitin-mediated degradation of p27 to increase p27 protein level. Our preliminary results showed a time- and dose-dependent upregulation of both p27 and p21 in response to combination treatment of 30uM of compound 34 and CDK2i (PF4091) (FIG.34). p27 and p21 are members of the CIP / KIP family sharing sequence homology at the N-terminal domain which allows them to bind to both the cyclin and CDK. Owning to a high level of homology in their primary structure, p21 and p27 are believed to inhibit their targets through similar mechanisms. Whether treatment-mediated p21 protein accumulation was a direct effect of compound 34 or an indirect effect from cell cycle arrest requires further investigation. In addition, the results demonstrated that pCDC6 reduction is most significant in response to combination therapy of compound 34 and PF4901 at 24hr compared to that of monotherapy of either compound 34 or PF4901, which correlated well with the enhanced anti-proliferative effect of the combination therapy in 0VCAR3 cells.

[0417] As illustrated in FIGs 35A-35B, compound 34 reduced cell viability of pancreatic ductal adenocarcinoma (PDAC) cell lines MIA PaCa-2 and PL45.

[0418] The two PDAC cell lines we used in this study contain different KRAS point mutation at codon 12. MIA PaCa-2 has a glycine to cysteine (G12C) mutation and PL45 has a glycine to aspartic acid (G12D) mutation. Both cell lines are susceptible to gemcitabine treatment (FIGs 35A and 35B); however, only PL45 is sensitive to RMC-9805, a first- in-class RAS-G12D (ON) inhibitor (Revolution Medicines), since it harbors G12D mutation (FIG. 35B). Treating MIA PaCa-2 and PL45 with compound 34 alone resulted in a dose-dependent inhibition of cell viability in both cell lines (FIGs 35A-35B).

[0419] Although the invention has been described with reference to the presently preferred embodiment, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

Claims

1. What Is Claimed Is:

1. A method of treating ovarian cancer in a subject comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a compound4.selected from5. 7.thereby treating ovarian cancer in the subject.

2. The method of claim 1, wherein the compound inhibits phosphorylation of p27.

3. The method of claim 1, wherein the compound inhibits CDK2 and CDK4 / 6.

4. The method of claim 1, wherein the compound inhibits ovarian cancer cell proliferation and / or decreases ovarian cancer cell viability.

5. The method of claim 1, wherein the compound increases ovarian cancer cell death.

6. The method of claim 1, further comprising administering to the subject an anti-cancer treatment.

7. The method of claim 6, wherein the anti-cancer treatment is selected from the group consisting of chemotherapy, radiation treatment, immunotherapy, resection of a tumor, and any combination thereof.

8. The method of claim 6, wherein the anti-cancer treatment is administered prior to, simultaneously with, or after administration of the pharmaceutical composition.

9. The method of claim 6, wherein the anti-cancer treatment is an anti-cancer agent selected from the group consisting of palbociclib, ribociclib, abemaciclib, osirmetinib, gefitinib, lapatinib, pantitumumab, vandetanib, necitumumab, vemurafenib, sorafenib tosylate, PLX-4720, dabrafenib, paclitaxel, cisplatin, docetaxol, carboplatin, vincristine, vinblastine, methotrexate, cyclophosphamide, CPT-11, 5 -fluorouracil, gemcitabine, estramustine, carmustine, adriamycin, etoposide, arsenic trioxide, irinotecan, Herceptin, vemurafenib, erlotinib, cetuximab, letrozole,fulvestrant, epolhilone derivatives, elacestrant, lasofoxifene, anastrozole, sotorasib, and adagrasib. trametinib, cobmetinib, Bay-293, everolimus, erolintib, cetaxumab, panitumumab, and afatinib, imatinib, sunitinib, panatinib, axitinib, foretinib, nintedanib, and amuvatinib, adavorsertib, pertuzumab, am-trastuzumab-deruxtecan, T-DM1 or ado -trastuzumab emtansine, pertuzumab / trastuzumab / hyaluronidase, neratinib), and tucatinib.

10. The method of claim 1, further comprising administering to the subject a second CDK2- selective inhibitor.

11. The method of claim 10, wherein the CDK2-selective inhibitor is PF-07104091.

12. The method of claim 1, wherein the subject is a human.

13. The method of claim 1, wherein administration of the pharmaceutical composition is intracutaneous, subcutaneous, intravenous, intraperitoneal, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transdermal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoidal, intraspinal, intrasternal, oral, sublingual, buccal, rectal, vaginal, nasal or ocular, or by infusion, inhalation, or nebulization.

14. A method of treating pancreatic cancer in a subject comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a24. 26.thereby treating pancreatic cancer in the subject.

15. The method of claim 14, wherein the compound inhibits phosphorylation of p27.

16. The method of claim 14, wherein the compound inhibits CDK2 and CDK4 / 6.

17. The method of claim 14, wherein the compound inhibits pancreatic cancer cell proliferation and / or decreases pancreatic cancer cell viability.

18. The method of claim 14, wherein the compound increases pancreatic cancer cell death.

19. The method of claim 14, further comprising administering to the subject an anti-cancer treatment.

20. The method of claim 19, wherein the anti-cancer treatment is selected from the group consisting of chemotherapy, radiation treatment, immunotherapy, resection of a tumor, and any combination thereof.

21. The method of claim 19, wherein the anti-cancer treatment is administered prior to, simultaneously with, or after administration of the pharmaceutical composition.

22. The method of claim 19, wherein the anti-cancer treatment is an anti-cancer agent selected from the group consisting of palbociclib, ribociclib, abemaciclib, osirmetinib, gefitinib, lapatinib, pantitumumab, vandetanib, necitumumab, vemurafenib, sorafenib tosylate, PLX-4720, dabrafenib, paclitaxel, cisplatin, docetaxol, carboplatin, vincristine, vinblastine, methotrexate, cyclophosphamide, CPT-11, 5 -fluorouracil, gemcitabine, estramustine, carmustine, adriamycin, etoposide, arsenic trioxide, irinotecan, Herceptin, vemurafenib, erlotinib, cetuximab, letrozole, fulvestrant, epolhilone derivatives, elacestrant, lasofoxifene, anastrozole, sotorasib, and adagrasib. trametinib, cobmetinib, Bay-293, everolimus, erolintib, cetaxumab, panitumumab, and afatinib, imatinib, sunitinib, panatinib, axitinib, foretinib, nintedanib, and amuvatinib, adavorsertib, pertuzumab, am-trastuzumab-deruxtecan-nxki, T-DM1 or ado-trastuzumab emtansine, pertuzumab / trastuzumab / hyaluronidase-zzxf, neratinib), and tucatinib.

23. The method of claim 14, wherein the subject is a human.

24. The method of claim 14, wherein administration of the pharmaceutical composition is intracutaneous, subcutaneous, intravenous, intraperitoneal, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transdermal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoidal, intraspinal, intrasternal, oral, sublingual, buccal, rectal, vaginal, nasal or ocular, or by infusion, inhalation, or nebulization.

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