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WO2025207582A1 - Agents de dégradation de la survivine pour le traitement ciblé du cancer - Google Patents

Agents de dégradation de la survivine pour le traitement ciblé du cancer

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Publication number
WO2025207582A1
WO2025207582A1 PCT/US2025/021273 US2025021273W WO2025207582A1 WO 2025207582 A1 WO2025207582 A1 WO 2025207582A1 US 2025021273 W US2025021273 W US 2025021273W WO 2025207582 A1 WO2025207582 A1 WO 2025207582A1
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WO
WIPO (PCT)
Prior art keywords
survivin
substituted phenyl
bromo
composition
phenyl
Prior art date
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Pending
Application number
PCT/US2025/021273
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English (en)
Inventor
Jian-ting ZHANG
Jingyuan Liu
Qingbin CUI
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University of Toledo
Original Assignee
University of Toledo
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Filing date
Publication date
Application filed by University of Toledo filed Critical University of Toledo
Publication of WO2025207582A1 publication Critical patent/WO2025207582A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
    • C07D241/36—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings condensed with carbocyclic rings or ring systems
    • C07D241/38—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings condensed with carbocyclic rings or ring systems with only hydrogen or carbon atoms directly attached to the ring nitrogen atoms
    • C07D241/40—Benzopyrazines
    • C07D241/44—Benzopyrazines with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the hetero ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/498—Pyrazines or piperazines ortho- and peri-condensed with carbocyclic ring systems, e.g. quinoxaline, phenazine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • Survivin is a homo-dimeric protein of 16.5-kDa containing a single Baculovirus IAP Repeat (BIR) domain, a zinc-finger fold, and an extended C-terminal helical coiled coil, and is a member of the inhibitor of apoptosis protein (IAP) family that is expressed only in cancer cells but not in adult normal tissues.
  • IAP apoptosis protein
  • each R is the same, and each R comprises a fluoro-substituted phenyl; a difluoro-substituted phenyl; a trifluoro-substituted phenyl; a chloro-substituted phenyl; a bromo- substituted phenyl; an iodo-substituted phenyl; a trifluoromethyl-substituted phenyl; a chloro-, fluoro- substituted phenyl; a di-bromo-substituted phenyl; a bromo-, fluoro-substituted phenyl; a bromo-, chloro- substituted phenyl; a bromo-, difluoro-substituted phenyl; or trimethylphenyl.
  • the compound is 7I-10: (7I-10) [0014] In certain embodiments, the compound is 7I-14: [0015] In certain from the group consisting of: , 64594-WO-PCT/TECH-2024-07 , , [0017] Further provided is a method of degrading survivin in a cell, the method comprising administering to a cell an effective amount of a survivin degrader compound and degrading survivin in the cell, wherein the survivin degrader compound comprises Formula I: Formula I wherein each R is independently a or triple substitution with a halogen, -NO 2 , - CF 3 , -CCl 3 , -CN, -COR 1 wherein R 1 is H or alkyl, -SO 3 H, -SO 2 CF 3 , or -NR 2 3 + wherein R 2 is alkyl; or a salt, 64594-WO-PCT/TECH-2024-07 stereoisomer, race
  • the cell is a cancer cell.
  • the cancer cell is a prostate cancer cell.
  • the cancer is breast cancer, colon cancer, lung cancer, pancreatic cancer, prostate cancer, ovarian cancer, or leukemia.
  • FIGS.2A Previous survivin inhibitors (FIG.2A), and non-limiting example survivin degrader compounds in accordance with the present disclosure (FIG.2B).
  • FIG.3 Scheme 1, showing drug design and synthesis of ten survivin degrader compounds in accordance with the present disclosure.
  • FIGS.4A-4B Cytotoxicity of 7I analogs 7I-1 to 7I-10.
  • FIG.4A shows a survival assay of C4-2 and PC-3 cells in the presence of different concentrations of 7I and its analogs 7I-1 to 7I-10.
  • FIG.4B shows IC50’s of 7I and 7I-1 to 7I-10 derived from survival curves shown in FIG.4A.
  • FIGS.5A-5C Analogs with mono-halogen substitutions at the 4-position on benzene rings and cytotoxicity.
  • FIG.5A shows the chemical structures of 7I-11 to 7I-14.
  • FIG.5B shows results of a cell viability assay of 7I-11, 7I-12, 7I-13, and 7I-14 compared with 7I in C4-2 and PC-3 cells.
  • FIG.5C shows the IC50’s of 7I and 7I-1 to 7I-10 derived from survival curves shown in FIG.5B.
  • FIGS.9A-9C Effect of –CH 3 substitution of –F in 7I-10 or –CF 3 in 7I-14 on cytotoxicity and survivin expression.
  • FIG.9A shows the chemical structures of 7I-24 and 7I-25.
  • FIG.9B shows the cytotoxicity and IC 50 of 7I-24 and -25 in C4-2 and PC-3 cells.
  • FIG.9C shows the effect of 7I-24 and 7I-25 64594-WO-PCT/TECH-2024-07 on survivin expression in C4-2 and PC-3 cells.
  • C4-2 and PC-3 cells were treated with 7I-10 or 7I-14 at 1 ⁇ M, or 7I-24 and 7I-25, at various concentrations for 48 hours followed by Western blot analyses of survivin and actin control.
  • FIGS.10A-10B Dose-dependent reduction of survivin protein in CRPC cells by 7I-10.
  • FIG.10A shows a western blot analysis of survivin and actin control in C4-2, PC-3, DU-145, and 22Rv-1 cells following treatments with different concentrations of 7I-10 for 48 hours.
  • FIG.10B shows a quantification of survivin level from FIG.10A of three independent experiments. One sample t-test was used to test for statistical significance, *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
  • FIG.11A-11B Dose-dependent reduction of survivin protein in CRPC cells by 7I-14.
  • FIG.12 Cytotoxicity IC50 of 7I-10 and 7I-14 in DU-145 (top left) and 22Rv-1 (bottom left) cells, and Pearson correlation analysis between cytotoxicity IC50 and the IC50 in reducing survivin expression of 7I-10 (top right) or 7I-14 (bottom right) in four CRPC cell lines. Correlation coefficient r and p-value were displayed.
  • FIGS.14A-14D Survivin overexpression decreased the sensitivity of 7I-10 and 7I-14.
  • FIGS.14A-14B show survivin-transfected C4-2 cells (FIG.14A) showed resistant to 7I-10 or 7I-14 (FIG. 14B) than vector-transfected cells.
  • FIGS.14C-14D show docetaxel-resistant DU145 cells (FIG.14C) showed resistance to 7I-10 or 7I-14 (FIG.14D) than parental DU145 cells.
  • FIGS.15A-15B Survivin-null cell line MC3T3-E1 is insensitive to survivin inhibition.
  • FIG.15A shows the Western blot analyses of survivin and actin control in PC-3, C4-2, and MC3T3-E1 cells.
  • FIG.15B shows 7I-10 and 7I-14 IC 50 values in these cell lines of 3 independent experiments.
  • FIGS.16A-16D Survivin half-life.
  • FIGS.16A-16B show the Western blot analyses of survivin and actin control in C4-2 (FIG.16A) and PC-3 (FIG.16B) cells following pre-treatments with 10 ⁇ M cycloheximide (CHX) and then treatments with 2 ⁇ M 7I-10 or 4 ⁇ M 7I-14 for different times.
  • FIG. 16C shows relative survivin level (%) from FIGS.16A-16B.
  • FIG.16D shows survivin half-life (t 1/2 ) in C4-2 and PC-3 cells determined from FIG.16C of 3 independent experiments. (*p ⁇ 0.05).
  • FIGS.17A-17B The effect of 7I-10 and 7I-14 on survivin mRNA level.
  • the figures show a western blot (FIG.17A) and RT-PCR analysis (FIG.17B) of survivin and actin control in C4-2 and PC-3 cells following treatment with 8 ⁇ M 7I-10 or 7I-14 for 6 hours.
  • the bar graphs in FIG.17B show fold change in survivin mRNA level.
  • FIGS.18A-18B Western blot analyses (FIG.18A) and quantification (FIG.18B) of survivin level in C4-2 and PC-3 cells following a pre-treatment with proteasome inhibitors, of MG132 (10 ⁇ M) or bortezomib (70 nM) for 1 hour and then co-treatment with 1 ⁇ M of 7I-10 (left) or 7I-14 (right) for 24 hours co-treatment.
  • ⁇ -Actin was used as a loading control. (p > 0.05, *p ⁇ 0.05, ***p ⁇ 0.001).
  • FIG.19 Mammalian two-hybrid assay.
  • FIGS.20A-20C 7I-10 and 7I-14 induction of apoptosis.
  • FIGS.20A-20B show Caspase-Glo 3/7 activity assay (Promega) in C4-2 (FIG.20A) and PC-3 (FIG.20B) cells following 7I-10 (left) or 7I-14 (right) treatments for 24 hours at indicated concentrations.
  • FIG.20C shows Western blot analyses of full- length and cleaved PARP in C4-2 (left) and PC-3 (right) cells.
  • FIGS.21A-21D Synergism between 7I-10 or 7I-14 with docetaxel in C4-2 cells.
  • FIGS. 21A-21B shows the 2D (FIG.21A) or 3D (FIG.21B) maps of the synergistic effects between 7I-10 and docetaxel in C4-2 cell.
  • FIGS.21C-21D show the 2D (FIG.21C) or 3D (FIG.21D) maps of the synergistic effects between 7I-14 and docetaxel in C4-2 cells.
  • FIGS.22C-22D show the 2D (FIG.22C) or 3D (FIG.22D) maps of the synergistic effects between 7I-14 and docetaxel in PC-3 cell.
  • the 3D graphs were generated by the SynergyFinder+, displaying (x-axis) docetaxel (nM), (y-axis) 7I-10 or 7I-14 (nM), and (z-axis) HSA synergy scores. Positive scores (red) indicate synergism, and negative scores (green) indicate antagonism.
  • FIGS.23A-23H In-vivo activity of 7I-14 and its synergism with docetaxel.
  • FIG.23F shows a Western blot analysis of survivin, cleaved caspase 3 (cCas3), and actin loading control in xenograft tumors.
  • FIGS.23G-23H show the wet weight of liver and lung (FIG.23G) as well as white blood cell count and hemoglobin level (FIG.23H) in the mice. *p ⁇ 0.05, **p ⁇ 0.01, and ***p ⁇ 0.001.
  • administering refers to bringing a patient, tissue, organ or cells in contact with a compound.
  • administration can be accomplished in vitro, i.e., in a test tube, or in vivo, i.e., in cells or tissues of living organisms, for example, humans.
  • the present disclosure encompasses administering the compounds useful in the present disclosure to a patient or subject.
  • a “patient” or “subject”, used equivalently herein, refers to a mammal, preferably a human, that either: (1) has a disorder remediable or treatable by administration of the compound; or (2) is susceptible to a disorder that is preventable by administering the compound.
  • the terms “effective amount” and “therapeutically effective amount” refer to the quantity of active therapeutic agent sufficient to yield a desired therapeutic response without undue adverse side effects such as toxicity, irritation, or allergic response.
  • the specific “effective amount” will vary with such factors as the particular condition being treated, the physical condition of the patient, the type of animal being treated, the duration of treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives. The optimum effective amounts can be readily determined by one of ordinary skill in the art using routine experimentation.
  • survivin is conventionally thought to be “undruggable”, the homo-dimerization interface of survivin can be targeted to induce the degradation of survivin. Because survivin exists as a homo-dimer, a small molecule compound that inhibits survivin dimerization may promote survivin degradation via proteasome and eliminate the protein, leading to spontaneous apoptosis.
  • inhibitors i.e., survivin degrader compounds
  • the survivin degrader compounds are 64594-WO-PCT/TECH-2024-07 also synergistic in combination with docetaxel, which is a commonly used anticancer drug, both in cell lines and in animal models.
  • docetaxel which is a commonly used anticancer drug, both in cell lines and in animal models.
  • the present inventors disclosed certain inhibitors of the homo-dimerization interface of survivin in U.S. Patent No.10,517,871 B2 and U.S. Patent Application Publication No. 2021/0299123 A1, which are incorporated herein by reference.
  • compositions of the present disclosure may comprise an effective amount of a survivin degrader compound described herein, and/or additional agents, dissolved or dispersed in a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable salts can also be prepared from the compounds by treatment with organic or inorganic acids, such as methanesulfonic acid, and hydrochloric acid.
  • the term “pharmaceutically acceptable salt” refers to a compound formulated from a base compound which achieves substantially the same pharmaceutical effect as the base compound.
  • the present disclosure further includes methods utilizing hydrates of the survivin degrader compounds.
  • the term “hydrate” includes but is not limited to hemihydrates, monohydrates, dihydrates, trihydrates and the like.
  • compositions disclosed herein can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, intraosseously, periprosthetically, topically, intramuscularly, subcutaneously, mucosally, intraosseosly, periprosthetically, in utero, orally, topically, locally, via inhalation (e.g., aerosol inhalation), by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the foregoing as would be known to one of ordinary skill in the art (see, for example, Remington’s Pharmaceutical Sciences, 2003, incorporated herein by reference).
  • inhalation e.g., aerosol inhalation
  • a composition herein and/or additional agent is formulated to be administered via an alimentary route.
  • Alimentary routes include all possible routes of administration in which the composition is in direct contact with the alimentary tract.
  • the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually.
  • these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft-shell gelatin capsules, they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
  • a composition described herein may be administered via a parenteral route.
  • parenteral includes routes that bypass the alimentary tract.
  • the pharmaceutical compositions disclosed herein may be administered, for example but not limited to, intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneous, or intraperitoneally (U.S. Patents 6,753,514, 6,613,308, 5,466,468, 5,543,158; 5,641,515, and 5,399,363 are each specifically incorporated herein by reference in their entirety).
  • Solutions of the compositions disclosed may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions may also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Patent 5,466,468, specifically incorporated herein by reference in its entirety). In some cases, the form should be sterile and should be fluid to the extent that easy injectability exists.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (i.e., glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
  • polyol i.e., glycerol, propylene glycol, liquid polyethylene glycol, and the like
  • suitable mixtures thereof and/or vegetable oils.
  • Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and/or by the use of surfactants.
  • Sterile injectable solutions are prepared by incorporating the compositions in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized compositions into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the compositions may be formulated for administration via various miscellaneous routes, for example, topical (i.e., transdermal) administration, mucosal administration (intranasal, vaginal, etc.), and/or via inhalation.
  • compositions for topical administration may include the compositions formulated for a medicated application such as an ointment, paste, cream, or powder.
  • Ointments include all oleaginous, adsorption, emulsion, and water-soluble based compositions for topical application, while creams and lotions are those compositions that include an emulsion base only.
  • Topically administered medications may contain a penetration enhancer to facilitate the adsorption of the active ingredients through the skin. Suitable penetration enhancers include glycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones, and laurocapram.
  • compositions for topical application include polyethylene glycol, lanolin, cold cream, and petrolatum, as well as any other suitable absorption, emulsion, or water-soluble ointment base.
  • Topical preparations may also include emulsifiers, gelling agents, and antimicrobial preservatives as necessary to preserve the composition and provide for a homogenous mixture.
  • Transdermal administration of the compositions may also comprise the use of a “patch.”
  • the patch may supply one or more compositions at a predetermined rate and in a continuous manner over a fixed period of time.
  • the compositions may be delivered by eye drops, intranasal sprays, inhalation, and/or other aerosol delivery vehicles.
  • compositions disclosed herein may be delivered via an aerosol.
  • aerosol refers to a colloidal system of finely divided solid or liquid particles dispersed in a liquefied or pressurized gas propellant.
  • the typical aerosol for inhalation consists of a suspension of active 64594-WO-PCT/TECH-2024-07 ingredients in liquid propellant or a mixture of liquid propellant and a suitable solvent.
  • Suitable propellants include hydrocarbons and hydrocarbon ethers.
  • Suitable containers will vary according to the pressure requirements of the propellant.
  • the survivin degrader compound(s) may be administered to the subject orally, topically, nasally, parenterally, paracancerally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, subcutaneously, intraperitoneally, intraventricularly, intracranially, intratumorally, or by pulmonary delivery.
  • the compounds and compositions herein can be used in combination therapies. That is, the compounds and compositions can be administered concurrently with, prior to, or subsequent to one or more other desired therapeutic or medical procedures or drugs.
  • the particular combination of therapies and procedures in the combination regimen will take into account compatibility of the therapies and/or procedures and the desired therapeutic effect to be achieved.
  • the survivin degrader compound is part of a combination therapy with an immunotherapeutic agent.
  • immunotherapeutic agents include nivolumab, pembrolizumab, rituximab, durvalumab, cemiplimab, and combinations thereof.
  • the survivin degrader compound is part of a combination therapy with a hormonal therapeutic agent.
  • Non-limiting examples of hormonal therapeutic agents include anastrozole, exemestane, letrozole, tamoxifen, raloxifene, fulvestrant, toremifene, goserelin, leuprolide, triptorelin, apalutamide, enzalutamide, darolutamide, bicalutamide, flutamide, nilutamide, abiraterone, ketoconazole, degarelix, medroxyprogesterone acetate, megestrol acetate, mitotane, and combinations thereof.
  • the survivin degrader compounds described herein are effective in suppressing tumor growth without apparent toxicity, and are easy to make with a one-step chemical synthesis.
  • the survivin degrader compounds directly target the survivin protein without effects on other proteins and, thus, are less likely to cause side effects.
  • 7I-10 and 7I-14 also synergized with docetaxel and selectively suppressed survivin expression by inhibiting survivin dimerization, leading to its degradation in the proteasome. Both 7I-10 and 7I-14 selectively cause survivin degradation without effect on other members of the IAP family. Furthermore, overexpression of ectopic survivin conferred resistance to both 7I-10 and 7I-14, indicating the on-target effects of both compounds. Finally, in the PC-3 cells xenograft model, 7I-14 effectively suppressed tumor growth and synergized with docetaxel without added toxicity to the animal.
  • FIG.14B shows that the C4-2/Survivin cells are significantly more resistant to both 7I-10 and 7I-14 compared with the control C4-2/Vec cells, indicating that survivin likely mediates the cytotoxicity of 7I-10 and 7I-14.
  • DU145 Doc docetaxel-resistant DU145 cell line
  • FIG.14C DU145 Doc 64594-WO-PCT/TECH-2024-07 cells express higher level of endogenous survivin compared with DU145 cells.
  • DU145 Doc cells are also significantly more resistant to 7I-10 and 7I-14 (FIG.14D).
  • 7I-10 and 7I-14 reduce survivin stability
  • these inhibitors selectively induced survivin loss.
  • a cycloheximide-chase experiment was performed to determine the inhibitor effect on survivin half-life.
  • FIGS.16A-16D both 7I-10 and 7I-14 dramatically reduced survivin half-life.
  • survivin mRNA was not changed by 7I-10 and 7I-14 under the same treatment condition (FIGS.17A-17B).
  • both 7I-10 and 7I-14 dose-dependently induced cleavage of PARP (FIG.20C), a substrate of executioner caspases during apoptosis. Based on these findings, it is apparent that 7I-10 and 7I-14 treatments induce spontaneous apoptosis of cancer cells. [00118] Both 7I-10 and 7I-14 synergize with docetaxel [00119] Docetaxel is one of the first line treatment for metastatic CRPC and survivin is known to contribute resistance to drugs including docetaxel.
  • the hetero- disubstitutions may render these compounds distinct 3D conformations due to different atom sizes or electron configuration of atomic orbital, resulting in different interactions and bonding with survivin.
  • 7I-13 with 4-iodo is more active than 7I with 4-fluoro also possibly due to its size, posing a different conformation and leading to different binding and affinity to survivin through the semidirectional halogen-bonding effect.
  • the strong activity of 7I-14 with 4-trifluoromethyl may also be due to its size in addition to its strong electron-withdrawing potential.
  • Methylene blue assay 2,000-4,000 cells/well were seeded in a 96-well plate, and cultured for 24 h before adding seven gradient concentrations of each compound for another 72 h. The medium was then removed, and cells were fixed with methanol for 30 min and subsequently stained with 100 ⁇ L of 1% methylene blue (diluted in 10 mM borate buffer) for 30 min.
  • Cells were then lysed in a modified RIPA buffer (150 mM NaCl, 50 mM Tris pH 7.4, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 5 mM EDTA pH 8.0) with a protease inhibitor cocktail and a phosphatase inhibitor cocktail (Roche, Indianapolis, IN, USA) for 30 min on ice.
  • the cell debris was removed by centrifugation at 16,000 ⁇ g for 15 min at 4 °C.
  • the concentration of proteins was measured using the Bio-Rad Protein Assay (Bio-Rad, herculues, USA). Protein lysates were analyzed by standard SDS-PAGE and transferred to PVDF membranes.
  • the docking experiments were performed by the default protocol. 64594-WO-PCT/TECH-2024-07 [00144] Mammalian two-hybrid reporter assay [00145] The two-hybrid mammalian reporter assay was established previously. Briefly, the assay was performed by using the MatchmakerTM Mammalian Assay Kit 2 (Takara Bio). During the establishment of the vectors, the coding region of survivin was cloned into the pM plasmid fusing with the GAL4-DNA binding domain (pM-Survivin) and into the pVP16 plasmid fusing with an activation domain (pVP16- Survivin).
  • pM-Survivin was digested by XbaI and NdeI or by EcoRI-HF and PvuII
  • pVP16-Survivin was digested by XbaI and NheI-HF or by EcoRI-HF and PvuII.
  • Survivin coding region contains PvuII cutting sites, but the vectors do not.
  • the reporter plasmid pG5SEAP was linearized by KpnI-HF and its correct size was confirmed.
  • Luminescent SEAP activity was measured by a tube luminometer (Sirius FB12 Luminometer, Berthold Technologies). Final results of relative SEAP activities were calculated by normalizing the SEAP activity to Renilla luciferase activity and normalizing again to the DMSO vehicle control of the vector:vector control.
  • RNA extraction and real-time PCR 64594-WO-PCT/TECH-2024-07 [00151] C4-2 or PC-3 cells were plated at 3 ⁇ 10 6 in 10 cm dishes overnight. The next day, 7I-10, 7I- 14, or the same volume of DMSO control was directly added to dishes to a final concentration of 4 ⁇ M.
  • each 20 ⁇ L reaction was composed of 2 ⁇ L diluted cDNA, 2 ⁇ L primers (forward and backward), 6 ⁇ L nuclease-free water, and 10 ⁇ L SYBR Green qPCR Kit (RadiantTM). Reaction conditions were set up according to the SYBR Green datasheet and melting curve analysis was conducted to verify specificity of the primers.
  • Example 1 [00160] Chemical Synthesis and Methods [00161] 7I analogs were synthesized as follows. Briefly, a mixture of 2,3-dichloroquinoxaline (1 equivalent) with corresponding halogen-substituted aniline (2.2 equivalents) in ethanol was refluxed for 18- 24 h. Then the solvent was removed under reduced pressure, after which, the solid was re-dissolved in ethyl acetate and washed with saturated NaHCO 3 and brine, dried over Na 2 SO 4 . Ethyl acetate was removed under reduced pressure to get the crude product which was then purified by column chromatograph.
  • N 2 ,N 3 -bis(4-iodophenyl)quinoxaline-2,3-diamine (7I-13) [00187] 1 H NMR (600 MHz, DMSO-d 6 ) ⁇ 9.10 (s, 2H), 7.79 – 7.70 (m, 8H), 7.57 (dd, J 6.1, 3.4 Hz, 2H), 7.41 – 7.35 (m, 2H). 13 C NMR (151 MHz, DMSO-d 6 ) ⁇ 141.44, 140.54, 137.66, 136.53, 126.00, 125.96, 123.08.

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Abstract

L'invention concerne des composés de dégradation de la survivine, et leurs procédés de fabrication et d'utilisation. Certains composés de dégradation de la survivine sont utiles pour traiter des cancers ou inhiber la croissance de cellules cancéreuses.
PCT/US2025/021273 2024-03-26 2025-03-25 Agents de dégradation de la survivine pour le traitement ciblé du cancer Pending WO2025207582A1 (fr)

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US63/569,842 2024-03-26

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