Conjugated compound comprising 13-cis-retinoic acid and ascorbic acid

Abstract

The present invention relates to a conjugate compound comprising 13-cis-retinoic acid and vitamin C, and its pharmaceutically acceptable salts thereof. The present invention also relates to a pharmaceutical composition comprising the compound and a pharmaceutically acceptable carrier. The present compound has a much better water solubility than that of retinoic acid, and thus reduces an effective dosage of retinoic acid and minimizes side effect. The present compound increases the efficiency and concentration of 13-cis-retinoic acid to across the cell membrane into the cell. The present compound is useful in treating cancer such as pancreatic cancer.

Description

1 / 19 Conjugated compound comprising 13-cis-retinoic acid and ascorbic acid Field of Invention

[001] The present invention relates to a conjugated compound comprising 13-cis-retinoic acid and vitamin C and pharmaceutically acceptable salts thereof. The present invention also relates to a pharmaceutical composition comprising the compound and a pharmaceutically acceptable carrier. Background of the Invention

[002] All-trans-retinoic acid (ATRA), also called tretinoin, and isotretinoin (13-cis-retinoic acid) are orally administered vitamin A derivatives. 13-Cis-retinoic acid and ATRA are promising compounds for the treatment of a variety of cancers due to their specific effects on cell proliferation, differentiation, and apoptosis, as well as their low toxicity. Retinoic acid receptors in human cell nuclei have been discovered by biochemists and have not mutated in cancerous cells; therefore, retinoic acids could potentially exert their anticancer effects on many malignancies. For example, treatment with 13-cis-retinoic acid has been found in children with high-risk neuroblastoma to reduce the risk of cancer recurrence after high-dose chemotherapy and stem cell transplantation. ATRA has been studied in combination with other drugs in various types of cancer and precancerous lesions.Several clinical trials using ATRA as part of combination therapy are currently underway [Kocher, HM, Basu, B., Froeling, FEM et al. Phase I clinical trial repurposing alltrans retinoic acid as a stromal targeting agent for pancreatic cancer. Nat Commun 11,4841 (2020)]. For example, ATRA with different interferons (IFN) has been shown to potentiate the effects of both drugs and lead to inhibition of cell growth and death in tumor cell lines. However, Petition 870250080277, dated 09 / 08 / 2025, p. 16 / 39 2 / 19 To unlock the therapeutic potential of retinoic acids, many studies emphasize the need for a better understanding of the mechanisms that block retinoic acid signaling and retinoic acid-regulated gene expression in cancers such as acute myeloid leukemia (AML). Combinational therapies targeting multiple gene silencing mechanisms may be the most effective strategy to reactivate ATRA-sensitive gene expression and AML cell differentiation, as well as mediate the anticancer activities of ATRA in general. Currently, the identification of protein classes that control gene expression through histone and DNA modifications is driving the development of new therapeutic agents, so-called epigenetic drugs, which alter chromatin structure. However, these epigenetic modifying drugs have been shown to be only partially effective against different types of cancer when used alone.

[003] Vitamin A is a fat-soluble vitamin and an essential nutrient for humans. It is a group of organic compounds that includes retinol, retinal (also known as retinaldehyde), retinoic acid, and several provitamin A carotenoids (primarily beta-carotene [β-carotene]). Retinoic acid is a metabolite of vitamin A1 (all-trans-retinol). Isotretinoin (13-cis-retinoic acid) is an orally administered derivative of vitamin A.

[004] Vitamin C (also known as ascorbic acid and ascorbate) is a water-soluble vitamin found in citrus fruits and other fruits and vegetables. Vitamin C is an essential nutrient involved in tissue repair, collagen formation, and the enzymatic production of certain neurotransmitters. Vitamin C is necessary for the functioning of several enzymes and is important for the function of the immune system. Vitamin C also functions as an antioxidant. Petition 870250080277, dated 08 / 09 / 2025, page 17 / 39 3 / 19 Brief Description of the Drawings

[005] Figure 1 shows examples of the synthesis of the conjugate compound of Formula A1 and A2 without a protecting group.

[006] Figure 2 shows examples of the synthesis of the conjugate compound of Formula A2 with a protecting group. The 2-methoxyethoxymethyl ether (MEM) protecting group is used as an illustration. Other protecting groups can also be used.

[007] Figure 3 shows an example of the synthesis of the conjugate compound of Formula A1 used in this study. Detailed Description of the Invention Definitions

[008] Pharmaceutically acceptable salts, as used herein, are salts that retain the desired biological activity of the original compound and do not confer undesirable toxicological effects. Pharmaceutically acceptable salt forms include various crystalline polymorphs as well as the amorphous form of the different salts. Pharmaceutically acceptable salts may be formed with metallic or organic counterions and include, but are not limited to, alkali metal salts such as sodium or potassium; alkaline earth metal salts such as magnesium or calcium; and ammonium or tetraalkylammonium salts, i.e., NX4+ (where X is C1-4). Abbreviations

[009] HATU, 1-[Bis(dimethylamino)methylene]-1H-1,2,3triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate DIPEA, N,N-Diisopropylethylamine DMAP, 4-Dimethylaminopyridine DCC, N,N'-Dicyclohexylcarbodiimide NHS, N-Hydroxysuccinimide THF, Tetrahydrofuran DMF, Dimethylformamide Ac, Acetate Petition 870250080277, dated 08 / 09 / 2025, page 18 / 39 4 / 19 ACN, Acetonitrile Bn, Benzyl ether GOOD, Benzyloxymethyl acetal Bz, Benzoate MEM, 2-methoxyethoxymethyl ether MEMCl, 2-Methoxyethoxymethyl chloride MOM, Methoxymethyl acetal MOP, Methoxypropyl acetal Nap, 2-Naphthylmethyl ether NMR, Nuclear Magnetic Resonance PMB, 4-methoxybenzyl ether TBDMS, Tert-butyldimethylsilyl ether TBDPS, Tert-butyldiphenylsilyl ether TBS, Tert-butyldimethylsilyl ether TES, Triethylsilicic ether THP, Tetrahydropyranyl Acetal TIPS, Triisopropylsilyl ether TMS, Trimethylsilyl ether Troc, 2,2,2-Trichloroethyl Carbonate Conjugated compound comprising 13-cis-retinoic acid and ascorbic acid.

[010] The present invention relates to conjugated compounds of Formula A1 and A2 comprising 13-cis-retinoic acid and vitamin C (ascorbic acid). The 13-cis-retinoic acid in the conjugated compound provides therapeutic activity and the vitamin C in the conjugated compound increases the water solubility of the compound and significantly reduces the effective dosage of 13-cis-retinoic acid in the compound for the treatment of a disease. Petition 870250080277, dated 09 / 08 / 2025, p. 19 / 39 5 / 19 Formula Al Formula A2 Preparation of compounds with formula Al and formula A2 [Oil]Figures 1 and 2 show examples of different schemes (with or without a protecting group) for the synthesis of the conjugated compound with formula Al and A2.

[012] As shown in Figure 1, to form retinoic acid chloride (compound 2), a solution of retinoic acid (1) is added with (COC1)2 and CH2CI2 in a catalytic amount of dimethylformamide (DMF) under stirring at room temperature. Subsequently, distillation is carried out under reduced pressure to remove unreacted (COC1)2 and DMF, to obtain retinoic acid chloride (compound 2).

[013] In some embodiments, the compounds represented by Formula A1 and Formula A2 can be synthesized directly from the combination of retinoic acid or retinoic acid chloride with vitamin C (Figure 1). For example, a vitamin C solution is mixed with retinoic acid (compound 1) or retinoic acid chloride (compound 2) in the presence of an N,N'-dicyclohexylcarbodiimide (DCC) catalytic system and 4 Petition 870250080277, dated 08 / 09 / 2025, page 20 / 39 6 / 19 dimethylaminopyridine (DMAP) or added with N-hydroxysuccinimide (NHS) at room temperature. After the reaction was complete, the solution was concentrated under reduced pressure to obtain the target compound in solid form.

[014] In some embodiments, the compound of Formula A2 can be synthesized using a protecting group (Figure 2). For example, isopropylidene ascorbic acid (compound 3) is dissolved in a dichloromethane solution and then added with N,N-diisopropylethylamine (DIPEA), followed by the addition of 2-methoxyethoxymethyl chloride (MEMCl), so as to form compound 4, in which the hydroxyl groups at C2 and C3 on the ascorbic acid ring are protected by the protecting groups of 2-methoxyethoxymethyl ether (MEM). Subsequently, compound 4 is dissolved in 80% ethanoic acid (HOAc) and reacted at 50°C for 2 hours. After the reaction is complete, the solution is concentrated under reduced pressure to remove the ethanoic acid. The prepared residue (compound 5) and retinoic acid chloride (compound 2) are subsequently dissolved in DMF and DCC and DMAP are added at room temperature. The mixture is then stirred for 2 hours to form compound 6.

[015] Subsequently, the protecting group can be removed by treatment with an appropriate deprotecting agent. For example, the removal of the MEM protecting groups in compound 6 was achieved by reacting compound 6 with anhydrous zinc bromide (ZnBr2) in CH2Cl2 or HCl / dioxane at room temperature for 2 hours. After the reaction was complete, the solution was concentrated and dried. The residue is then purified to give the compound of Formula A2.

[016] Suitable protecting groups for the methods presented include, but are not limited to, 2,2,2-trichloroethyl carbonate (Troc), 2-methoxyethoxymethyl ether (MEM), 2-naphthylmethyl ether (Nap), 4-methoxybenzyl ether (PMB), acetate (Ac), benzoate (Bz), benzyl ether (Bn), benzyloxymethyl acetal (BOM), methoxymethyl acetal (MOM), methoxypropyl acetal (MOP), ether Petition 870250080277, dated 08 / 09 / 2025, page 21 / 39 7 / 19 methyl, tetrahydropyranilic acetal (THP), triethylsilicic ether (TES), triisopropylsilicic ether (TIPS), trimethylsilicic ether (TMS), tert-butyldimethylsilicic ether (TBS or TBDMS) and tert-butyldiphenylsilicic ether (TBDPS). Pharmaceutical Compositions

[017] The present invention provides pharmaceutical compositions comprising one or more pharmaceutically acceptable carriers and a conjugate compound of Formula A1 or A2 or a pharmaceutically acceptable salt thereof.

[018] In one embodiment, the compound is incorporated into any acceptable carrier, including creams, gels, lotions, or other types of suspensions that can stabilize the compound and release it to the affected area through topical applications. In another embodiment, the pharmaceutical composition may be in a dosage form, such as tablets, capsules, granules, fine granules, powders, syrups, suppositories, injectable solutions, patches, or the like. The above pharmaceutical composition may be prepared by conventional methods.

[019] Pharmaceutically acceptable carriers, which are inactive ingredients, may be selected by those skilled in the art using conventional criteria. Pharmaceutically acceptable carriers include, but are not limited to, non-aqueous solutions, suspensions, emulsions, microemulsions, micellar solutions, gels, and ointments. Pharmaceutically acceptable carriers may also contain ingredients that include, but are not limited to, saline and aqueous electrolyte solutions; ionic and non-ionic osmotic agents, such as sodium chloride, potassium chloride, glycerol, and dextrose; pH adjusters and buffers, such as hydroxide, phosphate, citrate, acetate, borate salts; and trolamine; antioxidants such as salts, acids and / or bases of bisulfite, sulfite, metabisulfite, thiosulfite, ascorbic acid, acetylcysteine, cysteine, glutathione, butylated hydroxyanisole, butylated hydroxytoluene, Petition 870250080277, dated 08 / 09 / 2025, page 22 / 39 8 / 19 tocopherols and ascorbyl palmitate; surfactants such as lecithin, phospholipids including, but not limited to, phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol; poloxamers and poloxamines, polysorbates such as polysorbate 80, polysorbate 60 and polysorbate 20, polyethers such as polyethylene glycols and polypropylene glycols; polyvinyls such as polyvinyl alcohol and povidone; cellulose derivatives such as methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, carboxymethylcellulose and hydroxypropylmethylcellulose and salts thereof; petroleum derivatives such as mineral oil and white petrolatum; fats such as lanolin, peanut oil, palm oil, soybean oil; mono-, di- and triglycerides; Acrylic acid polymers, such as carboxypolymethylene gel and hydrophobically modified cross-linked acrylate copolymer, polysaccharides, such as dextrans, and glycosaminoglycans, such as sodium hyaluronate.Other pharmaceutically acceptable carriers include xanthan gum, carrageenan, Avicel RC-591 (a combination of microcrystalline cellulose and) and polyethylene glycol. Alternatively, the active compound may be dissolved or suspended in a pharmaceutically acceptable lipid formulation, such as those described by Kalepu et al. (Acta Pharmaceutica Sinica B, 3: 361-372, 2013), for example, vegetable oil, coconut oil, castor oil, etc.

[020] These pharmaceutically acceptable carriers can be preserved against bacterial contamination using well-known preservatives, these include, but are not limited to, benzalkonium chloride, ethylenediaminetetraacetic acid and its salts, benzethonium chloride, chlorhexidine, chlorobutanol, methylparaben, thimerosal and phenylethyl alcohol, or they can be formulated as a preservative-free formulation for single or multiple use.

[021] For example, a tablet formulation or a capsule formulation of the compound may contain other excipients Petition 870250080277, dated 08 / 09 / 2025, page 23 / 39 9 / 19 that do not exhibit bioactivity or reaction with the compound. The excipients in a tablet or capsule may include fillers, binders, lubricants and glidants, disintegrants, wetting agents, and release rate modifiers. Binders promote the adhesion of the formulation particles and are important for the tablet formulation. Examples of tablet or capsule excipients include, but are not limited to, carboxymethylcellulose, cellulose, ethylcellulose, hydroxypropylmethylcellulose, methylcellulose, karaya gum, starch, tragacanth gum, gelatin, magnesium stearate, titanium dioxide, poly(acrylic acid), and polyvinylpyrrolidone. For example, a tablet formulation may contain inactive ingredients such as colloidal silicon dioxide, crospovidone, hypromellose, magnesium stearate, microcrystalline cellulose, polyethylene glycol, sodium starch glycolate, and / or titanium dioxide.A capsule formulation may contain inactive ingredients such as gelatin, magnesium stearate, and / or titanium dioxide.

[022] For example, an adhesive formulation of the compound may comprise some inactive ingredients such as 1,3-butylene glycol, dihydroxyaluminum aminoacetate, disodium edetate, D-sorbitol, gelatin, kaolin, methylparaben, polysorbate 80, povidone, propylene glycol, propylparaben, sodium carboxymethylcellulose, sodium polyacrylate, tartaric acid, titanium dioxide, and purified water. An adhesive formulation may also contain a skin permeability enhancer such as lactate esters or diethylene glycol monoethyl ether.

[023] Topical formulations that include the compound may be in the form of gel, cream, lotion, liquid, emulsion, ointment, spray, solution, and suspension. Inactive ingredients in topical formulations include, but are not limited to (emollient / permeation enhancer), monoethyl ether of Petition 870250080277, dated 08 / 09 / 2025, page 24 / 39 10 / 19 diethylene glycol (emollient / permeation enhancer), DMSO (solubility enhancer), silicone elastomer (rheology / texture modifier), caprylic / capric triglyceride (emollient), octisalate (emollient / UV filter), silicone fluid (emollient / diluent), squalene (emollient), sunflower oil (emollient) and silicon dioxide (thickening agent).

[024] In one embodiment, the concentration of the conjugate compound comprising 13-cis-retinoic acid and vitamin C or pharmaceutically acceptable salts thereof in the pharmaceutical composition may be, but is not limited to, 0.1 μM to 10 mM, 0.1 μM to 1 mM, 0.1 μM to 500 μM, 0.1 μM to 250 μM, 0.1 μM to 100 μM, 0.1 μM to 50 μM, 1 μM to 10 mM, 1 μM to 1 mM, 1 μM to 500 μM, 1 μM to 250 μM, 1 μM to 100 μΜ, from 1 μΜ to 50 μΜ, from 10 μΜ to 10 mM, from 10 μΜ to 1 mM, from 10 μΜ to 500 μΜ, from 10 μΜ to 250 μΜ, from 10 μΜ to 100 μΜ or from 10 μΜ to 50 μΜ. Method of Use

[025] These conjugated compounds are useful for the treatment of cancer, such as pancreatic cancer, lung cancer, colorectal cancer, ovarian cancer, adrenal cancer, bone cancer, brain cancer, breast cancer, gallbladder cancer, head and neck cancer, kidney cancer, laryngeal cancer, liver cancer, prostate cancer, parathyroid cancer, skin cancer, stomach cancer and thyroid cancer. Additional examples of cancers suitable for treatment by the present conjugated compounds include cholangiocarcinoma, acute and chronic lymphocytic and granulocytic tumors, adenocarcinoma, adenoma, basal cell carcinoma, cervical dysplasia and carcinoma in situ, Ewing's sarcoma, squamous cell carcinoma, giant cell tumor, glioblastoma multiforme, hairy cell tumor, intestinal ganglioneuroma, hyperplastic corneal nerve tumor, islet cell carcinoma, Kaposi's sarcoma, leiomyoma, malignant carcinoid, Petition 870250080277, dated 08 / 09 / 2025, page 25 / 39 11 / 19 malignant melanoma, malignant hypercalcemia, Marfanoid habitus tumor, medullary carcinoma, metastatic skin carcinoma, mucosal neuroma, myeloma, mycosis fungoides, neuroblastoma, osteosarcoma, pheochromocytoma, polycythemia vera, primary brain tumor, small cell lung tumor, ulcerative and papillary squamous cell carcinoma, hyperplasia, seminoma, soft tissue sarcoma, retinoblastoma, rhabdomyosarcoma, renal cell tumor, topical skin lesion, reticular cell sarcoma, and Wilms' tumor. [02 6]The present method comprises a step of administering to a cancer patient a compound of Formula A1 or Formula A2, in an amount effective to treat the cancer. An effective amount, as used herein, is the amount effective in treating the cancer, improving the pathological condition or reducing the symptoms of the cancer.

[027] The pharmaceutical composition of the present invention can be applied by systemic administration or local administration. Local administration includes topical administration and inhalation. Systemic administration includes oral, parenteral (such as intravenous, intramuscular, subcutaneous or rectal) and other systemic routes. In systemic administration, the active compound first reaches the plasma and then distributes to the target tissues. Intravenous administration is a preferred route of administration for the present invention.

[028] The dosage of the composition may vary based on the extent of the cancer and the individual response of each patient. For systemic administration, plasma concentrations of the administered active compound may vary; but are generally 1x10-10-1x10-4 mol / liter and preferably 1x10-8-1x10-5 mol / liter.

[029] In one embodiment, the pharmaceutical composition is administered intravenously to the individual. The dosage for intravenous bolus injection or intravenous infusion is, Petition 870250080277, dated 08 / 09 / 2025, page 26 / 39 12 / 19 generally, from 0.03 to 20 mg / kg / day and, preferably, from 0.03 to 10 mg / kg / day.

[030] In one embodiment, the pharmaceutical composition is administered orally to the individual. The dosage for oral administration is generally at least 0.1 mg / kg / day and less than 100 mg / kg / day. For example, the dosage for oral administration is 0.1-100 or 0.5-50 mg / kg / day for a human individual. For example, the dosage for oral administration is 20-1000 mg / day or 100-2000 mg / day, and preferably 20-500, 25-200, 50-500, 50-200, 100-600, 100-400, 100-800, 200-800, 400-800, 400-1200, 500-2000 or 800-2000 mg / day for a human individual.

[031] In one embodiment, the pharmaceutical composition is administered subcutaneously to the individual. The dosage for subcutaneous administration is generally 0.3 to 20 mg / kg / day and preferably 0.3-3 mg / kg / day.

[032] In one embodiment, the pharmaceutical composition is administered by inhalation. Inhalation methods include liquid instillation, instillation as a pressurized fluid preparation via metered-dose inhaler or equivalent, or inhalation of an aerosolized solution via nebulizer, inhalation of dry powder, and directing soluble or dry material into the airstream during mechanical ventilation. Surface concentrations of the active compound administered by inhalation may vary, but are generally 1x10-10-1x10-4 mol / liter and preferably 1x10-8-1x10-5 mol / liter.

[033] Those skilled in the art will recognize that a wide variety of management mechanisms are also suitable for the present invention.

[034] The present method is useful in the treatment of a mammalian individual, such as humans, horses and dogs. The present invention is particularly useful in the treatment of humans.

[035] The following examples further illustrate the present Petition 870250080277, dated 09 / 08 / 2025, p. 27 / 39 13 / 19 invention. These examples are intended only to illustrate the present invention and should not be interpreted as limiting. EXAMPLES Example 1. Synthesis of the compound with Formula A1

[036] Figure 3 summarizes a synthesis scheme for compound A1.

[037] To a solution of (R)-5-((S)-1,2-dihydroxyethyl)-3,4-dihydroxyfuran-2(5H)-one (1,200 mg, 1.14 mmol), (2Z,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona2,4,6,8-tetraenoic acid (2,409 mg, 1.36 mmol) and DCC (351 mg, 1.70 mmol) in DMF (5 mL), DMAP (166 mg, 1.36 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours in a dark environment. After the reaction was complete, the mixture was diluted with water (10 mL) and extracted with EtOAc (10 mL x 3). The combined organic layers were concentrated under reduced pressure to obtain a residue. The residue was purified by prep-HPLC (column: Phenomenex luna C18 250 mm x 100 mm x 10 µm; mobile phase: [H2O (0.1% TFA-H2O)-ACN]; B%: 20%-50%, 30 min) to yield S171-BP (51.65 mg, 9.48% yield) as a yellow solid. All processes were performed in a dark environment with aluminum foil wrapping. The structure of Formula A1 was confirmed by NMR analysis and summarized below.

[038] MS (ESI): calculated mass for C26H34O7 458.55, m / z found 459.45 [M+H]+.

[039] 1H NMR (400 MHz, DMSO-de): δ 12.52 (brs, 1H), 7.62 (d, J = 15.2 Hz, 1H), 7.21-7.14 (m, 1H), 6.35-6.29 (m, 2H), 6.256.21 (m, 1H), 5.86 (s, 1H), 5.01-4.78 (m, 3H), 3.80 (t, J = 7.0 Hz, 2H), 3.47-3.45 (m, 2H), 1.69 (s, 3H), 2.017 (s, 5H), 1.693 (s, 3H), 1.58-1.56 (m, 1H), 1.45-1.44 (m, 1H), 1.02 (s, 6H). Example 2. Cytotoxicity of compound Formula A1 in cancer cell lines.

[040] Objectives: To test the cytotoxic effect of Formula A1 compounds on three cancer cell lines in vitro. Petition 870250080277, dated 08 / 09 / 2025, page 28 / 39 14 / 19 Cancer Cell Lines Cell line Tumor type@ Culture medium* A549 Non-Small Cell Lung Cancer PANC-1 Pancreatic Cancer DMEM Caco-2 Colorectal Cancer DMEM @All cell lines used in this study were derived from human patients. *All complete culture media were prepared with bovine serum antibiotics. Fetal cell reagents for cell culture and viability assay up to a final concentration of 10% and without viability. Item Full Name DMEM FBS DPBS Trypsin DMSO EtOH MTT Dulbecco Modified Eagle Medium Fetal bovine serum Dulbecco Buffered Saline Solution 0.25% Trypsin-EDTA Dimethyl sulfoxide Ethanol Cell Viability Reagent MTT Phosphate Procedures

[041] Cells were cultured in the indicated growth medium with 10% FBS and maintained in a humidified incubator at 37°C containing 5% CO2. The compound of Formula A2 was weighed and dissolved in a DMSO / EtOH mixture solvent at a ratio of 1 / 4 (v / v) to a final concentration of 200 mM on the day of treatment. One day before treatment, cells in the logarithmic growth phase were harvested, counted, and seeded in a 96-well plate at a density of 5 x 103 cells / 100 mL / well. After overnight culture, 100 mL of fresh medium prepared at the concentration was gently added to the assumed wells. Petition 870250080277, dated 08 / 09 / 2025, pp. 29 / 39 15 / 19 serially from 40 to 1000 μM to bring the working concentration from 20 to 500 μM subsequently. The control plate without Formula A2 underwent the same procedure as above, but with 100 mL of fresh medium prepared with the same volume of solvent added. All plates were gently mixed and incubated for the next 24 hours.

[042] On the day of cell viability detection, the tested wells received 50 mL of MTT working solution and were incubated at 37°C for 3 hours. Spectrophotometric absorbance readings were recorded at a wavelength of 540 nm. Readings from blank wells (wells containing only cell culture medium without cells) and negative control wells (wells with cells and culture medium without Formula A2 and solvent treatment) were recorded and used for calibration. After all OD540 readings were subtracted from the blank value, the percentage of cell viability was calculated using the following formula: Cell viability (%) = [OD540 (sample) / OD540 (negative control on sample plate)] / [OD540 (control) / OD540 (negative control on control plate)] x 100%.

[043] The concentration of Compound A that inhibited cell survival by 50% (IC50) was determined using an online tool, Quest Graph™ IC50 Calculator. AAT Bioquest, Inc., June 26, 2024, www.aatbio.com / tools / ic50-calculator. Results

[044] Cell viability calculated at different concentrations of Formula A1 was listed in Table 1-3. The values ​​were calculated from the average of 6 replicated wells. These cell viability values ​​were subsequently used to estimate the IC50 concentrations for each cell line (Table 4). Table 1. Cell viability of A549 vs. concentration of Formula A1 Petition 870250080277, dated 08 / 09 / 2025, pages 30 / 39 16 / 19 Concentration of Formula A1 (μM) Cell Viability of A549 0 100% 20 102% 50 94% 100 100% 150 80% 180 29% 230 63% 253 28% 500 11% Table 2. Cell viability of PANC-1 vs. Formula A1 concentration Concentration of Formula A2 (μM) Cell Viability of PANC-1 0 100% 20 69% 50 69% 100 54% 150 50% 180 16% 230 17% 253 19% 500 22% Table 3. Cell viability of Caco-2 vs. Formula A1 concentration Concentration (μM) of Formula A2 Cell Viability of Caco-2 0 100% 200 91% 300 58% 400 33% Petition 870250080277, dated 08 / 09 / 2025, pages 31 / 39 17 / 19 500 24% Table 4. IC50 of Formula A1 for cell lines A549, PANC-1 and Caco-2 Cell line IC50 (μM) A549 197.34 PANC-1 84.42 Caco-2 324.18 Table 5. Cell viability of A549 vs. 13-cis retinoic acid concentration Concentration of 13-cis RA (μM) Cell Viability of A549 0 100% 7.5 95% 15 78% 30 75% 60 73% 120 57% 240 50% 480 50% 960 51% Absolute IC50: 518.23 μM

[045] Using the A549 cell line for comparison, the IC50 of 13-cis retinoic acid is 518.23 μM; while the IC50 of Formula A1 is 197.34 μM, indicating that Formula A1 is more effective than 13-cis retinoic acid in inhibiting the growth of the A549 cell line. Example 3. Synthesis of the compound with formula A2 (Prophetic Example)

[046] Formula A2 is prepared according to Figure 2.

[047] To form retinoic acid chloride, a solution of (2Z,4E,6E,8E)-3,7—dimethyl—9—(2,6,6—trimethylcyclohex-1-en1-yl)nona-2,4,6,8-tetraenoic acid is added with (COCl)2 and CH2Cl2 in a catalytic amount of dimethylformamide (DMF) under stirring at room temperature. Subsequently, distillation is Petition 870250080277, dated 08 / 09 / 2025, pages 32 / 39 18 / 19 carried out under reduced pressure to remove unreacted (COCl)2 and DMF, in order to obtain retinoic acid chloride (compound 2).

[048] The compound of Formula A2 is synthesized using a protecting group. R)-5-((S)-1,2-dihydroxyethyl)-3,4-dihydroxyfuran2(5H)-one (1,500 mg, 1.14 mmol) is dissolved in a dichloromethane solution (5 ml) and then added with N,N-diisopropylethylamine (DIPEA), followed by the addition of 2-methoxyethoxymethyl chloride (300 mg, 1.32 mmol) (MEMCl). The reaction mixture is stirred at room temperature for 16 hours in a dark environment. After the reaction is complete, compound 4 is formed and its hydroxyl groups at C2 and C3 on the ascorbic acid ring are protected by the protecting groups of 2-methoxyethoxymethyl ether (MEM).

[049] Subsequently, compound 4 is dissolved in 80% ethanoic acid (HOAc) and reacted at 50°C for 4 hours. After the reaction is complete, the solution is concentrated under reduced pressure to remove the ethanoic acid. Compound 5 and the prepared retinoic acid chloride (compound 2) are then dissolved in DMF (5 mL) and added with DCC (172 mg, 1.19 mmol) and DMAP (210 mg, 1.36 mmol) at room temperature and stirred for 4 hours to form compound 6.

[050] Next, the protecting group is removed by treatment with an appropriate deprotecting agent, anhydrous zinc bromide (ZnBr2) in CH2Cl2 or HCl / dioxane at room temperature for 4 hours. After the reaction is complete, the mixture is diluted with water (10 mL) and extracted with EtOAc (10 mL x 3). The combined organic layers are concentrated under reduced pressure to obtain a residue. The residue is purified by prep-HPLC. The residue is then purified to yield the compound of Formula A2.

[051] It should be understood that the above describes preferred embodiments of the present invention and that modifications may be made without departing from the scope of the Petition 870250080277, dated 09 / 08 / 2025, pp. 33 / 39 19 / 19 present invention, as set forth in the claims. Petition 870250080277, dated 09 / 08 / 2025, pp. 34 / 39

Claims

1 / 1 CLAIMS 1. Compound characterized by having the structure of Formula A2 or Al, or a pharmaceutically acceptable salt thereof: Formula Al.

2. Compound according to claim 1, characterized in that it is Formula A2.

3. Compound according to claim 1, characterized in that it has the formula Al.

4. Pharmaceutical composition characterized in that it comprises the compound as defined in claim 1 and a pharmaceutically acceptable carrier thereof.

5. A method for treating cancer, characterized by comprising administering the compound as defined in claim 1 to an individual in need thereof.

6. Method, as defined in claim 3, characterized in that the compound is administered by intravenous administration.

7. Method, as defined in claim 3, characterized in that the cancer is pancreatic cancer, lung cancer, or colorectal cancer. Petition 870250080277, dated 08 / 09 / 2025, pp. 35 / 39