Method for producing an oxathiazine-like compound

Abstract

To provide the use of compounds for preparing pharmaceuticals for treating pancreatic cancer.SOLUTION: The present invention provides the use of a compound 2250 having the following structural formula, wherein the compound 2250 is used in combination with gemcitabine.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to new compounds, methods for producing new compounds, and their use.

Background Art

[0002] Oxathiazine-like compounds are known from Patent Document 1 and Patent Document 2.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0004] In order to provide compounds having stronger antitumor activity and antibacterial activity, less toxicity and side effects, and less resistance to the treatment of tumor cells or bacterial cells, new compounds and methods for producing such compounds are still needed in the art.

Means for Solving the Problems

[0005] According to the present invention, new oxathiazine-like compounds, methods for producing new oxathiazine-like compounds, compounds useful for the production of oxathiazine-like compounds, and their use are disclosed.

Brief Description of the Drawings

[0006]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Mode for Carrying Out the Invention

[0007] According to certain embodiments, the present invention relates to oxathiazine-like compounds, as well as derivatives thereof, and methods for preparing oxathiazine-like compounds and derivatives thereof, and compounds therefor.

[0008] Oxathiazine-like compounds and derivatives thereof according to certain embodiments of the present invention have antitumor activity, antibacterial activity, and / or other activities.

[0009] The method for producing an oxathiazine-like compound and its derivatives according to a specific embodiment of the present invention provides an advantageous method for producing compounds having antitumor activity, antibacterial activity, and / or other activities. In certain embodiments, the oxathiazine-like compounds and their derivatives are particularly useful in the treatment of cancers and tumors in subjects such as human patients. Thus, in certain embodiments, the present invention also relates to the treatment of cancers and tumors using the compounds described herein. For example, central nervous system cancers including glioblastoma, glioma, neuroblastoma, astrocytoma, and leptomeningeal metastatic cancer, colorectal cancer, rectal cancer, and colorectal cancer, ovarian cancer, breast cancer, prostate cancer, lung cancer, mesothelioma, melanoma, kidney cancer, liver cancer, pancreatic cancer, gastric cancer, esophageal cancer, bladder cancer, cervical cancer, gastric cardia cancer, gallbladder cancer, skin cancer, bone cancer, head and neck cancer, leukemia, lymphoma, lymphosarcoma, adenocarcinoma, fibrosarcoma, and their metastases are diseases considered for treatment according to certain embodiments of the present invention. Multidrug-resistant (MDR) tumors, including drug-resistant tumors such as solid tumors, non-solid tumors, and lymphomas, are also useful in certain embodiments using the compounds of the present invention. Currently, it is believed that all tumor cells can also be treated using the methods described herein.

[0010] Tumor stem cells (also called cancer stem cells (CSC)) are thought to be the main drivers of metastasis formation and tumor regrowth after resection.

[0011] In certain embodiments, the compounds of the present invention are particularly useful in the treatment of tumor stem cells in a subject.

[0012] In certain embodiments, the compounds of the present invention are particularly useful in the treatment of glioblastoma tumor stem cells in a subject.

[0013] In certain embodiments, the invention kills or inhibits the growth of tumor cells and / or CSCs by inhibiting oxidative stress, apoptosis and / or the growth of new blood vessels at the tumor site (anti-angiogenesis and anti-tubulogenesis). The main mechanism of action against the death of tumor cells and / or CSCs is oxidative stress. Also, tumor cells and / or CSCs can be killed by apoptosis according to the invention. At lower blood concentrations, the compounds according to the invention are effective in inhibiting the growth of tumor cells by their anti-angiogenic action and their anti-tubulogenic action, and these compounds are therefore useful for palliative treatment.

[0014] The oxathiazine-like compounds of the invention and their derivatives are metabolized much more slowly in the bloodstream than taurolidine and taurolutam. Therefore, lower doses of such compounds can be administered to patients to achieve similar effects.

[0015] Unexpectedly, it has been found that tumor cells respond by initiating a program of apoptotic cell death within minutes of exposure to taurolidine, as follows: 1. The main damage to tumor cells by taurolidine is an increase in reactive oxygen species (ROS), measured quantitatively by fluorescence. 2. The induction of oxidative stress by taurolidine as the first step is supported by the finding that the addition of reducing agents such as glutathione or N-acetylcysteine can prevent the anti-tumor action of taurolidine. 3. The damage caused by high ROS to the mitochondria of tumor cells results in the loss of their membrane potential and the release of apoptosis-inducing factor (AIF). 4. AIF is translocated to the nucleus, initiates the expression of apoptosis-promoting genes, produces blebbing of the plasma membrane as a hallmark of apoptosis, and causes chromatin condensation and DNA fragmentation. 5. In contrast to normal cells, tumor cells are very sensitive to oxidative stress. This explains the action of taurolidine on a wide range of tumor cells that does not damage normal cells.

[0016] Also, in certain embodiments, the compounds of the present invention are useful for the treatment of microbial infections in subjects such as human patients. Microbial infections that can be treated according to certain embodiments include bacterial infections, fungal infections, and / or viral infections.

[0017] Cancer patients tend to be susceptible to infections, and are particularly prone to microbial infections, especially during and / or after surgery.

[0018] In certain embodiments, the compounds of the present invention are utilized for treating a glioblastoma in a subject.

[0019] In certain embodiments, the compounds of the present invention are utilized for treating an S. aureus (Staphylococcus aureus) infection in a subject.

[0020] In certain embodiments, the compounds of the present invention are utilized according to the present invention for treating MRSA in a subject.

[0021] In certain embodiments, the compounds of the present invention are utilized according to the present invention for treating E. coli (Escherichia coli) in a subject.

[0022] In certain embodiments, the compounds of the present invention are utilized according to the present invention for treating H. pylori in a subject and / or for treating cancers (if any) associated with H. pylori in a subject.

[0023] In certain embodiments, the compounds of the present invention are utilized according to the present invention for treating HIV in a subject.

[0024] In certain embodiments, Formula I: [Chemical formula] (In the formula, R is H, alkyl, etc., for example, methyl, ethyl, propyl (e.g., isopropyl), benzyl, etc.), and the compound is used according to the present invention.

[0025] In certain embodiments, the new compound 2250 (tetrahydro-1,4,5-oxathiazine-4-dioxide or 1,4,5-oxathiazane-4-dioxide) is prepared and / or utilized according to the present invention. The FTIR spectrum of the compound 2250 prepared according to the present invention is shown in FIG. 8.

[0026] In certain embodiments, the new compound 2245 is prepared and / or utilized according to the present invention.

[0027] Compound 2250 prevents and treats gastric tumors, including tumors caused by or associated with H. pylori, or tumors as a result of metastasis to the stomach.

[0028] The amount of the compound required depends on the tumor size. In one embodiment, the present invention includes surgically reducing the tumor size and treating with one or more of the above compounds. The above compounds may be administered before, during, or after surgery to reduce the tumor. The compounds according to the present invention may be administered by any suitable method including, but not limited to, gels, capsules, tablets, intravenous injection (IV), intraperitoneal injection (IP), and / or directly to the tumor.

[0029] The gel may contain, for example, 2% - 4% (e.g., 3%) of the active compound of the present invention such as compound 2250 alone, or may be combined with and contain taurolidine / tauroglutamam for administration or presence alone, and may also be for topical administration. Such gels can be used to treat skin and oral tumors including squamous cell carcinomas of the mouth and skin. Also, such gels can be used to treat cervical cancer or cervical dysplasia by administering them as suppositories to the vagina or by syringe. The present invention may include combinations of suppositories retaining the active compound.

[0030] Examples of solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the composition provided is mixed with at least one inert pharmaceutically acceptable excipient and / or filler or bulking agent (such as starch, lactose, sucrose, glucose, mannitol, and silicic acid), binder (such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and acacia), humectant (such as glycerol), disintegrant (such as agar, calcium carbonate, potato starch, tapioca starch, alginic acid, certain silicates, and sodium carbonate), dissolution retardant (such as paraffin), absorption enhancer (such as quaternary ammonium compounds), wetting agent (such as cetyl alcohol and glycerol monostearate), absorbent (such as kaolin and bentonite clay), and lubricant (such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate), and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may contain a buffering agent.

[0031] The compounds of the present disclosure, particularly compound 2250, have been found to be very soluble in water. In certain embodiments, PVP is not necessary to increase solubility. For example, a 3.2% solution of 2250 is isotonic. This is an unexpected advantage over taurolidine.

[0032] The compounds of the present invention, such as compound 2250 (regardless of the presence or absence of taurolidine and / or taurultam), are particularly useful in surgical oncology because the compounds do not interfere with wound healing. Since other such antitumor drugs interfere with wound healing and promote suture dehiscence, the administration of other antitumor drugs must be delayed for at least five weeks after surgery. Such problems can be avoided by the compounds of the present invention, such as compound 2250, which can be administered during and immediately after surgery without the problems of wound healing or suture dehiscence.

[0033] Solid compositions of the same type may also be employed as fillers for soft gelatin capsules and / or hard filled gelatin capsules using excipients such as lactose, i.e., milk sugar, similar to high molecular weight polyethylene glycol, etc. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coating agents and shells such as enteric coating agents and other coating agents well known in pharmaceutical formulation technology. The above coating agents may optionally contain an opacifying agent and may be compositions that release only the composition(s) that are optionally provided with a delay at a specific part of the intestinal tract or compositions that preferentially release the composition. Examples of embeddable compositions include polymeric substances and waxes. Solid compositions of the same type may also be employed as fillers for soft gelatin capsules and hard filled gelatin capsules using excipients such as lactose, i.e., milk sugar, similar to high molecular weight polyethylene glycol, etc.

[0034] In certain embodiments, the capsule may comprise an excipient formulation comprising one or more of hydroxypropyl methylcellulose (HPMC), gelatin, and fish gelatin. In certain embodiments, the capsule may contain Compound 2250 together with tauroursodeoxycholic acid and / or taurine. The capsule may further contain one or more of lycopene, ellagic acid (polyphenol), curcumin, piperine, delphinidin, resveratrol, isothiocyanates such as sulforaphane, capsaicin, and piperlonguminine.

[0035] The active compounds of the present invention, such as Compound 2250, may be combined with compounds such as gemcitabine. This combination can be used for treating cancers such as pancreatic cancer. Also, tauroursodeoxycholic acid and / or taurine may be combined with gemcitabine, for example, for treating pancreatic cancer.

[0036] In certain embodiments, the nutritional cancer prevention and treatment product may contain 100 mg to 500 mg of compound 2250 alone, or 100 mg to 500 mg of taurolidine and / or taurolutam, and also one or more lycopene, such as 20 mg to 200 mg of ellagic acid (polyphenol), curcumin, piperine (20 mg to 200 mg), delphinidin, resveratrol, isothiocyanate (sulforaphane, capsaicin, and piperlonguminine) in combination.

[0037] Unexpectedly, since the above compound does not inhibit wound healing like other chemotherapeutic agents, it has been found that there may be a possibility of administering the above compound during and immediately after surgery.

[0038] Unexpectedly, it has been found that taurolidine, taurolutam, and oxathiazine-like compounds and their derivatives kill tumor stem cells, which is very rare among chemotherapeutic agents and is probably unknown. Typical chemotherapeutic agents, when effective against tumor stem cells, are generally only effective at very high doses that are extremely toxic to human patients.

[0039] Unexpectedly, it has been found that lower doses of taurolidine and / or taurolutam than required to kill tumor cells killed tumor stem cells.

[0040] Unexpectedly, it has been found that oxathiazine-like compounds and their derivatives have a half-life in human blood that is significantly longer than that of taurolidine and taurolutam. Therefore, these compounds are not removed from the patient's bloodstream so rapidly, and as a result, effectively delay the loss of drug efficacy caused by the body's clearance mechanism.

[0041] Unexpectedly, it has been found that when certain oxathiazine-like compounds and their derivatives are applied directly to tissues, they reduce the burning sensation, which is different from what was observed in patients treated with taurolidine.

[0042] Unexpectedly, oxathiazine-like compounds and their derivatives have been found to have a particularly advantageous combination of properties, including high water solubility, a wide range of administration routes including oral and intravenous injection (i.v.), extended stability and half-life, and a reduction in the side effect of burning sensation.

[0043] Thus, the half-life of Compound 2250 is greater than 24 hours in human blood, significantly longer than the half-life of taurolidine, which was found to be approximately 30 minutes using the same test.

[0044] In one embodiment, the invention includes treating a patient by administering Compound 2250 to the patient, which results in a baseline blood concentration of Compound 2250 within about 5 minutes of administration. The method includes maintaining the blood concentration of Compound 2250 in the patient at about 80% of the baseline blood concentration for about 20 hours.

[0045] In one embodiment, the invention includes maintaining the blood concentration of an anti-tumor compound in a patient at about 80% of the patient's baseline blood concentration for about 20 hours by administering a daily dose of Compound 2250 once daily to maintain the blood concentration at 80% of the baseline blood concentration.

[0046] The daily dose may be from about 0.1 g to about 100 g, such as from about 5 g to about 30 g. The daily dose may be administered in the form of an orally administrable composition. The daily dose may be administered in the form of capsules, tablets, or a pharmaceutically acceptable solution. The daily dose may be administered in a form containing Compound 2250 at a concentration of from about 0.01% weight / volume to about 3% weight / volume. The daily dose may be administered in a form containing Compound 2250 at a concentration of from about 0.01 μg / ml to about 1000 μg / ml. The daily dose may be administered in a form containing one or more solubilizing agents, such as polyhydric alcohols.

[0047] In certain embodiments, the above compound is administered in the composition at a concentration of about 0.01 μg / ml to about 1000 μg / ml. In certain embodiments, the above compound is administered in the composition at a concentration of about 1 μg / ml to about 100 μg / ml. In certain embodiments, the above compound is administered in the composition at a concentration of about 10 μg / ml to about 50 μg / ml. Also, the above composition may contain taurolidine and / or taurolutam at about 0.01 μg / ml to about 1000 μg / ml, about 1 μg / ml to about 100 μg / ml, or about 10 μg / ml to about 50 μg / ml.

[0048] In certain embodiments, the above compound is administered in the composition at a concentration of about 0.01% to about 3%. In certain embodiments, the above compound is administered in the composition at a concentration of about 0.1% to about 2.5%. In certain embodiments, the above compound is administered in the composition at a concentration of about 1% to about 2%. The above composition may further contain taurolidine and / or taurolutam at about 0.01% to about 3%, about 0.1% to about 2.5%, or about 1% to about 2%.

[0049] In one embodiment, the oxathiazine-like compound and its derivatives may be administered as a combination therapy using taurolidine and / or taurolutam to kill tumor stem cells. According to such an embodiment, unexpectedly, it has been found that the combination therapy requires a lower dose of the drug than that required to kill normal tumor cells in order to kill tumor stem cells.

[0050] In certain specific embodiments, the oxathiazine-like compound and its derivatives may be administered together with vitamin D3, which brings about an increase in the anti-tumor effect of the compound.

[0051] In one embodiment, the above compound is administered to a subject at a total daily dose of about 0.1 g to about 100 g, about 1 g to about 80 g, about 2 g to about 50 g, or about 5 g to about 30 g.

[0052] The effective amount of the above compound is in the range of about 0.1 mg / kg to 1000 mg / kg, preferably 150 mg / kg to 450 mg / kg per day, and most preferably 300 mg / kg to 450 mg / kg per day as the dosing unit.

[0053] As used herein, the term "pure" refers to a substance that is at least about 80% pure with respect to impurities and contaminants. In certain embodiments, the term "pure" refers to a substance that is at least about 90% pure with respect to impurities and contaminants. In certain specific embodiments, the term "pure" refers to a substance that is at least about 95% pure with respect to impurities and contaminants. In certain embodiments, the term "pure" refers to a substance that is at least about 99% pure with respect to impurities and contaminants. In certain embodiments, the term "pure" refers to a substance that is at least about 99.5% pure with respect to impurities and contaminants.

[0054] In certain specific embodiments, the compounds, compositions and methods of the present invention include the use of micronized compounds. In certain embodiments, the term "micronized" as used herein refers to a particle size in the range of about 0.005 microns to 100 microns. In certain specific embodiments, the term "micronized" as used herein refers to a particle size in the range of about 0.5 microns to 50 microns. In certain specific embodiments, the term "micronized" as used herein refers to a particle size in the range of about 1 micron to 25 microns. For example, the size of the drug particles may be about 1 micron, 5 microns, 10 microns, 15 microns, 20 microns, or 25 microns.

[0055] In certain embodiments, the compounds, compositions and methods of the invention include the use of nanoparticles. As used herein, the term "nanoparticle" refers to any particle having a diameter less than 1000 nanometers (nm). In certain embodiments, the nanoparticles have a diameter of less than 300 nm. In certain embodiments, the nanoparticles have a diameter of less than 100 nm. In certain embodiments, the nanoparticles have a diameter of less than 50 nm, for example, from about 1 nm to 50 nm. Formulations suitable for injection or infusion may include an isotonic solution containing one or more solubilizing agents, such as a polyhydric alcohol such as glucose, to provide a solution with increased compound concentration. Such solutions are described in European Patent No. 253662. The above solutions may be made isotonic with Ringer's solution or Ringer's lactate solution. The concentration of the compound in such solutions may range from 1 g / liter to 60 g / liter.

[0056] In certain embodiments, exemplary compounds and methods for making the compounds of the invention include the following.

Chemical formula

[0057] The above compounds may be in crystalline form, for example, after crystallization and / or after recrystallization in an alcohol, ketone, ester or combination thereof. For example, the compounds of the invention may be crystallized and / or recrystallized from an alcohol such as ethanol.

[0058] Exemplary compounds of the invention include the following.

Chemical formula

[0059] When used in the form of nanoparticles, the compounds of the invention have been found to achieve higher blood levels. In one embodiment, the invention includes Compound 2250 alone or in combination with taurolidine and / or taurolutam. For example, the invention includes nanoparticles of the compounds of the invention encapsulated in capsules.

[0060] In certain embodiments, the present invention also relates to derivatives of the above compounds having the activities described herein of the above compounds, for example, at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% or more of the above activities.

[0061] In certain embodiments, the present invention also relates to compositions comprising the compounds described herein, compositions comprising pharmaceutically acceptable solutions of the above compounds, and orally administrable compositions such as capsules and tablets comprising the above compositions.

[0062] In certain embodiments, the compounds of the present invention can be administered to a subject or patient locally or systemically by any suitable means, for example, in solution, for example, by intravenous drip.

Chemical formula

[0063] Starting materials: Isethionic acid, Especially Carbylsulfat, taurine, taurine amide, Cysteine, isethionic acid

[0064] Synthesis 1 I. a. Isethionic acid via Carbylsulfat

Chemical formula

[0065] b. Isethionic acid via taurine Biosynthesis via cysteine and taurine

Chemical formula

[0066] Chemical synthesis Ethylene oxide using bisulfite

[0067] II. Isethionic Amide HO-CH 2 -CH 2 -SO 2 -NH 2 a.

Chem.

[0068] b. Carbamyl Sulfate + NH 3

Chem.

[0069] Alternative chemical synthesis process for 2250 a) Sulfamic acid

Chem.

[0070] b) Paraformaldehyde, hexamethylenetetramine (hexamine, formin, urotropin)

[0071] c)

Chem.

[0072] d)

Chem.

[0073] e)

Chem.

[0074] f)

Chem.

[0075] g)

Chem.

[0076] h)

Chem.

[0077] Some alternative synthesis steps for 2250 and 2255 I. Starting materials 2250 / 2255 a.

Chem.

[0078] b. Carbamyl sulfate + H 2 O

Chem.

[0079] II. Reaction of amine with carbamyl sulfate

Chem.

[0080] III.

Chem.

[0081] Exemplary synthesis protocol I. Synthesis of 2244

Chem.

[0082] 2.15 g of pure 1907 was dissolved in 100 ml of ethyl acetate and catalyzed using 0.5 g of palladium / activated carbon. The solution was hydrogenated at room temperature and atmospheric pressure. The hydrogenation was completed after about 15 hours, and the hydrogen uptake was 450 ml.

[0083] After each hydrogenation, the reaction mixture was evacuated three times with nitrogen and then filtered through a filter aid (diatomaceous earth). The colorless and transparent ethyl acetate solution was concentrated to dryness on a rotary evaporator. Yield: 1.25 g, inoculated with crystallized 2244. Melting point: 42 °C - 44 °C IR: Corresponding to 2244, 99.3% pure.

[0084] II. Synthesis of 2244

Chemical formula

[0085] 5 g (0.023 mol) of 2264 / 1907 was boiled in 50 ml of concentrated HCl under reflux for 3 hours, then cooled to room temperature and separated in a separatory funnel with 30 ml of dichloromethane. The aqueous phase was evaporated and dried on a rotary evaporator. A yellow oil remained and slowly crystallized after inoculation with 2244 crystals. IR corresponds to substance 2244. Recrystallized from ethyl acetate. 0.7 g was obtained (24%). Melting point: 44 °C - 45 °C IR corresponds to the reference substance.

[0086] III. Synthesis of 2244

Chemical formula

[0087] 230 mg of 2269 was dissolved in 2 ml of NaOH (1N), and refluxed for 15 minutes using a reflux condenser. The clear solution was cooled to 20 °C and acidified with hydrochloric acid. The resulting precipitate was filtered under vacuum and dried. Yield: 110 mg Melting point: 114 °C - 116 °C. IR showed 99% benzoic acid as a by-product.

[0088] The acidic solution was concentrated to dryness on a rotary evaporator, and the solid was boiled with acetic ester. The ethyl acetate solution was filtered and concentrated to dryness under vacuum. Weight: 110 mg. The oil was contaminated with oil, and the IR peak for 2244 (isethionic acid amide) was unclear. 110 mg was recrystallized from acetic ester. Yield: 65 mg, Melting point: 43 °C - 45 °C IR corresponded to 52% of 2244.

[0089] IV. Synthesis of 2244

Chemical formula

[0090] 1.15 g of 2269 was dissolved in 10 ml of NaOH (1N), and refluxed for 15 minutes. The clear solution was cooled to 20 °C and acidified with hydrochloric acid. The resulting precipitate was filtered under vacuum and dried. Yield: 0.5 mg Melting point: 114 °C - 116 °C IR showed 82% benzoic acid by-product as a reference substance. Hydrolysis was not complete.

[0091] The acidic solution was concentrated to dryness on a rotary evaporator, and the solid was boiled with acetic ester. The ethyl acetate solution was filtered and concentrated to dryness under vacuum. Weight: 0.8 g. The oil was contaminated with oil, and the IR peak for 2244 (isethionic acid amide) was unclear. 0.8 g was recrystallized from acetic ester. Yield: 160 mg, Melting point: 43 °C - 45 °C IR corresponded to 26% at 2244.

[0092] V. Synthesis of 2244

Chemical formula

[0093] 215 g of 0.1 mol of 2264 and 1000 ml of concentrated hydrochloric acid (about 36%) were boiled together under reflux for 30 minutes. 2264 dissolved and an oil layer was present. The reaction mixture was cooled and transferred to a separating funnel where the oil was separated from the aqueous phase. The acidic aqueous solution that dissolved isethionic acid amide (2244) was concentrated at 50 °C using a rotary evaporator until it was almost dry. The yellow oily residue was placed in the refrigerator overnight and 32.3 g of transparent crystals were filtered under vacuum. Melting point 43 °C - 45 °C. IR: In oxygen, at the following wave numbers as shown in Figure 7, 655.82 cm -1 , 729.12 cm -1 , 844.85 cm -1 , 898.86 cm -1 , 947.08 cm -1 , 1003.02 cm -1 , 1060.88 cm -1 , 1134.18 cm -1 , 1236.41 cm -1 , 1288.49 cm -1 , 1317.43 cm -1 , 1408.08 cm -1 , 1572.04 cm -1 , 3105.5 cm -1 , 3209.66 cm -1 , 3313.82 cm -1 , and has peaks at 3427.62 cm -1 .

[0094] The mother liquor was concentrated until it was completely dry.

[0095] VI. Synthesis of 2244

Chem.

[0096] 21.5 g of 0.1 mol 2264 and 100 ml of concentrated hydrochloric acid (about 36%) were boiled together under reflux for 30 minutes. An oil layer was formed, and the reaction mixture was cooled in a separatory funnel, where the oil was separated from the aqueous phase. The acidic aqueous solution in which amide isethionate (2244) was dissolved was shaken twice with methylene chloride, the methylene chloride was separated, and the acidic aqueous solution was concentrated to dryness on a rotary evaporator at 50 °C. The yellow oily residue was placed in a refrigerator overnight to obtain 12.3 g of oil. Melting point: 41 °C - 43 °C. Analysis of the product showed that it corresponded to 99.8% of 2244 by IR.

[0097] Distillation experiment: 12.3 g was distilled under high vacuum. External temperature Internal temperature Vacuum 190 °C - 210 °C 183 °C - 186 °C 0.1 mm Weight: 9.3 g of oil that was solid at room temperature. Melting point: 43 °C - 45 °C.

[0098] VII. Synthesis of 2244

Chem.

[0099] 2.0 g of pure compound 1907 was dissolved in 200 ml of acetate ester, 0.5 g of palladium / activated carbon was added, and the mixture was autoclaved at 100 °C and hydrogenated at 50 °C. After a running time of 6 hours, the reaction mixture was cooled overnight and then filtered and concentrated to dryness under vacuum.

[0100] Weight: 1.7 g of oil - CH 2 Cl 2 After the addition and shaking of, it was left standing, - and then suction filtered to obtain a crystalline solid with a weight of 0.6 g and a melting point of about 40 °C. For analysis, 0.2 g of acetate ester was added twice for crystallization. Melting point 43 °C - 44 °C.

[0101] VIII. Synthesis of 2244 [Chemical formula]

[0102] After dissolving 2.15 g of pure 1907 in 100 ml of acetic acid - ethyl ester, it was added to 0.5 g of palladium / activated carbon. Then, the mixture was hydrogenated at room temperature and atmospheric pressure. The hydrogenation was completed after approximately 15 hours. The hydrogen absorption amount was approximately 450 ml. Then, the hydrogen was evacuated and flushed with nitrogen three times, and each reaction mixture was filtered through diatomaceous earth (celite). Ethyl acetate, which is a colorless and transparent solution, was evaporated to dryness using a rotary evaporator.

[0103] Weight: 1.25 g of oil crystallized after inoculating with crystals of 2244. Melting point: 42 °C - 44 °C IR: Corresponds to 99.3% of 2244.

[0104] IX. Synthesis of 2250 [Chemical formula]

[0105] 1.2 g of pure 2245 was dissolved in 150 ml of acetic acid that completely dissolves at 60 °C. 0.3 g of palladium / activated carbon was added, stirred at 75 °C, and the mixture was hydrogenated at atmospheric pressure.

[0106] The hydrogenation was stopped after 7 days. The hydrogen adsorption amount was approximately 480 ml.

[0107] The hydrogen was evacuated and purged with nitrogen three times. Then, the reaction mixture was filtered through a filter aid (diatomaceous earth) at 70 °C. The clear and warm glacial acetic acid solution was cooled to room temperature, and white crystals were suction - filtered. Weight: 0.74 g, Melting point: 225 °C - 227 °C IR: 2245 corresponds to the starting material.

[0108] The mother liquor was concentrated to dryness using a rotary evaporator. 0.38 g of impure material was extracted with ethyl acetate. The solution was concentrated.

[0109] Ethyl acetate soluble portion: a semi-solid substance obtained by sublimation; 0.15 g of semi-solid substance obtained by recrystallization from a few drops of water Yield: 70 mg, melting point: 95 °C - 98 °C IR corresponded to 98% at 2250.

[0110] X. One-step synthesis of sodium 2-benzyl ether ethanesulfonate in high yield

Chemical formula

[0111] 10.5 g of sodium 2-bromoethanesulfonate was added to a solution of 110 ml of benzyl alcohol and 1.15 g of sodium benzyl oxide.

[0112] Subsequently, the mixture was boiled 4 times under reflux. Then, the mixture was concentrated to dryness under vacuum and then boiled 3 times with ethyl alcohol. The alcohol was filtered off and concentrated to dryness.

[0113] The yield was 9.8 g, confirmed by UV and IR.

[0114] The sodium 2-benzyl ether ethanesulfonate in the obtained ethyl alcohol was boiled, filtered, and then the solution was cooled to crystallize pure sodium 2-benzyl ether ethanesulfonate crystals from the solution to obtain pure crystals.

[0115] XI. Synthesis of 2250

Chemical formula

[0116] 6.3 g of vinylsulfonamide (derived from 2258), 50 ml of concentrated formic acid, and 1.1 g of paraformaldehyde were combined with reflux for 2 hours to produce Compound 2250. Subsequently, the clear acidic solution was concentrated to dryness using a rotary evaporator.

[0117] The residue is 5.9 g of a pale yellow honey-like syrup. IR: Mixture of vinylsulfonamide and 2250 2 grams were sublimated to obtain a small amount of crystals. Sublimation of the semi-solid substance: IR: Corresponding to 98% of 2250.

[0118] XII. Synthesis of Vinylsulfonamide

Chemical formula

[0119] Formylisethionate chloride was placed in 50 ml of chloroform, transferred to a 350 ml sulfonation flask, and cooled to -10°C. Subsequently, 25% ammonia gas was introduced. After the introduction of ammonia gas, the chloroform / NH 3 was found to weigh 5 g. The above mixture was slowly stirred from -3°C to 2°C.

[0120] 20 ml of chloroform was added dropwise to 9.0 g of distilled 2249. NH 4 Cl immediately precipitated. Subsequently, ammonium chloride was filtered under vacuum, and the clear chloroform solution was concentrated using a rotary evaporator until dry. Yield: 6.3 g of a clear, thin oil. IR: 96% of CH 2 =CH-SO 2 -NH 2 (corresponding to vinylsulfonamide).

[0121] XIII. Synthesis of 2261

Chemical formula

[0122] 300 g (1.26 mol) of 2260 was weighed into a 750 ml multi-necked flask equipped with a KPG stirrer. 415 ml of trichloroethylene + phosphorus oxychloride (density corresponding to about 1.47), as well as 150 ml of phosphorus oxychloride and 5.7 ml of DMF were warmed to 105 °C while stirring. The mixture was reacted for 5 hours. 3 The solid was filtered off under vacuum, and the liquid was distilled under a water pump vacuum. The filter cake was washed with ethyl acetate. After distilling off trichloroethylene and phosphorus oxychloride, the wash-acetate was transferred to the flask and distilled again.

[0123] 250 g (1.07 mol - 85%) of a yellow liquid was collected. IR corresponded to 2261.

[0124] XIV. Synthesis of 2250 and 2255:

[0125]

Chemical formula

[0126] XV. New synthetic scheme for compound 2250 and related compounds: Starting materials:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0127] Compound (tetrahydro-oxathiazine-dioxide):

Chemical formula

[0128] Chemical intermediate Protecting group: Benzyl chloride

Chem.

[0129] Protecting group: Benzyl chloroformate

Chem.

[0130] XVI. Synthesis of precursor compounds

Chem.

[0131] Synthesis: 83.9 g of sodium vinylsulfonate was added to a solution of 400 ml of benzyl alcohol, and 0.5 g of sodium (catalytic amount) was added. The mixture was warmed to 150 °C with stirring until most of the sodium vinylsulfonate had dissolved in the solution. After 3 hours, the above mixture was cooled overnight and a thick solid crystallized. This solid was vacuum filtered, then suspended in ethyl alcohol and vacuum filtered to dryness. Yield: 94.0 g, IR: corresponding to the desired compound (61.2% pure).

[0132] XVII. Synthesis of 1905

Chem.

[0133] 60 grams of sodium vinyl sulfonate was added to a solution of 1000 ml of benzyl alcohol and 0.5 g of sodium. Then, the entire mixture was stirred and heated under reflux. After approximately 3 hours, the excess benzyl alcohol was removed by distillation under vacuum, and the residue was boiled with alcohol. The alcohol solution was filtered, concentrated, and crystallized to about 1 / 2. 37.3 g of a yellow cotton-like substance was obtained.

[0134] Also, the above procedure was repeated using 250 g of sodium vinyl sulfonate and 2 liters of benzyl alcohol, treated as described above, and about 208 g was crystallized.

[0135] Also, the above procedure was repeated using 100 g of sodium vinyl sulfonate and 1 liter of benzyl alcohol, treated as described above, and about 105 g was crystallized.

[0136] Also, the above procedure was repeated using 200 g of sodium vinyl sulfonate, treated as described above, and about 130 g was crystallized.

[0137] Synthesis of XVIII.1906

Chemical formula

[0138] 6.7 g of 1905 (recrystallized) was added to 50 ml of thionyl chloride and 1 ml of dimethylformamide. The sodium salt immediately dissolved, the mixture was heated to 40 °C - 50 °C, left standing overnight at 20 °C, and evacuated until concentrated (vacuumed). Yield: 9.8 g, which was added to 50 ml of 2N NaOH and stirred well. The NaOH solution was washed with CHCl 3 and then shaken with concentrated HCl to precipitate, captured with Na 2 SO 4 and then dried and distilled.

[0139] The above process was repeated using 208 g of 1905 mixed with 1000 ml of thionyl chloride and 10 ml of dimethylformamide. The above mixture was refluxed and the excess thionyl chloride was removed by distillation until dry. The yield was 250 g and it was treated as above.

[0140] XIX. Synthesis of 1907

Chemical formula

[0141] 9.8 g of 1906 was dissolved in chloroform (CHCl 3 )(turbid), concentrated in a part of 150 ml of concentrated ammonia water, and stirred. Stirring was continued for 3 hours while heating to 40 °C to 50 °C. Then, the mixture was dried and concentrated under vacuum. Yield: 3.1 g of a blackish oil

[0142] 3.1 g of the blackish oil was added to 50 ml of 2N NaOH and stirred well. The NaOH solution was washed with CHCl 3 , then shaken with concentrated HCl to precipitate, captured with Na 2 SO 4 , dried, and distilled. Yield: 2.5 g of an oil

[0143] For analysis, 0.5 g of the sample was concentrated at a temperature of 160 °C to a solid and crystallized three times from ethyl acetate / benzene. Melting point: 75 °C to 76 °C Molecular formula: C 9 H 13 NO 3 S MW: 215.2 Calculated value: C = 50.23%, H = 6.09%, N = 6.51%, S = 14.86% Measured value: C = 50.14%, H = 6.15%, N = 6.35%, S = 14.79%

[0144] XX. Synthesis of 1908

Chemical formula

[0145] 1.2 g of 1907 was dissolved in 200 ml of ethyl acetate, and 0.4 g of Pd activated carbon was added. The mixture was hydrogenated in a hydrogenated autoclave at 100 °C and 50 °C for 4 hours. The mixture was left under pressure at room temperature over the weekend. Then, the ethyl acetate solution was filtered and dried under vacuum. Yield: 1.1 g of oil.

[0146] Synthesis of XXI.1908

Chemical Structure

[0147] 2 grams of 1907 was dissolved in 200 ml of ethyl acetate, and 0.5 g of Pd / palladium / activated carbon was added. The mixture was hydrogenated in a high-pressure autoclave at 100 °C and 50 °C. After 6 hours, the reaction mixture was cooled overnight, then filtered and distilled under vacuum until dry to obtain the residual oil. Yield: 1.7 g of oil. CH 2 Cl 2 was added, stirred, allowed to stand for crystallization, and separated by suction under vacuum. Weight: 0.6 g, melting point about 40 °C. Analysis: 0.2 g was recrystallized twice from ethyl acetate. Melting point: 43 °C - 44 °C Molecular formula: C 2 H 7 NO 3 S MW: 125 Calculated values: C = 19.22%, H = 5.65%, N = 11.21%, S = 25.65% Measured values: C = 19.20%, H = 5.67%, N = 11.07%, S = 25.73%

[0148] Synthesis of XXII.1909 19.9 grams of 1906 was dissolved in 100 ml of chloroform and added to a solution of 23 grams of pure benzylamine and 200 ml of pure chloroform. Immediately, benzylamine hydrochloride precipitated and the reaction mixture warmed up. Then, the mixture was refluxed and the hydrochloride compound was separated by suction to obtain a clear CHCl 3 The mother liquor was placed under vacuum for drying.

[0149] Yield: 27 g of a yellowish transparent oil that slowly solidified.

[0150] 27 g of it was dissolved in about 20 ml of ethyl acetate and N-hexane (an appropriate amount) was added until the solution was almost turbid. The mixture was left overnight while cooling, and crystallization occurred. Yield: 9.2 g, Melting point: 50 °C - 53 °C

[0151] For analysis, 1 g in n-hexane was recrystallized three times. Melting point 56 °C - 57 °C.

[0152] Synthesis of XXIII.2260

Chemical formula

[0153] 0.675 mol of sodium isethionate salt (100.0 g) and 2.02 mol of benzyl chloride (233 mL) were mixed in a 750 mL multi-necked flask equipped with a KPG stirrer. The above mixture was heated at an internal temperature of 70 °C (external temperature of 95 °C), and then triethylamine (120 mL) was added dropwise over 1 hour to raise the external temperature to 125 °C and maintain it. Then, the external temperature rose to 140 °C and the internal temperature rose to 130 °C. Solids accumulated on the stirrer but returned to the suspension. Vapors of hydrochloric acid were generated.

[0154] After adding 30 mL of triethylamine dropwise, the reaction was allowed to proceed for an additional 1.5 hours. A sticky yellowish suspension was formed. After cooling the product to an internal temperature of 50 °C, 300 mL of water was added and the mixture was vigorously stirred for 20 minutes and transferred to a 2 L separatory funnel. The flask was then rinsed with 100 mL of water.

[0155] The combined aqueous phase was washed twice with 280 mL of dichloromethane.

[0156] The aqueous phase was maintained at 40 °C while adding KCl (about 130 g of KCl) until the solution was saturated. The mixture was filtered through pleated filter paper and stored in the refrigerator overnight.

[0157] The remaining solid was extracted and dried to obtain 30.85 g, with a yield of 17.9%. IR: An OH band similar to that of the precursor is present.

[0158] The mother liquor was treated again with KCl and stored in the refrigerator (35 °C - 40 °C) overnight.

[0159] The solid resulting from the second precipitation with KCl was filtered and dried, yielding 60.0 g = 34.9%, and the IR corresponds to the desired product.

[0160] Solid 1: Boiled with 150 mL of EtOH and filtered while hot.

[0161] By repeating the precipitation, boiling, and crystallization with KCl, 32 g of the product was obtained in a 19% yield.

[0162] XXIV. Synthesis of 2256

Chemical formula

[0163] 40 g of taurine amide hydrochloride, 18 g of sodium nitrite, and 300 ml of distilled water were boiled together under reflux until no more gas was generated. The resulting clear yellow solution was then cooled to 50 °C.

[0164] 30 ml of 1N NaOH was added to 10.5 g of acetaldehyde. The clear yellow solution was left under vacuum over the weekend to dry. The result was a rust-colored honey-like residue weighing 37.6 g, which was extracted with ethyl alcohol. The alcohol solution was filtered and concentrated to dryness using a rotary evaporator. The resulting thick oily residue was dissolved in ethyl acetate. The ethyl acetate solution was filtered and concentrated.

[0165] This yielded 30.7 g of a rust-colored, dense oil. White crystals were isolated from the dense oil. The melting point was approximately 114 °C to 116 °C.

[0166] The IR spectrum confirmed that the resulting compound had the structure of compound 2256.

Chemical formula

[0167] In certain embodiments, a sublimation apparatus equipped with laboratory glassware known in the art may be used in the sublimation technique for purifying the compounds according to the present invention. In certain embodiments, the sublimation vessel is heated under vacuum and under reduced pressure. The compound volatilizes, leaving non-volatile residual impurities, and condenses as the purified compound on a cooled surface. This cooled surface often takes the form of a cold finger. After heating is complete and the vacuum is released, the sublimed compound can be collected from the cooled surface.

[0168] In one embodiment, substituted derivatives of compound 2250 may be prepared. Examples of substituted derivatives of compound 2250 include the following.

Chemical formula

[0169] In the above formula, R can be H, alkyl, or aryl. In certain embodiments, R is C 1 ~C6 It is alkyl. In certain embodiments, R is methyl.

[0170] In certain embodiments, the derivative of compound 2250 is prepared according to the following reaction mechanism.

Chemical formula

[0171] In one embodiment, the present disclosure includes a method of killing tumor stem cells by administering to a subject in need thereof an effective amount of taurolidine, taurolutam, or a mixture thereof. The effective amount of taurolidine and / or taurolutam for killing tumor stem cells is less than the amount of taurolidine and / or taurolutam required to kill tumor cells.

[0172] In certain embodiments, taurolidine, taurolutam, or a mixture thereof is administered in a tumor stem cell killing composition at a concentration of about 0.01 μg / ml to about 500 μg / ml. In certain embodiments, taurolidine, taurolutam, or a mixture thereof is administered in a tumor stem cell killing composition at a concentration of about 0.1 μg / ml to about 100 μg / ml. In certain embodiments, taurolidine, taurolutam, or a mixture thereof is administered in a tumor stem cell killing effective composition at a concentration of about 10 μg / ml to about 50 μg / ml. Taurolidine is effective for killing tumor stem cells in in vitro tissue culture at 0.01 μg / ml.

[0173] In certain embodiments, taurolidine, taurolutam, or a mixture thereof is administered in a tumor stem cell killing composition at a concentration of about 0.001% to about 2%. In certain embodiments, taurolidine, taurolutam, or a mixture thereof is administered in a tumor stem cell killing composition at a concentration of about 0.01% to about 1.5%. In certain embodiments, taurolidine, taurolutam, or a mixture thereof is administered in a tumor stem cell killing effective composition at a concentration of about 0.1% to about 1%.

[0174] In one embodiment, taurine lysine, taurine lutam, or a mixture thereof is administered to a subject in need thereof at a total daily dose of about 0.01 g to about 50 g, about 0.1 g to about 30 g, about 0.5 g to about 10 g, or about 1 g to about 5 g for tumor stem cell killing.

[0175] The effective dose for tumor stem cell killing of taurine lysine, taurine lutam, or a mixture thereof is in the range of about 0.01 mg / kg to 500 mg / kg, preferably 1 mg / kg to 100 mg / kg per day, and most preferably 5 mg / kg to 50 mg / kg per day in terms of the dosing unit.

[0176] In another embodiment, the present disclosure may be used in combination with taurine lysine and / or taurine lutam, the following compounds:

Chemical formula

Chemical formula

Examples

[0177] Example 1: Antitumor activity of compound 2250 Introduction Based on the recognition that taurine lysine is a potent antitumor agent, analog 2250 was synthesized by Geistlich Pharma.

[0178] Materials and methods Chemical substances: The compound 2250 and the 2% solution of tauroursodeoxycholic acid were provided by Wolfhausen's Geistlich Pharma AG, the assignee of the present invention.

[0179] Cell lines: Similar to the human colorectal adenocarcinoma cell line SW480, the human glioblastoma cell line LN-229 was used as previously described (Rodak et al. 2005).

[0180] Cytotoxicity assay: Dissociated LN-229 cells were seeded in 96-well plates at a density of 10 4 cells per well in 100 μl of culture medium. Approximately 24 hours later, when the cells reached a culture density of 70% - 80%, the medium was changed and treatment with compound number 2250 (4.0 μg / ml - 1000 μg / ml), tauroursodeoxycholic acid (4.0 μg / ml - 1000 μg / ml), or standard medium was initiated. Triplicate cultures were prepared for each sample. After 24 hours of incubation at 25 °C, the remaining adherent viable cells were stained using crystal violet as described (Rodack et al. 2005). The cell viability was determined by measuring the absorbance at 540 nm. The results were expressed as the death rate given by the difference between 100% of the cells and the percentage of surviving cells. The EC 50 value corresponds to the concentration that induces 50% cell death.

[0181] Results Positive control: After incubating human glioblastoma cells (LN-229) with tauroursodeoxycholic acid for 24 hours, the concentration-dependent cytotoxicity was determined to have an EC 50 = 45 μg / ml (Table 1, Figure 1), and this value corresponded to previous results obtained using this cell line (Rodack et al. 2005).

[0182] Testing of 2250: When 2250 was incubated under the same experimental conditions as tauroursodeoxycholic acid, a similar concentration-dependent loss of cell viability was observed. The half-maximal effective concentration inducing cell death was EC 50 = 50 μg / ml. (Table 1, Figure 1).

[0183] The results regarding the cytotoxicity of SW480 cells are shown in Figure 2.

[0184] Discussion Compound 2250 presents a new path in the search for novel antitumor agents of the tauroursodeoxycholic acid type. Biologically, the compound is as potent as tauroursodeoxycholic acid. Chemically, the compound exhibits distinct characteristics compared to tauroursodeoxycholic acid. By substituting the NH group with an ether-oxygen, the bicyclic structure of tauroursodeoxycholic acid is avoided. Compound 2250 has a monocyclic structure and is a close structural analog of taurultam.

[0185] Mechanistically, the above results indicate that, since 2250 lacks a methoxy group, the antitumor activity of tauroursodeoxycholic acid is not dependent on the formation of methoxy derivatives. The above compound induces blebbing in tumor cells.

[0186] Summary Compound 2250 exhibits potent antitumor activity in vitro, as determined in human glioblastoma cells (cell line LN-229). Its efficacy (EC 50 = 45 μg / ml) is comparable to that of tauroursodeoxycholic acid (EC 50 = 50 μg / ml) tested in the same cell line.

[0187]

Table 1

[0188] The above values were measured in triplicate experiments, and the OD is the absorbance at 540 nm ± standard deviation (SD). High values correspond to high cell viability.

[0189] Example 2: When testing the new compound 2250 (tetrahydro-1,4,5-oxathiazine-4-dioxide), it was found to have a very high level of antibacterial activity against Staphylococcus aureus and Escherichia coli. The antibacterial activity against Staphylococcus aureus was approximately two times higher than that of taurolutam.

[0190] Example 3: When testing compound 2250 in the punch plate test, it was found to be very active against MRSA strains 188, 189, 193, 194, and 195.

[0191] By showing both antibacterial and antitumor activities, compound 2250 is particularly suitable for surgical oncology.

[0192] Example 4: When each compound identified as compound 2250, 2255, 2245, A1, A3, B1, B2, or B3 in this specification was tested against the cancer cell lines of the cancers identified in this specification, it was found to be active against such cell lines.

[0193] Example 5: When each compound identified as compound 2250, 2255, 2245, A1, A3, B1, B2, or B3 in this specification was administered to patients with the cancers identified in this specification, it was found to be effective in treating such cancers and safe for use in patients. When each of these compounds was administered together with vitamin D3, its derivatives, metabolites, or analogs, it was found that the combination increased the antitumor effect of the compound.

[0194] Example 6: The half-life of compound 2250 in human fresh blood was measured in vitro at 37°C by GC, PYE Unicam Series 204 FID. Baseline value: 49.0 ppm After 1 hour: 50.6 ppm After 2 hours: 47.6 ppm After 20 hours: 38.6 ppm - 39.0 ppm

[0195] Therefore, the half-life of Compound 2250 is over 24 hours in human blood, significantly longer than that of tauroursodeoxycholic acid, which was found to be approximately 30 minutes using the same test.

[0196] Example 7: Tissue samples of high-grade gliomas of WHO grade IV, newly diagnosed in patients (middle-aged, 54 ± 10 years old), were mechanically minced, enzymatically digested, and the dissociated cells were filtered. The isolated tumor cells were cultured as bulk cells. Cancer stem cells (CSCs) were isolated from the murine SMA560 glioma cell line or newly isolated human glioblastoma cells by neurosphere formation under neurosphere conditions (using Neurobasal medium).

[0197] Cytotoxicity assay Bulk glioma cells were cultured and incubated with tauroursodeoxycholic acid or taurultam for 24 or 48 hours as previously described (Rodak et al., J. Neurosurg. 102, 1055 - 1068, 2005). After culturing the CSCs for 7 days, they were exposed to tauroursodeoxycholic acid, taurultam, or temozolomide for 24 hours. The number of remaining adherent cells was stained (Crystal Violet or Alamar Blue) and quantified by absorbance measurement (540 nm). Cell survival was represented as the percentage of viable cells relative to the number of cells surviving in untreated control cultures. Results are given as % kill or the dose required for half-maximal cytotoxicity (EC 50 is given.

[0198] Results Cytotoxicity of tauroursodeoxycholic acid and taurultam against cancer cells and cancer stem cells derived from mice The mouse SMA560 glioma cell line was used to provide tumor bulk cells and CSCs. After incubation of SMA560 bulk cells with various concentrations of tauroursodeoxycholic acid and tauroursodeoxycholate (6.25 μg / ml, 12.5 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml), cytotoxicity was determined after 24 and 48 hours of incubation. For both tauroursodeoxycholic acid and tauroursodeoxycholate, there was no significant difference in drug efficacy between 24 and 48 hours of incubation time, and obvious dose-dependent cytotoxicity was observed (Figure 3A, Figure 3B). EC 50 values were 34.6 μg / ml for tauroursodeoxycholic acid and 19.3 μg / ml for tauroursodeoxycholate (Figure 3C).

[0199] Mouse CSCs were prepared from the SMA560 glioma cell line and cultured for 7 days. CSCs were treated with the same concentrations of tauroursodeoxycholic acid and tauroursodeoxycholate as above, and cytotoxicity was determined after 24 hours. As shown in Figure 4, both tauroursodeoxycholic acid and tauroursodeoxycholate showed dose-dependent cytotoxicity against murine CSCs with an EC 50 of 12.5 μg / ml for tauroursodeoxycholic acid and an EC 50 of 10 μg / ml for tauroursodeoxycholate. These values demonstrate for the first time that tauroursodeoxycholic acid and tauroursodeoxycholate are effective against CSCs.

[0200] Tauroursodeoxycholic acid and tauroursodeoxycholate induce cell death in human CSCs isolated from four different glioblastoma patients. CSCs were isolated from glioblastoma tissues resected from four patients. The same concentration range of tauroursodeoxycholic acid and tauroursodeoxycholate as above was applied, and cytotoxicity was measured 24 hours after incubation with the drug. All four glioblastoma CSCs (GBM3, 4, 5, and 6) tested were similarly sensitive to tauroursodeoxycholic acid and tauroursodeoxycholate (Figure 5A, Figure 5B). The average EC 50 value of tauroursodeoxycholic acid was 13 ± 2 μg / ml, and the EC 50The value was 11 ± 1.4 μg / ml (Table 2). In these experiments, the cytotoxic performance of tauroursodeoxycholic acid and tauroursodeoxycholic acid was compared with that of temozolomide (TIM) applied in the concentration range of 5 μM to 1000 μM (Figure 2C). The average EC of TMZ 50 value was 68.5 ± 26 μg / ml (Table 2). Interestingly, this concentration is much higher than the peak plasma level of TMZ (13.7 μg / ml) measured in patients (Portnow et al., Clin Cancer Res 15, 7092 - 7098, 2009).

[0201] The results demonstrated that both tauroursodeoxycholic acid and tauroursodeoxycholic acid are effective against CSCs, and this finding was established for glioma CSCs derived from two species, namely mice and humans.

[0202] Mouse CSCs were generated from a mouse glioma cell line (SMA560). Notably, based on the EC 50 values, CSCs were more sensitive to tauroursodeoxycholic acid and tauroursodeoxycholic acid than the corresponding glioma bulk cells (about 3 - fold for tauroursodeoxycholic acid and 2 - fold for tauroursodeoxycholic acid) (Figure 3, Figure 4).

[0203] Human CSCs newly isolated from four human glioblastoma patients were similarly very chemosensitive to both tauroursodeoxycholic acid and tauroursodeoxycholic acid. The EC 50 values for cytotoxicity were 13 ± 2 μg / ml and 11 ± 1.4 μg / ml, respectively (Table 2). These values demonstrate that human CSCs are more sensitive (about 3 - fold to 4 - fold) to tauroursodeoxycholic acid and tauroursodeoxycholic acid than human glioblastoma bulk cells showing EC 50 values in the range of 50 μg / ml (Rodak et al., J. Neurosurg., 102, 1055 - 68, 2005).

[0204]

Table 2

[0205] Example 8: Cancer stem cells derived from a murine glioma cell line and human cancer stem cells were tested with tauroursodeoxycholic acid and taurultam. Tauroursodeoxycholic acid and taurultam were as potent against cancer stem cells derived from a murine glioma cell line as they were against human cancer stem cells newly isolated from four glioblastoma patients (EC 50 = 13 ± 2 μg / ml for tauroursodeoxycholic acid, EC 50 = 11 ± 1.4 μg / ml for taurultam), conferring strong antitumor activity (EC 50 = 12.5 μg / ml for tauroursodeoxycholic acid, EC 50 = 10 μg / ml for taurultam).

[0206] Example 9 Antitumor effect on pancreatic stem cell-like multicellular spheroid cultures Multicellular spheroids are composed of tumor cells that grow in a three-dimensional structure that stimulates growth, microenvironmental conditions, and stem cell-like properties of actual tumors. The multicellular tumor spheroid (MCTS) model compensates for many of the deficiencies seen in monolayer cultures. Spheroids sized 200 μm to 500 μm generate chemical gradients of oxygen, nutrients, and degradation products while having morphological and functional characteristics similar to tumors. Thus, assays utilizing the MCTS model enable evaluation of drug penetration and are more predictive of success in vivo compared to monolayer cultures. The MCTS assay is a tumor model system of intermediate complexity between standard monolayers and in vivo tumors.

[0207] Pancreatic tumor cells (Panc Tu-1, BxPC-3, Mia Paca-2, ASPC1) and primary pancreatic tumor cells (Bo80) were seeded in an ultra-low attachment plate in a special stem cell medium.

[0208] After growing pancreatic tumor cells (ASPC1, Mia Paca-2, Panc TuI, BxPC-3) and primary pancreatic tumor cells (Bo80) in monolayer cultures, they were seeded on ultra-low attachment plates under the conditions of a special stem cell medium to form multicellular spheroids, and aggregates were excluded through a cell strainer.

[0209] In the tumor cell lines AsPC-1, BxPC-3, and HCT-116, half-maximal inhibition of cell survival was achieved with compound 2250 at 750 μM to 1000 μM. These effects were similar to those observed in the glioma cell line LN-229. The induction of cell death was due to apoptosis and necrosis (or perhaps necroptosis). The induction of this programmed cell death was blocked by the addition of the reducing agent N-acetylcysteine, and it was found that caspases were not involved. Therefore, there is a redox-directed mechanism of action.

[0210] The growth of pancreatic tumor cells (AsPC-1, BxPC-3, and HCT-116) was inhibited by compound 2250 with a half-maximal effect concentration of 300 μM, which is much lower than the concentration required to induce cytotoxicity.

[0211] As shown in Figure 8, multicellular pancreatic tumor (Panc TuI or BxPC-3) spheroids were tested as controls and samples treated with tauroursodeoxycholic acid (500 μM) or compound 2250 (1000 μM) over 48 hours (columns labeled A). After treatment, the whole cell suspensions were each passed through a 45-μm cell strainer again, and the residual aggregates were analyzed for stability (columns labeled B).

[0212] Figures 9A and 9B show the results of FACS analysis of the CD133 content of Panc TuI multicellular spheroid cultures. CD133 is a known and well-established stem cell marker. The results show that the amount of CD133-positive cells in the multicellular spheroid cultures of Panc TuI was enriched 10-fold compared to Panc TuI grown in monolayer culture (B). Isotype IgG was used as a negative control (A). The results demonstrate that tauroursodeoxycholic acid and compound 2250 have antitumor activity against pancreatic stem cells such as multicellular spheroid cultures.

[0213] Example 10 In Vivo Study of Tauroursodeoxycholic Acid and Compound 2250 as Antitumor Agents in Malignant Pancreatic Cancer The effects of tauroursodeoxycholic acid and compound 2250 were analyzed in nude mice (NMRI-Foxn1 nu / nu). 1×10 7 Tumor cells (PancTu-I and MiaPaca 2) were subcutaneously injected into the flank. The animals were randomized into three groups: a control group, an intraperitoneal injection treatment with tauroursodeoxycholic acid (TRD) group, and an intraperitoneal injection treatment with compound 2250 (NDTRLT) group.

[0214] Before starting the treatment, the tumors were grown to a size of 200 mm 3 The mice were treated every other day at 500 mg / kg * body weight (BW).

[0215] As shown in Figure 10A, the administration of tauroursodeoxycholic acid significantly reduced the MiaPaca2 tumor volume (by about 2-fold) compared to the control.

[0216] As shown in Figure 10B, the administration of compound 2250 significantly reduced the MiaPaca2 tumor volume (more than 3-fold) compared to the control.

[0217] As shown in Figure 10C, the administration of tauroursodeoxycholic acid significantly reduced the PancTuI tumor volume (by about 3-fold) compared to the control.

[0218] As shown in Figure 10D, administration of compound 2250 significantly decreased (by about 2-fold) the PancTuI tumor volume compared to the control.

[0219] The doses of tauroursodeoxycholic acid and compound 2250 applied showed no toxic effects on the mice under study. A significant decrease in tumor growth was obtained in any of the tumor cell line models.

[0220] Tumor growth (volume) was significantly decreased from the 9th day onwards (PancTuI) and from the 11th day onwards (MiaPaca2) compared to the control. The dose of 500 mg / kg administered intraperitoneally showed sufficient tolerance without any signs of obvious toxicity.

[0221] As shown in Figure 11A, when the xenograft model of the primary pancreatic tumor (Bo73) was observed for 15 days, it was found that administration of tauroursodeoxycholic acid slightly decreased the relative tumor volume compared to the control, and administration of compound 2250 further decreased the relative tumor volume compared to the control. However, probably due to the short study period and the slow tumor growth rate, the difference in tumor volume was not statistically relevant. In Figure 11B, when the xenograft model of the primary pancreatic tumor (Bo70) was observed for 23 days, it was observed that administration of tauroursodeoxycholic acid and compound 2250 significantly decreased the tumor volume compared to the control.

[0222] Administration of tauroursodeoxycholic acid and / or compound 2250, for example intraperitoneally, inhibits tumor growth in vivo.

Claims

Use of compound 2250 for preparing a medicament for treating pancreatic cancer, wherein the compound 2250 is used in combination with gemcitabine. 【Chemical 1】

2. The use according to claim 1, wherein the medicament is for oral administration, intravenous administration or topical administration.

3. The use according to claim 1, wherein the medicament is for oral administration.

4. The use according to claim 1, wherein the medicament is for intravenous administration.

5. The use according to claim 1, wherein the medicament is for topical administration.

6. The use according to claim 1, wherein the medicament contains 0.1 g to 100 g of the compound 2250 per daily dose.

7. The use according to claim 1, wherein the medicament contains 5 g to 30 g of the compound 2250 per daily dose.

8. The use according to claim 1, wherein the medicament is in the form of a gel, capsule, tablet or solution.

9. The use according to claim 1, wherein the compound 2250 is present in the medicament at a concentration of 0.01 μg / ml to 1000 μg / ml.

10. The use according to claim 1, wherein the compound 2250 is present in the medicament at a concentration of 0.01% w / v to 3% w / v.

11. A pharmaceutical composition for treating pancreatic cancer, comprising compound 2250, wherein the pharmaceutical composition is used in combination with gemcitabine. 【Chemical Formula 2】

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