Oxathiazine-like compound, and preparation method and application thereof

Abstract

The present application discloses a method of manufacturing a novel oxathiazine-like compound, a composition comprising the oxathiazine-like compound, and use of the oxathiazine-like compound and the composition comprising the oxathiazine-like compound in the preparation of a variety of medicaments. It was found that these compounds have significantly longer half-life compared to taurolidine and bovine sulfanilamide.

Description

[0001] This application is filed on December 17, 2015, with application number 201910974314.4 and invention name “ Thiazide-like compounds, preparation methods and uses thereof" (the application date is December 17, 2015, the application number is 201580072835.0, the invention name is "Preparation A divisional application of the invention patent application entitled "a method for producing thiazide-like compounds"). Technical Field

[0002] The present invention relates to novel compounds, processes for preparing the novel compounds and their uses. Background Art

[0003] Known from U.S. Pat. No. 3,202,657 and U.S. Pat. No. 3,394,109 Thiazide-like compounds.

[0004] There remains a need in the art for new compounds and methods of making such compounds to provide compounds with enhanced antitumor and antimicrobial activity, reduced toxicity and side effects, and reduced resistance of tumor or microbial cells to treatment. Summary of the invention

[0005] According to the present invention, a new Thiazide-like compounds, making new Thiazide-like compounds and methods and their uses. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1 The anti-tumor activity of one embodiment of the present invention in a cytotoxicity assay of LN-229 cells is graphically shown.

[0007] Figure 2 The anti-tumor activity of one embodiment of the present invention in a cytotoxicity assay of SW480 (human colon adenocarcinoma) cells is graphically shown.

[0008] Figure 3 A-3C Cytotoxicity induced in mouse SMA560 glioma cells after treatment with taurolidine and taurine (TT). Figure 3 A) and 48h( Figure 3 B), the cytotoxicity was assessed. Figure 3 C) The EC values ​​of taurolidine (34.6 μg / ml) and taurine (19.3 μg / ml) are given. 50 Data are presented as mean ± SD of three independent experiments.

[0009] Figure 4 The cytotoxicity induced by taurolidine and taurine (TT) in mouse SMA560 glioma cancer stem cells (CSCs). The data are presented as mean ± SD.

[0010] Figure 5 A-5C is for patients taking taurolidine ( Figure 5 A) and taurosulfanilamide (TT) Figure 5 B) or temozolomide ( Figure 5 C) Cytotoxicity induced in tumor stem cells isolated from four glioblastoma multiforme (GBM) patients (GBM#3, #4, #5 and #6) after 24 h of treatment. Data are presented as mean ± SD.

[0011] Figure 6 FTIR spectrum of compound 2244 prepared according to the present invention.

[0012] Figure 7 is the FTIR spectrum of compound 2250 prepared according to the present invention.

[0013] Figure 8 Shown are the results of a spheroid toxicity assay of multicellular pancreatic tumor (Panc TuI or BxPC-3) spheroids, where control, taurolidine-treated (500 μM) or compound 2250-treated (1000 μM) samples were treated for 48 hours (columns labeled A) and filtered to test the stability of residual aggregates (columns labeled B).

[0014] Fig.9A and 9B Figure 2 shows the results of FACS analysis of CD133 content in Panc TuI multicellular spheroid cultures.

[0015] Fig. 10A Shown are MiaPaCa2 tumor volumes after treatment with control or taurolidine. Fig. 10B MiaPaCa2 tumor volumes after treatment with control or compound 2250 are shown. Fig. 10C Shown are PancTu I tumor volumes after treatment with control or taurolidine. Fig. 10D PancTu I tumor volumes after treatment with control or Compound 2250 are shown.

[0016] Fig.11A This is a pancreatic primary tumor (Bo70) xenograft model that was observed for 15 days when treated with a control, taurolidine, or compound 2250. Fig. 11B This is a pancreatic primary tumor (Bo 70) xenograft model that was observed for 23 days when treated with control, taurolidine or compound 2250. DETAILED DESCRIPTION

[0017] According to certain embodiments, the present invention relates to Thiazide-like compounds and their derivatives and preparation Thiazide-like compounds and methods of their derivatives.

[0018] According to some embodiments of the present invention Thiazide-like compounds and their derivatives have antitumor activity, antimicrobial activity and / or other activities.

[0019] According to some embodiments of the present invention Thiazide-like compounds and their derivatives provide an advantageous method for producing compounds having anti-tumor activity, anti-microbial activity and / or other activities. In certain embodiments, Thiazide-like compounds and their derivatives are particularly useful in methods of treating cancer and tumors in subjects such as human patients. Therefore, in certain embodiments of the present invention, methods of treating cancer and tumors using the compounds described herein are also contemplated. Cancers such as central nervous system cancers including glioblastomas, gliomas, neuroblastomas, astrocytomas and carcinomatous meningitis, colon cancer, rectal cancer, 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, cardia cancer, gallbladder cancer, skin cancer, bone tumors, cancers of the head and neck, leukemia, lymphoma, lymphosarcoma, adenocarcinoma, fibrosarcoma and metastasis thereof, for example, are diseases contemplated for treatment according to certain embodiments of the present invention. Drug-resistant tumors, such as multidrug-resistant tumors (MDR), are also useful in certain embodiments of using the compounds of the present invention, including drug-resistant tumors such as solid tumors, non-solid tumors and lymphomas. It is currently believed that any tumor cell can be treated using the methods described herein.

[0020] Tumor stem cells, also known as cancer stem cells (CSCs), are considered to be the main factors in the formation of metastasis and tumor regeneration after surgery.

[0021] In certain embodiments, the compounds of the invention are particularly useful for the treatment of tumor stem cells in a subject.

[0022] In certain embodiments, the compounds of the invention are particularly useful for the treatment of glioblastoma tumor stem cells in a subject.

[0023] In certain embodiments, the present invention kills tumor cells and / or CSCs, or inhibits their growth, by oxidative stress, apoptosis and / or inhibiting the growth of new blood vessels at the tumor site (anti-angiogenesis and anti-lumenization). A major mechanism of action for killing tumor cells and / or CSCs is oxidative stress. Tumor cells and / or CSCs can also be killed by apoptosis according to the present invention. When the blood concentration is low, the compound according to the present invention effectively inhibits the growth of tumor cells by anti-angiogenesis and anti-lumenization, so that these compounds are useful in palliative treatment.

[0024] The present invention Thiazide-like compounds and their derivatives are metabolized much more slowly in the bloodstream than taurolidine and taurine, so lower doses of these compounds can be administered to patients to achieve similar effects.

[0025] It was unexpectedly discovered that within minutes of exposure to taurolidine, tumor cells responded by initiating an apoptotic cell death program as follows:

[0026] 1. The primary damage of taurolidine to tumor cells is the increase of reactive oxygen species (ROS), which is measured by fluorescence.

[0027] 2. Oxidative stress introduced by taurolidine as a major step is supported by the finding that the antitumor effect of taurolidine can be prevented by the addition of reducing agents such as glutathione or N-acetylcysteine.

[0028] 3. Increased ROS damage to tumor cell mitochondria leads to loss of their membrane potential and release of apoptosis-inducing factor (AIF).

[0029] 4. AIF translocates to the nucleus and initiates the expression of pro-apoptotic genes, leading to plasma membrane blebbing, chromatin condensation, and DNA fragmentation, which are hallmarks of apoptosis.

[0030] 5. Compared with normal cells, tumor cells are very sensitive to oxidative stress. This explains the effect of taurolidine on a wide range of tumor cells in addition to normal cells.

[0031] In certain embodiments, the compounds of the invention can also be used to treat microbial infections in subjects (eg, human patients). Microbial infections that can be treated according to certain embodiments include bacterial infections, fungal infections, and / or viral infections.

[0032] Cancer patients are prone to immunosuppression, which results in susceptibility to microbial infections, particularly during and / or after surgery.

[0033] In certain embodiments, compounds of the invention are used to treat glioblastoma in a subject.

[0034] In certain embodiments, compounds of the invention are used to treat a S. aureus infection in a subject.

[0035] In certain embodiments, compounds of the invention are used in accordance with the present invention to treat MRSA in a subject.

[0036] In certain embodiments, compounds of the invention are used in accordance with the present invention to treat E. coli in a subject.

[0037] In certain embodiments, compounds of the invention are used in accordance with the present invention to treat Helicobacter pylori (H. pylori) in a subject and / or one or more cancers associated with H. pylori in a subject.

[0038] In certain embodiments, compounds of the invention are used in accordance with the present invention to treat HIV in a subject.

[0039] In certain embodiments, compounds according to formula I are used according to the present invention, wherein R is H, alkyl, etc., such as methyl, ethyl, propyl (eg, isopropyl), benzyl, etc.

[0040]

[0041] In certain embodiments, novel compound 2250 (tetrahydro-1,4,5- Thiazide-4-dioxide or 1,4,5- The FTIR spectrum of compound 2250 prepared according to the present invention is as follows: Figure 8 shown.

[0042] In certain embodiments, novel compound 2245 is prepared and / or used according to the present invention.

[0043] Compound 2250 prevents and treats gastric tumors (including tumors caused by or associated with Helicobacter pylori) or tumors formed by metastasis to the stomach.

[0044] The amount of compound depends on the size of the tumor. In one embodiment, the present invention includes reducing the size of the tumor with surgery and treating with one or more compounds. The compound can be administered before, during or after surgery to reduce the tumor. Compounds according to the present invention can be administered with any suitable method, including but not limited to, by gel, capsule, tablet, IV (intravenous administration), IP (intra-film administration) and / or directly administered to the tumor.

[0045] The gel may contain, for example, 2-4% (e.g., 3%) of an active compound of the invention (e.g., compound 2250), either alone or in combination with taurolidine / tauramide (which may also be administered alone or present), and may be administered topically. This gel may be used to treat tumors of the skin and oral cavity, including squamous cell tumors of the oral cavity and skin. This gel may also be administered via a suppository to the vagina, or via a syringe, for the treatment of cervical cancer or cervical dysplasia. The present invention may include a combination of suppositories with an active compound.

[0046] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the provided composition is mixed with at least one inert, pharmaceutically acceptable excipient and / or filler or extender (such as starch, lactose, sucrose, glucose, mannitol and silicic acid), binder (such as carboxymethylcellulose, alginate, gelatin, polyvinyl pyrrolidone, sucrose and gum arabic), humectant (for example, glycerol), disintegrant (such as agar, calcium carbonate, potato starch, tapioca starch, alginic acid, certain silicates and sodium carbonate solution), solution retardant (such as paraffin), absorption promoter (such as quaternary ammonium compound), wetting agent (such as cetyl alcohol and monostearate glyceride), absorbent (such as kaolin, bentonite), 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 include a buffer.

[0047] The compounds of the present disclosure, particularly compound 2250, have been found to be very soluble in water. In certain embodiments, PVP is not required to increase solubility. For example, a 3.2% solution of 2250 is isotonic. This is an unexpected advantage relative to taurolidine.

[0048] The compounds of the present invention, such as Compound 2250 (with or without taurolidine and / or tauramide), are very useful in surgical oncology because the compounds do not interfere with wound healing. Administration of other antineoplastic drugs must be delayed until up to five weeks or more after surgery because other such antineoplastic drugs hinder wound healing and promote anastomotic leakage. This problem can be avoided using the compounds of the present invention, such as Compound 2250, which can be administered during and immediately after surgery without wound healing problems or leakage problems.

[0049] Solid compositions of similar types can be used as fillers in soft and / or hard filled gelatin capsules, which use excipients such as lactose or milk sugar and high molecular weight polyethylene glycol, etc. Tablets, dragees, capsules, pills and granules can be prepared with coatings and shells (such as enteric coatings and other coatings known in the pharmaceutical field). They can optionally contain opacifiers and can be such compositions that they only release one or more compositions provided in or preferentially in a certain part of the intestinal tract, and optionally release in a delayed manner. The embodiment of the embedding composition that can be used includes polymers and waxes. Solid compositions of similar types can be used as fillers in soft and / or hard filled gelatin capsules, which use excipients such as lactose or milk sugar and high molecular weight polyethylene glycol, etc.

[0050] In certain embodiments, the capsule may include an excipient formulation containing one or more of hydroxypropylmethylcellulose (HPMC), gelatin, and fish gelatin. In certain embodiments, the capsule may contain a combination of compound 2250 and taurolidine and / or tauramide. The capsule may optionally further include one or more of lycopene, ellagic acid (polyphenol), curcumin, piperine, delphinidin, resveratrol, isothiocyanates (such as sulforaphane), capsaicin, and piperonamide.

[0051] Active compounds of the invention, such as compound 2250, can be combined with compounds such as gemcitabine. Such combinations can be used to treat cancers such as pancreatic cancer. Taurolidine and / or taurine can also be combined with gemcitabine to treat, for example, pancreatic cancer.

[0052] In some embodiments, the nutritional cancer prevention and treatment product may contain only 100-500 mg of compound 2250 or a combination of 100-500 mg of compound 2250 and 100-500 mg of taurolidine and / or taurine, and one or more of the following: lycopene (e.g., 20-200 mg), ellagic acid (polyphenol), curcumin, piperine (20-200 mg), delphinidin, resveratrol, isothiocyanates (such as sulforaphane), capsaicin, and piperonamide.

[0053] Surprisingly, these compounds were found to be useful during and immediately after surgery because they do not inhibit wound healing like other chemotherapeutic agents.

[0054] It was unexpectedly found that taurolidine, taurine and The ability of thiazide-like compounds and their derivatives to kill cancer stem cells is highly unusual and perhaps unknown among chemotherapeutic agents. Typical chemotherapeutic agents, if they are effective against cancer stem cells, are generally only effective at very high doses, which are extremely toxic to human patients.

[0055] It has been unexpectedly discovered that the doses of taurolidine and / or taurine that kill cancer stem cells are lower than the doses required to kill tumor cells.

[0056] Unexpectedly discovered that Thiazide-like compounds and their derivatives have a significantly lower half-life in human blood than taurolidine and tauramide. Therefore, these compounds are not cleared as quickly from the patient's bloodstream, effectively delaying the loss of drug efficacy caused by the body's clearance mechanisms.

[0057] It was unexpectedly found that when applied directly to tissues, some Thiazide-like compounds and their derivatives reduce the burning sensation, unlike the burning sensation observed in patients treated with taurolidine.

[0058] Unexpectedly discovered that Thiazide-like compounds and their derivatives have a particularly advantageous combination of properties including high water solubility, flexible administration routes (including oral and intravenous injection), long stability and half-life, and reduced side effects of burning sensation.

[0059] Thus, compound 2250 has a half-life in human blood of greater than 24 hours, which is significantly higher than the half-life of taurolidine, which is thought to be about 30 minutes using the same assay.

[0060] In one embodiment, the invention comprises treating a patient by administering Compound 2250 to the patient, resulting in a baseline blood concentration of Compound 2250 within about 5 minutes of administration. The method comprises maintaining the blood concentration of Compound 2250 in the patient at about 80% of the baseline blood concentration for about 20 hours.

[0061] In one embodiment, the invention comprises maintaining a 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 a blood concentration of 80% of the baseline blood concentration.

[0062] The daily dose can be about 0.1 g to about 100 g, for example, about 5 g to 30 g. The daily dose can be administered in the form of an orally administrable composition. The daily dose can be administered in the form of a capsule, tablet, or pharmaceutically acceptable solution. The daily dose can be administered in the form of a compound 2250 containing a concentration of about 0.01 to about 3% w / v. The daily dose can be administered in the form of a compound 2250 containing a concentration of about 0.01 μg / ml to about 1000 μg / ml, and the daily dose can be administered in the form of one or more solubilizing agents (e.g., polyols).

[0063] In some embodiments, the compound can be administered in a composition at a concentration of about 0.01 to about 1000 μg / ml. In some embodiments, the compound can be administered in a composition at a concentration of about 1 to about 100 μg / ml. In some embodiments, the compound can be administered in a composition at a concentration of about 10 to about 50 μg / ml. The composition can also include about 0.01 to about 1000 μg / ml, about 1 to about 100 μg / ml, or about 10 to about 50 μg / ml of taurolidine and / or tauramide.

[0064] In some embodiments, the compound can be administered in a composition at a concentration of about 0.01 to about 3%. In some embodiments, the compound can be administered in a composition at a concentration of about 0.1 to about 2.5%. In some embodiments, the compound can be administered in a composition at a concentration of about 1% to about 2%. The composition can also additionally include about 0.01 to about 3%, about 0.1 to about 2.5%, or about 1 to about 2% of taurolidine and / or tauramide.

[0065] In one embodiment, Thiazide-like compounds and their derivatives can be administered as a combination therapy with taurolidine and / or taurine to kill tumor stem cells. According to this embodiment, it was unexpectedly found that the dose required for the combination therapy to kill tumor stem cells is lower than the dose required to kill normal tumors.

[0066] In certain embodiments, Thiazide-like compounds and their derivatives can be administered together with vitamin D3, which results in an increase in the antitumor effect of the compounds.

[0067] In one embodiment, the compound is administered to a subject at a total daily dose of 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.

[0068] The effective dosage of the compound is a dosage unit in the range of 0.1-1000 mg / kg, preferably in the range of 150-450 mg / kg / day, most preferably in the range of 300-450 mg / kg / day.

[0069] As used herein, the term pure refers to a substance that is at least about 80% pure with respect to impurities and contaminants. In some embodiments, the term pure refers to a substance that is at least about 90% pure with respect to impurities and contaminants. In certain embodiments, the term pure refers to a substance that is at least about 95% pure with respect to impurities and contaminants. In some embodiments, the term pure refers to a substance that is at least about 99% pure with respect to impurities and contaminants. In some embodiments, the term pure refers to a substance that is at least about 99.5% pure with respect to impurities and contaminants.

[0070] In certain embodiments, the compounds, compositions and methods of the present invention comprise 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 to 100 microns. In certain embodiments, the term "micronized" as used herein refers to a particle size in the range of about 0.5 to 50 microns. In certain embodiments, the term "micronized" as used herein refers to a particle size in the range of about 1 to 25 microns. For example, the drug particle size can be about 1, 5, 10, 15, 20 or 25 microns.

[0071] In certain embodiments, the compounds, compositions and methods of the present 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 some embodiments, the diameter of the nanoparticle is less than 300nm. In some embodiments, the diameter of the nanoparticle is less than 100nm. In some embodiments, the diameter of the nanoparticle is less than 50nm, for example, between about 1nm and 50nm. Preparations suitable for injection or infusion may include isotonic solutions containing one or more solubilizing agents (such as polyols such as glucose) to provide solutions with increased concentrations of the compound. Such solutions have been described in EP 253662B1. The solution can be rendered isotonic with Ringer's solution or lactated Ringer's solution. The concentration of the compound in this solution can be within the range of 1-60g / liter.

[0072] In certain embodiments, exemplary compounds and methods for making compounds of the invention include the following:

[0073]

[0074]

[0075] The compound may be in crystalline form, for example, after crystallization and / or recrystallization in an alcohol, ketone, ester or a combination thereof. For example, the compound of the invention may be crystallized or recrystallized from an alcohol such as ethanol.

[0076] Exemplary compounds of the present invention include the following:

[0077]

[0078] It has been found that when nanoparticles are used, the compounds of the claimed invention reach higher blood levels. In one embodiment, the present invention includes only compound 2250 alone or in combination with taurolidine and / or tauramide. For example, the present invention includes nanoparticles of the compounds of the present invention encapsulated in capsules.

[0079] In certain embodiments, the present invention also relates to derivatives of the above-mentioned compounds, which have, for example, the activity of the compound as described herein, for example, at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% or more of the activity.

[0080] In certain embodiments, the present invention also relates to compositions comprising the compounds described herein, including pharmaceutically acceptable solutions of the compounds, and orally administrable compositions, such as capsules and tablets containing the compositions.

[0081] In certain embodiments, the compounds of the invention may be administered to a subject or patient by an appropriate method (eg, in a solution, such as topically, systemically (eg, by intravenous infusion), etc.).

[0082] Synthesis of 2250

[0083]

[0084] Sublimation in vacuum at about 70-80℃

[0085] Starting Materials:

[0086] Isethionic acid

[0087] Ethylenesulfonic anhydride (Carbysulfat), taurine, taurine amide, especially cysteine, isethionic acid

[0088] Synthesis 1

[0089] I.

[0090] a. Synthesis of isethionic acid from divalent carbonyl sulfate

[0091]

[0092] b. Synthesis of isethionic acid from taurine

[0093] Through biochemical synthesis of cysteine ​​and taurine

[0094] ·

[0095] ·

[0096] Chemical synthesis

[0097] Ethylene oxide and bisulfite

[0098] II. Isethionamide

[0099] a.

[0100]

[0101] b. Ethylene sulfonic anhydride + NH3

[0102]

[0103] 2250Possible alternative chemical synthesis steps

[0104] a) Sulfamic acid

[0105]

[0106] b) Paraformaldehyde, hexamethylenetetramine (hexamethylenetetramine, formaldehyde amine (Formine), hexamethylenetetramine)

[0107] c)

[0108]

[0109] d)

[0110]

[0111] e)

[0112]

[0113] f)

[0114]

[0115]

[0116] g)

[0117]

[0118]

[0119] h)

[0120]

[0121] Several alternative synthetic steps of 2250 and 2255 I. Starting materials 2250 / 2255

[0122] a.

[0123] b. Ethylene sulfonic anhydride + H2O

[0124]

[0125] Synthesis of Sodium Isethionate from Ethylene Oxide + Sodium Bisulfite II. Reaction of Amines with Ethylene Sulfonic Anhydride

[0126]

[0127] III.

[0128]

[0129] Exemplary Synthetic Schemes

[0130] I. Synthesis of 2244

[0131]

[0132] 2.15g pure 1907 is dissolved in 100ml ethyl acetate and catalyzed by 0.5g activated carbon-supported palladium. The solution is hydrogenated under room temperature and atmospheric pressure. The hydrogenation is completed after about 15 hours and the absorption capacity of hydrogen is 450ml. The hydrogenation is removed 3 times, using nitrogen each time, and then the reaction mixture is filtered through a filter aid (diatomaceous earth). The transparent colorless ethyl acetate solution is concentrated and dried in a rotary evaporator.

[0133] Yield: 1.25 g, seeded with 2244 crystals from crystallization.

[0134] Melting point: 42-44℃.

[0135] IR: corresponds to 2244, purity 99.3%.

[0136] II. Synthesis of 2244

[0137]

[0138] 5 g (0.023 mol) of 2264 / 1907 were boiled in 50 ml of concentrated hydrochloric acid 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 in a rotary evaporator. A yellow oil was left, which slowly crystallized after seeding with 2244 crystals.

[0139] IR corresponds to substance 2244.

[0140] Recrystallized from ethyl acetate.

[0141] Yield 0.7 g (24%).

[0142] Melting point: 44-45℃.

[0143] Melting point: 44-45℃.

[0144] IR corresponds to the reference substance.

[0145] III. Synthesis of 2244

[0146]

[0147] wherein Ph is a phenyl group.

[0148] 230 mg of 2269 were dissolved in 2 ml of NaOH (1 N) and boiled under reflux with a reflux condenser for 15 minutes. The clear solution was cooled to 20° C. and acidified with hydrochloric acid. The resulting precipitate was filtered off and dried in vacuo.

[0149] Yield: 110 mg.

[0150] Melting point: 114-116℃.

[0151] IR showed 99% benzoic acid as a by-product.

[0152] The acidic solution was concentrated to dryness on a rotary evaporator and the solid was boiled with acetate.The ethyl acetate solution was filtered and concentrated to dryness in vacuo.

[0153] Weight: 110 mg. The oil was contaminated with oil and the IR peak of 2244 (hydroxyethylsulfonic acid amide) was unclean.

[0154] The 110 mg were recrystallized from acetate.

[0155] Yield: 65 mg, melting point: 43-45 ° C,

[0156] The IR corresponds to 2244 which is 52%.

[0157] IV. Synthesis of 2244

[0158]

[0159] wherein Ph is a phenyl group.

[0160] 1.15 g of 2269 were dissolved in 10 ml of NaOH (1 N) and boiled under reflux for 15 minutes. The clear solution was cooled to 20° C. and acidified with hydrochloric acid. The resulting precipitate was filtered off and dried in vacuo.

[0161] Yield: 0.5 mg.

[0162] Melting point: 114-116℃.

[0163] IR showed 82% benzoic acid byproduct as reference substance. Hydrolysis was incomplete.

[0164] The acidic solution was concentrated to dryness on a rotary evaporator and the solid was boiled with acetate.The ethyl acetate solution was filtered and concentrated to dryness in vacuo.

[0165] Weight: 0.8 g. The oil was contaminated with oil and the IR peak of 2244 (hydroxyethylsulfonic acid amide) was unclean.

[0166] This 0.8 g was recrystallized from acetate.

[0167] Yield: 160 mg, melting point: 43-45°C

[0168] The IR corresponds to 2244 at 26%.

[0169] Synthesis of V.2244

[0170]

[0171] 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. The 2264 decomposed and an oily layer was formed. The reaction mixture was cooled and transferred to a separatory funnel, where the oil was separated from the aqueous phase. The acidic aqueous solution of hydroxyethylsulfonic acid amide (2244) was concentrated to almost dryness in a rotary evaporator at 50°C. The yellow oily residue was placed in a refrigerator overnight and 32.3 g of clean crystals were filtered off in vacuo. Melting point 43-45°C. IR: Peaks in oxygen with the following wave numbers: 655.82, 729.12, 844.85, 898.86, 947.08, 1003.02, 1060.88, 1134.18, 1236.41, 1288.49, 1317.43, 1408.08, 1572.04, 3105.5, 3209.66, 3313.82, and 3427.62 cm -1 ,like Figure 7 shown.

[0172] The mother liquor was concentrated to complete dryness.

[0173] Synthesis of VI.2244

[0174]

[0175] 21.5 g of 0.1 Mol 2264 and 100 ml of concentrated hydrochloric acid (about 36%) are boiled together under reflux for 30 minutes. An oil layer forms and the reaction mixture is cooled in a separatory funnel, where the oil is separated from the aqueous phase. Isethionic acid amide (2244) is dissolved in the acidic aqueous solution and shaken twice with dichloromethane, the dichloromethane is separated and the acidic aqueous solution is concentrated to dryness at 50° C. in a rotary evaporator. Melting point: 41-43° C. Product analysis shows, by IR, corresponding to 99.8% of 2244.

[0176] Distillation experiment:

[0177] 12.3 g were distilled off in high vacuum:

[0178] External temperature Internal temperature Vacuum

[0179] 190-210℃ 183-186℃ 0.1mm

[0180] Weight: 9.3 g oil, which is solid at room temperature. Melting point: 43-45°C.

[0181] VII. Synthesis of 2244

[0182]

[0183] 2.0 g of pure compound 1907 were dissolved in 200 ml of acetate, 0.5 g of palladium / activated carbon were added, and the mixture was autoclaved at 100° C. and hydrogenated at 50° C. After running for 6 hours, the reaction mixture was cooled overnight, then filtered and concentrated to dryness in vacuo.

[0184] Weight: 1.7 g of oil - CH2Cl2 was added and shaken, then stopped - then suction and filtration gave a crystalline solid, weight: 0.6 g, melting point about 40°C.

[0185] For analysis, twice the amount of acetate (0.2 g) was added for crystallization. Melting point 43-44°C.

[0186] VIII. Synthesis of 2244

[0187]

[0188] 2.0 g of pure compound 1907 was dissolved in 100 ml of ethyl acetate, and then 0.5 g of palladium / activated carbon was added. The mixture was then hydrogenated at room temperature and atmospheric pressure. The hydrogenation was terminated after about 15 hours. The absorption of hydrogen was about 450 ml. The hydrogen was then discharged 3 times and flushed with nitrogen, and then each reaction mixture was filtered through diatomaceous earth (celite). The transparent, colorless solution, ethyl acetate, was evaporated to dryness on a rotary evaporator.

[0189] Weight: 1.25 g oil, crystallized after addition of 2244 crystals.

[0190] Melting point: 42-44℃.

[0191] IR: 2244 corresponding to 99.3%.

[0192] Synthesis of IX.2250

[0193]

[0194] 1.2 g of pure 2245 was dissolved in 150 ml of acetic acid and completely dissolved at 60° C. 0.3 g of palladium supported on activated carbon was added and stirred at 75° C. The compound was then hydrogenated at atmospheric pressure.

[0195] The hydrogenation was stopped after 7 days. The amount of hydrogen absorbed was about 480 ml.

[0196] The hydrogen was removed and purged with nitrogen three times.

[0197] The reaction mixture was then filtered through a filter aid at 70° C. (diatomaceous earth). The clear warm glacial acetic acid solution was cooled to room temperature and the white crystals were sucked off and filtered.

[0198] Weight: 0.74g, melting point: 225-227℃

[0199] IR: 2245 corresponds to the starting material.

[0200] The mother liquor was concentrated to dryness on a rotary evaporator.

[0201] Weight: 0.38 g of impure material was extracted with ethyl acetate.

[0202] The solution was concentrated.

[0203] Ethyl acetate soluble part: semi-solid material obtained by sublimation;

[0204] 0.15 g of a semisolid material was obtained which was recrystallized from a few drops of water.

[0205] Yield: 70 mg, melting point: 95-98°C.

[0206] The IR corresponds to 2250 which is 98%.

[0207] X. One-step synthesis of solid sodium 2-anisole ethane sulfonate with high yield

[0208]

[0209] 10.5 g of sodium 2-bromoethanesulfonate was added to a solution of 110 ml of benzylethanol and 1.15 g of sodium benzyl alcohol.

[0210] The mixture was then boiled four times under reflux. The mixture was then concentrated to dryness in vacuo and then boiled three times with ethanol. The alcohol was filtered and concentrated to dryness.

[0211] The yield was 9.8 g and was confirmed by UV and IR.

[0212] Pure crystals are obtained by crystallizing pure sodium 2-anisole ethane sulfonate from solution by boiling the resulting sodium 2-anisole ethane sulfonate in ethanol, filtering, and then cooling the solution.

[0213] Synthesis of XI.2250

[0214]

[0215] 6.3 g of vinylsulfonamide (from 2258), 50 ml of concentrated formic acid and 1.1 g of paraformaldehyde were combined at reflux for 2 hours to produce compound 2250. The clear acidic solution was then concentrated to dryness on a rotary evaporator.

[0216] The residue was 5.9 g of light yellow, honey-like syrup.

[0217] IR: A mixture of vinylsulfonamide and 2250, 2 g, was sublimed and a few crystals were obtained.

[0218] Subliming semisolid: IR: 2250 corresponding to 98%.

[0219] XII. Synthesis of Vinylsulfonamide

[0220]

[0221] Formyl isethionic chloride is placed in 50 ml of chloroform and placed in a 350 ml sulfonation flask and cooled to -10°C. Then 25% ammonia gas is introduced. After the introduction of ammonia gas, the weight of chloroform / NH3 is 5 g. The mixture is slowly stirred from -3°C to 2°C.

[0222] 20 ml of chloroform was added dropwise to 9.0 g of distilled 2249. NH4Cl precipitated immediately.

[0223] The ammonium chloride was then filtered off under vacuum and the clear chloroform solution was concentrated to dryness in a rotary evaporator.

[0224] Yield: 6.3 g of clear, thin oil.

[0225] IR: corresponds to 96% CH2=CH-SO2-NH2 (vinylsulfonamide)

[0226] XIII. Synthesis of 2261

[0227]

[0228] 300 g (1.26 mol) of 2260 were weighed into a 750 ml multi-necked flask equipped with a KPG stirrer.

[0229] 415 ml of trichloroethylene + phosphorus oxychloride (in 10% POCl3, density equivalent to about 1.47) and 150 ml of phosphorus oxychloride and 5.7 ml of DMF were stirred and heated to 105° C. The mixture was allowed to react for 5 hours.

[0230] The solid was filtered under vacuum and the liquid was distilled under water pump vacuum. The filter cake was rinsed with ethyl acetate. After the trichloroethylene and phosphorus oxychloride were distilled off, the washing acetic acid was transferred to a flask and also distilled.

[0231] 250 g (1.07 mol - 85%) of yellow liquid were collected. IR corresponded to 2261.

[0232] XIV. Synthesis of 2250 and 2255:

[0233]

[0234] XV. New Synthesis Scheme of Compound 2250 and Its Related Compounds:

[0235] Starting Materials:

[0236] 3-Hydroxypropane-1-sulfonic acid

[0237] 3-Hydroxy-propane-sulfonic acid-γ-sultone (1,3-propane sultone)

[0238] 3-Hydroxy-propane-2-sulfonic acid

[0239] 2-Hydroxy-propane-1-sulfonic acid

[0240] Compound (tetrahydro- Thiazide-dioxide):

[0241]

[0242] Chemical intermediates

[0243] Protecting group: benzyl chloride

[0244]

[0245] Protecting group: Benzyl chloroformate

[0246]

[0247] XVI. Synthesis of Precursor Compounds

[0248]

[0249] synthesis:

[0250] 83.9 g of sodium vinyl sulfonate are added to 400 ml of benzyl alcohol solution, 0.5 g of sodium is added (catalytic amount). The mixture is heated to 150° C. with stirring and most of the sodium vinyl sulfonate goes into solution. After 3 hours, the mixture is cooled overnight and a thick solid crystallizes. The solid is filtered under vacuum and then suspended in ethanol, filtered and dried.

[0251] Yield: 94.0 g, IR: corresponds to the expected compound (purity 61.2%).

[0252] XVII.1905 Synthesis

[0253]

[0254] 60 grams of sodium vinyl sulfonate were added to a solution of 1000 ml of benzyl alcohol and 0.5 g of sodium. The whole mixture was then stirred and heated under reflux. After about 3 hours, the excess benzyl alcohol was distilled and removed under vacuum, and the remainder was then boiled with ethanol. The ethanol solution was filtered, concentrated, and crystallized to about 1 / 2 to obtain 37.3 g of a yellow cotton-like substance.

[0255] The process was also repeated using 250 g of sodium vinyl sulfonate and 2 liters of benzyl alcohol as described above, and about 208 g were crystallized.

[0256] The process was also repeated using 100 g of sodium vinyl sulfonate and 1 liter of benzyl alcohol as described above, and about 105 g were crystallized.

[0257] The process was also repeated using 200 g of sodium vinyl sulfonate, treated as described above, and about 130 g were crystallized.

[0258] XVIII. Synthesis of 1906

[0259]

[0260] 6.7 g of 1905 (recrystallized) were added to 50 ml of thionyl chloride and 1 ml of dimethylformamide. The sodium salt dissolved immediately and the mixture was heated to 40-50°C and placed under vacuum at 20°C overnight until concentrated. Yield: 9.8 g, which was added to 50 ml of NaOH 2N and stirred well. The NaOH solution was washed with CHCl3, then shaken with concentrated hydrochloric acid to precipitate and captured with Na2SO4, then dried and distilled.

[0261] The process was repeated with 208 g of 1905 mixed with 1000 ml of thionyl chloride and 10 ml of dimethylformamide. The mixture was refluxed and the excess thionyl chloride was distilled off until dry. The yield was 250 g, which was processed as above.

[0262] Synthesis of XIX.1907

[0263]

[0264] 9.8 g of 1906 was dissolved in chloroform (CHCl3) (turbid) and concentrated in a portion of 150 ml of concentrated aqueous ammonia and stirred. Stirring was continued for 3 hours while heating to 40-50°C, then the mixture was dried and concentrated under vacuum.

[0265] Yield: 3.1 g dark oil

[0266] 3.1 g of the dark oil were added to 50 ml of NaOH 2N and stirred well. The NaOH solution was washed with CHCl3, then shaken with concentrated hydrochloric acid to precipitate and trapped with Na2SO4, then dried and distilled.

[0267] Yield: 2.5 g oil

[0268] For analysis, 0.5 g of a sample was condensed at 160° C., turned into a solid and crystallized three times from ethyl acetate / benzene.

[0269] Melting point: 75-76℃

[0270] Molecular formula: C9H 13 NO3S

[0271] MW: 215.2

[0272] Calculated: C = 50.23%, H = 6.09%, N = 6.51%, S = 14.86%

[0273] Actual: C = 50.14%, H = 6.15%, N = 6.35%, S = 14.79%

[0274] Synthesis of XX.1908

[0275]

[0276] 1.2 g of 1907 was dissolved in 200 ml of ethyl acetate and 0.4 g of palladium on activated carbon was added. The mixture was hydrogenated in a hydrogenation kettle at 100°C and 50°C for 4 hours. The mixture was left at room temperature and under pressure over the weekend. The ethyl acetate solution was then filtered and dried under vacuum.

[0277] Yield: 1.1 g oil.

[0278] XXI.1908 Synthesis

[0279]

[0280] 2 grams of 1907 were dissolved in 200 ml of ethyl acetate and 0.5 g of palladium / palladium / charcoal were added. The mixture was hydrogenated in an autoclave at 100° C. and 50° C. After 6 hours, the reaction mixture was left to cool overnight and then filtered and distilled under vacuum until it dried to a residual oil.

[0281] Yield: 1.7 g oil.

[0282] Add CH2Cl2, stir and let stand, crystallize, and separate by suction under vacuum. Weight: 0.6 g, melting point about 40°C.

[0283] analyze:

[0284] 0.2 g was recrystallized twice from ethyl acetate.

[0285] Melting point: 43-44°C

[0286] Molecular formula: C2H7NO3S

[0287] Atomic weight: 125

[0288] Calculated: C = 19.22%, H = 5.65%, N = 11.21%, S = 25.65%

[0289] Actual: C = 19.20%, H = 5.67%, N = 11.07%, S = 25.73%

[0290] XXII.1909 Synthesis

[0291] 19.9 grams of 1906 were dissolved in 100 ml of chloroform and a solution of 23 grams of pure benzylamine and 200 ml of pure chloroform was added. Immediately, the benzylamine hydrochloride precipitated and the reaction mixture warmed. The mixture was then refluxed and the hydrochloride compound was separated by suction, and the transparent CHCl3 mother liquor was then placed in vacuum drying.

[0292] Yield: 27 g of a yellow, transparent oil which slowly turned into a solid.

[0293] The 27 g was dissolved in about 20 ml of ethyl acetate and N-hexane (amount) was added to make the solution nearly cloudy. The mixture was left in the cold overnight, and then it crystallized.

[0294] Yield: 9.2 g, melting point: 50-53°C

[0295] For analysis, 1 g was recrystallized three times from N-hexane. Melting point 56-57°C.

[0296] Synthesis of XXIII.2260

[0297]

[0298] 0.675 mol of sodium isethionate (100.0 g) and 2.02 mol of benzyl chloride (233 mL) were mixed in a 750 mL multi-necked flask with a KPG stirrer. The mixture was heated at an internal temperature of 70° C. (external temperature 95° C.), and triethylamine (120 mL) was then added dropwise over one hour, and then the external temperature was raised and maintained to 125° C. Subsequently, the external temperature was raised to 140° C., and the internal temperature rose to 130° C. The solids gathered on the stirrer, but returned to a suspended state. Hydrochloric acid vapor was emitted.

[0299] 30 mL of triethylamine was added dropwise and then reacted for another 1.5 hours. A viscous yellow suspension was formed. The product was cooled to an internal temperature of 50°C, and then 300 mL of water was added and stirred vigorously for 20 minutes, and the mixture was transferred to a 2 L separatory funnel. Then, the flask was rinsed with 100 mL of water.

[0300] The combined aqueous phases were washed twice with 280 mL of dichloromethane.

[0301] The aqueous phase was kept at 40°C while KCl was added to the solution until saturated (about 130 g KCl). The mixture was filtered through a fluted filter and stored in a refrigerator overnight.

[0302] The remaining solid was extracted and dried to give 30.85 g, a yield of 17.9%.

[0303] IR: OH band is present, similar to the precursor.

[0304] The mother liquor was treated again with KCI and stored (at 35-40°C) in a refrigerator overnight.

[0305] The solid from the second precipitation with KCl was filtered off and dried, yielding 60 g = 34.9% and the IR corresponded to the desired product.

[0306] Solid 1: Boil with 150 mL EtOH and filter while hot.

[0307] By repeated precipitation with KCl, boiling and crystallization, 32 g of the product was obtained with a yield of 19%.

[0308] XXIV. Synthesis of 2256

[0309]

[0310] 40 g of tauramide hydrochloride, 18 g of sodium nitrite and 300 ml of distilled water were boiled together under reflux until no more gas was generated. The clear yellow solution was then cooled to 50°C.

[0311] 30 ml of 1N NaOH was added to 10.5 g of acetaldehyde. The clear yellow solution was left to dry under vacuum over the weekend. The result was a rusty red honey-like residue weighing 37.6 g, which was extracted with ethanol. The ethanol solution was filtered and concentrated on a rotary evaporator to dryness. The resulting thick oil residue was dissolved in ethyl acetate. The ethyl acetate solution was filtered and concentrated.

[0312] This gave 30.7 g of a thick oil of rust-like color. White crystals separated from the thick oil. Melting point about 114-116°C

[0313] IR spectroscopy confirmed that the obtained compound had the structure of compound 2256:

[0314]

[0315] In certain embodiments, a sublimation apparatus consisting of laboratory glassware known in the art can be used for sublimation techniques to purify compounds according to the present invention. In certain embodiments, the sublimation vessel is heated under vacuum and reduced pressure. The compound volatilizes and condenses on a cooled surface to purify the compound, leaving behind impurities of non-volatile residues. The cooled surface often appears in the form of cold fingers. After heating stops and the vacuum is released, the sublimated compound can be collected from the cooled surface.

[0316] In one embodiment, substituted derivatives of Compound 2250 may be prepared. Substituted derivatives of Compound 2250 include:

[0317]

[0318] Wherein R can be H or alkyl or aryl. In certain embodiments, R is C1 to C6 alkyl. In certain embodiments, R is methyl.

[0319] In certain embodiments, the derivative of compound 2250 is prepared according to the following reaction scheme:

[0320]

[0321] Hypotaurine

[0322]

[0323] In one embodiment, the present disclosure includes a method of killing tumor stem cells by administering to a subject in need thereof a tumor stem cell killing effective amount of taurolidine, tauramide, and mixtures thereof. The tumor stem cell killing effective amount of taurolidine and / or tauramide is less than the amount of taurolidine and / or tauramide required to kill tumor cells.

[0324] In some embodiments, taurolidine, tauramide, and mixtures thereof are administered in a tumor stem cell killing composition at a concentration of about 0.01 to about 500 μg / ml. In some embodiments, taurolidine, tauramide, and mixtures thereof are administered in a tumor stem cell killing composition at a concentration of about 0.1 to about 100 μg / ml. In some embodiments, taurolidine, tauramide, and mixtures thereof are administered in a tumor stem cell killing composition at a concentration of about 10 to about 50 μg / ml. 0.01 μg / ml of taurolidine effectively kills tumor stem cells in in vitro tissue culture.

[0325] In some embodiments, taurolidine, tauramide, and mixtures thereof are administered in a concentration of about 0.001 to about 2% in a tumor stem cell killing composition. In some embodiments, taurolidine, tauramide, and mixtures thereof are administered in a concentration of about 0.01 to about 1.5% in a tumor stem cell killing composition. In some embodiments, taurolidine, tauramide, and mixtures thereof are administered in a concentration of about 0.1 to about 1% in a tumor stem cell killing composition.

[0326] In one embodiment, taurolidine, taurine, and mixtures thereof are 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 to kill tumor stem cells.

[0327] The effective dosage of taurolidine, taurine and mixtures thereof for killing tumor stem cells is a dosage unit in the range of about 0.01-500 mg / kg per day, preferably a dosage unit of 1-100 mg / kg per day, and most preferably a dosage unit of 5-50 mg / kg per day.

[0328] In another embodiment, the disclosure includes a method of killing tumor stem cells by administering to a subject in need thereof a compound selected from the group consisting of:

[0329] wherein each R is independently H, alkyl or aryl,

[0330]

[0331] This can be used in combination with taurolidine and / or tauramide. This technology provides a method of killing tumor stem cells using at least two compounds with different half-lives, thereby expanding the pharmacokinetic effect obtained thereby. In one embodiment, compound 2250 can be used in combination with taurolidine and / or tauramide.

[0332] Example:

[0333] Embodiment 1:

[0334] Antitumor effect of compound 2250

[0335] introduce

[0336] Based on the recognition that taurolidine is a potent antitumor agent, Geistlich Pharma synthesized analog 2250.

[0337] Materials and methods

[0338] A solution of compound 2250 and 2% taurolidin was provided by the assignee of the present invention, Geistlich Pharma AG of Wolhusen.

[0339] Cell lines: The human glioma cell line LN-229 and the human colon adenocarcinoma cell line SW480 were used as described previously (Rodak et al. 2005).

[0340] Cytotoxicity assay: Isolated LN-229 cells were seeded in 96-well plates in 100 μl of culture medium at a density of 10 cells per well. 4 cells. Approximately 24 hours later, when cells reached 70-80% confluence, the medium was changed and treatment with compound #2250 (4-1000 μg / ml), taurolidine (4-1000 μg / ml) or standard medium was initiated. Three cultures were prepared for each sample. After incubation for 24 hours at 25°C, the remaining adherent surviving cells were stained with crystal violet as previously described (Rodack et al. 2005). Cell viability was determined by measuring the absorbance at 540 nm. The results were expressed as killing percentage, which is given by the difference between 100% cell survival and the percentage cell survival. EC 50 Values ​​correspond to the concentration causing 50% cell death.

[0341] result

[0342] Positive control: Human glioblastoma cells (LN-229) were incubated with taurolidine for 24 hours and then 50 =45 μg / ml to determine the concentration-dependent cytotoxicity (Table 1, Figure 1 ), the EC 50 Values ​​correspond to previous results obtained with this cell line (Rodack et al. 2005).

[0343] Testing of 2250: When 2250 was incubated under the same experimental conditions as taurolidine, a similar concentration-dependent loss of cell viability was observed. The half-maximal concentration that caused cell death, EC 50 =50μg / μl (Table 1, Figure 1 ).

[0344] The cytotoxicity results of SW480 cells are shown in Figure 2 shown.

[0345] discuss

[0346] Compound 2250 represents a new approach to the search for new antitumor drugs of the taurolidine type. Biologically, the compound is as effective as taurolidine. Chemically, the compound shows characteristics that are significantly different from taurolidine. The bicyclic structure of taurolidine is avoided by replacing the NH group with an ether-oxygen. Compound 2250 is a taurine analog with a monocyclic structure and a closed structure.

[0347] Mechanistically, the results suggest that the antitumor activity of taurolidine is unlikely to be due to the formation of methoxy derivatives because 2250 lacks a methoxy group. This compound caused blebbing of tumor cells.

[0348] Summarize

[0349] Compound 2250 showed potent in vitro antitumor activity as measured in human glioblastoma cells (cell line LN-229). Its potency (EC 50 =45 μg / ml) compared to taurolidine (EC 50 =50 μg / ml).

[0350] Table 1: Cytotoxicity of 2250 and taurolidine to LL-229 glioblastoma cells

[0351]

[0352] The values ​​were measured in replicates and the OD is the absorbance at 540 nm plus or minus the standard deviation (SD).

[0353] Embodiment 2:

[0354] The new compound 2250 (tetrahydro-1,4,5- Thiazide-4-dioxide) was tested and found to have very high antibacterial activity against Staphylococcus aureus and Escherichia coli. The antibacterial activity against Staphylococcus aureus was about twice as high as that of taurine.

[0355] Embodiment 3:

[0356] Compound 2250 was tested and found to be highly active against MRSA strains 188, 189, 193, 194 and 195 in the perforated plate assay.

[0357] By showing a combination of antibacterial and antitumor activities, compound 2250 is particularly suitable for tumor surgery.

[0358] Embodiment 4:

[0359] Each of the compounds 2250, 2255, 2245, A1, A3, B1, B2 or B3 identified herein was tested on cancer cell lines of the cancers identified herein and was found to be active against such cell lines.

[0360] Embodiment 5:

[0361] Each of the compounds 2250, 2255, 2245, A1, A3, B1, B2 or B3 identified herein was administered to a patient suffering from a cancer identified herein and was found to be effective in treating such cancer and safe for use in the patient. Each of the compounds was used in combination with vitamin D3, a derivative, metabolite or analog thereof, and the combination was found to increase the anti-tumor effect of the compound.

[0362] Embodiment 6:

[0363] The half-life of compound 2250 in fresh human blood was measured in vitro at 37°C by GC, PYE Unicam Series 204FID.

[0364] Base value: 49.0ppm

[0365] After 1 hour: 50.6ppm

[0366] After 2 hours: 47.6ppm

[0367] After 3 hours: 38.6-39.0ppm.

[0368] Thus, compound 2250 has a half-life in human blood of greater than 24 hours, which is significantly higher than the half-life of taurolidine, which is thought to be about 30 minutes using the same test.

[0369] Embodiment 7:

[0370] Tissue samples were obtained from newly diagnosed patients with high-grade brain glioma of WHO grade IV (54 ± 10 years old), which were mechanically minced, enzymatically digested and free cells were filtered. The isolated tumor cells were cultured as somatic cells. Cancer stem cells (CSCs) were isolated by neurospheres formed from the mouse SMA560 glioma cell line or from freshly isolated human glioblastoma cells under neurosphere conditions (using neural culture medium).

[0371] Cytotoxicity assay

[0372] As previously described (Rodak et al., J. Neurosurg. 102, 1055-1068, 2005), glioma tumor cells were cultured with taurolidine or tauramide for 24 hours or 48 hours. CSCs were cultured for 7 days and then exposed to taurolidine, tauramide or temozolomide for 24 hours. The remaining number of adherent cells was stained (crystal violet or Alamar blue) and quantified by measuring absorbance (540nm). Cell survival was expressed as the percentage of surviving cells relative to the number of cells in untreated control cultures. Results were expressed as % killing or as the EC of the dose required for half-maximal cytotoxicity. 50 Given.

[0373] result

[0374] Cytotoxicity of taurolidine and taurine against cancer cells and cancer stem cells from mice

[0375] The mouse SMA560 glioma cell line was used to provide tumor somatic cells and CSCs. After incubation of SMA560 somatic cells with various concentrations of taurolidine and tauramide (6.25, 12.5, 25, 50, 100, 200 μg / ml), cytotoxicity was determined after 24 and 48 hours of incubation. For taurolidine and tauramide, it was found that the cytotoxicity of the cells was significantly increased after 24 and 48 hours of incubation ( Figure 3 There was a significant dose-dependent cytotoxicity between A and B, but no significant difference in efficacy. 50 The value was 34.6 μg / ml and the EC 50 The value is 19.3μg / ml ( Figure 3 C).

[0376] Mouse CSCs were generated from the SMA560 glioma cell line and cultured for 7 days. The CSCs were treated with the same concentrations of taurolidine and taurine as described above and the cytotoxicity was determined after 24 hours. Figure 4 As shown in Figure 2, both taurolidine and taurine showed dose-dependent cytotoxicity. Taurolidine had an inhibitory effect on the EC of mouse CSCs. 50 The value is 12.5 μg / ml and the EC value of taurine for mouse CSCs 50 The value is 10 μg / ml. These values ​​are the first to demonstrate the efficacy of taurolidine and taurine against CSC.

[0377] Taurolidine and taurine pairs induced cell death in human CSCs isolated from four different patients.

[0378] CSCs were isolated from glioblastoma tissues resected in four patients. The same range of concentrations of taurolidine and tauramide were applied as above and cytotoxicity was measured after 24 hours of incubation with the drugs. All four glioblastoma CSCs tested (GBM#3, #4, #5, and #6) were similarly sensitive to taurolidine and tauramide ( Figure 5 A, B). Average EC of taurolidine 50 The value is 13±2μg / ml, and the EC of taurine 50 The value was 11 ± 1.4 μg / ml (Table 2). In these experiments, the cytotoxicity of taurolidine and taurine administered in a concentration range of 5 μM to 1000 μM was compared with that of temozolomide (TIM) ( Figure 5 C) Average EC of TMZ 50 The value was 68.5±26 μg / ml (Table 2). Interestingly, this concentration is much higher than the peak serum level of TMZ (13.7 μg / ml) measured in patients (Portnow et al., Clin Cancer Res 15, 7092-7098, 2009).

[0379] The results showed that taurolidine and taurine were effective against CSCs and this finding was established in glioma CSCs from two species, mice and humans.

[0380] Mouse CSCs were generated from a mouse glioma cell line (SMA560). 50 Compared with the corresponding glioma somatic cells, the CSCs were even more sensitive to taurolidine and tauramide (about 3-fold for taurolidine and 2-fold for tauramide) ( Figure 3 , 4 ).

[0381] Freshly isolated human CSCs from four human glioblastoma patients were equally highly chemosensitive to both taurolidine and tauramide. 50 The values ​​were 13 ± 2 ug / ml and 11 ± 1.4 μg / ml, respectively (Table 2). These values ​​indicate that, similar to their mouse CSC counterparts, human CSCs are more potent than human glioblastoma somatic cells (which show an EC range of 50 μg / ml). 50 values) are more sensitive to taurolidine and tauramide (approximately 3 to 4 times) (Rodak et al., J. Neurosurg., 102, 1055-68, 2005).

[0382] Table 2: Cytotoxicity caused by taurolidine (Tau), taurine (TT) or temozolomide (TMZ) in cancer stem cells (CSCs) derived from four glioblastoma patients. 50(μg / ml) = drug concentration causing 50% cell death compared to untreated control cultures in vitro.

[0383] #imgpt82#

[0384] Embodiment 8:

[0385] Taurolidine and taurine were tested against cancer stem cells derived from mouse glioma cell lines and human cancer stem cells. Taurolidine and taurine were found to be effective against mouse glioma cell lines (EC 50 =13±2μg / mI, EC for taurine 50 =10 μg / ml) and human cancer stem cells freshly isolated from four glioblastoma patients (to taurolidine EC 50 =μg / mI, EC of taurine 50 =11±1.4μg / ml) has strong anti-tumor activity.

[0386] Embodiment 9:

[0387] Antitumor effects on pancreatic stem cell-like multicellular spheroid cultures.

[0388] Multicellular spheroids consist of tumor cells grown in a three-dimensional structure that mimics the growth, microenvironmental conditions, and stem-like characteristics of real tumors. The multicellular tumor spheroid (MCTS) model overcomes many of the deficiencies seen in monolayer cultures. Spheroids at the 200–500 μm scale develop oxygen chemical gradients, nutrients, and metabolites while possessing tumor-like morphological and functional characteristics. Assays using the MCTS model therefore allow for the evaluation of drug penetration and the success of the assay in vivo is more predictable than with monolayer cultures. The MCTS assay is an intermediate level of complexity tumor model system between standard monolayers and in vivo tumors.

[0389] Pancreatic tumor cells (Panc Tu-1, BxPC-3, Mia Paca-2, ASPC1) and pancreatic primary tumor cells (Bo80) were seeded in ultra-low adhesion plates in a special stem cell culture medium.

[0390] Pancreatic tumor cells (ASPC1, Mia Paca-2, Panc TuI, BxPC-3) and pancreatic primary tumor cells (Bo80) were grown in monolayer culture and then seeded in ultra-low adhesion plates to form multicellular spheroids under special stem cell medium conditions and passed through a cell strainer to exclude aggregates.

[0391] Half-maximal inhibition of cell viability was achieved with 750-1000 μM of compound 2250 in tumor cell lines AsPC-1, BxPC-3, and HCT-116. These effects were similar to those observed in the glioma cell line LN-229. Induction of cell death was due to apoptosis and necrosis (most likely necroptosis). It was found that the addition of the reducing agent N-acetylcysteine ​​prevented this programmed cell death and that caspases were not involved. Thus, a redox-directed mechanism of action was observed.

[0392] Compound 2250 inhibited the growth of pancreatic tumor cells (AsPC-1, BxPC-3, and HCT-116) at a half-maximal concentration of 300 μM, which is much lower than the concentration required to cause cytotoxicity.

[0393] like Figure 8 As shown, multicellular pancreatic tumor (Panc TuI or BxPC-3) spheroids were tested as control, taurolidine-treated (500 μM), or compound 2250-treated (1000 μM) samples for 48 hours (column A). After treatment, each cell suspension was again passed through a 45 μm cell strainer to analyze the stability of residual aggregates (column B).

[0394] Fig.9A and 9B The results of FACS analysis of the content of CD133 in Panc TuI multicellular spheroid cultures are shown. CD133 is a well-known and confirmed marker of stem cells. The results show that the number of CD133 positive cells in multicellular spheroid cultures of pancreatic cancer Panc TuI is enriched 10 times compared to Panc TuI grown in monolayer culture (B). Isotype IgG is used as a negative control (A). The results show that taurolidine and compound 2250 have anti-tumor effects on pancreatic stem cells like multicellular spheroid cultures.

[0395] Example 10

[0396] In vivo study of taurolidine and compound 2250 as antitumor agents in malignant pancreatic cancer.

[0397] The effects of taurolidine and compound 2250 were analyzed in nude mice (NMRI-Foxn1nu / nu). 7 Tumor cells (PancTu-1 and MiaPaca 2) were injected subcutaneously into the flank. The animals were randomly divided into three groups: a control group; a group treated with taurolidine (TRD) intraperitoneally (ip) and a group treated with compound 2250 (NDTRLT) intraperitoneally (ip).

[0398] Before treatment, tumors grew to 200 mm3 Size. On alternate days, mice were treated with 500 mg / kg body weight (BW).

[0399] like Fig. 10A As shown, the administration of taurolidine significantly reduced the tumor volume of MiaPaCa2 (approximately 2-fold) compared with the control group.

[0400] like Fig. 10B As shown, administration of compound 2250 significantly reduced the tumor volume of MiaPaCa2 (approximately 3 times) compared with the control group.

[0401] like Fig. 10C As shown, the administration of taurolidine significantly reduced the tumor volume of PancTu I (approximately 3 times) compared with the control group.

[0402] like Fig. 10D As shown, administration of compound 2250 significantly reduced the tumor volume of PancTu I (approximately 2-fold) compared with the control group.

[0403] The doses of taurolidine and compound 2250 used in the study showed no toxic effects on mice. In both tumor cell line models, tumor growth was significantly reduced.

[0404] Compared to the control group, tumor growth (volume) was significantly reduced from day 9 (PancTuI) and day 11 (MiaPaCa2). The 500 mg / kg dose (ip) was well tolerated with no obvious signs of toxicity.

[0405] like Fig.11A As shown, a xenograft model of pancreatic primary tumor (Bo 73) was observed for 15 days and it was found that the administration of taurolidine slightly reduced the relative tumor volume compared with the control group, and compound 2250 further reduced the relative tumor volume compared with the control group. However, the difference in tumor volume was not statistically significant, which may be due to the short study time and slow tumor growth. Fig. 11B In a xenograft model of pancreatic primary tumor (Bo 73) was observed for 23 days, administration of taurolidine and compound 2250 significantly reduced the relative tumor volume compared with the control group.

[0406] Administration of taurolidine and / or Compound 2250, such as intraperitoneal administration, inhibits tumor growth in vivo.

Claims

1. Use of a composition for preparing a medicament for producing an anti-luminalization effect or an anti-angiogenic effect in a subject, the composition comprising a pharmaceutically acceptable excipient and one or more compounds selected from the following:

2. Use of a composition for preparing a medicament for producing oxidative stress on cancer stem cells or tumor cells, the composition comprising a pharmaceutically acceptable excipient and one or more compounds selected from the following:

3. Use of a composition for preparing a medicament for producing apoptosis on cancer stem cells or tumor cells, the composition comprising a pharmaceutically acceptable excipient and one or more compounds selected from the following:

4. The method according to any one of claims 1 to 3, wherein the drug is used to treat drug-resistant tumors, multidrug-resistant tumors (MDR), solid tumors, non-solid tumors or lymphomas.

5. The use according to any one of claims 1 to 3, wherein the drug is used to kill tumor stem cells.

6. The method of claim 1 , wherein the medicament is used to treat cancer of the central nervous system, glioma, glioma, neuroblastoma, astrocytoma, carcinomatous meningitis, colon cancer, rectal cancer, 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, cardia cancer, gallbladder cancer, skin cancer, bone cancer, cancer of the head and neck, leukemia, lymphoma, lymphosarcoma, adenocarcinoma, fibrosarcoma, squamous cell tumor or its metastasis.

7. The use according to claim 6, wherein the medicament is used to treat squamous cell tumors.

8. Compound 2250, Use in preparing medicine for treating squamous cell tumor.

9. A drug combination comprising one or more oxathiazide-like compounds and gemcitabine.

10. The pharmaceutical combination according to claim 9, wherein the oxathiazide-like compound is selected from: The pharmaceutical combination according to claim 9 , wherein the oxathiazide-like compound is Compound 2250.

12. The pharmaceutical combination according to claim 9, wherein the pharmaceutical combination is used to treat cancer, especially pancreatic cancer.

13. Use of one or more oxathiazine-like compounds and gemcitabine in the preparation of a pharmaceutical combination for treating cancer.

14. Use of one or more oxathiazine-like compounds in the preparation of a medicament for treating cancer in combination with gemcitabine.

15. The use according to claim 13 or 14, wherein the oxathiazine-like compound is selected from:

16. The use according to claim 13 or 14, wherein the oxathiazine-like compound is compound 2250.

17. The use according to claim 13 or 14, wherein the cancer is pancreatic cancer.

18. Use of a composition for preparing a medicament for treating a bacterial infection, a fungal infection and / or a viral infection in a subject, the composition comprising a pharmaceutically acceptable excipient and one or more compounds selected from the group consisting of:

19. The use according to claim 18, wherein the medicament is for treating HIV in a subject.

20. A method for producing compound 2260, comprising the following reaction:

21. A manufacturing (sodium 2-anisole ethane sulfonate), comprising the following reaction:

22. The method of claim 21, wherein the method comprises adding sodium 2-bromoethane sulfonate to a solution of benzyl alcohol and sodium benzyl alcohol to form a mixture, boiling the mixture to reflux four times, concentrating to dryness under vacuum, boiling with ethanol, filtering the ethanol, and concentrating to dryness to obtain solid sodium 2-anisoleethane sulfonate.

23. The method of claim 22, further comprising boiling the solid sodium 2-anisole ethane sulfonate in ethanol, filtering, and then cooling to obtain sodium 2-anisole ethane sulfonate crystals with improved purity.

24. A method for making a compound having the formula Ph-CH2-O-CH2-CH2-SO3Na, comprising the following reaction:

25. The method according to claim 24, comprising the following steps in order: Sodium vinyl sulfonate is added to a benzyl alcohol solution and a catalytic amount of sodium to form a mixture, the mixture is heated under stirring to dissolve most or all of the sodium vinyl sulfonate, the heated mixture is cooled to obtain crystals, the crystals are vacuum filtered, the filtered crystals are suspended in ethanol, the suspended crystals are vacuum filtered, and the vacuum filtered crystals are dried to obtain Ph-CH2-O-CH2-CH2-SO3Na.

26. A manufacturing The method comprises the following reaction:

27. The method according to claim 26, comprising the following steps in order: Sodium vinyl sulfonate is added to a benzyl alcohol solution and a trace amount of sodium, the mixture is stirred under reflux, the benzyl alcohol is distilled off under vacuum, the remainder is boiled with ethanol, the ethanol is filtered, the ethanol is concentrated, and the resulting reaction product is crystallized to obtain 28. A method for producing compound 2260, comprising the following reaction:

29. The method according to claim 28, comprising the following steps in order: Mixing sodium isethionate with benzyl chloride, heating the mixture, adding triethylamine dropwise to the mixture, cooling the mixture, adding water to the mixture and stirring to obtain an aqueous phase and an organic phase, separating the aqueous phase from the organic phase, washing the aqueous phase with dichloromethane, adding KCl to the aqueous phase, filtering, cooling the aqueous phase, extracting the remaining solid, treating the aqueous phase with KCl, cooling the aqueous phase, filtering the solid product from the aqueous phase, and drying the solid to obtain

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