HK1240209B - Processes for preparing oxathiazin-like compounds - Google Patents

Description

Method for preparing oxathiazine-like compounds

Background of the Invention

Technical Field

The present invention relates to novel compounds, methods for preparing the novel compounds and their uses.

Background Art

Oxathiazine-like compounds are known from US Pat. No. 3,202,657 and US Pat. No. 3,394,109.

There remains a need in the art for new compounds and methods of making such compounds that 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

According to the present invention, novel oxathiazine-like compounds, methods for preparing the novel oxathiazine-like compounds and uses thereof are disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 graphically shows the anti-tumor activity of one embodiment of the present invention in a cytotoxicity assay in LN-229 cells.

FIG2 graphically shows the anti-tumor activity of one embodiment of the present invention in a cytotoxicity assay of SW480 (human colon adenocarcinoma) cells.

Figures 3A-3C show cytotoxicity induced in mouse SMA560 glioma cells after treatment with taurolidine and tauramide (TT). Cytotoxicity was assessed after 24 hours (Figure 3A) and 48 hours (Figure 3B) of treatment. The lower panel (Figure 3C) shows the _EC50 values for taurolidine (34.6 μg/ml) and tauramide (19.3 μg/ml). Data are presented as mean ± SD of three independent experiments.

Figure 4 shows the cytotoxicity induced by taurolidine and taurine (TT) in mouse SMA560 glioma cancer stem cells (CSCs). Data are presented as mean ± SD.

Figures 5A-5C show the cytotoxicity induced in cancer stem cells isolated from four glioblastoma multiforme (GBM) patients (GBM#3, #4, #5, and #6) after 24 h of treatment with taurolidine (Figure 5A) and taurine (TT) (Figure 5B) or temozolomide (Figure 5C). Data are presented as mean ± SD.

FIG6 is the FTIR spectrum of compound 2244 prepared according to the present invention.

FIG7 is an FTIR spectrum of compound 2250 prepared according to the present invention.

Figure 8 shows 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 (column labeled A) and filtered to test the stability of residual aggregates (column labeled B).

Figures 9A and 9B show the results of FACS analysis of CD133 content in Panc TuI multicellular spheroid cultures.

Figure 10A shows the volume of MiaPaCa2 tumors after treatment with control or taurolidine. Figure 10B shows the volume of MiaPaCa2 tumors after treatment with control or compound 2250. Figure 10C shows the volume of PancTu I tumors after treatment with control or taurolidine. Figure 10D shows the volume of PancTu I tumors after treatment with control or compound 2250.

Figure 11A shows a pancreatic primary tumor (Bo70) xenograft model observed for 15 days after treatment with a control, taurolidine, or Compound 2250. Figure 11B shows a pancreatic primary tumor (Bo70) xenograft model observed for 23 days after treatment with a control, taurolidine, or Compound 2250.

DETAILED DESCRIPTION

According to certain embodiments, the present invention relates to oxathiazine-like compounds and derivatives thereof, and methods for preparing oxathiazine-like compounds and derivatives thereof.

According to certain embodiments of the present invention, the oxathiazine-like compounds and their derivatives have anti-tumor activity, antimicrobial activity and/or other activities.

The oxathiazine compounds and their derivatives according to certain embodiments of the present invention provide advantageous methods for producing compounds having anti-tumor activity, antimicrobial activity and/or other activities. In certain embodiments, the oxathiazine compounds and their derivatives are particularly useful in methods for treating cancers and tumors in subjects such as human patients. Therefore, in certain embodiments of the present invention, methods for treating cancers 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, leukemias, lymphomas, lymphosarcoma, adenocarcinomas, fibrosarcomas, and metastases thereof, for example, are diseases that are 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 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.

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

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

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

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-tubularization). 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 compounds according to the present invention effectively inhibit the growth of tumor cells through anti-angiogenesis and anti-tubularization effects, so that these compounds are useful in palliative treatment.

The oxathiazine compounds and their derivatives of the present invention are metabolized much more slowly in the bloodstream than taurolidine and tauramide. Therefore, lower doses of these compounds can be administered to patients to achieve similar effects.

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

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

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.

3. Increased ROS damages tumor cell mitochondria, leading to loss of membrane potential and release of apoptosis-inducing factor (AIF).

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.

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.

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

Cancer patients are prone to immunosuppression, which leads to susceptibility to microbial infections, particularly during and/or after surgery.

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

In certain embodiments, compounds of the invention are used to treat a Staphylococcus aureus infection in a subject.

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

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

In certain embodiments, compounds of the present 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.

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

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.

In certain embodiments, novel compound 2250 (tetrahydro-1,4,5-oxathiazine-4-dioxide or 1,4,5-oxathiazine-4-dioxide) is prepared and/or used according to the present invention. The FTIR spectrum of compound 2250 prepared according to the present invention is shown in FIG8 .

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

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

The amount of compound depends on the size of the tumor. In one embodiment, the present invention includes reducing tumor size with surgery and using one or more compound treatments. 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.

The gel may contain, for example, 2-4% (e.g., 3%) of an active compound of the invention (e.g., compound 2250), alone or in combination with taurolidine/tauramide (which may also be administered alone or in the presence of the active compound), 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 vaginally or by syringe for the treatment of cervical cancer or cervical dysplasia. The present invention may include a combination of suppositories with the active compound.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the compositions provided are mixed with at least one inert, pharmaceutically acceptable excipient and/or filler or extender (e.g., starch, lactose, sucrose, glucose, mannitol, and silicic acid), binder (e.g., carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and gum arabic), humectant (e.g., glycerol), disintegrant (e.g., agar, calcium carbonate, potato starch, tapioca starch, alginic acid, certain silicates, and sodium carbonate solution), solution retardant (e.g., paraffin), absorption accelerator (e.g., quaternary ammonium compounds), wetting agent (e.g., cetyl alcohol and glyceryl monostearate), absorbent (e.g., kaolin, bentonite), lubricant (e.g., 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.

The compounds of the present disclosure, particularly compound 2250, have been found to be highly 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 over taurolidine.

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 anti-tumor drugs must be delayed until up to five weeks or more after surgery because other such anti-tumor 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 issues.

Solid compositions of similar types can be used as fillers in soft and/or hard-filled gelatin capsules that use excipients such as lactose or milk sugar and high molecular weight polyethylene glycol. The solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared with coatings and shells (other coatings such as enteric coatings and pharmaceutical fields are well known). They can optionally comprise an emulsifier and can be such a composition 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 operable embedded composition includes polymers and waxes. Solid compositions of similar types can be used as fillers in soft and/or hard-filled gelatin capsules that use excipients such as lactose or milk sugar and high molecular weight polyethylene glycol.

In certain embodiments, the capsule may include an excipient formulation comprising one or more of hydroxypropyl methylcellulose (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 (a polyphenol), curcumin, piperine, delphinidin, resveratrol, isothiocyanates (e.g., sulforaphane), capsaicin, and piperonamide.

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.

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

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

The unexpected discovery that taurolidine, tauramide, and oxathiazine-like compounds and their derivatives can kill cancer stem cells is highly unusual and potentially unknown among chemotherapy agents. Typical chemotherapy agents, if effective against cancer stem cells, are generally only effective at very high doses, which can be highly toxic to human patients.

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.

Surprisingly, it was discovered that oxathiazide-like compounds and their derivatives have significantly shorter half-lives in human blood than taurolidine and tauramide. As a result, these compounds are cleared less rapidly from the patient's bloodstream, effectively delaying the loss of drug efficacy due to the body's clearance mechanisms.

It has been unexpectedly discovered that certain oxathiazide-like compounds and derivatives thereof, when applied directly to tissue, reduce the burning sensation unlike that observed in patients treated with taurolidine.

It has been unexpectedly discovered that oxathiazine-like compounds and their derivatives have a particularly advantageous combination of properties including high water solubility, flexible administration routes (including oral and intravenous), long stability and half-life, and reduced side effect of burning sensation.

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 approximately 30 minutes using the same assay.

In one embodiment, the present invention comprises treating a patient by administering Compound 2250 to the patient such that a baseline blood concentration of Compound 2250 is achieved 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.

In one embodiment, the invention comprises maintaining a blood concentration of an anti-tumor compound in a patient at approximately 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 at 80% of the baseline blood concentration.

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).

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.

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.

In one embodiment, the oxathiazide compounds and their derivatives can be administered as a combination therapy with taurolidine and/or taurine to kill cancer stem cells. According to this embodiment, it was unexpectedly discovered that the dose required for the combination therapy to kill cancer stem cells is lower than the dose required to kill normal tumors.

In certain embodiments, the oxathiazine-like compounds and their derivatives can be administered together with vitamin D3, which results in an increase in the anti-tumor effect of the compounds.

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.

An effective dose 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.

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.

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.

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 300 nm. In some embodiments, the diameter of the nanoparticle is less than 100 nm. In some embodiments, the diameter of the nanoparticle is less than 50 nm, for example, between about 1 nm and 50 nm. 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 compound concentrations. 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 such a solution can be within the range of 1-60 g/liter.

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

The compound may be in crystalline form, for example, after crystallization and/or recrystallization from 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.

Exemplary compounds of the present invention include the following:

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

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

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

In certain embodiments, the compounds of the present invention can be administered to a subject or patient by any suitable method (eg, in a solution, such as topically, systemically (eg, by intravenous infusion), etc.).

Synthesis of 2250

Sublimation in vacuum at about 70-80℃

Starting material:

Isethionic acid

Ethylenesulfonic anhydride (Carbysulfat), Taurine, Taurine amide,

Especially cysteine, isethionic acid

Synthesis 1

I.

a. Synthesis of isethionic acid from divalent carbonyl sulfate

b. Synthesis of isethionic acid from taurine

Through the biochemical synthesis of cysteine and taurine

●

●

Chemical synthesis

●Ethylene oxide and bisulfite

II. Isethionic acid amide

a.

b. Ethylenesulfonic anhydride + NH ₃

2250 Possible alternative chemical synthesis steps

a) Sulfamic acid

b) Paraformaldehyde, hexamethylenetetramine

(Hexamethylenetetramine, Formine, Hexamethylenetetramine)

c)

d)

e)

f)

g)

h)

Several optional synthetic steps for 2250 and 2255

I. Starting material 2250/2255

a.

b. Ethylenesulfonic anhydride + H ₂ O

Synthesis of Sodium Isethionate from Ethylene Oxide + Sodium Bisulfite

II. Reaction of Amines with Ethylenesulfonic Anhydride

III.

Exemplary synthetic schemes

Synthesis of I.2244

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

Yield: 1.25 g, seeded with 2244 crystals.

Melting point: 42-44℃.

IR: corresponding to 2244, purity 99.3%.

II. Synthesis of 2244

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 on a rotary evaporator and dried. This left a yellow oil which slowly crystallized after being seeded with 2244 crystals.

IR corresponds to substance 2244.

Recrystallized from ethyl acetate.

Yield 0.7 g (24%).

Melting point: 44-45℃.

Melting point: 44-45℃.

IR corresponds to the reference substance.

III. Synthesis of 2244

wherein Ph is a phenyl group.

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

Yield: 110 mg.

Melting point: 114-116℃.

IR showed that 99% of benzoic acid was a by-product.

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.

Weight: 110 mg. The oil was contaminated with oil and the IR peak of 2244 (isethionamide) was unclean.

These 110 mg were recrystallized from acetate.

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

The IR corresponds to 2244, which is 52%.

Synthesis of IV.2244

wherein Ph is a phenyl group.

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

Yield: 0.5 mg.

Melting point: 114-116℃.

IR showed 82% benzoic acid by-product as the reference substance. The hydrolysis was incomplete.

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.

Weight: 0.8 g. The oil was contaminated with oil and the IR peak of 2244 (isethionamide) was unclean.

This 0.8 g was recrystallized from acetate.

Yield: 160 mg, melting point: 43-45°C

The IR corresponds to 2244 at 26%.

Synthesis of V.2244

215 g (0.1 mol) of 2264 and 1000 ml (approximately 36%) of concentrated hydrochloric acid were boiled together under reflux for 30 minutes. The 2264 decomposed, leaving an oily layer. The reaction mixture was cooled and transferred to a separatory funnel, where the oil separated from the aqueous phase. The acidic aqueous solution containing the isethionic acid amide (2244) was concentrated to near dryness on a rotary evaporator at 50°C. The yellow oily residue was refrigerated overnight, and 32.3 g of clean crystals were filtered off in vacuo. Melting point 43-45°C. IR: peaks having the following wave numbers in oxygen: 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 , as shown in FIG. 7 .

The mother liquor was concentrated to complete dryness.

Synthesis of VI.2244

21.5 g (0.1 mol) of 2264 and 100 ml of concentrated hydrochloric acid (approximately 36%) were boiled together under reflux for 30 minutes. An oily layer formed and the reaction mixture was cooled in a separatory funnel, where the oil separated from the aqueous phase. Isethionic acid amide (2244) was dissolved in the acidic aqueous solution and shaken twice with dichloromethane. The dichloromethane was separated, and the acidic aqueous solution was concentrated to dryness on a rotary evaporator at 50°C. Melting point: 41-43°C. Product analysis by IR indicated 99.8% of 2244.

Distillation experiment:

12.3 g were distilled off in high vacuum:

External temperature Internal temperature Vacuum

190-210℃ 183-186℃ 0.1mm

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

Synthesis of VII.2244

2.0 g of pure compound 1907 was dissolved in 200 ml of acetate, 0.5 g of palladium/activated carbon was 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.

Weight: 1.7 g of oil - CH2Cl2 was added and shaken, then stopped - then suctioned and filtered to give a crystalline solid, weight: 0.6 g, melting point about 40°C.

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

VIII. Synthesis of 2244

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

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

Melting point: 42-44℃.

IR: 2244 corresponding to 99.3%.

Synthesis of IX.2250

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 mixture was then hydrogenated at atmospheric pressure.

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

The hydrogen was removed and purged with nitrogen three times.

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

Weight: 0.74g, melting point: 225-227℃

IR: 2245 corresponds to the starting material.

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

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

The solution was concentrated.

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

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

Yield: 70 mg, melting point: 95-98°C.

The IR corresponds to 2250 which is 98%.

X. One-step synthesis of solid sodium 2-anisoleethanesulfonate with high yield

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.

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.

The yield was 9.8 g and was confirmed by UV and IR.

Pure crystals are obtained by crystallizing pure sodium 2-anisoleethanesulfonate from solution by boiling the resulting sodium 2-anisoleethanesulfonate in ethanol, filtering, and then cooling the solution.

Synthesis of XI.2250

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.

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

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

Subliming semisolid: IR: 2250 corresponding to 98%.

XII. Synthesis of vinylsulfonamide

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

20 ml of chloroform was added dropwise to 9.0 g of distilled 2249. _{NH 4} Cl precipitated immediately.

The ammonium chloride was then filtered off under vacuum, and the clear chloroform solution was concentrated to dryness on a rotary evaporator.

Yield: 6.3 g of clear, thin oil.

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

Synthesis of XIII.2261

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

415 ml of trichloroethylene + phosphorus oxychloride (in 10% POCl ₃ , 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.

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.

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

XIV. Synthesis of 2250 and 2255:

XV. New Synthesis Scheme of Compound 2250 and Related Compounds:

Starting material:

3-Hydroxypropane-1-sulfonic acid

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

3-Hydroxy-propane-2-sulfonic acid

2-Hydroxy-propane-1-sulfonic acid

Compound (tetrahydro-oxathiazine-dioxide):

Chemical intermediates

Protecting group: benzyl chloride

Protecting group: benzyl chloroformate

XVI. Synthesis of Precursor Compounds

synthesis:

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

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

Synthesis of XVII.1905

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

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.

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.

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

XVIII. Synthesis of 1906

6.7 g of 1905 (recrystallized) was 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 then placed under vacuum at 20°C overnight until concentrated. Yield: 9.8 g. This was added to 50 ml of 2N NaOH and stirred thoroughly. The NaOH solution was washed with _CHCl₃ , then shaken with concentrated hydrochloric acid _to precipitate the solution, which was then captured with _Na₂SO₄ , dried, and distilled.

The process was repeated using 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.

Synthesis of XIX.1907

9.8 g of 1906 were 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, and then the mixture was dried and concentrated under vacuum.

Yield: 3.1 g dark oil

3.1 g of the dark oil were added to 50 ml of NaOH 2N and stirred thoroughly. The NaOH solution was washed with CHCl ₃ and then shaken with concentrated hydrochloric acid to precipitate and captured with Na ₂ SO ₄ , then dried and distilled.

Yield: 2.5g oil

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

Melting point: 75-76°C

Molecular formula: C ₉ H ₁₃ NO ₃ S

MW: 215.2

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

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

Synthesis of XX.1908

Dissolve 1.2 g of 1907 in 200 ml of ethyl acetate and add 0.4 g of palladium on activated carbon. Hydrogenate the mixture in a hydrogenation reactor at 100°C and 50°C for 4 hours. Leave the mixture at room temperature under pressure over the weekend. The ethyl acetate solution is then filtered and dried under vacuum.

Yield: 1.1 g oil.

Synthesis of XXI.1908

2 g 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, then filtered and distilled under vacuum until it dried to a residual oil.

Yield: 1.7 g oil.

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

analyze:

0.2 g was recrystallized twice from ethyl acetate.

Melting point: 43-44°C

Molecular formula: C ₂ H ₇ NO ₃ S

Atomic weight: 125

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

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

XXII.1909 Synthesis

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 CHCl mother liquor was then dried under vacuum.

Yield: 27 g of a yellow, clear oil which slowly turned into a solid.

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

Yield: 9.2 g, melting point: 50-53°C

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

Synthesis of XXIII.2260

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 equipped with a KPG stirrer. The mixture was heated to an internal temperature of 70°C (external temperature of 95°C), and triethylamine (120 mL) was then added dropwise over one hour. The external temperature was then raised to and maintained at 125°C. Subsequently, the external temperature was raised to 140°C, and the internal temperature rose to 130°C. Solids accumulated on the stirrer but returned to suspension. Hydrochloric acid vapor was emitted.

30 mL of triethylamine was added dropwise, and the reaction was continued for an additional 1.5 hours. A viscous yellow suspension 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. The mixture was then transferred to a 2 L separatory funnel. The flask was then rinsed with 100 mL of water.

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

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.

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

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

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

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.

Solid 1: Boil with 150 mL of EtOH and filter while hot.

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

Synthesis of XXIV.2256

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.

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 to dryness on a rotary evaporator. The resulting thick oily residue was dissolved in ethyl acetate. The ethyl acetate solution was filtered and concentrated.

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

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

In certain embodiments, a sublimation apparatus comprised 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 form a purified compound, leaving behind a non-volatile residue of impurities. The cooled surface often appears as a cold finger. After heating is stopped and the vacuum is released, the sublimated compound can be collected from the cooled surface.

In one embodiment, substituted derivatives of Compound 2250 can be prepared. Substituted derivatives of Compound 2250 include:

wherein R can be H or alkyl or aryl. In certain embodiments, R is C ₁ to C ₆ alkyl. In certain embodiments, R is methyl.

In certain embodiments, derivatives of compound 2250 are prepared according to the following reaction scheme:

Hypotaurine

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.

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.

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

In one embodiment, taurolidine, tauramide, 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.

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.

In another embodiment, the present 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:

wherein each R is independently H, alkyl or aryl,

This can be used in combination with taurolidine and/or tauramide. This technology provides a method for 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.

Example:

Example 1:

Antitumor effect of compound 2250

introduce

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

Materials and methods

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

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).

Cytotoxicity Assay: Dissociated LN-229 cells were seeded in 96-well plates in 100 μl of culture medium at a density of ¹⁰ cells per well. Approximately 24 hours later, when cells reached 70-80% confluency, the culture medium was replaced and treatment with Compound #2250 (4–1000 μg/ml), taurolidine (4–1000 μg/ml), or standard culture medium was initiated. Triplicate cultures were prepared for each sample. After a 24-hour incubation 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 absorbance at 540 nm. Results were expressed as percent kill, which is the difference between 100% cell survival and percent cell survival. The _EC50 value corresponds to the concentration that causes 50% cell death.

result

Positive control: After incubation of human glioblastoma cells (LN-229) with taurolidine for 24 hours, concentration-dependent cytotoxicity was determined with _EC50 = 45 μg/ml (Table 1, Figure 1), which corresponds to previous results obtained with this cell line (Rodack _et al. 2005).

Testing of 2250: When 2250 was incubated under the same experimental conditions as taurolidine, similar concentration-dependent cell viability loss was observed. The half-maximal concentration that caused cell death was _EC50 = 50 μg/μl (Table 1, Figure 1).

The cytotoxicity results of SW480 cells are shown in FIG2 .

discuss

Compound 2250 represents a new approach to the search for novel taurolidine-type antitumor drugs. Biologically, the compound is as effective as taurolidine. Chemically, it exhibits distinct characteristics from taurolidine. By replacing the NH group with an ether-oxygen, the bicyclic structure of taurolidine is avoided. Compound 2250 is a monocyclic and closed-loop tauramide analog.

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 causes blebbing of tumor cells.

Summarize

Compound 2250 exhibited potent in vitro antitumor activity as determined in human glioblastoma cells (cell line LN-229). Its potency ( _EC50 = 45 μg/ml) was comparable to that of taurolidine ( _EC50 = 50 μg/ml) tested in the same cell line.

Table 1: Cytotoxicity of 2250 and taurolidine against LL-229 glioblastoma cells

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

Example 2:

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

Example 3:

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

By displaying a combination of antibacterial and antitumor activities, compound 2250 is particularly suitable for tumor surgery.

Example 4:

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.

Example 5:

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

Example 6:

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

Reference value: 49.0ppm

After 1 hour: 50.6ppm

After 2 hours: 47.6ppm

After 3 hours: 38.6-39.0ppm.

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.

Example 7:

Tissue samples were obtained from patients (aged 54 ± 10 years) with newly diagnosed WHO grade IV high-grade gliomas. The tissue samples 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 forming neurospheres from the mouse SMA560 glioma cell line or from freshly isolated human glioblastoma cells under neurosphere conditions (using neural culture medium).

Cytotoxicity assay

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 subsequently 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). The survival of the cells was expressed as the percentage of the surviving cells relative to the number of cells in the untreated control cultures. The results were expressed as % killing rate or given as the _EC50 of the dose required for half-maximum cytotoxicity.

result

Cytotoxicity of taurolidine and taurine against cancer cells and cancer stem cells from mice

The mouse SMA560 glioma cell line was used to provide tumor somatic cells and CSCs. SMA560 cells were incubated with various concentrations of taurolidine and tauramide (6.25, 12.5, 25, 50, 100, and 200 μg/ml) and cytotoxicity was measured after 24 and 48 hours of incubation. For both taurolidine and tauramide, a clear dose-dependent cytotoxicity was observed between the 24 and 48 hours of incubation ( Figures 3A and B ), with no significant difference in potency. The EC ₅₀ value for taurolidine was 34.6 μg/ml, and the EC ₅₀ value for tauramide was 19.3 μg/ml ( Figure 3C ).

Mouse CSCs were generated from the SMA560 glioma cell line and cultured for 7 days. These CSCs were treated with the same concentrations of taurolidine and tauramide as described above, and cytotoxicity was measured 24 hours later. As shown in Figure 4, both taurolidine and tauramide exhibited dose-dependent cytotoxicity, with an _EC50 value of 12.5 μg/ml for taurolidine and ₁₀ μg/ml for tauramide against mouse CSCs. These values demonstrate for the first time that taurolidine and tauramide are effective against CSCs.

Taurolidine and taurine induced cell death in human CSCs isolated from four different patients.

CSCs were isolated from glioblastoma tissue removed from four patients. Taurolidine and tauramide were applied as above at the same concentration range and cytotoxicity was measured after 24 hours of incubation with the drug. All four glioblastoma CSCs tested (GBM#3, #4, #5, and #6) were similarly sensitive to taurolidine and tauramide (Fig. 5A, B). The average EC ₅₀ value of taurolidine was 13 ± 2 μg/ml, and the EC ₅₀ value of tauramide was 11 ± 1.4 μg/ml (Table 2). In these experiments, the cytotoxicity of taurolidine and tauramide with temozolomide (TIM) in a concentration range of 5 μM to 1000 μM was compared (Fig. 2C). The average EC ₅₀ value of TMZ was 68.5 ± 26 μg/ml (Fig. 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).

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

Mouse CSCs were generated from a mouse glioma cell line (SMA560). Remarkably, based on _EC50 values, these CSCs were even more sensitive to taurolidine and tauramide than the corresponding glioma somatic cells (approximately 3-fold for taurolidine and 2-fold for tauramide) (Figures 3 and 4).

Freshly isolated human CSCs from four human glioblastoma patients were equally highly chemosensitive to both taurolidine and tauramide. The EC ₅₀ values for cytotoxicity were 13 ± 2 μg/ml and 11 ± 1.4 μg/ml, respectively (Table 2). These values indicate that, similar to corresponding mouse CSCs, human CSCs are more sensitive (approximately 3 to 4 times) to taurolidine and tauramide than human glioblastoma somatic cells (which exhibit EC ₅₀ values in the 50 μg/ml range) (Rodak et al., J. Neurosurg., 102, 1055-68, 2005).

Table 2: Cytotoxicity caused by taurolidine (Tau), taurine (TT) or temozolomide (TMZ) in cancer stem cells (CSCs) derived from four glioblastoma patients. _EC50 (μg/ml) = drug concentration that causes 50% cell death compared to untreated control cultures in vitro.

Example 8:

Taurolidine and tauramide were tested against cancer stem cells derived from mouse glioma cell lines and human cancer stem cells. Taurolidine and tauramide were found to have strong antitumor activity against mouse glioma cell lines (EC ₅₀ for taurolidine = 13 ± 2 μg/mI, EC ₅₀ for tauramide = 10 μg/ml) and human cancer stem cells freshly isolated from four glioblastoma patients (EC ₅₀ for taurolidine = μg/mI, EC ₅₀ for tauramide = 11 ± 1.4 μg/ml).

Example 9:

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

Multicellular spheroids consist of tumor cells grown in a three-dimensional structure that mimics the growth, microenvironmental conditions, and stem-like characteristics of a true tumor. The multicellular tumor spheroid (MCTS) model overcomes many of the deficiencies seen in monolayer cultures. Spheroids, measuring 200–500 μm, develop chemical gradients of oxygen, nutrients, and metabolites while possessing tumor-like morphological and functional characteristics. Therefore, assays using the MCTS model allow for the assessment of drug penetration and are more predictable for in vivo success compared to monolayer cultures. MCTS assays represent an intermediate level of complexity between standard monolayers and in vivo tumors.

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

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

Compound 2250 achieved half-maximal inhibition of cell viability in the tumor cell lines AsPC-1, BxPC-3, and HCT-116 at doses of 750-1000 μM. These effects were similar to those observed in the glioma cell line LN-229. Cell death was induced by apoptosis and necrosis (most likely necroptosis). Addition of the reducing agent N-acetylcysteine was found to prevent this programmed cell death without involving caspases. Therefore, a redox-directed mechanism of action is suggested.

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.

As shown in Figure 8, multicellular pancreatic tumor (Panc TuI or BxPC-3) spheroids were tested for 48 hours as control, taurolidine-treated (500 μM), or compound 2250-treated (1000 μM) samples (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).

Figures 9A and 9B show the results of FACS analysis of CD133 content in Panc TuI multicellular spheroid cultures. CD133 is a well-known and well-established stem cell marker. The results show that the number of CD133-positive cells in Panc TuI multicellular spheroid cultures of pancreatic cancer is 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 taurolidine and compound 2250 have anti-tumor effects on pancreatic stem cells in multicellular spheroid cultures.

Example 10

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

The effects of taurolidine and compound 2250 were analyzed in nude mice (NMRI-Foxn1nu/nu). ^1x107 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 intraperitoneally (ip) with taurolidine (TRD); and a group treated intraperitoneally (ip) with compound 2250 (NDTRLT).

Tumors grew to a size of 200 mm ³ before treatment began. On alternate days, mice were treated with 500 mg/kg body weight (BW).

As shown in FIG10A , the administration of taurolidine significantly reduced the tumor volume of MiaPaCa2 (approximately 2-fold) compared with the control group.

As shown in FIG10B , administration of compound 2250 significantly reduced the tumor volume of MiaPaCa2 cells (approximately 3-fold) compared with the control group.

As shown in FIG10C , the administration of taurolidine significantly reduced the tumor volume of PancTu I (approximately 3-fold) compared with the control group.

As shown in FIG10D , administration of compound 2250 significantly reduced the tumor volume of PancTu I (approximately 2-fold) compared with the control group.

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

Compared to the control group, tumor growth (volume) was significantly reduced starting from day 9 (PancTuI) and day 11 (MiaPaCa2). The 500 mg/kg dose (i.p.) was well tolerated with no overt signs of toxicity.

As shown in Figure 11A, a pancreatic primary tumor (Bo 73) xenograft model was observed for 15 days and it was found that the administration of taurolidine slightly reduced the relative tumor volume compared to the control group, and compound 2250 further reduced the relative tumor volume compared to 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. In Figure 11B, a pancreatic primary tumor (Bo 73) xenograft model was observed for 23 days. Compared with the control group, the administration of taurolidine and compound 2250 greatly reduced the relative tumor volume.

Administration of taurolidine and/or Compound 2250, such as intraperitoneally, inhibits tumor growth in vivo.

Claims (10)

1. A method for preparing compound 2245 or compound 2250, comprising reacting compound 2244 as follows: or 2. The method of claim 1, wherein compound 2244 is produced by reacting compound 2264 as follows: A) B) C) Reaction C) includes the following steps in sequence: dissolving compound 2264 in acetate and adding palladium/activated carbon to form a reaction mixture; autoclaving the reaction mixture at 100°C; hydrogenating at 50°C; cooling overnight; filtering; and concentrating to dryness to obtain solid compound 2244; or D) Reaction D) includes the following steps in sequence: dissolving compound 2264 in acetate and adding palladium/activated carbon to form a reaction mixture, hydrogenating the mixture at room temperature and atmospheric pressure, removing hydrogen, filtering and drying to obtain solid compound 2244. 3. The method of claim 1, wherein compound 2244 is produced by reacting compound 2264 as follows: 4. The method according to claim 3, wherein the step of reacting compound 2264 further comprises boiling compound 2264 with concentrated HCl and refluxing to form a reaction mixture, cooling the reaction mixture and separating the oil phase and aqueous phase of the reaction mixture, concentrating the aqueous phase and cooling the oil residue to form crystals of 2244. 5. The method according to claim 3, wherein the step of reacting compound 2264 further comprises boiling compound 2264 with concentrated HCl and refluxing to form a reaction mixture, cooling the reaction mixture and separating the oil phase and the aqueous phase of the reaction mixture, dissolving the aqueous phase with dichloromethane, separating the dichloromethane, concentrating the aqueous phase and cooling to form an oil. 6. The method according to claim 3, wherein the step of reacting compound 2264 further comprises boiling compound 2264 with concentrated hydrochloric acid and refluxing to form a reaction mixture, cooling the reaction mixture, adding dichloromethane to form an aqueous phase and an oil phase, evaporating the aqueous phase, and seeding the oil phase with 2244 crystals to obtain 2244 crystals. 7. The method according to claim 2 or 3, wherein compound 2261 is reacted as follows to produce compound 2264: 8. The method according to claim 7, wherein compound 2260 is reacted to produce compound 2261 as follows: DMF is dimethylformamide, or 9. A method for preparing compound 2250, wherein the method comprises reacting compound 2244 by the following reaction: 10. A method for preparing compound 2250 or a mixture of compounds 2250 and 2255, wherein the method comprises reacting compound 2244 by the following reaction: