HUP0200376A2 - Thiaoloindolinone compounds, process for their preparation and pharmaceutical composions containing them and their use - Google Patents

Description

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DANUBIA Patent and Trademark Office Ltd.

Budapest

PUBLICATION COPY

Thiazoloindolinone compounds, process for their preparation and < i

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The invention relates generally to thiazoloindolinone compounds which are cyclin-dependent kinase II inhibitors and are useful, inter alia, for preventing/reducing the severity of epithelial cytotoxic effects (e.g. hair loss, palmoplantar syndrome or mucositis) resulting from chemotherapy and/or radiotherapy. The invention also relates to a process for the preparation of these compounds and to pharmaceutical compositions containing such compounds as active ingredients for preventing/reducing epithelial cytotoxic effects resulting from chemotherapy and/or radiotherapy.

Protein kinases play a critical role in the regulation of cell growth and differentiation and are key mediators of cellular signals that lead to the production of growth factors and cytokines (Schiessinger and Ullrich, Neuron 1992, 9, 383). Some of these kinases are listed below, but are not limited to: abl, ARaf, ATK, ATM, bcrabl, Blk, BRaf, Brk, Btk, CDK1, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, cfms, c-fms, c-kit, c-met, cRafl, CSF1R, CSK, c-src, EGFR, ErbB2, ErbB3, ErbB4, ERK,

95123-5194OE/Hoj

-2ERK1, ERK2, Fak, fes, FGFR1, FGFR2, FGFR3, FGFR4, FGFR5, Fgr, FLK-4, Fps, Frk, Fyn, GSK, gsk3a, gsk3b, Hck, IGF-1R, IKK, IKKI, IKK2, IKK3, INS-R, Integrin-linked kinase, Jak, JAKI, JAK2, JAK3, JNK, JNK, Lek, Lyn, MEK, MEK1, MEK2, p38, PDGFR, PIK, PKB1, PKB2, PKB3, PKC, PKCa, PKCb, PKCd, PKCe, PKCg, PKC1, PKCm, PKCz, PLK1, Polo-like kinase, PYK2, tie _h tie ₂ , TrkA, TrkB, TrkC, ULI3, UL97, VEGF-R1, VEGF-R2, Yes and Zap70. Protein kinases play a role in, for example, the following diseases: central nervous system disorders, such as Alzheimer's disease (Mandelkow, EM et al., FEBS Lett. 1992, 314, 315. Sengupta, A. et al., Mol. Cell. Biochem. 1997, 767,99), pain perception (Yashpal, KJ Neurosci. 1995, 15, 3263-72), inflammatory diseases, such as arthritis (Badger, J. Pharm. Exp. Ther. 1996, 279, 1453), psoriasis (Dvir, et al., J. Cell Biol. 1991, 113, 857), chronic obstructive pulmonary disease, bone diseases, such as osteoporosis (Tanaka et al., Nature, 1996, 303, 528), cancer (Hunter and Pines, Cell 1994, 79, 573), atherosclerosis (Hajjar and Pomerantz, FASEB J. 1992, 6, 2933), thrombosis (Salari, FEBS 1990, 263, 104), metabolic diseases such as diabetes (Borthwick, AC et al. Biochem. Biophys. Res. Commun. 1995, 210, 738), blood vessel proliferative disorders such as angiogenesis (Strawn et al., Cancer Res. 1996, 56, 3540; Jackson et al. J. Pharm. Exp. Ther. 1998, 284, 687), restenosis (Buchdunger et al., Proc. Nat. Acad. Sci USA 1991, 92, 2258), autoimmune diseases and transplant rejection (Bolen and Brugge, Ann. Rev. Immunol. 1997, 15, 371),

and infectious diseases, such as viral diseases (Littler, E. Nature 1992, 355, 160) or fungal infections (Lum, R. T., WO 9805335 A1 980212).

Chemotherapy and radiation therapy are routinely used to treat a variety of neoplastic conditions. Among the side effects associated with these treatments is that the patient may experience serious epithelial cell toxicity. The proliferative epithelial damage induced by chemotherapy often results in hair loss (alopecia), palmar-plantar syndrome, and mucositis; such side effects, especially mucositis, are also common consequences of radiation therapy. These side effects may vary in severity depending on the type of chemotherapy and/or radiation used, the dose, and the schedule of doses.

The invention relates to one or more different forms of the compound of formula (I), which are described in detail below.

The various forms of the compound of the invention provide a new opportunity to prevent or reduce the occurrence of epithelial cytotoxicity as a side effect of chemotherapy and/or radiation by administering one or more different forms of the above compound to patients receiving such treatment.

Epithelial cytotoxic side effects that can be prevented or reduced by the compounds of the invention include hair loss, palmar-plantar syndrome and/or mucositis induced by chemotherapy and/or radiation therapy.

-4The various forms of thiazoloindolinone compounds of formula (I) are further characterized by their melting point, X-ray diffraction crystallography, solubility in solvents, and their preparation process is also described.

The invention also relates to a process for preparing various forms of thiazolindoline compounds.

The invention also includes pharmaceutical compositions comprising (a) at least one form of a thiazoloindolinone compound as defined above in an amount effective to prevent/reduce the severity of epithelial cell toxicity as a side effect of chemotherapy and/or radiotherapy, (b) together with one or more pharmaceutically acceptable carriers, excipients or diluents.

Other aspects, features and advantages of the invention will become apparent in detail from the following description and claims.

The invention therefore relates to various forms of a thiazoloindolinone compound which are suitable for use as cyclin-dependent kinase inhibitors.

The thiazoloindolinone compound of the present invention is represented by formula (I) and is prepared in various forms, namely forms A, B and C.

The general preparation procedure is illustrated in Scheme 1 and in detail in the following examples. The product prepared according to Scheme 1 is 4-{[(7-oxo-6,7-dihydro-8H-[1,3]thiazolo[5,4-e]indol-8-ylidene)methyl]amino}-N-(4-pyridinylmethyl)benzenesulfonamide, the compound is recovered from the reaction medium and then recrystallized using specific solvent types to obtain the desired form of the compound.

The resulting compound form is then used as an active ingredient in pharmaceutical compositions that are administered topically or by other non-systemic administration to a patient (e.g., a mammal, such as a human patient) to prevent/reduce the severity of epithelial cytotoxic side effects (e.g., hair loss, palmar-plantar syndrome, and/or mucositis) induced by chemotherapy treatments and/or radiotherapy.

To prevent/reduce the severity of hair loss (baldness) induced by chemotherapy and/or radiation therapy, a suitable form of the thiazoloindolinone compound of the invention is administered topically to the body part subject to baldness, such as the head (e.g., scalp, eyebrow area, beard or mustache area, etc.).

For such topical application, the CDK2 inhibitor compound is formulated into a composition suitable for topical administration, wherein the compound is combined with a selected pharmaceutically acceptable carrier, diluent or excipient, in an amount of the compound selected to provide adequate prevention or reduction in the severity of the side effect of hair loss when used in accordance with the appropriate treatment regimen. The composition may be in any suitable dosage unit for administration.

The formulation and administration of compositions containing thiazoloindolinone compounds may be by any suitable method, for example, a liquid formulation for aerosol spray administration to the scalp of patients predisposed to chemotherapy-induced alopecia, or a dermal patch or dressing from which the CDK2 inhibitor is capable of being released and which is placed on the scalp at the affected area.

The compositions of the invention may also be in the form of washes, ointments, gels, foams, pastes, oils, creams or other suitable forms suitable for application to the body, for example to the scalp or other parts of the head for the prevention/reduction of the severity of baldness, the initial application being followed by massage, brushing or towelling to ensure that the composition is evenly distributed over the scalp.

Compositions of the CDK2 inhibitors of the invention suitable for topical administration may be, for example, a formulation described in the following literature: Tata, S., et al., Relative influence of Ethanol and Propylene Glycol Cosolvents on Deposition of Minoxidil into the Skin, Journal of Pharmaceutical Sciences, 83, No. 10, October 1994, 15081510. The composition of this composition may be, for example, the following: a 2% solution of the active ingredient in 60% ethanol, 20% propylene glycol and 20% water, this composition can be administered topically to the scalp.

Another formulation that can be used for topical administration of the CDK2 inhibitors of the invention is, for example, as described in U.S. Patent Nos. 5,849,733; 5,807,698; 5,625,031 and 5,486,509, the entire contents of which are incorporated herein by reference.

-7 Suitable compositions containing CDK2 inhibitors according to the invention also include, for example, lipid-based compositions (e.g. Hoffman, R. M. et al., Liposomes Can Specifically Target Entrapped Melanin To Hair Follicles in Histocultured Skin, In Vivo Cell. Dev. Biol., 29A: 192-194, March 1993, and Niemiec, S. M. et al., Influence of Nonionic Liposomal Composition on Topical Delivery of Peptide Drugs into Pilosebaceous Units: An in Vivo Study Using the Hamster Ear Model, Pharmaceutical Research, 12, No. 8, 1995, 1184-1188), and polymeric microsphere compositions, for example (Rolland, A. et al., Site-Specific Drug Delivery to Pilosebaceous Structures Using Polymeric Microspheres, Pharmaceutical Research, 10, No. 12, 1993, 1738-1744).

The compositions may be formulated to contain doses corresponding to the desired dosing regimen. The doses and dosing regimens for a given patient will be determined by one skilled in the art based on the characteristics of the chemotherapy and/or radiation therapy being administered.

Similar considerations apply to the administration of CDK2 inhibitors for the prevention/reduction of palmoplantar syndrome, administration may be topical to areas of the hand or foot at risk from chemotherapy and/or radiation treatment.

To prevent/reduce the severity of mucosal inflammation, cyclin-dependent kinase II inhibitors can be formulated into suitable topical formulations suitable for administration to the mucosa of the oral cavity.

-8- . ·:

• ·· ·· ·· ··*

Such a composition containing a cyclin-dependent kinase II inhibitor useful for mucositis can be formulated, for example, as described in the following literature: Cullinan, US 5,496,828, March 5, 1996, Methods of Inhibiting Ulcerative Mucositis. Suitable compositions comprise the active ingredient and excipients, diluents or carriers and may be in the form of tablets, capsules, sprays, mouthwashes, sublingual tablets, wafers, lozenges, lozenges, suspensions, powders, etc., suitable for delivery to the oral mucosa. An acceptable daily dose of a CDK2 inhibitor for combating epithelial cytotoxic side effects induced by chemotherapy and/or radiation may be 0.1-1000 mg/day, preferably 50-200 mg/day.

The cyclin-dependent kinase II inhibitor of the invention is preferably administered concurrently with chemotherapy and/or radiation (i.e., concurrently with or in close enough time to the chemotherapy and/or radiation to provide a preventive or ameliorating effect on the therapy-induced epithelial cytotoxic side effect that would otherwise occur without the CDK2 inhibitor). The chemotherapy and/or radiation may be of any type used in neoplastic conditions and/or other disease states and conditions. For example, the chemotherapy may include cycle-specific agents (e.g., cytosine arabinoside, ARA-C) or non-cycle-specific agents (e.g., cytoxan), alone or in combination with other agents.

-9In one embodiment of the invention, the cyclin-dependent kinase II inhibitor is administered 1-4 times per chemotherapy cycle as a cytoprotective agent to prevent/reduce the severity of epithelial cytotoxic side effects, such as hair loss, alopecia, palmar-plantar syndrome and/or mucositis.

In the prevention/reduction of chemotherapy-induced hair loss and baldness, the effect of a cyclin-dependent kinase II inhibitor is to temporarily suspend the hair follicle cell cycle by inhibiting cyclin-dependent kinase II activity. For such purposes, the inhibitor is administered in the form of a suitable topical preparation, preferably 1-2 times before and during the chemotherapy cycle.

In a preferred embodiment of the invention, the cyclin-dependent kinase II inhibitor is administered topically to prevent or reduce chemotherapy-induced hair loss, baldness, and the compound is selected based on compliance with the following characteristics:

(1) the IC50 value is less than 2.5 nmol, preferably less than 20 nmol against CDK2;

(2) the IC50 value is less than 1.5 pmol, preferably less than 5 pmol in the G1 checkpoint assay;

(3) show reproducible protection in a rat pup alopecia model when using a combination of at least two different cytotoxic agents, one of which is an alkylating agent (e.g., the combination includes: doxorubicin/cyclophosphamide (anthracycline/alkylating agent), etoposide (topoisomerase II inhibitor), taxol, etc.);

(4) at a topical dose of 10 mg/kg body weight, the plasma concentration should be less than 15 nmol and preferably, at a systemic application, an IC50 concentration of less than 0.01 for protection of the HT29 tumor cell line;

(5) the dermal irritation profile should be acceptable; and (6) it should be suitable for a given topical formulation (compatibility, bioavailability, etc.).

The appropriate form of the compound of the invention can be formulated into a topical formulation for use in doses and regimens suitable for preventing/reducing the severity of palmoplantar syndrome in the context of chemotherapy and/or radiation therapy.

In patients treated with chemotherapy and/or radiation, to prevent/reduce the severity of mucositis, a suitable form of the compound of the invention is preferably formulated into a topical preparation suitable for delivery to the oral mucosa, such as a mouthwash, sublingual tablet or lozenge.

The thiazoloindolinone compound of the invention is capable of crystallizing in more than one form, a property called polymorphism, and all such polymorphic forms (polymorphs) are within the scope of the invention. Polymorphism generally occurs in response to changes in temperature or pressure, or both, and may also occur by altering the crystallization process. Polymorphs are distinguished by different physical characteristics, typically X-ray diffraction patterns, solubility properties, and the melting point of the compound.

- 11 ...· ·..· ··.· '

The term "near" used in connection with melting point values, expressed in °C, means a value of ±2°C.

The reagents used in the process of the invention are commercially available or can be prepared as described in the literature. The physical data given for the compounds are consistent with the structures of the compounds. The 'H NMR spectra were recorded on a Varian Unity Plus NMR spectrophotometer at 300 or 40 MHz. The mass spectral data were recorded on a Micromass Platform II mass spectrometer (manufactured by Micromass Ltd., Altribcham, UK) using either atmospheric chemical ionization (APCI) or Electrospray Ionization (ESI). Analytical thin layer chromatography (TLC) was used to confirm the purity of certain intermediates that could not be isolated or were too unstable for full analysis, thus following the course of the reaction. Unless otherwise stated, silica gel (Merck Silica Gel 60 F254) was used. Unless otherwise noted, Merck Silica Gel 60 (230-400 mesh) silica gel and the indicated solvent system were used for column chromatography under pressure for purification of compounds.

Melting point values for each thiazoloindolinone compound were determined using a differential scanning calorimeter (TA model 2910). Sample weights were 1-3 mg in each case. The samples were placed in an aluminum corrugated vessel and purged with nitrogen at a flow rate of 40 ml/min. The heating rate was 10°C/min.

- 12Recrystallization of the given forms was performed in each case by dissolving the compound in the selected solvent, storing it at ambient temperature (e.g. 25°C) until the solvent evaporated. The remaining solid was then analyzed by X-ray diffraction.

To determine the non-solvent, the compound was dissolved in the selected solvent at 25°C, and then water or alcohol was added to precipitate the solid. The resulting solid was analyzed by X-ray diffraction.

For X-ray powder diffraction analysis, the powdered compound was sprinkled onto a quartz zero-background plate and scanned at 2 theta angles between 5° and 40° using a Scintag PADV or XDS2000 type instrument.

The different forms of the thiazoloindolinone compounds of the invention are designated as Forms A, B, and C, respectively.

The form

Form A of the thiazoloindolinone compound of formula (I) according to the invention was characterized by its melting point, X-ray diffraction crystallographic spectrum, solubility in solvent medium, anti-solvent nature and preparation method.

The melting point of Form A, as determined above, is 235°C.

When the thiazoloindolinone compound of the invention is dissolved in ethyl acetate and the solvent is cooled to 5°C, Form A precipitates.

The solubility of Form A (expressed in mg/ml) in polyethylene glycol 400, transcutol and methanol solvents is as follows:

- 13 Solvent

PEG 400 transcutol methanol

Solubility 4.76 mg/ml 3.15 mg/ml 0.30 mg/ml

In the slurry equilibrium study, acetone, n-butanol or methylene chloride is added to the Form A compound to form an equilibrated suspension at 25°C, which is then analyzed by powder X-ray diffraction, and no change in form occurs due to equilibration.

In the anti-solvent characterization, when the thiazoloindolinone compound of the invention is precipitated from dimethyl sulfoxide using water or alcohol as an anti-solvent, Form A is formed.

According to the X-ray diffraction study of Form A, the X-ray diffraction spectrum of this polymorphic modification is as shown in Figure 1 (A1 spectrum and A2 curve), where the given curves are derived from different doses of the compound. The X-ray diffraction values of Form A are summarized in Table 1.

Table 1

X-ray diffraction characteristics of the shape: 2 theta and relative intensity values

2-theta Relative intensity 2.84 5 3.12 3 3.46 4 3.58 3 3.64 3

3.80 4 3.97 5 6.49 40 7.08 79 10.75 14 12.01 100 12.78 54 14.19 16 15.37 4 16.44 36 17.38 13 17.69 36 18.00 33 18.76 13 19.52 36 20.77 4 21.19 9 21.36 15 21.59 10 21.80 11 22.79 39 23.12 75 24.00 23 24.61 14 25.42 31 27.78 24 28.92 12

31.90 4 35.82 3 35.99 4

Form B

Form B of the thiazoloindolinone compound of formula (I) is characterized by its melting point, X-ray crystallographic spectrum, solubility in solvents and method of preparation.

The melting point of Form B, as determined above, is 247°C.

The solubility of Form B in mg/ml in polyethylene glycol 400, transcutol and methanol solvents is as follows:

Solvent Solubility

PEG 400 4.5 mg/ml transcutol 2.77 mg/ml methanol 0.08 mg/ml

According to the X-ray diffractogram of Form B, this is a polymorphic modification with an X-ray diffraction spectrum as shown in Figure 1 (B spectrum). The X-ray diffraction values are summarized in Table 2.

Table 2

X-ray diffraction characteristics of form B: 2 theta and relative intensity values

2-theta Relative intensity 6.75 13 7.00 33 11.54 7 12.00 72 13.51 36 14.06 14 16.14 37 17.67 27 18.23 32 18.54 8 18.89 18 20.35 21 21.19 22 21.61 12 23.12 14 24.28 14 24.83 1 25.72 3 26.69 100 27.32 5 28.61 2 28.81 2 30.22 2 30.35 2

- 17·· ·· ····· ·· ·· ···« ⁴

30.77 1 31.08 1 31.67 1 33.62 2 35.15 1 35.70 4 35.97 1 36.94 2 37.64 1 38.36 3 40.99 1 42.47 1 42.68 3 44.88 1

C-shape

Form C of the thiazoloindolinone compound of formula (I) is characterized by its melting point, X-ray crystallographic spectrum, anti-solvent properties and method of preparation.

The melting point of Form C is determined as above to be 243°C. In the anti-solvent characterization, when the thiazoloindolinone compound of the invention is precipitated from n-methylpyrrolidone using water or alcohol as an anti-solvent, Form C is formed.

X-ray diffraction analysis of form C shows a modification with an X-ray diffraction spectrum as shown in Figure 2 (curve C). The X-ray diffraction values of form C are summarized in Table 3.

Table 3

X-ray diffraction characteristics of form C: 2 theta and relative intensity values

2-theta Relative intensity 12.88 21 13.64 49 14.66 22 16.63 9 17.69 76 18.08 17 18.68 43 19.15 87 19.70 9 20.27 39 20.60 28 20.96 37 22.04 100 24.02 58 24.68 31 25.37 17 26.64 34 28.69 30 29.60 29 30.30 7 31.10 10 31.52 12 31.84 10 33.16 9

33.62 8 35.77 6 36.41 15

The features of the invention are illustrated in more detail by the following non-limiting examples.

Example 1

Preparation of 4-Amino-N-(4-aminomethylpyridinyl)benzenesulfonamide

In a 250 ml round bottom flask, 50 ml anhydrous pyridine, 10.4 g (50.0 mmol) 4-(aminomethyl)pyridine and a magnetic stir bar were placed. The mixture was cooled to 0°C under nitrogen with stirring, and 12.8 g (55.0 mmol) N-acetylsulfanyl chloride was added. The resulting mixture was stirred at 0°C under nitrogen for 5 min, then allowed to warm to room temperature and stirred for an additional 16 h. The mixture was then concentrated to a thick solid and poured into approximately 500 g of ice water. The residue in the flask was rinsed into ice water with 25 ml of MeOH to precipitate the N-acetylsulfanylamide. The resulting precipitate was filtered, washed with excess water, and dried under vacuum at 50°C. The resulting solid was suspended in 75 ml of 1N hydrochloric acid and heated at 100°C until all the starting material was consumed. The mixture was then cooled, neutralized with ammonium hydroxide, the precipitate was filtered, and dried under vacuum at 50°C to give 5.78 g (43.9%) of the title compound.

*H NMR (DMSO-d ₆ ): δ 8.42 (d, 2H), 7.76 (t, 1H), 7.39 (d, 2H), 7.22 (d, 2H), 6.56 (d, 2H), 5.91 (s, 2H), 3.89 (d, 2H); APCI-MS m/z 264 (MH) ⁺

Example 2

Preparation of 6,8-Dihydro-7H-[1,3]thiazolo[5,4-e]indol-7-one

A 2 L three-necked round-bottomed flask was fitted with an internal thermometer, a 200 mL addition funnel, a magnetic stir bar and a stopper, the flask was flushed with nitrogen, 200 mL anhydrous THF and 15.2 g (0.100 mol) 6-aminobenzothiazole were added. The resulting mixture was stirred and cooled to -74°C with a dry ice/acetone bath. A solution of 11.0 g (0.103 mol) tert-butyl hypochlorite in 50 mL dichloromethane was then added over a period of approximately 15 min. The resulting solution was then stirred for a further 3 h at the temperature of the dry ice/acetone bath, and then 13.8 g (0.103 mol) ethyl methyl thioacetate in 50 mL dichloromethane was slowly added dropwise. The resulting solution was stirred for a further 3 hours at the dry ice/acetone temperature. Then 25.3 g (0.250 mol) of triethylamine dissolved in 50 ml of dichloromethane was added at the dry ice/acetone bath temperature and the mixture was stirred for 0.5 hour. The cooling bath was then removed, the mixture was allowed to warm to room temperature and then concentrated to a thick material. This thick oil was suspended in 200 ml of ether and 600 ml (0.25 mol) of hydrochloric acid and stirred for 24 hours. The resulting solid was filtered and stirred with water and ether. The solid was then suspended in cold MeOH, filtered and dried in vacuo for 16 hours to give 18.7 g.

-21 (79%) 8-(methylsulfanyl)-6,8-dihydro-7H-[1,3]thiazolo[5,4-e]indol-7-one is obtained:

1H NMR (DMSO-d ₆ ) δ 10.8 (s, 1H), 9.2 (s, 1H), 8.0 (d, 1H), 7.1 (d, 1H), 1.8 (s, 3H); APCI-MS m/z 235 (MH)'

A 500-mL Erlenmeyer flask was equipped with a stir bar, 8.1 g (0.034 mol) of 8-(methylsulfanyl)6,8-dihydro-7H-[1,3]thiazolo[5,4-e]indol-7-one and 100 mL of glacial acetic acid were weighed out. The resulting mixture was stirred until the starting material dissolved. The mixture was then diluted with 100 mL of THF, 325 mesh zinc metal was added, and the resulting heterogeneous mixture was stirred at 60°C for 2.5 hours. The material was filtered through a 1/2-inch pad of Celite, and the filter residue was washed with additional THF. The filtrates were combined, concentrated to give a wet solid, which was mixed with MeOH, air filtered and dried to give 4.52 g (70%) of the desired compound as a free flowing solid:

*H NMR (DMSO-d ₆ ): δ 10.5 (s, 1H), 9.1 (s, 1H), 7.9 (d, 1H), 7.0 (d, 1H), 3.6 (s, 2H); APCI-MS m/z 191

Example 3 Preparation of 8-[Ethoxymethylidene]-6H-[1,3]thiazolo[5,4-e]indol-7-one

A 250 ml round bottom flask was fitted with a stirring rod, 4.0 g (0.021 mol) of 6,8-dihydro-7H-[1,3]thiazolo[5,4-e]indol-7-one, 40 ml of glacial acetic acid and 17 g (0.105 mol) of diethoxymethyl acetate were weighed out. A reflux condenser was fitted to the flask and flushed with nitrogen. The mixture was then heated under reflux for 8 h and then cooled, the stirring rod being 22'··’ z.

The rod is removed and the mixture is concentrated. The resulting wet solid is then stirred with a solution of ether and ethanol, the mixture is filtered, the solid is washed with an ethanol/ether solution and dried in vacuo to give the title compound.

1H NMR (DMSO-d ₆ ): δ 10.5 (s, 1H), 9.1 (s, 1H), 7.8 (d, 1H), 7.7 (s, 1H), 7.0 (d, 1H), 4.5 (q, 2H), 1.4 (t, 3H); APCI-MS m/z 245 (MH)'

Example 4

Preparation of 4-{[(7-Oxo-6,7-dihydro-8H-[1,3]thiazolo[5,4-e]indol-8-ylidene)methyl]amino}-N-(4-pyridinylmethyl)benzenesulfonamide Form A

a) A mixture of 6.91 g (28.0 mmol) of 8-[ethoxymethylidene] 6H-[1,3]thiazolo[5,4-e]indol-7-one and 7.52 g (28.0 mmol) of 4-amino-N-(4-pyridinylmethyl)benzenesulfonamide in 150 ml of ethanol was heated under reflux for 22 hours. The resulting hot mixture was then filtered, the solid was air-dried and washed with 30 ml of hot ethanol. The filter cake was dried under high vacuum at room temperature for 48 hours to give the thiazoloindolinone compound as a yellow solid, yielding 11 g (85%). 'H NMR (DMSO-d ₆ ): δ 4.02 (d,J = 6.4 Hz, 2H) 7.11 (d,J = 8.4 Hz, 1H), 7.24 (d,J = 5.7 Hz, 2H), 7.56 (d,J = 8.8 Hz, 2H), 7.77 (d,J = 8.6 Hz, 2H), 7.81 (d,J = 8.4 Hz, 1H), 8.05 (d,J = 12.1 Hz, 1H), 8.24 (t,J = 6.3 Hz, 1H), 8.44 (d,J = 5.8 Hz, 2H), 9.26 (s, 1H), 10.92 (s, 1H), 11.15 (d,J = 12.1 Hz, 1H);

Elemental analysis for the compound with the formula C ₂ 2Hi7N ₅ S ₂ O · 0.25 H ₂ O:

Calcd: C, 56.46; H, 14.96; S, 13.70;

Found: C, 56.41; H, 3.72; N, 14.89; S, 13.77

b) A three-necked round-bottomed flask is equipped with a paddle stirrer, a thermometer and a condenser and 329 g (1.25 mol) of 8-[ethoxymethylidene]-6H-[1,3]thiazolo[5,4-e]indol-7-one, 293 g (1.19 mol) of 4-amino-N-(4-pyridinylmethyl)benzenesulfonamide and 15540 ml of absolute alcohol are weighed in. The resulting light orange slurry is heated at gentle reflux (77-78°C) for 27 hours to give a dark brick-red slurry. The brick-red solid is separated by filtration and washed with 1500 ml of hot ethanol. The solid filter cake is then stirred with a spatula, washed three times with 1000-1000 ml of boiling ethanol, and the solid is transferred to a drying tray and dried under vacuum at 50-60°C for 12 hours. The solid is then gently stirred and dried under vacuum at 60°C for a further 20 hours.

Elemental analysis for the compound with the formula C ₂ 2H]7N ₅ O ₃ S2 · 0.20 C ₂ H ₆ O:

Calcd: C, 56.91; H, 3.88; N, 14.81; S, 13.57;

Found: C, 56.22; H, 3.87; N, 14.77; S, 13.77

c) A 20 mm x 150 mm test tube is fitted with a magnetic stir bar and 0.123 g (0.5 mol) of 8-[ethoxymethylidene]-6H-[1,3]thiazolo[5,4-e]indol-7-one, 0.132 g (0.5 mmol) of 4-amino-N-(4-pyridinylmethyl)benzenesulfonamide and 5 ml of 95% ethanol are weighed. The mixture is then sealed with a rubber stopper (14/22) and vented with a 3 inch - 18 gauge syringe needle. The reaction vessel is then cooled to room temperature in an oil bath.

-24 °C, then the oil bath temperature was raised to 78 °C and maintained at this temperature for about 24 hours. The reaction vessel was then removed from the oil bath and allowed to cool to the touch. The precipitate formed was filtered off with suction, the solid was washed with excess 95% ethanol and collected by suction. The resulting solid was dried under vacuum at 40 °C for 2 hours and allowed to cool to give 52 mg (11%) of the title compound.

MS (M+H)=464.

Example 5 Preparation of 4-{[(7-Oxo-6,7-dihydro-8H-[1,3]thiazolo[5,4-e]indol-8-ylidene)methyl]amino}-N-(4-pyridinylmethyl)benzenesulfonamide Form B

a) A mixture of 51.0 g (207 mmol) of 8-[ethoxymethylidene]-6H-[1,3]thiazolo[5,4-e]indol-7-one and 57.1 g (217 mmol) of 4-amino-N-(4-pyridinylmethyl)benzenesulfonamide in 1.2 l of ethanol was heated under reflux for 22 hours, and the resulting mixture was concentrated to near dryness by distilling off the ethanol. The resulting concentrate was diluted with 800 ml of ethanol and heated at 80°C for 1 hour. The warm mixture was then filtered, the solid was washed with ethanol, and the filter cake was dried under high vacuum at room temperature to give the thiazoloindolinone compound as a yellow solid, 88.9 g (93%).

1H NMR (DMSO-d ₆ ): δ 4.02 (d,J - 6.4 Hz, 2H), 7.11 (d,J = 8.4 Hz, 1H), 7.24 (d,J = 5.7 Hz, 2H), 7.56 (d,J = 8.8 Hz, 2H), 7.77 (d,J = 8.6 Hz, 2H), 7.81 (d,J = 8.4 Hz, 1H), 8.05 (d,J

-25 = 12.1 Hz, 1H), 8.24 (t,J = 6.3 Hz, 1H), 8.44 (d,J - 5.8 Hz, 2H), 9.26 (s, 1H), 10.92 (s, 1H), 11.15 (d,J = 12.1 Hz, 1H);

Elemental analysis for the compound with the formula C ₂ 2H ₁₇ N5S ₂ O3 · 0.25 H ₂ O · 0.02 C ₂ H ₆ O:

Calcd: C, 56.45; H, 3.79; N, 14.93; S, 13.68;

Found: C, 56.47; H, 3.78; N, 14.71; S, 13.80

b) A magnetic stir bar is placed in a 250 ml round bottom flask, 1.98 g (8.05 mmol) of 8-[ethoxymethylidene]-6H-[1,3]thiazolo[5,4-e]indol-7-one and 2.16 g (8.2 mmol) of 4-amino-N-(4-pyridinylmethyl)benzenesulfonamide and 80 ml of 95% ethanol are weighed. The round bottom flask is equipped with a water-cooled reflux condenser, then the vessel is placed in an oil bath at room temperature and the temperature of the oil bath is raised while stirring and the mixture is heated at gentle reflux for about 36 hours. The reaction vessel is then removed from the oil bath, the mixture is filtered, and the solid is separated by suction. The resulting solid was washed with excess 95% ethanol and then filtered off with suction. The solid was dried under vacuum at room temperature for 10 days to give 2.785 g (74.7%) of the thiazoloindolinone compound.

MS (M+H) = 464

Example 6

Preparation of a mixture of dihydro-8H-[1,3]thiazolo[5,4-e]indol-8-ylidene)methyl]amino}-N-(4-pyridinylmethyl)benzenesulfonamide forms A and B

A magnetic stir bar is placed in a 250 ml round bottom flask, 4.11 g (17.12 mmol) of 8-[ethoxymethylidene-6H-[1,3]thiazolo[5,4-e]indol-7-one and 4.62 g (17.55 mmol) of 4-26...· ·..· ·..· ··.·· j.

-amino-N-(4-pyridinylmethyl)benzenesulfonamide and 150 ml of 95% ethanol were added. The round bottom flask was equipped with a water-cooled reflux condenser and then placed in a room temperature oil bath and the temperature of the oil bath was raised while stirring by heating the mixture at gentle reflux for 48 hours. The reaction vessel was then removed from the oil bath, the hot mixture was filtered, the solid was suction filtered, the resulting solid was washed with 95% alcohol and suction filtered. The solid was then dried under vacuum for 2 days at room temperature to give 5.143 g (64.8%) of the thiazoloindolinone compound.

MS (M+H) = 464

Example 7

The compound 4-(((7-oxo-6,7-dihydro-8H-[1,3]thiazolo[5,4-e]indol-8-ylidene)methyl)amino)-N-(4-pyridinylmethyl)benzenesulfonamide was subjected to crystallizations with different solvents and crystallization methods, which are summarized in the following Table 4.

-η Table 4

Crystallization experiments

Crystallization solvent Initial form Crystallization method Final form Note DMSO The form Evaporative at 25°C The form evaporation is very slow DMSO The form Anti-solvent water at 25°C The form DMSO The form Anti-solvent water at 37°C, then cooled to 25°C The form DMSO The form Anti-solvent alcohol at 25°C The form DMSO The form Anti-solvent alcohol at 37°C, then cooling to 25°C The form Pyridin The form Evaporation at 25°C The form Low solubility DMF Form B Evaporation at 25°C The form Ethyl acetate Form B Evaporation at 25°C The form Low solubility Ethyl acetate The form Cooling at 5°C The form (less than 0.06 mg/ml) N-methylpyrrolidone Form B Water as anti-solvent at 25°C Form C confirmed by PXRD n-Butanol The form Slurry equilibration at 25°C The form

Acetone The form Slurry equilibration at 25°C The form Methylene chloride The form Slurry equilibration at 25°C The form Tetrahydrofuran The form Evaporation at 25°C The form PEG 400 The form Water as anti-solvent at 25°C Form A + Form B PEG 400 The form Alcohol as an anti-solvent at 25°C Form A + Form B

The abbreviations in the table above have the following meanings:

DMSO = dimethylsulfoxide; DMF = dimethylformamide;

PEG 400 = polyethylene glycol with a molecular weight of 400;

PXRD = powder X-ray diffraction.

For relatively non-volatile solvents (DMSO, DMF) complete evaporation is very difficult at 25°C. The presence of the solvent is evident as an amorphous hump appears in the PXRD curve. Cooling at 5°C was less successful than other crystallization approaches due to the low solubility of 4-(((7-oxo-6,7-dihydro-8H-[1,3]thiazolo[5,4-e]indol-8-ylidene)methyl)amino)-N-(4-pyridinylmethyl)benzenesulfonamide under these conditions.

Although the solution according to the invention has been presented through various embodiments and features, it is obvious that other embodiments and modifications fall within the scope of the invention.

-29ations. Thus, the invention should be interpreted broadly and includes such variations, modifications and other embodiments within its scope, the scope of which is defined by the claims.

The present description and claims may be used as a basis for priority in any subsequent application. The claims of such subsequent applications may be directed to any feature or combination thereof described herein. It may be a product, composition, process or use and may include, without limitation, one or more of the following claims.

Claims (49)

-30Patent claims 1. A thiazoloindolinone compound of formula (I) having a melting point of around 235°C. 2-theta Relative intensity 2-theta Relative intensity 2.84 5 2-theta Relative intensity 2. The thiazoloindolinone compound according to claim 1, having an X-ray diffraction spectrum corresponding to the A1 and A2 spectra shown in Figure 1. 3.97 5 3.80 4 3.64 3 3.58 3 3.46 4 3.12 3 3. The thiazoloindolinone compound of claim 1, having the following solubility characteristics: Solvent Solubility PEG 400 4.76mg/ml transcutol 3.15mg/ml methanol 0.30mg/ml 4. The thiazoloindolinone compound according to claim 1, having the following X-ray diffraction characteristics: 5. A thiazoloindolinone compound prepared by dissolving the compound of formula (I) according to claim 1 in a crystallization solvent and then evaporating the solvent at ambient temperature, wherein the thiazoloindolinone compound is a crystalline solid and the crystallization solvent is one of dimethylformamide, dimethylsulfoxide, pyridine, ethyl acetate or tetrahydrofuran. 6.75 13 6. A compound formed by dissolving the compound of claim 1 in dimethylsulfoxide and then adding water or alcohol as a reagent to the resulting solution, said reagent being in an amount sufficient to precipitate the compound. 6.49 40 7.00 33 7. A compound prepared by dissolving the compound of claim 1 in ethyl acetate and then cooling the resulting solution, whereby said thiazoloindolinone compound precipitates. 7.08 79 8. 4-{[(7-oxo-6,7-dihydro-8H-[1,3]thiazolo[5,4-e]indol-8-ylidene)methyl]amino}-N-(4-pyridinylmethyl)benzenesulfonamide Form A. 9. A pharmaceutical composition comprising 4-{[(7-oxo-6,7-dihydro-8H-[1,3]thiazolo[5,4-e]indol-8-ylidene)methyl]amino}-N-(4-pyridinylmethyl)benzenesulfonamide Form A. 10. A pharmaceutical composition comprising 4-{[(7-oxo-6,7-dihydro-8H-[1,3]thiazolo[5,4-e]indol-8-ylidene)methyl]amino}-N(4-pyridinylmethyl)benzenesulfonamide Form A and which is suitable for preventing or reducing the severity of epithelial cell toxicity induced by chemotherapy and/or radiation therapy, by topically applying the composition to the epithelial site showing epithelial cell toxicity. 10.75 14 11.54 7 A pharmaceutical composition comprising a thiazoloindolinone compound according to any one of claims 1-8 together with one or more pharmaceutically acceptable carriers, excipients or diluents. 12.88 21 12.00 72 12. A thiazoloindolinone compound of formula (I) according to claim 1, having a melting point of about 247°C. 12.78 54 12.01 100 13.64 49 13.51 36 13. A thiazoloindolinone compound of formula (I) according to claim 1, having an X-ray diffraction spectrum as shown in Figure 1 B. 14.66 22 14.06 14 14. A thiazoloindolinone compound of formula (I) according to claim 1, having the following solubility characteristics: Solvent Solubility PEG 400 4.5 mg/ml transcutol 2.77 mg/ml methanol 0.08 mg/ml 14.19 16 15. A thiazoloindolinone compound of formula (I) according to claim 1, having the following X-ray diffraction characteristics: 15.37 4 16.63 9 16. 4-{[(7-oxo-6,7-dihydro-8H-[1,3]thiazolo[5,4-e]indol-8-ylidene)methyl]amino}-N-(4-pyridinylmethyl)benzenesulfonamide Form B. 16.14 37 16.44 36 17.69 76 17. A pharmaceutical composition comprising 4-{[(7-oxo-6,7-dihydro-8H-[1,3]thiazolo[5,4-e]indol-8-ylidene)methyl]amino}-N-(4-pyridinylmethyl)benzenesulfonamide form B. 17.67 27 17.69 36 17.38 13 18.68 43 18.08 17 18. A pharmaceutical composition comprising 4-{[(7-oxo-6,7-dihydro8H-[1,3]thiazolo[5,4-e]indol-8-ylidene)methyl]amino}-N-(4pyridinylmethyl)benzenesulfonamide form B and which is suitable for use in combination with chemotherapy and/or radiation therapy. 18.89 18 18.54 8 18.23 32 18.76 13 18.00 33 19.70 9 19.15 87 19. A pharmaceutical composition comprising a thiazoloindolinone compound according to any one of claims 12-16 together with one or more pharmaceutically acceptable carriers, excipients or diluents. 19.52 36 20.96 37 20.60 28 20.27 39 20. A thiazoloindolinone compound of formula (I) according to claim 1, having a melting point of about 243°C. 20.35 21 20.77 4 21. A thiazoloindolinone compound of formula (I) according to claim 1, having an X-ray diffraction spectrum as shown in Figure 2 C. 21.61 12 21.19 22 21.80 11 21.59 10 21.36 15 21.19 9 22.04 100 22. A thiazoloindolinone compound of formula (I) according to claim 1, having the following X-ray diffraction characteristics: 22.79 39 23. A thiazoloindolinone compound prepared by dissolving the compound of formula (I) according to claim 1 in N-methylpyrrolidone and then adding water or alcohol as an anti-solvent to the resulting solution, said anti-solvent being added in an amount necessary to precipitate said thiazoloindolinone compound. 23.12 14 23.12 75 24. 4-{[(7-oxo-6,7-dihydro-8H-[1,3]thiazolo[5,4-e]indol-8-ylidene)methyl]amino}-N-(4-pyridinylmethyl)benzenesulfonamide Form C. 24.68 31 24.02 58 24.83 1 24.28 14 24.61 14 24.00 23 25. A pharmaceutical composition comprising Form C of 4-{[(7-oxo-6,7-dihydro8H-[1,3]thiazolo[5,4-e]indol-8-ylidene)methyl]amino}-N-(4-pyridinylmethyl)benzenesulfonamide. 25.37 17 25.72 3 25.42 31 26. A pharmaceutical composition comprising 4-{[(7-oxo-6,7-dihydro8H-[1,3]thiazolo[5,4-e]indol-8-ylidene)-methyl]amino}-N-(4pyridinylmethyl)-benzenesulfonamide Form C and suitable for preventing or reducing the severity of epithelial cell toxicity induced by chemotherapy and/or radiation therapy, by topically applying the composition to the epithelial site showing epithelial cell toxicity. 26.64 34 26.69 100 27. A pharmaceutical composition comprising a thiazoloindolinone compound according to any one of claims 20-24 together with one or more pharmaceutically acceptable carriers, excipients or diluents. 27.32 5 27.78 24 28. A process for the preparation of a thiazoloindolinone compound of formula (I) according to claim 1, characterized in that at least one process step of the synthesis according to reaction scheme 1 is carried out, thus obtaining 4-{[(7-oxo-6,7-dihydro-8H-[1,3]thiazolo[5,4-e]indol-8-ylidene)methyl]amino}-N(4-pyridinylmethyl)benzenesulfonamide as the product; treating the resulting product to give 4-{[(7-oxo-6,7-dihydro-8H-[1,3]thiazolo[5,4-e]indol-8-ylidene)methyl]amino}-N(4-pyridinylmethyl)benzenesulfonamide Form A, B or C; and recovering said thiazoloindolinone compound. 28.69 30 28.81 2 28.61 2 28.92 12 29. The process according to claim 28, characterized in that in said treatment the product obtained is dissolved in a solvent, then the solvent is evaporated from the obtained solution and the thiazoloindolinone compound is recovered. 29.60 29 30. The process according to claim 29, characterized in that one of the following solvents is used as the solvent: 30.30 7 30.77 1 30.35 2 30.22 2 31. The process of claim 28, wherein the treatment comprises dissolving the product in a solvent and then cooling the resulting solution to precipitate said thiazoloindolinone compound. 31.84 10 31.52 12 31.10 10 31.67 1 31.08 1 31.90 4 32. The process of claim 31, wherein the solvent is ethyl acetate and said thiazoloindolinone compound is Form A. -32...· ·..· ·..· ··/· j. 33. The method according to claim 28, characterized in that the treatment comprises dissolving the product in a solvent and then adding an anti-solvent to the resulting solution, which is water or an alcohol, thereby precipitating the thiazoloindolinone compound. 33.62 8 33.16 9 33.62 2 34. The process of claim 33, wherein the solvent is dimethylsulfoxide and said thiazoloindolinone compound is Form A. -34·.?·..· ’*í* -υ 35. The process of claim 33, wherein the solvent is N-methylpyrrolidone and the anti-solvent is water and said thiazoloindolinone compound is form C. 35.77 6 - To prevent or reduce the severity of 35-dish induced epithelial cell toxicity, the preparation is applied topically to the epithelial site showing epithelial cell toxicity. 35.97 1 35.70 4 35.15 1 35.99 4 35.82 3 36. The method of claim 28, wherein said thiazoloindolinone compound is Form A. 36.41 15 36.94 2 37. The method of claim 28, wherein said thiazoloindolinone compound is Form B. -37*»*· ·.?·.·· ·’/* «Λ 37.64 1 38. The method of claim 28, wherein said thiazoloindolinone compound is in Form C. -38 .J··.?·*-”’’ «/. dimethylformamide, dimethylsulfoxide, pyridine, ethyl acetate or tetrahydrofuran and said thiazoloindolinone compound is in Form A. 38.36 3 -39···..-.: : 'ψ j Forms A, B and C for use in the prevention/reduction of the severity of symptoms of epithelial cytotoxicity occurring as a side effect of chemotherapy and/or radiation therapy. 39. 4-{[(7-oxo-6,7-dihydro-8H-[1,3]thiazolo[5,4-e]indol8-ylidene)methyl]amino}-N-(4-pyridinylmethyl)benzenesulfonamide -40- .: »·· ····<» for use in topical administration to the palms and/or feet in reducing symptoms. 40. Form A of 4-{[(7-oxo-6,7-dihydro-8H-[1,3]thiazolo[5,4-e]indol-8-ylidene)methyl]amino}-N-(4-pyridinylmethyl)benzenesulfonamide for use according to claim 39. 40.99 1 41. Form B of 4-{[(7-oxo-6,7-dihydro-8H-[1,3]thiazolo[5,4-e]indol-8-ylidene)methyl]amino}-N-(4-pyridinylmethyl)benzenesulfonamide for use according to claim 39. 42. Form C of 4-{[(7-oxo-6,7-dihydro-8H-[1,3]thiazolo[5,4-e]indol-8-ylidene)methyl]amino}-N-(4-pyridinylmethyl)benzenesulfonamide for use according to claim 39. 42.68 3 42.47 1 43. The use of claim 39, wherein the side effect of epithelial cytotoxicity is hair loss (baldness). 44. The use of claim 39, wherein the side effect of epithelial cytotoxicity is palmoplantar syndrome. 44.88 1 45. The use of claim 39, wherein the side effect of epithelial cytotoxicity is mucosal inflammation. 46. Use according to claim 39 of 4-{[(7-oxo-6,7-dihydro-8H-[1,3]thiazolo[5,4-e]indol-8-ylidene)methyl]amino}-N(4-pyridinylmethyl)benzenesulfonamide form A, B or C for the prevention/reduction of symptoms of chemotherapy-induced hair loss, baldness. 47. Use according to claim 39 of 4-{[(7-oxo-6,7-dihydro-8H-[1,3]thiazolo[5,4-e]indol-8-ylidene)methyl]amino}-N(4-pyridinylmethyl)benzenesulfonamide form A, B or C for the prevention/treatment of chemotherapy-induced palmar-plantar syndrome. 48 Use according to claim 39 for topical administration of 4-{[(7-oxo-6,7-dihydro-8H-[1,3]thiazolo[5,4-e]indol-8-ylidene)methyl]amino}-N(4-pyridinylmethyl)benzenesulfonamide form A, B or C to the oral cavity for the prevention/alleviation of symptoms of chemotherapy-induced mucositis. 49. Use according to claim 39 for topical application of 4-{[(7-oxo-6,7-dihydro-8H-[1,3]thiazolo[5,4-e]indol-8-ylidene)methyl]amino}-N(4-pyridinylmethyl)benzenesulfonamide form A, B or C to a body site at risk for the treatment of epithelial cytotoxicity induced by chemotherapy and/or radiation therapy. The authorized person: DANUBE Patent and Trademark Office Ltd. Gézán Olchváry is a patent attorney