HK1169408B - Cdk inhibitor salts - Google Patents

Description

Salts of CDK inhibitors

The present invention relates to novel water-soluble salts of crystalline, cdk inhibitors, processes for their preparation, hydrates and polymorphs of the novel salt forms, their use in therapy and pharmaceutical compositions containing them.

It is well known that the progression of the cell cycle is controlled by a series of checkpoints, also known as restriction points, which are regulated by a family of enzymes known as cyclin-dependent kinases (cdks). In turn, cdks themselves are regulated at many levels, e.g., binding to cyclin.

Coordinated activation and inactivation of different cyclin/cdk complexes is essential for normal progression through the cell cycle. The key G1-S and G2-M transitions are both controlled by distinct cyclin/cdk activity activations. In G1, cyclin D/cdk4 and cyclin E/cdk2 are both thought to mediate the onset of S-phase. Progression to S-phase requires the activity of cyclin A/cdk2, while initiation of mitosis requires activation of cyclin A/cdc2(cdk1) and cyclin B/cdc 2. General references to cyclins and cyclin-dependent kinases can be found, for example, in: kevin R.Webster et al, Exp.Opin.Inves t.drugs, 1998, Vol.7(6), 865-.

Checkpoint controls are defective in tumor cells, which may be due in part to dysregulation of cdk activity. For example, altered cyclin E and cdk expression has been observed in tumor cells, and deletion of the cdk inhibitor p27KIP gene in mice has been shown to result in higher incidence of cancer.

There is increasing evidence to support the notion that cdks are rate-limiting enzymes in cell cycle progression and therefore represent target molecules for therapeutic intervention. In particular, direct inhibition of cdk/cyclin kinase activity should help to limit the dysregulated proliferation of tumor cells.

Some pyrazoloquinazolines have been shown to be potent inhibitors of cyclin dependent kinase enzymes, particularly cdk 2. One of these compounds is currently being developed as an anticancer agent. cdk inhibitors are believed to block passage of cells from the G2/M phase of the cell cycle.

In a first aspect, it is an object of the present invention to provide a salt of compound 125 having the formula:

in this specification, unless otherwise indicated, compound 125 is 8- [4- (4-methyl-piperazin-1-yl) -phenylamino ] -1, 4, 4-trimethyl-4, 5-dihydro-1H-pyrazolo [4, 3-H ] quinazoline-3-carboxylic acid methylamide. It can be prepared as described in international patent application WO2004104007 published on 2.12.2004 and has protein kinase inhibitory activity and is therefore useful as an antitumor agent in therapy. In particular, example 58 of the above international patent application describes a preferred preparation of compound 125.

A specific synergistic composition of this compound with other antitumor agents is described and claimed in international patent application WO2007090794 published on 8/16/2007.

Compound 125 is a poorly water soluble compound having a solubility in water of less than 0.1 mg/ml. Solubility of compound 125 in 5% glucose solution was less than 0.1mg/ml, about 0.8mg/ml in 10% polysorbate 80 aqueous solution, about 8mg/ml in 50% polyethylene glycol 400 aqueous solution, and about 10mg/ml or higher when formulated as an HCl salt in situ.

In addition, the free base absorbs light moisture as it exhibits a maximum water absorption of about 2% at 90% Relative Humidity (RH) at 25 ℃.

The first free base obtained was converted to the trihydrochloride salt to improve the solubility of the compound, while allowing the drug to be formulated as an aqueous solution (solubility of about 10mg/ml in a 5% glucose solution) for early pharmacological and toxicological assessment (as described in example 59 of the above international patent application).

By addressing the problems of the earlier formulation methods, the resulting trihydrochloride salts are amorphous hygroscopic solids and are therefore not suitable for development into oral formulations.

Moisture absorption is a serious concern for pharmaceutical powders. For example, moisture has a significant impact on the physical, chemical, and manufacturing properties of drugs, excipients, and formulations. It is also a key factor in determining packaging, storage, handling and shelf life, and successful development requires a thorough understanding of the hygroscopic properties.

For example, a transition from anhydrous to hydrate form can be observed when the relative humidity exceeds a critical level and the water content in the solid increases rapidly. This affects not only the physical and pharmaceutical properties of the drug itself, but also its biopharmaceutical aspects. Furthermore, it is well known that the hydrate form is generally less soluble than the corresponding anhydrous form, and it also has a potentially adverse effect on the dissolution rate properties of the active compound itself and its absorption characteristics through the gastrointestinal tract. In the same way, a transition from the crystalline to the amorphous form can be observed in the presence of humidity, with potential drawbacks in terms of physical stability. For example, an amorphous active drug, if dissolved, can absorb relatively large amounts of water from the atmosphere until it dissolves, while its chemical stability is also affected because the thermodynamically activated amorphous structure is more susceptible to chemical degradation and chemical interaction with other chemical species. Thus, the performance and efficacy of both the formulation and the active ingredient can be significantly altered.

Thus, there is a need in therapy for a water-soluble salt of compound 125 that has lower hygroscopicity and good and reproducible biopharmaceutical properties, which salt can enable safer and effective oral administration.

The present inventors have solved the above technical problems by providing new salts and new crystal forms of salts of the compound 125 having improved physicochemical properties. In fact, the new salts are crystalline, less hygroscopic, rapidly soluble solids with high water solubility and, in addition to possessing all the other advantages, in particular the therapeutic advantages exhibited by the known forms of amorphous free base and trihydrochloride, substantially introduce the important advantages of handling, storage and formulation, etc.

Surprisingly, new salt and free base forms of compound 125 were found and confirmed as crystals. The properties of crystalline powders make such forms particularly suitable for drug development.

Drawings

The invention is described with reference to the following figures.

FIG. 1 shows an X-ray diffraction pattern for compound 125 free base and its crystalline salts, where the 2 θ angle (degrees) is recorded on the X-axis and the intensity (CPS) is recorded on the y-axis. In particular, the spectra are for compound 125 free base form I (a) and the following salts: trihydrochloride form I (B), L-malate form I (C), glycolate form I (D), malonate form I (E) salts.

FIG. 2 shows an X-ray diffraction pattern of compound 125 free base form I (A) and salts thereof: maleate II form (B), succinate I form (C), adipate I form (D) salts.

FIG. 3 shows an X-ray diffraction pattern of compound 125 free base form I (A) and salts thereof: dihydrochloride salt form I (B), L-lactate salt form I (C), methanesulfonate salt form I (D), phosphate salt form I (E), fumarate salt semi-crystalline form I (F) salts.

FIG. 4 shows an X-ray diffraction pattern of compound 125 free base form I (A) and salts thereof: form I (B) trihydrochloride, form I (C) dihydrochloride and form I (D) hydrochloride.

FIG. 5 shows X-ray diffraction patterns for compound 125 maleate form I (A) with a free base to counterion molar ratio of 0.5: 1, compound 125 maleate form II (B) with a free base to counterion molar ratio of 2: 1, compound 125 maleate form I (C) with a free base to counterion molar ratio of 2: 1, and compound 125 maleate form III (D) with a free base to counterion molar ratio of 1: 1.

FIG. 6 shows an X-ray diffraction pattern of Compound 125 glycolate salt form I.

FIG. 7 shows an X-ray diffraction pattern of compound 125 malonate type I.

Figure 8 shows an X-ray diffraction pattern of compound 125 trihydrochloride form I.

Figure 9 shows an X-ray diffraction pattern of compound 125 dihydrochloride salt form I.

Figure 10 shows an X-ray diffraction pattern of compound 125 hydrochloride form I.

FIG. 11 shows an X-ray diffraction pattern of compound 125 maleate form I with a 1: 1 molar ratio of free base to counterion.

FIG. 12 shows an X-ray diffraction pattern of compound 125 maleate form II with a 1: 1 molar ratio of free base to counterion.

FIG. 13 shows an X-ray diffraction pattern of compound 125 maleate form III with a 1: 1 molar ratio of free base to counterion.

Figure 14 shows an X-ray diffraction pattern of compound 125 free base form I.

Figure 15 shows an X-ray diffraction pattern of compound 125 free base form II obtained by precipitating a salt of compound 125 dissolved in a pH6.8 buffer solution.

Figure 16 shows the DSC thermogram for compound 125 free base form I (a) and the following salts: malonate type I (B), phosphate type I (C), mesylate type I (D), fumarate semi-crystalline type I (E), and L-malate type I (F). Thermograms record temperature (. degree. C.) on the x-axis, while heat flow (mW) on the y-axis.

Figure 17 shows the DSC thermogram for compound 125 free base form I (a) and the following salts: glycolate type I (B), adipate type I (C), L-lactate type I (D), succinate type I (E) and maleate type II (F) salts.

Figure 18 shows DSC thermograms for compound 125 trihydrochloride form I (a), dihydrochloride form I (B), and hydrochloride form I (C).

Figure 19 records DSC thermograms of the additionally dried 125L-lactate form I (a), succinate form I (B) and adipate form I (C) salts as described in example 6.

Figure 20 records a typical DSC thermogram for compound 125 maleate.

Figure 21 records a TGA thermogram of compound 125 maleate salt (a), and a TGA thermogram of compound 125 maleate salt equilibrated, for example, by a hygroscopicity test (DVS) similar to the method described in example 8. TGA thermograms record temperature (c) on the x-axis, while weight percent (%) is recorded on the y-axis.

In a first aspect, the present invention relates to novel salts of compound 125 and crystalline forms thereof selected from the group consisting of fumarate, L-malate, maleate, succinate, adipate, malonate, glycolate, phosphate, methanesulfonate, and L-lactate.

In another aspect, the present invention relates to a novel crystalline form of compound 125 selected from the hydrochloride, dihydrochloride, and trihydrochloride salts.

Such salts were found to be crystalline, making this form particularly useful for drug development.

Salts of these compounds 125 can be obtained by known analogous methods by adding the desired number of moles of solvent or aqueous solution of the counter ion to the free base dissolved in a suitable solvent. The solvent is preferably an organic (especially anhydrous) solvent, more preferably methanol, ethanol, dichloromethane and mixtures thereof. Precipitation or crystallization of the resulting salt may be facilitated by addition or reprocessing in an anhydrous non-polar solvent such as diethyl ether, n-hexane or cyclohexane, if desired.

According to the invention, the definition of salt also includes hydrates and polymorphs thereof.

In particular, the present invention relates to novel crystalline forms and hydrates of compound 125 maleate.

The term "hydrate" as used herein refers to a compound formed by solvation, wherein the solvent is water.

Next, in another aspect, the invention relates to a stable crystalline form of compound 125 as the free base.

It is another object of the present invention to provide a pharmaceutical composition comprising as an active ingredient any salt of compound 125 as defined above, a crystalline form or hydrate of compound 125 maleate, or a crystalline form of compound 125 in free base form, together with pharmaceutically acceptable excipients and/or carriers.

It is a further object of the present invention to provide the use of any salt of compound 125 as defined above, a crystalline form or hydrate of compound 125 maleate, or a crystalline form of compound 125 as free base, as a medicament, in particular as a CDK inhibitor.

It is another object of the present invention to provide a method of treating a mammal, including a human being, in need of CDK inhibition, said method comprising administering to said mammal a therapeutically effective amount of any salt of compound 125 as defined above, a crystalline form or hydrate of compound 125 maleate, or a crystalline form of compound 125 as free base.

In addition, the present invention relates to the use of any salt of compound 125 as defined above, a crystalline form or hydrate of compound 125 maleate salt, or a crystalline form of compound 125 in free base form, in a method of treatment of a mammal, including a human, suffering from a disease state treatable by CDK inhibition, which means cell proliferative disorders such as cancer, viral infections, autoimmune diseases and neurodegenerative disorders.

Thus, any salt of compound 125 as defined above, a crystalline form or hydrate of compound 125 maleate salt, or a crystalline form of compound 125 as a free base can be used alone or in combination with other therapeutic agents, for treating mammals, including humans, suffering from a disease state treatable by CDK inhibition, or for the preparation of medicaments needed for such treatment.

Accordingly, the present invention also provides the use of any salt of compound 125 as defined above, a crystalline form or hydrate of compound 125 maleate salt, or a crystalline form of compound 125 as the free base, for the preparation of a medicament for the treatment of a disease state treatable by CDK inhibition.

The term "treatable disease state" means that the treatment according to the invention results in a remission of the disease state, or at least an improvement of the condition and quality of life of the mammal receiving the treatment.

Examples of such disease states are in particular different cancers, which may include all types of cancer, hematopoietic cancers of myeloid or lymphoid lineage, tumors of mesenchymal origin, tumors of the central and peripheral nervous system, melanoma, mesothelioma, seminoma, teratocarcinoma, osteosarcoma and kaposi's sarcoma, and also cell proliferative diseases such as benign prostatic hypertrophy, familial adenomatosis, polyposis, neurofibromatosis, psoriasis, vascular smooth cell proliferation associated with atherosclerosis, pulmonary fibrosis, arthritis, glomerulonephritis, post-operative stenosis and restenosis, organ transplant rejection and host versus graft disease.

The effective dosage of the salt of compound 125 can vary depending on the disease, the severity of the condition, and the condition of the patient being treated. Therefore, the doctor always sets the optimum dose for each patient. In any case, an effective dose may range from about 20 mg/day to about 300 mg/day, preferably from about 50 mg/day to about 150 mg/day (calculated as the free base), as a single or multiple divided daily dose.

A salt of compound 125 as defined above, a crystalline form or hydrate of compound 125 maleate, or a crystalline form of compound 125 in free base form, is readily absorbed orally, and is therefore preferably administered orally.

Needless to say, the compounds of the invention may be administered via any route of administration, for example, by parenteral, topical, rectal and nasal routes.

Accordingly, in a first aspect, the present invention relates to the fumarate, L-malate, maleate, succinate, adipate, malonate, glycolate, phosphate, methanesulfonate, and L-lactate salts of compound 125.

Preferred salts of the invention are the L-malate, maleate, malonate, glycolate, phosphate and L-lactate salts of compound 125.

More preferred salts of compound 125 are the maleate, malonate and glycolate salts.

As previously mentioned, the present invention also relates to novel crystalline forms and hydrates of the compound 125 salt.

In another aspect, the invention relates to a novel crystalline form of compound 125 in free base form.

In yet another aspect, it was found that compound 125 maleate salt can be obtained as a crystalline solid having three different crystalline forms, referred to as form I, form II and form III.

Form I is a high melting crystalline form of the compound 125 maleate, characterized as a hydrate, having reversible uptake of about 1 mole of water under room conditions (e.g., 25 ℃/60% RH), and which can be converted to form III under the action of stress conditions (e.g., storage at 40 ℃/75% RH) of exposure to temperature and/or humidity.

Total absorption of about 3.0-3.5% at 25 ℃ and 90% Relative Humidity (RH) can be reversed by lowering RH to about 20% at the same temperature.

Form II is a high melting crystalline form of compound 125 that exhibits the property of retaining non-stoichiometric amounts of solvent (e.g., alcohols such as ethanol, butanol, propanol) in the crystal lattice and that can be converted to form I or III under the action of dry conditions or stress conditions of exposure to temperature and humidity (e.g., storage at 40 ℃/75% RH).

Form III is a high melting crystalline form of the compound 125 maleate, characterized as a hydrate, with reversible absorption of about 1 mole of water at room conditions (e.g., 25 ℃/60% RH). Absorption of about 3.0-3.5% at 25 ℃ and 90% Relative Humidity (RH) can be reversed by lowering RH to about 20% at the same temperature.

In yet another aspect, it was found that compound 125 maleate salt can be obtained as a crystalline solid in molar ratios of 0.5: 1, 1: 1 and 2: 1.

The compound 125 hydroxyacetate salt and the compound 125 malonate salt are slightly hygroscopic, both having a reversible water absorption of about 2.5% at 25 ℃/90% RH.

The compound 125 salt has good solubility, especially the solubility of maleate, malonate, and glycolate salts in 0.5% glucose solution is about 10mg/mL or more.

In addition to the advantage of high water solubility, the compound 125 salts, in particular the maleate, malonate and glycolate salts, are particularly suitable for reproducible production with a defined acid/base ratio.

This finding makes such salts particularly suitable for use in liquid formulations for oral administration, as well as for intravenous dosage forms.

Table 1-solid state characterization of the salt and free base forms of compound 125

Note (): unless otherwise stated, the salt is the free base and the counterion in a molar ratio of 1: 1.

Note (×): the recorded PXRD peaks were selected based on their higher intensity in the complete data set.

In a preferred embodiment, the substantially pure maleate form I of compound 125 having a 1: 1 molar ratio of free base to counterion has the X-ray diffraction pattern of FIG. 11.

Also highly preferred is form I maleate salt of compound 125 having a 1: 1 molar ratio of free base to counterion, which has an X-ray diffraction pattern of the type shown in FIG. 11 and a significant peak intensity at about the 2 θ values (degrees) set forth in Table 1.

In the sample without any other substance (other crystal form, excipient), diffraction peaks should be observable at approximately the 2 θ values (degrees) described in table 7.

In another preferred embodiment, the substantially pure maleate form II of compound 125 having a 1: 1 molar ratio of free base to counterion has the X-ray diffraction pattern of FIG. 12.

Also highly preferred is form II maleate salt of compound 125 having a 1: 1 molar ratio of free base to counterion, which has an X-ray diffraction pattern of the type shown in FIG. 12 and has a peak intensity at the 2 θ values (degrees) set forth in Table 1.

In the sample without any other substance (other crystal form, excipient), diffraction peaks should be observable at approximately the 2 θ values (degrees) described in table 8.

In another preferred embodiment, the substantially pure form III maleate salt of compound 125 having a 1: 1 molar ratio of free base to counterion has the X-ray diffraction pattern of FIG. 13.

Also highly preferred is the compound 125 maleate form III with a 1: 1 molar ratio of free base to counterion, having an X-ray diffraction pattern of the type shown in FIG. 13 and having peak intensities at the 2 θ values (degrees) set forth in Table 1.

In the sample without any other substance (other crystal form, excipient), diffraction peaks should be observable at approximately the 2 θ values (degrees) described in table 9.

In another preferred embodiment, the substantially pure form I hydroxyacetate of compound 125 having a 1: 1 molar ratio between the free base and counterion has the X-ray diffraction pattern shown in FIG. 6.

Form I of compound 125 glycolate having a 1: 1 molar ratio of free base to counterion is also highly preferred, having an X-ray diffraction pattern of the type shown in FIG. 6 and having peak intensities at the 2 θ values (degrees) set forth in Table 1.

In a sample without any other substance (other crystalline form, excipient), a diffraction peak should be observable at about the 2 θ value (degrees) described in table 2.

In another preferred embodiment, the substantially pure form I malonate of compound 125 with a 1: 1 molar ratio of free base to counterion has the X-ray diffraction pattern shown in FIG. 7.

Also highly preferred is compound 125 malonate type I with a 1: 1 molar ratio of free base to counterion, having an X-ray diffraction pattern of the type shown in FIG. 7 and having peak intensities at the 2 θ values (degrees) set forth in Table 1.

In the sample without any other substance (other crystal form, excipient), diffraction peaks should be observable at approximately the 2 θ values (degrees) described in table 3.

In another preferred embodiment, the substantially pure form I trihydrochloride of compound 125 has the X-ray diffraction pattern shown in FIG. 8.

Compound 125 trihydrochloride form I, having an X-ray diffraction pattern of the type shown in figure 8, which has peak intensities at the 2 theta values (degrees) set forth in table 1, is also highly preferred.

In the sample without any other substance (other crystal form, excipient), diffraction peaks should be observable at approximately the 2 θ values (degrees) described in table 4.

In another preferred embodiment, substantially pure dihydrochloride form I of compound 125 has the X-ray diffraction pattern shown in FIG. 9.

Compound 125 dihydrochloride salt form I, having an X-ray diffraction pattern of the type shown in fig. 9, which has peak intensities at the 2 theta values (degrees) described in table 1, is also highly preferred.

In samples without any other substance (other crystalline form, excipients), diffraction peaks should be observable at approximately the 2 θ values (degrees) described in table 5 below.

In another preferred embodiment, the substantially pure hydrochloride salt form I of compound 125 has the X-ray diffraction pattern shown in FIG. 10.

Compound 125 hydrochloride form I, having an X-ray diffraction pattern of the type shown in figure 10, which has peak intensities at the 2 theta values (degrees) described in table 1, is also highly preferred.

In the sample without any other substance (other crystal form, excipient), diffraction peaks should be observed at approximately 2 θ values (degrees) described in the following table 6.

In another preferred embodiment, substantially pure compound 125 free base form I has the X-ray diffraction pattern shown in FIG. 14.

Also highly preferred is compound 125 free base form I having an X-ray diffraction pattern of the type shown in figure 14, which has a peak intensity at the 2 theta values (degrees) set forth in table 1.

In the sample without any other substance (other crystal form, excipient), diffraction peaks should be observed at approximately 2 θ values (degrees) described in the following table 10.

In another preferred embodiment, substantially pure compound 125 free base form II has the X-ray diffraction pattern of FIG. 15.

Also highly preferred is compound 125 free base form I having an X-ray diffraction pattern of the type shown in figure 15, which has a peak intensity at the 2 theta values (degrees) set forth in table 1.

In the sample without any other substance (other crystal form, excipient), diffraction peaks should be observed at approximately 2 θ values (degrees) described in the following table 11.

In yet another aspect, it was found that compound 125 maleate salt could be prepared as a crystalline solid with a molar ratio of free base to counterion of 0.5: 1.

In another preferred embodiment, the substantially pure maleate form I of compound 125 having a free base to counterion mole ratio of 0.5: 1 has the X-ray diffraction pattern of code A in figure 5.

In yet another aspect, it was found that compound 125 maleate salt could be made as a crystalline solid with a molar ratio of free base to counterion of 2: 1.

Also highly preferred is the substantially pure maleate form I of compound 125 having a molar ratio of free base to counterion of 2: 1, which has the X-ray diffraction pattern of code C in FIG. 5.

In another preferred embodiment, the substantially pure maleate form II of compound 125 having a 2: 1 molar ratio of free base to counterion has the X-ray diffraction pattern of code B in FIG. 5.

By substantially pure is meant that the crystalline form of the invention has a purity of at least 90%. More preferably, the crystalline form of the invention has a purity of at least 95%, most preferably at least 99% by weight of the crystals of the acid addition salt or the free base of compound 125 are present in the crystalline form of the invention.

In a further aspect, involving solid state characterization by DSC, it was found that compounds 125 succinate, L-lactate, adipate, phosphate, mesylate, fumarate, and L-malate, characterized by PXRD as crystalline materials, exhibited complex DSC profiles. These salts undergo a thermal transition involving a desolvation/dehydration process and a subsequent melting of the desolvated/dehydrated form characterized by its DSC melting peak temperature. When, for example, degradation occurs, further thermal transitions may accompany.

In a further aspect, involving solid state characterization by DSC, compound 125, trihydrochloride, dihydrochloride, and hydrochloride, were also found to exhibit complex DSC profiles. These salts undergo a thermal transition involving the desolvation/dehydration process, and a subsequent characteristic involving melting by degradation and loss of HCl characterized by its DSC melting peak temperature.

In yet another aspect, involving solid state characterization by DSC, compound 125 malonate was also found to exhibit a complex DSC profile. This salt undergoes a thermal transition involving melting and subsequent degradation and evaporation of the counterion, possibly followed by crystallization of the free base and its subsequent melting, these characteristics being characterized by its DSC melting peak temperature.

It should be understood that the onset and/or peak temperature values of a DSC may vary somewhat slightly from machine to machine, from method to method, or from sample to sample, and thus the values cited herein should not be considered absolute. In fact, the observed temperature depends on the rate of temperature change and the sample preparation technique and the particular instrument used. The temperature values obtained by applying these different conditions may vary within a range of plus or minus about 4 c, which should be estimated and taken into account. The results are further described in table 1 and example 6.

According to another aspect of the present invention, pharmaceutical compositions for administration to mammals, including humans, may be formulated according to methods known in the art and in any pharmaceutical form known in the art.

For example, a pharmaceutical composition comprises a salt of compound 125 as defined herein, in association with a pharmaceutically acceptable diluent or carrier. The compositions of the present invention may be in the form of suppositories suitable for oral use (e.g., as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), topical use (e.g., as creams, ointments, gels, or aqueous or oily solutions or suspensions), administration via inhalation (e.g., as finely divided powders or liquid aerosols), administration via insufflation (e.g., as finely divided powders), or administration via parenteral administration (e.g., as sterile aqueous or oily solutions for intravenous, subcutaneous, intramuscular, or rectal administration).

The compositions of the invention can be obtained by conventional methods using conventional pharmaceutical excipients which are well known in the art.

Thus, a composition for oral use may comprise, for example, one or more coloring agents, sweetening agents, flavoring agents and/or preserving agents.

Suitable pharmaceutically acceptable excipients for formulation of a tablet formulation include, for example, fillers such as lactose, mannitol, microcrystalline cellulose, sodium carbonate, pregelatinized starch, calcium phosphate or calcium carbonate; granulating and disintegrating agents such as croscarmellose sodium, corn starch, crospovidone or sodium starch glycolate; binders such as starch, microcrystalline cellulose, povidone, sucrose; lubricants such as magnesium stearate, stearic acid, sodium stearyl fumarate, polyethylene glycol or talc; glidants such as colloidal silicon dioxide; preservatives such as ethyl or propyl paraben; and antioxidants such as ascorbic acid.

The tablet formulation may be uncoated or coated to modify its disintegration properties and subsequent absorption of the active ingredient in the gastrointestinal tract or to improve its stability and/or appearance, in either case using conventional coating agents and methods well known in the art.

Compositions for oral use may be in the form of hard capsules wherein the active ingredient is mixed with an inert solid diluent, for example calcium carbonate, calcium phosphate or kaolin, and include the excipients mentioned above for tablet formulations; or in the form of soft capsules wherein the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin, soybean oil, coconut oil, or preferably olive oil, or any other acceptable excipient. Compositions for oral use may also be in the form of hard capsules, wherein the active ingredient is formulated as a stable pharmaceutical solid or semisolid dispersion comprising the active ingredient together with, for example, a hydrophilic carrier, a water-soluble vitamin E derivative as an antioxidant and optionally other excipients. Aqueous suspensions generally contain the active ingredient in finely divided form together with one or more suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as lecithin or condensation products of alkylene oxides with fatty acids (e.g. polyoxyethylene stearate), or condensation products of ethylene oxide with long chain aliphatic alcohols (e.g. heptadecaethyleneoxycetanol), or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol (e.g. polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g. polyethylene sorbitan monooleate).

The aqueous suspension may also contain one or more preservatives (such as ethyl or propyl paraben), antioxidants (such as ascorbic acid), colorants, fragrances, and/or sweeteners (such as sucrose, saccharin or aspartame). Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may also contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol.

Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation.

These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.

Dispersible or lyophilized powders and granules suitable for the preparation of an aqueous suspension or solution by the addition of water generally comprise the active ingredient together with a dispersing or wetting agent, suspending agent and one or more preservatives.

Suitable dispersing or wetting agents and suspending agents are exemplified by those mentioned above.

Additional excipients, such as sweetening, flavoring and coloring agents, may also be present.

The pharmaceutical compositions of the present invention may also be in the form of oil-in-water emulsions.

The oily phase may be a vegetable oil, such as olive oil or arachis oil; or a mineral oil such as liquid paraffin or a mixture of any such materials.

Suitable emulsifiers may be, for example, naturally occurring gums such as gum acacia or gum tragacanth, naturally occurring phosphatides such as soy bean, lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides (e.g. sorbitan monooleate) and condensation products of the partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate.

The emulsion may also contain sweetening, flavoring and preservative agents.

Syrups and elixirs may be formulated with sweetening agents, such as glycerol, propylene glycol, sorbitol, aspartame or sucrose, and may also contain a demulcent, a preservative, an aromatic and/or a colouring agent.

The pharmaceutical compositions may also be in the form of sterile injectable aqueous or oleaginous suspensions, solutions, emulsions or special systems which may be formulated according to known methods using one or more suitable dispersing or wetting agents and suspending agents as already mentioned above.

The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in polyethylene glycol.

Suppositories can be prepared by mixing the active ingredient with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum and release the drug.

Suitable excipients include, for example, cocoa butter and polyethylene glycols.

Topical formulations, such as creams, ointments, gels, and aqueous or oily solutions or suspensions, are generally obtained by formulating the active ingredient with conventional, topically acceptable excipients or diluents using conventional methods well known in the art.

Compositions for administration via insufflation may be in the form of finely divided powders containing particles having a mean diameter of, for example, 30 μm or less, preferably 5 μm or less, and more preferably between μm and 1 μm, the powder itself comprising the active ingredient alone or diluted with one or more physiologically acceptable carriers such as lactose.

The powder for insufflation is then conveniently contained in a capsule containing, for example, 1 to 50mg of active ingredient and fitted with a turbo-inhalation device for use. Compositions for administration via inhalation may be in the form of conventional pressurized aerosols, which produce an aerosol of the active ingredient containing finely divided solids or liquid droplets.

Conventional aerosol propellants such as volatile fluorocarbons or hydrocarbons may be used in the preparation, and aerosol devices may conveniently dispense metered amounts of the active ingredient. An example of a composition for oral use in the form of a hard capsule is given in example 10.

Examples

The following examples illustrate the invention.

Temperatures are measured in degrees Celsius (. degree. C.).

Unless otherwise indicated, reactions or experiments were performed at room temperature.

For short:

RT: at room temperature

RH: relative humidity

PXRD: powder X-ray diffraction

DSC: differential scanning calorimetry

DVS: dynamic vapor adsorption

TGA: thermogravimetric analysis

Example 1: experiment for salt formation of Compound 125

One portion of compound 125 (about 500mg) was dissolved in 10mL of a 1: 1 mixture of methanol and dichloromethane at Room Temperature (RT) to give a nominal concentration of about 50 mg/mL.

Several salt formation experiments were then performed by adding a 1: 1 molar amount of counter ion to 0.7mL of a solution of the compound 125 free base at Room Temperature (RT).

The cooling crystallization experiment was carried out at-30 ℃ and the standing time was about 24-36 hours.

The resulting precipitate was collected via vacuum filtration and dried in vacuo at 40 ℃.

When no crystallization occurred, the solution was concentrated by evaporation at Room Temperature (RT) in a gentle stream of nitrogen and then precipitated.

In some cases, a further recrystallization step (e.g., triturating the compound in diethyl ether) is required to separate the crystalline or at least powdered sample from the gummy residue.

Drying was carried out at 40 ℃ under vacuum.

By passing¹Chemical identification of compound 125 and the acidic counterion was performed by H NMR (for description see example 9).

Example 2: gram-Scale preparation of Compound 125 glycolate, maleate and malonate

When preparing the glycolate and the maleate, the free base is dissolved in absolute ethanol under reflux, while the malonate is prepared under reflux using methanol.

After complete dissolution of the free base, 1 equivalent of an acidic counterion was added.

After a suitable period of reflux treatment in the reaction vessel, heating was discontinued to allow spontaneous cooling to Room Temperature (RT). During which the glycolic acid salt precipitates, while the precipitation of the maleate and malonate salts requires further cooling steps down to 0 ℃ and-20 ℃ respectively. The precipitated material was then filtered and dried in vacuo at 40 ℃ for at least 24 hours.

Example 3: scale-up preparation of compound 125 maleate

An amount of compound 125 free base was heated under reflux in anhydrous ethanol for 30 minutes with stirring to completely dissolve the starting material (concentration about 25 g/L).

After that, 1 equivalent of maleic acid was dissolved in ethanol (concentration about 315g/L) and added to the free base solution.

After 30 minutes of reflux to complete the salinization, the stirring was slowed and the heating was discontinued.

The mixture was spontaneously returned to Room Temperature (RT) overnight, allowing it to precipitate.

The next day, the suspension was cooled to 0 ℃, stirred at this temperature for 30 minutes, and then filtered using a glass fiber filter.

The reactor was then washed with mother liquor and the resulting suspension was filtered on an existing plate.

The material was then dried at 50 ℃ for 48 hours.

Example 4: solubility of Compound 125 salt and free base

The solubility of the compound 125 salt was determined by the following procedure, if no other conditions were specified: to obtain target concentrations of 10mg/mL or 20mg/mL, the media described below were added to known amounts of compound 125 salt or free base from evaporation of DMSO stock solutions in 96-well plates. The resulting formulation was shaken at Room Temperature (RT) for 30 minutes, filtered and analyzed by HPLC.

The results are reported below; the achievement of the target value (10mg/ml or 20mg/ml) is indicated by an indication of "or higher".

The solubility values of compound 125 glycolate in different aqueous media were determined and the results were as follows:

6.2mg/mL in 5% glucose solution; pH1.2 buffer solution (chloride buffer solution) and pH4.5 buffer solution (acetate buffer solution) of 10.0mg/mL or more; pH6.8 buffer solution (phosphate buffer solution) was 0.2 mg/mL.

The solubility value of compound 125 malonate in 5% glucose solution was 18.4 mg/mL.

The solubility value of compound 125 trihydrochloride form I in a 5% glucose solution is 10mg/mL or more.

The solubility value of compound 125 dihydrochloride in 5% glucose solution was 20mg/mL or more.

The solubility values of compound 125, maleate form III, in various aqueous media were determined and the results obtained were as follows:

10.0mg/mL or more in a 5% glucose solution; about 40.0mg/mL in buffer solution (acetate buffer) at pH 4.5; pH6.8 buffer solution (phosphate buffer solution) < 0.1 mg/mL.

The solubility of compound 125 maleate form III in the buffer solution was determined by adding 10mL of medium to 40mg of compound 125. The vials were mechanically shaken at 37 ℃ and protected from light. After 16 hours, samples were removed and analyzed for solubility by specific HPLC assays.

The solubility values of compound 125 free base in various aqueous media were determined and the results were as follows:

dissolved in 5% glucose solution to be less than 0.1 mg/mL; 7.2mg/mL in a 5% glucose solution of 50% polyethylene glycol 400; 0.8mg/mL in 10% polysorbate 80 in 5% glucose solution; the concentration of the hydrochloric acid in situ salt is 10mg/mL after being prepared.

Example 5: analysis results of powder X-ray diffraction (PXRD)

Compound 125 salt was characterized by powder X-ray diffraction (PXRD) by irradiating a powder sample between 5 ° and 34 ° 2 θ using a Thermo/ARL XTRA apparatus at room temperature with a cuka source (45kV, 40mA, 1.8 kW-ka 1 radiation, wavelength λ ═ 1.54060 angstroms).

The scan rate was 1.20 °/minute (0.020 ° steps with 1 second count per step).

In the X-ray diffraction diagram, the angle of diffraction 2 θ is plotted on the horizontal axis (X-axis) and the line intensity is plotted on the vertical axis (y-axis).

In the paragraph defining the X-ray powder diffraction peaks of the salt and free base crystalline forms of compound 125, the expression "… about … at the 2 theta angle indicated in the table uses the term" about "to indicate the precise location of the peak (i.e., the recited 2 theta angle value), which should not be construed as an absolute value, as one skilled in the art would appreciate that the precise location of the peak may vary slightly from machine to machine, from sample to sample, or due to slight variations in the measurement conditions utilized.

It is also stated in the preceding paragraph that the crystalline form of the salt and free base of compound 125 has an X-ray powder diffraction pattern "substantially" identical to the X-ray powder diffraction patterns shown in figures 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15, and has the substantially most pronounced peaks at the 2 theta angle values shown in tables 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11. It should be clear that the term "substantially" is also used herein to mean that the values of the angle 2 theta of the X-ray powder diffraction pattern vary slightly from machine to machine, from sample to sample, or due to slight variations in the measurement conditions utilized, and that the peak positions shown in the figures or quoted in the tables should likewise not be interpreted as absolute values.

In this connection, it is known in the prior art that X-ray powder diffraction patterns can be obtained with one or more measurement errors depending on the measurement conditions, such as the apparatus, the sample preparation or the machine used. In particular, it is generally known that the intensity in an X-ray powder diffraction pattern may fluctuate depending on the measurement conditions and sample preparation.

For example, those familiar with X-ray powder diffraction will appreciate that the relative intensities of peaks may be affected by particles, for example, greater than 30 microns in size and having an aspect ratio (aspect ratio) that is not uniform, which may affect analysis of a sample.

It will also be apparent to those skilled in the art that the position of the reflection can be affected by the exact height of the sample in the diffractometer and zero calibration of the diffractometer.

The surface flatness of the sample may also have a minor effect.

Thus, it should be clear to one skilled in the art that the diffractogram data presented herein should not be interpreted as absolute (for more information, see Fundamentals of Powder Diffraction and structural Characterization, Pecharsky and Zavalij, Kluwer academic publishers, 2003). Accordingly, it should be understood that the crystalline forms of the salt and the free base of compound 125 described in the present invention are not limited to crystals having the same X-ray powder diffraction pattern as that shown in fig. 1, and any crystals having substantially the same X-ray powder diffraction pattern as that shown in fig. 1 are within the scope of the present invention.

Substantial identity of the X-ray powder diffraction pattern can be determined by those skilled in the art familiar with X-ray powder diffraction.

In general, the measurement error of the diffraction angle in the X-ray powder diffraction pattern is about 0.5 degrees or less (or more suitably, about 0.2 degrees or less), and the degree of this measurement error should be taken into consideration when considering the X-ray powder diffraction patterns in fig. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15 and when interpreting the peak positions mentioned herein and in tables 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11.

Thus, when it is stated that, for example, the salt and the free base of compound 125 have an X-ray powder diffraction pattern with at least one particular peak at about 2 θ to 22.8 degrees (or any other mentioned angle), then this can be interpreted as 2 θ to 15.2 degrees plus or minus 0.5 degrees, or 2 θ to 15.2 degrees plus or minus 0.2 degrees.

Figures 1 to 5 record the powder X-ray diffraction patterns of the salt and free base of compound 125 isolated on a small scale as described in example 1.

The main X-ray diffraction peaks of the compound 125 glycolate (type I), malonate (type I), maleate (types I, II and III) are recorded in figures 7, 8, 9, 10, 11, 12 and 13, which record examples of the powder X-ray diffraction patterns of the compound 125 salt obtained on a larger scale according to examples 2, 3 and 4 (glycolate, malonate and maleate).

The main X-ray diffraction peak 2 θ angles for compound 125 glycolate (form I), malonate (form I), trihydrochloride (form I), dihydrochloride (form I), hydrochloride (form I), maleate (forms I, II, and III), compound 125 free base (forms I and II) are summarized in tables 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 below.

TABLE 2 Compound 125 glycolic acid salt

TABLE 3 Compound 125 malonate salt

TABLE 4 Compound 125 trihydrochloride, form I

TABLE 5 Compound 125 dihydrochloride

TABLE 6 Compound 125 hydrochloride

TABLE 7 Compound 125 maleate form I

TABLE 8 Compound 125 maleate form II

TABLE 9 Compound 125 maleate form III

TABLE 10 Compound 125 free base form I

TABLE 11 Compound 125 free base, form II

Example 6: analysis results of Differential Scanning Calorimetry (DSC)

DSC analysis was performed using a Perkin-Elmer DSC-7 apparatus. The DSC aluminum pan was loaded with about 2mg of sample and the analysis temperature ranged between 30 ℃ and a maximum of 300 ℃. The samples were analyzed in a nitrogen flow at a heating rate of 10 ℃/min.

Figures 16, 17, 18 record DSC thermograms of the salt and free base of compound 125 isolated on a small scale as described in example 1 and the hydrochloride salt obtained in different ratios.

Figure 19 records DSC thermograms of the L-lactate form I (a), succinate form I (B) and adipate form I (C) salts of compound 125 after small scale isolation and further drying treatment in vacuo at 65 ℃ as described in example 1. Comparison with the original DSC thermogram (recorded in figure 17) shows the nature of the hydrated form. In fact, it was observed that the DSC curves of L-lactate and adipate maintained an initial thermal behavior including thermal characteristics associated with desolvation and/or solid state transition. On the other hand, the succinate DSC curve shows a significant change upon drying by a new thermal transition.

It was observed that L-lactate and adipate were converted to anhydrous form and showed a single melting peak by the heat treatment procedure in DSC experiments.

Figure 20 records a typical DSC thermogram for compound 125 maleate salt obtained according to example 1 and characterized as forms I and III. With respect to compound 125 maleate, the observed melting endotherm is approximately 183 ℃ (peak temperature), showing a Δ Hf of approximately 65 joules/gram. The dehydration endotherm typically detected in the initial portion of the DSC thermogram depends on the equilibrium of the water uptake of the material.

It should be understood that the onset and/or peak temperature values of a DSC may vary slightly from machine to machine, from method to method, or from sample to sample, and thus the recited values should not be interpreted as absolute. In fact, the temperature observed will depend on the rate of temperature change as well as the sample preparation technique and the particular instrument used. Variations of plus or minus about 4 c in temperature values resulting from the application of these various conditions should be estimated and taken into account.

Example 7: results of thermogravimetric analysis (TGA)

TGA analysis was performed using a Perkin-Elmer TGA-7 apparatus. DSC aluminum pans were loaded with approximately 5-10mg of sample. The analysis temperature ranges between 30 ℃ and a maximum of about 200 ℃. The samples were analyzed in a nitrogen flow (to eliminate the oxidation and pyrolysis (pyrolitic) effects) at a heating rate of 2 ℃/min.

Figure 21 records a typical TGA thermogram of compound 125 maleate salt obtained according to example 1 and characterizes the behaviour of forms I and III when equilibrated after dehydration (a) and e.g. DVS sorption ramp (B). The weight loss step, detected within 60 ℃, is related to the dehydration endotherm usually detected in the initial part of the DSC thermogram, which generally depends on the equilibrium of the water uptake of the material.

Example 8: analysis results of Dynamic Vapor Sorption (DVS)

Samples of compound 125 salt and free base were tested for hygroscopicity using DVS 1000(SMS) to investigate the water uptake of these materials. The device is a "controlled atmosphere microbalance" that exposes a weighed sample to a stylized change in Relative Humidity (RH) at a constant and controlled temperature. From the measured parameters (weight, time and RH) recorded in the Excel worksheet, a hygroscopicity curve in the range of the test RH can be obtained.

The adsorption/desorption cycle between 0% and 90% RH can be performed at a controlled temperature of 25 ℃.

The gradual change in RH can be 10% and 3%; it was operated by software at the balance of the sample weight. This condition may be defined at a constant rate of change of weight percent (e.g., 0.005%/minute). The results of the experiments are reported in DVS isotherm reports and isotherm plots. Examples of the water uptake of compound 125 maleate during DVS adsorption temperature rise are summarized in table 12 below.

Table 12-compound 125 maleate DVS adsorption data

Relative to each otherHumidity (%)	Water absorption (%)
		0.0	0.0
10.0	0.1
		20.0	0.2
30.0	1.9
		40.0	2.5
50.0	2.7
		60.0	2.8
70.0	2.8
		80.0	2.9
90.0	3.1

Example 9: NMR identification analysis

¹H NMR experiment wasAt a constant temperature of 28 ℃ on a Varian Inova 500 spectrometer operating at 499.8 MHz. A small amount of each sample was dissolved in 0.75mL of DMSO-d6 and transferred to a 5mm NMR tube for subsequent analysis. This analysis enables confirmation of the expected chemical structure of both the molecule and the counterion.

Example 10: composition percentage of preparation for oral use

Composition (I)	Range%
		Compound 125	5-70
Lactose monohydrate	25-95
		Magnesium stearate	0.05-2.5
Colloidal silica	0.05-1

From the above data and examples, it should be clear to those skilled in the art that the novel salts of compound 125 described in the present invention are a new, improved and valuable tool in therapy.

Claims (3)

1. A crystalline form of a salt of compound 125 having the formula:

it has an X-ray powder diffraction (CuK α source) pattern comprising characteristic peaks at about the following 2 θ angle values:

-maleate form I: 5.3, 6.0, 11.9, 12.7, 13.5, 14.5, 17.9, 19.4, 20.9, 22.9, 23.2, 24.7;

-maleate form II: 4.8, 9.6, 11.6, 15.7, 16.0, 16.7, 19.3, 20.9, 21.3, 22.1, 23.3, 27.7;

-maleate form III: 6.0, 11.8, 12.3, 13.3, 14.3, 16.3, 17.8, 20.8, 22.8, 24.3, 26.4, 27.6;

malonate type I: 11.5, 12.4, 14.3, 15.8, 18.8, 20.9, 21.8, 22.7, 23.0, 24.8;

-hydroxyacetate salt form I: 6.6, 11.8, 12.2, 12.7, 16.1, 17.5, 19.4, 21.9, 23.6, 23.9, 25.9, 27.8.

2. A pharmaceutical composition comprising as active ingredient a crystalline form of a salt of compound 125 as defined in claim 1, together with pharmaceutically acceptable excipients and/or carriers.

3. Use of a crystalline form of a salt of compound 125 as defined in claim 1 for the preparation of a medicament for the treatment of a disease state treatable by CDK inhibition.