HK1166015B - Plk inhibitor salts - Google Patents

Description

Salts of PLK inhibitors

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

Cancer is a leading cause of death in humans; surgery, radiation and chemotherapy are useful methods for combating cancer.

PLK1 is a serine threonine kinase essential for the development of appropriate mitosis.

Expression of PLK1 was seen in all proliferative normal tissues, and its overexpression was observed in a range of tumors, including breast, prostate, ovarian, lung, stomach and colon. When RNAi leads to PLK1 depletion in cancer cells, inhibition of proliferation and reduced viability leads to cell cycle arrest with 4N DNA content, followed by the observation of apoptosis. Although 4 different PLKs family members are described in humans, inhibition of enzyme activity or depletion of PLK1 is sufficient to cause G2/M cell cycle blockade and apoptosis in tumor cell lines and tumor regression in xenograft models. Furthermore, for other PLKs, tumor suppressor function has been described and it has been reported that PLK2 and PLK 3-rather than PLK 1-are expressed in non-proliferative, differentiated post-mitotic cells, such as neurons, suggesting that PLK 1-specific compounds may have safer properties (see, e.g., Strebhardt K, et al, NatRev Cancer 2006; 6 (4): 321-30)

Inhibitors of mitosis for Cancer therapy are widely accepted clinical strategies for the treatment of a variety of human cancers (see, e.g., Jackson JR et al, Nature Reviews Cancer 2007; 7, 107-117). The taxanes (paclitaxel and docetaxel) and vinca alkaloids (vincristine and vinblastine) function as follows: stabilizing or destabilizing microtubules has catastrophic consequences in cell development through mitosis. They are first line treatments for several tumor types including hodgkin's disease, non-hodgkin's lymphoma, testicular cancer, neuronal cells and Wilms tumors (vinca alkaloids), and they are second line treatments in cisplatin-refractory ovarian cancer, breast cancer, lung and esophageal cancers (taxanes). However, due to the role of microtubules in the process, such as cell migration, phagocytosis and axonal transport, certain toxicities are often observed with these agents, such as peripheral neuropathy.

Pyrazoloquinazolines (Nerviano Medical Sciences Srl.) described and claimed in patent application WO2008074788 are potent inhibitors of PLK1 and are therefore suitable for the treatment of proliferative disorders, in particular cancer.

Compound 937 has the following formula:

is one of the compounds described and claimed in the above-mentioned patent application. The preparation thereof, pharmaceutical compositions containing it and the use thereof in medicine are also described and claimed.

The compound 937 is 1- (2-hydroxy-ethyl) -8- [5- (4-methyl-piperazin-1-yl) -2-trifluoromethoxy-phenylamino ] -4, 5-dihydro-1H-pyrazolo [4, 3-H ] quinazoline-3-carboxamide, and its preferred preparation is described in the above-mentioned patent application examples 38 and 40.

Compound 937 is a poorly water soluble crystalline compound that exhibits a solubility in water of less than 0.1 mg/ml.

The solubility of compound 937 was about 4mg/ml in a 5% glucose solution of 10% polysorbate 80, about 7mg/ml in a 5% glucose solution with aqueous 50% polyethylene glycol 400, and about 8-9mg/ml when formulated as the hydrochloride or mesylate in situ salt. The compound 937 is also suitable for aqueous methylcellulose (methocel) suspensions in the approximate range of 2-8 mg/ml.

The resulting free base is initially formulated as an in situ salt or methylcellulose suspension for use in early pharmacological and toxicological assessments. Although the problem of the early formulation method is solved, the formulation method applied is not suitable for the development of oral formulations.

Preclinical studies of the active pharmaceutical ingredient require the possibility of dissolving it in a suitable excipient. The general purpose is to render the compounds water soluble in order to be suitable for the preparation of injectable sterile formulations; fast dissolution of the drug substance contained in a solid dosage form (e.g. hard gelatin capsules) and immediate-release tablets also require sufficient water-solubility.

The poor solubility observed for compound 937 makes it difficult to formulate and administer.

In addition, the free base is slightly hygroscopic as it shows a maximum absorption of about 1% water at 90% Relative Humidity (RH) at 25 ℃.

Moisture absorption is also 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. Amorphous active drug substances, if they readily absorb moisture, can absorb relatively large amounts of water from the atmosphere until they dissolve, while their chemical stability is also compromised because thermodynamically activated amorphous structures are 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.

Furthermore, the powder bulk density of the free base is low, about 90 mg/ml.

Advantageous bulk properties, such as powder bulk density, are also desirable. Higher powder bulk density relates to more suitable particle morphology, allowing for improved flowability and resulting in improved properties of the pharmaceutical composition. Higher powder densities allow, for example, more suitable capsule formulation manufacturing methods or higher dosage strengths within similar capsule sizes, as well as the specific possibility of reducing the number and/or size of capsules to be administered, particularly during early clinical phases.

Thus, there is a need in therapy for water-soluble salts of compound 937 with good and reproducible physicochemical and bulk properties, such as sufficient solubility, low hygroscopicity, crystallinity and favourable bulk density of the powder, but also in the solid state and chemical stability under variable storage temperature, humidity and lighting conditions, ensuring proper biopharmaceutical behaviour and enabling safer and effective oral administration.

It is well known to the skilled person that salt formation does not always lead to enhanced solubility characteristics (see e.g.Shozo Miyazaki et al, national Journal of pharmaceuticals 1980; 6(1), 77-85) and that the behaviour of different salts in terms of solubility and/or dissolution and/or sensitive effect on counter-ions may vary with the nature of the respective drug-counter-ion pair.

Surprisingly, the present inventors have solved the above technical problem by providing new salts of the compound 937 with improved physicochemical and stacking properties, as well as new crystalline forms of the salts.

In fact, the new salts are crystalline, rapidly soluble solids with high water solubility; moreover, these salts are low or moderately hygroscopic and therefore introduce substantial advantages in handling, storage and formulation; finally, these salts have an unexpectedly greater bulk density of the powder than the free base, in addition to all other advantages, particularly the therapeutic advantages as the free base exhibits when formulated as an in situ salt or methylcellulose suspension.

Surprisingly, new salt forms of compound 937 were found and demonstrated to be crystalline as well as the free base form. The properties of crystalline powders make such forms particularly suitable for drug development.

Drawings

The invention is described with reference to the following figures.

Figure 1 shows an X-ray diffraction pattern of compound 937 free base and its crystalline salts, where the 2 θ angle (degrees (deg)) is recorded on the X-axis and the intensity (CPS) is recorded on the y-axis. In particular, the spectrum relates to the compound 937 free base form I (a) and to the following salts: l-tartrate form I (B), succinate form I (C), phosphate form I (D), mesylate form I (E), maleate form I (F), L-malate form I (G), hydrochloride form I (H), fumarate-half mole counterion form I (I), fumarate form I (J), citrate-half mole counterion form I (K), benzenesulfonate form I (L), L-aspartate-half mole counterion form I (M).

FIG. 2 shows an X-ray diffraction pattern of compound 937 free base form I (A) and salts thereof: l-tartrate form I (B), fumarate form I (C), L-malate form I (D).

Figure 3 shows an X-ray diffraction pattern of compound 937 fumarate salt form I.

Figure 4 shows an X-ray diffraction pattern of compound 937L-malate salt form I.

Figure 5 shows an X-ray diffraction pattern of compound 937L-tartrate form I.

FIG. 6 shows X-ray diffraction patterns of compound 937 maleate form I (A-B) and maleate form II (C).

FIG. 7 shows an X-ray diffraction pattern of compound 937 succinate form I (A) and succinate form II (B-C).

Figure 8 shows an X-ray diffraction pattern of compound 937 free base, form I.

Figure 9 shows the DSC thermogram of compound 937 free base form I (a) and the following salts: l-tartrate form I (B), succinate form I (C), phosphate form I (D), mesylate form I (E), maleate form I (F), L-malate form I (G), hydrochloride form I (H), fumarate-half mole counterion form I (I), fumarate form I (J), citrate form I (K), benzenesulfonate form I (L), L-aspartate-half mole counterion form I (M).

Thermograms record temperature (. degree. C.) on the x-axis, while heat flow (mW) on the y-axis.

FIG. 10 shows DSC thermograms of compound 937 free base form I (A1), free base form II (A2), L-tartrate form I (B), succinate form II (C-D), fumarate form I (E), maleate form I (F), maleate form II (G) and L-malate form I (H).

FIG. 11 shows the DSC thermograms of the compound 937 free base form I (A-B1) and free base form II (B2) in detail.

Figure 12 shows an X-ray diffraction pattern of batches of compound 937 free base form I (a-B), also characterized by the DSC characteristics reported in figure 11.

Figure 13 shows DVS characteristics (hygroscopicity determination) of compound 937 free base form I (a), fumarate salt form I (B), maleate salt form II (C), succinate salt form II (D), L-malate salt form I (E), L-tartrate salt form I (F).

Figure 14 shows an X-ray diffraction pattern of a scaled-up batch of compound 937 fumarate salt form I.

Figure 15 shows DSC characteristics of scaled-up batches of compound 937 fumarate salt form I.

Figure 16 shows an X-ray diffraction pattern of a scaled-up batch of compound 937 fumarate salt form II.

In a first aspect, the present invention relates to novel salts of compound 937 selected from the group consisting of L-tartrate, succinate, phosphate, mesylate, maleate, L-malate, hydrochloride, fumarate (half mole of counter ion), fumarate, citrate (half mole of counter ion), benzenesulfonate and L-aspartate (half mole of counter ion), and crystalline forms, solvates and hydrates thereof.

In particular, the present invention relates to novel salts of compound 937 selected from the group consisting of L-tartrate, succinate, maleate, L-malate and fumarate, and their crystalline forms, solvates and hydrates.

More particularly, the present invention relates to novel salts of compound 937 selected from the group consisting of L-tartrate, L-malate and fumarate salts, and crystalline forms, solvates and hydrates thereof.

Still more particularly, the present invention relates to novel crystalline forms of the compound 937 fumarate salt, and solvates and hydrates thereof.

Most particularly, the present invention relates to a solvate of the compound 937 fumarate salt.

Salts of these compounds 937 can be obtained by known analogous methods by adding the desired stoichiometric amount of a solvent or an 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 salts also includes their crystalline forms, solvates and hydrates thereof.

The term "solvate" as used herein means a compound formed by solvation, e.g. as a combination of solvent molecules and solute molecules or ions. Well-known solvent molecules include water, alcohols and other polar organic solvents. Alcohols include methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, and tert-butanol. Alcohols also include polymeric alcohols, such as polyalkylene glycols (e.g., polyethylene glycol, polypropylene glycol).

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

When reference is made to "solvates" and "hydrates" the present invention is intended to include both stoichiometric and non-stoichiometric "solvates" and "hydrates" unless otherwise indicated.

A stoichiometric solvate has a fixed ratio of solvent molecules to compound molecules. This is typically due to bonding interactions between the solvent and the compound molecule. In non-stoichiometric solvates, the solvent is not present in a fixed ratio to the compound molecule and can often vary. In non-stoichiometric solvates, the solvent is often present in the interstitial spaces or channels within the crystal lattice. Stoichiometric hydrates have a fixed ratio of water molecules to molecules of the compound. This is typically due to bonding interactions between the water and the compound molecules. In non-stoichiometric hydrates, water is not present in a fixed ratio to the compound molecule and can often vary. In non-stoichiometric hydrates, water is often present in the interstitial spaces or channels within the crystal lattice.

Next, in another aspect, the invention relates to a new stable crystalline form of the compound 937 as free base.

It is another object of the present invention to provide a pharmaceutical composition comprising as active ingredient any salt of the compound 937 as defined above, a crystalline form, solvate or hydrate of the compound 937 fumarate, or a crystalline form of the compound 937 as free base together with pharmaceutically acceptable excipients and/or carriers.

It is a further object of the present invention to provide any salt of the compound 937 as defined above, a crystalline form, solvate or hydrate of the compound 937 fumarate salt, or a crystalline form of the compound 937 as free base, for use as a medicament.

It is another object of the present invention to provide any salt of the compound 937 as defined above, a crystalline form, solvate or hydrate of the compound 937 fumarate salt, or a crystalline form of the compound 937 as free base, alone or in combination with other therapeutic agents, for use in the treatment of a mammal, including a human being, suffering from a disease state treatable by PLK inhibition.

Furthermore, the present invention relates to any salt of the compound 937 as defined above, a crystalline form, solvate or hydrate of the compound 937 fumarate salt, or a crystalline form of the compound 937 as free base, for use in the treatment of a mammal, including a human being, suffering from a disease state treatable by PLK inhibition, such as cell proliferation disorders, viral infections, autoimmune and neurodegenerative disorders.

Furthermore, the present invention relates to any salt of the compound 937 as defined above, a crystalline form, solvate or hydrate of the compound 937 fumarate salt, or a crystalline form of the compound 937 as free base, for use in the treatment of a mammal, including a human being, suffering from a disease state treatable by PLK inhibition, said disease state being characterized by a cell proliferative disorder being cancer.

It is another object of the present invention to provide a method of treating a mammal, including a human being, in need of PLK inhibition, said method comprising administering to said mammal a therapeutically effective amount of any salt of the compound 937 as defined above, a crystalline form, solvate or hydrate of the compound 937 fumarate salt, or a crystalline form of the compound 937 as free base.

Finally, another object of the present invention is to provide the use of any salt of the compound 937 as defined above, a crystalline form, solvate or hydrate of the compound 937 fumarate salt, or a crystalline form of the compound 937 as free base, alone or in combination with other therapeutic agents, in the manufacture of a medicament suitable for the treatment of a disease state treatable by PLK 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 specific different cancers, which may include cancers such as bladder cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, including small cell lung cancer, esophageal cancer, gallbladder cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer, prostate cancer, and skin cancer, including squamous cell carcinoma; hematopoietic cancers of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma and Burkett's lymphoma; hematopoietic cancers of the myeloid lineage, including acute and chronic myelogenous leukemias, myelodysplastic syndrome and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; tumors of the central and peripheral nervous system, including astrocytomas, neuroblastomas, gliomas, and schwannomas; other tumors, including melanoma, seminoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum, keratoacanthoma, follicular thyroid carcinoma, and kaposi's sarcoma. Examples of such disease states are also specific cell proliferation disorders such as, for example, benign prostate hyperplasia, familial adenomatous polyposis, neurofibromatosis, psoriasis, vascular smooth cell proliferation, which is associated with atherosclerosis, pulmonary fibrosis, arthritis, glomerulonephritis and post-operative stenosis and restenosis.

The effective dose of the compound 937 salts may vary according to the disease, the severity of the condition and the condition of the patient to be treated. Therefore, the doctor always sets the optimum dose for each patient. In any event, an effective dose may range from about 5 to about 500 mg/dose (calculated as the free base) 1 to 5 times per day.

A salt of the compound 937 as defined above, a crystalline form, solvate or hydrate of the compound 937 fumarate salt, or a crystalline form of the compound 937 as free base is readily absorbed orally, and thus it is preferred to administer it 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.

As another aspect, it has been found that compound 937 fumarate salt can be obtained as a crystalline solid in the form of a crystalline form referred to as form I. Form I is a high melting (high melting) crystalline form of compound 937 fumarate salt, which shows a moderate water uptake of 1.6% at 25 ℃/90% RH, which is reversible by lowering RH at a constant temperature of 25 ℃. (PXRD characteristics: FIG. 1J; DSC characteristics: FIG. 9J; DVS characteristics: FIG. 13B and Table 11-other references to PXRD and DSC characteristics are described in Table 1)

It has been found that the compound 937 fumarate salt can be obtained as a crystalline solid also in a stoichiometric ratio of 0.5: 1. (PXRD characteristics: FIG. 1I; DSC characteristics: FIG. 9I).

The compound 937 fumarate salt can be obtained as a crystalline solid in the form of a crystal form called form II (PXRD pattern: figure 16).

As another aspect, it has been found that the compound 937L-malate salt and the compound 937L-tartrate salt can be obtained as crystalline solids in crystalline forms, both referred to as form I. The compound 937L-malate salt and the compound 937L-tartrate salt were moderately hygroscopic, both showing a water uptake of about 4.7% and 5.8% at 25 ℃/90% RH, respectively. The water content is quite stable at ambient conditions and therefore both salts can be considered as stable hydrates (PXRD characteristics: L-malate salts FIG. 1G, L-tartrate salts FIG. 1B; DSC characteristics L-malate salts FIG. 9G, L-tartrate salts FIG. 9B; DVS characteristics: L-malate salts FIG. 13E, L-tartrate salts FIG. 13F; further references to PXRD and DSC characteristics are described in Table 1).

As another aspect, it has been found that the compound 937 maleate salt can be obtained as a crystalline solid in two different crystalline forms, referred to as form I and form II. Form I is a high melting point crystalline form of the compound 937 maleate salt characterized by a stably hydrated form. (PXRD characteristics: FIG. 1F; DSC characteristics: FIG. 9F; other references to PXRD and DSC characteristics are described in Table 1). Form II is a high melting point crystalline form of compound 937 maleate salt characterized by a moderately hygroscopic form (PXRD pattern: fig. 6C; DSC pattern: fig. 10G; DVS pattern: fig. 13C). Form II is converted to form I by exposure to stress conditions of temperature and moisture (stressed conditions), for example, stored at 40 deg.C/75% RH. (PXRD characteristics: FIG. 6B; DSC characteristics: FIG. 10F)

As another aspect, it has been found that the compound 937 succinate salt can be obtained as a crystalline solid in two different crystalline forms, referred to as form I and form II. Form I is a high melting point crystalline form of compound 937 succinate salt characterized as a methanol solvate (PXRD features: fig. 1C and 7A; DSC features: fig. 9C). Form II is a high melting crystalline form of the compound 937 succinate salt characterized by a hydrated form that is stable under ambient conditions and stress conditions of temperature and moisture, e.g., storage at 40 ℃/75% RH (PXRD characteristics: FIGS. 7B and 7C; DSC characteristics: FIGS. 10C and 10D; DVS characteristics: FIG. 13D).

Furthermore, it has been found that the compound 937 citrate salt can be obtained as a crystalline solid in a stoichiometric ratio of 0.5: 1 (PXRD characteristics: FIG. 1K; DSC characteristics: FIG. 9K) and as an amorphous solid in a stoichiometric ratio of 1: 1.

The compound 937 salt showed good solubility in water, in particular the fumarate, L-malate and L-tartrate (tartarate) salts had solubilities in water in the range of 1-4mg/ml (more details are reported in example 4).

In addition to exhibiting the advantage of sufficient aqueous solubility, the compound 937 salts, in particular fumarate, L-malate and L-tartrate, are also particularly suitable to be reproducibly prepared with a well-defined acid/base ratio. This finding makes these salts particularly suitable for liquid formulations for oral administration as well as for intravenous formulations.

Furthermore, the compound 937 salt showed an advantageous powder bulk density, in particular the fumarate, L-malate and L-tartrate salts, of more than 240mg/ml (more details are reported in example 12).

As already mentioned, the solid state properties of the salt and free base forms of compound 937 are illustrated as shown in table 1, along with a complete listing of the related PXRD and DSC diagrams.

Table 1-description of the solid state properties of the salts and free base forms of the compound 937 and figures/table references

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

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

In a preferred embodiment, the substantially pure form I fumarate salt of compound 937 has the X-ray diffraction pattern shown in FIG. 3, with significant peak intensities at approximately 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 1: 1 fumarate salt form II of compound 937 has the X-ray diffraction pattern shown in fig. 16, with significant peak intensities at approximately the 2 Θ values (degrees) described 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 10.

In another preferred embodiment, the substantially pure 1: 1L-malate salt form I of compound 937 has the X-ray diffraction pattern shown in fig. 4 with significant peak intensities at about the 2-theta values (degrees) described 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 4.

In another preferred embodiment, the substantially pure form I of the 1: 1L-tartrate salt of compound 937 has the X-ray diffraction pattern shown in FIG. 5 with significant peak intensities 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 3.

In another preferred embodiment, the substantially pure form I1: 1 maleate salt of compound 937 has an X-ray diffraction pattern as shown in FIG. 6A with significant peak intensities at approximately 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 5.

In another preferred embodiment, the substantially pure 1: 1 maleate form II of compound 937 has an X-ray diffraction pattern as shown in figure 6C, with significant peak intensities at approximately 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 6.

In another preferred embodiment, the substantially pure 1: 1 maleate form I (methanol solvate) of compound 937 has an X-ray diffraction pattern as shown in fig. 7A with significant peak intensities at approximately the 2 θ values (degrees) described 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 1: 1 maleate form II of compound 937 has an X-ray diffraction pattern as shown in figure 7C, with significant peak intensities at approximately 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 compound 937 free base form I has an X-ray diffraction pattern, shown in figure 8, with significant peak intensities at approximately 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.

The form I crystal structure can thus be characterized by DSC features 9A and 10a1 melting endotherms reported in fig. 9 and 10, and DSC features 11A and 11B1 reported in fig. 11. Another preferred embodiment is the compound 937 free base form II, which shows DSC characteristics 10a2 and 11B2 reported in figure 10 and figure 11, respectively. The higher melting point DSC peak corresponds to form II, as the free base batches characterized by the thermograms reported in fig. 9A and 10A share the same X-ray powder diffraction pattern as reported in fig. 12.

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 acid addition salt or the free base of the compound 937 is present as the crystalline form according to the invention.

In a further aspect, involving solid state characterization by DSC, compound 937 fumaric acid (half molar counter ion), the hydrochloride, mesylate and phosphate salts, characterized as crystalline material by PXRD, were found to exhibit complex DSC profiles (figure 9). 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.

PXRD and DSC results are further described in table 1 and examples 6 and 7.

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, the pharmaceutical composition comprises a salt of the compound 937 as defined herein, in association with a pharmaceutically acceptable diluent or carrier.

The compositions of the present invention may be in a form suitable for oral use. Examples of these forms are: tablets, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules. The compositions of the present invention may be in a form suitable for topical use. Examples of these forms are: creams, ointments, gels or aqueous or oily solutions or suspensions. The compositions of the invention may be in a form suitable for administration via inhalation, such as, for example, a finely divided powder or a liquid aerosol. The compositions of the invention may be in a form suitable for administration via insufflation, such as, for example, a finely divided powder. The compositions of the invention may be in a form suitable for administration via parenteral administration (e.g., such as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular) or in the form of suppositories for 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 tablet formulations 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 stearoyl fumarates, glyceryl behenate, polyethylene glycol or talc; glidants such as colloidal silicon dioxide; preservatives such as ethyl or propyl paraben; and antioxidants such as ascorbic acid.

Tablet formulations may be uncoated or coated to modify their disintegration properties and subsequent absorption of the active ingredient in the gastrointestinal tract to improve their stability and/or appearance. Uncoated or coated tablets require the use of conventional coating agents and methods well known in the art.

Compositions for oral use may be formulated in the form of hard capsules wherein a fill mixture is prepared with the active ingredient mixed with an inert solid diluent, for example calcium carbonate, calcium phosphate or kaolin, and including the excipients described above for tablet formulation; compositions for oral use may also be formulated in the form of soft gelatin capsules wherein a fill mixture is prepared with the active ingredient in admixture 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 are generally prepared by including the active ingredient in finely divided form, with the addition of dispersing or wetting agents such as lecithin, polyoxyethylene stearate or sorbitan monooleate, polyethylene sorbitan monooleate, such as suspending agents (e.g., such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone) as the active ingredient. The aqueous suspension may contain one or more suitable additives, such as preservatives, antioxidants, coloring agents, flavoring agents and/or sweetening agents, to provide a palatable oral preparation. Oily suspensions may be obtained by suspending the active ingredient in a vegetable oil, such as olive oil or sesame oil. Dispersible or lyophilised powders and granules suitable for the preparation of an aqueous suspension or solution by the addition of water comprise the active ingredient together with suitable excipients (extenders, dispersing or suspending agents and preservatives).

The formulations may also be in the form of sterile injectable suspensions, solutions, emulsions, which may be formulated according to known methods using suitable excipients, for example selected from the dispersing, wetting and suspending agents already mentioned above.

Topical formulations, such as creams, ointments, gels, and solutions or suspensions, may be obtained by formulating the active ingredient with conventional carrier excipients or diluents using conventional methods well known in the art.

Compositions for administration by insufflation may be in the form of finely divided powders containing particles of suitable average diameter, for example, 50 μm or less, the powder itself containing the active ingredient alone or diluted with a suitable carrier such as lactose.

Powders for insufflation are formulated in capsules containing appropriate amounts of the active ingredient to be used by means of a turbo-inhalation device. Compositions for administration via inhalation may be in the form of conventional pressurized aerosols configured to disperse the active ingredient into an aerosol containing finely divided solids or droplets.

Conventional aerosol propellants and devices may be used to disperse a defined amount of 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: small-scale preparation of compound 937 salts

One part of compound 937 free base (about 40 ÷ 50mg) was dissolved in 4 ÷ 5mL of a 2: 1 mixture of methanol and dichloromethane at RT to give a nominal concentration of about 10 mg/mL.

Several salt formation experiments were then performed by adding a 1: 1 stoichiometric amount of counter ion to 4 ÷ 5mL of the compound 937 free base solution at 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 RT in a gentle stream of nitrogen and further 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 937 and the acidic counter ion was performed by H NMR (for explanation see example 9).

Example 2: gram-scale preparation of the compound 937L-tartrate, succinate, fumarate, citrate, maleate and L-malate.

The free base (500mg, 0.939mmol) was dissolved in a 2: 1 mixture of dichloromethane: methanol (24mL) at room temperature under reflux, followed by the addition of 1 equivalent of the acidic counter ion dissolved in methanol, or in the case of fumaric acid, 1 equivalent of the acidic counter ion dissolved in 96% ethanol. The solution was reduced to 10mL under vacuum and then cooled to-20 ℃. The precipitated material was then filtered, washed with diethyl ether and finally dried under vacuum at 40 ℃ for at least 24 hours.

Example 3: scale-up preparation of compound 937 fumarate salt form I

An amount of compound 937 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).

About 1 equivalent of fumaric acid was then dissolved in ethanol (concentration about 29g/L) and added to the free base solution.

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

The mixture was cooled to about 5 ℃, stirred at this temperature for about 1 hour, filtered, washed with absolute ethanol, and then dried under vacuum at 35-40 ℃.

The resulting material was an ethanol solvate of compound 937 fumarate.

Example 4: scale-up preparation of compound 937 fumarate salt form II

An amount of compound 937 free base was stirred in water at RT and after 30 minutes about 1 equivalent of fumaric acid was added.

The suspension was stirred for 2 hours, then cooled to 4 ℃ and held at this temperature for 1 hour, before being filtered. The resulting material was washed on a filter with cold water and dried under vacuum at 35-40 ℃.

Example 5: solubility of compound 937 salts and free bases

The solubility determination of the compound 937 salt prepared as described in example 2 was performed by the following procedure: known amounts of the compound 937 salt and the free base were stirred in water at RT for 4 hours, assuming a target concentration of 10mg/ml with excess solids. The resulting formulation was filtered and analyzed by HPLC.

The results are reported below.

The solubility value of the compound 937 free base in water according to the above method is < 0.1 mg/mL.

The solubility value of the compound 937L-tartrate salt according to the above method in water is 1.9 mg/mL.

The solubility value of compound 937 succinate in water according to the above method is 5.8 mg/mL.

The solubility value of compound 937 maleate salt in water according to the above method was 3.1 mg/mL.

The solubility value of the compound 937L-malate salt in water according to the above method is 3.9 mg/mL.

The solubility value of compound 937 fumarate in water according to the above method was 0.7 mg/mL.

Example 6: analysis results of powder X-ray diffraction (PXRD)

Compound 937 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 section defining the X-ray powder diffraction peaks of the salt and free base forms of compound 937, the expression ". about" is used to indicate the exact position of the peak (i.e. the cited 2 θ angle values) at the 2 θ angles stated in the table, which should not be interpreted as absolute values, as the skilled person will understand that the exact position of the peak will vary slightly from machine to machine, from sample to sample, or due to slight variations in the measurement conditions employed.

It is also believed that in the preceding paragraphs, the crystalline forms of the salt and free base of compound 937 have an X-ray powder diffraction pattern that is "substantially" identical to the X-ray powder diffraction patterns shown in figures 1, 2, 3, 4, 5, 6, 7, 8, 12, 14, and 16, and have substantially the most pronounced peaks at the 2 θ angle values shown in tables 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. 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, and therefore 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 and/or sample preparation). 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 skilled in the art of X-ray powder diffraction will appreciate that the relative intensities of the peaks may be affected by particles, e.g., greater than 3 θ microns in size and non-uniform in aspect ratio (aspect ratio), which may affect analysis of the 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 also affects the results.

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). Thus, it should be understood that the crystalline forms of the salt and the free base of the compound 937 described in this invention are not limited to crystals having an X-ray powder diffraction pattern identical to, for example, the X-ray powder diffraction pattern shown in fig. 1, and crystals having any X-ray powder diffraction pattern substantially identical to, for example, the X-ray powder diffraction pattern 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, 12, 14 and 16 and when interpreting the peak positions mentioned herein and in tables 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

Thus, when it is stated that, for example, salts and the free base of compound 937 have an X-ray powder diffraction pattern with at least one particular peak at about 2 θ -20.1 degrees (or any other mentioned angle), then this can be interpreted as 2 θ -20.1 degrees plus or minus 0.5 degrees, or 2 θ -20.1 degrees plus or minus 0.2 degrees.

Figure 1 records the powder X-ray diffraction pattern of the salt and free base of compound 937 isolated on a small scale as described in example 1.

Figure 2 reports the salts of compound 937 obtained on a larger scale as described in example 2: examples of powder X-ray diffraction patterns of the salts of L-tartrate form I (B), fumarate form I (C) and L-malate form I (D).

The X-ray diffraction peaks for the compound 937 fumarate salt form I, L-malate salt form I, L-tartrate salt form I, maleate salt form I (A-B), maleate salt form II (C), succinate salt form I (A) and succinate salt form II (B-C) are reported in the figures 3, 4, 5, 6 and 7, respectively.

Figure 8 reports the X-ray diffraction peaks of compound 937 free base form I.

Figure 12 reports the X-ray diffraction peaks of compound 937 free base form I (a-B) which relates to the same batch characterized by the DSC characteristics reported in figure 9, figure 10 and figure 11 (DSC data are also discussed in example 6).

The main X-ray diffraction peak 2 theta angles for the compound 937 fumarate salt form I, L-tartrate salt form I, L-malate salt form I, maleate salt form I and maleate salt form II, succinate salt form I, succinate salt form II, free base and fumarate salt form II are reported in tables 2, 3, 4, 5, 6, 7, 8, 9 and 10, respectively.

A PXRD signature for a scaled-up batch of compound 937 fumarate salt form I obtained according to example 3 is reported in figure 14.

A PXRD signature for a scaled-up batch of compound 937 fumarate salt form II obtained according to example 4 is reported in figure 16.

Table 2-compound 937 fumarate salt form I.

TABLE 3 Compound 937L-tartrate form I

Table 4-compound 937L-malate salt form I.

TABLE 5 Compound 937 maleate form I

TABLE 6 Compound 937 maleate form II

Table 7-compound 937 succinate form I.

TABLE 8 Compound 937 succinate form II

TABLE 9 Compound 937 free base form I

Table 10-compound 937 fumarate salt form II.

Example 7: 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.

Figure 9 records the DSC thermogram of the salt and free base of compound 937 isolated on a small scale as described in example 1.

Figure 10 records the DSC thermogram of a compound 937 salt isolated at a higher scale as described in example 2 compared to the original free base and includes the features of known alternative crystalline forms.

In particular, DSC features 9A, 10a1, 11A and 11B1 relate to the free base form I, while DSC features 10a2 and 11B2 relate to form II.

DSC characteristics of a scaled-up batch of compound 937 fumarate salt form I obtained according to example 3 are reported in figure 15. The melting endotherm with decomposition was observed to be at about 260 ℃ (peak temperature). It should be understood that the onset and/or peak temperature values of the DSC may vary slightly from machine to machine or from sample to sample, and therefore, the values should not be considered absolute. In fact, the observed temperature will depend on the rate of temperature change as well as the sample preparation technique and the particular instrument employed. 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 8: results of thermogravimetric analysis (TGA)

TGA analysis was performed using a Perkin-Elmer TGA-7 apparatus. The aluminum pan was loaded with approximately 5-10mg of sample. The analysis temperature ranges between 30 ℃ and a maximum of about 250 ℃. The samples were analyzed in a nitrogen flow at a heating rate of 2 ℃/min.

Example 9: analysis results of Dynamic Vapor Sorption (DVS)

The water uptake of the compound 937 salt and the free base was studied by subjecting a sample of these materials to a hygroscopicity test using DVS 1000 (SMS). 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 progressive change in RH was 10%; 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.

DVS characteristics of compound 937, preferably salt, prepared as described in example 2 compared to the original free base are recorded in figure 13.

Examples of the water uptake of compound 937 fumarate during DVS adsorption ramp up are summarized in table 11 below.

TABLE 11 Compound 937 fumarate DVS adsorption data

Relative humidity (%)	Water absorption (%)
		0.0	0.0
10.0	0.1
		20.0	0.3
30.0	0.4
		40.0	0.6
50.0	0.9
		60.0	1.2
70.0	1.4
		80.0	1.5
90.0	1.6

Example 10: NMR identification analysis

¹The H NMR experiment was carried out at a constant temperature of 28 ℃,performed on a varianannova 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 5-mm NMR tube for subsequent analysis. This analysis enables confirmation of the expected chemical structure of both the molecule and the counter ion.

Example 11: composition percentage of preparation for oral use

Composition (I)	Range%
		Compound 937	4-20
Lactose monohydrate	45-55
		Pregelatinized starch	30-45
Glyceryl behenate	1-2

Example 12: bulk density of powder

Determination of the powder bulk density of the fumarate, L-malate and L-tartrate salts of compound 937 and the free base was carried out by hand filling hard gelatin capsules with the free-settling active ingredient.

Bulk density has been calculated by dividing the capsule fill weight, including the free-settling active ingredient, by the normal volume of a known hard gelatin capsule. The fill weight has been calculated as the difference between the gross weight of the filled capsule and the tare weight of the empty capsule.

The fumarate, L-malate and L-tartrate salts are characterized by a powder bulk density value of greater than 240mg/ml and a free base density of about 90 mg/ml.

From the above data and examples it should be clear to the skilled person that the novel salts of the compound 937 described in the present invention are a new, improved and valuable tool in therapy.

Claims (3)

1. Crystalline form I fumarate salt of compound 937 having the formula:

it is an ethanol solvate having an X-ray powder diffraction pattern comprising diffraction peaks shown in the following table 2:

2. a pharmaceutical composition comprising as active ingredient the fumarate salt of the compound 937 as defined in claim 1, together with pharmaceutically acceptable excipients and/or carriers.

3. Use of the fumarate salt of a compound 937 as defined in claim 1 for the preparation of a medicament for the treatment of a disease state treatable by PLK inhibition.