EA035560B1 - CRYSTALLINE FORMS OF 2-[(2S)-1-AZABICYCLO[2.2.2]OCT-2-YL]-6-(3-METHYL-1H-PYRAZOL-4-YL)THIENO[3,2-d]PYRIMIDIN-4(3H)-ONE HEMIHYDRATE - Google Patents

Abstract

The present invention relates to a crystalline form I of 2-[(2S)-1-azabicyclo[2.2.2]oct-2-yl]-6-(3-methyl-1H-pyrazol-4-yl)thieno[3,2-d]pyrimidin-4(3H)-one hemihydrate (compound 1) and/or tautomers thereof, wherein compound 1 has the structureprocesses for preparing a crystalline form of compound 1 and/or tautomers thereof; pharmaceutical compositions; methods of inhibiting a cell division cycle 7 in a mammal; and methods of treating a cell division cycle 7 mediated cancer in a mammal.

Description

The technical field to which the invention relates

The present invention relates to crystalline forms of hemihydrate 2 - [(2S) -1azabicyclo [2.2.2 | oct-2-yl | -6- (3-methyl-1H-pyrazol-4-yl) thieno [3.2L | pyrimidine-4 (3H) -one (compound 1) and / or its tautomers, where compound 1 has the structure

The invention also relates to methods for preparing crystalline forms of compound 1 and / or its tautomers. The invention further relates to pharmaceutical compositions containing crystalline compound 1 and / or its tautomers, to methods of inhibiting the cell division cycle 7 (Cdc7) protein in a mammal, including the administration of crystalline compound 1 and / or its tautomers, as well as to methods of treating cancer mediated protein of the cycle of cell division 7, in a mammal.

Cancer is characterized by abnormal cell proliferation with an impaired control mechanism. Most cancer cells grow faster than cells in normal tissues. During the cell division cycle, chromosome doubling is an essential factor, and DNA replication in the S phase of the cell division cycle is tightly regulated. Suppression of DNA replication has been confirmed to be an effective therapy in cancer treatment and, for example, DNA replication inhibitors such as hydroxyurea (HU), gemcitabine and active metabolites of 5-fluorouracil are widely used as therapeutic agents for cancer treatment in clinical practice. ...

Cdc7 is an extremely evolutionarily conserved serine / threonine kinase and is known to play an important role in initiating DNA replication. The kinase activity of Cdc7 is controlled by its binding to the activating partner. During the transition from the late stage of the G1 phase to the S phase, Cdc7 forms a complex with Dbf4 (also known as ASK) and phosphorylates the substrate Cdc7 to control the transition from the G1 phase to the S phase. Recent studies have reported that Cdc7 plays an important role in DNA replication as well as in DNA damage signaling pathways.

In recent years, Cdc7 has been viewed as an attractive target for cancer treatment. Increased expression of Cdc7 has been observed in many cancer cell lines and clinical tumors, including tumors associated with breast cancer, colorectal cancer, and lung cancer. In some cancer cell lines, an increase in the copy number of the activating factor, Dbf4, in chromosomes is observed. Interestingly, in the cancer cell line and the cell line of untransformed fibroblasts, different responses to suppression of Cdc7 expression using siRNA are observed. Suppression of Cdc7 expression when using siRNA causes S-phase blockade in cancer cell lines and induces apoptosis, while in normal cells it induces G1 phase blockade in a p53 activity-dependent manner. In addition, Cdc7 is activated in cells subjected to replicative stress, and the level of apoptosis induced by hydroxyurea and etoposide is increased in cells with downregulation of Cdc7. Accordingly, a Cdc7 inhibitor used alone or in combination with other chemotherapeutic agents would be suitable for the selective treatment of cancer.

Homma M. et al., US Patent No. 8,722,660 B2, pending national phase application PCT / JP2011 / 053303 (published as WO 2011/102399), discloses compounds that are effective inhibitors of Cdc7. The compounds are suitable for inhibiting the kinase activity of Cdc7 in vitro and in vivo and for treating disorders of cell proliferation, in particular cancer.

US Pat. No. 8,722,660 B2 further describes pharmaceutical compositions containing said compounds and methods of treating or treating diseases, disorders or conditions associated with the Cdc7 kinase, including proliferative diseases such as cancer.

Synthesis of 2 - [(2S) -1-azabicyclo [2.2.2] oct-2-yl] -6- (3-methyl-1H-pyrazol-4-yl) thieno [3,2-d] pyrimidine4 (3H) -one and / or its tautomers (untreated compound 1) was described in example 178 of US patent No. 8,722,660 B2. Large-scale production of a pharmaceutical composition poses many challenges for chemists and chemical engineers. Despite the fact that many of these tasks involve the use of a large number of reagents and control of reactions carried out on an industrial scale, the preparation and handling of the final active product pose specific problems associated with the nature of the final crystalline form, which is also called pharmaceutically active in the present description. substance. The active product must not only be obtained in high yield, be stable and available for rapid isolation, but it is also necessary to control the methods of production so as to efficiently and stably obtain the desired

- 1,035,560 crystalline form. The stability and purity of the crystalline form in a pharmaceutical preparation must be monitored at every stage of the manufacturing process, including synthesis, isolation, bulk storage, pharmaceutical formulation, and long-term storage.

The pharmaceutically active substance used to prepare pharmaceutical compositions should be as pure as possible and its long-term storage stability should be confirmed under various environmental conditions. These properties help to prevent the appearance of undesirable degradation products in pharmaceutical compositions, since degradation products can potentially be toxic or may simply lead to a decrease in the activity of the composition.

One of the main problems in the industrial production of pharmaceutical compounds is that the active substance must have a stable crystalline polymorph, which will provide stable technological parameters and pharmaceutical characteristics. When an unstable crystalline form is used during preparation and / or storage, changes can occur in the crystalline polymorph, leading to quality control problems and producing heterogeneous compositions. These changes can affect the reproducibility of the production process, and this leads to the fact that the resulting formulations may not meet the high and stringent quality requirements for formulations or pharmaceutical compositions. For this reason, in general, it should be noted that any changes in the solid form of a pharmaceutical composition that can improve its physical and chemical stability provide significant advantages over less stable forms of the same drug. In addition, it is extremely important to develop a reliable production process that ensures stable production of the active substance. The presence of several crystalline forms with close solubility values creates difficulties in the industrial production of pharmaceutical compounds.

If a compound crystallizes from a solution or suspension, crystallites with different spatial structural lattices can form, this property is called polymorphism. Each of these crystalline forms is called a polymorph. Although polymorphs of a given substance have the same chemical composition, they may differ from each other in one or more physical properties, such as solubility, dissociation, true density, melting point, crystal shape, compression characteristics, flow properties and / or stability. in solid form.

As described generally above, the polymorphic characteristics of drugs can be extremely important in pharmacology. Differences in physical properties exhibited by polymorphs affect practical characteristics such as storage stability, compression characteristics and density (which is important in the preparation of formulations and products), as well as the dissolution rate (which is an important factor in determining bioavailability). Stability differences can be caused by changes in chemical activity (for example, different rates of oxidation, as a result of which a dosage form containing one polymorph is discolored faster than a form containing another polymorph), mechanical changes (for example, tablets crumble on storage if a kinetically more beneficial polymorph is converted into a thermodynamically more stable polymorph) or both types of changes (for example, tablets of one polymorph are more susceptible to destruction at high humidity). In addition, the physical properties of the crystal can be important during processing. For example, one polymorph may have an increased tendency to form solvates, which leads to aggregation of the solid form and makes it difficult to handle the solid. Alternatively, the polymorphs may differ from one another in shape and particle size distribution, which makes it difficult to filter the pharmaceutically active substance to remove impurities.

While formulations having improved chemical and physical properties are desirable, there is no means to predictably obtain new dosage forms (eg, polymorphs and other new crystalline forms) of existing molecules for inclusion in these formulations. These new forms could provide consistency of physical properties in various environments associated with the production and use of the compositions. Thus, there is a need for new forms of drugs that would be suitable for inhibiting the kinase activity of Cdc7 in vitro and in vivo, and for the treatment of disorders of cell proliferation, in particular cancer, and other disorders associated with the kinase activity of Cdc7, and would also have properties suitable for industrial production and incorporation into compositions.

The present invention relates to crystalline hemihydrate 2 - [(2S) -1-azabicyclo [2.2.2] oct2-yl] -6- (3-methyl-1H-πyrazol-4-yl) thieno [3,2-d] πyrimidine -4 (3H) -one (compound 1) and / or its tautomers, where compound 1 has the structure

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These forms have properties suitable for industrial production, incorporation into pharmaceutical formulations and / or storage. The present invention also relates to pharmaceutical compositions containing crystalline compound 1 and / or its tautomers; and methods of using said compound, including the treatment of certain diseases, disorders or conditions such as described herein.

In some embodiments, the invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent; and crystalline compound 1 and / or its tautomers.

Some embodiments of the invention relate to methods of treating a subject in need of a Cdc7 kinase inhibitor by administering an effective amount of crystalline Compound 1 and / or its tautomers.

Some embodiments of the invention relate to said processes, where the compound is crystalline Form I of compound 1. Some additional embodiments of the invention relate to said processes, where the compound is crystalline Form A of compound 1. Some additional embodiments of the invention relate to said processes, where the compound is a mixture of crystalline form I of compound 1 and crystalline form A of compound 1.

Some embodiments of the invention relate to methods for preparing crystalline forms of compound 1 and / or its tautomers. Some embodiments of the invention relate to said processes, where the compound is crystalline Form I of compound 1. Some additional embodiments of the invention relate to said processes, where the compound is crystalline Form A of compound 1. Some additional embodiments of the invention relate to said processes, where the compound is a mixture of crystalline form I of compound 1 and crystalline form A of compound 1.

Brief Description of Drawings

In the following description, XRPD refers to X-ray powder diffraction, tnMR refers to solid state nuclear magnetic resonance spectroscopy, and ORTEP refers to the Oak Ridge thermal ellipsoid program.

FIG. 1 is an XRPD pattern of Crystalline Form 1 of Compound 1.

FIG. 2 shows the ¹³ C tnMR spectrum of crystalline Form I of compound 1.

FIG. 3 is an XRPD pattern of Crystalline Form A of Compound 1.

FIG. 4 shows the ¹³ C tnMR spectrum of crystalline form A of compound 1.

FIG. 5 is an XRPD pattern overlay of Crystalline Form 1 of Compound 1 and Crystalline Form A of Compound 1.

FIG. 6 is an overlay of the ¹³ C tnMR spectrum of crystalline Form 1 of compound 1 and crystalline Form A of compound 1.

FIG. 7 shows the solubility of crystalline form I of compound 1 and crystalline form A of compound 1 in a water / DMSO mixture at 60 ° C.

FIG. 8 is an ORTEP image of the crystal structure of crystal Form I of compound 1, where hydrogen atoms are not indicated.

FIG. 9 is an ORTEP image of the crystal structure of crystal form A of compound 1, where hydrogen atoms are not indicated.

Detailed description

Definitions and abbreviations

It should be understood that unless otherwise indicated, the terms used above and below in the present description have the following meanings.

In the present specification, weight% and weight% are used interchangeably to describe weight percentages.

As used herein, the terms crystalline compound, compound, and crystalline form of the compound are used interchangeably.

As used herein, the terms crystalline form of compound 1 and crystalline compound 1 are used interchangeably.

As used herein, the terms crude formulation of compound 1 and

- 3 035560 crude compound 1 refers to 2 - [(2S) -1-azabicyclo [2.2.2] oct-2-yl] -6- (3-methyl-1H-pyrazol4-yl) thieno [.3.2L | pyrimidine -4 (.3H) -one and / or its tautomers. The synthesis of crude compound 1 was described in Example 178 in US Pat. No. 8,722,660 B2. As a non-limiting example, tautomerization of the untreated compound 1 can occur at the pyrazole and pyrimidine groups of the untreated compound 1. Specific examples of tautomerization that can occur in the untreated compound 1 include

Specific non-limiting examples of the isomeric structures of the included tautomers are

2 - [(2S) -1-azabicyclo [2.2.2] oct-2-yl] -6- (3-methyl-1H-pyrazol-4-yl) thieno [3,2-d] pyrimidine-4 (3H )-he,

2 - [(2S) -1-azabicyclo [2.2.2] oct-2-yl] -6- (5-methyl-1H-pyrazol-4-yl) thieno [3,2-d] pyrimidine-4 (3H )-he.

As used herein, the terms Form I and Crystalline Form I of Compound 1 are used interchangeably to describe crystal Form I of the hemihydrate 2 - [(2S) -1-azabicyclo [2.2.2] oct-2yl] -6- (3-methyl-1H-pyrazole -4-yl) thieno [3,2D] pyrimidin-4 (3H) -one (compound 1) and / or its tautomers, which can be characterized in some embodiments by the data shown in FIG. 1, 2, 7 and 8.

As used herein, the terms Form A and Crystalline Form A of Compound 1 are used interchangeably to describe crystal Form A of the hemihydrate 2 - [(2S) -1-azabicyclo [2.2.2] oct-2yl] -6- (3-methyl-1H-pyrazole -4-yl) thieno [3,2D] pyrimidin-4 (3H) -one (compound 1) and / or its tautomers, which can be characterized in some embodiments by the data shown in FIG. 3, 4, 7 and 9.

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As used herein, the terms Form A / I and Crystalline Form A / I of Compound 1 are used interchangeably to describe a mixture of Crystalline Form A of Compound 1 and Crystalline Form I of Compound 1.

As used herein, a crystal refers to a solid in which its constituent atoms, molecules or ions are packed in a regularly ordered repeating three-dimensional network having a highly ordered chemical structure. For the purposes of this application, the terms crystalline form and polymorph are synonymous; the terms allow differentiation between crystals that have different properties (eg, different XRPD patterns, different DSC scans, or different ¹³ C solid state NMR spectra).

As used herein, substantially crystalline refers to solid forms of compound 1 and / or its tautomers that have at least a particular crystalline phase content in percent by weight. Specific percentages by weight include 10, 20, 30, 40, 50, 60, 70, 75, 80, 85, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5 and 99.9%. In some embodiments, substantially crystalline refers to solid forms that contain at least 70% crystalline phase. In some embodiments, substantially crystalline refers to solid forms that contain at least 80% crystalline phase. In some embodiments, substantially crystalline refers to solid forms that contain at least 85% crystalline phase. In some embodiments, substantially crystalline refers to solid forms that contain at least 90% crystalline phase. In some embodiments, substantially crystalline refers to solid forms that contain at least 95% crystalline phase.

When used in the present description, the term hydrate refers to a solvate where the solvent molecule is H ₂ O and is contained in a specified stoichiometric amount, which is at least 1: 1 water: 2 - [(2S) -1-azabicyclic [2.2.2 ] oct-2-yl] -6- (3-methyl-1H-pyrαzol-4yl) thieno [3,2-d] pyrimigin-4 (3H) -one and / or its tautomers, and includes, for example, monohydrates, dihydrates and trihydrates.

When used in the present description, the term hemihydrate refers to a solvate where the solvent molecule is H ₂ O and is contained in a specific stoichiometric amount, which is 0.5: 1 water: 2 - [(2S) -1-azabicyclo [2.2.2] oct-2-yl] -6- (3-methyl-1H-purazol-4-yl) thieno [3,2d] purimugin-4 (3H) -one and / or its tautomers.

As used herein, the term dosage refers to the amount of a particular compound contained in a dosage form.

As used herein, the term mixture refers to the combined elements of a mixture regardless of the phase state of the combination (eg, liquid or liquid / crystalline).

As used herein, the term seeding refers to the addition of crystalline material to a solution or mixture to initiate crystallization.

As used herein, the term D10, which describes the particle size distribution, refers to the diameter of the particles in which 10% of the mass of the sample are particles having a diameter less than the D10 diameter.

As used herein, the term D50, which describes the particle size distribution, refers to the particle diameter at which 50% of the mass of the sample is particles having a diameter less than the D50 diameter.

As used herein, the term D90, which describes the particle size distribution, refers to the diameter of the particles in which 90% of the mass of the sample is particles having a diameter less than the D90 diameter.

It should be understood that when used in the present description of the phrase crystalline compound 1 and / or its tautomers, etc. mean crystalline compound 1 and all its tautomeric forms. As a non-limiting example, tautomerization of crystalline compound 1 can occur in the pyrazole and pyrimidine groups of crystalline compound 1. Specific examples of tautomerization that can occur in compound 1 include

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Specific non-limiting examples of the isomeric structures of the included tautomers are

2 - [(^) - 1-azabicyclo [2.2.2 | oct-2-yl | -6- (3-methyl-1H-pyrazol-4-yl) thieno [3.2 hemihydrate d] pyrimidine-4 (3H) - She and

hemihydrate 2 - [(2S) -1-azabicyclo [2.2.2] oct-2-yl] -6- (5-methyl-1H-pyrazol-4-yl) thieno [3,2d] pyrimidine-4 (3H) -it.

In one aspect, the present invention relates to crystalline hemihydrate 2 [(2S) -1-azabicyclo [2.2.2] oct-2-yl] -6- (3-methyl-1H-pyrazol-4-yl) thieno [3, 2-d] pyrimidin-4 (3H) -one (compound 1) and / or its tautomers, where compound 1 has the structure

This description provides a variety of characteristic data describing cry

- 6 035560 Steel Forms of Compound 1. However, it should be understood that not all of this information is necessary for a person skilled in the art to determine the possible presence of a particular form in a given composition. Determination of a particular shape can be carried out using any piece of characteristic data that a person skilled in the art can consider to be sufficient to confirm the presence of a particular shape, for example, even a single distinctive peak may be sufficient for a person skilled in the art to determine the presence of the specified shape. specific form.

At least one of the crystalline forms according to the present invention has properties that make the solid forms suitable for industrial preparation of a pharmaceutical formulation. At least one of the crystalline forms can provide desirable physical and chemical properties, including low hygroscopicity and chemical and optical stability. As a non-limiting example, crystalline Form I of compound 1 and crystalline Form A of compound 1 are not hygroscopic at 25 or 40 ° C. As a further non-limiting example, crystalline Form I of compound 1 and crystalline Form A of compound 1 are chemically and optically stable at 60 ° C / 75% relative humidity (outdoor) for at least eight weeks. As a further non-limiting example, crystalline Form I of compound 1 and crystalline Form A of compound 1 are chemically and optically stable at 80 ° C (unopened) for at least eight weeks.

Some embodiments of the invention relate to solid forms of compound 1 and / or its tautomers, where the solid form has at least a specific content of the crystalline phase in percent by weight. In some embodiments, the solid form of Compound 1 and / or its tautomers is substantially crystalline. Non-limiting examples of crystalline or substantially crystalline form of compound 1 include crystalline form I of compound 1 and crystalline form A of compound 1. Some embodiments of the invention also relate to the solid form of compound 1 and / or its tautomers, where the solid form has at least a specific the content of the crystalline phase in percent by weight, and thus one or more of these crystalline forms are not included in the particular content in solid form in percent by weight. Specific percentages by weight include 10, 20, 30, 40, 50, 60, 70, 75, 80, 85, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5 and 99.9%. If the solid form has a specific content of the crystalline phase in percent by weight, the remainder of the solid form is an amorphous form of compound 1 and / or its tautomers.

Other embodiments of the invention relate to compound 1 and / or its tautomers, which are in crystalline form or substantially crystalline form. Crystalline compound 1 may have a specific content by weight of the crystalline form. Specific percentages by weight include 10, 20, 30, 40, 50, 60, 70, 75, 80, 85, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5 and 99.9%. If Compound 1 and / or its tautomers have a specific percentage by weight of said crystalline form, the remainder of Compound 1 is some combination of the amorphous form of Compound 1 and one or more crystalline forms of Compound 1, except for the specified crystalline form. In some embodiments, the solid form of Compound 1 and / or its tautomers contains at least 95% by weight of the crystalline form. In some embodiments, the solid form of Compound 1 and / or its tautomers contains at least 90% by weight of the crystalline form. In some embodiments, the solid form of Compound 1 and / or its tautomers contains at least 85% by weight of the crystalline form. In some embodiments, the solid form of Compound 1 and / or its tautomers contains at least 80% by weight of the crystalline form.

In the following description of solid forms of compound 1, embodiments of the invention may be described with reference to a particular crystalline form of compound 1 and / or its tautomers, which may be characterized by one or more of the properties discussed herein. Crystalline form data can also be used to describe a mixture of different crystal forms that may be contained in the crystalline form of compound 1. However, specific crystalline forms of compound 1 can also be characterized by one or more polymorph properties such as described herein whether or not associated with a particular crystalline form.

X-ray powder diffraction (XRPD) crystallographic analysis is commonly used to analyze a particular crystal form. It should be understood that if, in the specification and claims, the crystalline form of Compound 1 and / or its tautomers is identified by one or more characteristic XRPD peaks expressed in 2θ angles, then each of the 2θ values is defined as the stated value ± 0.2 degrees. It should be noted that the values in the XRPD spectrum (in particular, values with a small angle (and a large d)) may shift slightly depending on the degree of fineness of the sample.

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It should be understood that if, in the specification and claims, the crystalline form of compound 1 and / or its tautomers is identified by one or more characteristic XRPD peaks expressed in interplanar spacing (d), then each d value is defined as the stated value ± 0.2 angstroms ...

It should be understood that if in the description and claims crystalline form of Compound 1 and / or its tautomers identified by one or more characteristic peaks ^{of 13} C-NMR, expressed in ppm, then each of the values in ppm is defined as the specified value ± 0.5 ppm ...

In some embodiments, the present invention provides a crystalline compound, hemihydrate 2 - [(2S) -1-azabicyclo [2.2.2] oct-2-yl] -6- (3-methyl-1H-pyrazol-4-yl) thieno [3,2d] pyrimidin-4 (3H) -one (compound 1) and / or its tautomers, where compound 1 has the structure

about

In some embodiments, crystalline compound 1 is characterized by an X-ray powder diffraction pattern having characteristic peaks expressed in degrees 2θ at 4.1 ± 0.2, 8.1 ± 0.2, 12.2 ± 0.2, and 16.3 ± 0.2. In additional embodiments, crystalline compound 1 is characterized by an X-ray powder diffraction pattern having characteristic peaks expressed in degrees 2θ at 4.1 ± 0.2, 8.1 ± 0.2, 12.2 ± 0.2, 16.3 ± 0.2, 17.8 ± 0.2, 26.4 ± 0.2 and 27.0 ± 0.2.

In some other embodiments, crystalline compound 1 is characterized by an X-ray powder diffraction pattern having characteristic interplanar spacing (d) peaks, expressed in angstroms, at 21.6 ± 0.2, 10.9 ± 0.2, 7.3 ± 0, 2 and 5.4 ± 0.2. In additional embodiments, crystalline compound 1 is characterized by an X-ray powder diffraction pattern having characteristic interplanar spacing (d) peaks, expressed in angstroms, at 21.6 ± 0.2, 10.9 ± 0.2.7.3 ± 0.2 , 5.4 ± 0.2, 5.0 ± 0.2, 3.4 ± 0.2 and 3.2 ± 0.2.

In some other embodiments, crystalline compound 1 has a solid ¹³ C NMR spectrum including characteristic peaks expressed in ppm at 159.7 ± 0.5, 158.4 ± 0.5, 145.1 ± 0.5, 140. 6 ± 0.5, 135.5 ± 0.5, 121.2 ± 0.5, 114.5 ± 0.5, 60.4 ± 0.5, 53.8 ± 0.5, 46.5 ± 0.5 and 14.5 ± 0.5.

In some embodiments, crystalline compound 1 is crystalline Form I of compound 1. FIG. 1 shows an X-ray powder diffraction (XRPD) pattern of crystalline Form I of Compound 1.

In some embodiments, Form I is characterized by an X-ray powder diffraction pattern having characteristic peaks expressed in degrees 2θ at 15.2 ± 0.2, 17.8 ± 0.2, and 27.6 ± 0.2. In some embodiments, Form I is characterized by an X-ray powder diffraction pattern having characteristic peaks expressed in degrees 2θ at 4.1 ± 0.2, 8.1 ± 0.2, 12.2 ± 0.2, 15.2 ± 0.2, 16.3 ± 0.2, 17.8 ± 0.2 and 27.6 ± 0.2. In some embodiments, Form I is characterized by an X-ray powder diffraction pattern having characteristic peaks expressed in degrees 2θ at 4.1 ± 0.2, 8.1 ± 0.2, 12.2 ± 0.2, 15.2 ± 0.2, 16.3 ± 0.2, 17.8 ± 0.2, 19.0 ± 0.2 and 27.6 ± 0.2. In some embodiments, Form I is characterized by an X-ray powder diffraction pattern having characteristic peaks expressed in degrees 2θ at 4.1 ± 0.2, 8.1 ± 0.2, 12.2 ± 0.2, 16.3 ± 0.2, 17.8 ± 0.2, 19.0 ± 0.2, 20.4 ± 0.2, 26.4 ± 0.2, 27.0 ± 0.2, 27.6 ± 0, 2 and 30.4 ± 0.2.

In some embodiments, Form I is characterized by an X-ray powder diffraction pattern having characteristic interplanar spacing (d) peaks, expressed in angstroms, at 5.8 ± 0.2, 5.0 ± 0.2, and 3.2 ± 0.2. In some embodiments, Form I is characterized by an X-ray powder diffraction pattern having characteristic interplanar spacing (d) peaks expressed in angstroms at 21.6 ± 0.2, 10.9 ± 0.2, 7.3 ± 0.2, 5.8 ± 0.2, 5.4 ± 0.2, 5.0 ± 0.2, and 3.2 ± 0.2. In some embodiments, Form I is characterized by an X-ray powder diffraction pattern having characteristic interplanar spacing (d) peaks expressed in angstroms at 21.6 ± 0.2, 10.9 ± 0.2, 7.3 ± 0.2, 5.8 ± 0.2, 5.4 ± 0.2, 5.0 ± 0.2, 4.3 ± 0.2, 3.4 ± 0.2, 3.3 ± 0.2, 3, 2 ± 0.2 and 2.9 ± 0.2.

In some embodiments, Form 1 is characterized by an x-ray diffractogram

- 8,035560 powder diffraction, essentially as shown in FIG. 1.

FIG. 2 shows the ¹³ C tnNMR spectrum of crystalline Form I of compound 1.

In some embodiments, Form I has a solid state ¹³ C NMR spectrum including characteristic ppm peaks at 32.2 ± 0.5, 30.2 ± 0.5, 29.3 ± 0.5, 28.2 ± 0.5, 25.3 ± 0.5 and 24.8 ± 0.5. In some embodiments, Form I has a solid ¹³ C NMR spectrum including characteristic peaks, expressed in ppm, at 159.7 ± 0.5, 158.4 ± 0.5, 145.1 ± 0.5, 144.8 ± 0.5, 140.6 ± 0.5, 135.7 ± 0.5, 135.3 ± 0.5, 121.2 ± 0.5, 114.7 ± 0.5, 114.5 ± 0, 5, 60.4 ± 0.5, 53.8 ± 0.5, 46.5 ± 0.5, 32.2 ± 0.5, 30.2 ± 0.5, 29.3 ± 0.5, 28.2 ± 0.5, 25.3 ± 0.5, 24.8 ± 0.5 and 14.5 ± 0.5.

In some embodiments, Form I has a solid state ¹³ C NMR spectrum substantially as depicted in FIG. 2.

FIG. 8 shows an ORTEP image of the crystal structure of crystal Form I of compound 1, where hydrogen atoms are not indicated.

In some embodiments, Form I is characterized by colorless crystals. In some embodiments, Form I is characterized by prismatic crystals. In some embodiments, Form I is characterized by a monoclinic crystal system. In some embodiments, Form I has a P2i space group (# 4). In some embodiments, Form I is characterized by the lattice parameters of the single crystal: a = 6.2263 (1) A; b = 43.5007 (8) A; c = 6.7944 (2) A; β = 117.207 (2) °; and V = 1636.65 (6) A ³ . In some other embodiments, Form I is characterized by any of the crystallographic parameters listed in Example 14.

In some embodiments, Form I is characterized by an ORTEP depiction where no hydrogen atoms are indicated, essentially as shown in FIG. 8.

In some embodiments, Form I is characterized by a low frequency Raman spectroscopy spectrum including characteristic peaks at 17.4 ± 0.4 cm ⁻¹ and at about 11 cm ⁻¹ representing a shoulder peak.

In some embodiments, the solubility of Form I in a water / DMSO mixture at 60 ° C is substantially the same as depicted in FIG. 7.

In some embodiments, Form I is not hygroscopic at 25 ° C. In some embodiments, Form I is not hygroscopic at 40 ° C. In some embodiments, Form I is not hygroscopic at temperatures ranging from 25 ° C to 40 ° C.

In some embodiments, Form I is chemically and optically stable at 80 ° C (unopened) for at least eight weeks. In some embodiments, Form I is chemically and optically stable at 60 ° C / 75% relative humidity (outdoors) for at least eight weeks.

In some embodiments, crystalline Compound 1 is Crystal Form A of Compound 1.

FIG. 3 shows an X-ray powder diffraction (XRPD) pattern of crystalline Form A of Compound 1.

In some embodiments, Form A is characterized by an X-ray powder diffraction pattern having characteristic peaks expressed in degrees 2θ at 17.6 ± 0.2, 18.2 ± 0.2, and 19.7 ± 0.2. In some embodiments, Form A is characterized by an X-ray powder diffraction pattern having characteristic peaks expressed in degrees 2θ at 4.1 ± 0.2, 8.1 ± 0.2, 12.2 ± 0.2, 16.3 ± 0.2, 17.6 ± 0.2, 18.2 ± 0.2 and 19.7 ± 0.2. In some embodiments, Form A is characterized by an X-ray powder diffraction pattern having characteristic peaks expressed in degrees 2θ at 4.1 ± 0.2, 8.1 ± 0.2, 12.2 ± 0.2, 16.3 ± 0.2, 17.6 ± 0.2, 18.2 ± 0.2, 19.7 ± 0.2, 26.5 ± 0.2, 27.1 ± 0.2 and 29.9 ± 0, 2. In some embodiments, Form A is characterized by an X-ray powder diffraction pattern having characteristic peaks expressed in degrees 2θ at 4.1 ± 0.2, 8.1 ± 0.2, 12.2 ± 0.2, 16.3 ± 0.2, 17.6 ± 0.2, 18.2 ± 0.2, 19.7 ± 0.2, 24.1 ± 0.2, 26.5 ± 0.2, 27.1 ± 0, 2 and 29, 9 ± 0.2.

In some embodiments, Form A is characterized by an X-ray powder diffraction pattern having characteristic interplanar spacing (d) peaks, expressed in angstroms, at 5.1 ± 0.2, 4.9 ± 0.2, and 4.5 ± 0.2. In some embodiments, Form A is characterized by an X-ray powder diffraction pattern having characteristic interplanar spacing (d) peaks, expressed in angstroms, at 21.5 ± 0.2, 10.9 ± 0.2, 7.3 ± 0.2, 5.5 ± 0.2, 5.1 ± 0.2, 4.9 ± 0.2 and 4.5 ± 0.2. In some embodiments, Form A is characterized by an X-ray powder diffraction pattern having characteristic interplanar spacing (d) peaks, expressed in angstroms, at 21.5 ± 0.2, 10.9 ± 0.2, 7.3 ± 0.2, 5.5 ± 0.2, 5.1 ± 0.2, 4.9 ± 0.2, 4.5 ± 0.2, 3.4 ± 0.2, 3.3 ± 0.2 and 3, 0 ± 0.2. In some embodiments, Form A is characterized by an X-ray powder diffraction pattern having characteristic interplanar spacing (d) peaks, expressed in angstroms, at 21.5 ± 0.2, 10.9 ± 0.2, 7.3 ± 0.2, 5.5 ± 0.2, 5.1 ± 0.2, 4.9 ± 0.2, 4.5 ± 0.2, 3.7 ± 0.2, 3.4 ± 0.2, 3, 3 ± 0.2 and 3.0 ± 0.2.

- 9 035560

In some embodiments, Form A has an X-ray powder diffraction pattern substantially as shown in FIG. 3.

FIG. 4 shows the ^{B * * * * 13} C mNMR spectrum of crystalline form A of compound 1.

In some embodiments, Form A has a solid ¹³ C NMR spectrum including characteristic ppm peaks at 32.1 ± 0.5, 30.8 ± 0.5, 28.8 ± 0.5, and 25.2 ± 0.5. In some embodiments, Form A has a solid ¹³ C NMR spectrum including characteristic peaks, expressed in ppm, at 159.7 ± 0.5, 158.5 ± 0.5, 144.8 ± 0.5, 140.6 ± 0.5, 135.6 ± 0.5, 121.0 ± 0.5, 114.6 ± 0.5, 60.3 ± 0.5, 53.4 ± 0.5, 46.6 ± 0, 5, 32.1 ± 0.5, 30.8 ± 0.5, 28.8 ± 0.5, 25.2 ± 0.5, and 14.5 ± 0.5.

In some embodiments, Form A has a solid state ¹³ C NMR spectrum substantially as depicted in FIG. 4.

FIG. 9 shows an ORTEP image of the crystal structure of crystal form A of compound 1, where no hydrogen atoms are indicated.

In some embodiments, Form A is characterized by colorless crystals. In some embodiments, Form A is characterized by lamellar crystals. In some embodiments, Form A is characterized by a monoclinic crystal system. In some embodiments, Form A has space group C2 (# 5). In some embodiments, Form A has lattice parameters: a = 12.031 (2) A; b = 6.2460 (8) A; c = 21.947 (4) A; β = 95.26 (1) °; and V = 1642.3 (4) A ³ . In some other embodiments, Form A has any of the crystallographic parameters listed in Example 14.

In some embodiments, Form A is characterized by an ORTEP image where no hydrogen atoms are indicated, essentially as shown in FIG. nine.

In some embodiments, Form A is characterized by a low frequency Raman spectroscopy spectrum including characteristic peaks at 15.6 ± 0.4 cm ^-1 .

In some embodiments, the solubility of Form A in a water / DMSO mixture at 60 ° C is substantially as depicted in FIG. 7.

In some embodiments, Form A is not hygroscopic at 25 ° C. In some embodiments, Form A is not hygroscopic at 40 ° C. In some embodiments, Form A is not hygroscopic at temperatures ranging from 25 ° C to 40 ° C.

In some embodiments, Form A is chemically and optically stable at 80 ° C (unopened) for at least eight weeks. In some embodiments, Form A is chemically and optically stable at 60 ° C / 75% RH (outdoors) for at least eight weeks.

In some embodiments, crystalline Compound 1 is a mixture of Crystalline Form I of Compound 1 and Crystalline Form A of Compound 1.

Methods of use and pharmaceutical compositions

Compound 1 and / or its tautomers and crystalline forms are suitable agents for the prevention or treatment of cancer. Accordingly, Compound 1 can be used to suppress excessive or abnormal Cdc7 activity in mammals (eg, mice, rats, hamsters, rabbits, cats, dogs, cattle, sheep, monkeys, humans).

In some embodiments, the present invention provides a pharmaceutical composition comprising crystalline 2- [(2S) -1-azabicyclo [2.2.2] oct-2-yl] -6- (3-methyl-1Hpyrazol-4-yl) thieno hemihydrate [3,2D] pyrimidin-4 (3H) -one (compound 1) and / or its tautomers, where compound 1 has the structure:

In some embodiments, the present invention provides a pharmaceutical composition comprising crystalline Form I of compound 1. In some embodiments, the present invention provides a pharmaceutical composition comprising crystalline Form A of Compound 1. In some embodiments, the present invention provides a pharmaceutical composition comprising a mixture of crystalline Form I compound 1 and crystal form A of compound 1.

In some embodiments, the present invention provides a method for inhibiting a 7 cell cycle protein in a mammal, comprising administering an effective amount of crystalline compound 1 and / or its tautomers, or a pharmaceutical composition comprising

- 10 035560 crystalline compound 1 and / or its tautomers.

In some embodiments, the present invention provides a method for treating cancer in a mammal, wherein said cancer is mediated by a cell cycle protein 7, comprising administering an effective amount of crystalline compound 1 and / or its tautomers, or a pharmaceutical composition comprising crystalline compound 1 and / or its tautomers.

In some embodiments, crystalline Compound 1 and / or tautomers thereof, or a pharmaceutical composition comprising crystalline Compound 1 and / or tautomers thereof, is suitable for the treatment of cancer. Non-limiting examples of cancers include colorectal cancer (eg, colorectal cancer, rectal cancer, anal cancer, familial colorectal cancer, hereditary non-polyposis colorectal cancer, gastrointestinal stromal tumor), lung cancer (eg, non-small cell lung cancer, small cell lung cancer, malignant mesothelioma ), mesothelioma, pancreatic cancer (eg, ductal pancreatic cancer, pancreatic endocrine tumor), oropharyngeal cancer, laryngeal cancer, esophageal cancer, gastric cancer (eg, papillary adenocarcinoma, mucinous adenocarcinoma, adenosquamous carcinoma, intestinal carcinoma) small bowel cancer, breast cancer (eg, infiltrating intraductal carcinoma, non-infiltrating intraductal carcinoma, inflammatory breast cancer), ovarian cancer (eg, ovarian epithelial carcinoma, extragonadal germ cell tumor, ovarian germ cell tumor, ovarian tumor) low potential malignancy), testicular tumor, prostate cancer (eg, hormone-dependent prostate cancer, hormone-independent prostate cancer), liver cancer (eg, hepatic cell carcinoma, primary liver cancer, extrahepatic bile duct cancer), thyroid cancer (eg, medullary carcinoma thyroid), kidney cancer (eg, renal cell carcinoma, transitional cell carcinoma of the renal pelvis and urinary duct), uterine cancer (eg, cervical cancer, uterine cancer, uterine sarcoma), brain tumor (eg, medulloblastoma, glioma, pineal astrocytoma , pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, pituitary adenoma), retinoblastoma, skin cancer (eg, basal cell tumor, malignant melanoma), sarcoma (eg, rhabdomyosarcoma, leiomyosarcoma, soft tissue sarcoma), urinary bladder cancer cancer (eg, multiple myeloma, leukemia, malignant lymphoma, Hodgkin's lymphoma, chronic proliferative bone marrow disease), cancer of unknown primary site), cancer growth inhibitory agent, cancer metastasis inhibitor, apoptosis promoter, etc.

In some embodiments, crystalline Compound 1 and / or tautomers thereof, or a pharmaceutical composition comprising crystalline Compound 1 and / or tautomers thereof, is suitable for the treatment of hematologic cancer, breast cancer, colorectal cancer, lung cancer, pancreatic cancer, etc.

Crystalline compound 1 and / or its tautomers, or a pharmaceutical composition containing the crystalline form of compound 1 and / or its tautomers, can be administered orally.

In some embodiments, a pharmaceutical composition comprising crystalline Compound 1 and / or its tautomers is formulated as an oral dosage form. In some additional embodiments, a pharmaceutical composition comprising crystalline Compound 1 and / or tautomers thereof is administered orally.

Examples of oral dosage forms for the pharmaceutical composition of the present invention include an oral preparation such as a capsule.

In some embodiments, the pharmaceutical composition comprises crystalline Form I of Compound 1.

In some embodiments, the pharmaceutical composition comprises crystalline Form A of Compound 1.

In some embodiments, the pharmaceutical composition comprises Crystalline Form I of Compound 1 or Crystalline Form A of Compound 1 and further comprises an excipient. In some embodiments, said filler is mannitol or lactose. In some embodiments, the filler is present in an amount ranging from 49 to 90% by weight of the pharmaceutical composition.

In some embodiments, the pharmaceutical composition comprises Crystalline Form I of Compound 1 or Crystalline Form A of Compound 1 and further comprises a lubricant. In some embodiments, said glidant is colloidal silicon dioxide. In some embodiments, said lubricant is present in an amount in the range of 1 to 4% by weight based on the weight of the pharmaceutical composition.

In some embodiments, the pharmaceutical composition comprises a mixture of Crystalline Form I of Compound 1 and Crystalline Form A of Compound 1.

In some embodiments, the pharmaceutical composition comprises crystalline compound 1 and / or its tautomers and at least one excipient selected from mannitol and lactose.

In some embodiments, the pharmaceutical composition comprises crystalline

- 11 035560 compound 1 and / or its tautomers and mannitol, where mannitol is contained in an amount in the range from 49 to 90 wt% based on the weight of the pharmaceutical composition.

In some embodiments, the pharmaceutical composition comprises crystalline compound 1 and / or its tautomers and lactose, where lactose is present in an amount ranging from 49 to 90% by weight of the pharmaceutical composition.

In any of the above embodiments, the pharmaceutical composition according to the present invention containing compound 1 and / or its tautomers further comprises colloidal silicon dioxide. In some embodiments, the colloidal silicon dioxide is present in an amount ranging from 1 to 4% by weight of the pharmaceutical composition.

In some embodiments, the pharmaceutical composition comprising crystalline Compound 1 and / or tautomers thereof is in the form of a capsule.

In some embodiments, said capsule further comprises at least one filler and at least one glidant. In some embodiments, said filler is mannitol. In some embodiments, said glidant is colloidal silicon dioxide.

In some embodiments, the pharmaceutical composition containing crystalline compound 1 and / or its tautomers, in the form of a capsule contains from 5 to 15 wt.% Crystalline form of compound 1 and / or its tautomers, from 85 to 95 wt.% Filler and from 0, 5 to 2% glidant based on the weight of the capsule excluding the capsule shell. In some embodiments, the filler is mannitol. In some embodiments, the glidant is colloidal silicon dioxide. In some embodiments, the capsule shell is 20 to 30% by weight based on the total weight of the capsule.

In some embodiments, the crystalline form is Crystal Form I of Compound 1. In some embodiments, the crystalline form is Crystal Form A of Compound 1. In some embodiments, the crystalline form is a mixture of Crystal Form A of Compound 1 and Crystal Form I of Compound 1.

Despite the fact that the amount of crystalline compound 1 and / or its tautomers in the pharmaceutical composition according to the present invention may vary depending on the type of pharmaceutical composition, in general it ranges from 0.01 to 99.9 wt%, for example from 2 to 85 wt%, such as 5 to 70 wt%, based on the weight of the pharmaceutical composition as a whole.

Although the amount of additives in the pharmaceutical composition according to the present invention may vary depending on the type of pharmaceutical composition, it is generally in the range from 1 to 99.9 wt%, for example from 10 to 90 wt%, based on the weight the pharmaceutical composition as a whole.

The crystalline forms according to the present invention are stable and have low toxicity and can be used safely. Although the daily dose may vary depending on the condition and body weight of the mammal, the route of administration, etc., for example, the crystalline form according to the present invention can be orally administered in the form of a pharmaceutical composition described in this application to a mammal for treating cancer ... In some embodiments, the dose administered to an adult (body weight about 60 kg) is 30 mg orally once or twice a day.

General synthesis methods

The development of the methods for the preparation of the crystalline forms described in this application has been particularly difficult due to the similar values of the relative solubility of the crystalline forms. Indeed, the development of reliable and stable production processes according to the present invention required a deep understanding of the crystallization characteristics of the compound and the thermodynamics of the process. In one aspect, crystalline Form I of Compound 1 is prepared by a method comprising the step of recrystallizing a crude preparation of Compound 1 or its tautomers from a mixture containing DMSO and at least one other solvent selected from water, acetonitrile and acetone.

In some embodiments, the mixture contains DMSO and water. In some embodiments, the mixture comprises DMSO: water solvents in a volume ratio of 10: 1 to 12: 1. In some embodiments, DMSO is present in an amount ranging from 9.6 to 10.6 ml DMSO / g of Compound 1. In some embodiments, the water concentration ranges from 0 to 8 wt% based on the total amount of DMSO and water.

In some embodiments, the mixture contains DMSO and acetone. In some embodiments, the mixture comprises DMSO: acetone solvents in a 10: 1 volume ratio. In some embodiments, the mixture contains DMSO, acetone, and water. In some embodiments, the mixture comprises DMSO: acetone: water solvents in a 2.4: 1: 1 volume ratio.

In another aspect, crystalline Form I of compound 1 is prepared by a process comprising suspending crystalline Form A of compound 1 or a mixture of crystalline Form A of compound 1 and crystalline Form I of compound 1 in a mixture containing DMSO and water.

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In some embodiments, the slurry is mixed at about 300 rpm. In some embodiments, the slurry is heated to a temperature in the range of 55 to 65 ° C. In some embodiments, the slurry is agitated for a period of time ranging from about 12 to 26 hours. In some embodiments, the slurry containing crystalline Form A of Compound 1 and the mixture containing DMSO and water are passed through a wet mill.

In another aspect of the present invention, crystalline Form A of Compound 1 is prepared by a process comprising the step of recrystallizing a crude formulation of Compound 1 from a mixture containing DMSO, ethanol and water.

In some embodiments, the mixture contains solvents DMSO: ethanol: water in a 1.6: 1: 3.5 volume ratio.

In another aspect, crystalline Form A of Compound 1 is prepared by a process comprising the step of recrystallizing a crude preparation of Compound 1 from a solution containing at least one solvent selected from methanol, ethanol, isopropyl alcohol, acetone, ethyl acetate, acetonitrile, or toluene.

In another aspect, crystalline Form A of Compound 1 is prepared by a process comprising the step of recrystallizing a crude formulation of Compound 1 from a mixture containing water and at least one solvent selected from methanol, ethanol, isopropyl alcohol, tetrahydrofuran, and trifluoroethanol.

In another aspect, crystalline Form A of Compound 1 is prepared by a process comprising the step of recrystallizing a crude preparation of Compound 1 from a mixture containing water and ethanol.

In another aspect, crystalline Form A of Compound 1 is prepared by a process comprising the step of recrystallizing a crude formulation of Compound 1 from a mixture containing heptane and at least one solvent selected from methanol, ethanol, isopropyl alcohol, or butan-2-one.

In another aspect, crystalline Form A of Compound 1 is prepared by a process comprising the step of recrystallizing a crude formulation of Compound 1 from a mixture containing diisopropyl ether and at least one solvent selected from methanol, ethanol, isopropyl alcohol, or butan-2-one.

In another aspect, the present invention provides a method for preparing a compound according to any of several embodiments described herein, comprising (i) (A1-1a) stirring compound 1 in DMSO to form a solution and heating the solution to a temperature in the range from 50 to 60 ° C;

(ii) (A1-1b) optionally filtering the solution;

(iii) (A1-2) adding water preheated to a temperature in the range of 50 to 60 ° C to the solution to obtain a mixture while maintaining the internal temperature of the mixture in the range of 50 to 60 ° C;

(iv) (A1-3) seeding the mixture obtained in step (A1-2) with crystalline Form I of compound 1 to obtain a first seeded mixture;

(v) (A1-4) adding water preheated to a temperature in the range of 50 to 60 ° C to the first seeded mixture to obtain a second seeded mixture while maintaining the internal temperature of the second seeded mixture in the range of 50 up to 60 ° C and mixing the second mixture with the seeded at a temperature in the range from 50 to 60 ° C;

(vi) (A1-4) aging the second seeded mixture obtained in step (A1-4) to provide crystalline Form I of Compound 1.

In some embodiments, the total amount of solvent in any of steps (A1-1a) to (A1-4) ranges from 9.6 to 10.6 ml solvent / g of compound 1, wherein the solvent contains DMSO and water.

In some embodiments, the concentration of water in any of steps (A1-1a) to (A1-4) ranges from 0 to 8 wt% based on the total amount of water and DMSO.

In some embodiments, the water concentration in step (A1-3) ranges from 3.5 to 4.3 wt% based on the total amount of water and DMSO.

In some embodiments, the seed crystal size distribution in step (A1-3) has a D10 ranging from 2 to 6 μm, a D50 ranging from 9 to 32 μm, or a D90 ranging from 32 to 62 μm.

In some embodiments, the amount of seed crystals added in step (A1-3) ranges from 0.5 to 1.0 wt% based on the total compound 1.

Examples of

Abbreviations:

DMF - dimethylformamide;

DSC - differential scanning calorimetry;

DMSO - dimethyl sulfoxide;

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EtOAc - ethyl acetate;

EtOH is ethanol;

IPE - isopropyl ether;

MeOH - methanol;

MEK - methyl ethyl ketone;

THF - tetrahydrofuran;

TMS = tetramethylsilane;

HRMS - high resolution mass spectrometry;

% e.i. - enantiomeric excess,%;

h - hour;

min - minute;

m / z is the ratio of mass to charge;

MS - mass spectrum;

NMR - nuclear magnetic resonance;

RP LC-MS - reversed-phase liquid chromatography-mass spectrometry;

CT - room temperature;

XRPD - X-ray Powder Diffraction;

s - singlet;

t is a triplet;

m - multiplet;

lat. - wide;

J is the constant of spin-spin interaction;

rpm - revolutions per minute;

% - percent by weight (unless otherwise indicated).

In the present description, butan-2-one is also referred to as methyl ethyl ketone (abbreviated MEK).

In the following examples, the ratios indicated for mixed solvent systems are volume ratios unless otherwise indicated. In addition,% means% by weight unless otherwise indicated.

General ways

Proton nuclear magnetic resonance ('H-NMR).

Proton ( ¹ H) nuclear magnetic resonance spectra were obtained on a Varian Mercury 300 spectrometer in DMSO-de at 600 MHz.

Solid-state nuclear magnetic resonance on carbon-13 nuclei ( ¹³ C mNMR).

Carbon-13 ( ¹³ C) solid state nuclear magnetic resonance spectra were obtained on a Bruker Avance III 500 MHz spectrometer equipped with a 4 mm HF / X CPMAS double resonance probe. Spectra were collected using cross-polarization protons / carbon-13 at a frequency of 12.5 kHz, contact time was 700 ms, relaxation delay 10 s, spin decoupling SPINAL64 100 kHz. The spectrum was processed using the line broadening algorithm to 10 Hz before performing the Fourier transform. TMS was used as an internal standard for calibrating chemical shift values. Chemical shifts are reported relative to TMS using the carbonyl carbon in glycine (176.70 ppm) as secondary standard.

X-ray Powder Diffraction (XRPD).

X-ray powder diffraction (XRPD) patterns were obtained on a Bruker AXS D8 Advance X-ray Diffractometer using Cu Ka radiation at 40 kV and 40 mA. Approximately 100 mg sample was carefully leveled in the center of a 50 mm diameter VeroWhitePlus sample holder for powder diffraction analysis.

The sample was continuously scanned from 2.9 to 35 ° 2θ using fixed coupled angles 2θ / θ in 0.025 ° 2θ steps, the acquisition time was 0.4 s / step. The sample was analyzed at ambient conditions, and all data were analyzed using EVA software version 9.0.

In some experiments, X-ray powder diffraction patterns were obtained using a Rigaku Ultima IV (Rigaku, Tokyo, Japan) with Cu Ka irradiation generated at 50 mA and 40 kV. The sample was placed on a silicone plate at room temperature. Data was collected in the range from 2 ° to 35 ° (2θ) in 0.02 ° steps and scan rate of 6 ° / min.

Water content.

Water content was determined using a Karl Fischer moisture meter (Hiranuma Moisture Meter AQ-7 manufactured by Hiranuma Sangyo Co., Ltd.).

X-ray diffraction of a single crystal.

X-ray diffraction data were acquired on a Rigaku R-AXIS RAPID (Rigaku, Tokyo, Japan) with Cu Ka monochromatic graphite radiation. Data was collected at 25 ° C. Crystal structures were determined by direct methods and refined using the full-matrix least squares method. Aniso

- 14 035560 tropic refinement of the position of atoms other than hydrogen and the hydrogen atoms were positioned according to geometric constraints and the position was refined using the rider model. All calculations were performed using the Crystal Structure crystallographic software (Rigaku, Tokyo, Japan), except for direct calculations for determining and refining the structure, which were performed using SHELXL-97. Packing charts were obtained using Mercury.

Raman spectroscopy.

Low-frequency Raman spectra were acquired on RXN1 systems with an air-cooled CCD detector (Kaiser Optical Systems, Inc., Ann Arbor, MI, USA) equipped with a SureBlock TRUMICRO module (Ondax Inc., Monrovia, CA USA) with an excitation wavelength laser 976 nm. Data was collected using a 10x objective lens, shutter speed was 10 s. The Raman shift values were calibrated against sulfur.

Example 1. Recrystallization of crystalline form A of compound 1 from various solutions.

Investigated the recrystallization of crystalline form A of compound 1 under conditions of using various solvents. As shown in table. 1, crystalline Form A of compound 1 was dissolved in 12 solvents. Water, heptane or IPE was added as an anti-solvent to keep the solution saturated. Recrystallization in heptane provided sufficient solids for analysis after gradual cooling. XRPD and THF / DSC analyzes indicated that the solids obtained from MEK or THF / IPE were amorphous (Table 1). Crystals prepared from THF, trifluoroethanol, THF / heptane, chloroform / heptane, chloroform / IPE, and trifluoroethanol / IPE were characterized as solvates by XRPD and TGA / DSC analysis. Crystals from all other solvents were XRPD analysis as Crystalline Form A of Compound 1.

X-ray powder diffraction patterns were obtained using a Rigaku Ultima IV (Rigaku, Tokyo, Japan) with Cu Ka irradiation generated at 50 mA and 40 kV. The sample was placed on a silicone plate at room temperature. Data was collected in the range from 2 ° to 35 ° (2θ) in 0.02 ° steps and scan rate of 6 ° / min.

Table 1. General description of the recrystallization study

Appraisal rate at 55 ° C; mg / ml) Crystalline form Bose supplements Anti-solvent Vol Heptane IPE Methanol 3 3.3 Form A Form A Form A Ethanol 10.5 Form A Form A Form A Form A IPA 6, 7 Form A Form L Form A Form A Acetone <2 Form A _ AND) -AND) _ aj IEC 2, 9 Amorphous Form A Form A Ethylletate <2 Form A - a J - and) Acetonitrile <2 Form A -and; - ci / Toluene <2 Form A - Ά) - Ά) Formamide 10.0 -------1 - Chloroform 2, 8 - <- Solvo ' ^- ' Solpa ^t THF 0, 1 Solvat Form A Solvat Amorphous Trifluoroethane > 100 Solvat Form A Solvat

a) - not carried out due to low solubility

b) - not formed after slow cooling c) - pasty residue

Example 2. Obtaining crystalline form A of compound 1 using a mixture of DMSO / ethanol / water.

Crude Compound 1 was prepared according to the method described in Example 178 in US Pat. No. 8,722,660 B2.

The crude compound 1 (300 g) was suspended in DMSO (1560 ml) and ethanol (930 ml). Then the suspension was dissolved by heating to a temperature of 75 to 85 ° C. After complete dissolution, the mixture was filtered to remove dust and the residue was washed with a mixed solution of DMSO (1040 ml) and ethanol (620 ml). The filtrate and wash solution after filtration to remove dust were combined and stirred at 75 to 85 ° C. After making sure there was no precipitate, water (5580 ml) was added dropwise over 1 hour or longer at the indicated temperature. After completion of the dropwise addition, the mixture was stirred at the same temperature for 1 hour or longer. After deposition

15,035,560 crystals were left to cool the mixture to 20-30 ° C and stirred for 2 hours or longer. After stirring, the crystals were collected by filtration and washed sequentially with water (3000 ml) and acetone (1500 ml) to obtain wet crystals. The obtained wet crystals were dried under reduced pressure at 60 ° C to give compound 1 as crystals (crystal form A of compound 1, 207.3 g, yield 69.1%). The resulting crystals of Crystalline Form A of Compound 1 (193.3 g) were pulverized in a jet mill to obtain a crystalline powder (ground product, Crystals of Form A, 188.9 g). The resulting crystals contained 2.5% water and were characterized by XRPD diffractogram having specific peaks of d (or d-distance) values at 21.5, 10.9, 7.3, 5.4, 5.0, 4.9, 4.5, 3.7, 3.4, 3.3 and 3.0 A.

1H NMR (600 MHz, DMSO-de) δ 1.39-1.48 (m, 2H), 1.48-1.58 (m, 2H), 1.75 (t, J = 11.1 Hz, 1H), 1.87 (br B, 1H), 2.24-2.36 (m, 1H), 2.46 (s, 3H), 2.53-2.65 (m, 2H), 2 , 80-2.97 (m, 1H), 3.07 (t, J = 11.3 Hz, 1H), 3.91 (t, J = 8.9 Hz, 1H), 7.44 (s, 1H), 8.04 (br ^, 1H).

Analytical calculation for Ci ₇ H ₂₀ N ₅ Oi, ₅ S: C, 58.27; H, 5.75; N, 19.98; Oh, 6.85; S, 9.15.

Experiment: C, 58.22; H, 5.80; N, 20.03; S, 9.09.

X-ray powder diffraction patterns were obtained using a Rigaku Ultima IV (Rigaku, Tokyo, Japan) with Cu α irradiation generated at 50 mA and 40 kV. The sample was placed on a silicone plate at room temperature. Data was collected in the range from 2 ° to 35 ° (2θ) in 0.02 ° steps and scan rate of 6 ° / min.

Example 3. Obtaining crystalline form I of compound 1 using a mixture of DMSO / acetone.

Crude Compound 1 was prepared according to the method described in Example 178 in US Pat. No. 8,722,660 B2. The crude compound 1 (35 g) was suspended in DMSO (105 ml). Then the suspension was dissolved by heating to a temperature of 70 to 80 ° C. After complete dissolution, the mixture was filtered to remove dust and the residue was washed with DMSO (70 ml). The filtrate and wash solution after filtration to remove dust were combined and stirred at 70 to 80 ° C. After making sure that there was no sediment, the mixture was left to cool to 50-60 ° C. After cooling, acetone (1750 ml) was added dropwise over 1 hour or longer at the indicated temperature. After completion of the dropwise addition, the mixture was stirred at the same temperature for 1 hour or longer. After precipitation of crystals, the mixture was left to cool to 20-30 ° C and stirred for 1 hour or longer. After stirring, the crystals were collected by filtration and washed with acetone (350 ml) to give wet crystals. The resulting wet crystals were dried under reduced pressure at 60 ° C. to give compound 1 as crystals (crystal form I of compound 1, 26.5 g, yield 75.7%). The resulting crystals of Crystalline Form I of Compound 1 (21.5 g) were pulverized in a jet mill to obtain a crystalline powder (ground product, Crystals of Form I, 18.2 g). The resulting crystals contained 2.6% water and were characterized by XRPD diffractogram having specific peaks of d (or d-distance) values at 21.6, 10.9, 7.2, 5.8, 5.4, 5.0, 4.3, 3.4, 3.3, 3.2 and 2.9 A.

1H NMR (600 MHz, DMSO-de) δ 1.39-1.48 (m, 2H), 1.48-1.59 (m, 2H), 1.75 (t, J = 11.1 Hz, 1H), 1.87 (br ^, 1H), 2.25-2.36 (m, 1H), 2.46 (s, 3H), 2.53-2.65 (m, 2H), 2, 81-2.97 (m, 1H), 3.07 (t, J = 11.3 Hz, 1H), 3.91 (t, J = 8.9 Hz, 1H), 7.44 (s, 1H ), 8.04 (br ^, 1H).

Analytical calculation for Ci ₇ H ₂₀ N ₅ Oi, ₅ S: C, 58.27; H, 5.75; N, 19.98; Oh, 6.85; S, 9.15.

Experiment: C, 58.22; H, 5.73; N, 19.84; S, 9.12.

X-ray powder diffraction patterns were obtained using a Rigaku Ultima IV (Rigaku, Tokyo, Japan) with Cu α irradiation generated at 50 mA and 40 kV. The sample was placed on a silicone plate at room temperature. Data was collected in the range from 2 ° to 35 ° (2θ) in 0.02 ° steps and scan rate of 6 ° / min.

Example 3-2. Preparation of Crystalline Form I of Compound 1 Using DMSO / Acetonitrile.

Crude Compound 1 was prepared according to the method described in Example 178 in US Pat. No. 8,722,660 B2. The crude compound 1 (1 g) was suspended in DMSO (5 ml). Then the suspension was dissolved by heating to a temperature of 75 to 80 ° C. After complete dissolution, acetonitrile (50 ml) was added dropwise over 1 hour or longer at the indicated temperature. The mixture was left to cool to 60-70 ° C and stirred at this temperature for 1 hour or longer. After precipitation of crystals, the mixture was left to cool to 20-30 ° C and stirred for 1 hour or longer. After stirring, the crystals were collected by filtration and washed with acetonitrile (10 ml) to give wet crystals. The resulting wet crystals were dried under reduced pressure at 60 ° C to give compound 1 as crystals (crystal form I of compound 1, 862 mg, yield 86.2%).

Example 3-3. Preparation of Crystalline Form A of Compound 1 Using Ethanol / Water.

Crude compound 1 was prepared according to the method described in example 178 in the patent

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US No. 8722660 B2.

The crude compound 1 (9.53 g) was suspended in a solution of ethanol and water (100/1, v / v, 400 ml). Then the suspension was heated to a temperature of 75 to 85 ° C. A solution of ethanol water (100/1, v / v, 380 ml) was added slowly at the same temperature to obtain a solution. The solution was left to cool to 20-30 ° C and stirred for 16 hours. The crystals were collected by filtration and washed with ethanol (60 ml) to give compound 1 as crystals (crystal form A of compound 1, 7.73 g, yield 81.1 %). The resulting crystals contained 2.6% water and were characterized by XRPD diffractogram having specific peaks for d (or d-distance) values at 22.1, 11.0, 7.3, 5.5, 5.1, 4.9, 4.5, 3.7, 3.4, 3.3 and 3.0 A.

1H NMR (300 MHz, DMSOO δ 1.37-1.61 (m, 4H), 1.69-1.91 (m, 2H), 2.23-2.33 (m, 1H), 2.46 (s, 3H), 2.54-2.67 (m, 2H), 2.77-2.94 (m, 1H), 3.00-3.14 (m, 1H), 3.91 (t , J = 8.9 Hz, 1H), 7.44 (s, 1H), 8.03 (br B, 1H), 12.24 (br B, 1H).

Analytical calculation for Ci ₇ H ₂₀ N ₅ Oi, 5S: C, 58.26; H, 5.75; N, 19.98.

Experiment: C, 58.09; H, 5.69; N, 19.84.

Example 4. Obtaining crystalline form I of compound 1 using a mixture of DMSO / acetone / water.

Crude Compound 1 was prepared according to the method described in Example 178 in US Pat. No. 8,722,660 B2. The crude compound 1 (30 g) was dissolved in DMSO (300 ml) at 20-30 ° C. After complete dissolution, the mixture was filtered to remove dust, and a filtered solution was obtained. The residue was washed with DMSO (60 ml) and a wash solution was obtained. A mixed solution of acetone (150 ml) and water (150 ml) was stirred at 45-55 ° C and 45 ml of the filtered solution was added dropwise over 1030 min at this temperature. After completion of the dropwise addition, the mixture was stirred at the same temperature for 10 minutes. To the mixture was added dropwise 15 ml of the filtered solution over 3-10 minutes at the same temperature. After completion of the dropwise addition, the mixture was stirred at the same temperature for 1 hour or longer. After the crystals were precipitated, the remainder of the filtered solution was added dropwise over 1 to 2 hours at the indicated temperature. After completion of the addition, the wash solution was added dropwise at the same temperature. After completion of the dropwise addition, the mixture was stirred at the same temperature for 1 hour. After stirring, the mixture was allowed to cool to 20-30 ° C. and stirred for 1 hour or longer. After stirring, the crystals were collected by filtration and washed sequentially with water (150 ml) and acetone (150 ml) to obtain wet crystals. The resulting wet crystals were dried under reduced pressure at 60 ° C to give compound 1 as crystals (crystal form I of compound 1, 25.9 g, yield 86.3%).

Example 5. Conversion of crystalline form A / I of compound 1 to crystalline form I of compound 1 in suspension.

A mixture of crystalline form A of compound 1 and crystalline form I of compound 1, which is referred to herein as crystalline form A / I of compound 1, (5 g) was suspended in a mixture of DMSO (60 ml) and water (5.15 ml). Then the suspension was heated to a temperature of 55 to 65 ° C and stirred for 12 hours or longer. Thereafter, some of the solids were collected by filtration. It was confirmed by XRPD measurements that Form A / I of Compound 1 was converted to Form I of Compound 1. After the conversion was confirmed, the mixture was allowed to cool to 20-30 ° C for 3 hours and stirred at that temperature for 1 hour or longer. Thereafter, some of the solids were collected by filtration. According to XRPD measurements, it was confirmed that the crystalline Form I of Compound 1 was obtained as crystals. After identification, a mixture of acetone (25 ml) and water (25 ml) was added dropwise over 30 minutes or longer at 20-30 ° C. After completion of the dropwise addition, the mixture was stirred at the same temperature for 1 hour or longer. After stirring, the crystals were collected by filtration and washed sequentially with water (25 ml) and acetone (25 ml) to obtain wet crystals. The resulting wet crystals were dried under reduced pressure at 60 ° C to obtain compound 1 as crystals (crystal form I of compound 1, 4.7 g, yield 94.0%).

X-ray powder diffraction patterns were obtained using a Rigaku Ultima IV (Rigaku, Tokyo, Japan) with Cu α irradiation generated at 50 mA and 40 kV. The sample was placed on a silicone plate at room temperature. Data was collected in the range from 2 ° to 35 ° (2θ) in 0.02 ° steps and scan rate of 6 ° / min.

Example 6. Crystalline forms of compound 1 at various concentrations and water content.

To investigate the effect of (i) the concentration of compound 1 and (ii) the content of water on the crystalline form of compound 1, the following screen was used.

Crystalline form A of compound 1 was completely dissolved in 5 ml DMSO / g crystal form A of compound 1 at 80 ° C and filtered through a membrane filter with a pore size of 0.22 μm. Then part of the resulting solution was diluted 4/3, 2 or 3 times with DMSO. Compound 1 solutions were added to acetone having different percentages of water at 55 ° C. The solutions were incubated at 55 ° C

- 17,035560 for 1 hour and stirred at 300 rpm, cooling to 25 ° C at a rate of 3 ° C / h. Collected a precipitate from each solution and investigated the crystalline form of the precipitates in XRPD analysis. X-ray powder diffraction patterns were obtained using a Rigaku Ultima IV (Rigaku, Tokyo, Ja pan) with Cu Ka irradiation generated at 50 mA and 40 kV. The sample was placed on a silicone plate at room temperature. Data was collected in the range from 2 ° to 35 ° (2θ) in 0.02 ° steps and scan rate of 6 ° / min. The results are shown in table. 2.

Table 2. Crystalline forms at various concentrations and water content

Final Water content in acetone before adding DMSO solution concentration [t, v / v)

0 C, 10.2 0.3 0.5 3.7 ten 1.5 2 7 five 7 ten 2 0 ¹ thirty 50 18.2 I I I I I A + I A + I A A A A A A A A A + I 13.6 Sill 1 1 1 A + l A + l L + 1 L + 1 L + 1 1 1 1 s, 1 S I I I I I I I I I I I - I I I 6, 1 3 3 3 3 I I I I 1 I I 1 - I I 3

A: Received crystals of crystalline form A of compound 1

I: Received crystals of crystal form I of compound 1

A + I: Received a mixture of crystal form A of compound 1 and crystal form I of compound 1

S: no solid precipitated

The results obtained demonstrate that at high concentrations of compound 1 (i.e. 18.2 g / L), pure crystalline Form I of compound 1 does not crystallize unless the water content in acetone before the addition of the DMSO solution is not low enough (i.e., less 0.7% (v / v)).

According to the screening results, the appropriate water content was determined in additional studies to optimize the preparation of crystalline form I of compound 1. Then, compound 1 in DMSO solution (10 ml DMSO / g of compound 1) was added dropwise to a mixture of acetone and water (10 ml of acetone / water mixture per g connections 1) at 50 ° C at different speeds. Based on the results shown below in table. 3, it was determined that the rate of addition of compound 1 in DMSO solution should be slow enough to obtain crystalline Form I of compound 1. Under these conditions, rapid addition promoted the formation of crystalline form A of compound 1. Table 3. Crystalline forms of compound 1 obtained under various conditions adding

Water content Crystalline Method of addition in acetone (%, _nd form v / v) Output Add: 10 min 20 Form A 82.4% Add: 60 min 20 Form A 80.9 + 1. Add. 1 revolution: 10 min 2. Move: 1 hour 20 Form Ά / Ι * 3. Add. 9 rev .: 10 min 77.8% 1. Add. 1 revolution: 10 min 2 0 Form I 2. Move: 1 hour 79.2%

3. Ext. 9 rpm: 65 min 1. Ext. 1.5 rpm: 15 min 2. Trans.: 1 hour 50 Form I 90.2% 3. Ext. 8.5 rpm: 60 min

* Form A / I denotes a mixture of Crystalline Form I of Compound 1 and Crystalline Form A of Compound 1

Example 7. Solubility of crystalline form I of compound 1 and crystalline form A of compound 1 in 200 mM phosphate buffer (pH 6.8).

To a glass test tube was added about 2 mg of crystalline Form A of compound 1 obtained in example 2 or crystalline form I of compound 1 obtained in example 3-2, and 2 ml of 200 mM phosphate buffer (pH 6.8). The test tube was vigorously shaken and incubated at 10, 20, 30 or 40 ° C for 20 h. After incubation, the suspension was centrifuged and the supernatant was filtered through a membrane filter with a pore size of 0.22 μm. Filtered solutions were diluted 2-fold with 50 mM sodium perchlorate buffer (pH 2.5) / acetonitrile (1/1, v / v). Determined the concentration by HPLC. The results are shown in table. 4.

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Table 4. Solubility of crystalline form I of compound 1 and crystalline form A of compound 1 in 200 mM phosphate buffer (pH 6.8) Solubility (μg / ml)

Temperature (° C) ten 20 thirty 40 Form A 102 104 115 136 Form I 63 71 80 99

Example 8. Recrystallization of Crystalline Form I of Compound 1.

Crystalline Form I of compound 1 (9.5 kg, 97.1% ee) was dissolved in DMSO (85.5 L, 9.0 ml DMSO / g of compound 1). The solution was heated to 60 ° C. The reaction mixture was filtered for fine purification, maintaining the temperature of the mixture at about 60 ° C. The lanes along which the compound passed were washed with DMSO (6.7 L, 0.7 ml DMSO / g of compound 1). Combine the compound 1 solution and the wash solution. Then the solution of compound 1 was heated to 60 ° C. To a solution of compound 1 was added water preheated to 60 ° C (4.0 L, 0.43 ml water / g of compound 1) over 1 h while maintaining the internal temperature at 60 ° C. The reaction mixture was seeded with crystalline Form I of compound 1 (106.0 g, 1.1 wt%). The reaction mixture was stirred at 60 ° C for 1 h. Water preheated to 60 ° C (3.9 L, 0.41 ml of water / g of compound 1) was added to the suspension of compound 1 for 3 h, maintaining the temperature of the mixture at 60 ° C. After the addition of water was complete, the slurry was stirred at 60 ° C for at least 4.5 hours. A sample of the slurry was taken and filtered to give crystals. Analyzed crystals by XRPD. If not all of the crystals in the sample were crystalline Form I of compound 1, then stirring was continued. Samples were taken, then analyzed every 6 hours while crystalline Form A of Compound 1 was retained in the crystal samples by XRPD analysis. The reaction mixture was cooled to 25 ° C for 5 hours. The solids were filtered off and washed twice with 3 ml of 2/1 acetone: water (28.5 L) per g of solid. Dry the solids in vacuo at 60 ° C for 18 hours to obtain 7.3 kg (76.8% yield) of the desired product. The target product was obtained as a white solid. HPLC analysis showed the resulting product to be> 99% pure and ee was shown on chiral HPLC. 99.1%. It was confirmed by XRPD analysis that the resulting product was Crystalline Form I of Compound 1.

X-ray powder diffraction (XRPD) patterns were obtained on a Bruker AXS D8 Advance X-ray Diffractometer using Cu Ka radiation at 40 kV and 40 mA. Approximately 100 mg sample was carefully leveled in the center of a 50 mm diameter VeroWhitePlus sample holder for powder diffraction analysis. The sample was continuously scanned from 2.9 to 35 ° 2θ using fixed coupled angles 2θ / θ in 0.025 ° 2θ steps, the acquisition time was 0.4 s / step. The sample was analyzed at ambient conditions, and all data were analyzed using EVA software version 9.0.

Example 9 Conversion of Form A / I Crystalline of Compound 1 to obtain Crystalline Form I of Compound 1 in suspension.

DMSO (240 ml, 3380 mmol, 12 ml DMSO / g crystal form A / I compound 1) and water (20.6 ml, 1140 mmol, 1 ml water / g crystal form A / I compound 1) were added to a 500 ml reactor OptiMax. ). Then, crystalline form A / I of compound 1 (20.00 g, 58.58 mmol) was added to the reactor. The resulting suspension was stirred at 300 rpm and heated to 60 ° C. After stirring for 26 h, a sample of the slurry was taken, filtered while hot and washed with a minimum amount of 1: 1 acetone: water to give approximately 100 mg of a solid which was analyzed by XRPD and shown to be crystalline Form I of Compound 1 The bulk of the slurry was cooled to 22 ° C over 6 hours and stirred at 22 ° C overnight. The next morning, the suspension was filtered and washed twice with 60 ml of a 2: 1 mixture of acetone: water. The precipitate was dried for 72 h at 45 ° C under vacuum. XRPD analysis of the solid, dried from the bulk of the slurry, indicated that it was crystalline Form I of Compound 1.

X-ray powder diffraction (XRPD) patterns were obtained on a Bruker AXS D8 Advance X-ray Diffractometer using Cu Ka radiation at 40 kV and 40 mA. Approximately 100 mg sample was carefully leveled in the center of a 50 mm diameter VeroWhitePlus sample holder for powder diffraction analysis. The sample was continuously scanned from 2.9 to 35 ° 2θ using fixed coupled angles 2θ / θ in 0.025 ° 2θ steps, the acquisition time was 0.4 s / step. The sample was analyzed at ambient conditions, and all data were analyzed using EVA software version 9.0.

Example 10 Conversion of Crystalline Form A / I of Compound 1 to obtain Crystalline Form I of Compound 1 in suspension using wet milling.

DMSO (240 ml, 3380 mmol, 12 ml DMSO / g crystal form A / I of compound 1) and water (20.6 ml, 1140 mmol, 1 ml water / g crystal form A / I of compound 1). Then, crystalline form A / I of compound 1 (20.00 g, 58.58 mmol) was added to the reactor.

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The resulting slurry was placed in a wet mill (which was set at 10,000 rpm) for 1 minute. The slurry was then passed once through a wet mill and transferred back to the OptiMax reactor. Then the suspension was heated to 60 ° C. After stirring for 17.5 h at 60 ° C, a sample of the slurry was taken, filtered while hot, and washed with a minimum amount of 1: 1 acetone: water to give approximately 100 mg of a solid which was analyzed by XRPD and was shown to represent is crystalline Form I of compound 1. The suspension was cooled to 22 ° C over 6 hours and stirred at 22 ° C overnight. The next morning, the suspension was filtered and washed twice with 60 ml of a 2: 1 mixture of acetone: water. The precipitate was dried for 72 h at 45 ° C under vacuum. XRPD analysis of the solid, dried from the bulk of the slurry, indicated that it was crystalline Form I of Compound 1.

X-ray powder diffraction (XRPD) diffraction patterns were obtained on a diffractometer

Bruker AXS D8 Advance X-ray Diffractometer using Cu Ka radiation at 40 kV and 40 mA. Approximately 100 mg sample was carefully leveled in the center of a 50 mm diameter VeroWhitePlus sample holder for powder diffraction analysis. The sample was continuously scanned from 2.9 to 35 ° 2Θ using fixed coupled angles 2θ / θ in 0.025 ° 2θ steps, the acquisition time was 0.4 s / step. The sample was analyzed at ambient conditions, and all data were analyzed using EVA software version 9.0.

Example 11. Dissolution of crystalline form I of compound 1 and crystalline form A of compound 1 in a mixture of water / DMSO at 60 ° C.

A DMSO / water solution with a known water content was added to a glass tube and heated to 60 ° C. Crystals of crystalline form I of compound 1 or crystalline form A of compound 1 were added to a test tube to obtain a suspension. The suspension was stirred on a magnetic stirrer and incubated for 24 h at 60 ° C. After incubation, the suspension was filtered at 60 ° C. A 10 μl sample of the filtrate was taken and diluted with 990 μl acetonitrile / water (1/1, v / v). The concentration of compound 1 in the sample was determined by HPLC. At 60 ° C., the solubility of crystalline Form I of compound 1 in a water / DMSO mixture was lower than the solubility of crystalline Form A of compound 1 in a mixture of water / DMSO (FIG. 7). The results of the experiments to determine the solubility are shown below in table. 5 and 6.

Table 5. Solubility of crystalline Form I of compound 1 in a mixture of water / DMSO at 60 ° C% water by volume Solubility (mg / ml)

0134.0

3108.0

492.15

4.574.80

665.82

834.82

Table 6. Solubility of Crystalline Form A of Compound 1 in Water / DMSO at 60 ° C % water by volume Solubility (mg / ml) 0 153, 8 6 73, 4 8.5 51.0 8.7 44, 0 9.8 38.5 10.7 32.1 11.3 28, 6 11.8 25.7

Example 12 Screening of Excipients for Pharmaceutical Compositions Containing Crystalline Form I of Compound 1.

The compatibility of the following excipients with crystalline Form I of compound 1 was evaluated:

lactose (FlowLac 100®; Mutchler, Inc; Harrington Park, NJ, USA);

mannitol (Pearlitol 100SD®, Roquette America, Inc; Geneva, IL, USA);

starch 1500 (StarCap 1500®, Colorcon, Inc; Harleysville, PA, USA);

anhydrous dicalcium phosphate (A-Tab®, Innophos, Inc; Cranbury, NJ, USA).

Since the epimerization of compound 1 is catalyzed in the presence of an acid, 10% L-glutamic acid was added to the test mixtures to accelerate the study to determine the stability. The compositions of the compositions used in the study are described in table. 7. To assess the stability, the mixtures were stored under accelerated failure conditions at 60 ° C / 75% rel. ow. in two weeks. A mixture containing no filler and containing an equivalent amount of acid was used as

- 20 035560 controls in a stability study. The results of the compatibility study are shown in table. 8.

Table 7. Compositions of mixtures for screening excipients

Amount (%, mass / mass)

Component Supplier Fl F2 F3 F4 F5 F6

Crystalline Form I

connections 1 F.I.S. 10.0 10.0 10.0 10.0 10.0 50, 0 (crushed naya) Aerosil 200 L- Evonik Industries ten ten ten ten 1, o ten glutamines th acid SigmaAldrich 10.0 one hundred 10.0 10.0 10.0 49.0 Mannit Roquette 79.0 - - - - -

Mutchler,

- 79.0-

Inc

Colorcon,

- -79.0

Inc

Innophus - - - 79.0 Asahi. - - - -79.0

Lactose (Flowlac 100)

Starch 1500

Dicalcium Phosphate Anhydrous (A-tab)

MKTs RN101

Kaser Corp

MCC = microcrystalline cellulose

Table 8. Results of an accelerated study of excipients to determine stability

Storage conditions, 60 ° C / 75% rel. ow

Component Study start Week 1 on open air 2 weeks in closed conditions 2 weeks outdoors No. Enant. comp Qty. comp (%) Enant. (%) Qty. comp. (%) Enant. (%) Qty. comp (%) Enant. (%) (%) (%) MKTs PH101 Christ. form I of compound I 1 / L- 101.6 1.59 90, 6 3.79 nt NT nt nt glut. acid (50/50) (F6) Mannit 100.5 1.62 99, 3 1, 68 NT NT nt nt (F1) Lactose 98.0 1.60 98.7 2, 02 101.0 2, 03 nt 2.25 (F2) Starch 97.1 1.61 95, 8 1, 95 99.5 2, 02 nt 2.08 (F3) Phosphate 95.2 1.60 93, 2 2, 19 95.8 1, 82 nt 2.52 dicalcium (F4) 97.1 1.60 99, 6 3.81 NT NT nt nt

MCC = microcrystalline cellulose NT = not tested

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For MCC PH101 and dicalcium phosphate, a significant increase in the content of chiral impurities was observed after 1 week, and they were not investigated after 2 weeks. For mannitol, lactose and starch, an acceptable increase in the content of chiral impurities was observed after 2 weeks.

Example 13 Preparation of a capsule formulation of crystalline Form I of compound 1.

To obtain a capsule formulation of crystalline Form I of Compound 1, mannitol, colloidal silicon dioxide and crystalline Form I of Compound 1 were weighed and added to a V-blender. The powders were then mixed in a V-blender for 5 minutes at 25 rpm. The resulting mixture was passed through a cone mill equipped with a 032R sieve (810 μm) for grinding the crystalline Form I of compound 1 and the excipients of mannitol and colloidal silicon dioxide. The mixture, after passing through a cone mill, was mixed in a V-blender for 5 minutes at 25 rpm. The homogeneity of the mixture was evaluated within the framework of technological control. The final mixture was collected and encapsulated in capsules by the manual encapsulation method. The capsules were polished using hand and measuring equipment. The polished capsules were examined for the content of metal impurities, their weight was checked and sorted within the framework of technological control. Weighed sorted capsules were bulk packed in a high density polyethylene (HDPE) drum lined with two layers of polyethylene bags.

Typical formulas for preparing about 45455 capsules containing about 10 mg dose of crystalline Form I of Compound 1, about 45,000 capsules containing about 25 mg dose of crystalline Form I of Compound 1, and about 28125 capsules containing about 80 mg dose of crystalline Form I of Compound 1 , are given in table. 9. As used herein, the dosage or dose in a capsule is expressed by the approximate weight of the active drug substance (crystalline Form I of Compound 1).

Table 9. Manufacturing formula for obtaining capsules containing crystalline Form I of compound 1

Ingredient Quantity for 45455 capsules with Quantity per 45,000 capsules with a dose of 25 mg Quantity for 28125 capsules with a dose of 80 mg dose 10 MG Crystalline form I of compound 1 ^a 454.55 r 1125.00 g 2250.00 g Mannitol ^s Colloidal 4495.45 r 3330.00 g 2205.00 g Silicon Dioxide Shells 50,00 r 45.00 g 45.00 g capsules 1727.27 r 1710.00 g 1350.00 g

a Actual weight was adjusted according to drug and moisture assay results to achieve the target activity of each capsule ^b Actual amount was adjusted according to the amount of crystalline Form I of Compound 1 used to achieve the target activity of each capsule

Example 14. X-ray diffraction of a single crystal.

The crystallographic parameters of the crystalline forms of compound 1 are given in table. 10. In the crystal structure of the crystal form of compound 1, determined by the X-ray method, the water molecule is located along the axis of symmetry of the 2nd order. Thus, the asymmetric unit cell contains the molecule 2 - [(2S) -1-azabcyclo [2.2.2] oct-2-yl] -6- (3-methyl-1H-pyrazol-4yl) thieno [3,2-d ] pyrimigin-4 (3H) -one and half of one water molecule. On the other hand, the asymmetric unit cell in the crystal structure of crystal form I of compound 1 contains two molecules 2 - [(2S) -1-azabicyclo [2.2.2] oct-2-yl] -6- (3-methyl-1H-pyrazole -4-yl) thieno [3,2-d] pyrimigin4 (3I) -one with different conformations and one water molecule. Asymmetric unit cells suggest that crystalline form A of compound 1 and crystalline form I of compound 1 are hemihydrates.

Oak Ridge Thermal Ellipsoid Program (ORTEP) images for Crystalline Form I of Compound 1 and Crystalline Form A of Compound 1 are shown in FIG. 8 and 9 respectively.

Table 10. Crystallographic parameters of crystals of compound 1

Crystalline form

Form A

Form I

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Temperature 298 C 298 C Molecular Formula C ₁₇ H ₁₉ N ₅ OS'0.5 H ₂ O C ₁₇ H ₁₉ N ₅ OS · 0.5 H ₂ O Molecular 349.43 349.43 weight Color and shape colorless, colorless, crystals lamellar prismatic Crystalline system Parameters Monoclinic monoclinic a = 12.031 (2) A 6.2263 (1) A gratings ^B 6.2460 (8) A 43.5007 (8) A ^C _ 21.947 (4) A 6.7944 (2) A P 95.26 (1) ° 117.207 (2) ° V = 1642.3 (4) 1636, 65 (6) A ³ Spatial C2 (# 5) P2 ₇ (# 4) Group Z 4 4 R (Ι> 2.00σ (I)) 0.1253 0.0594 R " 0.3662 0.1502 Flack χ 0.04 (7) 0, 004 (18)

In addition, X-ray structural analysis was also performed using another crystal of higher quality, calculations to refine the structure were performed using SHELXL-2014. The hydrogen atom of water was successfully identified on the new difference Fourier map, whereas on previous maps its location could not be determined due to poor data quality. The hydrogen atoms of the methyl group and water were positioned on the differential Fourier map, and others were positioned according to geometric constraints. The positions of all hydrogen atoms were refined using the rider model. The final optimized parameters are shown in table. eleven.

Table 11. Optimized crystallographic parameters of crystals of compound 1 Form A Form I Temperature 298 C 298 C Molecular Formula C ₂₇ H ₁₉ N ₅ OS-0.5H ₂ O S ₂₇ N ₁₉ C ₅ OZ-0.5N ₂ O Molecular mass Crystal system 350.44 monoclinic 350.44 monoclinic a (a) 12.0363 (4) 6.23273 (11) B (A) 6.2487 (2) 43.4973 (8) C (A) 21.9410 (7) 6.79041 (13) β (°) 95, 381 (7) 117.212 (8) V (A ³ ) 1642.95 (10) 1637.18 (13) Space group C2 P2 ₂ Ζ 4 4 R (I> 2σ (Ι)) 0.0692 0.0372 Rw 0.2193 0.0927 Flack 0.136 (11) 0.030 (8)

Example 15 Preparation of Crystalline Form I of Compound 1 from Untreated Compound 1

Crude compound 1 (16.0 kg) was dissolved in DMSO (144 L, 9.0 ml DMSO / g compound 1). The solution was heated to 60 ° C. The reaction mixture was filtered for fine purification, maintaining the temperature of the mixture at about 60 ° C. The lanes along which the compound passed were washed with DMSO (11.2 L, 0.7 ml DMSO / g of compound 1). Combine the compound 1 solution and the wash solution. Then the solution of compound 1 was heated to 60 ° C. Water preheated to 60 ° C was added to a solution of compound 1 (6.9

- 23,035,560 L, 0.43 ml water / g of compound 1) for 1 h, maintaining the internal temperature at 60 ° C. The reaction mixture was seeded with crystalline Form I of compound 1 (190.0 g, 1.2 wt%). The reaction mixture was stirred at 60 ° C for 1 h. Water preheated to 60 ° C (6.5 L, 0.41 ml of water / g of compound 1) was added to the suspension of compound 1 for 3 h, maintaining the temperature of the mixture at 60 ° C. After the addition of water was complete, the slurry was stirred at 60 ° C for at least 4.5 hours. A sample of the slurry was taken and filtered to give crystals. Analyzed crystals by XRPD. If not all of the crystals in the sample were crystalline Form I of compound 1, then stirring was continued. Samples were taken, then analyzed every 6 hours while crystalline Form A of Compound 1 was retained in the crystal samples by XRPD analysis. The reaction mixture was cooled to 25 ° C for 5 h. The solids were filtered off and washed twice with 3 volumes of 2/1 acetone: water (48 L). Dry the solids in vacuo at 60 ° C for 18 hours to obtain 9.5 kg (59.0% yield) of the desired product. The target product was obtained as a white solid. HPLC analysis showed the resulting product to be> 99% pure and ee was shown on chiral HPLC. 97.1%. It was confirmed by XRPD analysis that the resulting product was Crystalline Form I of Compound 1.

Although the above invention has been described in some detail for clarity and better understanding, these specific examples should be considered as illustrative and non-limiting. After studying the present description, it will be clear to those skilled in the art that various changes in form and some particular aspects can be made without departing from the true scope of the invention, which is defined in the appended claims, but not in specific examples.

Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. In case of conflict, preference is given to the present invention, including the above definitions.

Claims (17)

CLAIM 1. Crystalline form I of the compound, hemihydrate 2 - [(2S) -1-azabicyclo [2.2.2] oct-2-yl] -6- (3methyl-1H-pyrazol-4-yl) thieno [3,2-d ] pyrimidin-4 (3H) -one (compound 1) and / or its tautomers, where compound 1 has the structure O which is characterized by an X-ray powder diffraction pattern having characteristic peaks expressed in degrees 2θ measured using Cu Ka radiation generated at 50 mA and 40 kV at 4.1 ± 0.2, 8.1 ± 0.2, 12 , 2 ± 0.2, 15.2 ± 0.2, 16.3 ± 0.2, 17.8 ± 0.2, and 27.6 ± 0.2. 2. Crystalline form I of the compound according to claim 1, characterized in that said crystalline form is characterized by an X-ray powder diffraction pattern having characteristic peaks expressed in degrees 2θ at 4.1 ± 0.2, 8.1 ± 0.2, 12.2 ± 0.2, 15.2 ± 0.2, 16.3 ± 0.2, 17.8 ± 0.2, 19.0 ± 0.2 and 27.6 ± 0.2. 3. Crystalline form I of the compound according to claim 1, characterized in that said crystal form is characterized by an X-ray powder diffraction pattern having characteristic peaks of interplanar spacing (d), expressed in angstroms, at 21.6 ± 0.2, 10.9 ± 0.2, 7.3 ± 0.2, 5.8 ± 0.2, 5.4 ± 0.2, 5.0 ± 0.2 and 3.2 ± 0.2. 4. Crystalline form I of the compound according to claim 1, characterized in that said crystal form is characterized by an X-ray powder diffraction pattern having characteristic peaks of interplanar spacing (d), expressed in angstroms, at 21.6 ± 0.2, 10.9 ± 0.2, 7.3 ± 0.2, 5.8 ± 0.2, 5.4 ± 0.2, 5.0 ± 0.2, 4.3 ± 0.2, 3.4 ± 0, 2, 3.3 ± 0.2, 3.2 ± 0.2 and 2.9 ± 0.2. 5. Crystalline Form I of the compound of claim 1, wherein said crystal form has an X-ray powder diffraction pattern substantially as shown in FIG. 1. 6. Crystalline form I of the compound according to claim 1, characterized in that said crystalline form is characterized by a solid-state ¹³ C NMR spectrum, including characteristic peaks expressed in ppm at 32.2 ± 0.5, 30.2 ± 0.5, 29.3 ± 0.5, 28.2 ± 0.5, 25.3 ± 0.5 and 24.8 ± 0.5. - 24 035560 7. Crystalline form I of the compound according to claim 1, characterized in that said crystalline form is characterized by a solid state ¹³ C NMR spectrum including characteristic peaks expressed in ppm at 159.7 ± 0.5, 158.4 ± 0.5, 145.1 ± 0.5, 144.8 ± 0.5, 140.6 ± 0.5, 135.7 ± 0.5, 135.3 ± 0.5, 121.2 ± 0.5, 114, 7 ± 0.5, 114.5 ± 0.5, 60.4 ± 0.5, 53.8 ± 0.5, 46.5 ± 0.5, 32.2 ± 0.5, 30.2 ± 0.5, 29.3 ± 0.5, 28.2 ± 0.5, 25.3 ± 0.5, 24.8 ± 0.5 and 14.5 ± 0.5. 8. Crystalline Form I of the compound of claim 1, wherein said crystalline form has a solid-state ¹³ C NMR spectrum substantially as shown in FIG. 2. 9. A pharmaceutical composition for the treatment of cancer, containing crystalline Form I of compound 1 according to any one of claims 1 to 8. 10. A method of inhibiting cell cycle 7 (Cdc7) protein in a mammal, comprising administering an effective amount of crystalline Form I of compound 1 according to any one of claims 1 to 8 or a pharmaceutical composition according to claim 9 to the mammal. 11. A method of treating cancer in a mammal, wherein said cancer is mediated by a cell cycle 7 protein, comprising administering an effective amount of crystalline Form I of a compound according to any one of claims 1 to 8 or a pharmaceutical composition according to claim 9 to the mammal. 12. A method of obtaining crystalline Form I of a compound according to any one of claims 1 to 8, comprising: i) (A1-1a) stirring compound 1 in DMSO to obtain a solution and heating the solution to a temperature in the range of 50 to 60 ° C; ii) (A1-1b) filtering the solution; iii) (A1-2) adding water preheated to a temperature in the range of 50 to 60 ° C to the solution to obtain a mixture while maintaining the internal temperature of the mixture in the range of 50 to 60 ° C; iv) (A1-3) seeding the mixture obtained in step (A1-2) with crystalline Form I of compound 1 to obtain a first seeded mixture; v) (A1-4) Adding water preheated to a temperature in the range of 50 to 60 ° C to the first seeded mixture to obtain a second seeded mixture while maintaining the internal temperature of the second seeded mixture in the range of 50 to 60 ° C and mixing the second seeded mixture at a temperature in the range from 50 to 60 ° C; vi) (A1-4) aging the second seeded mixture obtained in step (A1-4) to provide crystalline Form I of Compound 1. 13. The method according to claim 12, characterized in that the total amount of DMSO in any of the stages (A1-1a) to (A1-4) is in the range from 9.6 to 10.6 ml DMSO / g of compound 1. 14. The method according to claim 12 or 13, characterized in that the concentration of water in any of the stages from (A11a) to (A1-4) is in the range from 0 to 8 wt.% Relative to the total amount of water and DMSO. 15. A method according to any one of claims 12-14, characterized in that the concentration of water in step (A1-3) is in the range from 3.5 to 4.3 wt.% Of the total amount of water and DMSO. 16. A method according to any one of claims 12-15, characterized in that the size distribution of the seed crystals in step (A1-3) is characterized by a D10 value in the range from 2 to 6 μm, D50 in the range from 9 to 32 μm or D90 in range from 32 to 62 microns. 17. A method according to any one of claims 12-16, characterized in that the amount of seed crystals added in step (A1-3) is in the range from 0.5 to 1.0 wt.% Of the total amount of compound 1.