EA045568B1 - SALT AND CRYSTAL FORMS 4-AMINO-5-(6-(4-METHYLPIPERAZIN-1-YL)-1H-BENZO[D]IMIDAZOL-2-YL)THIENO[2,3-B]PYRIDINE-6(7H)- SHE - Google Patents

Description

Link to related applications

This application claims priority to U.S. Provisional Application No.

63/022,867, filed May 11, 2020. The entire contents of the above application are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Hematopoietic precursor kinase 1 (HPK1) is a Ste20 serine/threonine kinase restricted to hematopoietic cells. It has been reported that HPK1 may represent a novel target for cancer immunotherapy (Sawasdikosol et al., Immunol Res. 2012 Dec; 54(1-3):262-5). In particular, targeted disruption of HPK1 alleles causes increased production of Th1 cytokines in T cells in response to contact with the TCR. HPK1(-/-) T cells proliferate faster than their haplotype-matched wild-type counterparts and are resistant to prostaglandin E2 (PGE(2))-mediated suppression. Most strikingly, mice given adoptive transfer of HPK1 (-/-)T cells became resistant to lung tumor growth. In addition, loss of HPK1 by dendritic cells (DCs) confers superior antigen presentation capabilities on them, allowing HPK1(−/−) DCs to elicit a more potent antitumor immune response when used as a cancer vaccine.

US Pat. No. 10,501,474, the entire disclosure of which is incorporated herein by reference, discloses potent HPK1 inhibitors. The structure of one of the inhibitors disclosed in US Patent No. 10,501,474, which is designated here as compound (I), is given below:

Compound (I).

Chemical name of compound (I): 4-amino-5-(6-(4-methylpiperazin-1-yl)-1H-benzo[d]imidazol2-yl)thieno[2,3-b]pyridin-6(7H) -He.

Successful development of pharmaceutically active agents such as compound (I) typically requires the identification of a solid form with properties allowing rapid isolation and purification after synthesis, that is suitable for large-scale production, can be stored for long periods of time with minimal water absorption, degradation or conversion to other solid forms that are suitable for the preparation of compositions and can be readily absorbed after administration to a subject (eg, soluble in water and gastric juice).

Brief Disclosure of the Present Invention

The present invention relates to the tartrate of compound (I), in which the molar ratio between compound (I) and tartaric acid is 1:1. Due to the presence of two carboxyl groups in tartaric acid and several basic nitrogen atoms in compound (I), different stoichiometric ratios are possible. For example, compound (I) with tartaric acid can form both a 1:1 salt and a 1:0.5 salt. Compound (I) 1:1 tartrate is referred to herein as Compound (I) 1:1 tartrate or Compound (I) 1:1 tartaric acid salt.

It was also found that the tartrate of compound (I) 1:1 can crystallize under strictly defined conditions to obtain non-hygroscopic crystalline forms (see example 6). The tartrate also has improved solubility in water and simulated gastric juices (see Example 7 and Table 7), has a long shelf life (see Example 8) and is suitable for large-scale synthesis (see Example 5).

Salts with thirteen different acids were screened (see examples 1-3) at different molar ratios of compound (I): acid. Among the 20 salt forms obtained (Examples 1 and 2), only the monohydrochloride, mesylate, tartrate and maleate had moderate to good crystallinity as determined by X-ray powder diffraction (XRPD). Further evaluation of these four salts in various solvent systems shows that the crystallinity of the mesylate and maleate is moderate (see Example 3). In addition, various polymorphic forms of monohydrochloride, mesylate, tartrate and maleate have been isolated using different solvent systems. It is noteworthy that the dihydrochloride has no or very low crystallinity, as shown in Examples 1 and 4.

Compared to the monohydrochloride, the 1:1 tartrate of compound (I) has the additional advantage of being non-hygroscopic. Additionally, as shown below in Example 9, compared to the free base and mono-HCl salt, the tartrate of compound (I) 1:1 in crystalline form resulted in improved plasma concentrations in dogs after oral administration. This is a significant advantage since the new solid form can be administered orally to provide effective plasma drug levels.

In one aspect, the present invention relates to the tartrate of compound (I), in which the molar

- 1 045568 the ratio between compound (I) and tartaric acid is 1:1.

In another aspect, the present invention relates to a pharmaceutical composition containing Compound (I) tartrate 1:1 and a pharmaceutically acceptable carrier or diluent.

Another aspect of the present invention relates to a method of treating a subject suffering from cancer, comprising administering to the subject an effective 1:1 amount of Compound (I) tartrate disclosed herein, or a corresponding pharmaceutical composition.

The present invention provides a method of treating a subject suffering from cancer, comprising administering to the subject an effective amount of Compound (I) tartrate 1:1 or a corresponding pharmaceutical composition and an effective amount of a second anti-cancer agent. In one embodiment, the second anticancer agent is a chemotherapeutic agent, a targeted therapeutic agent, radiation, or surgery.

The present invention also provides a method of treating a subject with cancer, comprising administering to the subject an effective amount of Compound (I) 1:1 tartrate disclosed herein, or a corresponding pharmaceutical composition, and an effective amount of an immunomodulatory agent, such as a checkpoint inhibitor (eg, anti-PD-1 antibody, anti-CTLA-4 antibody, or anti-PD-L1 antibody) or an inhibitor of tryptophan oxidation (eg, an IDO1, IDO2, or TDO2 inhibitor). In one embodiment, the immunomodulatory agent is an anti-PD-1 antibody.

In one embodiment, Compound (I) tartrate 1:1 or a corresponding pharmaceutical composition is administered together with an effective amount of one or more other anticancer drugs, preferably in combination with a PD-1 inhibitor. In one embodiment, the PD-1 inhibitor is nivolumab, pembrolizumab, pidilizumab, BMS 936559, MPDL3280A, MSB0010718C, or MEDI4736. In one specific embodiment, the PD-1 inhibitor is nivolumab. In yet another specific embodiment, the PD-1 inhibitor is pembrolizumab.

The present invention also provides the use of the 1:1 tartrate of compound (I) disclosed herein or a corresponding pharmaceutical composition containing the same in any of the methods described above. In one embodiment, the invention provides Compound (I) 1:1 tartrate or a pharmaceutical composition containing the same for use in any of the methods described herein. Another embodiment of the invention provides the use of Compound (I) 1:1 tartrate or a pharmaceutical composition containing it for the manufacture of a medicament for any of the methods described herein.

Brief description of the figures

In fig. 1 shows X-ray powder diffraction patterns (XRPD) of compound (I) hydrochloride 1:1 obtained in various examples. The lower spectrum refers to the hydrochloride obtained in example 1 (batch 1). The middle spectrum is for the hydrochloride prepared in Example 3 using IPA:water (Batch 2). The upper spectrum refers to the hydrochloride obtained in example 3 using acetone (batch 3).

In fig. 2 shows X-ray powder diffraction patterns (XRPD) of compound (I) mesylate 1:1 obtained in various examples. The lower spectrum refers to the mesylate obtained in example 2 (batch 1). The middle spectrum is for the mesylate produced in Example 3 using IPA:water (Batch 2). The upper spectrum refers to the mesylate obtained in example 3 using acetone (batch 3).

In fig. 3 shows X-ray powder diffraction patterns (XRPD) of the maleate of compound (I) 1:1 obtained in various examples. The lower spectrum refers to the maleate obtained in example 2 (batch 1). The middle spectrum is for the maleate produced in Example 3 using IPA:water (Batch 2). The upper spectrum refers to the maleate obtained in example 3 using acetone (batch 3).

In fig. Figure 4 shows the X-ray powder diffraction pattern (XRPD) of compound (I) dihydrochloride 1:2.

In fig. 5 shows thermograms obtained as a result of thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) of tartrate of compound (I) 1:1 obtained in example 5.

In fig. 6 shows the X-ray powder diffraction pattern (XRPD) of the tartrate of compound (I) 1:1 obtained in Example 5.

Detailed Disclosure of the Present Invention

The present invention relates to a new tartrate of compound (I) (ie 1:1 tartrate), as well as its polymorphic forms.

In one embodiment of the invention, the tartrate of compound (I) (ie, 1:1 tartrate) is crystalline.

In the context of the present invention, the term crystalline refers to a solid having a crystalline structure in which the individual molecules are characterized by a highly homogeneous, regular, fixed chemical configuration. The crystalline salt of compound (I) may be crystals of a single crystalline form of the salt of compound (I) or a mixture of crystals of different single crystalline forms. Monocrystalline form means that the salt of compound (I) is in the form of a single crystal or a plurality of crystals, in which all the crystals have

- 2 045568 the same crystalline form.

For the crystalline forms of compound (I) disclosed herein, at least a certain percentage (by weight) of the 1:1 tartrate of compound (I) is in monocrystalline form. Specific weight percentages include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99 ,5%, 99.9% or 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, 95%-100%, 70-80%, 80-90%, 90-100% mass percent of the salt of compound (I) are in monocrystalline form. It should be understood that all values and ranges between these values and ranges are intended to be embraced by the present invention.

When a crystalline salt of a compound (I) is characterized by a certain percentage of one specific crystalline salt form of a compound (I), the remainder consists of an amorphous form and/or crystalline forms other than one or more of said specific forms. Examples of single crystalline forms include Compound (I) 1:1 tartrate having one or more of the properties described herein.

The crystalline salts of compound (I) disclosed herein exhibit intense, unique XRPD patterns with sharp peaks corresponding to peak angles of 29 and a flat baseline, indicating highly crystalline material (eg, see FIG. 6). The XRPD X-ray patterns disclosed in this application were obtained with a copper radiation source (Cu Κα1 λ= 1.54179 A).

Characteristics of crystalline forms of tartrate of compound (I) 1:1

In one embodiment of the invention, the tartrate of compound (I) 1:1 is characterized by an X-ray powder diffraction pattern that contains peaks at 2θ values of 11.9°, 15.4°, 16.9° and 17.2° ± 0.2°. In another embodiment of the invention, the 1:1 tartrate of compound (I) is characterized by an X-ray powder diffraction pattern that contains at least three peaks at 2θ values selected from 11.9°, 15.4°, 16.9°, 17.2° and 25.6° ± 0.2°. In another embodiment of the invention, the tartrate of compound (I) 1:1 is characterized by an X-ray powder diffraction pattern that contains peaks at 2θ values of 11.9°, 15.4°, 16.9°, 17.2° and 25.6° ± 0 ,2°. In yet another embodiment of the invention, the tartrate of compound (I) 1:1 is characterized by an X-ray powder diffraction pattern that contains peaks at 2θ values: 11.9°, 14.0°, 15.4°, 16.9°, 17.2°, 25.6°, 26.3 and 30.7° ± 0.2°. In yet another embodiment of the invention, the tartrate of compound (I) 1:1 is characterized by an X-ray powder diffraction pattern that contains peaks at 2θ values: 11.9°, 14.0°, 15.4°, 16.9°, 17.2°, 22.1°, 25.6°, 26.3°, 30.7° and 34.0° ± 0.2°. In another embodiment of the invention, Compound (I) 1:1 tartrate exhibits the X-ray powder pattern indicated above and additionally contains peaks at 2θ values of 8.7° and 12.9° ± 0.2°. In yet another embodiment, Compound (I) 1:1 tartrate exhibits an X-ray powder diffraction pattern substantially similar to FIG. 6.

As used herein, a powder X-ray diffraction pattern is substantially similar to a diffraction pattern in a [particular] figure when at least 90%, e.g., at least 95%, at least 98%, or at least 99% of the signals expressed in 2θ, in two diffraction patterns are the same ± 0.2°. When determining similarity, one skilled in the art will appreciate that there may be differences in signal intensities and/or positions in XRPD diffraction patterns even for the same crystalline form. Thus, those skilled in the art will recognize that the maximum signal values in XRPD diffraction patterns (in degrees two-theta (°2θ) reported herein) generally mean that the reported value is ±0.2 degrees 2θ of the reported value, taking into account accepted in the art dispersion, as described below.

It is well known in the field of crystallography that for any given crystalline form, the angular position of the peak may vary slightly due to factors such as temperature change, sample bias, and the presence or absence of an internal standard. In the present disclosure, the variability of the angular peak position is ±0.2° in 2θ. In addition, the relative peak intensities for a given crystal form may vary due to differences in crystal sizes and non-random crystal orientation when preparing samples for XRPD analysis. It is well known in the art that this variability will take into account the above factors without preventing unambiguous identification of the crystalline form.

In another embodiment of the invention, the tartrate of compound (I) 1:1 has a critical phase transition temperature determined using a differential scanning calorimeter (DSC) of 189±2°C.

In another embodiment of the invention, Compound (I) 1:1 tartrate is characterized by a hygroscopicity measurement where water absorption is less than 4% (eg, 2% or 1%) by weight of the tartrate at 90% relative humidity (RH); or less than 2% (for example, 1% or 0.5%) by weight of the tartrate at 60% relative humidity; or less than 1% (for example, 0.5% or 0.1%) by weight of the tartrate at 30% relative humidity. In one embodiment, the water absorption is less than 0.1% by weight of the tartrate at a relative humidity (RH) of 30%. Hygroscopicity at different

- 3 045568 relative humidity (RH) was measured under the following conditions:

i) drying 0.5 to 1.5 mg of tartrate under a nitrogen atmosphere at 0% relative humidity for 2 hours;

ii) increasing or decreasing relative humidity in 10% increments from 0% to 90% and then to 0%;

iii) maintaining relative humidity at each step until the change in mass compared to the mass of the original tartrate is less than 0.01% per minute (%/min), provided that the minimum and maximum duration of time at each step is 10 minutes and 180 min respectively; and iv) determining the mass of the tartrate at the desired relative humidity (eg 90%, 60% or 30%), with steps i)-iv) being carried out at 25°C.

Hygroscopicity is measured using standard methods, for example, those described in G. Zografi and M. J. Kontny, Sorting of water by solids in Physical Characterization of Pharmaceutical Solids, ed. HG Brittain, Marcel Dekker, New York, NY (1995), pp. 385-418, or the procedure described in Example 6 of the present invention.

Characteristics of crystalline forms of monohydrochloride of compound (I) 1:1.

In one embodiment of the invention, the 1:1 monohydrochloride of compound (I) is a single crystalline form characterized by a powder X-ray diffraction pattern substantially similar to the upper spectrum in FIG. 1. Pharmaceutical compositions

The pharmaceutical compositions of the present invention contain Compound (I) 1:1 tartrate or a crystalline form thereof as described herein and one or more pharmaceutically acceptable carriers or diluents. The term pharmaceutically acceptable carrier refers to a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid excipient, diluent, excipient, solvent or encapsulating material involved in carrying or transporting any subject composition or component thereof. Each media must be acceptable in the sense that it is compatible with the composition in question and its components and does not cause harm to the subject. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository wax; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic solution; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances used in pharmaceutical compositions.

The compositions of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or through an implanted reservoir. The term parenteral in the context of the present invention includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion. In one embodiment, the compositions of the invention are administered orally, intraperitoneally or intravenously. Sterile injectable forms of the compositions of the present invention may be an aqueous or oily suspension. Said suspensions can be prepared in accordance with methods known in the art, using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example a solution in 1,3-butanediol. Acceptable carriers and solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile non-volatile oils are usually used as a solvent or suspending medium.

Any soft, fixed oil can be used for this purpose, including synthetic mono or diglycerides. Both fatty acids such as oleic acid and its glyceride derivatives and natural pharmaceutically acceptable oils such as olive oil or castor oil, especially in their polyoxyethylene variants, are suitable for the preparation of injectable preparations. These oily solutions or suspensions may also contain a long chain alcohol diluent or dispersant, such as carboxymethylcellulose or similar dispersing agents commonly used in pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants such as Tween, Spans and other emulsifiers or bioavailability enhancers that are commonly used in the manufacture of pharmaceutically acceptable solid, liquid or other dosage forms may also be used in the formulation.

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The pharmaceutically acceptable compositions of the invention can be administered orally in any suitable oral dosage form, including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of oral tablets, commonly used carriers include lactose and corn starch. Lubricants such as magnesium stearate are also commonly added. For oral administration in capsule form, suitable diluents include lactose and dried corn starch. When aqueous suspensions are required for oral administration, the active ingredient is combined with emulsifying and suspending agents. Certain sweeteners, flavors or colors may also be added if desired.

Alternatively, pharmaceutically acceptable compositions of the invention can be administered as suppositories for rectal administration. Suppositories can be prepared by mixing the agent with a suitable non-irritant excipient that is solid at room temperature but liquid at rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.

Pharmaceutically acceptable compositions of the invention can also be applied topically, especially when the target of treatment is areas or organs that are readily accessible for topical application, including diseases of the eye, skin or lower intestinal tract. Suitable topical preparations can easily be prepared for each of these areas or organs. Topical administration to the lower intestinal tract can be done using rectal suppositories (see above) or in a form suitable for enema. Transdermal patches can also be used for topical application.

For topical use, pharmaceutically acceptable compositions may be formulated as a suitable ointment containing the active ingredient suspended or dissolved in one or more vehicles. Carriers for topical administration of the compounds of this invention include, but are not limited to, the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutically acceptable compositions may be formulated as a suitable lotion or cream containing the active ingredients suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to: mineral oil, sorbitan monostearate, polysorbate 60, cetyl ether wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

Pharmaceutically acceptable compositions of the invention can also be administered by nasal aerosol or inhalation. Such compositions are prepared in accordance with methods well known in the pharmaceutical formulation art and may be formulated as solutions in saline using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons and/or other conventional solubilizing or dispersing agents. agents.

The amount of the compounds of the present invention that can be combined with the carrier to form a dosage form composition will vary depending on the subject being treated, the particular route of administration, and other factors determined by the person administering the dosage form. Dosages

The toxicity and therapeutic efficacy of the salt of compound (I) or its crystalline form described herein can be determined using standard pharmaceutical techniques in cell cultures or experimental animals. LD ₅₀ - represents the dose lethal to 50% of the population. ED ₅₀ - represents the dose therapeutically effective for 50% of the population. The ratio of doses causing toxic and therapeutic effects (LD ₅₀ /ED ₅₀ ) represents the therapeutic index. Preferred is a salt of compound (I) or a crystalline form thereof which has a high therapeutic index. A salt of compound (I) or a crystalline form thereof described herein may also be used and exhibit toxic side effects, in which case care should be taken to develop a delivery system that targets such salt or crystalline form to the affected tissue site to minimize potential damage to uninfected cells and thereby reduce side effects.

Data obtained from cell culture assays and animal studies can be used to determine dosage ranges for use in humans. The dosage of such salts or crystalline forms may be within a circulating concentration range that includes the ED50 with little or no toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration used. For any salt of compound (I) or crystalline form thereof described herein, a therapeutically effective dose may be initially assessed based on cell culture analysis. The dose can be adjusted in animal models to achieve a range of circulating plasma concentrations that includes the IC ₅₀ (ie, the concentration of test compound at which half-maximal inhibition of symptoms is achieved) determined in cell culture. Such information can be used to more accurately determine doses appropriate for humans. Plasma levels can be measured, for example, using high performance liquid chromatography.

It should also be understood that the specific dosage and treatment regimen for any particular subject will depend on a variety of factors, including, but not limited to, the following: potency of the particular compound used, age, body weight, general health, sex, diet, time of administration, rate of elimination , the combination of medications, the opinion of the treating physician, and the severity of the specific disease being treated. The amount in the composition of the salt of compound (I) or crystalline form of the present invention will also depend on the particular compound included in the composition.

Methods of treatment.

The subject is a mammal, preferably a human, but may also be an animal in need of veterinary treatment, for example, domestic animals (e.g., dogs, cats, etc.), farm animals (e.g., cows, sheep, pigs, horses, etc.) and laboratory animals (for example, rats, mice, guinea pigs, etc.).

As used herein, the term treatment of a subject suffering from cancer includes achieving, partially or substantially, one or more of the following: arresting growth, reducing the spread of cancer (eg, reducing tumor size), inhibiting the growth rate of cancer, alleviating or improving a clinical symptom or score cancer-related (such as tissue or serum components), or increasing the life expectancy of the subject; and reducing the likelihood of cancer recurrence.

The term "effective amount" means an amount that, when administered to a subject, produces beneficial or desired results, including clinical results, such as inhibiting, suppressing, or reducing cancer (eg, as measured by clinical symptoms or cancer cell counts) in the subject compared to a control.

In general, the effective amount of a compound provided herein will vary depending on various factors, such as the particular drug or compound, pharmaceutical formulation, route of administration, type of disease or disorder, characteristics of the subject or host being treated, and similar but , however, can be determined by one skilled in the art in the course of routine activities. The effective amount of the compound of the present invention can be easily determined by one skilled in the art by conventional methods known in the art.

In one embodiment, the effective amount of a compound provided herein ranges from about 0.1 to about 1000 mg/kg body weight, alternatively from about 1 to about 500 mg/kg body weight. In another embodiment of the invention, the effective amount of a compound provided herein is in the range from about 0.5 to about 5000 mg/m ² , alternatively from about 5 to about 2500 mg/m ² and in another embodiment from about 50 to approximately 1000 mg/ ^m2 . One skilled in the art will appreciate that certain factors may influence the dosage required to effectively treat a subject suffering from cancer or reduce the likelihood of cancer recurrence. These factors include, but are not limited to: severity of the disease or disorder, previous treatment, general health and/or age of the subject and other existing medical conditions.

A regimen of treating a subject with an effective amount of a compound of the present invention may consist of a single administration or, alternatively, include a series of administrations. For example, compound (I) tartrate 1:1 can be administered at least once a week. However, in another embodiment of the invention, the compound can be administered to a subject from about once a week to once a day for a given treatment. The length of the treatment period depends on many factors, such as the severity of the disease, the age of the subject, the concentration and activity of the compounds of the present invention, or a combination thereof. It should also be understood that the effective dose of a compound used for treatment or prophylaxis may increase or decrease over the course of a particular treatment or prophylaxis regimen. Dosage changes may occur, the need for which becomes apparent using standard diagnostic tests known in the art. In some cases, continuous administration may be required.

compound (I), its salts and crystalline forms disclosed herein inhibit HPK1. Thus, in general, the compounds described herein are useful for treating diseases or conditions associated with such kinases.

In one embodiment, the invention provides a method of inhibiting HPK1 activity in a subject in need of inhibiting HPK1 activity, comprising administering to the subject an effective amount of Compound (I) or a tartrate thereof (e.g., Compound (I) 1:1 tartrate), crystalline form, or pharmaceutical composition described in this document.

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Due to its activity against HPK1, compound (I) or its tartrate (e.g., compound (I) 1:1 tartrate), crystalline form or pharmaceutical composition described herein can be used to treat a subject with a condition associated with aberrant HPK1 activity.

In one embodiment, the condition associated with aberrant HPK1 activity is cancer.

Cancers that can be treated (including reducing the likelihood of recurrence) by the methods in accordance with the present invention include breast cancer, colorectal cancer, lung cancer, ovarian cancer, uterine cancer, prostate cancer, leukemia, lymphomas, brain cancer (including multiforme glioblastoma and neuroblastoma), head and neck cancer, pancreatic cancer, melanoma, hepatocellular carcinoma, kidney cancer and soft tissue sarcomas. In one embodiment, the cancer is breast cancer, colon cancer and ovarian cancer. In one embodiment, the cancer is selected from leukemia, acute myeloid leukemia, chronic myelogenous leukemia, breast cancer, brain cancer, colon cancer, colorectal cancer, head and neck cancer, hepatocellular carcinoma, lung adenocarcinoma, metastatic melanoma, pancreatic cancer gland cancer, prostate cancer, ovarian cancer and kidney cancer. In one embodiment, the cancer is lung cancer, colon cancer, brain cancer, neuroblastoma, prostate cancer, melanoma, glioblastoma multiforme or ovarian cancer. In another embodiment, the cancer is lung cancer, breast cancer, colon cancer, brain cancer, neuroblastoma, prostate cancer, melanoma, glioblastoma multiforme or ovarian cancer. In yet another embodiment, the cancer is breast cancer, colon cancer and lung cancer. In another embodiment of the invention, the cancer is breast cancer. In yet another embodiment, the cancer is a basal subtype breast cancer or luminal subtype B breast cancer. In yet another embodiment, the cancer is a basal subtype of breast cancer. In yet another embodiment, the basal subtype of breast cancer is ER (estrogen receptor), HER2, and PR (progesterone receptor) negative breast cancer. In yet another embodiment of the invention, the cancer is a soft tissue cancer. Soft tissue cancer is an industry-accepted term that covers tumors originating from any soft tissue of the body. Such soft tissues connect, support or surround various structures and organs of the body, including but not limited to the following: smooth muscle, skeletal muscle, tendon, fibrous tissue, adipose tissue, blood and lymphatic vessels, perivascular tissue, nerves, mesenchymal cells and synovial tissue . Thus, soft tissue cancer can be cancer of adipose tissue, muscle tissue, nerve tissue, joint tissue, blood vessels, lymphatic vessels and fibrous tissue. Soft tissue cancer can be benign or malignant. Generally, malignant soft tissue tumors are called sarcomas or soft tissue sarcomas. There are many types of soft tissue tumors, including lipoma, lipoblastoma, hibernoma, liposarcoma, leiomyoma, leiomyosarcoma, rhabdomyoma, rhabdomyosarcoma, neurofibroma, schwannoma (neurilemoma), neuroma, malignant schwannoma, neurofibrosarcoma, neurogenic sarcoma, nodular tenosynovitis, synovial sarcoma, hemangioma, glomus tumor, hemangiopericytoma, hemangioendothelioma, angiosarcoma, Kaposi's sarcoma, lymphangioma, fibroma, elastofibroma, superficial fibromatosis, fibrous histiocytoma, fibrosarcoma, fibromatosis, dermatofibrosarcoma protuberans (DFSP), malignant fibrous histiocytoma (MFH), myxoma, granular cell tumor, malignant mesenchymal tumor, alveolar soft tissue sarcoma, epithelioid sarcoma, clear cell sarcoma and desmoplastic small cell tumor. In a specific embodiment, the soft tissue cancer is a sarcoma selected from the group consisting of fibrosarcoma, gastrointestinal sarcoma, leiomyosarcoma, dedifferentiated liposarcoma, pleomorphic liposarcoma, malignant fibrous histiocytoma, round cell sarcoma and synovial sarcoma.

The present disclosure also relates to methods of treating a subject with a disease, comprising administering to the subject an effective amount of a compound represented by structural formula (I) in combination with an effective immunomodulatory therapy (also referred to as immunotherapy). Immunotherapy is the treatment of a disease using an immunomodulatory agent to induce, enhance, or suppress an immune response. Immunotherapy designed to induce or enhance an immune response is classified as activation immunotherapy, whereas immunotherapy that reduces or suppresses the response is classified as suppressive immunotherapy. The disease described here is cancer.

Immunomodulatory therapies, used alone or in combination approaches, include

i) immune checkpoint blocking inhibitors, including, but not limited to, anti-CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) antibodies (eg, ipilimumab); agents that interfere with the interaction of PD-1/PD-L1 and PD-L2, such as nivolumab (Opdivo - Bristol Myers

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Squibb), pembrolizumab (Keytruda, KM-3475, Merck), pidilizumab (CT-011, Cure Tech), BMS 936559 (BMS), and MPDL3280A (Roche); and other receptors that inhibit the immune response, such as anti-CD47 antibody;

ii) cell therapy (including but not limited to dendritic cell therapy (eg, Sipuleucel T (Provenge)) and adoptive T cell therapy), iii) vaccination strategies;

iv) adoptive T-cell therapy;

v) agents that prevent metabolic inhibition of the immune response, including inhibitors of indoleamine 2,3-dioxygenase (eg, INCB024360 (Incyte), 1-methyl-D-tryptophan, indoximod (NewLink Genetics)) or arginase; and vi) cytokine-based therapies, eg interferons (particularly type I interferon) and interleukins (eg interleukin-2). In one embodiment, the immunomodulatory agent used for immunomodulatory therapy is a PD-1 inhibitor, for example an anti-PD1 antibody.

Programmed cell death protein 1, also known as PD-1 and CD279 (cluster of differentiation 279), is a protein encoded by the PDCD1 gene in humans. PD-1 is a cell surface receptor that belongs to the immunoglobulin superfamily and is expressed on T cells and pro-B cells. PD-1 binds two ligands, PD-L1 and PD-L2, which are both members of the B7 family.

PD-1 and its ligands play an important role in suppressing the immune system by preventing T cell activation, which in turn reduces autoimmunity and promotes self-tolerance. The inhibitory effect of PD-1 is achieved through a dual mechanism of promoting apoptosis (programmed cell death) in antigen-specific T cells in the lymph nodes while reducing apoptosis in regulatory T cells (suppressor T cells).

PD-1 inhibitors used in the present invention include, but are not limited to, nivolumab, pembrolizumab, pidilizumab, BMS 936559, MPDL3280A, MSB0010718C or MEDI4736. These include antibodies BMS 936559, MPDL3280A, MSB0010718C and MEDI4736, which bind PD-L1 ligand. Nivolumab and pembrolizumab are approved by the Food and Drug Administration (FDA) to treat unresectable or metastatic melanoma that no longer responds to other drugs.

Vaccination strategies include antimicrobial immunotherapy, which involves vaccination, which is involved in activating the immune system to respond to an infectious agent.

Adoptive T-cell therapy uses cytotoxic T-cell responses to attack cancer cells. T cells that are naturally or genetically engineered to react to a patient's cancer are produced in vitro and then transplanted back into the cancer patient. One study using autologous tumor-infiltrating lymphocytes showed efficacy in treating patients with metastatic melanoma. This can be achieved by extracting T cells found in a patient's tumor that have been trained to attack the cancer cells. Such T cells are called tumor-infiltrating lymphocytes (TILs) and are subsequently stimulated to proliferate in vitro using high concentrations of IL-2, anti-CD3, and alloreactive feeder cells. These T cells are then transplanted back into the patient along with exogenous IL-2 to further enhance their anticancer activity

The present invention also provides methods for treating a subject with cancer comprising administering to the subject an effective amount of Compound (I) or a tartrate thereof (e.g., Compound (I) 1:1 tartrate), a crystalline form thereof, or a pharmaceutical composition described herein, in combination with effective anticancer therapy. In one embodiment, the cancer is a metastatic cancer. Metastatic cancer is cancer that has spread from its original site to other parts of the body.

The anticancer therapy described herein involves coadministration of an effective amount of a second anticancer agent along with a disclosed HPK-1 inhibitor. An anticancer agent is a compound that, when administered in an effective amount to a subject suffering from cancer, involves achieving, in part or substantially, one or more of the following results: growth arrest, reduction in the extent of cancer (eg, reduction in tumor size), inhibition of growth rate cancer and reducing or improving a clinical symptom or indicator associated with cancer (such as tissue or serum components), or increasing the life expectancy of the subject.

Anticancer agents suitable for use in the methods described herein include any anticancer agents approved for the treatment of cancer. In one embodiment, the anticancer agent includes, but is not limited to, a targeting antibody, an angiogenesis inhibitor, an alkylating agent, an antimetabolite, a vinca alkaloid, a taxane, a podophyllotoxin, a topoisomerase inhibitor, a hormonal antitumor agent, and other anticancer agents. In one embodiment of the invention, the anticancer agent is

- 8 045568 is a PD-1 inhibitor, for example an anti-PD1 antibody.

In one embodiment, anticancer agents that may be used in the methods described herein include paclitaxel, docetaxel, 5fluorouracil, trastuzumab, lapatinib, bevacizumab, letrozole, goserelin, tamoxifen, cetuximab, panitumumab, gemcitabine, capecitabine, irinotecan, Oxaliplatin, carboplatin, cisplatin, doxorubicin, epirubicin, cyclophosphamide, methotrexate, vinblastine, vincristine, melphalan, cytarabine, etoposide, daunorubicin, bleomycin, mitomycin and adriamycin and combinations thereof, but are not limited to the above.

In one embodiment, the anticancer agent and Compound (I) or its tartrate (eg, Compound (I) 1:1 tartrate), crystalline form thereof, or pharmaceutical composition described herein are administered simultaneously. When administered simultaneously, the anticancer agent and the compound may be administered in the same composition or in different compositions. Alternatively, the compound and the additional anticancer agent are administered separately at different times.

The following examples are intended to be illustrative and are not intended to limit the scope of the invention in any way.

experimental part

Abbreviations:

¹ H proton aq. water

w. wide

DCM dichloromethane

DVS Dynamic vapor sorption

Eq. hour equivalent

HPLC high performance liquid chromatography

IPA isopropanol

LC-MS liquid chromatography coupled with mass spectroscopy MeOH methanol min minutes

NMR nuclear magnetic resonance

PLM polarized light microscopy

RH relative humidity

Tgoosh room temperature

TGA thermogravimetric analysis

THF tetrahydrofuran

UPLC ultra-performance liquid chromatography

XRPD X-ray powder diffractometry

Analysis conditions.

X-ray powder diffractometry (XRPD).

XRPD analysis was performed using a Bruker D8 Advance X-ray powder diffractometer. The options for XRPD are listed below.

Reflection Mode Options X-ray wavelength SiKa, 1.54179 A, X-ray tube parameters 40 kV, 40 mA Scanning area (°2Θ) 4-40 Sample rotation speed 15 rpm Scan speed 10 deg/min

Thermogravimetric analysis (TGA).

2-5 mg of material was weighed in an open platinum crucible and loaded into a TA Q5000IR thermogravimetric analyzer. The sample was then heated at a rate of 10°C/min from 25°C to 350°C/400°C.

Differential scanning calorimetry (DSC).

0.5-1 mg of material was weighed into an aluminum DSC crucible and sealed loosely with an aluminum lid. The crucible containing the sample was then loaded into a TA Instruments Q2000. As soon as received

- 9 045568 stable heat flow characteristic at 25°C, the sample and reference were heated to 350°C at a rate of 10°C/min and the resulting heat flow characteristic was monitored. ^1H nuclear magnetic resonance spectroscopy ( ^1H -NMR).

Nuclear magnetic resonance measurements were recorded on a Bruker Avance DRX 400 instrument at 400 MHz and room temperature using DMSO-d ₆ or CD ₃ OD as solvent without internal standard.

HPLC/UPLC methods.

A representative method used to measure solubility is summarized in Table. 1. The representative method used to assess stability is summarized in Table. 2.

Table 1

UPLC method used to measure solubility

Column options ACQUITY UPLC® HSS PFP 1.8 microns, 2.1*100 mm Column temperature 60°C Mobile phase A 0.05% THF in water Mobile phase B acetonitrile Flow rate 0.8 ml/min Time % mobile phase A % mobile phase B (min) 0.00 90 10 Gradient Profile 4.00 10 90 5.00 10 90 6.5 90 10 Wavelength 235 nm detector Injection volume 0.5 µl Solvent for Acetonitrile/water flushing needles Dilution Acetonitrile/water (50/50 by volume)

table 2

HPLC method used to evaluate stability_______

Column options Waters XBridge Phenyl (150 * 4.6 mm, 3.5 microns) Column temperature 30°C Mobile phase A 0.05% THF in water Mobile phase B 0.05% THF in acetonitrile Flow rate 1.5 ml/min Gradient Profile Time (min) % mobile phase A % mobile phase B 0.00 95 5 20 5 95 20.01 95 5 27 95 5 Detector wavelength 235 nm Injection volume 5 µl Needle cleaning solvent Acetonitrile/water Dilution Acetonitrile/water (50/50 by volume)

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Examples

Example 1: Initial screening of salts.

Initial salt screening was performed using 1 or 2 equivalents _{of HCl, H2SO4 and H3PO4 in} a 2:1 mixture of dichloromethane (DCM) and methanol (MeOH). Approximately 100 mg 4-amino-5(6-(4-methylpiperazin-1-yl)-1H-benzo[d]imidazol-2-yl)thieno[2,3-b]pyridin-6(7H)-one ( I) dissolved in DCM and MeOH (2:1) at 50°C. 1 or 2 equivalents of acid were added and the resulting solution was stirred at room temperature for 3 days, and the resulting salt samples were analyzed by XRPD.

Using HCl (1 eq.), H2SO4 (2 eq.) and H ₃ PO ₄ (1 eq.) salts with low crystallinity were obtained. Using HCl (2 eq.) and H2SO4 (1 eq.) amorphous salts were obtained. The diffraction pattern of mono-HCl salt (batch 1) is shown in Fig. 1.

Example 2: Extended salt screening.

Extended salt screening was performed using 0.5, 1 and/or 2 equivalents of H ₃ PO ₄ , methanesulfonic acid, p-toluenesulfonic acid, citric acid, malic acid, fumaric acid, lactic acid, tartaric acid, succinic acid, benzoic acid and maleic acid in a mixture of THF and MeOH (2:1). A total of 16 salts were prepared, and the results are summarized in Table. 3.

Approximately 100 mg 4-amino-5-(6-(4-methylpiperazin-1-yl)-1H-benzo[d]imidazol-2yl)thieno[2,3-b]pyridin-6(7H)-one (I ) was dissolved in a 2:1 mixture of THF and MeOH at 50°C. 0.5, 1 or 2 equivalents of acid were added and the resulting solution was stirred at room temperature overnight. Samples that resulted in solids were centrifuged, and samples that did not precipitate were dried at room temperature. All samples were characterized by ¹ H NMR, PLM and XRPD.

All 16 isolated salts showed varying degrees of birefringence. However, only the 1:1 mesylate, 1:1 maleate, and 1:1 tartrate salts showed moderate to good crystallinity as measured by XRPD. Diffraction patterns of 1:1 mesylate (batch 1) and 1:1 maleate (batch 1) obtained using this method are shown in FIG. 2 and fig. 3 respectively. It is noteworthy that, as shown in table. 3 below, 0.5 equivalents of tartaric acid did not produce the desired hemitartrate (1:0.5) and was therefore not studied further.

Table 3 of salts prepared using 11 pharmaceutically acceptable acids in the extended salt screening

Number Acid Molar ratio acid: Compound (I) Used in the experiment In the separated salt 1 Phosphoric acid 1.05:1 1.0:1 2 Methanesulfonic acid 1.05:1 0.9:1 3 Methanesulfonic acid 2.1:1 2:1 4 p-Toluenesulfonic acid 1.05:1 0.9:1 5 p-Toluenesulfonic acid 2.1:1 2.1:1 6 Lemon acid 1.05:1 1.0:1 7 Lemon acid 0.55:1 0.6:1 8 L-malic acid 1.05:1 1.0:1 9 Fumaric acid 1.05:1 1.1:1 10 Fumaric acid 0.53:1 0.5:1 AND Lactic acid 1.05:1 1.0:1 12 L-tartaric acid 1.05:1 1.0:1 13 L-tartaric acid 0.53:1 0.7:1 14 succinic acid 1.05:1 1.0:1 15 Benzoic acid 1.05:1 1.1:1 16 Maleic acid 1.05:1 1.1:1

Example 3. Preparation of selected salts.

Hydrochloride, mesylate, tartrate and maleate were prepared separately using 2 different systems

- 11 045568 solvents: acetone and isopropyl alcohol/water (IPA/water) (95:5).

Approximately 100 mg 4-amino-5-(6-(4-methylninerasin-1-yl)-1H-benzo[d]imidαzol-2-yl)thieno[2,3b]pyridin-6(7H)-one (I ) were suspended in acetone (2 ml) or IPA/water (95:5, 2 ml). The suspension was stirred at 50°C. Acid (1 eq, 0.5 mol/L) was added and the mixture was stirred overnight. The salt was then isolated and dried at 30°C in vacuum. The resulting salts were characterized using TGA, DSC and XRPD.

The two solvent systems resulted in different polymorphic forms of hydrochloric acid salts (IPA: batch 2; acetone: batch 3), mesylates (IPA: batch 2; acetone: batch 3) and maleates (IPA: batch 2; acetone: batch 3). Diffraction patterns of the isolated polymorphic forms of HCl salts, mesylates and maleates are shown in Fig. 1, fig. 2 and fig. 3 respectively. As shown in XRPD diffraction patterns, the crystallinity of mesylates and maleates is moderate.

One 1:1 tartrate polymorph of compound (I) was isolated using the two solvent systems described in Example 3.

Example 4. Preparation of 4-amino-5-(6-(4-methylpiperαzin-1-yl)-1H-benzo[d]imidazol-2yl)thieno[2,3-b]pyridin-6(7H)-one dihydrochloride (I).

Approximately 15 g (40.95 mmol) 4-amino-5-(6-(4-methylpiperazin-1-yl)-1H-benzo[d]imidazol-2yl)thieno[2,3-b]pyridine-6- (7H)-one (I) was suspended in a mixture of DCM (300 ml) and MeOH (450 ml). At room temperature, 2M HCl in Et ₂ O (45 ml, 90.15 mmol) was slowly added. The resulting mixture was stirred at room temperature for 60 minutes. The solvent was then removed in vacuo and the resulting solid triturated with Et ₂ O (120 ml) and filtered to give the dihydrochloride salt as a brown precipitate. The dihydrochloride salt was characterized using XRPD and NMR. The XRPD diffraction pattern is shown in Fig. 4, where it can be seen that the crystallinity of the resulting dihydrochloride was very low.

Example 5. Preparation of 4-amino-5-(6-(4-methylpiperazin-1-yl)-1H-benzo[d]imidazol-2yl)thieno[2,3-b]pyridin-6(7H)-one tartrate (I).

4-Amino-5-(6-(4-methylpiperazin-1-yl)-1H-benzo[d]imidazol-2-yl)thieno[2,3-b]pyridin-6(7H)-one (4, 5 kg) was dissolved in an aqueous solution of acetic acid (44 kg water, 1.78 kg acetic acid) at a temperature of 55 to 60°C. The solution was stirred for 15-120 minutes. In a separate reactor, an aqueous solution of L-(+)-tartaric acid was prepared by adding the acid (1.98 kg) to water (14-18 kg) at a temperature of 20 to 30°C, and stirring the solution for 15-60 minutes. Then, an acetic acid solution containing compound (I) was slowly added to an aqueous solution of L-(+)-tartaric acid at a temperature of 55 to 60°C. Optionally, seed crystals were added. The resulting mixture was left to crystallize at a temperature of 55 to 60°C for 12-24 hours. Then the mixture was cooled to 20-25°C and stirred for 8-16 hours. After this, the precipitated product was collected, washed with ethanol and dried at temperature from 40 to 60°C for 3-24 hours in vacuum to obtain the required product. The title compound was characterized by 1H NMR, DSC, TGA and XRPD. The DSC and TGA results are shown in Fig. 5. XRPD diffraction pattern is shown in Fig. 6, and the results are presented in table. 4.

Table 4. List of the main peaks and their relative intensity in the XRPD diffraction pattern of 4-amino-5-(6-(4-methylpiperazin-1-yl)-1H-benzo[d]imidazol-2-yl)thieno[2,3-] tartrate b]pyridin-6(7H)one (tartrate of compound (I) 1:1).

_____________________________________Table 4

Angle (20) Relative intensity (%) 8.7 7.2 ID 49.9 12.9 h,o 14.0 33.6 15.4 64.2 16.9 100 17.2 49.6 22.1 28.7 25.6 41.2 26.3 26.9 30.7 29.1 34.0 28.7

1H NMR (400 MHz, DMSO-dS): 12.65 (broad s, 1H), 10.65 (broad s, 1H), 8.00 (broad s, 1H),

- 12 045568

7.68 (d, J=5.6 Hz, 1H), 7.49 (wide s, 1H), 7.20-7.17 (m, 2H), 6.92 (d, J=2.5 Hz, 1H), 6.71 (wide s, 4H),

4.16 (s, 2H), 3.22 (wide s, 4H), 2.88 (wide s, 4H), 2.52 (s, 3H).

Example 6. Measurement of hygroscopicity of 4-amino-5-(6-(4-methylpiperazin-1-yl)-1H-benzo[d]imidazol-2yl)thieno[2,3-b]pyridin-6(7H)-one (I) and its salts.

The hygroscopicity of the test compound was studied using a DVS device. The experimental parameters are given in table. 5. The results of measuring hygroscopicity are presented in table. 6.

Table 5

DVS device parameters

Total flow Temperature Solvent Control mode Action gas (cm ³ ) oven (°C) preheating 200 25 Water Open loop Didn't carry out Drying 2 hours in N ₂ (0% RH) Method stages Mode DMDT Cycle: 0% RH - 90% RH - 0% RH Step: 10% Dm/dt criteria: <0.01%/min. Sampling rate: 1 sec Minimum duration: 10 min. Maximum duration: 180 min.

Table 6

Hygroscopicity measurement results

Compound Mass change (%) Absorption water at relative humidity 30% Absorption water at relative humidity 60% Absorption water at relative humidity 90% Free base 0.5 0.9 26 Tartrate 0.06 o, s 0.7 Monohydrochloride (lot 3) 3.7 6.1 38.3

Example 7. Solubility of 4-amino-5-(6-(4-methylpiperazin-1-yl)-1H-benzo[d]imidazol-2-yl)thieno[2,3b]pyridin-6(7H)-one ( I) and its salts.

Test compounds were prepared in 3 different media as described below.

Samples prepared in water: Approximately 30-40 mg of test material was weighed into a glass vial. 1 ml of water was added. The sample was stirred for 1 h at ambient temperature and analyzed by HPLC after 24 h.

Samples prepared in simulated gastric fluid (SGF) buffer: Approximately 40 mg of test material was weighed into a glass vial. 4 ml of SGF buffer was added to obtain a final concentration of 10 mg/ml. The sample was stirred for 24 h at 37°C and analyzed by HPLC after 24 h.

Samples prepared in fasting simulated intestinal fluid (FaSSIF) buffer: approximately 8 mg of test material was weighed into a glass vial. 4 ml of FassiF buffer was added to obtain a final concentration of 2 mg/ml. The sample was stirred for 24 hours at 37°C and analyzed by HPLC after 24 hours.

Samples were stirred for 1 h at ambient temperature or 24 h at 37°C. The samples were then centrifuged and the resulting supernatants were analyzed by HPLC to determine solubility. The solubility of compound (I) and its salts in various media is given in table. 7.

- 13 045568

Table 7

Solubility of compound (I) and its salts in various media

Compound Solubility (mg/ml) water SGF FaSSIF Free base o, s 0.5 0.09 Tartrate - 9.0 0.02 Monohydrochloride (lot 3) 10.2 9.0 0.22 Dihydrochloride 3.3 - -

Example 8. Stability of 4-amino-5-(6-(4-methylpiperazin-1-yl)-1H-benzo[d]imidazol-2yl)thieno[2,3-b]pyridin-6(7H)-one tartrate (I).

The purity and stability of 4-amino-5-(6-(4-methylpiperazin-1-yl)-1Hbenzo[d]imidazol-2-yl)thieno[2,3-b]pyridin-6(7H)-one tartrate were determined (I). Stability samples were prepared and stored under 3 different storage conditions: 2-8°C/60%RH, 25°C/60%RH and 40°C/75%RH. Stability samples were removed from their storage conditions during each time period and allowed to equilibrate with environmental conditions before analysis. Appearance was assessed visually, purity and total impurities were analyzed by HPLC, and crystalline form was characterized by XRPD. The results are shown in table. 8.

- 14 045568

Table 8. Data on the long-term stability of 4-amino-5-(6-(4-methylpiperazin-1-yl)1H-benzo[d]imida-zol-2-yl)thieno[2,3-b]pyridin-6 tartrate (7H)-one (tartrate of compound (I) 1:1).

Table 8

Conditions storage Parameter Originally 1 month 3 months 6 months 12 months 5 °C/ 60% RH Appearance Total amount of impurities Light brown solid 0.03% Light brown solid 0.03% Light brown solid 0.0% Light brown solid 0.03% Light brown solid 0.03% Purity 100% 100% 100% 100% 100% Crystal form Yes Not studied Not studied Not studied Like in the initial stage External view Light brown solid Light brown solid Light brown solid Light brown solid Light brown solid 25°C/60% General RH quantity 0.03% 0.02% 0.03% 0.03% 0.03% impurities Purity 100% 100% 100% 100% 100% Crystal form Yes Not studied Not studied Not studied Like in the initial stage 40°C/75% RH External view Light brown solid Light brown solid Light brown solid Light brown solid Light brown solid General quantity 0.03% 0.02% 0.03% 0.03% 0.03% impurities Purity 100% 100% 100% 100% 100% Crystal form Yes Not studied Not studied Not studied Like in the initial stage

Example 9. Pharmacokinetic analyzes of 4-amino-5-(6-(4-methylpiperαzin-1-yl)-1H-benzo[d]imidazol2-yl)thieno[2,3-b]pyridin-6(7H)-one (I) and its salts.

A single dose of compound (I), compound (I) monohydrochloride 1:1 (lot 3), compound (I) dihydrochloride and compound (I) tartrate 1:1 in powder form in a capsule was administered to male beagles at a dose of 125 mg/kg or 150 mg/kg. Blood samples were collected over 24 hours and plasma levels of Compound (I) were analyzed by LC/MS. The results are presented in table. 9.

-

Claims (12)

Table 9. Pharmacokinetic parameters after oral administration of beagles 4-amino-5-(6-(4methylpiperazin-1-yl)-1H-benzo[d]imidazol-2-yl)thieno[2,3-b]-pyridin-6( 7H)-she (compound (I)) and its salts. Table 9 Solid form Free base Monohydrochloride (lot 3) Dihydrochloride Tartrate Oral dose (mg/kg) 125 125 125 150 Smack, (ng/ml) 301 547 530 514 AUCo-tlast (ng -h/ml) 2264 4354 6862 6550 CLAIM 1. Tartrate of compound (I) represented by the following structural formula: where the molar ratio between compound (I) and tartaric acid is 1:1. 2. Tartrate according to claim 1, characterized in that the tartrate is crystalline. 3. Tartrate according to claim 1, characterized in that at least 90 wt.% of the tartrate is in monocrystalline form. 4. Tartrate according to any one of claims 1 to 3, characterized in that the tartrate is characterized by a powder X-ray diffraction pattern that contains peaks at 11.9°, 15.4°, 16.9° and 17.2°±0.2 degrees 2θ . 5. Tartrate according to any one of claims 1 to 3, characterized in that the tartrate is characterized by a powder X-ray diffraction pattern that contains at least three peaks selected from peaks at 11.9°, 15.4°, 16.9°, 17. 2° and 25.6°±0.2 degrees 2θ. 6. Tartrate according to any one of claims 1 to 3, characterized in that the tartrate is characterized by a powder X-ray diffraction pattern that contains peaks at 11.9°, 14.0°, 15.4°, 16.9°, 17.2°, 25.6°, 26.3 and 30.7°±0.2 degrees 2θ. 7. Tartrate according to any one of claims 1 to 3, characterized in that the tartrate is characterized by a powder X-ray diffraction pattern that contains peaks at 11.9°, 14.0°, 15.4°, 16.9°, 17.2°, 22.1°, 25.6°, 26.3, 30.7° and 34.0° ± 0.2 degrees 2θ. 8. Tartrate according to any one of claims 4-7, characterized in that the tartrate is characterized by a powder X-ray diffraction pattern, which additionally contains peaks at 8.7° and 12.9°±0.2 degrees 2θ. 9. Tartrate according to any one of claims 1 to 8, characterized in that the tartrate is characterized by a critical phase transition temperature of 189±2°C, measured using a differential scanning calorimeter (DSC). 10. Tartrate according to any one of claims 1 to 9, characterized in that the water absorption is less than 4% by weight of the tartrate at a relative humidity (RH) of 90%, measured under the following conditions: i) drying 0.5 to 1.5 mg of tartrate under a nitrogen atmosphere at 0% relative humidity for 2 hours; ii) increasing or decreasing relative humidity in 10% increments from 0% to 90% and then to 0%; iii) maintaining the relative humidity at each step until the change in mass compared to the mass of the original tartrate is less than 0.01%/min, provided that the minimum and maximum duration of time at each step is 10 minutes and 180 minutes, respectively; and iv) determining the mass of the tartrate at a relative humidity of 90%, with steps i)-iv) being carried out at 25°C. 11. Tartrate according to claim 10, characterized in that the water absorption is less than 1% of the tartrate mass at a relative humidity (RH) of 90%. 12. Tartrate according to any one of claims 1 to 11, characterized in that the water absorption is less than 1% of the mass of the tartrate at a relative humidity of 30% (RH), measured under the following conditions: i) drying 0.5 to 1.5 mg of tartrate under a nitrogen atmosphere at 0% relative humidity for 2 hours; ii) increasing or decreasing relative humidity in 10% increments from 0% to 90% and then to 0%; iii) maintaining the relative humidity at each step until the change in mass compared to the mass of the original tartrate is less than 0.01%/min, provided that the minimum and maximum duration of time at each step is 10 minutes and 180 minutes, respectively; and iv) determining the mass of tartrate at a relative humidity of 30%, and steps i)-iv) are carried out -