WO2023241552A1

WO2023241552A1 - Salt and/or crystal form for compounds as casein kinase inhibitors

Info

Publication number: WO2023241552A1
Application number: PCT/CN2023/099851
Authority: WO
Inventors: Guanglong WU; Yuan Liu; Hanping Wang; Qing Ma; Enxing ZHOU
Original assignee: Gritscience Biopharmaceuticals Co., Ltd
Priority date: 2022-06-14
Filing date: 2023-06-13
Publication date: 2023-12-21

Abstract

The present disclosure provides salt and/or crystal form for compounds, especially for compounds as casein kinase inhibitors.

Description

Salt and/or Crystal Form for Compounds as Casein Kinase Inhibitors

TECHNICAL FIELD

The present application relates to salt and/or crystal form for compounds as casein kinase inhibitors, a pharmaceutical composition comprising said salt and/or crystal form, uses of said salt and/or crystal form and pharmaceutical compositions, and methods for preparing said salt and/or crystal form.

BACKGROUND OF THE INVENTION

The compound HY-B is 2- (8- (3- (4-fluorophenyl) -1-methyl-1H-pyrazol-4-yl) imidazo [1, 2-b] pyridazin-2-yl) propan-2-ol, which is a potent inhibitor of the casein kinase. However, no crystalline form of it has been reported yet.

The present application includes the unexpected discovery of novel solid forms of HY-B. The novel salt and/or crystal form of HY-B disclosed herein have surprising and useful properties.

SUMMARY OF THE INVENTION

The present disclosure provides salt and/or crystal form for compounds as casein kinase inhibitors.

The present disclosure also provides a process for preparing the salt and/or crystal form for compounds as casein kinase inhibitors.

The present disclosure also provides a pharmaceutical composition contains the salt and/or crystal form for compounds as casein kinase inhibitors and a pharmaceutically acceptable carrier or excipient.

The present disclosure also provides a method for inhibiting casein kinase includes a step of administering to a subject in need thereof an effective amount of the salt and/or crystal form for compounds as casein kinase inhibitors.

Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCES

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWING

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are employed, and the accompanying drawings (also “figure” and “FIG. ” herein) , of which:

Figure 1-1 shows structure of HY-B.

Figure 2-1 shows XRPD patterns of starting materials (823129-01-A/B) ;

Figure 2-2 shows TGA/DSC curves of starting material (823129-01-A) ;

Figure 2-3 shows 1H NMR spectrum of starting material (823129-01-A) ;

Figure 2-4 shows UPLC chromatogram of starting material (823129-01-A) ;

Figure 2-5 shows TGA/DSC curves of starting material (823129-01-B) ;

Figure 2-6 shows 1H NMR spectrum of starting material (823129-01-B) ;

Figure 2-7 shows UPLC chromatogram of starting material (823129-01-B) ;

Figure 3-1 shows XRPD pattern of re-prepared sulfate Type A (823129-12-A) ;

Figure 3-2 shows TGA/DSC curves of re-prepared sulfate Type A (823129-12-A) ;

Figure 3-3 shows 1H NMR spectrum of re-prepared sulfate Type A (823129-12-A) ;

Figure 3-4 shows UPLC chromatogram of re-prepared sulfate Type A (823129-12-A) ;

Figure 3-5 shows XRPD pattern of re-prepared maleate Type B (823129-13-A) ;

Figure 3-6 shows TGA/DSC curves of re-prepared maleate Type B (823129-13-A) ;

Figure 3-7 shows 1H NMR spectrum of re-prepared maleate Type B (823129-13-A) ;

Figure 3-8 shows UPLC chromatogram of re-prepared maleate Type B (823129-13-A) ;

Figure 3-9 shows XRPD pattern of re-prepared tosylate Type A (823129-14-A) ;

Figure 3-10 shows TGA/DSC curves of re-prepared tosylate Type A (823129-14-A) ;

Figure 3-11 shows 1H NMR spectrum of re-prepared tosylate Type A (823129-14-A) ;

Figure 3-12 shows UPLC chromatogram of re-prepared tosylate Type A (823129-14-A) ;

Figure 4-1 shows DVS plot of freebase Type A (823129-01-A) ;

Figure 4-2 shows XRPD overlay of freebase Type A (823129-01-A) before and after DVS;

Figure 4-3 shows DVS plot of sulfate Type A (823129-12-A) ;

Figure 4-4 shows XRPD overlay of sulfate Type A (823129-12-A) before and after DVS;

Figure 4-5 shows DVS plot of maleate Type B (823129-13-A) ;

Figure 4-6 shows XRPD overlay of maleate Type B (823129-13-A) before and after DVS;

Figure 4-7 shows DVS plot of tosylate Type A (823129-14-A) ;

Figure 4-8 shows XRPD overlay of tosylate Type A (823129-14-A) before and after DVS;

Figure 4-9 shows Plots of kinetic solubility;

Figure 4-10 shows XRPD overlay of freebase Type A (823129-01-A) after stability evaluation;

Figure 4-11 shows XRPD overlay of sulfate Type A (823129-12-A) after stability evaluation;

Figure 4-12 shows XRPD overlay of maleate Type B (823129-13-A) after stability evaluation;

Figure 4-13 shows XRPD overlay of tosylate Type A (823129-14-A) after stability evaluation;

Figure 5-1 shows XRPD overlay of HY-B maleate polymorphs;

Figure 5-2 shows VT-XRPD patterns of maleate Type B (823129-13-A) ;

Figure 5-3 shows XRPD pattern of maleate Type C (823129-27-A) ;

Figure 5-4 shows TGA/DSC curves of maleate Type C (823129-27-A) ;

Figure 5-5 shows 1H NMR spectrum of maleate Type C (823129-27-A) ;

Figure 5-6 shows UPLC chromatogram of maleate Type C (823129-27-A) ;

Figure 5-7 shows XRPD pattern of maleate Type D (823129-35-A9) ;

Figure 5-8 shows TGA/DSC curves of maleate Type D (823129-35-A9) ;

Figure 5-9 shows 1H NMR spectrum of maleate Type D (823129-35-A9) ;

Figure 6-1 shows XRPD overlay of the solids from competitive slurry for 3 days (I/II) ;

Figure 6-2 shows XRPD overlay of the solids from competitive slurry for 3 days (II/II) ;

Figure 6-3 shows XRPD overlay of the solids from competitive slurry for another 1 day (I/II) ;

Figure 6-4 shows XRPD overlay of the solids from competitive slurry for another 1 day (I/II) ;

Figure 7-1 shows XRPD overlay of maleate Type B (823129-13-A) after stability evaluation;

Figure 9-1 shows XRPD patterns of HCl salt Type A/B/C (823129-03-B1/D1/A2) ;

Figure 9-2 shows TGA/DSC curves of HCl salt Type A (823129-03-B1) ;

Figure 9-3 shows 1H NMR spectrum of HCl salt Type A (823129-03-B1) ;

Figure 9-4 shows TGA/DSC curves of HCl salt Type B (823129-03-D1) ;

Figure 9-5 shows 1H NMR spectrum of HCl salt Type B (823129-03-D1) ;

Figure 9-6 shows TGA/DSC curves of HCl salt Type C (823129-03-A2) ;

Figure 9-7 shows 1H NMR spectrum of HCl salt Type C (823129-03-A2) ;

Figure 9-8 shows XRPD pattern of sulfate Type A (823129-03-B3) ;

Figure 9-9 shows TGA/DSC curves of sulfate Type A (823129-03-B3) ;

Figure 9-10 shows 1H NMR spectrum of sulfate Type A (823129-03-B3) ;

Figure 9-11 shows XRPD patterns of maleate Type A/B (823129-03-A4/B4) ;

Figure 9-12 shows TGA/DSC curves of maleate Type A (823129-03-A4) ;

Figure 9-13 shows 1H NMR spectrum of maleate Type A (823129-03-A4) ;

Figure 9-14 shows TGA/DSC curves of maleate Type B (823129-03-B4) ;

Figure 9-15 shows 1H NMR spectrum of maleate Type B (823129-03-B4) ;

Figure 9-16 shows XRPD pattern of tosylate Type A (823129-03-B9) ;

Figure 9-17 shows TGA/DSC curves of tosylate Type A (823129-03-B9) ;

Figure 9-18 shows 1H NMR spectrum of tosylate Type A (823129-03-B9) ;

Figure 9-19 shows XRPD pattern of mesylate A (823129-03-A11) ;

Figure 9-20 shows TGA/DSC curves of mesylate Type A (823129-03-A11) ;

Figure 9-21 shows 1H NMR spectrum of mesylate Type A (823129-03-A11) ;

Figure 9-22 shows XRPD patterns of oxalate Type A/B (823129-03-B12/C12) ;

Figure 9-23 shows TGA/DSC curves of oxalate Type A (823129-03-B12) ;

Figure 9-24 shows 1H NMR spectrum of oxalate Type A (823129-03-B12) ;

Figure 9-25 shows TGA/DSC curves of oxalate Type B (823129-03-C12) ;

Figure 9-26 shows 1H NMR spectrum of oxalate Type B (823129-03-C12) ;

Figure 9-27 shows XRPD patterns of HBr salt Type A/B (823129-03-A13/B13) ;

Figure 9-28 shows TGA/DSC curves of HBr salt Type A (823129-03-A12) ;

Figure 9-29 shows 1H NMR spectrum of HBr salt Type A (823129-03-A13) ;

Figure 9-30 shows TGA/DSC curves of HBr salt Type B (823129-03-B13) ;

Figure 9-31 shows 1H NMR spectrum of HBr salt Type B (823129-03-B13) ;

Figure 9-32 shows XRPD overlay of residual solids from solubility test of freebase Type A in SGF;

Figure 9-33 shows XRPD overlay of residual solids from solubility test of freebase Type A in FaSSIF;

Figure 9-34 shows XRPD overlay of residual solids from solubility test of freebase Type A in FeSSIF;

Figure 9-35 shows XRPD overlay of residual solids from solubility test of freebase Type A in water;

Figure 9-36 shows XRPD overlay of residual solids from solubility test of sulfate Type A in SGF;

Figure 9-37 shows XRPD overlay of residual solids from solubility test of sulfate Type A in FaSSIF;

Figure 9-38 shows XRPD overlay of residual solids from solubility test of sulfate Type A in FeSSIF;

Figure 9-39 shows XRPD overlay of residual solids from solubility test of sulfate Type A in water;

Figure 9-40 shows XRPD overlay of residual solids from solubility test of maleate Type B in SGF;

Figure 9-41 shows XRPD overlay of residual solids from solubility test of maleate Type B in FaSSIF;

Figure 9-42 shows XRPD overlay of residual solids from solubility test of maleate Type B in FeSSIF;

Figure 9-43 shows XRPD overlay of residual solids from solubility test of maleate Type B in water;

Figure 9-44 shows XRPD overlay of residual solids from solubility test of tosylate Type A in SGF;

Figure 9-45 shows XRPD overlay of residual solids from solubility test of tosylate Type A in FaSSIF;

Figure 9-46 shows XRPD overlay of residual solids from solubility test of maleate Type B in FeSSIF;

Figure 9-47 shows XRPD overlay of residual solids from solubility test of tosylate Type A in water;

Figure 9-48 shows XRPD pattern of re-prepared maleate Type B (823129-24-A) ;

Figure 9-49 shows TGA/DSC curves of re-prepared maleate Type B (823129-24-A) ;

Figure 9-50 shows 1H NMR spectrum of re-prepared maleate Type B (823129-24-A) ;

Figure 9-51 shows UPLC chromatogram of re-prepared maleate Type B (823129-24-A) ;

Figure 9-52 shows XRPD pattern of re-prepared tosylate Type A (823129-25-A) ;

Figure 9-53 shows TGA/DSC curves of re-prepared tosylate Type A (823129-25-A) ;

Figure 9-54 shows 1H NMR spectrum of re-prepared tosylate Type A (823129-25-A) ;

Figure 9-55 shows UPLC chromatogram of re-prepared tosylate Type A (823129-25-A) .

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

As used herein, the term “treating” , unless otherwise indicated, generally refers to reversing, alleviating the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. The term “treatment” , as used herein, unless otherwise indicated, generally refers to the act of treating as "treating" is defined immediately above. The term “treating” may also include adjuvant and neo-adjuvant treatment of a subject.

As used herein, the term “preventing” unless otherwise indicated, generally refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It may be understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words may be also expressly disclosed.

As used herein, the term “pharmaceutically acceptable salt” generally refers to a salt that may be pharmaceutically acceptable and that may possess the desired pharmacological activity of the parent compound. Such salts may include: acid addition salts, formed with inorganic acids or formed with organic acids or basic addition salts formed with the conjugate bases of any of the inorganic acids wherein the conjugate bases comprise a cationic component.

As used herein, the term “pharmaceutically acceptable carrier” generally refers to aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles may include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like) , carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity may be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms may be made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly (orthoesters) and poly (anhydrides) . Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release may be controlled. Depot injectable formulations may be also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues. The injectable formulations may be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers may include sugars such as lactose. Desirably, at least 95%by weight of the particles of the active ingredient may have an effective particle size in the range of 0.01 to 10 micrometers.

As used herein, the term “prodrug” generally refers to a compound that is metabolized, for example hydrolyzed or oxidized, in the host to form the compound of the present invention. Typical examples of prodrugs may include compounds that have biologically labile protecting groups on a functional moiety of the active compound. Prodrugs may include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, dedcylated, phosphorylated, dephosphorylated to produce the active compound.

As used herein, the term “casein kinase” generally refers to a protein having an activity of catalyzing the serine/threonine-selective phosphorylation of proteins. This activity may be referred to as “casein kinase activity” . The Gene ID for gene encoding casein kinase may be 1453 or 1454.

As used herein, the term “subject” generally refers to an animal, which may include, but not limited to, cattle, pigs, sheep, chicken, turkey, buffalo, llama, ostrich, dogs, cats, and humans, and the subject may be a human. It may be contemplated that in the method of treating a subject thereof of the sixth embodiment can be any of the compounds either alone or in combination with another compound of the present invention.

As used herein, the term “effective amount” generally refers to an amount of an agent or a compound being administered which will treat a disease or disorder, or some or all of the symptom. The result can be reduction and/or alleviation of the signs, symptoms, or causes of a disease or disorder, or any other desired alteration of a biological system. For example, an “effective amount” for therapeutic uses is the amount of the composition including a compound as disclosed herein required to provide a clinically significant decrease in a disease or disorder symptoms without undue adverse side effects.

As used herein, the term “administering” generally refers to the compound may be administered by any appropriate route, for example, orally, parenterally, intravenously, intradermally, subcutaneously, or topically, in liquid or solid form.

As used herein, the term “formula” may be hereinafter referred to as a “compound (s) of the invention” . Such terms are also defined to include all forms of the compound of formula, including hydrates, solvates, isomers, crystalline and non-crystalline forms, isomorphs, polymorphs, and metabolites thereof. For example, the compounds of formula, or pharmaceutically acceptable salts thereof, may exist in unsolvated and solvated forms. When the solvent or water is tightly bound, the complex may have a well-defined stoichiometry independent of humidity. When, however, the solvent or water is weakly bound, as in channel solvates and hygroscopic compounds, the water/solvent content may be dependent on humidity and drying conditions. In such cases, non-stoichiometry will be the norm.

The compounds of “formula” may have asymmetric carbon atoms. The carbon-carbon bonds of the compounds of formula may be depicted herein using a solid line, a solid wedge, or a dotted wedge. The use of a solid line to depict bonds to asymmetric carbon atoms may be meant to indicate that all possible stereoisomers (e.g. specific enantiomers, racemic mixtures, etc. ) at that carbon atom are included. The use of either a solid or dotted wedge to depict bonds to asymmetric carbon atoms may be meant to indicate that only the stereoisomer shown is meant to be included. It is possible that compounds of the present application may contain more than one asymmetric carbon atom. In those compounds, the use of a solid line to depict bonds to asymmetric carbon atoms may be meant to indicate that all possible stereoisomers are meant to be included. For example, unless stated otherwise, it may be intended that the compounds of formula can exist as enantiomers and diastereomers or as racemates and mixtures thereof. The use of a solid line to depict bonds to one or more asymmetric carbon atoms in a compound of formula and the use of a solid or dotted wedge to depict bonds to other asymmetric carbon atoms in the same compound may be meant to indicate that a mixture of diastereomers is present.

The compounds of the present application (e.g., the compounds of formula) may exist as clathrates or other complexes. Included within the scope of the invention are complexes such as clathrates, drug-host inclusion complexes wherein, in contrast to the aforementioned solvates, the drug and host may be present in stoichiometric or non-stoichiometric amounts. Also included may be complexes of formula containing two or more organic and/or inorganic components which may be in stoichiometric or non-stoichiometric amounts. The resulting complexes may be ionized, partially ionized, or non-ionized. For a review of such complexes, see J. Pharm. Sci., 64 (8) , 1269-1288 by Haleblian (August 1975) .

Stereoisomers of formula may include cis and trans isomers, optical isomers such as R and S enantiomers, diastereomers, geometric isomers, rotational isomers, conformational isomers, and tautomers of the compounds of formula, including compounds exhibiting more than one type of isomerism; and mixtures thereof (such as racemates and diastereomeric pairs) . Also included may be acid addition or base addition salts wherein the counterion is optically active, for example, D-lactate or L-lysine, or racemic, for example, DL-tartrate or DL-arginine.

When any racemate crystallizes, crystals of two different types are possible. The first type is the racemic compound (true racemate) referred to above wherein one homogeneous form of crystal is produced containing both enantiomers in equimolar amounts. The second type is the racemic mixture or conglomerate wherein two forms of crystal are produced in equimolar amounts each comprising a single enantiomer.

The compounds of formula may exhibit the phenomena of tautomerism and structural isomerism. For example, the compounds of formula may exist in several tautomeric forms, including the enol and imine forms, and the keto and enamine forms, and geometric isomers and mixtures thereof. All such tautomeric forms may be included within the scope of compounds of formula. Tautomers may exist as mixtures of a tautomeric set in solution. In solid form, usually one tautomer predominates. Even though one tautomer may be described, the present invention includes all tautomers of the compounds of formula.

The present invention also includes isotopically-labeled compounds, which are identical to those recited in formula above, but for the fact that one or more atoms may be replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that may be incorporated into compounds of formula include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, such as, but not limited to, ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F, and ³⁶Cl. Certain isotopically-labeled compounds of formula, for example those into which radioactive isotopes such as ³H and ¹⁴C are incorporated, may be useful in drug and/or substrate tissue distribution assays. Tritiated, i.e., ³H, and carbon-14, i.e., ¹⁴C, isotopes may be particularly used for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium, i.e., ²H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be used in some circumstances. Isotopically-labeled compounds of formula may generally be prepared by carrying out the procedures disclosed in the Schemes and/or in the Examples below, by substituting an isotopically-labeled reagent for a non-isotopically-labeled reagent.

The compounds of the present application may be used in the form of salts derived from inorganic or organic acids. Depending on the particular compound, a salt of the compound may be advantageous due to one or more of the salt's physical properties, such as enhanced pharmaceutical stability in differing temperatures and humidity, or a desirable solubility in water or oil. In some instances, a salt of a compound also may be used as an aid in the isolation, purification, and/or resolution of the compound.

In one aspect, the present application provides a crystalline form of compound HY-B, 2- (8-(3-(4-fluorophenyl) -1-methyl-1H-pyrazol-4-yl) imidazo [1, 2-b] pyridazin-2-yl) propan-2-ol, wherein said crystalline form exhibits an X-ray powder diffraction pattern having one or more characteristic peaks expressed in degrees 2-theta selected from the group consisting of:

(1) 18.8±0.3 degrees, 19.3±0.3 degrees, 21.8±0.3 degrees;

(2) 8.2±0.3 degrees, 18.3±0.3 degrees, 23.4±0.3 degrees;

(3) 11.3±0.3 degrees, 18.5±0.3 degrees, 26.1±0.3 degrees;

(4) 12.6±0.3 degrees, 18.3±0.3 degrees, 18.9±0.3 degrees;

(5) 10.5±0.3 degrees, 18.4±0.3 degrees, 28.8±0.3 degrees;

(6) 9.5±0.3 degrees, 10.8±0.3 degrees, 18.3±0.3 degrees;

(7) 9.2±0.3 degrees, 18.4±0.3 degrees, 24.7±0.3 degrees;

(8) 13.3±0.3 degrees, 18.0±0.3 degrees, 20.1±0.3 degrees;

(9) 6.5±0.3 degrees, 9.7±0.3 degrees, 19.2±0.3 degrees;

(10) 9.5±0.3 degrees, 19.1±0.3 degrees, 24.5±0.3 degrees;

(11) 10.4±0.3 degrees, 11.9±0.3 degrees, 16.9±0.3 degrees;

(12) 14.5±0.3 degrees, 21.2±0.3 degrees, 23.0±0.3 degrees;

(13) 8.0±0.3 degrees, 10.3±0.3 degrees, 18.9±0.3 degrees; and

(14) 11.9±0.3 degrees, 18.3±0.3 degrees, 20.3±0.3 degrees.

For example, wherein said crystalline form further exhibits an X-ray powder diffraction pattern having one or more characteristic peaks expressed in degrees 2-theta selected from the group consisting of:

(1) 10.1±0.3 degrees, 12.5±0.3 degrees, 24.0±0.3 degrees;

(2) 12.0±0.3 degrees, 15.4±0.3 degrees, 16.5±0.3 degrees;

(3) 8.5±0.3 degrees, 8.9±0.3 degrees, 25.6±0.3 degrees;

(4) 8.2±0.3 degrees, 17.0±0.3 degrees, 21.8±0.3 degrees;

(5) 20.3±0.3 degrees, 24.8±0.3 degrees, 27.4±0.3 degrees;

(6) 14.3±0.3 degrees, 20.7±0.3 degrees, 25.2±0.3 degrees;

(7) 10.3±0.3 degrees, 18.0±0.3 degrees, 24.1±0.3 degrees;

(8) 13.8±0.3 degrees, 20.4±0.3 degrees, 25.3±0.3 degrees;

(9) 18.6±0.3 degrees, 19.5±0.3 degrees, 23.5±0.3 degrees;

(10) 17.2±0.3 degrees, 18.4±0.3 degrees, 24.9±0.3 degrees;

(11) 15.2±0.3 degrees, 19.5±0.3 degrees, 20.7±0.3 degrees;

(12) 9.8±0.3 degrees, 17.5±0.3 degrees, 20.4±0.3 degrees;

(13) 14.0±0.3 degrees, 16.1±0.3 degrees, 24.3±0.3 degrees; and

(14) 24.7±0.3 degrees, 27.3±0.3 degrees, 28.6±0.3 degrees.

For example, wherein said crystalline form exhibits an X-ray powder diffraction pattern having one or more characteristic peaks expressed in degrees 2-theta selected from the group consisting of:

(1) 10.7±0.3 degrees, 20.3±0.3 degrees, 25.2±0.3 degrees;

(2) 9.5±0.3 degrees, 16.8±0.3 degrees, 21.7±0.3 degrees;

(3) 20.8±0.3 degrees, 22.7±0.3 degrees, 23.7±0.3 degrees;

(4) 9.4±0.3 degrees, 12.0±0.3 degrees, 25.3±0.3 degrees;

(5) 11.9±0.3 degrees, 12.9±0.3 degrees, 28.2±0.3 degrees;

(6) 10.1±0.3 degrees, 17.3±0.3 degrees, 23.9±0.3 degrees;

(7) 14.3±0.3 degrees, 19.0±0.3 degrees, 22.1±0.3 degrees;

(8) 19.7±0.3 degrees, 23.0±0.3 degrees, 24.0±0.3 degrees;

(9) 11.2±0.3 degrees, 25.0±0.3 degrees, 27.7±0.3 degrees;

(10) 21.4±0.3 degrees, 23.7±0.3 degrees, 25.6±0.3 degrees;

(11) 7.6±0.3 degrees, 24.7±0.3 degrees, 26.5±0.3 degrees;

(12) 10.1±0.3 degrees, 13.7±0.3 degrees, 22.6±0.3 degrees;

(13) 15.4±0.3 degrees, 18.5±0.3 degrees, 22.9±0.3 degrees; and

(14) 14.6±0.3 degrees, 20.8±0.3 degrees, 23.3±0.3 degrees.

(1) 18.8±0.3 degrees, 19.3±0.3 degrees, 21.8±0.3 degrees, 10.1±0.3 degrees, 12.5±0.3 degrees, 24.0±0.3 degrees, 10.7±0.3 degrees, 20.3±0.3 degrees, 25.2±0.3 degrees;

(2) 8.2±0.3 degrees, 18.3±0.3 degrees, 23.4±0.3 degrees, 12.0±0.3 degrees, 15.4±0.3 degrees, 16.5±0.3 degrees, 9.5±0.3 degrees, 16.8±0.3 degrees, 21.7±0.3 degrees;

(3) 11.3±0.3 degrees, 18.5±0.3 degrees, 26.1±0.3 degrees, 8.5±0.3 degrees, 8.9±0.3 degrees, 25.6±0.3 degrees, 20.8±0.3 degrees, 22.7±0.3 degrees, 23.7±0.3 degrees;

(4) 12.6±0.3 degrees, 18.3±0.3 degrees, 18.9±0.3 degrees, 8.2±0.3 degrees, 17.0±0.3 degrees, 21.8±0.3 degrees, 9.4±0.3 degrees, 12.0±0.3 degrees, 25.3±0.3 degrees;

(5) 10.5±0.3 degrees, 18.4±0.3 degrees, 28.8±0.3 degrees, 20.3±0.3 degrees, 24.8±0.3 degrees, 27.4±0.3 degrees, 11.9±0.3 degrees, 12.9±0.3 degrees, 28.2±0.3 degrees;

(6) 9.5±0.3 degrees, 10.8±0.3 degrees, 18.3±0.3 degrees, 14.3±0.3 degrees, 20.7±0.3 degrees, 25.2±0.3 degrees, 10.1±0.3 degrees, 17.3±0.3 degrees, 23.9±0.3 degrees;

(7) 9.2±0.3 degrees, 18.4±0.3 degrees, 24.7±0.3 degrees, 10.3±0.3 degrees, 18.0±0.3 degrees, 24.1±0.3 degrees, 14.3±0.3 degrees, 19.0±0.3 degrees, 22.1±0.3 degrees;

(8) 13.3±0.3 degrees, 18.0±0.3 degrees, 20.1±0.3 degrees, 13.8±0.3 degrees, 20.4±0.3 degrees, 25.3±0.3 degrees, 19.7±0.3 degrees, 23.0±0.3 degrees, 24.0±0.3 degrees;

(9) 6.5±0.3 degrees, 9.7±0.3 degrees, 19.2±0.3 degrees, 18.6±0.3 degrees, 19.5±0.3 degrees, 23.5±0.3 degrees, 11.2±0.3 degrees, 25.0±0.3 degrees, 27.7±0.3 degrees;

(10) 9.5±0.3 degrees, 19.1±0.3 degrees, 24.5±0.3 degrees, 17.2±0.3 degrees, 18.4±0.3 degrees, 24.9±0.3 degrees, 21.4±0.3 degrees, 23.7±0.3 degrees, 25.6±0.3 degrees;

(11) 10.4±0.3 degrees, 11.9±0.3 degrees, 16.9±0.3 degrees, 15.2±0.3 degrees, 19.5±0.3 degrees, 20.7±0.3 degrees, 7.6±0.3 degrees, 24.7±0.3 degrees, 26.5±0.3 degrees;

(12) 14.5±0.3 degrees, 21.2±0.3 degrees, 23.0±0.3 degrees, 9.8±0.3 degrees, 17.5±0.3 degrees, 20.4±0.3 degrees, 10.1±0.3 degrees, 13.7±0.3 degrees, 22.6±0.3 degrees;

(13) 8.0±0.3 degrees, 10.3±0.3 degrees, 18.9±0.3 degrees, 14.0±0.3 degrees, 16.1±0.3 degrees, 24.3±0.3 degrees, 15.4±0.3 degrees, 18.5±0.3 degrees, 22.9±0.3 degrees; and

(14) 11.9±0.3 degrees, 18.3±0.3 degrees, 20.3±0.3 degrees, 24.7±0.3 degrees, 27.3±0.3 degrees, 28.6±0.3 degrees, 14.6±0.3 degrees, 20.8±0.3 degrees, 23.3±0.3 degrees, ±0.3 degrees. As used herein when used in reference to a degree 2-theta value refers to the stated value ±0.3 degrees 2-theta. As used herein when used in reference to a degree 2-theta value refers to the stated value ±0.2 degrees 2-theta. As used herein when used in reference to a degree 2-theta value refers to the stated value ±0.1 degrees 2-theta.

For example, wherein said crystalline form exhibits mass loss in the TGA (thermogravimetric) analysis selected from the group consisting of:

(1) mass loss of 1.3%up to 120 ℃;

(2) mass loss of 0.4%up to 120 ℃;

(3) mass loss of 0.7%up to 120 ℃;

(4) mass loss of 5.7%up to 120 ℃;

(5) mass loss of 9.7%up to 120 ℃;

(6) mass loss of 16.7%up to 120 ℃;

(7) mass loss of 14.2%up to 120 ℃;

(8) mass loss of 8.7%up to 120 ℃;

(9) mass loss of 1.8%up to 120 ℃;

(10) mass loss of 6.8%up to 120 ℃;

(11) mass loss of 4.1%up to 120 ℃;

(12) mass loss of 6.8%up to 120 ℃;

(13) mass loss of 0.6%up to 120 ℃; and

(14) mass loss of 8.3%up to 120 ℃.

For example, wherein said crystalline form exhibits endotherm and/or exotherm peak in the DSC (differential scanning calorimetry) analysis selected from the group consisting of:

(1) 135.3 ℃ (endotherm) ;

(2) 134.6 ℃ (endotherm) ;

(3) 136.5 ℃ (endotherm) ;

(4) 127.7 ℃ (endotherm) ;

(5) 115.3 ℃ (endotherm) ;

(6) 99.4 ℃ (endotherm) ;

(7) 112.3 ℃ (endotherm) ;

(8) 121.9 ℃ (endotherm) ;

(9) 151.8 ℃ (endotherm) ;

(10) 91.8 ℃ (endotherm) , 122.4 ℃ (endotherm) ;

(11) 113.8 ℃ (endotherm) ;

(12) 102.1 ℃ (endotherm) , 118.4 ℃ (endotherm) ;

(13) 155.7 ℃ (endotherm) ; and

(14) 125.5 ℃ (endotherm) .

For example, wherein said crystalline form exhibits salt stoichiometry in the UPLC-IC (ultra-performance liquid chromatography-ion chromatography) analysis selected from the group consisting of (molar rate of acid and freebase) :

(1) 0.5 (molar rate of maleic acid and freebase) ;

(2) 1 (molar rate of maleic acid and freebase) ;

(3) 1 (molar rate of maleic acid and freebase) ;

(4) 1.5 (molar rate of maleic acid and freebase) ;

(5) 1 (molar rate of hydrochloric acid and freebase) ;

(6) 1 (molar rate of hydrochloric acid and freebase) ;

(7) 1.2 (molar rate of hydrochloric acid and freebase) ;

(8) 1.1 (molar rate of sulphuric acid and freebase) ;

(9) 1 (molar rate of p-methylbenzene sulfonic acid and freebase) ;

(10) 1 (molar rate of methanesulfonic acid and freebase) ;

(11) 1.1 (molar rate of oxalic acid and freebase) ;

(12) 1.1 (molar rate of oxalic acid and freebase) ;

(13) 1 (molar rate of hydrobromic acid and freebase) ; and

(14) 1 (molar rate of hydrobromic acid and freebase) .

For example, wherein said crystalline form comprises no residual solvent or comprises residual solvent (weight%) selected from the group consisting of: 2.9 weight%of ACN; and 0.5 weight%of IPA.

For example, wherein said crystalline form exhibits purity rate in the LC-MS (liquid chromatography–mass spectrometry) analysis selected from the group consisting of: 99.50 area%; and 98.37 area%.

For example, wherein said crystalline form has solubility selected from the group consisting of: 2.8 mg/mL for SGF, 0.17 mg/mL for FaSSIF, 0.11 mg/mL for FeSSIF, 0.77 mg/mL for H₂O; 5.0 mg/mL for SGF, 0.68 mg/mL for FaSSIF, 0.12 mg/mL for FeSSIF, 3.1 mg/mL for H₂O; and 2.4 mg/mL for SGF, 0.21 mg/mL for FaSSIF, 0.12 mg/mL for FeSSIF, 1.3 mg/mL for H₂O.

For example, wherein said crystalline form exhibits hygroscopicity in the DVS (Dynamic vapor sorption) analysis at 25 ℃ and 80%RH (room humidity) selected from the group consisting of: 0.03%, 9.1%, and 1.2%.

In one aspect, the present application provides an acid addition salt of crystalline form of the present application, or a pharmaceutically acceptable salt, prodrug, or metabolite thereof, or a solvate or hydrate of any of the foregoing.

In one aspect, the present application provides a process of preparing crystalline form of the present application, the process is selected from the group consisting of:

(1) mixing compound HY-A and equimolar maleic acid in IPA, followed by slurry at RT for 3 days;

(2) mixing compound HY-A and maleic acid in 5 mL IPAc, followed by slurry at RT for 3 days;

(3) compound HY-A is ground for 3～5 min;

(4) solution vapor diffusion in CH₂Br₂/n-Hexane at RT for 4 days using compound HY-A;

(5) mixing compound HY-A and equimolar HCl in IPAc, followed by slurry at RT for 3 days;

(6) mixing compound HY-A and equimolar HCl in ACN/H₂O (19: 1, v/v) , followed by slurry at RT for 3 days;

(7) mixing compound HY-A and HCl (molar ratio of 1: 2, FB/acid) in IPA for slurry at RT for 3 days followed by evaporation at RT

(8) mixing compound HY-A equimolar H₂SO₄ in IPAc, followed by slurry at RT for 3 days

(9) mixing compound HY-A and equimolar p-toluenesulfonic acid in IPAc, followed by slurry at RT for 3 days;

(10) mixing compound HY-A and methanesulfonic acid (molar ratio of 1: 2, FB/acid) in IPA, followed by slurry at RT for 3 days;

(11) mixing compound HY-A and equimolar oxalic acid in IPAc, followed by slurry at RT for 3 days;

(12) mixing compound HY-A and equimolar oxalic acid in MIBK, followed by slurry at RT for 3 days;

(13) mixing compound HY-A and equimolar HBr in IPA, followed by slurry at RT for 3 days; and

(14) mixing compound HY-A and equimolar HBr in IPAc, followed by slurry at RT for 3 days.

In one aspect, the present application provides a pharmaceutical composition, the pharmaceutical composition comprises crystalline form of the present application and/or acid addition salt of crystalline form of the present application, or a pharmaceutically acceptable salt, prodrug, or metabolite thereof, or a solvate or hydrate of any of the foregoing, and a pharmaceutically acceptable carrier.

In one aspect, the present application provides a kit, the kit comprises crystalline form of the present application, acid addition salt of crystalline form of the present application and/or pharmaceutical composition of the present application, or a pharmaceutically acceptable salt, prodrug, or metabolite thereof, or a solvate or hydrate of any of the foregoing, and a pharmaceutically acceptable carrier.

In one aspect, the present application provides a method for inhibiting casein kinase (CK) activity, said method comprising administering to a subject in need thereof an effective amount of crystalline form of the present application, acid addition salt of crystalline form of the present application and/or pharmaceutical composition of the present application, or a pharmaceutically acceptable salt, prodrug, or metabolite thereof, or a solvate or hydrate of any of the foregoing and/or kit of the present application. For example, the casein kinase (CK) may be selected from the group consisting of casein kinase I alpha (CK1α) , casein kinase I delta (CK1δ) and casein kinase I epsilon (CK1ε) . For example, the method may be selected from the group consisting of an in vitro method, an ex vivo method, and an in vivo method.

In another embodiment, the present application provides use crystalline form of the present application, acid addition salt of crystalline form of the present application and/or pharmaceutical composition of the present application, or a pharmaceutically acceptable salt, prodrug, or metabolite thereof, or a solvate or hydrate of any of the foregoing and/or kit of the present application in the preparation of a drug and/or a kit for use in inhibiting casein kinase (CK) activity. For example, the casein kinase (CK) may be selected from the group consisting of casein kinase I alpha (CK1α) , casein kinase I delta (CK1δ) and casein kinase I epsilon (CK1ε) . For example, the method may be selected from the group consisting of an in vitro method, an ex vivo method, and an in vivo method.

In another embodiment, the present application provides crystalline form of the present application, acid addition salt of crystalline form of the present application and/or pharmaceutical composition of the present application, or a pharmaceutically acceptable salt, prodrug, or metabolite thereof, or a solvate or hydrate of any of the foregoing and/or kit of the present application for use in inhibiting casein kinase (CK) activity. For example, the casein kinase (CK) may be selected from the group consisting of casein kinase I alpha (CK1α) , casein kinase I delta (CK1δ) and casein kinase I epsilon (CK1ε) . For example, the method may be selected from the group consisting of an in vitro method, an ex vivo method, and an in vivo method.

In another aspect, the present application provides a method for preventing and/or treating a disease or disorder, said method comprising administering to a subject in need thereof an effective amount of the compound of the present application, or a pharmaceutically acceptable salt, prodrug, or metabolite thereof, or a solvate or hydrate of any of the foregoing. For example, the disease or disorder may be selected from the group consisting of neurological disease and psychiatric disease. For example, the disease or disorder may be selected from the group consisting of mood disorder, sleep disorder, and circadian disorder. For example, the disease or disorder may be selected from the group consisting of depressive disorder and bipolar disorder.

In another embodiment, the present application provides use crystalline form of the present application, acid addition salt of crystalline form of the present application and/or pharmaceutical composition of the present application, or a pharmaceutically acceptable salt, prodrug, or metabolite thereof, or a solvate or hydrate of any of the foregoing and/or kit of the present application in the preparation of a drug and/or a kit for use in preventing and/or treating a disease or disorder. For example, the disease or disorder may be selected from the group consisting of neurological disease and psychiatric disease. For example, the disease or disorder may be selected from the group consisting of mood disorder, sleep disorder, and circadian disorder. For example, the disease or disorder may be selected from the group consisting of depressive disorder and bipolar disorder.

In another embodiment, the present application provides crystalline form of the present application, acid addition salt of crystalline form of the present application and/or pharmaceutical composition of the present application, or a pharmaceutically acceptable salt, prodrug, or metabolite thereof, or a solvate or hydrate of any of the foregoing and/or kit of the present application for use in preventing and/or treating a disease or disorder. For example, the disease or disorder may be selected from the group consisting of neurological disease and psychiatric disease. For example, the disease or disorder may be selected from the group consisting of mood disorder, sleep disorder, and circadian disorder. For example, the disease or disorder may be selected from the group consisting of depressive disorder and bipolar disorder.

In another embodiment, the present application provides compositions comprising a compound of the present application or a pharmaceutically acceptable salt, prodrug, or metabolite thereof, or a solvate or hydrate of any of the foregoing, and optionally a pharmaceutically acceptable carrier.

The compounds of the application may be administered orally. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, or buccal or sublingual administration may be employed by which the compound enters the blood stream directly from the mouth.

In some cases, the compounds of the present application may also be administered directly into the blood stream, into muscle, or into an internal organ. Suitable means for parenteral administration may include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular and subcutaneous. Suitable devices for parenteral administration may include needle (including microneedle) injectors, needle-free injectors and infusion techniques.

The compounds of the present application may also be administered topically to the skin or mucosa, that is, dermally or transdermally. In some cases, the compounds of the present application may also be administered intranasally or by inhalation. In some cases, the compounds of the present application may be administered rectally or vaginally. In another embodiment, the compounds of the present application may also be administered directly to the eye or ear.

The dosage regimen for the compounds and/or compositions containing the compounds is based on a variety of factors, including the type, age, weight, sex and medical condition of the patient; the severity of the condition; the route of administration; and the activity of the particular compound employed. Thus, the dosage regimen may vary widely. Dosage levels of the order from about 0.01 mg to about 100 mg per kilogram of body weight per day may be useful in the treatment of the above-indicated conditions.

Suitable subjects according to the present invention include mammalian subjects. Mammals according to the present invention may include, but are not limited to, canine, feline, bovine, caprine, equine, ovine, porcine, rodents, lagomorphs, primates, and the like, and encompass mammals in utero. In one embodiment, humans are suitable subjects. Human subjects may be of either gender and at any stage of development.

In another embodiment, the present application provides use of one or more compounds of the present application for the preparation of a medicament for the treatment of the conditions recited herein.

For the treatment of the conditions referred to above, the compounds of the present application may be administered as compound per se. Alternatively, pharmaceutically acceptable salts may be suitable for medical applications because of their greater aqueous solubility relative to the parent compound.

In another embodiment, the present application provides compositions. Such compositions may comprise a compound of the present application presented with a pharmaceutically acceptable carrier. The carrier may be a solid product, a liquid, or both, and may be formulated with the compound as a unit-dose composition, for example, a tablet, which can contain from 0.05%to 95%by weight of the active compounds. A compound of the present application may be coupled with suitable polymers as targetable drug carriers. Other pharmacologically active substances may also be present.

The compounds of the present invention may be administered by any suitable route, maybe in the form of a pharmaceutical composition adapted to such a route, and in a dose effective for the treatment intended. The active compounds and compositions, for example, may be administered orally, rectally, parenterally, or topically.

The compounds of the present application may be used, alone or in combination with other therapeutic agents, in the treatment of various conditions or disease states. The compound (s) of the present application and other therapeutic agent (s) may be administered simultaneously (either in the same dosage form or in separate dosage forms) or sequentially.

The administration of two or more compounds “in combination” may mean that the two compounds are administered closely enough in time that the presence of one alters the biological effects of the other. The two or more compounds may be administered simultaneously, concurrently or sequentially. Additionally, simultaneous administration may be carried out by mixing the compounds prior to administration or by administering the compounds at the same point in time but at different anatomic sites or using different routes of administration.

The phrases “concurrent administration, ” “co-administration, ” “simultaneous administration, ” and “administered simultaneously” may mean that the compounds are administered in combination.

Examples

The following examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc. ) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair (s) ; kb, kilobase (s) ; pl, picoliter (s) ; s or sec, second (s) ; min, minute (s) ; h or hr, hour (s) ; aa, amino acid (s) ; nt, nucleotide (s) ; i.m., intramuscular (ly) ; i.p., intraperitoneal (ly) ; s.c., subcutaneous (ly) ; and the like.

Example 1 Preparation of Compounds

Preparation of Compound

The synthetic scheme of HY-B

Step 1: methyl 8-bromo-6-chloroimidazo [1, 2-b] pyridazine-2-carboxylate

To a stirred mixture of 4-bromo-6-chloropyridazin-3-amine (5 g, 24 mmol, 1.0 equiv) and methyl 3-bromo-2-oxopropanoate (17 g, 96 mmol, 4.0 equiv) in DME (50 mL) at room temperature under air atmosphere. The reaction stirred for 16 h at 90 ℃. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (3: 1) to afford methyl 8-bromo-6-chloroimidazo [1, 2-b] pyridazine-2-carboxylate (5.7 g, 81%) as an off-white solid. ¹H NMR (300 MHz, CDCl₃) δ 8.50 (s, 1H) , 7.47 (s, 1H) , 4.00 (s, 3H) . LC/MS (ESI, m/z) : [ (M + 1) ] ⁺ = 290, 292.

Step 2: 6-chloro-8- [3- (4-fluorophenyl) -1-methylpyrazol-4-yl] imidazo [1, 2-b] pyridazine-2-carboxylate

To a stirred mixture of methyl 8-bromo-6-chloroimidazo [1, 2-b] pyridazine-2-carboxylate (1 g, 3.4 mmol, 1.0 equiv) and 3- (4-fluorophenyl) -1-methyl-4- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyrazole (1 g, 3.4 mmol, 1.0 equiv) in toluene (15 mL) were added Cs₂CO₃ (2.3 g, 6.9 mmol, 2.0 equiv) and Pd (dppf) Cl₂ CH₂Cl₂ (280 mg, 0.34 mmol, 0.1 equiv) at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 2 h at 100 ℃ under nitrogen atmosphere. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (1: 2) to afford methyl 6-chloro-8- [3- (4-fluorophenyl) -1-methylpyrazol-4-yl] imidazo [1, 2-b] pyridazine-2-carboxylate (390 mg, 29%) as a light brown solid. ¹H NMR (400 MHz, CDCl₃) δ 8.87 (s, 1H) , 8.43 (d, J = 0.8 Hz, 1H) , 7.51 (dd, J = 8.6, 5.6 Hz, 2H) , 7.16 (t, J = 8.6 Hz, 2H) , 6.79 (s, 1H) , 4.05 (s, 3H) , 4.01 (s, 3H) . LC/MS (ESI, m/z) : [ (M + 1) ] ⁺ = 386.

Step 3:

To a solution of methyl 6-chloro-8- [3- (4-fluorophenyl) -1-methylpyrazol-4-yl] imidazo [1, 2-b] pyridazine-2-carboxylate (390 mg, 1.0 mmol, 1.0 equiv) in EA (10 mL) was added Pd/C (97 mg, 10%) under nitrogen atmosphere. The mixture was hydrogenated at 50 ℃ for 16 h under hydrogen atmosphere using a hydrogen tyre. The resulting mixture was filtered and the filter cake was washed with MeOH (3 x 10 mL) . The filtrate was concentrated under reduced pressure. The residue was purified by Prep-HPLC with the following conditions (Column: X Select CSH Prep C18 OBD Column, , 19*250 mm, 5 um; Mobile Phase A: water (0.05%FA) , Mobile Phase B: ACN; Flow rate: 25 mL/min; Gradient: 30 B to 55 B in 7 min, 254/220 nm; RT₁: 6.47 min; Injection Volumn: 0.4 ml; Number Of Runs: 8) to afford methyl 8- [3- (4-fluorophenyl) -1-methylpyrazol-4-yl] imidazo [1, 2-b] pyridazine-2-carboxylate (29.5 mg, 8%) as an off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.84 (s, 1H) , 8.52 (s, 1H) , 8.12 (d, J = 4.9 Hz, 1H) , 7.52-7.47 (m, 2H) , 7.15-7.09 (m, 2H) , 6.77 (d, J = 5.0 Hz, 1H) , 4.04 (s, 3H) , 4.01 (s, 3H) . LC/MS (ESI, m/z) : [ (M + 1) ] ⁺ = 352.

Step 4:

To a stirred mixture of methyl 8- [3- (4-fluorophenyl) -1-methylpyrazol-4-yl] imidazo [1, 2-b] pyridazine-2-carboxylate (100 mg, 0.285 mmol, 1.00 equiv) in THF (10.00 mL) was added MeMgBr (0.60 mL, 1.800 mmol, 6.32 equiv) dropwise at 0 ℃ under air atmosphere. The reaction was reacted for 1 hour at room temperature. The reaction was monitored by LCMS, then quenched by NHCl₄ aq., concentrated under reduced pressure. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, ACN in 0.1%FA aq., 35%to 55%gradient in 15 min; detector, UV 254 nm. This resulted in 2- [8- [3- (4-fluorophenyl) -1-methylpyrazol-4-yl] imidazo [1, 2-b] pyridazin-2-yl] propan-2-ol (29.5 mg, 29.50%) as a light yellow solid. ¹H NMR (300 MHz, Chloroform-d) δ 8.80 (s, 1H) , 8.08 (s, 1H) , 7.89 (s, 1H) , 7.53 (dd, J = 8.6, 5.4 Hz, 2H) , 7.14 (t, J = 8.7 Hz, 2H) , 6.76 (s, 1H) , 4.08 (s, 3H) , 1.74 (s, 6H) . LC/MS (ESI, m/z) : [ (M + 1) ] ⁺ =352.2.

Assay of biological activities

The CK1δ kinase assay was performed with a buffer (40 μL, pH 7.5) containing 50 mM Tris, 10 mM MgCl₂, 1 mM dithiothreitol, 100 μg/mL BSA with 10 μM ATP, 2nM wild type CK1δ, and 42 μM peptide substrate PLSRTLpSVASLPGL (Flotow et al., 1990) in the presence of 1 μL of a CK1δinhibitor (e.g., a compound of the present application) or 4%DMSO (e.g., as control) . The reaction mixture was incubated for 85 min at 25 ℃; detection was carried out as described for the Kinase-Glo Assay (Promega) . Luminescent output was measured on the Perkin Elmer Envision plate reader (PerkinElmer, Waltham, MA) .

Bmal1-dLuc or Per2-dLuc U2OS cells were suspended in the culture medium (DMEM supplemented with 10%fetal bovine serum, 0.29 mg/mL L-glutamine, 100 units/mL penicillin, and 100 mg/mL streptomycin) and plated onto 96-well white solid-bottom plates at 200 μL (10,000 cells) per well. After 2 days, 100 μL of the explant medium (DMEM supplemented with 2%B27, 10 mM HEPES, 0.38 mg/mL sodium bicarbonate, 0.29 mg/mL L-glutamine, 100 units/mL penicillin, 100 mg/mL streptomycin, 0.1 mg/mL gentamicin, and 1 mM luciferin, pH 7.2) was dispensed to each well, followed by the application of 1 μL of a compound of the present application (dissolved in DMSO; final concentration was 0.7%in DMSO) . The plate was covered with an optically clear film and set to microplate reader (Infinite M200, Tecan) . The luminescence was recorded every 1 h for 3-4 days. The period parameter was obtained from the luminescence rhythm by curve fitting program CellulaRhythm or MultiCycle (Actimetrics) , both of which generated similar results.

The CK1δ inhibition results (EC50) of HY-B is 0.4 μM. The present disclosure provides HY-B as potent inhibitor of casein kinase.

Assay of Drug Transport

1. Preparation of Caco-2 Cells

1) 50 μL and 25 mL of cell culture medium were added to each well of the Transwell insert and reservoir, respectively. And then the HTS transwell plates were incubated at 37 ℃, 5%CO₂ for 1 hour before cell seeding.

2) Caco-2 cells were diluted to 6.86х10⁵ cells/mL with culture medium and 50 μL of cell suspension were dispensed into the filter well of the 96-well HTS Transwell plate. Cells were cultivated for 14-18 days in a cell culture incubator at 37 ℃, 5%CO₂, 95%relative humidity. Cell culture medium was replaced every other day, beginning no later than 24 hours after initial plating.

2. Preparation of Stock Solutions

10 mM stock solutions of test compounds were prepared in DMSO. The stock solutions of positive controls were prepared in DMSO at the concentration of 10 mM. Digoxin and propranolol were used as control compounds in this assay.

3. Assessment of Cell Monolayer Integrity

1) Medium was removed from the reservoir and each Transwell insert and replaced with prewarmed fresh culture medium.

2) Transepithelial electrical resistance (TEER) across the monolayer was measured using Millicell Epithelial Volt-Ohm measuring system (Millipore, USA) .

3) The Plate was returned to the incubator once the measurement was done.

The TEER value was calculated according to the following equation:

TEER measurement (ohms) *Area of membrane (cm²) = TEER value (ohm·cm²)

TEER value should be greater than 230 ohm·cm², which indicates the well-qualified Caco-2 monolayer.

4. Assay Procedures

1) The Caco-2 plate was removed from the incubator and washed twice with pre-warmed HBSS (10 mM HEPES, pH 7.4) , and then incubated at 37 ℃ for 30 minutes.

2) The stock solutions of control compounds and test compounds were diluted in DMSO to get 1 mM solutions and then diluted with HBSS (10 mM HEPES, pH 7.4) get 5 μM working solutions. The final concentration of DMSO in the incubation system was 0.5%.

3) To determine the rate of drug transport in the apical to basolateral direction. 125 μL of 5 μM working solution of control compound and test compounds were added to the Transwell insert (apical compartment) , and transfer 50 μL sample (D0 sample) immediately from the apical compartment to a new 96-well plate. Fill the wells in the receiver plate (basolateral compartment) with 235 μL of HBSS (10 mM HEPES, pH 7.4) .

4) To determine the rate of drug transport in the basolateral to apical direction. 285 μL of 5 μM working solution of control compound and test compounds were to the receiver plate wells (basolateral compartment) , and transfer 50 μL sample (D0 sample) immediately from the basolateral compartment to a new 96-well plate. Fill the wells in the Transwell insert (apical compartment) with 75 μL of HBSS (10 mM HEPES, pH 7.4) . The assay was performed in duplicate.

5) The plates were incubated at 37 ℃ for 2 hours.

6) At the end of the incubation, 50 μL samples from donor sides (apical compartment for Ap→Bl flux, and basolateral compartment for Bl→Ap) and receiver sides (basolateral compartment for Ap→Bl flux, and apical compartment for Bl→Ap) were transferred to wells of a new 96-well plate, followed by the addition of 4 volume of cold methanol containing appropriate internal standards (IS) . Samples were Vortexed for 5 minutes and then centrifuged at 3, 220 g for 40 minutes. An aliquot of 100 μL of the supernatant was mixed with an appropriate volume of ultra-pure water before LC-MS/MS analysis.

7) To determine the Lucifer Yellow leakage after 2 hour transport period, stock solution of Lucifer yellow was prepared in water and diluted with HBSS (10 mM HEPES, pH 7.4) to reach the final concentration of 100 μM. 100 μL of the Lucifer yellow solution was added to each Transwell insert (apical compartment) , followed by filling the wells in the receiver plate (basolateral compartment) with 300 μL of HBSS (10 mM HEPES, pH 7.4) . The plates were Incubated at 37 ℃ for 30 mins. 80 μL samples were removed directly from the apical and basolateral wells (using the basolateral access holes) and transferred to wells of new 96 wells plates. The Lucifer Yellow fluorescence (to monitor monolayer integrity) signal was measured in a fluorescence plate reader at 485 nM excitation and 530 nM emission.

5. Data Analysis

The apparent permeability coefficient (P_app) , in units of centimeter per second, can be calculated for Caco-2 drug transport assays using the following equation:
P_app = (V_A× [drug] _acceptor) / (Area×Time× [drug] _{initial, donor})

Where V_A is the volume (in mL) in the acceptor well, Area is the surface area of the membrane (0.143 cm² for Transwell-96 Well Permeable Supports) , and time is the total transport time in seconds.

The efflux ratio will be determined using the following equation:
Efflux Ratio=P_app (B-A) /P_app (A-B)

Where P_app (B-A) indicates the apparent permeability coefficient in basolateral to apical direction, and P_app (A-B) indicates the apparent permeability coefficient in apical to basolateral direction.

The recovery can be determined using the following equation:
Recovery%= (V_A× [drug] _acceptor+V_D× [drug] _donor) / (V_D× [drug] _{initial, donor})

Where V_A is the volume (in mL) in the acceptor well (0.235 mL for Ap→Bl flux, and 0.075 mL for Bl→Ap) , V_D is the volume (in mL) in the donor well (0.075 mL for Ap→Bl flux, and 0.235 mL for Bl→Ap)

The leakage of Lucifer Yellow, in unit of percentage (%) , can be calculated using the following equation:
%LY leakage = 100× [LY] _acceptor/ ( [LY] _donor+ [LY] _acceptor)

LY leakage of <1%is acceptable to indicate the well-qualified Caco-2 monolayer.

The P_app (B-A) , P_app (A-B) and Efflux ratio of HY-B is summarized below.

Assay of Intrinsic Clearance

1. The master solution was prepared according to below.

2. Three separated experiments were performed as follows. a) With NADPH: 10 μL of 20 mg/mL liver microsomes and 40 μL of 10 mM NADPH were added to the incubations. The final concentrations of microsomes and NADPH were 0.5 mg/mL and 1 mM, respectively. b) Without NADPH: 10 μL of 20 mg/mL liver microsomes and 40 μL of ultra-pure H₂O were added to the incubations. The final concentration of microsomes was 0.5 mg/mL. c) Heat-inactivated microsomes without NADPH: 10 μL of 20 mg/mL heat-inactivated liver microsomes and 40 μL of ultra-pure H₂O were added to the incubations. The final concentration of microsomes was 0.5 mg/mL.

3. The reaction was started with the addition of 4 μL of 200 μM test compound solution or control compound solution at the final concentration of 2 μM and carried out at 37 ℃.

4. Aliquots of 50 μL were taken from the reaction solution at 0, 15, 30, 45 and 60 min. The reaction was stopped by the addition of 4 volumes of cold acetonitrile with IS (100 nM alprazolam, 200 nM labetalol, 200 nM caffeine and 2 μM ketoprofen) . Samples were centrifuged at 3, 220 g for 40 minutes. Aliquot of 100 μL of the supernatant was mixed with 100 μL of ultra-pure H₂O and then used for LC-MS/MS analysis.

5. Data Analysis

All calculations were carried out using Microsoft Excel.

Peak areas were determined from extracted ion chromatograms. The slope value, k, was determined by linear regression of the natural logarithm of the remaining percentage of the parent drug vs. incubation time curve.

The in vitro half-life (in vitro t_1/2) was determined from the slope value:

in vitro t_1/2 = - (0.693/k)

Conversion of the in vitro t_1/2 (min) into the in vitro intrinsic clearance (in vitro CL_int, in μL/min/mg protein) was done using the following equation (mean of duplicate determinations) :

in vitro CL_int = (0.693 *volume of incubation (μl) ) / (in vitro t_1/2 *amount of proteins (mg) )

Conversion of the in vitro t_1/2 (min) into the scale-up unbound intrinsic clearance (Scale-up CL_int, in mL/min/kg) was done using the following equation (mean of duplicate determinations) :

The Scaling Factors for Intrinsic Clearance Prediction in Liver Microsomes are summarized below.

a. Iwatsubo et al, Davies and Morris, 1993, 10 (7) pp 1093-1095.

b. Barter et al, 2007, Curr Drug Metab, 8 (1) , pp 33-45; Iwatsubo et al, 1997, JPET, 283 pp 462-469.

The Papp (B-A) , Papp (A-B) and Efflux ratio are summarized below.

The HY-B has a desirable intrinsic clearance property.

The purpose of this application was to perform a salt screening for compound HY-B and a polymorph screening for a selected salt to identify a proper form for further development.

Using HY-B freebase Type A as material (201328-029-P1, 823129-01-A) , a salt screening was performed under 52 different conditions using 11 acids in 4 solvent systems (Two molar ratios were used for two acids) . A total of 12 crystalline salt hits were obtained during screening, which were characterized by X-ray powder diffraction (XRPD) , thermo gravimetric analysis (TGA) and differential scanning calorimetry (DSC) . The salt stoichiometry was determined using ultra performance liquid chromatography (UPLC) combined with ion chromatography (IC) or ¹H solution nuclear magnetic resonance (¹H NMR) . Approximate solubility was tested for the salt hits in water at RT. Based on the approximate solubility and characterization results (small TGA weight loss and neat endothermic signal on DSC curve) , sulfate Type A, maleate Type B and tosylate Type A were selected for re-preparation.

The three re-prepared salt forms and freebase Type A were used for salt evaluation, including hygroscopicity, kinetic solubility and solid stability. The results of salt evaluation were summarized in Table 1-1.

Hygroscopicity evaluated using dynamic vapor sorption (DVS) , indicated that the water uptake of freebase Type A, sulfate Type A, maleate Type B and tosylate Type A at 25 ℃/80%RH was 0.02%, 9.1%, 0.03%and 1.2%, respectively. After DVS test, no form change was observed for all samples.

For the kinetic solubility results in FeSSIF, similar solubility was observed among freebase Type A and 3 salt hits (The solubility at 24 hrs was ～0.12 mg/mL) . In SGF, FaSSIF and water, the solubility of all 3 salts was higher than freebase Type A, and sulfate Type A exhibited the highest solubility (The 24-h solubility was 5.0 mg/mL, 0.68 mg/mL and 3.1 mg/mL, respectively) . Maleate Type B and tosylate Type A showed similar solubility. Form change was observed for all forms after solubility test.

The results of solid stability evaluation showed no form change or obvious purity decrease for all samples after stored at 25 ℃/60%RH or 40 ℃/75%RH up to 12 days.

Based on the salt evaluation results, no obvious difference of stability was observed among the 3 salt hits and freebase Type A. The solubility of sulfate Type A was high, however, sulfate Type A was hygroscopic, which might indicate a risk in further development. The solubility of maleate Type B and tosylate Type A was higher than freebase Type A and the hygroscopicity was lower than sulfate Type A. Therefore, maleate Type B and tosylate Type A were used for PK study for comparison with freebase Type A. Based on the PK results, the bioavailability of both salt hits were higher than freebase Type A, and no obvious difference was observed between the two salt hits. Considering the safety class of coformers (Maleic acid was Class 1, p-toluenesulfonic acid and was Class 2. ) , and the hygroscopicity of maleate Type B was lower than tosylate Type A, maleate was selected as the proper salt for further polymorph screening study.

Using HY-B maleate material as material (N210824-073-01, 823129-27-A. ) Based on XRPD result, the sample was crystalline, and thus named as maleate Type C. 106 polymorph screening experiments were performed, using methods of anti-solvent addition, slow evaporation, slow cooling, slurry (RT and 50 ℃) , temperature cycling, solid vapor diffusion, solution vapor diffusion, polymer induced crystallization and grinding. Based on the XRPD characterization results, a total of four polymorphs were obtained, including hemi-maleate Type A (anhydrate) , mono-maleate Type B and C (anhydrates) , and sesquialter-maleate Type D (anhydrate or hydrate) . The characterization results were summarized in Table 1-2.

The inter-conversion relationship between mono-maleate anhydrates Type B and Type C was investigated via competitive slurry in different solvents at RT and 50 ℃. Maleate Type B was obtained in all experiments, which indicated that Type B was more thermodynamically stable than Type C from RT to 50 ℃ and thus maleate Type B was selected for polymorph evaluation. Since hygroscopicity, kinetic solubility and solid stability under conditions of 25 ℃/60%RH and 40 ℃/75%RH were performed during salt evaluation, solid stability at 80 ℃ for 24 hrs was performed in polymorph evaluation. The results showed no form change or obvious purity decrease for maleate Type B after stored at 80 ℃ for 24 hrs.

Based on all the evaluation results, mono-maleate Type B was selected as the proper solid form for further development.

Table 1-1 Summary of salt evaluation

Table 1-2 Summary of characterization for HY-B maleate polymorphs

*: Only maleate Type B and Type C were mono-maleate.

Example 2

Characterization of Starting Materials

Two batches of HY-B materials were received for this application (201328-029-P1, 823129-01-A; 201328-007, 823129-01-B) , which were used for salt screening and re-preparation/evaluation for salts. The materials were characterized by XRPD, TGA, DSC, ¹H NMR and UPLC purity. XRPD results in Figure 2-1 showed both materials were the same form, which was named as freebase Type A.

The TGA/DSC curves of material (823129-01-A) were displayed in Figure 2-2, which showed a weight loss of 0.3%up to 150 ℃ and an endotherm at 176.5 ℃ (onset) . The ¹H NMR spectrum was displayed in Figure 2-3. UPLC results (Table 2-1 and Figure 2-4) showed the purity of material (823129-01-A) was 98.32 area%.

The TGA/DSC curves of material (823129-01-B) were displayed in Figure 2-5, which showed a weight loss of 0.6%up to 150 ℃ and an endotherm at 176.2 ℃ (onset) . The ¹H NMR spectrum was displayed in Figure 2-6. UPLC results (Table 2-2 and Figure 2-7) showed the purity of material (823129-01-B) was 96.41 area%.

Approximate solubility of material (823129-01-A) was determined in 21 solvents/co-solvents at RT. Approximately 2 mg of sample was added into a 3-mL glass vial. Solvents in Table 2-3 were then added stepwise into the vials until the solids were dissolved visibly or a total volume of 2 mL was reached. Solubility results summarized in Table 2-3 were used to guide the solvent selection in salt screening experiment design.

Table 2-1 Impurity summary of material (823129-01-A)

Table 2-2 Impurity summary of material (823129-01-B)

Table 2-3 Approximate solubility of material (823129-01-A)

Example 3

Salt Screening

Summary of Salt Screening

Using HY-B freebase Type A (823129-01-A) as material, a total of 52 salt screening experiments were performed using 11 acids in 4 solvent systems (two charge ratios were used for 2 acids) . Around 20 mg of freebase Type A (823129-01-A) and corresponding acid were added in 0.5 mL solvent followed by slurry at RT for 3 days. The clear samples were transferred to stir at 5 ℃, which were transferred to stir at -20 ℃ if no solid was obtained. Then, anti-solvent addition was used for the clear solutions and evaporation at RT was used if still no solid was obtained. The resulting suspension was centrifuged to retrieve the solids for XRPD test and the results were summarized in Table 3-1. A total of 12 crystalline salt hits were obtained from salt screening, which were characterized by XRPD, TGA, DSC and NMR or UPLC/IC. The detailed results of salt hit characterization were listed in Example 9.2.

Table 3-1 Summary of salt screening

^a: The clear samples were transferred to stir at 5 ℃ for 3 hrs;
^b: The clear samples were transferred to stir at 5 ℃ for 3 hrs, and stir at -20 ℃ for 6 hrs;
^c: The clear samples were transferred to stir at 5 ℃ for 3 hrs, stir at -20 ℃ for 6 hrs followed
by addition of anti-solvent (MTBE) ;
^d: The clear samples were transferred to stir at 5 ℃ for 3 hrs, stir at -20 ℃ for 6 hrs followed
by addition of anti-solvent (MTBE) and evaporation at RT under vacuum;
^e: The gel samples were transferred to stir with temperature cycling (50 ℃ ～5 ℃, 0.01 ℃/min,
2 cycles) .

Approximate Solubility in Water

In order to preliminarily evaluate the solubility of each salt hit, approximate solubility test was performed for salt hits from salt screening in water. Approximately 2 mg of sample was added into a glass vial. Solvents were then added stepwise into the vials (100, 500 or 1000 μL for each step) until the solids were dissolved visibly or a total volume of 10 mL was reached. Solubility results summarized in Table 3-2.

Table 3-2 Summary of approximate solubility for sat hits

*: Precipitation was observed after the pH was adjusted to 2.26 using 0.1 M NaOH.
^#: Suspension was observed after magnetically slurry (750 rpm) overnight.

Re-preparation of Candidate Salts

Based on the approximate solubility in water and characterization results (small TGA weight loss, neat DSC endotherm) , safety class and usage frequency of salt coformers, sulfate Type A, maleate Type B and tosylate Type A were selected to be re-prepared.

All the salt forms were re-prepared via slurry on 350 mg scale (based on freebase) . The re-prepared samples were characterized by XRPD, TGA, DSC, ¹H NMR or UPLC/IC, which were summarized in Table 3-3. The re-prepared salt forms were used for salt evaluation.

Table 3-3 Summary of characterization for re-prepared salts

Sulfate

Sulfate Type A (823129-12-A) was re-prepared via adding 349.9 mg freebase Type A (823129-01-A) in 5 mL IPAc followed by addition of 261.5 μL 4 M H₂SO₄ (acid/FB=1.05) and slurry at RT for 27 hrs (1000 rpm) . The solid was obtained via centrifugation and vacuum drying at RT. The XRPD pattern was displayed in Figure 3-1. The TGA/DSC curves were displayed in Figure 3-2, which showed a weight loss of 7.7%up to 120 ℃ and an endotherm at 121.8 ℃ (peak) . The ¹H NMR result in Figure 3-3 showed no residual IPAc was detected. UPLC results (Table 3-4 and Figure 3-4) showed the purity was 99.56 area%. UPLC/IC results showed the molar ratio of acid/FB was 1.1.

Table 3-4 Impurity summary of re-prepared sulfate Type A (823129-12-A)

Maleate

Maleate Type B (823129-13-A) was obtained via adding 350.1 mg freebase Type A (823129-01-A) and 115.9 mg maleic acid in 5 mL IPAc at RT for 3 days (1000 rpm) . The solid was obtained via vacuum drying at RT after centrifugation. The XRPD pattern was displayed in Figure 3-5. The TGA/DSC curves were displayed in Figure 3-6, which showed a weight loss of 0.4%up to 120 ℃ and an endotherm at 134.6 ℃ (peak) . The ¹H NMR result in Figure 3-7 showed the molar ratio of acid/FB was 1.0 and no residual IPAc was detected. UPLC results (Table 3-5 and Figure 3-8) showed the purity was 99.51 area%.

Table 3-5 Impurity summary of re-prepared maleate Type B (823129-13-A)

Tosylate

Tosylate Type A (823129-14-A) was re-prepared via adding 350.0 mg freebase Type A (823129-01-A) and 189.6 mg p-toluenesulfonic acid in 5 mL IPAc followed by slurry at RT for 3 days (1000 rpm) . The solid was obtained via vacuum drying at RT after centrifugation. The XRPD pattern was displayed in Figure 3-9. The TGA/DSC curves were displayed in Figure 3-10, which showed a weight loss of 0.4%up to 120 ℃ and an endotherm at 150.0 ℃ (peak) . The ¹H NMR result in Figure 3-11 showed the molar ratio of acid/FB was 1.0 and the molar ratio of IPAc/API was 0.02 (0.4 wt%) . UPLC results (Table 3-6 and Figure 3-12) showed the purity was 98.91 area%.

Table 3-6 Impurity summary of re-prepared tosylate Type A (823129-14-A)

Example 4

Salt Evaluation

Three re-prepared salt samples (sulfate Type A, maleate Type B and tosylate Type A) were used for salt evaluation along with freebase Type A, including hygroscopicity, kinetic solubility and solid stability.

Hygroscopicity

DVS test was performed for freebase Type A and 3 salt hits to evaluate the hygroscopicity. For sulfate Type A, DVS test started from ambient humidity (～40 %RH) and it started from 0%RH for other samples. DVS isotherm plots were collected at 25 ℃ between 0%RH and 95%RH. XRPD characterization was performed for the samples after DVS test. The DVS evaluation results were summarized in Table 4-1. The DVS plots and XRPD results were shown from Figure 4-1 to Figure 4-8. Based on the results, the water uptake of freebase Type A, sulfate Type A, maleate Type B and tosylate Type A at 25 ℃/80%RH was 0.02%, 9.1%, 0.03%and 1.2%. No form change was observed for all samples after DVS test.

Table 4-1 Summary of DVS evaluation

Kinetic Solubility

Kinetic solubility was measured for freebase Type A and 3 re-prepared salt hits in water and three bio-relevant media.

The material was added into H₂O, SGF, FaSSIF or FeSSIF with solid loading of 5 mg/mL (10 mg/mL in SGF, and 12 mg/L for sulfate in water) followed by rolling at 37 ℃ at 25 rpm for 1, 2, 4 and 24 hrs. For each time point, centrifugation and filtration (0.45 μm PTFE filter) were performed. Solubility by UPLC and pH were tested for supernatants. Solids were tested by XRPD. The results were summarized in Table 4-2 and the solubility plots were displayed in Figure 4-9.

Based on the results, freebase Type A and 3 salt showed similar solubility in FeSSIF (～0.12 mg/mL at 24 hrs) . In SGF, FaSSIF and water, the solubility of all 3 salts was higher than that of freebase Type A, and sulfate Type A exhibited the highest solubility (The 24-h solubility was 5.0 mg/mL, 0.68 mg/mL and 3.1 mg/mL, respectively) . Maleate Type B and tosylate Type A showed similar solubility. Form change was observed for all forms after solubility test.

Table 4-2 Summary of kinetic solubility test

S: solubility (mg/mL) .
-: The sample was clear and XRPD test was not performed.
FC: Form change. ^a: Freebase Type A; ^b: New form I; ^c: Freebase Type A+peaks; ^d: New form
II; ^e: New form I+ freebase Type A.

Solid Stability

Freebase Type A and 3 re-prepared salt hits were stored under the conditions of 25 ℃/60%RH and 40 ℃/75%RH for 12 days for solid stability evaluation. The physical and chemical stability were evaluated by XRPD and UPLC purity, respectively. The results were summarized in Table 4-3 and the XRPD results were shown from Figure 4-10 to Figure 4-13. The results showed no form change or obvious purity decrease after stored under two conditions for 12 days.

Table 4-3 Summary of solid stability evaluation

Salt Selection

Based on the salt evaluation results, no obvious difference of stability was observed among the 3 salt hits and freebase Type A. The solubility of sulfate Type A was high, however, sulfate Type A was hygroscopic, which might indicate high risk in further development. The solubility of maleate Type B and tosylate Type A was higher than freebase Type A and the hygroscopicity was lower than sulfate Type A. Therefore, maleate Type B and tosylate Type A were used for PK study for comparison with freebase Type A. Based on the PK results, the bioavailability of both salt hits were higher than freebase Type A, and no obvious difference was observed between the two salt hits. Considering the safety class of coformers (Maleic acid was Class 1, p-toluenesulfonic acid and was Class 2. ) , and the hygroscopicity of maleate Type B was lower than tosylate Type A, maleate was selected as the lead salt for further polymorph screening study.

Example 5

Polymorph Screening for Maleate

Based on the PK results, mono-maleate was selected for polymorph screening. Using HY-B maleate Type C as material, a total of 106 polymorph screening experiments were performed, including anti-solvent addition, slow evaporation, slow cooling, slurry (RT and 50 ℃) , temperature cycling, solid vapor diffusion, solution vapor diffusion, polymer induced crystallization and grinding. Based on the XRPD results of polymorph screening, a total of four polymorphs were obtained. The characterization was summarized in Table 5-1, and XRPD overlay was displayed in Figure 5-1. The detailed procedures of polymorph screening experiments referred to appendix 9.6.

Table 5-1 Characterization summary of maleate polymorphs

*: Only maleate Type B and Type C were mono-maleate.

Maleate Type A

Maleate Type A (823129-03-A4) was obtained from salt screening. The preparation and characterization results referred to appendix 9.2.3. Due to the small TGA weight loss, maleate Type A was postulated to be an anhydrate.

Maleate Type B

Maleate Type B (823129-13-A) was obtained via mixing 350.1 mg freebase Type A (823129-01-A) and 115.9 mg maleic acid in 5 mL IPAc followed by slurry at RT for 3 days (1000 rpm) . The characterization results referred to Example. The VT-XRPD (variable-temperature XRPD) results (Figure 5-2) showed no form change was observed for maleate Type B after N₂ purge for 20 min, heated to 100 ℃ under N₂ purge (The peak shifts were postulated to be due to the swell of lattice) , cooled to 30 ℃ under N₂ purge or exposed to air. Due to the small TGA weight loss and VT-XRPD results, maleate Type B was postulated to be an anhydrate.

Maleate Type C

Maleate Type C (N210824-073-01, 23129-27-A) was tested. The XRPD pattern was shown in Figure 5-3. The TGA/DSC curves were displayed in Figure 5-4, which showed a weight loss of 0.7%up to 120 ℃, and an endotherm at 136.5 ℃ (peak) . The ¹H NMR result in Figure 5-5 showed the molar ratio of acid/FB was 1.0, the molar ratio of residual IPAc/API was 0.05 (1.1 wt%) . UPLC results (Table 5-2 and Figure 5-6) showed the purity was 98.37 area%. Due to the small TGA weight loss, maleate Type C was postulated to be an anhydrate.

Table 5-2 Impurity summary of maleate Type C (823129-27-A)

Maleate Type D

Maleate Type D (823129-35-A9) was obtained via solution vapor diffusion in CH₂Br₂/n-Hexane at RT for 4 days using material (823129-27-A) . The XRPD pattern was displayed in Figure 5-7. The TGA/DSC curves were displayed in Figure 5-8, which showed a weight loss of 5.7%up to 120 ℃, and an endotherm at 127.7 ℃ (peak) . The ¹H NMR result in Figure 5-5 showed the molar ratio of acid/FB was 1.5 and no residual CH₂Br₂ or n-Hexane was detected. Therefore, maleate Type D was postulated to be an anhydrate or hydrate. Due to the limited amount, no further study was performed.

Example 6

Inter-conversion Relationship Study

Two anhydrates of mono-maleate (maleate Type B/C) were observed in this application. In order to investigate the inter-conversion relationship between the anhydrates, competitive slurry was performed at RT and 50 ℃.

Mono-maleate Type C (823129-27-A) was added into IPAc, Anisole and MEK. After slurry at RT and 50 ℃ for 2 hrs (1000 rpm) for filtration. Around 6 mg of maleate Type B and C were added into the saturated solution for slurry at RT and 50 ℃ for 3 days (1000 rpm) . And then around 0.5 mg hemi-maleate Type A was added for slurry at RT and 50 ℃ for 1 day (1000 rpm) . The solid was tested by XRPD.

The results were summarized in Table 6-1 and the XRPD results were shown from Figure 6-1 to Figure 6-4. Maleate Type B was obtained in all experiments, which indicated that Type A was more stable than Type C from RT to 50 ℃.

Table 6-1 Summary of competitive slurry between anhydrates

*: Yellow samples was observed after slurry for 3 days.
^#: Hemi-maleate Type A was added into the suspension samples after slurry for 3 days, and
then the samples were stirred for another 1 day.

N/A: Clear and yellow solution was obtained after slurry for 1 day. Yellow gel sample was obtained after vacuum drying at RT, and thus XRPD test was not performed.

Example 7

Polymorph Evaluation

Mono-maleate Type B was more stable anhydrous form from RT to 50 ℃ than Type C, which was selected for evaluation. During the salt screening, evaluation, including hygroscopicity, kinetic solubility, solid stability at 25 ℃/60%RH and 40 ℃/75%RH were performed for maleate Type B (Refer to Example) , and thus only the solid stability at 80 ℃ for 24 hrs was performed for maleate Type B in this Example. The results were summarized in Table 7-1 and the XRPD results were displayed in Figure 7-1. The results showed no form change or obvious purity change was observed for maleate Type B after stored at 80 ℃ for 24 hrs.

Table 7-1 Summary of solid stability evaluation

Example 8

Using HY-B freebase Type A as material, a salt screening was performed under 52 different conditions using 11 acids in 4 solvent systems (Two molar ratios were used for two acids) . A total of 12 crystalline salt hits were obtained during screening. Based on the approximate solubility and characterization results, sulfate Type A, maleate Type B and tosylate Type A were selected for re-preparation. The three re-prepared salt forms and freebase Type A were used for salt evaluation, including hygroscopicity, kinetic solubility and solid stability.

Based on the salt evaluation results, the solubility of sulfate Type A was high, however, sulfate Type A was hygroscopic, which might indicate high risk in further development. The solubility of maleate Type B and tosylate Type A was higher than freebase Type A and the hygroscopicity was lower than sulfate Type A. The PK results showed the bioavailability of both salt hits were higher than freebase Type A, and no obvious difference was observed between the two salt hits. Considering the safety class of conformer and solid state properties of three salts, maleate was selected as the lead salt for further polymorph screening study.

Using HY-B maleate Type C, 106 polymorph screening experiments were performed. Based on the XRPD characterization results, a total of four polymorphs were obtained, including hemi-maleate Type A (anhydrate) , mono-maleate Type B and C (anhydrates) , and sesquialter-maleate Type D (anhydrate or hydrate) . The inter-conversion relationship between mono-anhydrate anhydrates Type B and Type C was investigated via competitive slurry in different solvents at RT and 50 ℃. Maleate Type B was obtained in all experiments, which indicated that Type B was more thermodynamically stable than Type C from RT to 50 ℃. The stability results showed no form change or obvious purity decrease for maleate Type B after stored at 80 ℃ for 24 hrs.

Example 9

Abbreviation for Solvent Used

The solvent abbreviations are listed in Table 9-1.

Table 9-1 Solvent abbreviation list

Characterization of Salt Hits

A total of 12 salt hits were obtained from salt screening, which were characterized by XRPD, TGA and DSC. The salt stoichiometry was determined using UPLC/IC or ¹H NMR. All the characterization results were summarized in Table 9-2.

Table 9-2 Characterization summary of salt hits

HCl Salt

HCl salt Type A/B (823129-03-B1/D1) were obtained via mixing freebase Type A (823129-01-A) and equimolar HCl in IPAc and ACN/H₂O (19: 1, v/v) , respectively, followed by slurry at RT for 3 days. HCl salt Type C (823129-03-A2) was obtained via mixing freebase Type A (823129-01-A) and HCl (molar ratio of 1: 2, FB/acid) in IPA for slurry at RT for 3 days followed by evaporation at RT. The XRPD patterns were displayed in Figure 9-1.

The TGA/DSC curves of HCl salt Type A (823129-03-B1) were displayed in Figure 9-2, which showed a weight loss of 9.7%up to 120 ℃ and an endotherm at 115.3 ℃ (peak) . ¹H NMR result (Figure 9-3) showed no residual IPAc was detected. UPLC/IC results showed the molar ratio was 1.0 (acid/FB) .

The TGA/DSC curves of HCl salt Type B (823129-03-D1) were displayed in Figure 9-4, which showed a weight loss of 16.7%up to 120 ℃ and an endotherm at 99.4 ℃ (peak) . ¹H NMR result (Figure 9-5) showed the molar ratio of ACN/API was 0.3 (2.9 wt%) . UPLC/IC results showed the molar ratio was 1.0 (acid/FB) .

The TGA/DSC curves of HCl salt Type C (823129-03-A2) were displayed in Figure 9-6, which showed a weight loss of 14.2%up to 120 ℃ and an endotherm at 112.3 ℃ (peak) . ¹H NMR result (Figure 9-7) showed the molar ratio of IPA/API was 0.04 (0.9 wt%) . UPLC/IC results showed the molar ratio was 1.2 (acid/FB) .

Sulfate

Sulfate Type A (823129-03-B3) were obtained via mixing freebase Type A (823129-01-A) equimolar H₂SO₄ in IPAc followed by slurry at RT for 3 days. The XRPD pattern was displayed in Figure 9-8. The TGA/DSC curves of sulfate Type A (823129-03-B3) were displayed in Figure 9-9, which showed a weight loss of 8.7%up to 120 ℃, and an endotherm at 121.9 ℃ (peak) . ¹H NMR result (Figure 9-10) showed no residual IPAc was detected. UPLC/IC results showed the molar ratio was 1.1 (acid/FB) .

Maleate

Maleate Type A/B (823129-03-A4/B4) was obtained via mixing freebase Type A (823129-01-A) and equimolar maleic acid in IPA and IPAc, respectively, followed by slurry at RT for 3 days. The XRPD patterns were displayed in Figure 9-11.

The TGA/DSC curves of maleate Type A (823129-03-A4) were displayed in Figure 9-12, which showed a weight loss of 1.3%up to 120 ℃ and an endotherm at 135.3 ℃ (peak) . The ¹H NMR result in Figure 9-13 showed the molar ratio of acid/FB was 0.5 and no residual IPA was detected.

The TGA/DSC curves of maleate Type B (823129-03-B4) were displayed in Figure 9-14, which showed a weight loss of 0.9%up to 120 ℃ and an endotherm at 134.4 ℃ (peak) . The ¹H NMR result in Figure 9-15 showed the molar ratio of acid/FB was 1.1 and no residual IPA was detected.

Tosylate

Tosylate Type A (823129-03-B9) was obtained via mixing freebase Type A (823129-01-A) and equimolar p-toluenesulfonic acid in IPAc followed by slurry at RT for 3 days. The XRPD pattern was displayed in Figure 9-16. The TGA/DSC curves of tosylate Type A (823129-03-B9) were displayed in Figure 9-17, which showed a weight loss of 1.8%up to 120 ℃ and an endotherm at 151.8 ℃ (peak) . The ¹H NMR result in Figure 9-18 showed the molar ratio of acid/FB was 1.0 and no residual IPAc was detected.

Mesylate

Mesylate Type A (823129-03-A11) was obtained via mixing freebase Type A (823129-01-A) and methanesulfonic acid (molar ratio of 1: 2, FB/acid) in IPA followed by slurry at RT for 3 days. The XRPD pattern was displayed in Figure 9-19. The TGA/DSC curves of mesylate Type A (823129-03-A11) were displayed in Figure 9-20, which showed a weight loss of 6.8%up to 120 ℃ and two endotherms at 91.8 and 122.4 ℃ (peak) . The ¹H NMR result in Figure 9-21 showed the molar ratio of acid/FB was 1.0 and no residual IPA was detected.

Oxalate

Oxalate Type A/B (823129-03-B12/C12) were obtained via mixing freebase Type A (823129-01-A) and equimolar oxalic acid in IPAc and MIBK, respectively, followed by slurry at RT for 3 days. The XRPD patterns were displayed in Figure 9-22.

The TGA/DSC curves of oxalate Type A (823129-03-B12) were displayed in Figure 9-23, which showed a weight loss of 4.1%up to 120 ℃ and an endotherm at 113.8 ℃ (peak) . The ¹H NMR result in Figure 9-24 showed no residual IPAc was detected. UPLC/IC results showed the molar ratio was 1.1 (acid/FB) .

The TGA/DSC curves of oxalate Type B (823129-03-C12) were displayed in Figure 9-25, which showed a weight loss of 6.8%up to 120 ℃ and two endotherms at 102.1 and 118.4 ℃ (peak) . The ¹H NMR result in Figure 9-26 showed no residual MIBK was detected. UPLC/IC results showed the molar ratio was 1.1 (acid/FB) .

HBr Salt

HBr salt Type A/B (823129-03-A13/B13) were obtained via mixing freebase Type A (823129-01-A) and equimolar HBr in IPA and IPAc, respectively, followed by slurry at RT for 3 days. The XRPD patterns were displayed in Figure 9-27.

The TGA/DSC curves of HBr salt Type A (823129-03-A13) were displayed in Figure 9-28, which showed a weight loss of 0.6%up to 120 ℃ and an endotherm at 155.7 ℃ (peak) . The ¹H NMR result in Figure 9-29 showed no residual IPA was detected. UPLC/IC results showed the molar ratio was 1.0 (acid/FB) .

The TGA/DSC curves of HBr salt Type B (823129-03-B13) were displayed in Figure 9-30, which showed a weight loss of 8.3%up to 120 ℃ and an endotherm at 125.5 ℃ (peak) . The ¹H NMR result in Figure 9-31 showed no residual IPAc was detected. UPLC/IC results showed the molar ratio was 1.0 (acid/FB) .

Preparation of Bio-relevant Media

Simulated Gastric Fluid (SGF)

200 mg of NaCl and 100 mg of Triton X-100 were weighed into a 100-mL volumetric flask. Purified water was added to dissolve the solid. 136 μL HCl (12M) was added after the solid was dissolved completely, and pH was then adjusted to 1.8 using 1 M HCl or 1 M NaOH. Purified water was added to the volume.

Fasted-State Simulated Intestinal Fluid (FaSSIF)

0.17 g of anhydrous NaH₂PO₄, 0.021 g of NaOH and 0.31 g of NaCl were weighed into a 50-mL volumetric flask. ～48 mL purified water was added to dissolve the solid. The pH was adjusted to 6.5 using 1 M HCl or 1 M NaOH. Purified water was then added to the volume. 22 mg of SIF powder was added into a 10-mL volumetric flask followed by addition of the prepared buffer to the volume.

Fed-State Simulated Intestinal Fluid (FeSSIF)

0.41 mL of acetic acid, 202 mg of NaOH and 594 mg of NaCl were weighed into a 50-mL volumetric flask. ～48 mL purified water was added to dissolve the solid. The pH was adjusted to 5.0 using 1 M HCl or 1 M NaOH. Purified water was then added to the volume. 0.56 g of SIF powder was added into a 10-mL volumetric flask followed by addition of the prepared buffer to the volume.

XRPD Result of the Solids from Kinetic Solubility Measurement

The XRPD results of the residual solid from kinetic solubility measurement were displayed from Figure 9-32 to Figure 9-47.

Re-preparation of Candidate Salt

Maleate

Maleate Type B (823129-24-A) was re-prepared via adding 370.0 mg freebase Type A (823129-01-A) and 122.5 mg maleic acid in 6 mL IPAc followed by addition of seed (823129-13-A) and slurry at RT for 27 hrs (1000 rpm) . The solid was obtained via centrifugation and vacuum drying overnight at RT. The XRPD pattern was displayed in Figure 9-48. The TGA/DSC curves were displayed in Figure 9-49, which showed a weight loss of 0.4%up to 120 ℃ and an endotherm at 133.6 ℃ (peak) . The ¹H NMR result in Figure 9-50 showed the molar ratio of acid/FB was 1.0 and no residual IPAc was observed. UPLC results (Table 9-3 and Figure 9-51) showed the purity was 99.46 area%.

Table 9-3 Impurity summary of re-prepared maleate Type B (823129-24-A)

Tosylate

Tosylate Type A (823129-14-A) was re-prepared via adding 370.1 mg freebase Type A (823129-01-A) and 200.6 mg p-toluenesulfonic acid in 6 mL IPAc followed by addition of seed (823129-14-A) and slurry at RT for 27 hrs (1000 rpm) . The solid was obtained via centrifugation and vacuum drying overnight at RT. The XRPD pattern was displayed in Figure 9-52. The TGA/DSC curves were displayed in Figure 9-53, which showed a weight loss of 2.5%up to 120 ℃ and an endotherm at 150.6 ℃ (peak) . The ¹H NMR result in Figure 9-54 showed the molar ratio of acid/FB was 1.0 and the molar ratio of IPAc/API was 0.02 (0.4 wt%) . UPLC results (Table 9-4 and Figure 9-55) showed the purity was 98.87 area%.

Table 9-4 Impurity summary of re-prepared tosylate Type A (823129-25-A)

Polymorph Screening

Based on the approximate solubility of maleate Type C (823129-27-A) in Table 9-5, a total of 106 polymorph screening experiments were performed for maleate using different crystallization or solid transformation methods. The methods utilized and results were summarized in Table 9-6.

Table 9-5 Approximate solubility of maleate Type C (823129-27-A)

Table 9-6 Summary of polymorph screening experiments

Anti-solvent Addition

A total of 20 anti-solvent addition experiments were carried out. About 20 mg of maleate Type C (823129-27-A) was dissolved in 0.2～2.0 mL solvent to obtain a clear solution and the solution was magnetically stirred (～1000 rpm) followed by addition of anti-solvent until precipitant appeared or the total amount of anti-solvent reached 10.0 mL. The samples without precipitate were transferred to slurry at 5 ℃. After slurry, clear solutions were further transferred to slurry at -20 ℃ and then transferred to RT for evaporation if still clear. The solids were isolated for XRPD analysis. Results in Table 9-7 showed that maleate Type A/B/C/A+B/A+freebase Type/freebase Type A/gel were obtained.

Table 9-7 Summary of anti-solvent addition experiments

*: The solid was obtained via slurry at 5 ℃;
^#: The solid was obtained via evaporation at RT.

Slow Evaporation

Slow evaporation experiments were performed under 10 conditions. Briefly, ～20 mg of maleate Type C (823129-27-A) was dissolved in 1.0～3.0 mL of solvent in a 3-mL glass vial. The samples were filtered using 0.45 μm PTFE filter and the resulting solutions were subjected to slow evaporation at RT with vials sealed byand poked with 4 pinholes. The solids were isolated for XRPD analysis and the results summarized in Table 9-8 indicated that maleate Type A/B/C/A+freebase Type A/B+peak/gel were was obtained.

Table 9-8 Summary of slow evaporation experiments

Slow Cooling

Slow cooling experiments were conducted in 10 solvent systems. About 20 mg of maleateType C (823129-27-A) was suspended in 1.0～2.0 mL of solvent in a 3-mL glass vial at RT. The suspension was then heated to 50 ℃, equilibrated for about 30 min and filtered to a new vial. Filtrates were slowly cooled down to 5 ℃ at a rate of 0.1 ℃/min. The obtained solids were kept isothermal at 5 ℃. If no solid was obtained, the sample was transferred to -20 ℃. If still no solid was obtained, the sample was transferred to RT for evaporation. The solids were tested by XRPD. Results summarized in Table 9-9 indicated that maleate Type B/C/B+C/B+D were obtained.

Table 9-9 Summary of slow cooling experiments

*: The solid was obtained via evaporation at RT;
^#: The solid was obtained at -20 ℃.

Slurry at RT

About 20 mg of maleate Type C (823129-27-A) was suspended in 0.5 mL of solvent in an HPLC glass vial. After the suspension was stirred magnetically (1000 rpm) for 5 days at RT, the remaining solids were centrifuged for XRPD analysis. Results summarized in Table 9-10 indicated that maleate Type A/B/C/A+C/A+freebase Type A were generated.

Table 9-10 Summary of slurry conversion experiments at RT

*: Clear solution was obtained at RT, which was transferred to slurry at 5 ℃ for 16 hrs and
solid was obtained.

Slurry at 50 ℃

About 20 mg of maleate Type C (823129-27-A) was suspended in 0.5 mL of solvent in an HPLC glass vial. After the suspension was stirred (1000 rpm) for about 5 days at 50 ℃, the remaining solids were centrifuged for XRPD analysis. Results summarized in Table 9-11 indicated that maleate Type A/B/B/freebase Type A were generated.

Table 9-11 Summary of slurry conversion experiments at 50 ℃

*: Clear solution was obtained after slurry at 50 ℃ for 6 days, which was transferred to RT for
evaporqation and solid was obtained.

Temperature Cycling

About 20 mg of maleate Type C (823129-27-A) was suspended in 0.5 mL of solvent in an HPLC glass vial. After slurry (1000 rpm) with temperature cycling (50 ℃～5 ℃, 0.1 ℃/min, 2 cycles) , the remaining solids were centrifuged for XRPD analysis. Results summarized in Table 9-12 indicated that maleate Type B/C/B+freebase Type A were obtained.

Table 9-12 Summary of temperature cycling experiments

Solid Vapor Diffusion

Solid vapor diffusion experiments were conducted using 6 different solvents. Approximately 20 mg of maleate Type C (823129-27-A) was weighed into a 3-mL vial, which was placed into a 20-mL vial with 4 mL of volatile solvent. The 20-mL vial was sealed with a cap and kept at RT for 6 days allowing solvent vapor to interact with sample. The solids were tested by XRPD and the results summarized in Table 9-13 showed that maleate Type A/B/C were obtained.

Table 9-13 Summary of solid vapor diffusion experiments

Solution Vapor Diffusion

13 solution vapor diffusion experiments were conducted. Approximate 20 mg of meleate Type C (823129-27-A) was dissolved in 0.2～1.6 mL of appropriate solvent to obtain a clear solution in a 3-mL vial. This solution was then placed into a 20-mL vial with 3 mL of volatile solvent. The 20-mL vial was sealed with a cap and kept at RT allowing sufficient time for organic vapor to interact with the solution. If clear solution was obtained, the sample was transferred to store at 5 ℃. If still no solid was obtained, the samples was transferred to RT for evaporation. The precipitants were isolated for XRPD analysis. The results summarized in Table 9-14 showed that maleate Type A/B/C/D/freebase Type A/gel were obtained.

Table 9-14 Summary of solution vapor diffusion experiments

*: The solid was obtained via evaporation at RT.
^#: The solid was obtained at 5 ℃.

Polymer Induced Crystallization

Polymer induced crystallization experiments were performed under 10 conditions. Briefly, ～20 mg of maleate Type C (823129-27-A) was dissolved in 1.0～2.0 mL of solvent in a 3-mL glass vial. The resulting solutions were filtered using 0.45 μm PTFE filter followed by addition of ～2 mg polymer. The vials were sealed byand poked with 4 pinholes for slow evaporation at RT. The solids were isolated for XRPD analysis and the results summarized in Table 9-15 indicated that maleate Type A/B/C/A+B/B+peak/freebase Type A were obtained.

Table 9-15 Summary of polymer induced crystallization experiments

Polymer mixture A: polyvinyl pyrrolidone (PVP) , polyvinyl alcohol (PVA) , polyvinylchloride (PVC) , polyvinyl acetate (PVAC) , hypromellose (HPMC) , methyl cellulose (MC) (mass ratio of 1: 1: 1: 1: 1: 1) .

Polymer mixture B: polycaprolactone (PCL) , polyethylene glycol (PEG) , polymethyl methacrylate (PMMA) sodium alginate (SA) , and hydroxyethyl cellulose (HEC) (mass ratio of 1: 1: 1: 1: 1) .

Grinding

Grinding experiments were performed with or without solvent addition. Approximate 20 mg of maleate Type C (823129-27-A) was weighed into the mortar. 10 μL solvent was added into the mortar. The solids were ground for 3～5 min. The solids were isolated for XRPD analysis. Results summarized in Table 9-16 showed that only maleate Type C was obtained.

Table 9-16 Summary of grinding experiments

Instruments and Methods

XRPD

For XRPD analysis, PANalytical X'Pert3 X-ray powder diffract meters were used. The XRPD parameters used are listed in Table 9-17.

Table 9-17 Parameters for XRPD test

TGA and DSC

TGA data were collected using a TA Q5000/Discovery 5500 TGA from TA Instruments. DSC was performed using a TA Discovery 2500 DSC from TA Instruments. Detailed parameters used are listed in Table 9-18.

Table 9-18 Parameters for TGA and DSC test

DVS

DVS was measured via a SMS (Surface Measurement Systems) DVS Intrinsic. The relative humidity at 25 ℃ were calibrated against deliquescence point of LiCl, Mg (NO₃) ₂ and KCl. Parameters for DVS test are listed in Table 9-19.

Table 9-19 Parameters for DVS test

Solution NMR

Solution NMR was collected on Bruker 400M NMR Spectrometer using DMSO-d₆.

UPLC/IC

Waters H-Class UPLC were utilized for purity, solubility and stability test, and detailed chromatographic conditions are listed in Table 9-20 and Table 9-21. IC parameters were listed in Table 9-22.

Table 9-20 UPLC methods for solubility test

Table 9-21 UPLC methods for purity test

Table 9-22 IC parameters for stoichiometric test

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

Crystalline form of compound HY-B, 2- (8- (3- (4-fluorophenyl) -1-methyl-1H-pyrazol-4-yl) imidazo [1, 2-b] pyridazin-2-yl) propan-2-ol, wherein said crystalline form exhibits an X-ray powder diffraction pattern having one or more characteristic peaks expressed in degrees 2-theta selected from the group consisting of:

(1) 18.8±0.3 degrees, 19.3±0.3 degrees, 21.8±0.3 degrees;

(2) 8.2±0.3 degrees, 18.3±0.3 degrees, 23.4±0.3 degrees;

(3) 11.3±0.3 degrees, 18.5±0.3 degrees, 26.1±0.3 degrees;

(4) 12.6±0.3 degrees, 18.3±0.3 degrees, 18.9±0.3 degrees;

(5) 10.5±0.3 degrees, 18.4±0.3 degrees, 28.8±0.3 degrees;

(6) 9.5±0.3 degrees, 10.8±0.3 degrees, 18.3±0.3 degrees;

(7) 9.2±0.3 degrees, 18.4±0.3 degrees, 24.7±0.3 degrees;

(8) 13.3±0.3 degrees, 18.0±0.3 degrees, 20.1±0.3 degrees;

(9) 6.5±0.3 degrees, 9.7±0.3 degrees, 19.2±0.3 degrees;

(10) 9.5±0.3 degrees, 19.1±0.3 degrees, 24.5±0.3 degrees;

(11) 10.4±0.3 degrees, 11.9±0.3 degrees, 16.9±0.3 degrees;

(12) 14.5±0.3 degrees, 21.2±0.3 degrees, 23.0±0.3 degrees;

(13) 8.0±0.3 degrees, 10.3±0.3 degrees, 18.9±0.3 degrees; and

(14) 11.9±0.3 degrees, 18.3±0.3 degrees, 20.3±0.3 degrees.
The crystalline form of claim 1, wherein said crystalline form further exhibits an X-ray powder diffraction pattern having one or more characteristic peaks expressed in degrees 2-theta selected from the group consisting of:

(1) 10.1±0.3 degrees, 12.5±0.3 degrees, 24.0±0.3 degrees;

(2) 12.0±0.3 degrees, 15.4±0.3 degrees, 16.5±0.3 degrees;

(3) 8.5±0.3 degrees, 8.9±0.3 degrees, 25.6±0.3 degrees;

(4) 8.2±0.3 degrees, 17.0±0.3 degrees, 21.8±0.3 degrees;

(5) 20.3±0.3 degrees, 24.8±0.3 degrees, 27.4±0.3 degrees;

(6) 14.3±0.3 degrees, 20.7±0.3 degrees, 25.2±0.3 degrees;

(7) 10.3±0.3 degrees, 18.0±0.3 degrees, 24.1±0.3 degrees;

(8) 13.8±0.3 degrees, 20.4±0.3 degrees, 25.3±0.3 degrees;

(9) 18.6±0.3 degrees, 19.5±0.3 degrees, 23.5±0.3 degrees;

(10) 17.2±0.3 degrees, 18.4±0.3 degrees, 24.9±0.3 degrees;

(11) 15.2±0.3 degrees, 19.5±0.3 degrees, 20.7±0.3 degrees;

(12) 9.8±0.3 degrees, 17.5±0.3 degrees, 20.4±0.3 degrees;

(13) 14.0±0.3 degrees, 16.1±0.3 degrees, 24.3±0.3 degrees; and

(14) 24.7±0.3 degrees, 27.3±0.3 degrees, 28.6±0.3 degrees.
The crystalline form of any one of claims 1-2, wherein said crystalline form exhibits an X-ray powder diffraction pattern having one or more characteristic peaks expressed in degrees 2-theta selected from the group consisting of:

(1) 10.7±0.3 degrees, 20.3±0.3 degrees, 25.2±0.3 degrees;

(2) 9.5±0.3 degrees, 16.8±0.3 degrees, 21.7±0.3 degrees;

(3) 20.8±0.3 degrees, 22.7±0.3 degrees, 23.7±0.3 degrees;

(4) 9.4±0.3 degrees, 12.0±0.3 degrees, 25.3±0.3 degrees;

(5) 11.9±0.3 degrees, 12.9±0.3 degrees, 28.2±0.3 degrees;

(6) 10.1±0.3 degrees, 17.3±0.3 degrees, 23.9±0.3 degrees;

(7) 14.3±0.3 degrees, 19.0±0.3 degrees, 22.1±0.3 degrees;

(8) 19.7±0.3 degrees, 23.0±0.3 degrees, 24.0±0.3 degrees;

(9) 11.2±0.3 degrees, 25.0±0.3 degrees, 27.7±0.3 degrees;

(10) 21.4±0.3 degrees, 23.7±0.3 degrees, 25.6±0.3 degrees;

(11) 7.6±0.3 degrees, 24.7±0.3 degrees, 26.5±0.3 degrees;

(12) 10.1±0.3 degrees, 13.7±0.3 degrees, 22.6±0.3 degrees;

(13) 15.4±0.3 degrees, 18.5±0.3 degrees, 22.9±0.3 degrees; and

(14) 14.6±0.3 degrees, 20.8±0.3 degrees, 23.3±0.3 degrees.
The crystalline form of any one of claims 1-3, wherein said crystalline form exhibits an X-ray powder diffraction pattern having one or more characteristic peaks expressed in degrees 2-theta selected from the group consisting of:

(1) 18.8±0.3 degrees, 19.3±0.3 degrees, 21.8±0.3 degrees, 10.1±0.3 degrees, 12.5±0.3 degrees, 24.0±0.3 degrees, 10.7±0.3 degrees, 20.3±0.3 degrees, 25.2±0.3 degrees;

(2) 8.2±0.3 degrees, 18.3±0.3 degrees, 23.4±0.3 degrees, 12.0±0.3 degrees, 15.4±0.3 degrees, 16.5±0.3 degrees, 9.5±0.3 degrees, 16.8±0.3 degrees, 21.7±0.3 degrees;

(3) 11.3±0.3 degrees, 18.5±0.3 degrees, 26.1±0.3 degrees, 8.5±0.3 degrees, 8.9±0.3 degrees, 25.6±0.3 degrees, 20.8±0.3 degrees, 22.7±0.3 degrees, 23.7±0.3 degrees;

(4) 12.6±0.3 degrees, 18.3±0.3 degrees, 18.9±0.3 degrees, 8.2±0.3 degrees, 17.0±0.3 degrees, 21.8±0.3 degrees, 9.4±0.3 degrees, 12.0±0.3 degrees, 25.3±0.3 degrees;

(5) 10.5±0.3 degrees, 18.4±0.3 degrees, 28.8±0.3 degrees, 20.3±0.3 degrees, 24.8±0.3 degrees, 27.4±0.3 degrees, 11.9±0.3 degrees, 12.9±0.3 degrees, 28.2±0.3 degrees;

(6) 9.5±0.3 degrees, 10.8±0.3 degrees, 18.3±0.3 degrees, 14.3±0.3 degrees, 20.7±0.3 degrees, 25.2±0.3 degrees, 10.1±0.3 degrees, 17.3±0.3 degrees, 23.9±0.3 degrees;

(7) 9.2±0.3 degrees, 18.4±0.3 degrees, 24.7±0.3 degrees, 10.3±0.3 degrees, 18.0±0.3 degrees, 24.1±0.3 degrees, 14.3±0.3 degrees, 19.0±0.3 degrees, 22.1±0.3 degrees;

(8) 13.3±0.3 degrees, 18.0±0.3 degrees, 20.1±0.3 degrees, 13.8±0.3 degrees, 20.4±0.3 degrees, 25.3±0.3 degrees, 19.7±0.3 degrees, 23.0±0.3 degrees, 24.0±0.3 degrees;

(9) 6.5±0.3 degrees, 9.7±0.3 degrees, 19.2±0.3 degrees, 18.6±0.3 degrees, 19.5±0.3 degrees, 23.5±0.3 degrees, 11.2±0.3 degrees, 25.0±0.3 degrees, 27.7±0.3 degrees;

(10) 9.5±0.3 degrees, 19.1±0.3 degrees, 24.5±0.3 degrees, 17.2±0.3 degrees, 18.4±0.3 degrees, 24.9±0.3 degrees, 21.4±0.3 degrees, 23.7±0.3 degrees, 25.6±0.3 degrees;

(11) 10.4±0.3 degrees, 11.9±0.3 degrees, 16.9±0.3 degrees, 15.2±0.3 degrees, 19.5±0.3 degrees, 20.7±0.3 degrees, 7.6±0.3 degrees, 24.7±0.3 degrees, 26.5±0.3 degrees;

(12) 14.5±0.3 degrees, 21.2±0.3 degrees, 23.0±0.3 degrees, 9.8±0.3 degrees, 17.5±0.3 degrees, 20.4±0.3 degrees, 10.1±0.3 degrees, 13.7±0.3 degrees, 22.6±0.3 degrees;

(13) 8.0±0.3 degrees, 10.3±0.3 degrees, 18.9±0.3 degrees, 14.0±0.3 degrees, 16.1±0.3 degrees, 24.3±0.3 degrees, 15.4±0.3 degrees, 18.5±0.3 degrees, 22.9±0.3 degrees; and

(14) 11.9±0.3 degrees, 18.3±0.3 degrees, 20.3±0.3 degrees, 24.7±0.3 degrees, 27.3±0.3 degrees, 28.6±0.3 degrees, 14.6±0.3 degrees, 20.8±0.3 degrees, 23.3±0.3 degrees, ±0.3 degrees.
The crystalline form of any one of claims 1-4, wherein said crystalline form exhibits mass loss in the TGA (thermogravimetric) analysis selected from the group consisting of:

(1) mass loss of 1.3%up to 120 ℃;

(2) mass loss of 0.4%up to 120 ℃;

(3) mass loss of 0.7%up to 120 ℃;

(4) mass loss of 5.7%up to 120 ℃;

(5) mass loss of 9.7%up to 120 ℃;

(6) mass loss of 16.7%up to 120 ℃;

(7) mass loss of 14.2%up to 120 ℃;

(8) mass loss of 8.7%up to 120 ℃;

(9) mass loss of 1.8%up to 120 ℃;

(10) mass loss of 6.8%up to 120 ℃;

(11) mass loss of 4.1%up to 120 ℃;

(12) mass loss of 6.8%up to 120 ℃;

(13) mass loss of 0.6%up to 120 ℃; and

(14) mass loss of 8.3%up to 120 ℃.
The crystalline form of any one of claims 1-5, wherein said crystalline form exhibits endotherm and/or exotherm peak in the DSC (differential scanning calorimetry) analysis selected from the group consisting of:

(1) 135.3 ℃ (endotherm) ;

(2) 134.6 ℃ (endotherm) ;

(3) 136.5 ℃ (endotherm) ;

(4) 127.7 ℃ (endotherm) ;

(5) 115.3 ℃ (endotherm) ;

(6) 99.4 ℃ (endotherm) ;

(7) 112.3 ℃ (endotherm) ;

(8) 121.9 ℃ (endotherm) ;

(9) 151.8 ℃ (endotherm) ;

(10) 91.8 ℃ (endotherm) , 122.4 ℃ (endotherm) ;

(11) 113.8 ℃ (endotherm) ;

(12) 102.1 ℃ (endotherm) , 118.4 ℃ (endotherm) ;

(13) 155.7 ℃ (endotherm) ; and

(14) 125.5 ℃ (endotherm) .
The crystalline form of any one of claims 1-6, wherein said crystalline form exhibits salt stoichiometry in the UPLC-IC (ultra-performance liquid chromatography-ion chromatography) analysis selected from the group consisting of (molar rate of acid and freebase) :

(1) 0.5 (molar rate of maleic acid and freebase) ;

(2) 1 (molar rate of maleic acid and freebase) ;

(3) 1 (molar rate of maleic acid and freebase) ;

(4) 1.5 (molar rate of maleic acid and freebase) ;

(5) 1 (molar rate of hydrochloric acid and freebase) ;

(6) 1 (molar rate of hydrochloric acid and freebase) ;

(7) 1.2 (molar rate of hydrochloric acid and freebase) ;

(8) 1.1 (molar rate of sulphuric acid and freebase) ;

(9) 1 (molar rate of p-methylbenzene sulfonic acid and freebase) ;

(10) 1 (molar rate of methanesulfonic acid and freebase) ;

(11) 1.1 (molar rate of oxalic acid and freebase) ;

(12) 1.1 (molar rate of oxalic acid and freebase) ;

(13) 1 (molar rate of hydrobromic acid and freebase) ; and

(14) 1 (molar rate of hydrobromic acid and freebase) .
The crystalline form of any one of claims 1-7, wherein said crystalline form comprises no residual solvent or comprises residual solvent (weight%) selected from the group consisting of: 2.9 weight%of ACN; and 0.5 weight%of IPA.
The crystalline form of any one of claims 1-8, wherein said crystalline form exhibits purity rate in the LC-MS (liquid chromatography–mass spectrometry) analysis selected from the group consisting of: 99.50 area%; and 98.37 area%.
The crystalline form of any one of claims 1-9, wherein said crystalline form has solubility selected from the group consisting of: 2.8 mg/mL for SGF, 0.17 mg/mL for FaSSIF, 0.11 mg/mL for FeSSIF, 0.77 mg/mL for H₂O; 5.0 mg/mL for SGF, 0.68 mg/mL for FaSSIF, 0.12 mg/mL for FeSSIF, 3.1 mg/mL for H₂O; and 2.4 mg/mL for SGF, 0.21 mg/mL for FaSSIF, 0.12 mg/mL for FeSSIF, 1.3 mg/mL for H₂O.
The crystalline form of any one of claims 1-10, wherein said crystalline form exhibits hygroscopicity in the DVS (Dynamic vapor sorption) analysis at 25 ℃ and 80%RH (room humidity) selected from the group consisting of: 0.03%, 9.1%, and 1.2%.
An acid addition salt of crystalline form of any one of claims 1-11, or a pharmaceutically acceptable salt, prodrug, or metabolite thereof, or a solvate or hydrate of any of the foregoing.
A process of preparing crystalline form of any one of claims 1-11, the process is selected from the group consisting of:

(1) mixing compound HY-A and equimolar maleic acid in IPA, followed by slurry at RT for 3 days;

(2) mixing compound HY-A and maleic acid in 5 mL IPAc, followed by slurry at RT for 3 days;

(3) compound HY-A is ground for 3～5 min;

(4) solution vapor diffusion in CH₂Br₂/n-Hexane at RT for 4 days using compound HY-A;

(5) mixing compound HY-A and equimolar HCl in IPAc, followed by slurry at RT for 3 days;

(6) mixing compound HY-A and equimolar HCl in ACN/H₂O (19: 1, v/v) , followed by slurry at RT for 3 days;

(7) mixing compound HY-A and HCl (molar ratio of 1: 2, FB/acid) in IPA for slurry at RT for 3 days followed by evaporation at RT

(8) mixing compound HY-A equimolar H₂SO₄ in IPAc, followed by slurry at RT for 3 days

(9) mixing compound HY-A and equimolar p-toluenesulfonic acid in IPAc, followed by slurry at RT for 3 days;

(10) mixing compound HY-A and methanesulfonic acid (molar ratio of 1: 2, FB/acid) in IPA, followed by slurry at RT for 3 days;

(11) mixing compound HY-A and equimolar oxalic acid in IPAc, followed by slurry at RT for 3 days;

(12) mixing compound HY-A and equimolar oxalic acid in MIBK, followed by slurry at RT for 3 days;

(13) mixing compound HY-A and equimolar HBr in IPA, followed by slurry at RT for 3 days; and

(14) mixing compound HY-A and equimolar HBr in IPAc, followed by slurry at RT for 3 days.
A pharmaceutical composition, the pharmaceutical composition comprises crystalline form of any one of claims 1-11 and/or acid addition salt of crystalline form of claim 12, or a pharmaceutically acceptable salt, prodrug, or metabolite thereof, or a solvate or hydrate of any of the foregoing, and a pharmaceutically acceptable carrier.
A kit, the kit comprises crystalline form of any one of claims 1-11, acid addition salt of crystalline form of claim 12 and/or pharmaceutical composition of claim 14, or a pharmaceutically acceptable salt, prodrug, or metabolite thereof, or a solvate or hydrate of any of the foregoing, and a pharmaceutically acceptable carrier.
A method for inhibiting casein kinase (CK) activity, said method comprising providing crystalline form of any one of claims 1-11, acid addition salt of crystalline form of claim 12 and/or pharmaceutical composition of claim 14, or a pharmaceutically acceptable salt, prodrug, or metabolite thereof, or a solvate or hydrate of any of the foregoing and/or kit of claim 15.
The method of claim 16, wherein said casein kinase (CK) is selected from the group consisting of casein kinase I alpha (CK1α) , casein kinase I delta (CK1δ) and casein kinase I epsilon (CK1ε) .
The method of any one of claims 16-17, wherein said method is selected from the group consisting of an in vitro method, an ex vivo method, and an in vivo method.
A method for preventing and/or treating a disease or disorder, said method comprising administering to a subject in need thereof an effective amount of crystalline form of any one of claims 1-11, acid addition salt of crystalline form of claim 12 and/or pharmaceutical composition of claim 14, or a pharmaceutically acceptable salt, prodrug, or metabolite thereof, or a solvate or hydrate of any of the foregoing and/or kit of claim 15.