Patent Application
20240309018
This application claims the priority of:
The present disclosure relates to a class of thiazololactamospiroheterocyclic compounds and use thereof in the manufacture of a medicament for treating related diseases. Specifically, the present disclosure relates to a compound represented by formula (I) and a pharmaceutically acceptable salt thereof.
Ras/Raf/MEK/ERK pathway is a classical mitogen activated protein kinase (MAPK) signaling cascade pathway, is involved in the signaling of various growth factors, cytokines, mitogens and hormone receptors after activation, and is one of the most important signaling pathways for controlling cell growth, differentiation and survival.
Studies have shown that abnormal activation of Ras/Raf/MEK/ERK pathway caused by mutation or amplification is a determinant of various cancers. In human tumors, the incidence of RAS mutation is about 22%, the incidence of BRAF mutation is about 7%, and the incidence of MEK mutation is about 1%. Therefore, key node proteins on this pathway have become important targets for the treatment of cancers (Cancer Discov. 2019, 9, 329-341). Currently, a number of BRAF inhibitors and MEK1/2 inhibitors, as well as their combination regimens, have been approved by the US FDA for the treatment of melanoma, BRAFV600E mutant non-small cell lung cancer and other cancers. However, the use of BRAF and MEK inhibitors for these upstream nodes can rapidly lead to a problem of drug resistance due to mutation or pathway reactivation, greatly limiting their clinical application. Extracellular regulated protein kinases (ERK) (especially ERK1 and ERK2 kinases) are major players and downstream key nodes in the Ras/Raf/MEK/ERK pathway, and their over-activation can be found in many human cancers. ERK, as the terminal signaling kinase of this pathway, has not yet been found to have mutations that lead to drug resistance. Therefore, a drug targeting ERK kinase is expected to overcome the problem of drug resistance caused by the treatment with upstream target inhibitors, and become a more potential therapeutic strategy. But so far, research on ERK inhibitors is still in the clinical phase, and no ERK inhibitors have been approved for marketing as drugs.
In summary, there is an urgent need to develop a safe and effective ERK inhibitor drug to meet the need of treatment of a tumor.
The present disclosure provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,
The present disclosure provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,
In some embodiments of the present disclosure, the above-mentioned R1 and R2 are each independently selected from H, CH3 and CH2CH3, wherein the CH3 and CH2CH3 are optionally substituted with 1, 2 or 3 Ra, and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the above-mentioned R1 and R2 are each independently selected from H, CH3, CHF2, CD3 and CH2CH3, and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the above-mentioned R4, R5, R6, and R7 are each independently selected from H, F, Cl, Br, I and CH3, wherein the CH3 is optionally substituted with 1, 2 or 3 Rc, and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the above-mentioned R4, R5, R6, and R7 are each independently selected from H, F, Cl, Br, I and CH3, and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the above-mentioned Ra is selected from F, Cl, Br, I, CH3 and OCH3, wherein the CH3 and OCH3 are optionally substituted with 1, 2 or 3 R, and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the above-mentioned Ra is selected from CH3 and OCH3, and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the above-mentioned ring A is selected from
wherein the
are optionally substituted with 1, 2 or 3 Rd, and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the above-mentioned ring A is selected from
and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the above-mentioned structural moiety
is selected from
and other variables are as defined in the present disclosure.
The present disclosure also includes some embodiments that are obtained by combining any of the above-mentioned variables.
In some embodiments of the present disclosure, the above-mentioned compound or a pharmaceutically acceptable salt thereof is disclosed, wherein the compound is selected from:
The present disclosure also provides a compound represented by the following formula or a pharmaceutically acceptable salt thereof,
The present disclosure also provides use of the above-mentioned compound or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating a solid tumor.
The present disclosure provides crystal form A of WX001, which is characterized by an X-ray powder diffraction pattern having characteristic diffraction peaks at 2θ angles of: 10.2080±0.2000°, 18.8429±0.2000°, 20.6217±0.2000°;
In some embodiments of the present disclosure, the X-ray powder diffraction pattern of the above-mentioned crystal form A, expressed in 2θ angle, comprises at least 4 or 5 diffraction peaks selected from: 10.2080±0.2000°, 18.8429±0.2000°, 20.6217±0.2000°, 25.0767±0.2000°, and 25.4797±0.2000°.
In some embodiments of the present disclosure, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at 2θ angles of: 10.2080±0.2000°, 18.8429±0.2000°, 20.6217±0.2000°, 25.0767±0.2000°, 25.4797±0.2000°.
In some embodiments of the present disclosure, the X-ray powder diffraction pattern of the above-mentioned crystal form A, expressed in 2θ angle, comprises at least 6, 7 or 8 diffraction peaks selected from: 10.2080±0.2000°, 15.2687±0.2000°, 17.6747±0.2000°, 18.8429±0.2000°, 20.6217±0.2000°, 21.0531±0.2000°, 25.0767±0.2000°, and 25.4797±0.2000°.
In some embodiments of the present disclosure, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at 2θ angles of: 10.2080±0.2000°, 15.2687±0.2000°, 17.6747±0.2000°, 18.8429±0.2000°, 20.6217±0.2000°, 21.0531±0.2000°, 25.0767±0.2000°, 25.4797±0.2000°.
In some embodiments of the present disclosure, the X-ray powder diffraction pattern of the above-mentioned crystal form A, expressed in 2θ angle, comprises at least 9, 10, 11 or 12 diffraction peaks selected from: 10.2080±0.2000°, 14.4684±0.2000°, 15.2687±0.2000°, 17.6747±0.2000°, 18.8429±0.2000°, 20.6217±0.2000°, 21.0531±0.2000°, 21.5713±0.2000°, 22.0420±0.2000°, 22.4540±0.2000°, 25.0767±0.2000°, and 25.4797±0.2000°.
In some embodiments of the present disclosure, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at 2θ angles of: 10.2080±0.2000°, 14.4684±0.2000°, 15.2687±0.2000°, 17.6747±0.2000°, 18.8429±0.2000°, 20.6217±0.2000°, 21.0531±0.2000°, 21.5713±0.2000°, 22.0420±0.2000°, 22.4540±0.2000°, 25.0767±0.2000°, 25.4797±0.2000°.
In some embodiments of the present disclosure, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at 2θ angles of: 10.2080±0.2000°, 18.8429±0.2000°, and/or 20.6217±0.2000°, and/or 9.4003±0.2000°, and/or 10.4856±0.2000°, and/or 14.4684±0.2000°, and/or 15.0133±0.2000°, and/or 15.2687±0.2000°, and/or 15.6003±0.2000°, and/or 15.9518±0.2000°, and/or 16.6214±0.2000°, and/or 17.6747±0.2000°, and/or 17.9514±0.2000°, and/or 18.4703±0.2000°, and/or 19.1531±0.2000°, and/or 19.6571±0.2000°, and/or 21.0531±0.2000°, and/or 21.2894±0.2000°, and/or 21.5713±0.2000°, and/or 22.0420±0.2000°, and/or 22.4540±0.2000°, and/or 23.1098±0.2000°, and/or 24.5027±0.2000°, and/or 25.0767±0.2000°, and/or 25.4797±0.2000°, and/or 25.8919±0.2000°, and/or 26.3255±0.2000°, and/or 26.9544±0.2000°, and/or 28.3997±0.2000°, and/or 29.0345±0.2000°, and/or 29.3507±0.2000°, and/or 33.5390±0.2000°, and/or 34.2457±0.2000°, and/or 37.9776±0.2000°.
In some embodiments of the present disclosure, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at 2θ angles of: 10.2080°, 10.4856°, 14.4684°, 15.0133°, 15.2687°, 15.9518°, 16.6214°, 17.6747°, 17.9514°, 18.4703°, 18.8429°, 19.1531°, 20.6217°, 21.0531°, 21.2894°, 21.5713°, 22.0420°, 22.4540°, 25.0767°, 25.4797°, 26.3255°, 26.9544°.
In some embodiments of the present disclosure, the XRPD pattern of the above-mentioned crystal form A is substantially as shown in
In some embodiments of the present disclosure, the analysis data of the XRPD pattern of the above-mentioned crystal form A is as shown in Table 1:
In some embodiments of the present disclosure, the above-mentioned crystal form A has a differential scanning calorimetry curve having an onset of an endothermic peak at 241.0±3.0° C.
In some embodiments of the present disclosure, the DSC curve of the above-mentioned crystal form A is as shown in
In some embodiments of the present disclosure, the above-mentioned crystal form A has a thermogravimetric analysis curve having a weight loss of up to 0.83% at 150.0±3.0° C.
In some embodiments of the present disclosure, the TGA curve of the above-mentioned crystal form A is as shown in
The present disclosure also provides use of the above-mentioned crystal form A in the manufacture of a medicament for the treatment of a solid tumor.
The compounds of the present disclosure exhibit superior inhibitory activity against ERK1 and ERK2 enzymes: the compounds of the present disclosure exhibit superior inhibitory activity against HT29 cell proliferation: the compounds of the present disclosure have good solubility under different pH conditions: the compounds of the present disclosure have excellent pharmacokinetic properties and tumor inhibitory effect.
Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A specific term or phrase should not be considered indefinite or unclear in the absence of a particular definition, but should be understood in the conventional sense. When a trade name appears herein, it is intended to refer to its corresponding commodity or active ingredient thereof.
The term “pharmaceutically acceptable” is used herein in terms of those compounds, materials, compositions, and/or dosage forms, which are suitable for use in contact with human and animal tissues within the scope of reliable medical judgment, with no excessive toxicity, irritation, allergic reaction or other problems or complications, commensurate with a reasonable benefit/risk ratio.
The term “pharmaceutically acceptable salt” means a salt of compounds disclosed herein that is prepared by reacting the compound having a specific substituent disclosed herein with a relatively non-toxic acid or base. When compounds disclosed herein contain a relatively acidic functional group, a base addition salt can be obtained by bringing the compound into contact with a sufficient amount of base in a pure solution or a suitable inert solvent. When compounds disclosed herein contain a relatively basic functional group, an acid addition salt can be obtained by bringing the compound into contact with a sufficient amount of acid in a pure solution or a suitable inert solvent. Some specific compounds disclosed herein contain both basic and acidic functional groups and can be converted to any base or acid addition salt.
The pharmaceutically acceptable salt disclosed herein can be prepared from the parent compound that contains an acidic or basic moiety by conventional chemical methods. Generally, such salt can be prepared by reacting the free acid or base form of the compound with a stoichiometric amount of an appropriate base or acid in water or an organic solvent or a mixture thereof.
Unless otherwise specified, the term “isomer” is intended to include geometric isomers, cis- or trans-isomers, stereoisomers, enantiomers, optical isomers, diastereomers, and tautomers.
Compounds disclosed herein may be present in a specific geometric or stereoisomeric form. The present disclosure contemplates all such compounds, including cis and trans isomers, (−)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereoisomer, (D)-isomer, (L)-isomer, and a racemic mixture and other mixtures, for example, a mixture enriched in enantiomer or diastereoisomer, all of which are encompassed within the scope disclosed herein. The substituent such as alkyl may have an additional asymmetric carbon atom. All these isomers and mixtures thereof are encompassed within the scope disclosed herein.
Unless otherwise specified, the term “enantiomer” or “optical isomer” means stereoisomers that are in a mirrored relationship with each other.
Unless otherwise specified, the term “cis-trans isomer” or “geometric isomer” is produced by the inability of a double bond or a single bond between ring-forming carbon atoms to rotate freely.
Unless otherwise specified, the term “diastereomer” means a stereoisomer in which two or more chiral centers of are contained in a molecule and is in a non-mirrored relationship between molecules.
Unless otherwise specified, “(+)” means dextroisomer, “(−)” means levoisomer, and “(+)” means racemate.
Unless otherwise specified, a wedged solid bond () and a wedged dashed bond (
) indicate the absolute configuration of a stereocenter: a straight solid bond (
) and a straight dashed bond (
) indicate the relative configuration of a stereocenter: a wavy line (
) indicates a wedged solid bond (
) or a wedged dashed bond (
); or a wavy line (
) indicates a straight solid bond (
) and a straight dashed bond (
).
Unless otherwise specified, the term “tautomer” or “tautomeric form” means that different functional groups in an isomer are in dynamic equilibrium and can be rapidly converted into each other at room temperature. If tautomers are possible (as in solution), a chemical equilibrium of tautomers can be achieved. For example, proton tautomers (also known as prototropic tautomers) include interconversions by proton transfer, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions by recombination of some bonding electrons. A specific example of keto-enol tautomerization is interconversion between two tautomers pentane-2,4-dione and 4-hydroxypent-3-en-2-one.
Unless otherwise specified, the term “enriched in one isomer”, “isomer enriched”, “enriched in one enantiomer” or “enantiomeric enriched” means that the content of one isomer or enantiomer is less than 100%, and the content of the isomer or enantiomer is 60% or more, or 70% or more, or 80% or more, or 90% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, or 99.5% or more, or 99.6% or more, or 99.7% or more, or 99.8% or more, or 99.9% or more.
Unless otherwise specified, the term “isomer excess” or “enantiomeric excess” means the difference between the relative percentages of two isomers or two enantiomers. For example, if one isomer or enantiomer is present in an amount of 90% and the other isomer or enantiomer is present in an amount of 10%, the isomer or enantiomeric excess (ee value) is 80%.
Optically active (R)- and (S)-isomer, or D and L isomer can be prepared using chiral synthesis or chiral reagents or other conventional techniques. If one kind of enantiomer of certain compound disclosed herein is to be obtained, the pure desired enantiomer can be obtained by asymmetric synthesis or derivative action of chiral auxiliary followed by separating the resulting diastereomeric mixture and cleaving the auxiliary group. Alternatively, when the molecule contains a basic functional group (such as amino) or an acidic functional group (such as carboxyl), the compound reacts with an appropriate optically active acid or base to form a salt of the diastereomeric isomer which is then subjected to diastereomeric resolution through the conventional method in the art to afford the pure enantiomer. In addition, the enantiomer and the diastereoisomer are generally isolated through chromatography which uses a chiral stationary phase and optionally combines with a chemical derivative method (for example, carbamate generated from amine).
Compounds disclosed herein may contain an unnatural proportion of atomic isotopes at one or more of the atoms that make up the compounds. For example, a compound may be labeled with a radioisotope such as tritium (3H), iodine-125 (125I) or C-14 (14C). For another example, hydrogen can be replaced by heavy hydrogen to form a deuterated drug. The bond between deuterium and carbon is stronger than that between ordinary hydrogen and carbon. Compared with undeuterated drugs, deuterated drugs have advantages of reduced toxic side effects, increased drug stability, enhanced efficacy, and prolonged biological half-life of drugs. All changes in the isotopic composition of compounds disclosed herein, regardless of radioactivity, are included within the scope of the present disclosure.
The term “optional” or “optionally” means that the subsequent event or condition may occur but not requisite, that the term includes the instance in which the event or condition occurs and the instance in which the event or condition does not occur.
The term “substituted” means one or more than one hydrogen atom(s) on a specific atom are substituted by a substituent, including deuterium and hydrogen variants, as long as the valence of the specific atom is normal and the substituted compound is stable. When the substituent is oxo (i.e., ═O), it means two hydrogen atoms are substituted. Positions on an aromatic ring cannot be substituted by oxo. The term “optionally substituted” means an atom can be substituted by a substituent or not, unless otherwise specified, the species and number of the substituent may be arbitrary so long as being chemically achievable.
When any variable (such as R) occurs in the constitution or structure of the compound more than once, the definition of the variable at each occurrence is independent. Thus, for example, if a group is substituted by 0-2 R, the group can be optionally substituted by up to two R, wherein the definition of R at each occurrence is independent. Moreover, a combination of the substituent and/or the variant thereof is allowed only when the combination results in a stable compound.
When the number of a linking group is 0, such as —(CRR)0—, it means that the linking group is a single bond.
When the number of a substituent is 0, it means that the substituent does not exist. For example, -A-(R)0 means that the structure is actually-A.
When a substituent is vacant, it means that the substituent does not exist. For example, when X is vacant in A-X, the structure of A-X is actually A.
When one of variables is a single bond, it means that the two groups linked by the single bond are connected directly. For example, when L in A-L-Z represents a single bond, the structure of A-L-Z is actually A-Z.
When the bond of a substituent can be cross-linked to two or more atoms on a ring, such substituent can be bonded to any atom on the ring. For example, a structural moiety
represents the substituent R thereof can be substituted at any site on cyclohexyl or cyclohexadiene. When an enumerated substituent does not indicate through which atom it is linked to the substituted group, such substituent can be bonded through any of its atoms. For example, a pyridyl group as a substituent may be linked to the substituted group through any one of carbon atoms on the pyridine ring.
When an enumerated linking group does not indicate its linking direction, its linking direction is arbitrary. For example, when the linking group L in
is -M-W—, the -M-W— can be linked to the ring A and the ring B in the same direction as the reading order from left to right to constitute
or can be linked to the ring A and the ring B in the reverse direction as the reading order from left to right to constitute
A combination of the linking groups, substituents and/or variants thereof is allowed only when such combination can result in a stable compound.
Unless otherwise specified, when a group has one or more connectable sites, any one or more sites of the group can be connected to other groups through chemical bonds. Where the connection position of the chemical bond is variable, and there is H atom(s) at a connectable site(s), when the connectable site(s) having H atom(s) is connected to the chemical bond, the number of H atom(s) at this site will correspondingly decrease as the number of the connected chemical bond increases, and the group will become a group of corresponding valence. The chemical bond between the site and other groups can be represented by a straight solid bond (), or a wavy line (
) a straight dashed bond (
). For example, the straight solid bond in —OCH3 indicates that the group is connected to other groups through the oxygen atom in the group; the straight dashed bond in
indicates that the group is connected to other groups through two ends of the nitrogen atom in the group: the wavy line in
indicates that the group is connected to other groups through the 1- and 2-carbon atoms in the phenyl group:
indicates that any connectable site on the piperidinyl group can be connected to other groups through one chemical bond, including at least four connection ways,
even if a H atom is drawn on —N—,
still includes the connection way of
it's just that when one chemical bond is connected, the H at this site will be reduced by one, and the group will become the corresponding monovalent piperidinyl group.
Unless otherwise specified, the number of atoms on a ring is generally defined as the number of ring members, e.g., “5-7 membered ring” refers to a “ring” of 5-7 atoms arranged circumferentially.
Unless otherwise specified, the term “C1-3 alkyl” is used to indicate a linear or branched saturated hydrocarbon group consisting of 1 to 3 carbon atoms. The C1-3 alkyl group includes C1-2 and C2-3 alkyl groups and the like. It may be monovalent (e.g., methyl), divalent (e.g., methylene) or multivalent (e.g., methenyl). Examples of C1-3 alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.
Unless otherwise specified, the term “C1-3 alkoxy” refers to an alkyl group containing 1 to 3 carbon atoms and attached to the remainder of a molecule by an oxygen atom. The C1-3 alkoxy group includes C1-2, C2-3, C3 and C2 alkoxy groups, and the like. Examples of C1-3 alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), and the like.
Compounds disclosed herein can be prepared by a variety of synthetic methods well known to those skilled in the art, including the following enumerated embodiment, the embodiment formed by the following enumerated embodiment in combination with other chemical synthesis methods, and equivalent replacement well known to those skilled in the art. Alternative embodiments include, but are not limited to examples disclosed herein.
Throughout this specification, a reference to “an embodiment” or “embodiments” or “in another embodiment” or “in some embodiments” means that a specifically referenced element, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. Accordingly, a phrase “in one embodiment” or “in an embodiment” or “in another embodiment” or “in some embodiments” appearing in various places throughout this specification is not necessarily all referring to the same embodiment. Furthermore, a specific element, structure, or characteristic may be combined in any suitable manner in one or more embodiments.
In the present disclosure, Exo Up in a DSC curve indicates upward exotherm.
The structures of compounds disclosed herein can be confirmed by conventional methods well known to those skilled in the art. If the present disclosure relates to an absolute configuration of a compound, the absolute configuration can be confirmed by conventional techniques in the art, such as single crystal X-Ray diffraction (SXRD). In a single crystal X-Ray diffraction (SXRD), the diffraction intensity data of a cultivated single crystal are collected using a Bruker D8 venture diffractometer with a light source of CuKα radiation in a scanning mode of φ/ω scan; after collecting the relevant data, the crystal structure is further analyzed by the direct method (Shelxs97) to confirm the absolute configuration.
The present disclosure will be described in detail through examples below. These examples do not mean any limitation to the present disclosure.
All solvents used in the present disclosure are commercially available and can be used without further purification.
Solvents used in the present disclosure are commercially available.
The following abbreviations are used in the present disclosure: aq represents aqueous: eq represents equivalent or equivalence: DCM represents dichloromethane: PE represents petroleum ether: DMSO represents dimethyl sulfoxide: EtOAc represents ethyl acetate; EtOH represents ethanol: MeOH represents methanol: BOC represents tert-butoxycarbonyl, which is an amine protecting group: r.t. represents room temperature: O/N represents overnight; THF represents tetrahydrofuran: Boc2O represents di-tert-butyl dicarbonate: TFA represents trifluoroacetic acid: DIPEA represents diisopropylethylamine: iPrOH represents 2-propanol: mp represents melting point.
Compounds are named according to general naming principles in the art or by ChemDraw® software, and commercially available compounds are named with their vendor directory names.
Instrument model: X'Pert3 X-ray diffractometer of PANalytical
Test method: About 10 mg of sample was used for XRPD detection.
Instrument model: TA 2500 differential scanning calorimeter
Instrument model: TA 5500 thermogravimetric analyzer
Dynamic vapor adsorption (DVS) curves were collected on DVS Intrinsic plus of Surface Measurement Systems (SMS) company. The relative humidity at 25° C. was corrected with the deliquescent points of LiCl, Mg(NO3)2 and KCl.
The present disclosure is described in detail below by means of examples. However, it is not intended that these examples have any disadvantageous limitations to the present disclosure. The present disclosure has been described in detail herein, and embodiments are also disclosed herein. It will be apparent to those skilled in the art that various changes and modifications may be made to the embodiments disclosed herein without departing from the spirit and scope disclosed herein.
To a reaction flask were added A-1-1 (150 g, 635.36 mmol, 1 eq), calcium chloride (70.51 g, 635.36 mmol, 1 eq), tetrahydrofuran (500 mL) and ethanol (1000 mL). Sodium borohydride (48.07 g, 1.27 mol, 2 eq) was added under nitrogen, and the mixture was reacted at 20° C. for 15 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure. The concentrate was diluted with 15% citric acid aqueous solution (4000 mL), and extracted with ethyl acetate (4000 mL×3). The organic phases were combined, washed with saturated brine (2000 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to dryness to give a crude product. The crude product was purified by column chromatography to give A-1-2. 1H NMR (400 MHZ, DMSO-d6) δ (ppm)=7.47 (s, 1H), 5.58 (br s, 1H), 4.52 (s, 2H).
To a reaction flask were added A-1-2 (102 g, 525.64 mmol, 1 eq) and 2-methyltetrahydrofuran (1000 mL). The atmosphere was replaced with nitrogen gas. The mixture was cooled to −70° C., and lithium diisopropylamide (2 M, 525.64 mL, 2.0 eq) was slowly added dropwise. The mixture was stirred at −70° C. for 30 minutes. Then, a solution of A-1-3 (138.18 g. 788.46 mmol, 1.5 eq) in 2-methyltetrahydrofuran (400 mL) was slowly added dropwise, and the mixture was reacted at −70° C. for another 1 hour. After the reaction was completed, the reaction solution was quenched with saturated aqueous ammonium chloride solution (2000 mL), and extracted with ethyl acetate (2000 mL×4). The layers were separated. The organic phases were combined, washed with saturated brine (1000 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to dryness to give a crude product. The crude product was first purified by column, and then purified by slurrying with methyl tert-butyl ether to give A-1-4. 1H NMR (400 MHZ, DMSO-d6) δ (ppm)=6.41 (s, 1H), 5.43 (t, J=5.6 Hz, 1H), 5.00-4.84 (m, 4H), 4.38 (d, J=5.6 Hz, 2H), 1.12 (s, 9H).
To a reaction flask were added A-1-4 (50 g, 135.39 mmol, 1 eq), azodicarboxylic acid dipiperidide (40.99 g, 162.47 mmol, 1.2 eq) and tetrahydrofuran (500 mL). The atmosphere was replaced with nitrogen gas. The mixture was cooled to 0° C., and a solution of tributylphosphine (32.87 g, 162.47 mmol, 40.09 mL, 1.2 eq) in tetrahydrofuran (100 mL) was slowly added dropwise. The mixture was reacted at 0° C. for 1 hour. After the reaction was completed, water (500 mL) and saturated brine (500 mL) were added to the reaction solution in sequence. The mixture was extracted with ethyl acetate (500 mL×2). The layers were separated. The organic phases were combined, washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to dryness to give a crude product. The crude product was slurried with 500 mL of methyl tert-butyl ether, and filtered. The filtrate was collected, and concentrated to give a crude product. The crude product was slurried with 50 mL of n-hexane, and filtered. The filter cake was collected and dried to give A-1-5. 1H NMR (400 MHZ, DMSO-d6) δ (ppm)=5.29 (d, J=7.5 Hz, 1H), 4.88-4.75 (m, 3H), 4.60 (d, J=12.9 Hz, 1H), 4.22 (d, J=12.9 Hz, 1H), 1.26 (s, 9H).
To a reaction flask were added A-1-5 (29 g, 82.55 mmol, 1 eq), tetrahydrofuran (250 mL) and water (50 mL). The atmosphere was replaced with nitrogen gas. Iodine (2.10 g, 8.26 mmol, 1.66 mL, 0.1 eq) was added, and the mixture was reacted at 50° C. for 18 hours. Then additional iodine (2.10 g, 8.26 mmol, 1.66 mL, 0.1 eq) was added, and the mixture was reacted at 50° C. for another 6 hours. After the reaction was completed, a solution of crude A-1-6 was obtained and used directly in the next step.
To the solution containing crude A-1-6 was added sodium carbonate (17.50 g, 165.11 mmol, 2 eq). The atmosphere was replaced with nitrogen gas. Di-tert-butyl carbonate (27.03 g, 123.83 mmol, 28.45 mL, 1.5 eq) was added. The mixture was reacted at 20° C. for 12 hours. After the reaction was completed, the reaction solution was poured into water (200 mL), and then extracted with ethyl acetate (300 mL×3). The layers were separated. The organic phases were combined, washed with saturated brine (300 mL×3), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography to give A-1-7. 1H NMR (400 MHZ, DMSO-d6) δ (ppm)=5.46 (d, J=5.9 Hz, 1H), 5.32 (d, J=6.3 Hz, 1H), 4.64 (d, J=6.1 Hz, 1H), 4.55 (d, J=5.8 Hz, 1H), 4.49 (d, J=9.4 Hz, 2H), 1.53 (s, 9H).
To a reaction flask were added A-1-7 (26.5 g, 76.32 mmol, 1 eq), glacial acetic acid (1.37 g, 22.90 mmol, 1.31 mL, 0.3 eq) and acetonitrile (260 mL). The atmosphere was replaced with nitrogen gas. The mixture was heated to 50° C., and a solution of sodium chlorite (32.48 g, 305.28 mmol, 85% purity, 4 eq) in water (70 mL) was added dropwise. After the addition was completed, the mixture was reacted at 50° C. for another 12 hours. Then, additional sodium chlorite (8.97 g, 99.21 mmol, 1.3 eq) and glacial acetic acid (458.31 mg, 7.63 mmol, 436.49 μL, 0.1 eq) were added, and the mixture was reacted at 50° C. for another 6 hours. After the reaction was completed, the reaction solution was quenched with aqueous saturated sodium sulfite solution (150 mL), and water (90 mL) was added. The mixture was left to stand, and the organic phase was separated. The aqueous phase was extracted with ethyl acetate (90 mL). The combined organic phase was washed with saturated brine (90 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to give a crude product. The crude product was slurried with ethyl acetate: n-hexane (1:5, 120 mL) with stirring, and filtered. The filter cake was collected and dried to give A-1-8. 1H NMR (400 MHZ, CDCl3) δ (ppm)=5.61 (d, J=6.6 Hz, 2H), 4.76 (d, J=6.6 Hz, 2H), 1.66 (s, 9H).
To a dried reaction flask were added A-1-8 (10 g, 27.68 mmol, 1 eq) and dichloromethane (100 mL). Trifluoroacetic acid (41.04 g, 359.90 mmol, 26.65 mL, 13 eq) was added at 0° C. The mixture was reacted at 0° C. for 0.5 hours. After the reaction was completed, the reaction solution was slowly poured into aqueous saturated sodium bicarbonate solution (1000 mL), and adjusted to pH of 7˜8. The mixture was extracted with dichloromethane (1000 mL×3). The layers were separated. The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give A-1. 1H NMR (400 MHZ, DMSO-d6) δ (ppm)=9.59 (s, 1H), 4.99-4.78 (m, 4H).
To a reaction flask were added sodium hydroxide (590.8 g, 14.8 mol, 1.05 eq), ice water (20 L), and B-1-1 (2000.00 g, 14.07 mol, 1 eq). Then methyl iodide (2495.80 g, 17.59 mol, 1.25 eq) was added and the mixture was reacted at 25° C. for 2 hours. After the reaction was completed, 6N glacial hydrochloric acid aqueous solution was slowly added into the reaction flask to adjust the pH to 6˜7. The mixture was stirred for 0.5 hours, and filtered. The filter cake was collected. Acetonitrile (500 mL) was added to the filter cake. The mixture was stirred for 0.5 hours, and filtered. The filter cake was collected, and dried by baking to give B-1-2. 1H NMR (400 MHZ, DMSO-d6) δ (ppm)=12.69 (br s, 1H), 7.74 (br s, 1H), 2.45 (s, 3H), 1.86 (s, 3H).
To a reaction flask were added acetonitrile (15 L) and B-1-2 (1500.00 g, 9.60 mol, 1 eq) at 25° C. Then phosphorus oxychloride (1840.00 g, 12.0 mol, 1.25 eq) was added. The mixture was slowly heated to 62° C., and reacted at 62° C. for 12 hours. The reaction solution was poured into water (10.5 L), and solid sodium bicarbonate was added to adjust the pH to 6-7. The mixture was extracted with ethyl acetate (10.5 L), and the layers were separated to give an organic phase. The organic phase was washed with saturated brine (7.5 L), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give B-1-3. 1H NMR (400 MHZ, DMSO-d6) δ (ppm)=8.54 (s, 1H), 2.50 (s, 3H), 2.22 (s, 3H).
To a reaction flask were added B-1-3 (100 g, 572.57 mmol, 1 eq), water (24.76 g, 1.37 mol, 24.76 mL, 2.4 eq) and acetonitrile (1000 mL). The atmosphere was replaced with nitrogen gas. Sodium iodide (571.59 g, 3.81 mol, 6.66 eq) and trimethylchlorosilane (186.61 g, 1.72 mol, 218.00 mL, 3 eq) were added in sequence, and the mixture was reacted at 20° C. for 14 hours. After the reaction was completed, dichloromethane (800 mL) and water (1200 mL) were added to the reaction solution in sequence. Then solid sodium bicarbonate was added to adjust the pH to 6-7. The layers were separated, and the aqueous phase was extracted once with dichloromethane (500 mL). The organic phases were combined, washed successively with aqueous saturated sodium sulfite solution (500 mL) and saturated brine (500 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give a crude product. n-Heptane (0.5 L) was added to the crude product, and the mixture was stirred for 1 hour. The mixture was filtered, and the filter cake was collected to give B-1. 1H NMR (400 MHZ, DMSO-d6) § (ppm)=8.34 (s, 1H), 2.48 (s, 3H), 2.21 (s, 3H).
To a reaction flask were added A-1 (500 mg, 1.92 mmol, 1 eq), WX001-1 (427.54 mg, 2.30 mmol, 1.2 eq) and N′N-dimethylformamide (3 mL). The atmosphere was replaced with nitrogen gas. Cesium carbonate (935.92 mg, 2.87 mmol, 1.5 eq) was added, and the mixture was reacted at 25° C. for 16 hours. After the reaction was completed, the reaction solution was poured into water (20 mL), and the mixture was extracted with ethyl acetate (30 mL×3). The layers were separated. The organic phases were combined, washed with saturated brine (30 mL×3), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by thin layer chromatography on silica gel plate to give WX001-2. LCMS (m/z): 366, 368 [M+H]+.
In a pre-dried reaction flask, the atmosphere was replaced with nitrogen, and wet palladium on carbon (0.1 g, 819.15 μmol, 10% purity, 1 eq) and ethanol (20 mL) were added. Then WX001-2 (300 mg, 819.15 μmol, 1 eq) was added. The atmosphere was replaced with nitrogen three times, and the mixture was stirred to react at 50° C. and 50 Psi for 24 hours. After the reaction was completed, the reaction solution was filtered, and the filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by thin layer chromatography on silica gel plate to give WX001-3. 1H NMR (400 MHZ, CDCl3) δ (ppm)=8.96 (s, 1H), 7.58 (t, J=7.6 Hz, 1H), 7.19 (d, J=7.6 Hz, 1H), 7.09 (d, J=7.5 Hz, 1H), 5.26 (d, J=7.6 Hz, 2H), 5.14 (s, 2H), 4.80 (d, J=7.6 Hz, 2H), 2.55 (s, 3H).
To a dried reaction flask were added WX001-3 (60 mg, 208.81 μmol, 1 eq), tetrahydrofuran (1 mL) and zinc chloride solution (0.7 M, 298.31 μL, 1 eq). The mixture was cooled to −78° C., and lithium hexamethyldisilazide (1 M, 417.63 μL, 2 eq) was added. The mixture was reacted at 20° C. for 1 hour to give reaction solution 1.
A mixture of B-1 (55.57 mg, 208.81 μmol, 1 eq) and tetrakis(triphenylphosphine)palladium (7.24 mg, 6.26 μmol, 0.03 eq) in N′N-dimethylacetamide (1 mL) was heated to 50° C. under nitrogen, and then reaction solution I was added dropwise. After the addition was completed, the mixture was reacted at 50° C. for another 1 hour. After the reaction was completed, the reaction solution was poured into water (5 mL), and then extracted with dichloromethane (30 mL×3). The layers were separated. The organic phases were combined, washed with saturated brine (30 mL×3), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by thin layer chromatography on silica gel plate to give WX001-4. LCMS (m/z): 426.0 [M+H]+.
To a reaction flask were added WX001-4 (100 mg, 235.00 μmol, 1 eq), acetonitrile (1 mL) and water (0.5 mL). The atmosphere was replaced with nitrogen gas three times, and potassium monopersulfate (288.95 mg, 470.01 μmol, 2 eq) was added. The mixture was reacted at 20° C. for 14 hours. After the reaction was completed, the reaction solution was poured into a saturated sodium thiosulfate aqueous solution (5 mL), and the mixture was extracted with dichloromethane (30 mL×3). The layers were separated. The organic phases were combined, washed successively with saturated aqueous sodium bicarbonate solution (20 mL) and saturated brine (30 mL×3), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by thin layer chromatography on silica gel plate to give WX001-5. LCMS (m/z): 458.0 [M+H]+.
To a dried reaction flask were added WX001-5 (40 mg, 87.43 μmol, 1 eq), C-1 (16.98 mg, 174.85 μmol, 2 eq) and tetrahydrofuran (0.5 mL). The atmosphere was replaced with nitrogen gas. The mixture was cooled to 0° C., and lithium hexamethyldisilazide (1 M, 166.11 μL, 1.9 eq) was added dropwise. The mixture was reacted at 0° C. for 1 hour. After the reaction was completed, the reaction solution was poured into water (5 mL), and the mixture was extracted with dichloromethane (30 mL×3). The layers were separated. The organic phases were combined, washed with saturated brine (30 mL×3), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by preparative high performance liquid phase chromatography (column: Waters Xbridge BEH C18 100*25 mm*5 um; mobile phase: [water (10 mM ammonium bicarbonate)-acetonitrile]: B (acetonitrile) %: 20%-50%, 10 min) to give WX001. 1H NMR (400 MHZ, DMSO-d6) δ=9.67 (br s, 1H), 8.62 (s, 1H), 7.64 (t, J=7.7 Hz, 1H), 7.43 (d, J=1.8 Hz, 1H), 7.14 (dd, J=2.9, 7.7 Hz, 2H), 6.37 (d, J=1.5 Hz, 1H), 5.12 (d, J=7.2 Hz, 2H), 5.02 (s, 2H), 4.85 (d, J=7.2 Hz, 2H), 3.74 (s, 3H), 2.57 (s, 3H), 2.42 (s, 3H): LCMS (m/z): 475.0 [M+H]+.
Tetrahydrofuran (12 L) and I-1-1 (1200 g, 5.69 mol) were added into a reaction kettle. Tetramethylethylenediamine (661.59 g, 5.69 mol) was slowly added into the reaction kettle. The atmosphere was replaced with nitrogen gas. The mixture was cooled to −70° C. (internal temperature). Lithium diisopropylamide (2 M, 6.83 L) was slowly added dropwise, and the mixture was stirred at −70° C. for 0.5 hours. A solution of I-1-2 (1.76 kg, 9.39 mol) in tetrahydrofuran (4.8 L) was slowly added dropwise (1.5 hours), and the mixture was reacted at −70° C. for 1 hour. After the reaction was completed, water (6 L) was added dropwise to the reaction solution to quench the reaction mixture. The reaction bottle was washed with water (2.4 L). The mixture was combined, and stirred. The mixture was allowed to warm up to 10° C. The layers were separated, and the aqueous phase was extracted with ethyl acetate (6 L). The remaining aqueous phase was adjusted to pH 3˜4 with potassium bisulfate aqueous solution (15.6 L), and then extracted three times with ethyl acetate (6 L). The organic phases were combined and washed with saturated brine (6 L). The organic phase was dried with 1200 g of anhydrous sodium sulfate (m/m=1:1), and concentrated under reduced pressure at 45° C. to give a crude product. Dichloromethane (2.4 L) and isopropyl ether (7.2 L) were added to the crude product and the mixture was stirred at room temperature for half an hour. The mixture was filtered and the filter cake was collected to give I-1-3. 1H NMR (400 MHZ, DMSO-d6) δ=13.18 (br s, 1H), 6.20 (br s, 1H), 4.93-4.82 (m, 4H), 1.09 (s, 9H).
Dichloromethane (1330 mL), I-1-3 (1330 g, 1.99 mol, crude product), and 4-dimethylaminopyridine (41.37 g, 338.61 mmol) were added to the reaction kettle in sequence. N,N′-carbonyldiimidazole (419.86 g, 2.59 mol) was added in batches, and the mixture was reacted at 50° C. for 36 hours. After the reaction was completed, 2 N hydrochloric acid aqueous solution (5.32 L) was added to the reaction solution to adjust the pH to 3-4. The mixture was stirred for 0.5 hours, and then water (5.32 L) was added. The mixture was concentrated to remove the organic solvent. The residue was filtered to give a filter cake. The filter cake was stirred with sodium bicarbonate aqueous solution (5.32 L) for 0.5 hours, then filtered, and washed with water (2.66 L). The filter cake was collected. Absolute ethanol (10.64 L) was added to the crude product. The mixture was stirred for 2 hours, and filtered. The filter cake was washed with absolute ethanol (1.3 L). The filter cake was collected and dried to give I-1-4. 1H NMR (400 MHZ, DMSO-d6) δ=9.58 (br s, 1H), 4.94-4.84 (m, 4H).
To a reaction flask were added I-1-4 (283 g, 1.03 mol), cesium carbonate (505.36 g, 1.55 mol) and N,N′-dimethylformamide (2800 mL). The atmosphere was replaced with nitrogen gas. I-1-5 (221.24 g, 1.19 mol) was added, and the mixture was reacted at 20° C. for 12 hours. After the reaction was completed, the reaction solution was slowly poured into ice water (14 L). The mixture was stirred for 1 hour, and filtered. The filter cake was collected. Methyl tert-butyl ether (5 L) was added to the crude product and the mixture was stirred for 2 hours, and filtered. The filter cake was collected, and dried to give I-1-6. 1H NMR (400 MHZ, DMSO-d6) δ=7.63 (t, J=7.7 Hz, 1H), 7.13 (dd, J=2.8, 7.7 Hz, 2H), 5.04 (d, J=7.4 Hz, 2H), 4.98 (s, 2H), 4.86 (d, J=7.4 Hz, 2H), 2.41 (s, 3H).
Three reactions were carried out in parallel. To a reaction flask were added I-1-6 (183 g, 468.60 mmol) and tetrahydrofuran (2745 mL). The atmosphere was replaced with nitrogen gas. Diisopropylethylamine (181.69 g. 1.41 mol) and diethyl phosphite (194.14 g. 1.41 mol) were slowly added dropwise at 20° C., and the mixture was reacted at 40° C. for 16 hours. After the reaction was completed, the reaction solution was diluted with water (915 mL), and the mixture was extracted with dichloromethane (1830 mL*3). The organic phase was washed with saturated brine (915 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated at 45° C., and dried to give a crude product. The crude product was added to a mixed solvent of methyl tert-butyl ether and n-hexane (total 1098 mL, volume ratio 1:5), and the mixture was stirred for 1 hour. The mixture was filtered, and the filter cake was collected and dried. The filter cake was added to water (5400 mL), and the mixture was stirred for 1 hour, and filtered. The filter cake was collected, and dried to give I-1. 1H NMR (400 MHZ, DMSO-d6) δ=9.32 (s, 1H), 7.64 (t, J=7.6 Hz, 1H), 7.13 (t, J=8.4 Hz, 2H), 5.10 (d, J=7.4 Hz, 2H), 4.99 (s, 2H), 4.81 (d, J=7.5 Hz, 2H), 2.42 (s, 3H).
To two reaction flasks were respectively added a solution of I-1 (120 g, 403.05 mmol) and zinc chloride (0.7 M, 575.79 mL) in tetrahydrofuran (1200 mL). The atmosphere was replaced with nitrogen gas. The mixture was cooled to 0° C., and lithium hexamethyldisilazide (1 M, 806.11 mL) was slowly added dropwise. The mixture was allowed to warm up to 20° C., and then stirred for 1 hour to give reaction solution 1. To another reaction flask were added B-1 (107.25 g, 403.05 mmol), tetrakis(triphenylphosphine)palladium (13.97 g, 12.09 mmol) and N,N′-dimethylformamide (600 mL), and the reaction solution was heated to 50° C. to give reaction solution 2. Reaction solution 1 was slowly added dropwise into reaction solution 2, and the mixture was reacted at 50° C. for 1 hour. After the reaction was completed, the reaction mixture was quenched with 0.1M ethylenediamine tetraacetic acid disodium salt (10800 mL) and stirred for 30 min. n-Heptane (4800 mL) was added and the mixture was stirred for 0.5 hours. The mixture was filtered, and the filter cake was collected and dried to give a crude product. The crude product was slurried with ethanol (7200 mL) at 20° C. for 2 hours, and filtered. The filter cake was collected, and dried to give I-3. 1H NMR (400 MHZ, CDCl3) δ=8.54 (s, 1H), 7.54 (t, J=7.2 Hz, 1H), 7.19 (d, J=7.4 Hz, 1H), 7.07 (d, J=7.3 Hz, 1H), 5.29 (d. J=7.5 Hz, 2H), 5.11 (s, 2H), 4.84 (d, J=7.6 Hz, 2H), 2.73 (s, 3H), 2.66 (s, 3H), 2.52 (s, 3H).
To a reaction flask were added I-3 (120 g, 282.00 mmol), acetonitrile (110 mL) and water (550 mL). The atmosphere was replaced with nitrogen gas. Potassium monopersulfate (329.40 g, 535.81 mmol) was added, and the mixture was reacted at 30° C. for 12 hours. After the reaction was completed, 200 mL of ice-water mixture was added to the reaction solution. Then saturated sodium bicarbonate aqueous solution and saturated sodium thiosulfate aqueous solution were added (600 mL each), followed by 600 mL of water. The mixture was filtered, and the filter cake was collected and dried to give a crude product. 600 mL of absolute ethanol was added to the crude product, and the mixture was stirred for 1 hour. The mixture was filtered, and the filter cake was collected to give I-4. 1H NMR (400 MHZ, CDCl3) δ=8.90 (s, 1H), 7.55 (t, J=7.7 Hz, 1H), 7.20 (d, J=7.7 Hz, 1H), 7.06 (d, J=7.7 Hz, 1H), 5.31 (d, J=7.6 Hz, 2H), 5.11 (s, 2H), 4.84 (d, J=7.6 Hz, 2H), 3.44 (s, 3H), 2.92 (s, 3H), 2.50 (s, 3H).
Step 7: Synthesis of the crystal form A of WX001.
To a reaction flask were added I-4 (47 g, 102.73 mmol), C-1 (25.94 g, 267.09 mmol), dichloromethane (470 mL) and tetrahydrofuran (470 mL). The atmosphere was replaced with nitrogen gas. Lithium hexamethyldisilazide (1 M, 246.54 mL, 2.4 eq) was added dropwise at −5° C. (controlling the internal temperature at −5-3° C.), and the mixture was reacted at 0° C. for 0.5 hours. After the reaction was completed, the mixture was quenched with deionized water (470 mL). The mixture was concentrated to remove organic solvent, and filtered. The filter cake was collected. The filter cake was stirred with deionized water (1000 mL) at room temperature for 30 minutes, and filtered. The filter cake was collected. The filter cake was stirred with acetonitrile (1000 mL) at room temperature for 30 min, and filtered. The filter cake was collected to give the crystal form A of WX001. 1H NMR (400 MHZ, CDCl3) δ=8.44 (s, 1H), 7.60-7.50 (m, 2H), 7.18 (d, J=7.4 Hz, 1H), 7.06 (d, J=7.6 Hz, 1H), 6.88 (s, 1H), 6.42 (d, J=1.9 Hz, 1H), 5.27 (d, J=7.6 Hz, 2H), 5.12 (s, 2H), 4.85 (d, J=7.6 Hz, 2H), 3.85 (s, 3H), 2.69 (s, 3H), 2.52 (s, 3H). The XRPD pattern of the crystal form A of WX001 is shown in
About 20 mg of the crystal form A of WX001 in each portion was weighed into a 3-mL vial, and about 4 mL of solvent was added to a 20-mL vial. The 3-mL (open) vial was placed in the 20-mL vial, and the 20-mL vial was sealed. The samples adsorbed the solvent and partially dissolved, or were allowed to stand at room temperature for 7 days, and then the solids were collected and tested by XRPD. The test results are shown in Table 7.
About 20 mg of the crystal form A of WX001 in each portion was weighed into a 3-mL vial, and dissolved in 1.2˜1.8 mL of solvent. About 4 mL of anti-solvent was added to a 20-mL vial. The 3-mL (open) vial containing a clear solution was placed in the 20-mL vial, and then the 20-mL vial was sealed and allowed to stand at room temperature. The resulting solid was collected and tested by XRPD. The test results are shown in Table 8.
15˜20 mg of the crystal form A of WX001 was weighed into a 3-mL vial, and dissolved in 1.0˜3.0 mL of solvent. The vial was sealed with a sealing film, and 4 pinholes were punched in the sealing film. The solution was left to stand at room temperature to slowly volatilize. The resulting solid was collected and tested by XRPD. The test results are shown in Table 9.
15˜35 mg of the crystal form A of WX001 in each portion was weighed into a 3-mL vial, and dissolved in 1.0˜3.0 mL of solvent. The solution was stirred and equilibrated at 50° C. for about 3.5 hours, and then filtered. The supernatant was collected. The resulting supernatant was placed in a biochemical incubator, cooled from 50° C. to 5° C. at 0.1° C./min, and then maintained at a constant temperature of 5° C. The precipitated solids were collected and tested by XRPD. The test results are shown in Table 10.
5. Stirring with Temperature Cycle
About 25 mg of the crystal form A of WX001 in each portion was weighed into an HPLC vial, and 0.5 mL of solvent was added respectively. The resulting suspension was subjected to a temperature cycle program (the sample was heated to 50° C. and then cooled to 5° C. at a rate of 0.1° C./min; then this cycle was repeated; finally, the sample was maintained at 5° C.) with magnetic stirring (1000 rpm). The solids were collected by centrifugation, and tested by XRPD. The test results are shown in Table 11.
About 25 mg of the crystal form A of WX001 in each portion was weighed into an HPLC vial, and 0.5 mL of solvent was added respectively. The obtained turbid liquid was magnetically stirred (1000 rpm) at room temperature for 3 days. The solids were collected by centrifugation, and tested by XRPD. The test results are shown in Table 12.
About 25 mg of the crystal form A of WX001 in each portion was weighed into an HPLC vial, and 0.5 mL of solvent was added respectively. The obtained suspension was magnetically stirred (1000 rpm) at 50° C. for 3 days. The solids were collected by centrifugation, and tested by XRPD. The test results are shown in Table 13.
About 15 mg of the crystal form A of WX001 was respectively weighed into a 20 mL vial, and 0.7˜1.0 mL of solvent was added to completely dissolve the solid. The anti-solvent was added dropwise to the clear solution while stirring (1000 rpm) until solid precipitated or until the total volume of anti-solvent reached 10 mL. Samples without solid precipitation were stirred at 5° C. until solid precipitated. Clear samples were then stirred at −20° C. until solid precipitated. Still clear samples were then volatilized at room temperature. The precipitated solid was separated and tested by XRPD. The results are shown in Table 14.
SMS DVS Advantage dynamic vapor sorption apparatus
10˜30 mg of the crystal form A of WX001 was weighed and put into a DVS sample pan for testing.
The DVS pattern of the crystal form A of WX001 is shown in
The weight increase of the crystal form A of WX001 by moisture absorption at 25° C. and 80% RH was 0.1134%, indicating that the crystal form A of WX001 has almost no hygroscopicity.
12 portions of samples of the crystal form A of WX001 were weighed in parallel, and each portion was about 5 mg. The sample was placed at the bottom of an HPLC vial to form a thin layer. The samples were placed in a chamber at a constant temperature and humidity of 60° C./75% RH conditions and in a 92.5% RH desiccator. The bottles were sealed with a sealing film. Some small holes were punched in the sealing film to ensure that the samples were fully in contact with the ambient air. The caps of the sample bottles placed at 60° C. under light and light-shielding conditions (samples under light-shielding conditions were wrapped in tin foil) were tighten. The test results are shown in Table 15 below:
#ICH conditions
Conclusion: the crystal form A of WX001 has good stability.
The ability of the compound to inhibit ERK1 and ERK2 kinase activity was measured.
20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), 0.02% Brij35, 0.02 mg/mL bovine serum albumin (BSA), 0.1 mM Na3VO4, 2 mM dithiothreitol (DTT), 1% DMSO.
The assay compound was dissolved in 100% DMSO to prepare a stock solution of specific concentration. The compound was serially diluted in DMSO solution using Integra Viaflo Assist smart pipette.
Conclusion: The compound of the present disclosure exhibits excellent inhibitory activity against ERK1 kinase.
Conclusion: The compound of the present disclosure exhibits excellent inhibitory activity against ERK2 kinase.
The ability of the compound to inhibit the proliferation of HT29 tumor cells was measured.
The assay compound was dissolved in 100% DMSO to prepare 10 mM stock solution.
Conclusion: The compound of the present disclosure exhibits excellent inhibitory activity on HT29 cell proliferation.
Female BALB/c mice were used as assay animals to determine the blood concentration of the compound and evaluate the pharmacokinetic behavior after a single administration.
Four healthy adult female BALB/c mice were selected, wherein 2 mice were in an intravenous injection group and 2 mice were in an oral group. The vehicle in the intravenous injection group was 5% DMSO+95% (20% HP-β-CD). The compound to be assayed was mixed with an appropriate amount of vehicle for intravenous injection, vortexed and sonicated to prepare a clear solution of 0.5 mg/mL. The clear solution was filtered by a microporous membrane, and then ready for use. The vehicle in the oral group was 5% DMSO+95% (20% HP-β-CD). The compound to be assayed was mixed with the vehicle, vortexed and sonicated to prepare a solution of 0.3 mg/mL. Mice were administered 1 mg/kg intravenously or 3 mg/kg orally, and then whole blood was collected for a certain period. Plasma was prepared. The drug concentration was analyzed by LC-MS/MS method, and the pharmacokinetic parameters were calculated by Phoenix WinNonlin software (Pharsight, USA).
Note: DMSO: dimethyl sulfoxide: HP-β-CD: hydroxypropyl-β-cyclodextrin.
Conclusion: The compound of the present disclosure exhibits excellent oral exposure and bioavailability.
The solubility of the compound was determined to evaluate the solubility property of the compound.
Conclusion: The compound of the present disclosure has good solubility under different pH conditions.
The anti-tumor effect of WX001 was evaluated using a subcutaneous xenograft tumor model of human melanoma A375 cells in nude mouse.
Animals were reared in IVC (independent air supply system, and constant temperature and humidity) cages (3 animals per cage) in SPF grade of animal room at a temperature of 20-26° C. and a humidity of 40-70%;
Cage: The cage was made of polycarbonate, and had a volume of 375 mm×215 mm×180 mm. The bedding material was corncob, which was replaced once a week:
Food: Assay animals had free access to food (dry pelleted food sterilized by irradiation) throughout the assay period:
Drinking water: Assay animals had free access to sterilized water:
Cage identification: The animal information card for each cage should indicate the number, gender, strain, date of receipt, assay numbering of administration schedule, group, and start date of the assay of animals in the cage:
Animal identification: Assay animals were identified by ear tags.
Conclusion: WX001 can inhibit tumor growth in a dose-dependent manner at three doses of 12.5 mg/kg, 25 mg/kg and 50 mg/kg. During the administration, the body weight of animals is not observed to decrease significantly, and the tolerance is good.
Male SD rats were used as assay animals. After a single administration, the plasma concentration of the compound was measured and the pharmacokinetic behavior was evaluated.
Six healthy adult male SD rats were selected, wherein 3 rats were in an intravenous injection group and 3 rats were in an oral group. The vehicle in the intravenous injection group was 5% DMSO+95% (20% HP-β-CD). The compound to be assayed was mixed with an appropriate amount of vehicle for intravenous injection, vortexed and sonicated to prepare a clear solution of 0.2 mg/mL. The clear solution was filtered by a microporous membrane, and then ready for use. The vehicle in the oral group was 5% DMSO+95% (20% HP-β-CD). The compound to be assayed was mixed with the vehicle, vortexed and sonicated to prepare a solution of 1 mg/mL. SD rats were administered 1 mg/kg intravenously or 10 mg/kg orally, and then whole blood was collected for a certain period. Plasma was prepared. The drug concentration was analyzed by LC-MS/MS method, and the pharmacokinetic parameters were calculated by Phoenix WinNonlin software (Pharsight, USA).
Note: DMSO: dimethyl sulfoxide: HP-β-CD: hydroxypropyl-β-cyclodextrin.
Conclusion: The compound of the present disclosure exhibits excellent oral exposure and bioavailability.
Male cynomolgus monkeys were used as assay animals. After a single administration, the plasma concentration of the compound was measured and the pharmacokinetic behavior was evaluated.
Five healthy adult male cynomolgus monkeys were selected, wherein 2 animals were in an intravenous injection group, and 3 animals were in an oral group. The vehicle in the intravenous injection group was 5% DMSO+95% (20% HP-β-CD). The compound to be assayed was mixed with an appropriate amount of vehicle for intravenous injection, and dissolved with stirring to prepare a clear solution of 0.4 mg/mL. The clear solution was filtered by a microporous membrane, and then ready for use. The vehicle in the oral group was 5% DMSO+95% (20% HP-β-CD). The compound to be assayed was mixed with the vehicle, and dissolved with stirring to prepare a solution of 0.3 mg/mL. Cynomolgus monkeys were administered 1 mg/kg intravenously or 3 mg/kg orally, and then whole blood was collected for a certain period. Plasma was prepared. The drug concentration was analyzed by LC-MS/MS method, and the pharmacokinetic parameters were calculated by Phoenix WinNonlin software (Pharsight, USA).
Note: DMSO: dimethyl sulfoxide: HP-β-CD: hydroxypropyl-β-cyclodextrin.
Conclusion: The compound of the present disclosure exhibits excellent oral exposure and bioavailability.
Male beagle dogs were used as assay animals. After a single administration, the plasma concentration of the compound was measured and the pharmacokinetic behavior was evaluated.
Five healthy adult male beagle dogs were selected, wherein 2 were in an intravenous injection group, and 3 were in an oral group. The vehicle in the intravenous injection group was 5% DMSO+95% (20% HP-β-CD). The compound to be assayed was mixed with an appropriate amount of vehicle for intravenous injection, and dissolved with stirring to prepare a clear solution of 0.4 mg/mL. The clear solution was filtered by a microporous membrane, and then ready for use. The vehicle in the oral group was 5% DMSO+95% (20% HP-β-CD). The compound to be assayed was mixed with the vehicle, and dissolved with stirring to prepare a solution of 0.3 mg/mL. Cynomolgus monkeys were administered 1 mg/kg intravenously or 3 mg/kg orally, and then whole blood was collected for a certain period. Plasma was prepared. The drug concentration was analyzed by LC-MS/MS method, and the pharmacokinetic parameters were calculated by Phoenix WinNonlin software (Pharsight, USA).
Note: DMSO: dimethyl sulfoxide: HP-β-CD: hydroxypropyl-β-cyclodextrin.
Conclusion: The compound of the present disclosure exhibits excellent oral exposure and bioavailability.
A fully automated patch-clamp method was used to assay the effect of the compound on hERG potassium channel (human Ether-a-go-go Related Gene potassium channel) current.
CHO-hERG cells were cultured in a 175 cm2 culture flask. When cells grew to a density of 60˜80%, the culture medium was removed. Cells were washed once with 7 mL of PBS (Phosphate Buffered Saline), and then 3 mL of Detachin was added for digestion. After the digestion was completed, 7 mL of culture medium was added for neutralization, and then the mixture was centrifuged. The supernatant was aspirated, and then 5 mL of culture medium was added to resuspend the cells and ensure that the cell density was 2˜5×106/mL.
Extracellular solution formulation (mM): 140 NaCl, 5 KCl, 1 CaCl2), 1.25 MgCl2, 10 HEPES and 10 Glucose: the formulation was adjusted to pH of 7.4 with NaOH.
Intracellular solution formulation (mM): 140 KCl, 1 MgCl2, 1 CaCl2), 10 EGTA and 10 HEPES: the formulation was adjusted to pH of 7.2 with KOH.
The single-cell high-impedance sealing and whole-cell pattern formation processes were all automatically performed by a Qpatch instrument. After obtaining the whole-cell recording mode, the cells were clamped at −80 mV. The cells were applied successively with a pre-voltage of −50 mV for 50 milliseconds and a depolarizing stimulus of ±40 mV for 5 seconds, then repolarized to −50 mV for 5 seconds, and then back to −80 millivolts. This voltage stimulation was applied every 15 seconds, and recorded for 2 minutes. The extracellular solution was then given, and recorded for 5 minutes. Then a drug administration process started. The compound concentration started from the lowest assay concentration, and each assay concentration was given for 2.5 minutes. After all concentrations were given successively, a positive control compound 3 M Cisapride was given. At least 3 cells were assayed for each concentration (n≥3).
20.00 mM mother liquor of the compound was diluted with DMSO. 10 μL of the mother liquor of the compound was added to 20 μL of DMSO solution, and serially diluted 3-fold to 6 DMSO concentrations. 4 μL of the compound with 6 DMSO concentrations was respectively added to 396 μL of the extracellular solution, and serially diluted 100-fold to 6 intermediate concentrations. 80 μL of the compound with 6 intermediate concentrations was respectively added to 320 μL of the extracellular solution, and serially diluted 5-fold to the final concentration to be assayed. The highest assayed concentration was 40 μM, and there was a total of 6 concentrations: 40, 13.3, 4.4, 1.48, 0.494 and 0.165 μM, respectively. The DMSO content in the final assay concentration did not exceed 0.2%. This concentration of DMSO had no effect on the hERG potassium channel. All dilutions in the compound preparation were performed by a Bravo instrument.
Assay data were analyzed by GraphPad Prism 5.0 software.
The inhibitory effect of Cisapride with multiple concentrations on the hERG channel was measured as a positive control.
Conclusion: The compound of the present disclosure has a weak inhibitory effect on hERG potassium channel current, thereby lowering the risk of cardiotoxicity and improving safety.
The binding degree of the assay compound to human/mouse/rat/canine/monkey plasma albumin was studied.
Conclusion: The compound of the present disclosure has moderate plasma protein binding.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202110722003.6 | Jun 2021 | CN | national |
| 202111673614.2 | Dec 2021 | CN | national |
| 202210693548.3 | Jun 2022 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/102031 | 6/28/2022 | WO |
