Patent Application
20240335462
The application claims the right of the following priorities: CN202110801875.1, application date: Jul. 15, 2021; CN202110875486.3, application date: Jul. 30, 2021; CN202111162655.5, application date: Sep. 30, 2021; CN202111273381.7, application date: Oct. 29, 2021; CN202210039225.2, application date: Jan. 13, 2022; CN202210260637.9, application date: Mar. 16, 2022; CN202210731484.1, application date: Jun. 24, 2022.
The present disclosure relates to a new sulfur/phosphorus-containing aryl compound and a use thereof, and specifically relates to a compound of formula (V) and a pharmaceutically acceptable salt thereof.
JAK is a category of non-receptor tyrosine kinases, comprising four subtypes: JAK1, JAK2, JAK3, and TYK2. The JAK-STAT signaling pathway mediated by them is related to cell proliferation, differentiation, apoptosis, and immune regulation. The JAK-STAT pathway is essential for immune responses, and during inflammation, the overactivation of JAK conversely promotes the progression of the disease. As research into the mechanism of the JAK-STAT pathway in various autoimmune diseases deepens, JAK inhibitors, as the latest type of targeted autoimmune drugs, have gradually received marketing approval for rheumatoid arthritis (RA), psoriatic arthritis (PsA), and atopic dermatitis, with more indications in late-stage clinical development, including ankylosing spondylitis (AS), ulcerative colitis (UC), Crohn's disease, and others. Research has revealed that TYK2 mediates the signaling of IL-6, IL-10, IL-12, IL-23, and type I interferon, encompassing the key cytokines IL-12 and IL-23, which are currently considered critical in the progression of psoriasis. Consequently, TYK2 inhibitors are considered to be important targets for treating a substantial population with another autoimmune disease—psoriasis. However, as of now, no TYK2 inhibitors have been approved.
BMS-986165, a selective allosteric TYK2 inhibitor, is the most advanced candidate compound in this field. Its phase III trials have demonstrated clinical efficacy comparable to first-line biologics, significantly superior to the oral standard therapy apremilast (a PDE4 inhibitor). Moreover, it has a low incidence of adverse reactions, with a discontinuation rate due to adverse events lower than that of both the apremilast group and the placebo group. The mechanism of action of BMS-986165 is unique. Unlike other JAK inhibitors, it targets the JH2 pseudokinase domain of TYK2. However, similar to orthosteric inhibitors, it suppresses the kinase activity of TYK2, thereby exerting its effect. Notably, it achieves high kinase selectivity (>1000-fold). This demonstrates the substantial clinical application potential of high-selectivity TYK2 inhibitors in the treatment of psoriasis and other targets, signifying considerable clinical application value.
The present disclosure provides a compound of formula (V) or a pharmaceutically acceptable salt thereof,
and the —P(═O)(C1-3 alkyl)2, —P(═O)(C3-5 cycloalkyl)2, —S(═O)nC1-4 alkyl, —S(═O)nC1-3 alkylamino, —S(═O))n-4- to 5-membered heterocycloalkyl, —S(═O))nNH2, —S(═O)(═NR)C1-4 alkyl, —S(═O)(═NR)C1-3 alkylamino, —S(═O)(═NR)C3-5 cycloalkyl,
are each independently and optionally substituted by 1, 2, or 3 halogens;
The present disclosure provides a compound of formula (V) or a pharmaceutically acceptable salt thereof,
and the —P(═O)(C1-3 alkyl)2, —P(═O)(C3-5 cycloalkyl)2, —S(═O)nC1-4 alkyl, —S(═O)nC1-3 alkylamino, —S(═O))n-4- to 5-membered heterocycloalkyl, —S(═O))nNH2, —S(═O)(═NR)C1-4 alkyl, —S(═O)(═NR)C1-3 alkylamino, —S(═O)(═NR)C3-5 cycloalkyl,
are each independently and optionally substituted by 1, 2, or 3 halogens;
In some embodiments of the present disclosure, the L is selected from —NH— and —NHC(═O)—, and other variables are as defined in the present disclosure. In some embodiments of the present disclosure, the R1 is selected from OCH3, and the OCH3 is optionally substituted by 1, 2, or 3 Ra, and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the R1 is selected from OCH3 and OCF3, and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the R1 is selected from OCH3, and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the R2 is selected from H and CH3, and the CH3 is optionally substituted by 1, 2, or 3 Rb, and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the R2 is selected from H, CH3, and CD3, and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the R3 is selected from
and other variables are as defined in the present disclosure. In some embodiments of the present disclosure, the ring C is selected from phenyl, pyridyl, pyrimidinyl, pyridazinyl, and pyrazinyl, and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the structural moiety
is selected from
and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the structural moiety
is selected from
and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the Re is selected from F and CH3, and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the R5 is selected from CH3, cyclopropyl, imidazolyl, pyrazolyl, and pyridyl, and the CH3, cyclopropyl, imidazolyl, pyrazolyl, and pyridyl are each independently and optionally substituted by 1, 2, or 3 Re, and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the R5 is selected from CH3,
and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the structural moiety
is selected from
and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the compound or the pharmaceutically acceptable salt thereof is provided, and the compound is selected from:
and the —P(═O)(C1-3 alkyl)2, —P(═O)(C3-5 cycloalkyl)2, —S(═O)nC1-3 alkyl, —S(═O)nC1-3 alkylamino, —S(═O))n-4- to 5-membered heterocycloalkyl, —S(═O))nNH2, —S(═O)(═NR)C1-3 alkyl, —S(═O)(═NR)C1-3 alkylamino, —S(═O)(═NR)C3-5 cycloalkyl,
are each independently and optionally substituted by 1, 2, or 3 halogens;
In some embodiments of the present disclosure, the R1 is selected from OCH3, and the OCH3 is optionally substituted by 1, 2, or 3 Ra, and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the R1 is selected from OCH3 and OCF3, and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the R2 is selected from H and CH3, and the CH3 is optionally substituted by 1, 2, or 3 Rb, and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the R2 is selected from H, CH3, and CD3, and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the R3 is selected from
and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the R3 is selected from
and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the structural moiety
is selected from
and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the structural moiety
is selected from
and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the structural moiety
is selected from
and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the R5 is selected from CH3, cyclopropyl, imidazolyl, pyrazolyl, and pyridyl, and the CH3, cyclopropyl, imidazolyl, pyrazolyl, and pyridyl are each independently and optionally substituted by 1, 2, or 3 Re, and other variables are as defined in the present disclosure. In some embodiments of the present disclosure, the R5 is selected from CH3,
and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the structural moiety
is selected from
and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the compound or the pharmaceutically acceptable salt thereof is provided, and the compound is selected from
In some embodiments of the present disclosure, the compound or the pharmaceutically acceptable salt thereof is provided, and the compound is selected from
In some embodiments of the present disclosure, the R1 is selected from OCH3, and the OCH3 is optionally substituted by 1, 2, or 3 Ra, and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the R1 is selected from OCH3 and OCF3, and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the R2 is selected from H and CH3, and the CH3 is optionally substituted by 1, 2, or 3 Rb, and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the R2 is selected from CH3, and the CH3 is optionally substituted by 1, 2, or 3 Rb, and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the R2 is selected from H, CH3, and CD3, and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the R2 is selected from CH3 and CD3, and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the R3 is independently selected from
and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the R3 is independently selected from
and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the structural moiety
is selected from
and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the structural moiety
is selected from
and other variables are as defined in the present disclosure.
In some embodiment of the present disclosure, the structural moiety
is selected from
and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the structural moiety
is selected from
and other variables are as defined in the present disclosure.
The present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof,
In some embodiments of the present disclosure, the R1 is selected from OCH3, and the OCH3 is optionally substituted by 1, 2, or 3 Ra, and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the R1 is selected from OCH3 and OCF3, and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the R2 is selected from CH3, and the CH3 is optionally substituted by 1, 2, or 3 Rb, and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the R2 is selected from CH3 and CD3, and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the ring C is selected from
and other variables are as defined in the present disclosure.
In some embodiments of the present disclosure, the ring C is selected from
and other variables are as defined in the present disclosure. In some embodiments of the present disclosure, the structural moiety
is selected from
and other variables are as defined in the present disclosure.
There are still some embodiments of the present disclosure which are obtained by any combination of the above variables.
In some embodiments of the present disclosure, the compound or the pharmaceutically acceptable salt thereof is selected from:
The present disclosure provides a compound of the following formula or a pharmaceutically acceptable salt thereof,
In some embodiments of the present disclosure, the compound or the pharmaceutically acceptable salt thereof is selected from:
The compound of the present disclosure acts on the JH2 pseudokinase domain of TYK2 and exhibits a good inhibitory effect on TYK2 kinase. The compound of the present disclosure shows high inhibitory activity against cell proliferation in the Ba/F3-FL-TYK2-E957D cell line, which has a point mutation in the TYK2 gene. In human PBMC cells, the compound of the present disclosure demonstrates strong inhibitory activity against the TYK2 signaling pathway activated by IFN-α stimulation, and also exhibits weak inhibitory activity against the JAK1/2 signaling pathway activated by IL-6 stimulation, the JAK2/2 signaling pathway activated by GM-CSF stimulation, and the JAK1/3 signaling pathway activated by IL-2 stimulation, thereby showing high selectivity. The compound of the present disclosure possesses excellent pharmacokinetic properties. The compound of the present disclosure has a significant dose-dependent inhibitory effect on the release of IFNγ in mice induced by IL-12/IL-18. Additionally, the compound of the present disclosure has a significant alleviating effect on colitis and psoriasis.
Unless otherwise specified, the following terms and phrases when used herein 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 ordinary sense. When a trading 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, an allergic reaction, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
The term “pharmaceutically acceptable salt” refers to a salt of the compound of the present disclosure that is prepared by reacting the compound having a specific substituent of the present disclosure with a relatively non-toxic acid or base. When the compound of the present disclosure contains a relatively acidic functional group, a base addition salt can be obtained by contacting the compound with a sufficient amount of base in a pure solution or a suitable inert solvent. When the compound of the present disclosure contains a relatively basic functional group, an acid addition salt can be obtained by contacting the compound with a sufficient amount of acid in a pure solution or a suitable inert solvent. Certain specific compounds of the present disclosure contain both basic and acidic functional groups, thus can be converted to any base or acid addition salt.
The pharmaceutically acceptable salt of the present disclosure can be prepared from the parent compound that contains an acidic or basic moiety by conventional chemical method. 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 a geometric isomer, a cis-trans isomer, a stereoisomer, an enantiomer, an optical isomer, a diastereoisomer, and a tautomeric isomer.
The compounds of the present disclosure may exist in specific geometric or stereoisomeric forms. The present disclosure contemplates all such compounds, including cis and trans isomers, (−)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereoisomers, (D)-isomers, (L)-isomers, racemic mixtures, and other mixtures thereof, such as enantiomers or diastereomer enriched mixtures, all of which are within the scope of the present disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl. All these isomers and their mixtures are included within the scope of the present disclosure.
Unless otherwise specified, the term “enantiomer” or “optical isomer” refers to stereoisomers that are mirror images of each other.
Unless otherwise specified, the term “cis-trans isomer” or “geometric isomer” is caused by the inability to rotate freely of double bonds or single bonds of ring-forming carbon atoms.
Unless otherwise specified, the term “diastereomer” refers to a stereoisomer in which a molecule has two or more chiral centers and the relationship between the molecules is not mirror images.
Unless otherwise specified, “(+)” refers to dextrorotation, “(−)” refers to levorotation, and “(±)” refers to racemic.
Unless otherwise specified, the absolute configuration of a stereogenic center is represented by a wedged solid bond () and a wedged dashed bond (
), and the relative configuration of a stereogenic center is represented by a straight solid bond (
) and a straight dashed bond (
), a wave line (
) is used to represent a wedged solid bond (
) or a wedged dashed bond (
), or the wave line (
) is used to represent a straight solid bond (
) or a straight dashed bond (
).
Unless otherwise specified, the terms “enriched in one isomer”, “enriched in isomers”, “enriched in one enantiomer”, or “enriched in enantiomers” refer to the content of one of the isomers or enantiomers is less than 100%, and the content of the isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.
Unless otherwise specified, the term “isomer excess” or “enantiomeric excess” refers to the difference between the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90%, and the content of the other isomer or enantiomer is 10%, the isomer or enantiomer excess (ee value) is 80%.
Optically active (R)- and (S)-isomers, or D and L isomers can be prepared using chiral synthesis, chiral reagents, or other conventional techniques. If one kind of enantiomer of certain compound of the present disclosure is to be obtained, it can be obtained by asymmetric synthesis or derivative action of chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as amino) or an acidic functional group (such as carboxyl), a salt of a diastereoisomer is formed with an appropriate optically active acid or base, and then diastereomeric resolution is performed by conventional methods known in the art, and then the pure enantiomer is recovered. In addition, the enantiomer and the diastereoisomer are generally separated through chromatography which uses a chiral stationary phase and optionally combines with a chemical derivative method (such as carbamate generated from amine).
The compound of the present disclosure may contain an unnatural proportion of atomic isotopes at one or more than one atom that constitutes the compound. For example, the compound can be radiolabeled with a radioactive isotope, such as tritium (3H), iodine-125 (125), or C-14 (14C). For another example, deuterated drugs can be formed by replacing hydrogen with deuterium, the bond formed by deuterium and carbon is stronger than that of ordinary hydrogen and carbon, compared with non-deuterated drugs, deuterated drugs have the advantages of reduced toxic and side effects, increased drug stability, enhanced efficacy, extended biological half-life of drugs, etc. All isotopic variations of the compound of the present disclosure, whether radioactive or not, are encompassed 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 on a specific atom is substituted by the substituent, including deuterium and hydrogen variables, as long as the valence of the specific atom is normal and the substituted compound is stable. When the substituent is an oxygen (i.e., ═O), it means two hydrogen atoms are substituted. Positions on an aromatic ring cannot be substituted with a ketone.
The term “optionally substituted” means an atom can be substituted by a substituent or not, unless otherwise specified, the type and number of the substituent may be arbitrary as 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 to 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 a substituent is 0 in number, it means that the substituent is absent. In the case of -A-(R)0, the structure is actually -A.
When a substituent is vacant, it means that the substituent is absent, for example, when X is vacant in A-X, the structure of A-X is actually A.
When one of the variables is selected from 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, this substituent can be bonded to any atom on this ring. For example, the structural moiety
indicates that the substituent R can be substituted at any position on cyclohexyl or cyclohexadiene. When the enumerative substituent does not indicate by which atom it is linked to the group to be substituted, such substituent can be bonded by any atom thereof. For example, when pyridyl acts as a substituent, it can be linked to the group to be substituted by any carbon atom on the pyridine ring.
When the enumerative linking group does not indicate the direction for linking, the direction for linking is arbitrary, for example, the linking group L contained in
is -M-W—, then -M-W— can link ring A and ring B to form
in the direction same as left-to-right reading order, and form
in the direction contrary to left-to-right reading order. A combination of the linking groups, substituents and/or variables thereof is allowed only when such combination can result in a stable compound.
Unless otherwise specified, when a group has one or more than one linkable site, any one or more than one site of the group can be linked to other groups through chemical bonds. When the linking site of the chemical bond is not positioned, and there is an H atom at the linkable site, then the number of H atoms at the site will decrease correspondingly with the number of the chemical bonds linking thereto so as to meet the corresponding valence. The chemical bond between the site and other groups can be represented by a straight solid bond (), a straight dashed bond (
), or a wavy line
For example, the straight solid bond in —OCH3 means that it is linked to other groups through the oxygen atom in the group; the straight dashed bond in
means that it is linked to other groups through the two ends of the nitrogen atom in the group; the wave line in
means that the phenyl group is linked to other groups through carbon atoms at position 1 and position 2;
means that it can be linked to other groups through any linkable sites on the piperidinyl by one chemical bond, including at least four types of linkage, including
Even though the H atom is drawn on the —N—,
still includes the linkage of
merely when one chemical bond was connected, the H of this site will be reduced by one to the corresponding monovalent piperidinyl.
Unless otherwise specified, the number of atoms in a ring is usually defined as the number of ring members, for example, “5- to 7-membered ring” refers to a “ring” in which 5 to 7 atoms are arranged around.
Unless otherwise specified, the term “C1-4 alkyl” refers to a linear or branched saturated hydrocarbon group consisting of 1 to 4 carbon atoms. The C1-4 alkyl includes C1-2, C1-3, C2-3 alkyl, etc.; it can be monovalent (such as methyl), divalent (such as methylene), or multivalent (such as methine). Examples of C1-4 alkyl include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, s-butyl, and t-butyl), etc.
Unless otherwise specified, the term “C1-3 alkyl” refers to a linear or branched saturated hydrocarbon group consisting of 1 to 3 carbon atoms. The C1-3 alkyl includes C1-2, C2-3 alkyl, etc.; it can be monovalent (such as methyl), divalent (such as methylene), or multivalent (such as methine). Examples of C1-3 alkyl include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), etc.
Unless otherwise specified, the term “C1-3 alkoxy” refers to an alkyl group containing 1 to 3 carbon atoms that are connected to the rest of the molecule through an oxygen atom. The C1-3 alkoxy includes C1-2, C2-3, C3, C2 alkoxy, etc. Examples of C1-3 alkoxy include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), etc.
Unless otherwise specified, the term “C1-3 alkylamino” refers to an alkyl group containing 1 to 3 carbon atoms that are connected to the rest of the molecule through an amino group. The C1-3 alkylamino includes C1-2, C3, C2 alkylamino, etc. Examples of C1-3 alkylamino include, but are not limited to, —NHCH3, —N(CH3)2, —NHCH2CH3, —N(CH3)CH2CH3, —NHCH2CH2CH3, —NHCH2(CH3)2, etc.
Unless otherwise specified, “C3-5 cycloalkyl” refers to a saturated cyclic hydrocarbon group consisting of 3 to 5 carbon atoms, which is a monocyclic system, and the C3-5 cycloalkyl includes C3-4, C4-5 cycloalkyl, etc.; it can be monovalent, divalent, or multivalent. Examples of C3-5 cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, etc.
Unless otherwise specified, the term “4- to 5-membered heterocycloalkyl” by itself or in combination with other terms refers to a saturated monocyclic group consisting of 4 to 5 ring atoms, wherein 1, 2, 3, or 4 ring atoms are heteroatoms independently selected from O, S, and N, and the rest are carbon atoms, wherein nitrogen atoms are optionally quaternized, and nitrogen and sulfur heteroatoms can be optionally oxidized (i.e., NO and S(O)p, p is 1 or 2). In addition, with regard to the “4- to 5-membered heterocycloalkyl”, a heteroatom may occupy the connection position of the heterocycloalkyl with the rest of the molecule. The 4- to 5-membered heterocycloalkyl includes 4-membered and 5-membered heterocycloalkyl. Examples of 4- to 5-membered heterocycloalkyl include, but are not limited to, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrothiophenyl (including tetrahydrothiophen-2-yl, tetrahydrothiophen-3-yl, etc.), tetrahydrofuranyl (including tetrahydrofuran-2-yl, etc.), etc.
Unless otherwise specified, the terms “5- to 6-membered heteroaryl ring” and “5- to 6-membered heteroaryl” in the present disclosure can be used interchangeably, and the term “5- to 6-membered heteroaryl” refers to a monocyclic group consisting of 5 to 6 ring atoms with a conjugated π-electron system, wherein 1, 2, 3, or 4 ring atoms are heteroatoms independently selected from O, S, and N, and the rest are carbon atoms, wherein nitrogen atoms are optionally quaternized, and nitrogen and sulfur heteroatoms can be optionally oxidized (i.e., NO and S(O)p, p is 1 or 2). The 5- to 6-membered heteroaryl can be linked to the rest of the molecule through a heteroatom or a carbon atom. The 5- to 6-membered heteroaryl includes 5-membered and 6-membered heteroaryl. Examples of the 5- to 6-membered heteroaryl include, but are not limited to, pyrrolyl (including N-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, etc.), pyrazolyl (including 2-pyrazolyl, 3-pyrazolyl, etc.), imidazolyl (including N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, etc.), oxazolyl (including 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, etc.), triazolyl (including 1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, 1H-1,2,4-triazolyl, 4H-1,2,4-triazolyl, etc.), tetrazolyl, isoxazolyl (including 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, etc.), thiazolyl (including 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, etc.), furyl (including 2-furyl, 3-furyl, etc.), thienyl (including 2-thienyl, 3-thienyl, etc.), pyridyl (including 2-pyridyl, 3-pyridyl, 4-pyridyl, etc.), pyrazinyl, or pyrimidinyl (including 2-pyrimidinyl, 4-pyrimidinyl, etc.).
The structure of the compounds of the present disclosure can be confirmed by conventional methods known to those skilled in the art, and if the present disclosure involves an absolute configuration of a compound, then the absolute configuration can be confirmed by means of conventional techniques in the art. For example, in the case of single crystal X-ray diffraction (SXRD), diffraction intensity data are collected from the cultured single crystal using a Bruker D8 venture diffractometer with CuKα radiation as the light source and scanning mode: φ/ω scan, and after collecting the relevant data, the crystal structure is further analyzed by direct method (Shelxs97), so that the absolute configuration can be confirmed.
The compounds of the present disclosure can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by their combination with other chemical synthesis methods, and equivalent alternatives known to those skilled in the art, with preferred embodiments including, but not limited to, the examples of the present disclosure.
The solvent used in the present disclosure is commercially available.
In the present disclosure, the following abbreviations are used: aq stands for water; eq stands for equivalent; DCM stands for dichloromethane; PE stands for petroleum ether; DMSO stands for dimethyl sulfoxide; EtOAc stands for ethyl acetate; EtOH stands for ethanol; MeOH stands for methanol; DMF stands for N,N-dimethylformamide; Cbz stands for benzyloxycarbonyl, which is an amine protecting group; Boc stands for tert-butoxycarbonyl, which is an amine protecting group; r.t. stands for room temperature; O/N stands for overnight; THE stands for tetrahydrofuran; Boc2O stands for di-tert-butyl dicarbonate; TFA stands for trifluoroacetic acid; HCl stands for hydrochloric acid; mp stands for melting point; Pd(dppf)Cl2 stands for [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II); Pd(dppf)Cl2·CH2Cl2 stands for [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) dichloromethane complex; TEA stands for triethylamine; Xantphos stands for 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene; Pd2(dba)3 stands for tris(dibenzylideneacetone)dipalladium(0); EDCI stands for 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; HOBt stands for 1-hydroxybenzotriazole; NMP stands for N-methyl-2-pyrrolidone; DIPEA stands for N,N-diisopropylethylamine; LiHMDS stands for lithium bis(trimethylsilyl)amide; Pd2(dba)3·CHCl3 stands for tris(dibenzylideneacetone)dipalladium(0)-chloroform adduct.
The compounds of the present disclosure are named according to the conventional naming principles in the art or by ChemDraw® software, and the commercially available compounds use the supplier catalog names.
The present disclosure is described in detail by the examples below, but it does not mean that there are any adverse restrictions on the present disclosure. The present disclosure has been described in detail herein, and its specific examples have also been disclosed; for one skilled in the art, it is obvious to make various modifications and improvements to the examples of the present disclosure without departing from the spirit and scope of the present disclosure.
Compound A-1-1 (10 g, 49.49 mmol, 1 eq), bis(pinacolato)diboron (18.85 g, 74.24 mmol, 1.5 eq), and potassium acetate (14.57 g, 148.48 mmol, 3 eq) were dissolved in 1,4-dioxane (200 mL). After replacing with nitrogen three times, Pd(dppf)Cl2·CH2Cl2 (2.02 g, 2.47 mmol, 0.05 eq) was added, and the mixture was stirred at 100° C. for 2 hours. Water (200 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (100 mL*2). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (V/V, ethyl acetate/petroleum ether=0 to 25%) to obtain compound A-1. 1HNMR (400 MHz, CDCl3) δ: 7.13-7.11 (m, 1H), 6.93 (t, J=15.2, 7.6 Hz, 1H), 6.88-6.85 (m, 1H), 3.81 (s, 3H), 1.36 (s, 12H); LCMS m/z=250.1 [M+H]+.
Compound A-2-1 (18.5 g, 96.35 mmol, 1 eq) was dissolved in DCM (200 mL). Oxalyl chloride (15.90 g, 125.26 mmol, 10.96 mL, 1.3 eq) was then added to the mixture, and DMF (352.15 mg, 4.82 mmol, 370.68 μL, 0.05 eq) was added thereto. The reaction was carried out at 25° C. for 16 hours. The reaction system was then concentrated, added with dichloromethane (50 mL), concentrated again, and directly used in the next step to obtain compound A-2-2.
Compound A-2-2 (20 g, 95.04 mmol, 1 eq) was added to DCM (300 mL). Deuterated methylamine hydrochloride (5.36 g, 76.03 mmol, 0.8 eq) was then added. After cooling the mixture to 0° C., DIPEA (36.85 g, 285.11 mmol, 49.66 mL, 3 eq) was added, and the reaction was carried out at 20° C. for 16 hours. After the reaction was completed, a saturated ammonium chloride aqueous solution (100 mL) was added to the system for extraction and phase separation. The aqueous phase was then extracted once with dichloromethane (60 mL). The organic phases were combined, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. tert-Butyl methyl ether (100 mL) was added to the crude product. The mixture was stirred for 2 hours, filtered, and the filter cake was concentrated under reduced pressure to obtain compound A-2. 1H NMR (400 MHz, DMSO-d6) δ: 8.59 (s, 1H), 8.48 (s, 1H), 7.91 (s, 1H); LCMS m/z=208.1 [M+1]+.
Compound A-3-1 (20 g, 96.61 mmol, 1 eq) was dissolved in acetonitrile (100 mL) and water (15 mL). Lithium bromide (25.17 g, 289.84 mmol, 7.28 mL, 3 eq) and DIPEA (37.46 g, 289.84 mmol, 50.48 mL, 3 eq) were added thereto. The mixture was stirred and reacted at 20° C. for 3 hours. The system was filtered, and the filter cake was rinsed with acetonitrile (50 mL). The filter cake was collected to obtain compound A-3-2. LCMS m/z=192.9 [COOH+1]+.
Compound A-3-2 (10 g, 50.27 mmol, 1 eq) was dissolved in DCM (150 mL). Oxalyl chloride (8.93 g, 70.38 mmol, 6.16 mL, 1.4 eq) was then added to the mixture, and DMF (183.72 mg, 2.51 mmol, 193.39 μL, 0.05 eq) was added thereto. The reaction was carried out at 20° C. for 4 hours. The reaction system was concentrated, then dichloromethane (30 mL) was added, and the mixture was further concentrated. The concentrated mixture was added to dichloromethane (150 mL), and deuterated methylamine hydrochloride (3.37 g, 47.76 mmol, 0.95 eq) was added. After cooling to 0° C., DIPEA (19.49 g, 150.83 mmol, 26.27 mL, 3 eq) was added. The reaction was carried out at 20° C. for 16 hours. After the reaction was completed, water (150 mL) was added to the system for extraction and phase separation. The aqueous phase was then extracted once with dichloromethane (150 mL). The organic phases were combined, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether=0 to 20%) to obtain compound A-3. LCMS m/z=209.0 [M+1]+.
Compound A-4-1 (3 g, 12.48 mmol, 1 eq), dimethylphosphine oxide (1.02 g, 13.10 mmol, 1.05 eq), potassium phosphate tribasic (3.97 g, 18.72 mmol, 1.5 eq), and Xantphos (721.98 mg, 1.25 mmol, 0.1 eq) were dissolved in 1 4-dioxane (30 mL). After replacing with nitrogen three times, palladium acetate (280.13 mg, 1.25 mmol, 0.1 eq) was added. The mixture was stirred at 120° C. for 2 hours. The reaction mixture was filtered, and the filter cake was rinsed with ethyl acetate (30 mL*2). The filtrate was extracted with water (30 mL), and the organic phase was dried over anhydrous sodium sulfate and then concentrated. The crude product was purified by silica gel column chromatography (methanol/dichloromethane=0 to 20%) to obtain compound A-4. LCMS m/z=191.0 [M+H]+.
Compound A-5-1 (10 g, 35.35 mmol, 1 eq), dimethylphosphine oxide (2.76 g, 35.35 mmol, 1 eq), triethylamine (4.18 g, 41.36 mmol, 5.76 mL, 1.17 eq), and Xantphos (204.53 mg, 353.48 μmol, 0.01 eq) were dissolved in 1 4-dioxane (50 mL) and THF (50 mL). After replacing with nitrogen three times, tris(dibenzylideneacetone)dipalladium(0) (161.84 mg, 176.74 μmol, 0.005 eq) was added. The mixture was stirred at 15° C. for 2 hours. The reaction mixture was added with water (50 mL) and extracted with ethyl acetate (100 mL*2). The organic phase was dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (methanol/dichloromethane=0 to 9%) to obtain compound A-5. LCMS m/z=232.8 [M+H]+.
To synthesize reference examples 6 and 7 listed in Table 1, follow the synthetic steps of reference example 5, replacing dimethylphosphine oxide in step 1 with fragment 2, and replacing A-5-1 with fragment 1.
A-8-1 (1.38 g, 10.00 mmol, 1.29 mL, 1 eq) was dissolved in THE (30 mL). The mixture was cooled to −70° C., and a solution of cyclopropylmagnesium bromide in tetrahydrofuran (0.5 M, 3.09 g, 30 mmol, 60 mL, 3 eq) was added dropwise thereto. The mixture was stirred for 2 hours, then slowly warmed to 25° C., and stirred for 3 hours. The mixture was then cooled to 0° C., added with 0.5 M hydrochloric acid aqueous solution (40 mL) to quench the reaction, and then extracted with ethyl acetate (30 mL*3). The organic phases were combined, washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain compound A-8-2. LCMS m/z=131.2 [M+H]+.
2-Bromo-5-iodopyridine (800 mg, 2.82 mmol, 1 eq), A-8-2 (366.69 mg, 2.82 mmol, 1 eq), and Xantphos (163.05 mg, 281.80 μmol, 0.1 eq) were dissolved in 1,4-dioxane (14 mL). Triethylamine (5.64 mmol, 5.64 mL, 2 eq) was added, followed by replacement with nitrogen. Finally, the catalyst Pd2(dba)3 (258.05 mg, 281.80 μmol, 0.1 eq) was added, followed by replacement with nitrogen. The reaction mixture was heated to 75° C. and stirred for 12 hours. After the reaction was completed, the reaction mixture was cooled, added with 20 mL of water, and extracted with dichloromethane (30 mL*2). The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (dichloromethane:methanol=100:0 to 50:1) to obtain compound A-8. LCMS m/z=285.9 [M+1]+.
To synthesize reference examples 9 and 10 listed in Table 2, follow the synthetic steps of reference example 8, replacing cyclopropylmagnesium bromide in step 1 with fragment 1, and replacing 2-bromo-5-iodopyridine in step 2 with fragment 2.
Compound A-3-2 (7 g, 35.19 mmol, 1 eq) was dissolved in DCM (105 mL). Oxalyl chloride (6.25 g, 49.27 mmol, 4.31 mL, 1.4 eq) was then added to the mixture, and DMF (128.60 mg, 1.76 mmol, 135.37 μL, 0.05 eq) was added thereto. The reaction was carried out at 20° C. for 3 hours. The reaction system was concentrated, then dichloromethane (80 mL) was added, and the mixture was further concentrated. The concentrated mixture was added to dichloromethane (150 mL), and methylamine hydrochloride (2.26 g, 33.43 mmol, 0.95 eq) was added. After cooling to −60° C., DIPEA (13.64 g, 105.57 mmol, 18.39 mL, 3 eq) was added. The reaction was carried out at 20° C. for 10 hours. After the reaction was completed, water (80 mL) was added to the system for extraction and phase separation. The aqueous phase was then extracted with dichloromethane (100 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether=0 to 20%) to obtain compound A-11. LCMS m/z=206.0 [M+1]+.
Compound A-2-2 (8.77 g, 41.67 mmol, 1 eq) was dissolved in DCM (100 mL). Methylamine hydrochloride (2.81 g, 41.67 mmol, 1 eq) was then added to the mixture. After the mixture was cooled to 0° C., DIPEA (16.16 g, 125.01 mmol, 21.78 mL, 3 eq) was added thereto. The reaction was carried out at 20° C. for 12 hours. After the reaction was completed, water (50 mL) was added to the system for extraction and phase separation. The aqueous phase was then extracted with dichloromethane (50 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound A-12. LCMS m/z=205.0 [M+1]+.
Compound A-13-1 (6 g, 24.00 mmol, 1 eq) and stannous chloride dihydrate (21.66 g, 95.99 mmol, 4 eq) were dissolved in ethyl acetate (200 mL). The mixture was stirred at 70° C. for 1 hour. After cooling to room temperature, the reaction mixture was added with water (200 mL), and extracted with ethyl acetate (100 mL*2). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (V/V, ethyl acetate/petroleum ether=0 to 10%) to obtain compound A-13-2. LCMS m/z=219.7 [M+H]+.
Compound A-13-2 (2 g, 9.09 mmol, 1 eq), bis(pinacolato)diboron (3.46 g, 13.63 mmol, 1.5 eq), and potassium acetate (2.68 g, 27.27 mmol, 3 eq) were dissolved in 1,4-dioxane (20 mL). After replacing with nitrogen three times, Pd(dppf)Cl2·CH2Cl2 (371.13 mg, 0.45 mmol, 0.05 eq) was added, and the mixture was stirred at 100° C. for 18 hours. After cooling to room temperature, the reaction mixture was added with water (200 mL) and extracted with ethyl acetate (100 mL*2). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (V/V, ethyl acetate/petroleum ether=0 to 25%) to obtain compound A-13. LCMS m/z=267.9 [M+H]+.
Compound A-14-1 (1 g, 6.71 mmol, 1 eq) was dissolved in N,N-dimethylformamide (10 mL), and then sodium 2-propanethiolate (724.39 mg, 7.38 mmol, 1.1 eq) was added thereto. The mixture was stirred at 15° C. for 1 hour. The reaction mixture was added with water (10 mL) and extracted with ethyl acetate (10 mL*2). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound A-14. LCMS m/z=189.2 [M+H]+.
Compound A-15-1 (1 g, 5.20 mmol, 1 eq) was dissolved in N,N-dimethylformamide (20 mL), and then sodium 2-propanethiolate (2.91 g, 29.62 mmol, 5.7 eq) was added thereto. The mixture was stirred at 15° C. for 1 hour. The reaction mixture was added with water (20 mL) and extracted with ethyl acetate (20 mL*2). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound A-15. LCMS m/z=232.0 [M+H]+.
Compound 1-1 (280 mg, 637.15 μmol, 1 eq) was dissolved in DMF (7 mL), and then sodium thiomethoxide (178.63 mg, 2.55 mmol, 162.39 μL, 4 eq) was added thereto. The mixture was stirred at 40° C. for 2 hours. The reaction mixture was added with water (10 mL) and ethyl acetate (10 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (10 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain compound 1-2. LCMS m/z=468.1 [M+H]+.
Compound 1-2 (40 mg, 85.55 μmol, 1 eq) was dissolved in DCM (2 mL). The mixture was cooled to 0° C. and added with 3-chloroperoxybenzoic acid (26.05 mg, 128.33 μmol, purity of 85%, 1.5 eq). The reaction was carried out at 20° C. for 2 hours. 3-chloroperoxybenzoic acid was then added thereto (8.68 mg, 42.78 μmol, purity of 85%, 0.5 eq). The reaction was carried out for another 2 hours. The system was added with water (10 mL) and ethyl acetate (10 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (10 mL). The organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was separated by preparative high performance liquid chromatography (chromatographic column: Phenomenex Luna 80*30 mm*3 m; mobile phase: A (water containing 0.04% hydrochloric acid) and B (acetonitrile); gradient: B %: 5% to 35%, 8 minutes) to obtain hydrochloride of compound WX-001 and hydrochloride of compound WX-002.
Hydrochloride of WX-001: 1H NMR (400 MHz, CD3OD) δ: 9.40 (s, 2H), 8.34 (s, 1H), 7.99-7.96 (m, 1H), 7.67-7.66 (m, 1H), 7.46 (t, J=8.0 Hz, 1H), 6.54 (s, 1H), 3.82 (s, 3H), 3.35 (s, 3H), 1.81-1.70 (m, 1H), 1.11-1.01 (m, 4H); LCMS m/z=500.1 [M+H]+.
Hydrochloride of WX-002: 1H NMR (400 MHz, CD3OD) δ: 9.21 (s, 2H), 8.36 (s, 1H), 7.93-7.91 (m, 1H), 7.66-7.64 (m, 1H), 7.44 (t, J=8.0 Hz, 1H), 6.56 (s, 1H), 3.78 (s, 3H), 3.06 (s, 3H), 1.81-1.72 (m, 1H), 1.11-1.01 (m, 4H); LCMS m/z=484.1 [M+H]+.
Compound 1-2 (25 mg, 53.47 μmol, 1 eq) was dissolved in MeOH (0.2 mL), and then (diacetoxyiodo)benzene (51.67 mg, 160.41 μmol, 3 eq) and ammonium acetate (16.49 mg, 213.88 μmol, 4 eq) were added thereto. The reaction was carried out at 25° C. for 2 hours. The system was added with water (5 mL) and ethyl acetate (5 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (5 mL). The organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was purified by silica gel thin-layer chromatography (V:V:V, EtOAc:DCM:MeOH=5:5:1) to obtain compound WX-003. 1H NMR (400 MHz, CDCl3) δ: 10.37 (s, 1H), 9.34 (s, 2H), 9.13 (s, 1H), 8.25 (s, 1H), 8.05 (s, 1H), 7.66 (d, J=8.0 Hz, 2H), 7.25 (t, J=8.0 Hz, 1H), 6.65 (s, 1H), 3.85 (s, 3H), 3.25 (s, 3H), 1.60-1.56 (m, 1H), 1.06-1.02 (m, 2H), 0.88-0.83 (m, 2H); LCMS m/z=499.2 [M+H]+.
Compound 4-1 (200 mg, 847.15 μmol, 1 eq), compound A-1 (211.04 mg, 847.15 μmol, 1 eq), and sodium carbonate (269.37 mg, 2.54 mmol, 3 eq) were dissolved in DMF (4 mL) and water (0.8 mL). After replacing with nitrogen three times, Pd(dppf)Cl2·CH2Cl2 (69.18 mg, 84.71 μmol, 0.1 eq) was added thereto. The reaction was carried out at 80° C. for 12 hours. The reaction mixture was added with water (10 mL) and ethyl acetate (10 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (10 mL). The organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated to obtain compound 4-2. LCMS m/z=279.0 [M+H]+.
Compound 4-2 (180 mg, 646.72 μmol, 1 eq) and compound A-2 (161.47 mg, 776.07 mol, 1.2 eq) were added to THE (4.5 mL). After cooling to 0° C., the mixture was added with LiHMDS (1 M, 1.94 mL, 3.0 eq). The reaction was carried out at 20° C. for 2 hours. After the reaction was completed, the reaction mixture was added with water (20 mL) and ethyl acetate (20 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (20 mL). The organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated to obtain compound 4-3. LCMS m/z=450.1 [M+H]+.
Compound 4-3 (270 mg, 600.10 μmol, 1 eq) and cyclopropanecarboxamide (1.28 g, 15.00 mmol, 25 eq) were added to 1,4-dioxane (20 mL) and NMP (4 mL). Cesium carbonate (586.57 mg, 1.80 mmol, 3 eq) and Xantphos (52.08 mg, 90.01 μmol, 0.15 eq) were then added thereto. After replacing with nitrogen three times, Pd2(dba)3 (82.43 mg, 90.01 μmol, 0.15 eq) was added. The reaction was carried out at 120° C. for 18 hours. After the reaction was completed, the reaction mixture was added with water (30 mL) and ethyl acetate (30 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (30 mL). The organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether=0 to 100%) to obtain compound WX-004. 1H NMR (400 MHz, CDCl3) δ: 10.55 (s, 1H), 9.22 (s, 1H), 8.74 (s, 1H), 8.30 (s, 1H), 8.23-8.18 (m, 2H), 8.10 (s, 1H), 7.60 (d, J=8.0 Hz, 2H), 7.30 (t, J=8.0 Hz, 1H), 6.55 (s, 1H), 3.57 (s, 3H), 3.17 (s, 3H), 1.59-1.55 (m, 1H), 1.09-1.05 (m, 2H), 0.91-0.86 (m, 2H); LCMS m/z=499.1 [M+H]+.
To synthesize example 7 listed in Table 3, follow the synthesis steps of example 4, replacing 4-1 in step 1 with fragment 7-1 in the table below.
1H NMR (400 MHz, MeOD) δ: 8.34 (s, 1H), 8.06 (d, J = 8.0 Hz, 2H), 7.88 (d, J = 8.0 Hz, 2H), 7.56-7.53 (m, 1H), 7.49-7.46 (m, 1H), 7.41 (t, J = 8.0 Hz, 1H), 6.68 (s, 1H), 3.47 (s, 3H), 3.19 (s, 3H), 1.80-1.76 (m, 1H), 1.12- 1.09 (m, 2H), 1.06-1.02 (m, 2H); LCMS m/z = 498.1 [M + H]+. Purification method: preparative high performance liquid chromatography (chromatographic column: Phenomenex Luna C18 80 * 40 mm * 3 μm; mobile phase: A (water containing 0.04% hydrochloric acid) and B (acetonitrile); gradient: B %: 23% to 42%, 6 minutes).
To synthesize example 26 listed in Table 4, follow the synthesis steps of example 4, replacing 4-1 in step 1 with fragment 7-1 in the table below, replacing A-1 with A-13, and replacing A-2 in step 2 with A-12.
1H NMR (400 MHz, CDCl3) δ: 12.45 (s, 1H), 11.32 (s, 1H), 8.27 (s, 1H), 8.19 (s, 1H), 7.97 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 8.4 Hz, 2H), 7.17 (dd, J = 3.0, 8.6 Hz, 1H), 6.94 (dd, J = 3.0, 8.4 Hz, 2H), 3.31 (s, 3H), 3.06 (s, 3H), 3.06 (d, J = 4.8 Hz, 3H), 1.91-1.87 (m, 1H), 1.06-0.99 (m, 2H), 0.96-0.89 (m, 2H); LCMS m/z = 513.1 [M + 1]+. Purification method: preparative high performance liquid chromatography (chromatographic column: Welch Xtimate C18 100 * 40 mm * 3 μm; mobile phase: A (water containing 0.075% trifluoroacetic acid) and B (acetonitrile); gradient: B %: 26% to 56%, 8 minutes).
Compound 1-1 (0.45 g, 1.02 mmol, 1 eq) was dissolved in DMF (5 mL). Cesium carbonate (500.45 mg, 1.54 mmol, 1.5 eq) and benzyl mercaptan (254.37 mg, 2.05 mmol, 239.97 μL, 2 eq) were added thereto, and the mixture was stirred at 40° C. for 1 hour. The reaction mixture was added with water (10 mL) and ethyl acetate (10 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (10 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain compound 5-2. LCMS m/z=544.3 [M+H]+.
Compound 5-2 (200 mg, 367.88 μmol, 1 eq) was dissolved in acetic acid (2 mL) and water (0.6 mL). The mixture was then cooled to 0° C., and N-chlorosuccinimide (221.06 mg, 1.66 mmol, 4.5 eq) was added thereto. The mixture was stirred at 20° C. for 2 hours. Sodium sulfate was added to the system for drying, and the system was directly used in the next step. Compound 5-3 was obtained. LCMS m/z=520.0 [M+H]+.
Compound 5-3 (50 mg, 96.16 μmol, 1 eq) was dissolved in THF (2 mL). The mixture was then cooled to −20° C., and dimethylamine hydrochloride (392.06 mg, 4.81 mmol, 440.52 L, 50 eq) and triethylamine (1.46 g, 14.42 mmol, 2.01 mL, 150 eq) were added thereto. The reaction was carried out at 20° C. for 2 hours. After the reaction was completed, the reaction mixture was added with water (10 mL) and ethyl acetate (10 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (10 mL). The organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was separated by preparative high performance liquid chromatography (chromatographic column: Waters Xbridge BEH C18 100*30 mm*10 m; mobile phase: A (water containing 10 mM ammonium bicarbonate) and B (acetonitrile); gradient: B %: 30% to 65%, 10 minutes) to obtain compound WX-005. 1H NMR (400 MHz, CDCl3) δ:10.44 (s, 1H), 9.18 (s, 2H), 8.30 (s, 1H), 8.26 (s, 1H), 8.12 (s, 1H), 7.75-7.69 (m, 2H), 7.33 (t, J=8.0 Hz, 1H), 6.20 (s, 1H), 3.87 (s, 3H), 2.87 (s, 6H), 1.54-1.51 (m, 1H), 1.11-1.08 (m, 2H), 0.92-0.87 (m, 2H); LCMS m/z=529.2 [M+H]+.
Compound 5-3 (95.6 mg, 183.85 μmol, 1 eq) was dissolved in THE (2 mL). The mixture was then cooled to −20° C., and tetrahydropyrrole (13.08 mg, 183.85 μmol, 15.35 μL, 1 eq) and triethylamine (2.79 g, 27.58 mmol, 3.84 mL, 150 eq) were added thereto. The reaction was carried out at 20° C. for 2 hours. After the reaction was completed, the reaction mixture was added with water (10 mL) and ethyl acetate (10 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (10 mL). The organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was separated by preparative high performance liquid chromatography (chromatographic column: Phenomenex Luna C18 80*30 mm*3 m; mobile phase: A (water containing 0.04% hydrochloric acid) and B (acetonitrile); gradient: B %: 15% to 45%, 8 minutes) to obtain hydrochloride of compound WX-006. 1H NMR (400 MHz, CD3OD) δ: 9.30 (s, 2H), 8.35 (s, 1H), 7.98-7.95 (m, 1H), 7.68-7.65 (m, 1H), 7.47-7.43 (m, 1H), 6.58 (s, 1H), 3.81 (s, 3H), 3.39-3.32 (m, 4H), 1.88-1.86 (m, 4H), 1.85-1.70 (m, 1H), 1.12-1.08 (m, 2H), 1.04-1.02 (m, 2H); LCMS m/z=555.2 [M+H]+.
Compound A-5 (1 g, 4.29 mmol, 1 eq), compound A-1 (1.07 g, 4.29 mmol, 1 eq), and potassium phosphate tribasic (1.82 g, 8.58 mmol, 2 eq) were dissolved in water (4 mL) and 1,4-dioxane (20 mL). After replacing with nitrogen three times, Pd(dppf)Cl2·CH2Cl2 (350.43 mg, 429.11 μmol, 0.1 eq) was added. The mixture was stirred at 80° C. for 2 hours. The reaction mixture was added with water (10 mL) and extracted with ethyl acetate (20 mL*2). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (methanol/dichloromethane=0 to 10%) to obtain compound 8-1. 1HNMR (400 MHz, CDCl3) δ: 7.81-7.71 (m, 4H), 7.02-6.98 (m, 1H), 6.81-6.72 (m, 2H), 3.95 (s, 2H), 3.40 (s, 3H), 1.81 (s, 3H), 1.78 (s, 3H); LCMS m/z=276.1 [M+H]+.
Compound 8-1 (200 mg, 726.53 μmol, 1 eq) and compound A-3 (151.88 mg, 726.53 mol, 1 eq) were dissolved in THE (10 mL). LiHMDS (1 M, 2.91 mL, 4 eq) was added. The mixture was stirred at 15° C. for 1 hour. The reaction mixture was added with water (10 mL) and extracted with ethyl acetate (20 mL*2). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (methanol/dichloromethane=0 to 10%) to obtain compound 8-2. LCMS m/z=448.1 [M+H]+.
Compound 8-2 (0.1 g, 223.28 μmol, 1 eq), cyclopropanecarboxamide (475.05 mg, 5.58 mmol, 25 eq), cesium carbonate (218.25 mg, 669.84 μmol, 3 eq), and Xantphos (12.92 mg, 22.33 μmol, 0.1 eq) were dissolved in dioxane (5 mL). After replacing with nitrogen three times, Pd2(dba)3 (20.45 mg, 22.33 μmol, 0.1 eq) was added. The mixture was stirred at 120° C. for 4 hours. The reaction mixture was added with water (5 mL) and extracted with ethyl acetate (10 mL*3). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was separated by preparative high performance liquid chromatography (chromatographic column: Phenomenex Luna C18 80*40 mm*3 m; mobile phase: A (water containing 0.04% hydrochloric acid) and B (acetonitrile); gradient: B %: 24% to 46%, 7 minutes) to obtain hydrochloride of compound WX-008. 1HNMR (400 MHz, CD3OD) δ: 7.94-7.88 (m, 2H), 7.84-7.81 (m, 2H), 7.58-7.55 (m, 1H), 7.51-7.48 (m, 1H), 7.43-7.39 (m, 1H), 6.95 (s, 1H), 3.47 (s, 3H), 1.86 (s, 3H), 1.83 (s, 3H), 1.68-1.61 (m, 1H), 1.18-1.06 (m, 4H); LCMS m/z=497.1 [M+H]+.
To synthesize example 27 listed in Table 5, follow the synthesis steps of example 8, replacing A-1 in step 1 with A-13.
1H NMR (400 MHz, CDCl3) δ: 11.20 (s, 1H), 8.91 (br s, 1H), 8.28 (s, 1H), 8.07 (s, 1H), 7.77-7.65 (m, 4H), 7.16 (dd, J = 2.9, 9.2 Hz, 1H), 6.80 (dd, J = 2.9, 8.7 Hz, 1H), 3.33 (s, 3H), 1.73 (d, J = 12.8 Hz, 6H), 1.66- 1.60 (m, 1H), 1.11- 1.02 (m, 2H), 0.93-0.87 (m, 2H); LCMS m/z = 537.1 [M + Na]+. Purification method: preparative high performance liquid chromatography (chromatographic column: Phenomenex Luna C18 80 * 40 mm * 3 μm; mobile phase: A (water containing 0.04% hydrochloric acid) and B (acetonitrile); gradient: B %: 24% to 46%, 7 minutes).
Compound 1-1 (50 mg, 113.78 μmol, 1 eq) was dissolved in DMF (1 mL), and then sodium 2-propanethiolate (44.67 mg, 455.11 μmol, 4 eq) was added thereto. The reaction was carried out at 40° C. for 2 hours. The reaction mixture was added with water (10 mL) and ethyl acetate (10 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (10 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate:petroleum ether=10% to 30%) to obtain compound 9-1. LCMS m/z=496.2 [M+H]+.
Compound 9-1 (56 mg, 112.99 μmol, 1 eq) was added to MeOH (1 mL), and (diacetoxyiodo)benzene (109.18 mg, 338.98 μmol, 3 eq) and ammonium acetate (34.84 mg, 451.97 μmol, 4 eq) were added thereto. The reaction was carried out at 25° C. for 2 hours. The system was added with water (10 mL) and ethyl acetate (10 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (10 mL). The organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was purified by preparative high performance liquid chromatography (column: Welch Xtimate C18 100*40 mm*3 m; mobile phase: A (water containing 0.075% trifluoroacetic acid) and B (acetonitrile); gradient: B %: 23 to 63%, 8 minutes) to obtain trifluoroacetate of compound WX-009. 1H NMR (400 MHz, CDCl3) δ: 11.97-11.77 (m, 1H), 11.60-11.42 (m, 1H), 9.33 (s, 2H), 8.73-8.65 (m, 1H), 8.73-8.65 (m, 1H), 8.01-7.93 (m, 1H), 7.67-7.62 (m, 1H), 7.46-7.40 (m, 1H), 3.90-3.83 (m, 3H), 3.52-3.41 (m, 3H), 1.93-1.83 (m, 1H), 1.52-1.38 (m, 6H), 1.13-1.07 (m, 2H), 1.05-0.95 (m, 2H); LCMS m/z=527.2 [M+H]+.
Compound 10-1 (997.91 mg, 5.94 mmol, 2 eq), compound A-1-1 (600 mg, 2.97 mmol, 1 eq), and potassium phosphate tribasic (1.89 g, 8.91 mmol, 3 eq) were added to 1,4-dioxane (24 mL). After replacing with nitrogen, Pd(dppf)Cl2·CH2Cl2 (242.51 mg, 296.96 μmol, 0.1 eq) was added. The reaction was carried out at 100° C. for 3 hours. The reaction mixture was added with water (40 mL) and ethyl acetate (40 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (40 mL). The organic phases were combined, washed twice with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether=0 to 20%) to obtain compound 10-2. LCMS m/z=246.1 [M+H]+.
Compound 10-2 (300 mg, 1.22 mmol, 1 eq) and compound A-2 (279.86 mg, 1.35 mmol, 1.1 eq) were added to THE (6 mL). After cooling to 0° C., the mixture was added with LiHMDS (1 M, 4.89 mL, 4 eq). The reaction was carried out at 20° C. for 2 hours. After the reaction was completed, the reaction was quenched by methanol. The reaction mixture was added with water (20 mL) and ethyl acetate (20 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (20 mL). The organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated to obtain compound 10-3. LCMS m/z=417.1 [M+H]+.
Compound 10-3 (300 mg, 719.53 μmol, 1 eq) and cyclopropanecarboxamide (1.53 g, 17.99 mmol, 25 eq) were added to 1,4-dioxane (2 mL), NMP (0.4 mL), and water (0.4 mL). Cesium carbonate (703.31 mg, 2.16 mmol, 3 eq) and Xantphos (62.45 mg, 107.93 μmol, 0.15 eq) were then added thereto. After replacing with nitrogen, Pd2(dba)3 (98.83 mg, 107.93 μmol, 0.15 eq) was added. After replacing with nitrogen, the reaction was carried out at 120° C. for 3 hours. After the reaction was completed, the reaction mixture was added with water (10 mL) and ethyl acetate (10 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (10 mL). The organic phases were combined, washed twice with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether=0 to 100%) to obtain compound 10-4. LCMS m/z=466.1 [M+H]+.
Compound 10-4 (200 mg, 429.57 μmol, 1 eq) was added to methanol (4 mL), and then (diacetoxyiodo)benzene (415.09 mg, 1.29 mmol, 3 eq) and ammonium acetate (132.45 mg, 1.72 mmol, 4 eq) were added thereto. The reaction was carried out at 25° C. for 3 hours. The system was added with water (10 mL) and ethyl acetate (10 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (10 mL). The organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was purified by preparative thin-layer chromatography on a silica gel plate (ethyl acetate:dichloromethane :methanol=5:5:1) to obtain compound 10-5. 1H NMR (400 MHz, CDCl3) δ: 10.47 (s, 1H), 8.27 (s, 1H), 8.18 (s, 2H), 8.06 (d, J=8.0 Hz, 2H), 7.81 (d, J=8.0 Hz, 2H), 7.56 (d, J=8.0 Hz, 1H), 7.26 (t, J=8.0 Hz, 1H), 7.11-7.08 (m, 1H), 6.20 (s, 1H), 3.45 (s, 3H), 3.18 (s, 3H), 2.76 (s, 1H), 1.57-1.52 (m, 1H), 1.11-1.08 (m, 2H), 0.92-0.87 (m, 2H); LCMS m/z=497.2 [M+H]+.
Compound 10-5 (40 mg, 80.55 μmol, 1 eq) was dissolved in 1,4-dioxane (3 mL), and then Cu(OAc)2 (21.95 mg, 120.82 μmol, 1.5 eq) and pyridine (15.29 mg, 193.32 μmol, 15.60 L, 2.4 eq) were added thereto. The mixture was stirred in an open container for 10 minutes, and then added with methylboronic acid (9.64 mg, 161.10 μmol, 2 eq). The reaction was carried out at 100° C. for 12 hours. After the reaction was completed, the reaction mixture was added with water (10 mL) and ethyl acetate (10 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (10 mL). The organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was separated by preparative high performance liquid chromatography (chromatographic column: Phenomenex Luna C18 80*30 mm*3 m; mobile phase: A (water containing 0.04% hydrochloric acid) and B (acetonitrile); gradient: B %: 10% to 30%, 8 minutes) to obtain hydrochloride of compound WX-010. 1H NMR (400 MHz, CDCl3) δ: 10.47 (s, 1H), 8.27-8.24 (m, 2H), 8.19 (s, 1H), 7.94 (d, J=8.0 Hz, 2H), 7.82 (d, J=8.0 Hz, 2H), 7.57-7.55 (m, 1H), 7.28-7.24 (m, 1H), 7.12-7.10 (m, 1H), 6.19 (s, 1H), 3.47 (s, 3H), 3.14 (s, 3H), 2.72 (s, 3H), 1.56-1.53 (m, 1H), 1.12-1.08 (m, 2H), 0.92-0.87 (m, 2H); LCMS m/z=511.2 [M+H]+.
Compound 11-1 (3.83 g, 28.90 mmol, 3.58 mL, 0.9 eq), compound A-1 (8 g, 32.11 mmol, 1 eq), and potassium phosphate tribasic (20.45 g, 96.34 mmol, 3 eq) were added to 1,4-dioxane (120 mL) and water (24 mL). After replacing with nitrogen, Pd(dppf)Cl2·CH2Cl2 (1.57 g, 1.93 mmol, 0.06 eq) was added. The reaction was carried out at 100° C. for 6 hours. The reaction mixture was added with water (100 mL) and ethyl acetate (100 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (100 mL). The organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether=0 to 20%) to obtain compound 11-2. LCMS m/z=220.0 [M+H]+.
Compound 11-2 (3.0 g, 13.69 mmol, 1 eq) and compound A-11 (3.67 g, 17.79 mmol, 1.3 eq) were added to THE (30 mL). After cooling to −60° C., the mixture was added with LiHMDS (1 M, 41.06 mL, 3 eq). The reaction was carried out at 20° C. for 2 hours. After the reaction was completed, the reaction was quenched by methanol (10 mL). The reaction mixture was added with water (50 mL) and ethyl acetate (50 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (50 mL). The organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated to obtain compound 11-3. LCMS m/z=389.0 [M+H]+.
Compound 11-3 (4.2 g, 10.80 mmol, 1 eq) and cyclopropanecarboxamide (9.19 g, 108.03 mmol, 10 eq) were added to NMP (80 mL). Cesium carbonate (10.56 g, 32.41 mmol, 3 eq) and Xantphos (937.62 mg, 1.62 mmol, 0.15 eq) were then added thereto. After replacing with nitrogen, Pd2(dba)3 (1.48 g, 1.62 mmol, 0.15 eq) was added. After replacing with nitrogen, the reaction was carried out at 140° C. for 2 hours. After the reaction was completed, the reaction mixture was added with water (100 mL) and ethyl acetate (100 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (100 mL). The organic phases were combined, washed twice with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (methanol/dichloromethane=0 to 10%) to obtain compound 11-4. LCMS m/z=438.1 [M+H]+.
Compound 11-4 (300 mg, 685.83 μmol, 1 eq) was dissolved in DMF (5 mL), and then sodium 2-propanethiolate (100.96 mg, 1.03 mmol, 1.5 eq) was added thereto. The reaction was carried out at 60° C. for 2 hours. The reaction mixture was added with water (10 mL) and ethyl acetate (10 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (10 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain compound 11-5. LCMS m/z=494.1 [M+H]+.
Compound 11-5 (400 mg, 810.41 μmol, 1 eq) was added to MeOH (30 mL), and then (diacetoxyiodo)benzene (783.08 mg, 2.43 mmol, 3 eq) and ammonium acetate (249.86 mg, 3.24 mmol, 4 eq) were added thereto. The reaction was carried out at 25° C. for 2 hours. The system was added with water (10 mL) and ethyl acetate (10 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (10 mL). The organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was separated by preparative high performance liquid chromatography (chromatographic column: Phenomenex Luna C18 80*30 mm*3 m; mobile phase: A (water containing 0.04% hydrochloric acid) and B (acetonitrile); gradient: B %: 45% to 45%, 8 minutes). The obtained solution was concentrated under vacuum at 40° C. to remove acetonitrile, then adjusted to alkalinity (pH=8) with saturated sodium bicarbonate, and extracted with dichloromethane (30 mL*3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under vacuum and dried to obtain compound WX-011. LCMS m/z=525.2 [M+H]+.
WX-011 was subjected to resolution by SFC (column: DAICEL CHIRALCEL OD-H (250 mm*30 mm, 5 m); mobile phase: A (CO2) and B (ethanol containing 0.1% ammonia water); gradient: B %=65% to 65%, 15 minutes) to obtain WX-011A and WX-011B.
WX-011A: 1H NMR (400 MHz, CDCl3) δ: 11.07 (s, 1H), 10.05 (s, 1H), 9.28 (s, 2H), 8.25 (s, 1H), 8.13-8.12 (m, 1H), 7.80 (d, J=8.0 Hz, 1H), 7.64 (d, J=8.0 Hz, 1H), 7.33 (t, J=8.0 Hz, 1H), 3.83 (s, 3H), 3.38-3.33 (m, 1H), 3.03 (s, 3H), 1.91-1.88 (m, 1H), 1.41 (t, J=8.0 Hz, 6H), 1.10-1.08 (m, 2H), 0.92-0.89 (m, 2H); LCMS m/z=525.2 [M+H]+. SFC detection method: (column: Chiralpak AD-3, 3 μm, 0.46 cm id×5 cm L; mobile phase: A (CO2) and B (EtOH containing 0.1% isopropylamine); gradient: B %=50 to 50%, 5 minutes; flow rate: 4.0 mL/min; wavelength: 220 nm; pressure: 100 bar), with Rt of 2.538 min, and a chiral isomer excess of 100%.
WX-011B: 1H NMR (400 MHz, CDCl3) δ: 11.07 (s, 1H), 9.88 (s, 1H), 9.28 (s, 2H), 8.25 (s, 1H), 8.10-8.09 (m, 1H), 7.80 (d, J=8.0 Hz, 1H), 7.64 (d, J=8.0 Hz, 1H), 7.33 (t, J=8.0 Hz, 1H), 3.84 (s, 3H), 3.36-3.33 (m, 1H), 3.03 (s, 3H), 1.85-1.82 (m, 1H), 1.41 (t, J=8.0 Hz, 6H), 1.11-1.09 (m, 2H), 0.93-0.91 (m, 2H); LCMS m/z=525.2 [M+H]+. SFC detection method: (column: Chiralpak AD-3, 3 μm, 0.46 cm id×5 cm L; mobile phase: A (CO2) and B (EtOH containing 0.1% isopropylamine); gradient: B %=50 to 50%, 5 minutes; flow rate: 4.0 mL/min; wavelength: 220 nm; pressure: 100 bar), with Rt of 3.011 min, and a chiral isomer excess of 98.60%.
Compound WX-011A (790 mg, 1.51 mmol, 1 eq) was dissolved in MeCN (6 mL), and HCl/EtOAc (4 M, 1.13 mL, 3 eq) was added thereto. The mixture was stirred until clear. Water (50 mL) was then added to the system to obtain hydrochloride of compound WX-011A. 1H NMR (400 MHz, CD3OD) δ: 9.42 (s, 2H), 8.08 (dd, J=1.6, 8.0 Hz, 1H), 7.78 (dd, J=8.0 Hz, 1H), 7.51 (t, J=8.0 Hz, 1H), 6.95 (s, 1H), 3.88 (s, 3H), 3.86-3.77 (m, 1H), 3.02 (s, 3H), 1.91-1.78 (m, 1H), 1.47 (dd, J=6.8, 12.4 Hz, 6H), 1.19-1.04 (m, 4H); LCMS m/z=525.0 [M+H]+.
Compound 12-1 (3 g, 17.05 mmol, 4.97 mL, 1 eq), A-1 (5.10 g, 20.46 mmol, 1.2 eq), and potassium carbonate (4.71 g, 34.09 mmol, 2 eq) were dissolved in 1,4-dioxane (50 mL) and water (10 mL). After replacing with nitrogen three times, Pd(dppf)Cl2 (1.25 g, 1.70 mmol, 0.1 eq) was added thereto. After replacing with nitrogen once more, the mixture was heated to 80° C. and stirred for 4 hours. The reaction mixture was added with water (50 mL) and extracted with ethyl acetate (50 mL*2). The organic phases were combined and washed with saturated brine (30 mL*2). The organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether=0 to 30%) to obtain compound 12-2. LCMS m/z=219.1 [M+1]+.
Under a nitrogen atmosphere, compound 12-2 (2 g, 9.16 mmol, 1 eq) was added to THE (5 mL). A-11 (1.89 g, 9.16 mmol, 1 eq) was added thereto, and the mixture was stirred at 25° C. (room temperature) until dissolved. LiHMDS (1 M, 22.91 mL, 2.5 eq) was slowly added dropwise thereto, and the mixture was stirred at 25° C. (room temperature) for another 1 hour. The reaction mixture was added with saturated ammonium chloride aqueous solution (20 mL) and water (50 mL), and extracted with ethyl acetate (50 mL*2). The organic phases were combined and washed with saturated brine (50 mL*2). The organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether=0 to 50%) to obtain compound 12-3. LCMS m/z=387.9 [M+1]+.
Compound 12-3 (800 mg, 2.06 mmol, 1 eq) and cyclopropanecarboxamide (1.76 g, 20.63 mmol, 10 eq) were added to 1,4-dioxane (3 mL) and NMP (0.3 mL). Cesium carbonate (2.69 g, 8.25 mmol, 4 eq) and Xantphos (358.10 mg, 618.88 μmol, 0.15 eq) were then added thereto. After replacing with nitrogen three times, Pd2(dba)3 (320.30 mg, 309.44 μmol, 0.15 eq) was added. The reaction was carried out at 130° C. for 18 hours. After cooling to room temperature, the reaction mixture was added with water (30 mL) and ethyl acetate (30 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (30 mL). The organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether=0 to 50%) to obtain compound 12-4. LCMS m/z=437.3 [M+1]+.
Compound 12-4 (600 mg, 1.37 mmol, 1 eq) was dissolved in DMF (1 mL), and then sodium 2-propanethiolate (539.69 mg, 5.50 mmol, 4 eq) was added thereto. The reaction was carried out at 40° C. for 2 hours. The reaction mixture was added with water (10 mL) and ethyl acetate (10 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (10 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether=0 to 50%) to obtain compound 12-5. The crude product was directly used in the next step without purification. LCMS m/z=493.2 [M+1+.
12-5 (350 mg, 710.53 μmol, 1 eq) was added to MeOH (20 mL), and (diacetoxyiodo)benzene (686.57 mg, 2.13 mmol, 3 eq) and ammonium acetate (219.08 mg, 2.84 mmol, 4 eq) were added thereto. The reaction was carried out at 25° C. for 2 hours. The reaction mixture was added with water (20 mL) and ethyl acetate (20 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (20 mL). The organic phases were combined, washed with saturated sodium chloride aqueous solution (20 mL), dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (methanol/dichloromethane=0 to 3%) to obtain compound WX-012. 1H NMR (400 MHz, CDCl3) δ: 11.16 (s, 1H), 9.21 (d, J=2.4 Hz, 1H), 8.53 (br dd, J=2.5, 6.5 Hz, 1H), 8.29-8.14 (m, 4H), 7.69 (dd, J=1.5, 7.8 Hz, 1H), 7.57 (dd, J=1.4, 7.9 Hz, 1H), 7.38-7.32 (m, 1H), 3.59-3.52 (m, 1H), 3.56 (s, 3H), 3.39-3.29 (m, 1H), 3.07 (d, J=5.0 Hz, 3H), 2.82 (br s, 1H), 1.69-1.61 (m, 1H), 1.39 (dd, J=6.8, 13.3 Hz, 6H), 1.18-1.07 (m, 2H), 1.00-0.93 (m, 2H); LCMS m/z=524.1 [M+1]+.
WX-012 was subjected to resolution by SFC (column: DAICEL CHIRALPAK AD (250 mm*30 mm, 10 m); mobile phase: A (CO2) and B (isopropanol containing 0.1% ammonia water); gradient: B %=50% to 50%, 15 minutes) to obtain WX-012A and WX-012B.
WX-012A: 1H NMR (400 MHz, CDCl3) δ: 11.16 (s, 1H), 9.22 (d, 2.4 Hz, 1H), 8.54 (br s, 1H), 8.30-8.15 (m, 4H), 7.70 (dd, J=1.5, 7.8 Hz, 1H), 7.58 (dd, J=1.5, 8.0 Hz, 1H), 7.42-7.32 (m, 1H), 3.57 (s, 3H), 3.40-3.30 (m, 1H), 3.08 (d, J=5.2 Hz, 3H), 2.84 (br s, 1H), 1.70-1.62 (m, 1H), 1.40 (dd, J=6.8, 13.3 Hz, 6H), 1.17-1.12 (m, 2H), 1.02-0.95 (m, 2H); LCMS m/z=524.0 [M+H]+. SFC detection method: (column: Chiralpak AD-3, 3 μm, 0.46 cm id×15 cm L; mobile phase: A (CO2) and B (isopropanol containing 0.05% diethylamine); gradient: B %=40%, 8 minutes; flow rate: 2.5 mL/min; wavelength: 220 nm; pressure: 100 bar), with Rt of 4.941 min, and a chiral isomer excess of 99.66%.
WX-012B: 1H NMR (400 MHz, CDCl3) δ: 11.15 (s, 1H), 9.22 (d, J=2.4 Hz, 1H), 8.61 (s, 1H), 8.28-8.19 (m, 2H), 8.21-8.13 (m, 2H), 7.70 (dd, J=1.4, 7.7 Hz, 1H), 7.58 (dd, J=1.6, 7.9 Hz, 1H), 7.36 (t, J=7.9 Hz, 1H), 3.57 (s, 3H), 3.39-3.29 (m, 1H), 3.08 (d, J=5.0 Hz, 3H), 2.84 (br s, 1H), 1.66 (br d, J=4.0 Hz, 1H), 1.40 (dd, J=6.8, 13.3 Hz, 6H), 1.17-1.11 (m, 2H), 1.01-0.94 (m, 2H); LCMS m/z=524.0 [M+H]+. SFC detection method: (column: Chiralpak AD-3, 3 μm, 0.46 cm id×15 cm L; mobile phase: A (CO2) and B (Isopropanol containing 0.05% diethylamine); gradient: B %=40%, 8 minutes; flow rate: 2.5 mL/min; wavelength: 220 nm; pressure: 100 bar), with Rt of 6.054 min, and a chiral isomer excess of 98.66%.
Compound WX-011 (20 mg, 38.12 μmol, 1 eq) was added to 1,4-dioxane (2 mL), and then Cu(OAc)2 (20.77 mg, 114.37 μmol, 3 eq) and pyridine (7.24 mg, 91.50 μmol, 7.39 μL, 2.4 eq) were added thereto. The mixture was stirred in an open container for 10 minutes, and then added with methylboronic acid (6.85 mg, 114.37 μmol, 3 eq). The reaction was carried out at 100° C. for 3 hours. The system was added with water (10 mL) and ethyl acetate (10 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (10 mL). The organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was separated by preparative high performance liquid chromatography (chromatographic column: Phenomenex Luna C18 80*30 mm*3 m; mobile phase: A (water containing 0.0400 hydrochloric acid) and B (acetonitrile); gradient: B %0: 15% to 30%, 8 minutes) to obtain a crude product. The crude product was further purified by preparative thin-layer chromatography on a silica gel plate (ethyl acetate:dichloromethane:methanol=5:5:1) to obtain compound WX-013. 1HNMR (400 MHz, CD3CD) δ: 9.31 (s, 2H), 8.10-8.06 (m, 1H), 7.79-7.73 (m, 1H), 7.50 (t, J=8.0 Hz, 2H), 3.83 (s, 3H), 3.03 (s, 3H), 2.92-2.90 (m, 1H), 2.81 (s, 3H), 1.83-1.82 (i, 1H), 1.50 (d, J=8.0 Hz, 3H), 1.40 (d, J=8.0 Hz, 3H), 1.14-1.08 (in, 4H); LCMS m/z=539.2 [M+H]+.
To synthesize example 14 listed in Table 6, follow the synthesis steps of example 13, replacing WX-011 in step 1 with WX-012.
1H NMR (400 MHz, CDCl3) δ: 13.31 (br s, 1H), 11.90 (s, 1H), 9.21 (s, 1H), 8.58-8.39 (m, 2H), 8.31 (d, J = 8.5 Hz, 1H), 7.92 (dd, J = 1.4, 7.9 Hz, 1H), 7.88-7.81 (m, 1H), 7.58 (dd, J = 1.3, 7.8 Hz, 1H), 7.49-7.42 (m, 1H), 4.31-4.19 (m, 1H), 3.60 (s, 3H), 3.08 (d, J = 5.3 Hz, 3H), 2.82 (s, 3H), 2.11-2.01 (m, 1H), 1.62 (d, J = 6.4 Hz, 3H), 1.33 (d, J = 6.4 Hz, 3H), 1.15-1.09 (m, 2H), 1.08-1.02 (m, 2H); LCMS m/z = 538.1 [M + 1]+. Purification method: preparative high performance liquid chromatography (chromatographic column: Welch Xtimate C18 100 * 40 mm * 3 μm; mobile phase: A (water containing 0.04% trifluoroacetic acid) and B (acetonitrile); gradient: B %: 13% to 43%, 8 minutes).
Compound WX-011 (100 mg, 190.62 μmol, 1 eq) was added to 1,4-dioxane (2 mL), and then Cu(OAc)2 (103.87 mg, 571.87 μmol, 3 eq) and pyridine (36.19 mg, 457.50 μmol, 36.93 μL, 2.4 eq) were added thereto. The mixture was stirred in an open container for 10 minutes, and then added with ethylboronic acid (21.13 mg, 285.94 μmol, 1.5 eq). The reaction was carried out at 100° C. for 4 hours. The system was added with water (10 mL) and ethyl acetate (10 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (10 mL). The organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was separated by preparative high performance liquid chromatography (chromatographic column: Phenomenex Luna C18 80*30 mm*3 m; mobile phase: A (water containing 0.04% hydrochloric acid) and B (acetonitrile); gradient: B %: 20% to 40%, 8 minutes) to obtain hydrochloride of compound WX-015. 1H NMR (400 MHz, CDCl3) δ: 11.07 (s, 1H), 9.16 (s, 2H), 9.04 (s, 1H), 8.21 (s, 1H), 8.14-8.12 (m, 1H), 7.84 (d, J=8.0 Hz, 1H), 7.64 (d, J=8.0 Hz, 1H), 7.36 (t, J=8.0 Hz, 1H), 3.86 (s, 3H), 3.48-3.14 (m, 2H), 3.03 (s, 3H), 1.57-1.54 (m, 1H), 1.44 (d, J=8.0 Hz, 3H), 1.34 (d, J=8.0 Hz, 3H), 1.26-1.24 (m, 3H), 1.10-0.93 (m, 4H); LCMS m/z=553.2 [M+H]+.
To synthesize example 16 listed in Table 7, follow the synthesis steps of example 15, replacing WX-011 in step 1 with WX-012.
Compound 11-2 (2.0 g, 9.12 mmol, 1 eq) and compound A-12 (2.06 g, 10.04 mmol, 1.1 eq) were added to THE (20 mL). After cooling to −60° C., the mixture was added with LiHMDS (1 M, 27.37 mL, 3 eq). The reaction was carried out at 20° C. for 2 hours. After the reaction was completed, the reaction was quenched by methanol (10 mL). The reaction mixture was added with water (50 mL) and ethyl acetate (50 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (50 mL). The organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated to obtain compound 17-1. LCMS m/z=388.0 [M+H]+.
Compound 17-1 (0.52 g, 1.34 mmol, 1 eq) and cyclopropanecarboxamide (1.14 g, 13.41 mmol, 10 eq) were added to NMP (7 mL). Cesium carbonate (1.31 g, 4.02 mmol, 3 eq) and Xantphos (116.38 mg, 201.14 μmol, 0.15 eq) were then added thereto. After replacing with nitrogen, Pd2(dba)3 (184.19 mg, 201.14 μmol, 0.15 eq) was added. After replacing with nitrogen, the reaction was carried out at 140° C. for 2 hours. After the reaction was completed, the reaction mixture was added with water (30 mL) and ethyl acetate (30 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (30 mL). The organic phases were combined, washed twice with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (methanol/dichloromethane=0 to 10%) to obtain compound 17-2. LCMS m/z=437.1 [M+H]+.
Compound 17-2 (500 mg, 1.15 mmol, 1 eq) was dissolved in DMF (5 mL). Cesium carbonate (559.91 mg, 1.72 mmol, 1.5 eq) and benzyl mercaptan (284.58 mg, 2.29 mmol, 268.48 μL, 2 eq) were added. The mixture was stirred at 40° C. for 2 hours. The reaction mixture was added with water (10 mL) and ethyl acetate (10 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (10 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain compound 17-3. LCMS m/z=541.2 [M+H]+.
Compound 17-3 (400 mg, 739.87 μmol, 1 eq) was dissolved in acetic acid (4 mL) and water (0.5 mL). The mixture was then cooled to 0° C., and N-chlorosuccinimide (395.18 mg, 2.96 mmol, 4 eq) was added thereto. The mixture was stirred at 25° C. for 4 hours. Sodium sulfate was added to the system for drying, and the system was directly used in the next step. Compound 17-4 was obtained. LCMS m/z=517.0 [M+H]+.
Compound 17-4 (190 mg, 367.54 μmol, 1 eq) was added to THE (2 mL). The mixture was then cooled to −60° C., and dimethylamine hydrochloride (1.50 g, 18.38 mmol, 50 eq) and triethylamine (5.58 g, 55.13 mmol, 7.67 mL, 150 eq) were added. The reaction was carried out at 20° C. for 12 hours. The system was added with water (10 mL) and ethyl acetate (10 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (10 mL). The organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was separated by preparative high performance liquid chromatography (chromatographic column: Phenomenex Luna C18 80*30 mm*3 m; mobile phase: A (water containing 0.04% hydrochloric acid) and B (acetonitrile); gradient: B %: 17% to 37%, 7 minutes) to obtain hydrochloride of compound WX-017. 1H NMR (400 MHz, CDCl3) δ: 10.35 (s, 1H), 9.17 (s, 2H), 8.27 (s, 2H), 8.12 (s, 1H), 7.72-7.68 (m, J=8.0 Hz, 1H), 7.33 (t, J=8.0 Hz, 1H), 6.20 (s, 1H), 3.87 (s, 3H), 3.03 (s, 3H), 2.86 (s, 6H), 1.68-1.53 (m, 1H), 1.20-1.18 (m, 2H), 0.90-0.86 (m, 2H); LCMS m/z=526.1 [M+H]+.
Compound 17-4 (190 mg, 367.54 μmol, 1 eq) was added to THE (2 mL). The mixture was then cooled to −60° C., and azetidine hydrochloride (1.72 g, 18.38 mmol, 50 eq) and triethylamine (5.58 g, 55.13 mmol, 7.67 mL, 150 eq) were added. The reaction was carried out at 20° C. for 12 hours. The system was added with water (10 mL) and ethyl acetate (10 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (10 mL). The organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was separated by preparative high performance liquid chromatography (chromatographic column: Phenomenex Luna C18 80*30 mm*3 m; mobile phase: A (water containing 0.04% hydrochloric acid) and B (acetonitrile); gradient: B %: 17% to 37%, 7 minutes) to obtain hydrochloride of compound WX-018. 1H NMR (400 MHz, CDCl3) δ 10.35 (s, 1H), 9.19 (s, 2H), 8.27 (s, 2H), 8.09 (s, 1H), 7.71-7.67 (m, 2H), 7.33 (t, J=8.0 Hz, 1H), 6.22 (s, 1H), 3.93-3.91 (m, 4H), 3.85 (s, 3H), 2.98 (s, 3H), 2.22-2.19 (s, 2H), 1.51-1.49 (m, 1H), 1.05-1.03 (m, 2H), 0.90-0.86 (m, 2H); LCMS m/z=538.2 [M+H]+.
Compound 17-2 (200 mg, 458.26 μmol, 1 eq) was dissolved in DMF (7 mL), and then sodium 2-propanethiolate (179.90 mg, 1.83 mmol, 4 eq) was added thereto. The reaction was carried out at 40° C. for 16 hours. After cooling to room temperature, the reaction mixture was added with water (10 mL) and ethyl acetate (10 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (10 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product of compound 19-1. The crude product was directly used in the next step without purification. 1H NMR (400 MHz, CDCl3) δ: 10.30 (s, 1H), 8.85 (s, 2H), 8.36-8.25 (m, 2H), 8.12 (s, 1H), 7.63 (dd, J=7.8, 15.6 Hz, 2H), 6.24 (br s, 1H), 3.80 (s, 3H), 3.44 (quin, J=6.7 Hz, 1H), 3.01 (d, J=4.4 Hz, 3H), 1.58-1.50 (m, 1H), 1.38 (d, J=6.5 Hz, 6H), 1.13-1.05 (m, 2H), 0.92-0.82 (m, 2H); LCMS m/z=493.1 [M+H]+.
Compound 19-1 (100 mg, 203.01 μmol, 1 eq) was dissolved in DCM (2 mL). The mixture was then cooled to 0° C. and added with 3-chloroperoxybenzoic acid (52.55 mg, 304.51 mol, purity of 85%, 1.5 eq). The reaction was carried out at 20° C. for 16 hours. The system was added with water (10 mL) and ethyl acetate (10 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (10 mL). The organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was separated by preparative high performance liquid chromatography (chromatographic column: Phenomenex Luna 80*40 mm*3 m; mobile phase: A (water containing 0.04% hydrochloric acid) and B (acetonitrile); gradient: B %: 5% to 35%, 8 minutes). The obtained solution was concentrated under vacuum at 40° C. to remove acetonitrile, then adjusted to alkalinity (pH=8) with saturated sodium bicarbonate, and extracted with dichloromethane (30 mL*3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under vacuum and dried to obtain compound WX-019. 1H NMR (400 MHz, CDCl3) δ: 10.39 (s, 1H), 9.26 (s, 2H), 8.31-8.24 (m, 1H), 8.17-8.11 (m, 1H), 7.80-7.70 (m, 2H), 7.39-7.31 (m, 1H), 6.14-6.07 (m, 1H), 3.89 (s, 3H), 3.37-3.25 (m, 1H), 3.04 (d, J=4.8 Hz, 2H), 1.59-1.52 (m, 1H), 1.43 (d, 6H), 1.32-1.27 (m, 2H), 1.12-1.07 (m, 2H). LCMS m/z=525.1 [M+H]+.
Under a nitrogen atmosphere, compound 12-1 (1 g, 4.58 mmol, 1.35 eq) and compound A-12 (695.62 mg, 3.39 mmol, 1 eq) were dissolved in THE (5 mL). The mixture was stirred at 25° C. (room temperature). LiHMDS (1 M, 8.48 mL, 2.5 eq) was slowly added dropwise thereto. The mixture was stirred at 25° C. (room temperature) for another 1 hour. The reaction mixture was added with saturated ammonium chloride aqueous solution (20 mL) and water (50 mL), and extracted with ethyl acetate (50 mL*2). The organic phases were combined and washed with saturated brine (50 mL*2). The organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether=0 to 50%) to obtain compound 20-1. LCMS m/z=387.0 [M+1]+.
Compound 20-1 (1.27 g, 3.28 mmol, 1 eq) and cyclopropanecarboxamide (2.79 g, 32.83 mmol, 10 eq) were added to a mixed solvent of 1,4-dioxane (2 mL) and NMP (0.5 mL). Cesium carbonate (3.21 g, 9.85 mmol, 3 eq) and Xantphos (284.97 mg, 492.49 μmol, 0.15 eq) were then added thereto. After replacing with nitrogen three times, Pd2(dba)3 (450.99 mg, 492.49 μmol, 0.15 eq) was added. The reaction was carried out at 130° C. for 18 hours. After cooling to room temperature, the reaction mixture was added with water (30 mL) and ethyl acetate (30 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (30 mL). The organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether=0 to 50%) to obtain compound 20-2. LCMS m/z=436.1 [M+1]+.
Compound 20-2 (500 mg, 1.15 mmol, 1 eq) was dissolved in DMF (5 mL), and then sodium 2-propanethiolate (450.76 mg, 4.59 mmol, 4 eq) was added thereto. The reaction was carried out at 40° C. for 2 hours. The reaction mixture was added with water (10 mL) and extracted with ethyl acetate (10 mL*2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether=0 to 50%) to obtain compound 20-3. LCMS m/z=492.1 [M+1]+.
Compound 20-3 (100.20 mg, 203.83 μmol, 1 eq) was dissolved in DCM (10 mL). The mixture was then cooled to 0° C. and added with 3-chloroperoxybenzoic acid (82.76 mg, 407.65 mol, purity of 85%, 2 eq). The reaction was carried out at 25° C. for 2 hours. After the reaction was completed, the reaction mixture was added with water (30 mL) and ethyl acetate (30 mL) for extraction and phase separation. The aqueous phase was then extracted with ethyl acetate (30 mL). The organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain a crude product. The crude product was separated by preparative high performance liquid chromatography (chromatographic column: Phenomenex C18 150*40 mm*5 m; mobile phase: A (water containing 0.04% hydrochloric acid) and B (acetonitrile); gradient: B %: 15% to 45%, 10 minutes) to obtain hydrochloride of compound WX-020. 1H NMR (400 MHz, DMSO-d6) δ: 11.85 (br s, 1H), 11.03 (s, 1H), 9.12 (d, J=1.6 Hz, 1H), 9.06 (br s, 1H), 8.51 (s, 1H), 8.36 (dd, J=2.3, 8.4 Hz, 1H), 8.19 (d, J=8.4 Hz, 1H), 7.73 (br d, J=7.8 Hz, 1H), 7.62 (br d, J=7.5 Hz, 1H), 7.41 (t, J=7.9 Hz, 1H), 7.31 (br s, 1H), 3.53 (s, 3H), 3.46-3.30 (m, 1H), 2.82 (d, J=4.4 Hz, 3H), 1.99-1.89 (m, 1H), 1.24 (d, J=6.8 Hz, 6H), 0.99-0.85 (m, 4H); LCMS m/z=524.1 [M+1]+.
Compound 20-3 (1.8 g, 3.66 mmol, 1 eq) was dissolved in ethanol (18 mL). At 0° C., a solution of potassium hydrogenperoxomonosulphate (3.38 g, 5.49 mmol, 1.5 eq) in water (18 mL) was added thereto. The mixture was then gradually returned to 25° C., and stirred and reacted for 16 hours. After the reaction was completed, the reaction mixture was added with saturated sodium bicarbonate (15 mL) and sodium sulfite solution (15 mL) to quench, and extracted with ethyl acetate (30 mL*2). The organic phases were combined, washed with saturated sodium chloride solution, filtered, and the filtrate was dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (dichloromethane:methanol=100:0 to 100:2) to obtain a crude product. The crude product was stirred in methanol (5 mL) at 60° C. for 1 hour, and filtered. The filter cake was stirred in acetone (5 mL) at 60° C. for another 1 hour, and filtered. The filter cake was dried under vacuum to obtain compound WX-020. 1H NMR (400 MHz, DMSO-d6) δ: 10.79 (brs, 1H), 10.73 (brs, 1H), 9.09 (d, J=1.8 Hz, 1H), 8.64 (br d, J=4.5 Hz, 1H), 8.53 (s, 1H), 8.33 (dd, J=2.4, 8.4 Hz, 1H), 8.17 (d, J=8.4 Hz, 1H), 8.07 (s, 1H), 7.60-7.51 (m, 2H), 7.38-7.21 (m, 1H), 3.69-3.57 (m, 1H), 3.51 (s, 3H), 2.80 (d, J=4.4 Hz, 3H), 2.03-1.93 (m, 1H), 1.23 (d, J=6.8 Hz, 6H), 0.83-0.74 (m, 4H); LCMS m/z=524.0 [M+1]+.
Compound WX-020 (0.4 g, 763.94 μmol, 1 eq) was added to acetone (4 mL), and the reaction mixture was stirred. Dilute sulfuric acid aqueous solution (0.25 M, 7.64 mL, 2.5 eq) was then added thereto. The reaction mixture was heated to 55° C., stirred for 30 minutes, and then naturally cooled to room temperature. The reaction mixture was filtered. The filter cake was washed with a small amount of ethyl acetate (10 mL), and dried under vacuum to obtain sulfate of WX-020. 1H NMR (400 MHz, DMSO-d6) δ: 11.33 (brs, 1H), 10.93 (brs, 1H), 9.11 (d, J=1.8 Hz, 1H), 8.87 (brs, 1H), 8.48 (s, 1H), 8.36 (dd, J=2.4, 8.4 Hz, 1H), 8.17 (d, J=8.4 Hz, 1H), 7.70 (br d, J=7.8 Hz, 1H), 7.61 (dd, J=1.2, 8.0 Hz, 1H), 7.39 (t, J=7.9 Hz, 1H), 3.61 (quin, J=6.8 Hz, 1H), 3.54 (s, 3H), 2.82 (d, J=4.4 Hz, 3H), 1.93-1.87 (m, 1H), 1.24 (d, J=6.8 Hz, 6H), 0.92-0.86 (m, 4H); LCMS m/z=523.9 [M+1]+.
Compound A-8 (300 mg, 1.05 mmol, 1 eq) and A-1 (261.21 mg, 1.05 mmol, 1 eq) were added to a mixed solvent of 1,4-dioxane (16 mL) and water (1 mL). Cesium carbonate (434.76 mg, 3.15 mmol, 3 eq) was then added thereto. After replacing with nitrogen, Pd(dppf)Cl2 (76.72 mg, 104.86 μmol, 0.1 eq) was finally added thereto. Under a nitrogen atmosphere, the reaction mixture was stirred at 90° C. for 6 hours. After cooling to room temperature, the reaction mixture was added with water (25 mL) and extracted with ethyl acetate (35 mL*2). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain a crude product. The crude product was purified by silica gel column chromatography (methanol:dichloromethane=0 to 30%) to obtain compound 21-1. LCMS m/z=329.1 [M+1]+.
Compound 21-1 (90 mg, 274.10 μmol, 1 eq) and compound A-11 (56.47 mg, 274.10 mol, 1 eq) were dissolved in THE (10 mL). Under a nitrogen atmosphere, LiHMDS (1 M, 1.10 mL, 4 eq) was slowly added thereto at 0° C. The mixture was then warmed to 25° C. and stirred for 2 hours. After the reaction was completed, the reaction mixture was added with methanol (8 mL) and then concentrated under reduced pressure. The reaction mixture was then added with water (20 mL) and extracted with dichloromethane (30 mL*2). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain a crude product. The crude product was purified by silica gel column chromatography (methanol:dichloromethane=0 to 40%) to obtain compound 21-2. LCMS m/z=498.1 [M+1]+
Compound 21-2 (50 mg, 100.42 μmol, 1 eq), cyclopropanecarboxamide (213.65 mg, 2.51 mmol, 25 eq), and Xantphos (8.72 mg, 15.06 μmol, 0.15 eq) were dissolved in a mixed solvent of 1.4-dioxane (6 mL) and NMP (1 mL). Cesium carbonate (98.16 mg, 301.26 μmol, 3 eq) was added thereto. The system was replaced with nitrogen. Pd2(dba)3 (13.79 mg, 15.06 mol, 0.15 eq) was finally added thereto. Under a nitrogen atmosphere, the mixture was stirred at 130° C. for 12 hours. After cooling to room temperature, the reaction mixture was added with water (10 mL) and extracted with dichloromethane (20 mL*2). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain a crude product. The crude product was purified by silica gel column chromatography (dichloromethane:methanol=100:0 to 30:1) to obtain compound WX-021. 1H NMR (400 MHz, CDCl3) δ: 11.15 (s, 1H), 9.13 (dd, J=1.4, 5.4 Hz, 1H), 8.89 (br s, 1H), 8.28 (s, 1H), 8.23-8.16 (m, 2H), 8.10 (br d, J=7.3 Hz, 1H), 7.66 (dd, J=1.4, 8.0 Hz, 1H), 7.56 (d, J=8.0 Hz, 1H), 7.37-7.32 (m, 1H), 3.58 (s, 3H), 3.08 (d, J=5.0 Hz, 3H), 1.74-1.65 (m, 1H), 1.20-0.95 (m, 14H); LCMS m/z=547.1 [M+1]+.
To synthesize examples 22, 23, and 24, follow the synthesis steps of example 21, replacing A-8 in step 1 with fragments in the table below.
1H NMR (400 MHz, CDCl3) δ: 11.06 (s, 1H), 8.84 (dd, J = 1.1, 4.4 Hz, 1H), 8.73-8.63 (m, 1H), 8.17 (s, 1H), 8.11- 8.02 (m, 3H), 7.60-7.57 (m, 1H), 7.48-7.45 (m, 1H), 7.25 (t, J = 7.9 Hz, 1H), 3.48 (s, 3H), 2.99 (d, J = 5.0 Hz, 3H), 2.07-1.86 (m, 4H), 1.63-1.59 (m, 1H), 1.18-1.09 (m, 6H), 1.07- 1.03 (m, 2H), 0.89-0.84 (m, 2H); LCMS m/z = 523.2 [M + H]+.
1H NMR (400 MHz, CDCl3) δ: 11.14 (br s, 1H), 9.23 (br s, 1H), 8.34 (s, 1H), 8.21-8.11 (m, 1H), 7.79-7.73 (m, 4H), 7.54-7.48 (m, 1H), 7.31-7.23 (m, 1H), 7.22-7.18 (m, 1H), 3.43 (s, 3H), 3.07 (d, J = 5.0 Hz, 3H), 2.44- 2.31 (m, 2H), 1.81-1.64 (m, 1H), 1.30- 1.24 (m, 6H), 1.18-1.08 (m, 8H), 0.98-0.92 (m, 2H); LCMS m/z = 550.1 [M + H]+.
1H NMR (400 MHz, CDCl3) δ: 11.14 (s, 1H), 9.02 (br s, 1H), 8.32 (s, 1H), 8.16 (br d, J = 4.8 Hz, 1H), 7.93-7.87 (m, 2H), 7.74 (dd, J = 2.5, 8.3 Hz, 2H), 7.51 (dd, J = 1.4, 7.9 Hz, 1H), 7.31- 7.25 (m, 1H), 7.19 (dd, J = 1.5, 7.8 Hz, 1H), 3.45 (s, 3H), 3.07 (d, J = 5.0 Hz, 3H), 1.75-1.65 (m, 1H), 1.17-0.87 (m, 14H); LCMS m/z = 546.2 [M + H]+.
To synthesize example 25 listed in Table 9, follow the synthesis steps of example 21, replacing A-8 in step 1 with A-6, and A-11 in step 2 with A-12.
1H NMR (400 MHz, CDCl3) δ: 10.47 (s, 1H), 8.30-8.17 (m, 3H), 7.76 (d, J = 6.0 Hz, 4H), 7.59-7.53 (m, 1H), 7.26 (s, 1H), 7.13 (s, 1H), 6.34-6.21 (m, 1H), 3.45 (s, 3H), 3.04 (d, J = 4.8 Hz, 3H), 1.96 (br d, J = 9.8 Hz, 4H), 1.59-1.55 (m, 1H), 1.23-1.14 (m, 6H), 1.13-1.06 (m, 2H), 0.95-0.86 (m, 2H); LCMS m/z = 521.1 [M + H]+.
Compound 11-4 (100 mg, 228.61 μmol, 1 eq) was added to DMSO (1 mL), and then sodium sulfide (35.68 mg, 457.22 μmol, 19.18 μL, 2 eq) was added thereto. The reaction was carried out at 70° C. for 2 hours, and the mixture was cooled to room temperature to obtain a crude solution of compound 28-1. The crude solution was directly used in the next step without purification. LCMS m/z=452.1 [M+H]+.
The crude DMSO solution of compound 28-1 (theoretical content of 108 mg, 228.10 mol, 1 eq) obtained in the previous step was dissolved in DMF (1 mL). Potassium carbonate (45.67 mg, 330.44 μmol, 1.45 eq) and 1-bromo-4-chloropropane (52.31 mg, 305.08 μmol, 35.11 μL, 1.34 eq) were added thereto, and the mixture was stirred at 25° C. for 10 minutes. The reaction mixture was added with water (3 mL) and extracted twice with ethyl acetate (6 mL). The organic phase was washed once with saturated brine (5 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain compound 28-2. LCMS m/z=528.2 [M+H]+.
Compound 28-2 (0.86 g, 1.63 mmol, 1 eq) was dissolved in dichloromethane (20 mL). 3-Chloroperoxybenzoic acid (330.66 mg, 1.63 mmol, purity of 85%, 1 eq) was added thereto at 0° C. The reaction was carried out at 25° C. for 0.5 hours. The reaction mixture was washed once with 10% sodium thiosulfate aqueous solution (20 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (methanol/dichloromethane=0 to 10%) to obtain compound 28-3. LCMS m/z=544.2 [M+H]+.
Compound 28-3 (0.27 g, 496.30 μmol, 1 eq) was dissolved in DMF (9 mL), and potassium tert-butoxide (111.38 mg, 992.60 μmol, 2 eq) was added thereto at 25° C. The mixture was stirred for 1 hour. The reaction mixture was added with saturated ammonium chloride aqueous solution (9 mL) and extracted with dichloromethane (15 mL*2). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (methanol/dichloromethane=0 to 10%) to obtain compound 28-4. LCMS m/z=508.1 [M+H]+.
Compound 28-4 (0.15 g, 295.53 μmol, 1 eq) and trifluoroacetamide (33.41 mg, 295.53 mol, 1 eq) were dissolved in dichloromethane (7.5 mL). (Diacetoxyiodo)benzene (142.78 mg, 443.29 μmol, 1.5 eq), magnesium oxide (47.64 mg, 1.18 mmol, 13.31 μL, 4 eq), and rhodium acetate (26.12 mg, 59.11 μmol, 0.2 eq) were added thereto. The mixture was stirred at 40° C. for 1 hour. The reaction mixture was added with water (5 mL), and settled for phase separation. The organic phase was washed with 10% sodium thiosulfate aqueous solution (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by preparative thin-layer chromatography on a silica gel plate (dichloromethane:methanol=10:1) to obtain compound 28-5. LCMS m/z=619.1 [M+H]+.
Compound 28-5 (10 mg, 16.17 μmol, 1 eq) was dissolved in methanol (1 mL), and potassium carbonate (2.23 mg, 16.17 μmol, 1 eq) was added thereto. The mixture was stirred at 25° C. for 10 minutes. The reaction mixture was added with water (1 mL) and extracted with dichloromethane (2 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by preparative thin-layer chromatography on a silica gel plate (dichloromethane:methanol=10:1) to obtain compound WX-028. 1H NMR (400 MHz, CDCl3) δ: 11.19 (s, 1H), 9.34 (s, 2H), 8.54-8.24 (m, 2H), 7.84-7.82 (m, 1H), 7.72 (d, J=7.2 Hz, 1H), 7.41-7.37 (m, 1H), 3.90 (s, 3H), 3.06 (d, J=4.8 Hz, 3H), 2.71-2.65 (m, 1H), 2.41-2.24 (m, 1H), 1.14-1.06 (m, 8H); LCMS m/z=523.2 [M+H]+.
The crude DMSO solution of compound 28-1 (theoretical content of 108 mg, 228.10 mol, 1 eq) was dissolved in DMF (1 mL). Potassium carbonate (45.67 mg, 330.44 μmol, 1.45 eq) and 1-bromo-4-chlorobutane (52.31 mg, 305.08 μmol, 35.11 μL, 1.34 eq) were added thereto, and the mixture was stirred at 25° C. for 10 minutes. The reaction mixture was added with water (3 mL) and extracted with ethyl acetate (6 mL*2). The organic phase was washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 29-1. LCMS m/z=542.2 [M+H]+.
Compound 29-1 (120 mg, 221.38 μmol, 1 eq) was dissolved in methanol (5 mL), and (diacetoxyiodo)benzene (213.92 mg, 664.14 μmol, 3 eq) and ammonium acetate (51.19 mg, 664.14 μmol, 3 eq) were added thereto. The mixture was stirred at 25° C. for 10 minutes. The reaction mixture was extracted with dichloromethane (10 mL) and water (5 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (methanol/dichloromethane=0 to 10%) to obtain compound 29-2. LCMS m/z=573.2 [M+H]+.
Compound 29-2 (35 mg, 61.08 μmol, 1 eq) was dissolved in DMF (1 mL), and sodium hydride (9.77 mg, 244.30 μmol, purity of 60%, 4 eq) was added thereto at 0° C. The mixture was stirred at 25° C. for 0.5 hours. The reaction mixture was added with saturated ammonium chloride aqueous solution (2 mL), then added with water (1 mL), and extracted with dichloromethane (3 mL*2). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was separated by preparative high performance liquid chromatography (chromatographic column: Phenomenex Luna 80*30 mm*3 m; mobile phase: A (water containing 0.04% hydrochloric acid) and B (acetonitrile); gradient: B %: 10% to 40%, 8 minutes) to obtain hydrochloride of compound WX-29. 1HNMR (400 MHz, CD3OD) δ: 9.64 (s, 2H), 8.12-8.09 (m, 1H), 7.83-7.81 (m, 1H), 7.51 (t, J=8.0 Hz, 1H), 7.10 (s, 1H), 4.31-4.21 (m, 1H), 4.12-4.08 (m, 1H), 3.89 (s, 3H), 3.85-3.73 (m, 2H), 3.00 (s, 3H), 2.66-2.55 (m, 2H), 2.26-2.15 (m, 1H), 2.09-2.02 (m, 1H), 1.92-1.84 (m, 1H), 1.16-1.05 (m, 4H); LCMS m/z=537.3 [M+H]+.
Compound 11-1 (1.24 g, 9.36 mmol, 1.16 mL, 1 eq), A-13 (2.5 g, 9.36 mmol, 1 eq), and potassium carbonate (2.59 g, 18.72 mmol, 2 eq) were dissolved in a mixed solvent of 1,4-dioxane (80 mL) and water (15 mL). After replacing with nitrogen three times, Pd(dppf)Cl2 (684.86 mg, 936.00 μmol, 0.1 eq) was added thereto. Under a nitrogen atmosphere, the mixture was heated to 80° C. and stirred for 4 hours. After cooling to room temperature, the reaction mixture was added with saturated ammonium chloride aqueous solution (100 mL) and water (100 mL), and then extracted with ethyl acetate (100 mL*2). The organic phase was washed with saturated brine (100 mL*2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether=10 to 30%) to obtain compound 30-1. LCMS m/z=238.0 [M+1]+.
Under a nitrogen atmosphere, compound 30-1 (1.8 g, 3.79 mmol, purity of 50%, 1 eq) and A-12 (777.96 mg, 3.79 mmol, 1 eq) were added to THE (10 mL), and the mixture was stirred until dissolved. The mixture was cooled to 0° C., and LiHMDS (1 M, 9.49 mL, 2.5 eq) was added dropwise thereto. The mixture was then returned to 25° C. and stirred for another 2 hours. After the reaction was completed, the reaction mixture was added with water (100 mL) and extracted with ethyl acetate (50 mL*2). The organic phases were combined, washed with saturated brine (50 mL*2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (methanol/dichloromethane=3%) to obtain compound 30-2. LCMS m/z=406.0 [M+1]+.
Compound 30-2 (1.18 g, 2.91 mmol, 1 eq) and cyclopropanecarboxamide (6.19 g, 72.70 mmol, 25 eq) were added to 1,4-dioxane (80 mL) and NMP (2 mL). Cesium carbonate (2.84 g, 8.72 mmol, 3 eq) and Xantphos (252.39 mg, 436.19 μmol, 0.15 eq) were then added thereto. After replacing with nitrogen three times, Pd2(dba)3 (399.43 mg, 436.19 μmol, 0.15 eq) was added. The mixture was refluxed and reacted at 130° C. for 18 hours. After the reaction was completed, the reaction mixture was added with water (100) and extracted with ethyl acetate (50 mL*2). The organic phases were combined, washed with saturated sodium chloride aqueous solution (50 mL*2), dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (methanol/dichloromethane=3%) to obtain compound 30-3. LCMS m/z=455.1 [M+1]+.
30-3 (200 mg, 440.11 μmol, 1 eq) was dissolved in DMF (10 mL), and sodium 2-propanethiolate (86.39 mg, 880.23 μmol, 2 eq) was added thereto. The reaction was carried out at 40° C. for 16 hours. After the reaction was completed, the reaction mixture was added with water (10 mL) and ethyl acetate (10 mL) for extraction and phase separation. The aqueous phase was then extracted once with ethyl acetate (10 mL). The organic phases were combined, washed once with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain a crude product of compound 30-4. The crude product was directly used in the next step without purification. LCMS m/z=511.2 [M+1]+.
Compound 30-4 (200 mg, 391.71 μmol, 1 eq) was added to a mixed solution of ethanol (10 mL) and water (5 mL), and then potassium hydrogenperoxomonosulphate (361.21 mg, 587.56 μmol, 1.5 eq) was added thereto. The reaction was carried out at 25° C. for 2 hours. After the reaction was completed, the reaction mixture was added with saturated sodium bisulfite aqueous solution (10 mL), and extracted with ethyl acetate (20 mL*2). The organic phase was washed with saturated brine (10 mL*2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was separated by preparative high performance liquid chromatography (chromatographic column: Welch Xtimate C18 100*40 mm*3 m; mobile phase: A (water containing 0.075% trifluoroacetic acid) and B (acetonitrile); gradient: B %: 13% to 43%, 8 minutes). The obtained solution was concentrated under vacuum at 40° C. to remove acetonitrile, then adjusted to alkalinity (pH=8) with saturated sodium bicarbonate, and extracted with dichloromethane (30 mL*3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain compound WX-030. 1H NMR (400 MHz, CDCl3) δ: 10.47 (s, 1H), 9.17 (s, 2H), 8.53 (br s, 1H), 8.22 (s, 1H), 8.12 (s, 1H), 7.38 (d, J=9.0 Hz, 2H), 6.31-6.23 (m, 1H), 3.80 (s, 3H), 3.28-3.18 (m, 1H), 2.94 (d, J=4.8 Hz, 3H), 1.51-1.44 (m, 1H), 1.34 (d, J=6.8 Hz, 6H), 1.12-0.96 (m, 2H), 0.87-0.80 (m, 2H); LCMS m/z=543.2 [M+1]+.
Compound 11-2 (2 g, 9.12 mmol, 1 eq) and sodium 2-propanethiolate (906.59 mg, 9.24 mmol, 1.5 eq) were dissolved in DMF (20 mL), and the mixture was reacted at 40° C. for 1 hour. The reaction mixture was added with ethyl acetate (20 mL) and water (20 mL). The aqueous phase was then extracted once with ethyl acetate (20 mL), and the organic phase was washed three times with saturated sodium chloride aqueous solution (20 mL), dried over anhydrous sodium chloride, and concentrated under reduced pressure to obtain compound 31-1. LCMS m/z=276.1 [M+H]+.
Compound 31-1 (0.8 g, 2.91 mmol, 1 eq) and compound A-3-2 (577.90 mg, 2.91 mmol, 1 eq) were dissolved in THE (16 mL), and LiHMDS (1 mol/L, 8.72 mL, 3 eq) was added thereto at −60° C. The mixture was warmed to 25° C. and reacted for 3 hours. The reaction mixture was added with methanol (5 mL) to quench, then added with 2 mol/L hydrochloric acid solution to adjust the pH to 7 to 8, and added with ethyl acetate (20 mL) and water (20 mL). The aqueous phase was then extracted once with ethyl acetate (20 mL). The organic phase was washed once with saturated sodium chloride aqueous solution (20 mL), dried over anhydrous sodium sulfate, and concentrated. The obtained crude product was subjected to silica gel column chromatography (methanol/dichloromethane=0 to 10%) to obtain compound 31-2. LCMS m/z=432.1 [M+H]+.
Compound 31-2 (0.46 g, 1.07 mmol, 1 eq) and cyclopropanecarboxamide (906.42 mg, 10.65 mmol, 10 eq) were dissolved in 1,4-dioxane (16 mL). Cesium carbonate (867.55 mg, 2.66 mmol, 2.5 eq) was then added thereto. Under a nitrogen atmosphere, Pd2(dba)3 (30.53 mg, 33.34 μmol, 0.1 eq) and Xantphos (38.58 mg, 66.68 μmol, 0.2 eq) were added thereto. The reaction was carried out at 120° C. for 4 hours. The reaction system was cooled to 20 to 30° C. The reaction mixture was added with 2 mol/L hydrochloric acid solution to adjust the pH to 3, and added with dichloromethane (20 mL) and water (20 mL). The aqueous phase was then extracted once with dichloromethane (20 mL). The organic phase was washed once with saturated sodium chloride aqueous solution (20 mL), dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. The obtained crude product was subjected to silica gel column chromatography (methanol/dichloromethane=0 to 10%) to obtain compound 31-3. LCMS m/z=481.0 [M+H]+.
Compound 30-3 (0.42 g, 524.41 μmol, purity of 60%, 1 eq) was dissolved in MeOH (10 mL). Ammonium acetate (160.41 mg, 2.08 mmol, 4 eq) and (diacetoxyiodo)benzene (502.71 mg, 1.56 mmol, 3 eq) were then added thereto, and the reaction was carried out at 25° C. for 4 hours. The reaction mixture was added with ethyl acetate (10 mL) and water (10 mL). The aqueous phase was then extracted once with ethyl acetate (10 mL). The organic phase was washed with saturated sodium chloride aqueous solution (10 mL*3), dried over anhydrous sodium sulfate, and then concentrated. Compound 31-4 was obtained. LCMS m/z=512.2 [M+H]+. The crude product was directly used in the next step without purification.
Compound 31-4 (450 mg, 175.94 μmol, purity of 20%, 1 eq) and ammonium chloride (470.55 mg, 8.80 mmol, 50 eq) were dissolved in DMF (10 mL). HATU (200.69 mg, 527.81 mol, 3 eq) and DIPEA (27.29 mg, 211.12 μmol, 36.77 μL, 1.2 eq) were then added thereto. The reaction was carried out at 25° C. for 3 hours. The reaction mixture was added with ethyl acetate (10 mL) and water (10 mL). The aqueous phase was then extracted once with ethyl acetate (10 mL). The organic phase was washed with saturated sodium chloride aqueous solution (10 mL*3), dried over anhydrous sodium sulfate, and then concentrated. The obtained crude product was subjected to silica gel column chromatography (methanol/dichloromethane=0 to 10%), and then separated by preparative high performance liquid chromatography (chromatographic column: Phenomenex Luna 80*30 mm*3 m; mobile phase: A (water containing 0.04% hydrochloric acid) and B (acetonitrile); gradient: B %: 10% to 35%) to obtain hydrochloride of compound WX-031. 1H NMR (400 MHz, CD3OD) δ: 9.47 (s, 2H), 8.10 (dd, J=0.8, 8.0 Hz, 1H), 7.78 (dd, J=0.9, 7.9 Hz, 1H), 7.50 (t, J=8.0 Hz, 1H), 6.91 (s, 1H), 4.12-4.04 (m, 1H), 3.88 (s, 3H), 1.86-1.80 (m, 1H), 1.52 (dd, J=8.0, 18.8 Hz, 6H), 1.18-1.08 (m, 4H); LCMS m/z=511.1 [M+H]+.
Compound 12-2 (1.12 g, 5.13 mmol, 1 eq) was dissolved in DMF (10 mL), and then sodium 2-propanethiolate (2.01 g, 20.53 mmol, 4 eq) was added thereto. The reaction was carried out at 40° C. for 2 hours. The reaction mixture was added with water (10 mL) and ethyl acetate (10 mL) for extraction and phase separation. The aqueous phase was then extracted once with ethyl acetate (10 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether=10%) to obtain compound 32-1. LCMS m/z=275.1 [M+1]+.
Under a nitrogen atmosphere, compound 32-1 (250 mg, 911.14 μmol, 1 eq) and A-3-2 (181.25 mg, 911.14 μmol, 1 eq) were added to THF (10 mL). The mixture was stirred at 25° C., and LiHMDS(1 M, 2.28 mL, 2.5 eq) was slowly added dropwise thereto. The mixture was stirred at 25° C. for another 2 hours. The reaction mixture was added with saturated ammonium chloride aqueous solution (20 mL) and water (50 mL), and extracted with ethyl acetate (50 mL*2). The organic phases were combined, washed with saturated brine (50 mL*2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (methanol/dichloromethane=10%) to obtain compound 32-2. LCMS m/z=431.0 [M+1]+.
Compound 32-2 (240 mg, 556.96 μmol, 1 eq) and cyclopropanecarboxamide (1.19 g, 13.92 mmol, 25 eq) were added to a mixed solvent of 1,4-dioxane (10 mL) and NMP (1 mL). Cesium carbonate (544.41 mg, 1.67 mmol, 3 eq) and Xantphos (48.34 mg, 83.54 μmol, 0.15 eq) were then added thereto. After replacing with nitrogen three times, Pd2(dba)3 (76.50 mg, 83.54 μmol, 0.15 eq) was added. The mixture was stirred and reacted at 130° C. for 18 hours. After cooling to room temperature, the reaction mixture was added with water (30 mL) and ethyl acetate (30 mL) for extraction and phase separation. The aqueous phase was then extracted once with ethyl acetate (30 mL). The organic phases were combined, washed once with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain a crude product. The crude product was purified by silica gel column chromatography (methanol/dichloromethane=3%) to obtain compound 32-3. LCMS m/z=480.1 [M+1]+.
Compound 32-3 (187 mg, 389.95 μmol, 1 eq) was added to methanol (1 mL), and then (diacetoxyiodo)benzene (376.80 mg, 1.17 mmol, 3 eq) and ammonium acetate (120.23 mg, 1.56 mmol, 4 eq) were added thereto. The reaction was carried out at 25° C. for 2 hours. The reaction mixture was added with water (10 mL) and ethyl acetate (10 mL) for extraction and phase separation. The aqueous phase was then extracted once with ethyl acetate (10 mL). The organic phases were combined, washed once with saturated sodium chloride aqueous solution (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain a crude product of 32-4. LCMS m/z=511.1 [M+1]+. The crude product was directly used in the next step without purification.
Compound 32-4 (199 mg, 389.76 μmol, 1 eq) was dissolved in DMF (10 mL). Ammonium chloride (1.04 g, 19.49 mmol, 50 eq), HATU (222.30 mg, 584.65 μmol, 1.5 eq), and DIPEA (151.12 mg, 1.17 mmol, 203.67 μL, 3 eq) were then sequentially added thereto. The reaction was carried out at 25° C. for 1 hour. The reaction system was added with water (10 mL) and ethyl acetate (10 mL) for extraction and phase separation. The aqueous phase was then extracted once with ethyl acetate (10 mL). The organic phases were combined, washed once with saturated sodium chloride aqueous solution (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain a crude product. The crude product was separated by preparative high performance liquid chromatography (chromatographic column: Xtimate C18, 150*40 mm*3 m; mobile phase: A (water containing 0.04% hydrochloric acid) and B (acetonitrile); gradient: B %: 15% to 45%, 10 minutes) to obtain hydrochloride of compound WX-032. 1H NMR (400 MHz, DMSO-d6) δ: 11.49 (brs, 1H), 11.12 (brs, 1H), 9.27-9.08 (m, 1H), 8.67-8.52 (m, 1H), 8.48-8.36 (m, 1H), 8.28-8.17 (m, 1H), 8.12-8.08 (m, 1H), 7.98-7.92 (m, 1H), 7.73-7.59 (m, 2H), 7.44-7.36 (m, 1H),), 3.97-3.84 (m, 1H), 3.54-3.52 (m, 3H), 2.16-2.00 (m, 1H), 1.41-1.25 (m, 6H), 0.93-0.78 (m, 4H); LCMS m/z=510.1[M+1]+.
Under a nitrogen atmosphere, compound A-2-1 (627.52 mg, 3.27 mmol, 1 eq) and 31-1 (900 mg, 3.27 mmol, 1 eq) were added to tetrahydrofuran (10 mL), and the mixture was stirred until dissolved. LiHMDS (1 M, 8.17 mL, 2.5 eq) was slowly added dropwise at 25° C., and then the mixture was stirred at 25° C. for another 1 hour. After the reaction was completed, the reaction mixture was added with water (30 mL) and ethyl acetate (30 mL) for extraction and phase separation. The aqueous phase was then extracted once with ethyl acetate (30 mL). The organic phases were combined, washed once with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain a crude product. The crude product was purified by silica gel column chromatography (methanol/dichloromethane=0 to 10%) to obtain compound 33-1. 1H NMR (400 MHz, DMSO-d6) δ: 11.39 (br s, 1H), 8.94 (s, 2H), 8.65 (s, 1H), 7.563-7.55 (m, 2H), 7.31 (t, J=8.0 Hz, 1H), 6.85 (s, 1H), 3.77-3.69 (m, 1H), 3.66 (s, 3H), 1.31 (d, J=6.8 Hz, 6H); LCMS m/z=431.1 [M+1]+.
Compound 33-1 (500 mg, 1.16 mmol, 1 eq), cyclopropanecarboxamide (2.47 g, 29.01 mmol, 25 eq), and Xantphos (201.42 mg, 348.10 μmol, 0.3 eq) were sequentially added to a mixed solvent of NMP (1 mL) and 1,4-dioxane (10 mL). The reaction mixture was replaced with nitrogen three times, and cesium carbonate (1.51 g, 4.64 mmol, 4 eq) and Pd2(dba)3·CHCl3 (180.16 mg, 174.05 μmol, 0.15 eq) were added thereto. After replacing with nitrogen three times, the reaction mixture was heated to reflux (external temperature of 130° C.) and reacted for 16 hours. After cooling to room temperature, the reaction mixture was added with water (20 mL) and ethyl acetate (20 mL) for extraction and phase separation. The aqueous phase was then extracted once with ethyl acetate (10 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain a crude product. The crude product was purified by silica gel column chromatography (methanol/dichloromethane=0 to 10%) to obtain compound 33-2. LCMS m/z=480.1 [M+1]+.
Compound 33-2 (150 mg, 312.79 μmol, 1 eq) was dissolved in DCM (2 mL). 3-Chloroperoxybenzoic acid (80.97 mg, 469.19 μmol, 1.5 eq) was added thereto. The reaction was carried out at 20° C. for 16 hours. After the reaction was completed, the reaction mixture was added with water (20 mL) and ethyl acetate (20 mL) for extraction and phase separation. The aqueous phase was then extracted once with ethyl acetate (10 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product of 33-3. LCMS m/z=512.1 [M+1]+. The crude product was directly used in the next step without purification.
Compound 33-3 (140 mg, 273.68 μmol, 1 eq) and ammonium chloride (731.97 mg, 13.68 mmol, 50 eq) were added to DMF (10 mL). HATU (312.18 mg, 821.04 μmol, 3 eq) and DIPEA (212.23 mg, 1.64 mmol, 286.02 μL, 6 eq) were sequentially added thereto. The mixture was stirred and reacted at 25° C. for 1 hour. The reaction system was added with water (10 mL) and ethyl acetate (10 mL) for extraction and phase separation. The aqueous phase was then extracted once with ethyl acetate (10 mL). The organic phases were combined, washed once with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain a crude product. The crude product was separated by preparative high performance liquid chromatography (chromatographic column: Welch Xtimate C18 100*40 mm*3 m; mobile phase: A (water containing 0.075% trifluoroacetic acid) and B (acetonitrile); gradient: B %: 10% to 40%, 8 minutes). The obtained solution was concentrated under vacuum at 40° C. to remove acetonitrile, then adjusted to alkalinity (pH=8) with saturated sodium bicarbonate, and extracted with dichloromethane (30 mL*3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain compound WX-033. 1H NMR (400 MHz, CDCl3) δ: 10.67 (br s, 1H), 9.27 (s, 2H), 8.38 (s, 1H), 8.16-8.04 (m, 1H), 7.82 (d, J=8.4 Hz, 1H), 7.73 (d, J=6.8 Hz, 1H), 7.40-7.34 (m 1H), 5.83 (brs, 2H), 3.85 (s, 2H), 3.38-3.24 (m, 1H), 1.74-1.65 (m, 1H), 1.44 (d, J=6.8 Hz, 6H), 1.13-1.06 (m, 2H), 0.95-0.89 (m, 2H); LCMS m/z=511.2 [M+1]+.
Under a nitrogen atmosphere, 32-1 (250 mg, 911.14 μmol, 1 eq) was added to THF (10 mL), then A-2-1 (174.94 mg, 911.14 μmol, 1 eq) was added thereto, and the mixture was stirred until dissolved. LiHMDS (1 M, 2.28 mL, 2.5 eq) was added dropwise thereto at 25° C., and the mixture was stirred for another 2 hours. After the reaction was completed, the reaction mixture was added with saturated ammonium chloride aqueous solution (20 mL) and water (50 mL), and extracted with ethyl acetate (50 mL*2). The organic phases were combined, washed with saturated brine (50 mL*2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain a crude product. The crude product was purified by silica gel column chromatography (methanol/dichloromethane=10%) to obtain compound 34-1. LCMS m/z=430.0 [M+1]+.
Compound 34-1 (550 mg, 1.28 mmol, 1 eq) and cyclopropanecarboxamide (2.72 g, 31.98 mmol, 25 eq) were added to a mixed solvent of 1,4-dioxane (20 mL) and NMP (2 mL). Cesium carbonate (1.25 g, 3.84 mmol, 3 eq) and Xantphos (111.04 mg, 191.90 μmol, 0.15 eq) were then added thereto. After replacing with nitrogen three times, Pd2(dba)3 (175.72 mg, 191.90 μmol, 0.15 eq) was added. The mixture was reacted at 130° C. for 18 hours. After the reaction was cooled to room temperature, the reaction mixture was added with water (30 mL) and ethyl acetate (30 mL) for extraction and phase separation. The aqueous phase was then extracted once with ethyl acetate (30 mL). The organic phases were combined, washed once with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain a crude product. The crude product was purified by silica gel column chromatography (methanol/dichloromethane=3%) to obtain compound 34-2. LCMS m/z=479.2 [M+1]+.
Compound 34-2 (430 mg, 898.52 μmol, 1 eq) was dissolved in dichloromethane (10 mL). The mixture was then cooled to 0° C. and added with 3-chloroperoxybenzoic acid (364.84 mg, 1.80 mmol, purity of 85%, 2 eq). The reaction was then carried out at 25° C. for 2 hours. After the reaction was completed, the reaction mixture was added with water (30 mL) and ethyl acetate (30 mL) for extraction and phase separation. The aqueous phase was then extracted once with ethyl acetate (30 mL). The organic phases were combined, washed once with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain a crude product of 34-3. LCMS m/z=511.1 [M+1]+. The crude product was directly used in the next step without further purification.
Compound 34-3 (458 mg, 897.05 μmol, 1 eq) was added to DMF (10 mL). Ammonium chloride (2.40 g, 44.85 mmol, 50 eq), HATU (511.63 mg, 1.35 mmol, 1.5 eq), and DIPEA (347.81 mg, 2.69 mmol, 468.74 μL, 3 eq) were then added thereto. The reaction was carried out at 25° C. for 1 hour. After the reaction was completed, the reaction mixture was added with water (10 mL) and ethyl acetate (10 mL) for extraction and phase separation. The aqueous phase was then extracted once with ethyl acetate (10 mL). The organic phases were combined, washed once with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain a crude product. The crude product was separated by preparative high performance liquid chromatography (chromatographic column: Welch Xtimate C18 100*40 mm*3 m; mobile phase: A (water containing 0.075% trifluoroacetic acid) and B (acetonitrile); gradient: B %: 15 to 45%, 8 minutes) to obtain trifluoroacetate of compound WX-034. 1H NMR (400 MHz, DMSO-d6) δ: 11.34-10.93 (m, 2H), 9.22-9.00 (m, 1H), 8.59 (s, 1H), 8.50-8.23 (m, 2H), 8.17 (d, J=8.4 Hz, 1H), 7.77-7.56 (m, 4H), 7.42-7.30 (m, 1H), 3.65-3.58 (m, 1H), 3.52 (s, 3H), 2.01-1.87 (m, 1H), 1.24 (d, J=6.8 Hz, 6H), 0.93-0.79 (m, 4H); LCMS m/z=510.1 [M+1]+.
Compound 21-1 (100 mg, 304.56 μmol, 1 eq) and A-3-2 (60.58 mg, 304.56 μmol, 1 eq) were dissolved in THE (10 mL). LiHMDS (1 M, 1.22 mL, 4 eq) was added dropwise thereto at 0° C., and then the mixture was stirred and reacted at 25° C. for 2 hours. The reaction mixture was added with 8 mL of methanol, concentrated under reduced pressure, then added with water (20 mL), and extracted with dichloromethane (30 mL*2). The organic phase was washed with saturated sodium chloride solution (25 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain a crude product. The crude product was purified by silica gel column chromatography (dichloromethane:methanol=100:0 to 100:5) to obtain compound 35-1. LCMS m/z=485.0 [M+1]+.
Compound 35-1 (50 mg, 103.12 μmol, 1 eq), cyclopropanecarboxamide (219.40 mg, 2.58 mmol, 25 eq), and Xantphos (8.95 mg, 15.47 μmol, 0.15 eq) were dissolved in a mixed solvent of 1,4-dioxane (1 mL) and NMP (0.5 mL). Cesium carbonate (100.80 mg, 309.36 μmol, 3 eq) was then added thereto. After replacing with nitrogen, Pd2(dba)3 (14.16 mg, 15.47 μmol, 0.15 eq) was added thereto. After replacing with nitrogen, the reaction mixture was heated to reflux and reacted for 12 hours. After the reaction was completed, the reaction mixture was cooled to room temperature, and concentrated under reduced pressure to remove the organic solvent. The crude product was purified by silica gel column chromatography (dichloromethane:methanol=100:0 to 1:1) to obtain compound 35-2. LCMS m/z=534.1 [M+1]+.
Compound 35-2 (30 mg, 56.23 μmol, 1 eq), ammonium chloride (150.39 mg, 2.81 mmol, 50 eq), and HATU (32.07 mg, 84.35 μmol, 1.5 eq) were dissolved in DMF (2 mL). DIPEA (21.80 mg, 168.69 μmol, 29.38 μL, 3 eq) was then added thereto. The mixture was stirred and reacted at 25° C. for 12 hours. After the reaction was completed, the reaction mixture was added with water (6 mL) and extracted with ethyl acetate (10 mL*2). The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain a crude product. The crude product was purified by preparative high performance liquid chromatography (chromatographic column: Welch Xtimate C18 100*40 mm*3 m; mobile phase: A (water containing 0.075% trifluoroacetic acid) and B (acetonitrile); gradient: B %: 8 to 38%, 8 minutes) to obtain trifluoroacetate of compound WX-035. 1H NMR (400 MHz, CDCl3) δ: 13.05 (br s, 1H), 11.53 (s, 1H), 9.06 (d, J=4.4 Hz, 1H), 8.40 (s, 1H), 8.19-8.13 (m, 1H), 7.96 (br d, J=8.0 Hz, 1H), 7.71 (d, J=7.8 Hz, 1H), 7.61 (br s, 1H), 7.45 (d, J=6.8 Hz, 1H), 7.35-7.30 (m, 1H), 5.81 (br s, 1H), 3.48 (s, 3H), 2.00-1.93 (m, 1H), 1.06-0.87 (m, 14H); LCMS m/z=533.2 [M+1]+.
Compound A-1 (2 g, 8.03 mmol, 1 eq), compound A-14 (1.36 g, 7.23 mmol, 0.9 eq), and potassium phosphate tribasic (5.11 g, 24.09 mmol, 3 eq) were dissolved in 1,4-dioxane (20 mL) and water (10 mL). After replacing with nitrogen three times, Pd(dppf)Cl2·CH2Cl2 (393.38 mg, 481.71 μmol, 0.06 eq) was added. The mixture was stirred at 100° C. for 2 hours. The reaction mixture was added with water (10 mL) and extracted with ethyl acetate (20 mL*2). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether=0 to 50%) to obtain compound 36-1. 1HNMR (400 MHz, CDCl3) δ: 7.83-7.80 (m, 1H), 7.30-7.29 (m, 1H), 7.28-7.27 (m, 1H), 7.08-7.04 (m, 1H), 6.87 (dd, J=8.0, 1.6 Hz, 1H), 4.39-4.33 (m, 1H), 3.53 (s, 3H), 1.51 (d, J=6.8 Hz, 6H); LCMS m/z=276.1 [M+H]+.
Compound 36-1 (1 g, 3.63 mmol, 1 eq) and compound A-3-2 (830.74 mg, 4.18 mmol, 1.15 eq) were dissolved in isopropanol (10 mL) and water (3 mL). Zinc acetate (799.56 mg, 4.36 mmol, 1.2 eq) was added thereto. The mixture was stirred at 80° C. for 16 hours. The reaction mixture was added with water (30 mL), filtered, and the filter cake was collected to obtain compound 36-2. LCMS m/z=432.2 [M+H]+.
Compound 36-2 (1 g, 2.32 mmol, 1 eq), cyclopropanecarboxamide (788.19 mg, 9.26 mmol, 4 eq), cesium carbonate (1.51 g, 4.63 mmol, 2 eq), and Xantphos (133.97 mg, 231.54 mol, 0.1 eq) were dissolved in dioxane (10 mL). After replacing with nitrogen three times, Pd2(dba)3 (212.02 mg, 231.54 μmol, 0.1 eq) was added. The mixture was stirred at 120° C. for 3 hours. The reaction mixture was added with water (10 mL) and extracted with dichloromethane (10 mL*2). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was added with tert-butyl methyl ether (20 mL), stirred at 20° C. for 1 hour, and then filtered. The filter cake was collected to obtain compound 36-3. LCMS m/z=481.3 [M+H]+.
Compound 36-3 (1.42 g, 2.96 mmol, 1 eq) was dissolved in NMP (10 mL) and acetonitrile (5 mL). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (793.08 mg, 4.14 mmol, 1.4 eq), 1-hydroxybenzotriazole (199.65 mg, 1.48 mmol, 0.5 eq), methylamine hydrochloride (199.52 mg, 2.96 mmol, 1 eq), and methylimidazole (727.85 mg, 8.87 mmol, 706.65 μL, 3 eq) were then added thereto. The mixture was stirred at 65° C. for 1 hour. The reaction mixture was added with water (10 mL) and extracted twice with dichloromethane (10 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether=0 to 50%) to obtain compound 36-4. LCMS m/z=494.2 [M+H]+.
Compound 36-4 (0.7 g, 1.42 mmol, 1 eq) was dissolved in methanol (14 mL). (Diacetoxyiodo)benzene (1.14 g, 3.55 mmol, 2.5 eq) and ammonium acetate (273.30 mg, 3.55 mmol, 2.5 eq) were added thereto, and the mixture was stirred at 15° C. for 2 hours. The reaction mixture was added with water (15 mL) and extracted with dichloromethane (15 mL*2). The organic phase was washed with 5% sodium thiosulfate aqueous solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (methanol/ethyl acetate=0 to 10%) to obtain compound WX-036. Compound WX-036 was subjected to resolution by SFC (column: Chiralpak UH (250 mm*30 mm, 10 m); mobile phase: A (CO2) and B (ethanol containing 0.1% ammonia water); gradient: B %=50% to 50%, 15 minutes) to obtain compound WX-036A and compound WX-036B.
WX-036A: 1HNMR (400 MHz, CDCl3) δ: 11.14 (s, 1H), 8.94 (s, 1H), 8.40-8.38 (m, 1H), 8.27-8.23 (m, 2H), 8.19-8.14 (m, 1H), 7.85-7.83 (m, 1H), 7.62 (dd, J=8.0, 1.6 Hz, 1H), 7.42-7.38 (m, 1H), 4.08-4.01 (m, 2H), 3.56 (s, 3H), 3.07 (d, J=5.2 Hz, 3H), 1.75-1.69 (m, 1H), 1.48 (d, J=7.0 Hz, 3H), 1.42 (d, J=6.8 Hz, 3H), 1.15-1.11 (m, 2H), 0.98-0.93 (m, 2H); LCMS m/z=525.2 [M+H]+. SFC (column: Chiralpak IH-3, 3 μm, 0.46 cm id×5 cm L; mobile phase: A (CO2) and B (EtOH containing 0.1% isopropylamine); gradient: B %=5 to 50%, 3 minutes; flow rate: 3.4 mL/min; wavelength: 220 nm; pressure: 100 bar, Rt=1.615 min) with a chiral isomer excess of 100%.
WX-036B: 1HNMR (400 MHz, CDCl3) δ: 11.14 (s, 1H), 8.92 (s, 1H), 8.40-8.38 (m, 1H), 8.27-8.22 (m, 2H), 8.19-8.17 (m, 1H), 7.84 (dd, J=8.0, 1.6 Hz, 1H), 7.62 (dd, J=8.0, 1.6 Hz, 1H), 7.42-7.38 (m, 1H), 4.08-4.01 (m, 1H), 3.56 (s, 3H), 3.07 (d, J=5.2 Hz, 3H), 1.74-1.69 (m, 2H), 1.48 (d, J=6.8 Hz, 3H), 1.42 (d, J=7.2 Hz, 3H), 1.15-1.11 (m, 2H), 0.98-0.94 (m, 2H); LCMS m/z=525.2 [M+H]+. SFC (column: Chiralpak IH-3, 3 μm, 0.46 cm id×5 cm L; mobile phase: A (CO2) and B (EtOH containing 0.1% isopropylamine); gradient: B %=5 to 50%, 3 minutes; flow rate: 3.4 mL/min; wavelength: 220 nm; pressure: 100 bar, Rt=1.771 min) with a chiral isomer excess of 99.38%.
Compound A-1 (2 g, 8.03 mmol, 1 eq), compound A-15 (1.12 g, 4.82 mmol, 0.6 eq), and potassium phosphate tribasic (5.11 g, 24.09 mmol, 3 eq) were dissolved in dioxane (20 mL) and water (10 mL). After replacing with nitrogen three times, Pd(dppf)Cl2·CH2Cl2 (393.38 mg, 481.71 μmol, 0.06 eq) was added. The mixture was stirred at 100° C. for 1.5 hours. The reaction mixture was added with water (10 mL) and extracted twice with ethyl acetate (20 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether=0 to 50%) to obtain compound 37-1. 1HNMR (400 MHz, CDCl3) δ: 8.67-8.65 (m, 1H), 7.76 (dd, J=8.4, 2.4 Hz, 1H), 7.23-7.21 (m, 1H), 7.02-6.98 (m, 1H), 6.79-6.71 (m, 2H), 4.08-3.98 (m, 3H), 3.44 (s, 3H), 1.45 (d, J=6.8 Hz, 6H); LCMS m/z=275.0 [M+H]+.
Compound 37-1 (950 mg, 3.46 mmol, 1 eq) and compound A-3-2 (757.60 mg, 3.81 mmol, 1.1 eq) were dissolved in isopropanol (9.5 mL) and water (3.2 mL). Zinc acetate (762.32 mg, 4.15 mmol, 1.2 eq) was then added thereto. The mixture was stirred at 80° C. for 16 hours. The reaction mixture was added with water (30 mL), filtered, and the filter cake was collected to obtain compound 37-2. LCMS m/z=431.0 [M+H]+.
Compound 37-2 (1 g, 2.32 mmol, 1 eq), cyclopropanecarboxamide (790.00 mg, 9.28 mmol, 4 eq), and cesium carbonate (1.51 g, 4.64 mmol, 2 eq) were dissolved in dioxane (10 mL). After replacing with nitrogen three times, Xantphos (134.28 mg, 232.07 μmol, 0.1 eq) and Pd2(dba)3 (212.51 mg, 232.07 μmol, 0.1 eq) were added. The mixture was stirred at 120° C. for 3 hours. The reaction mixture was added with water (10 mL) and extracted with dichloromethane (10 mL*2). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was added with tert-butyl methyl ether (20 mL), stirred at 20° C. for 1 hour, and then filtered. The filter cake was collected to obtain compound 37-3. LCMS m/z=480.3 [M+H]+.
Compound 37-3 (1.06 g, 2.21 mmol, 1 eq) was dissolved in NMP (10 mL) and acetonitrile (5 mL). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (593.23 mg, 3.09 mmol, 1.4 eq), 1-hydroxybenzotriazole (149.33 mg, 1.11 mmol, 0.5 eq), methylamine hydrochloride (149.24 mg, 2.21 mmol, 1 eq), and N-methylimidazole (544.42 mg, 6.63 mmol, 528.57 μL, 3 eq) were then added thereto. The mixture was stirred at 65° C. for 1 hour. The reaction mixture was added with water (10 mL) and extracted with dichloromethane (10 mL*2). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether=0 to 50%) to obtain compound 37-4. LCMS m/z=493.2 [M+H]+.
Compound 37-4 (0.9 g, 1.83 mmol, 1 eq) was dissolved in methanol (18 mL). (Diacetoxyiodo)benzene (1.47 g, 4.57 mmol, 2.5 eq) and ammonium acetate (352.08 mg, 4.57 mmol, 2.5 eq) were added thereto, and the mixture was stirred at 15° C. for 2 hours. The reaction mixture was added with water (15 mL) and extracted with dichloromethane (15 mL*2). The organic phase was washed with 5% sodium thiosulfate aqueous solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (methanol/ethyl acetate=0 to 10%) to obtain compound WX-037. Compound WX-037 was subjected to resolution by SFC (column: Chiralpak UH (250 mm*30 mm, 10 m); mobile phase: A (CO2) and B (methanol containing 0.1% ammonia water); gradient: B %=45% to 45%, 10 minutes) to obtain WX-037A and WX-037B.
WX-037A: 1HNMR (400 MHz, CDCl3) δ: 11.14 (s, 1H), 8.97-8.96 (m, 1H), 8.84-8.75 (m, 1H), 8.26 (s, 1H), 8.22-8.16 (m, 3H), 7.55 (dd, J=8.0, 1.6 Hz, 1H), 7.35-7.31 (m, 1H), 7.23-7.21 (m, 1H), 3.83-3.76 (m, 1H), 3.46 (s, 3H), 3.07 (d, J=5.2 Hz, 3H), 1.73-1.69 (m, 1H), 1.42 (d, J=7.2 Hz, 3H), 1.36 (d, J=6.8 Hz, 3H), 1.15-1.12 (m, 2H), 0.99-0.94 (m, 2H); LCMS m/z=524.2 [M+H]+. SFC (column: Chiralpak IH-3, 3 μm, 0.46 cm id×10 cm L; mobile phase: A (CO2) and B (MeOH containing 0.1% isopropylamine); gradient: B %=10% to 50%, 4 minutes; flow rate: 3.4 mL/min; wavelength: 220 nm; pressure: 100 bar), with Rt of 2.795 min, and a chiral isomer excess of 100%.
WX-037B: 1HNMR (400 MHz, CDCl3) δ: 11.15 (s, 1H), 9.03-8.78 (m, 2H), 8.38-8.08 (m, 4H), 7.56-7.53 (m, 1H), 7.35-7.31 (m, 1H), 7.22-7.21 (m, 1H), 3.85-3.76 (m, 1H), 3.46 (s, 3H), 3.07 (d, J=4.4 Hz, 3H), 1.74-1.69 (m, 1H), 1.42 (d, J=6.0 Hz, 3H), 1.42 (d, J=6.4 Hz, 3H), 1.14-1.13 (m, 2H), 0.99-0.94 (m, 2H); LCMS m/z=524.2 [M+H]+. SFC (column: Chiralpak IH-3, 3 μm, 0.46 cm id×10 cm L; mobile phase: A (CO2) and B (MeOH containing 0.1% isopropylamine); gradient: B %=10% to 50%, 4 minutes; flow rate: 3.4 mL/min; wavelength: 220 nm; pressure: 100 bar), with Rt of 2.983 min, and a chiral isomer excess of 98.50%.
Under a nitrogen atmosphere, compound 36-1 (1.82 g, 6.61 mmol, 1 eq) and A-12 (1.36 g, 6.61 mmol, 1 eq) were sequentially added to tetrahydrofuran (50 mL), and the mixture was stirred until dissolved. LiHMDS (1 M, 16.52 mL, 2.5 eq) was slowly added dropwise thereto at 0° C., and the mixture was stirred at 25° C. for another 2 hours. After the reaction was completed, the reaction mixture was added with water (100 mL) and extracted with ethyl acetate (100 mL*2). The organic phases were combined, washed with saturated brine (100 mL*2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (dichloromethane:methanol=100:3) to obtain compound 38-1. LCMS m/z=444.0 [M+1]+.
Compound 38-1 (2.1 g, 4.73 mmol, 1 eq) and cyclopropanecarboxamide (4.03 g, 47.30 mmol, 10 eq) were dissolved in dioxane (40 mL) and NMP (2 mL). Cesium carbonate (4.62 g, 14.19 mmol, 3 eq) and Xantphos (410.55 mg, 709.54 μmol, 0.15 eq) were then added thereto. After replacing with nitrogen three times, Pd2(dba)3 (649.74 mg, 709.54 μmol, 0.15 eq) was added. The mixture was stirred at 130° C. for 16 hours. After the reaction was completed, the reaction mixture was cooled to room temperature, then added with water (40 mL), and extracted with ethyl acetate (40 mL*2). The organic phase was washed with saturated brine (40 mL*2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (dichloromethane:methanol=10:1) to obtain compound 38-2. LCMS m/z=493.1 [M+1]+.
Compound 38-2 (200 mg, 406.02 μmol, 1 eq) was added to a mixed solution of ethanol (10 mL) and water (5 mL), and potassium hydrogenperoxomonosulphate (374.41 mg, 609.02 mol, 1.5 eq) was added thereto. The reaction was carried out at 25° C. for 2 hours. The reaction mixture was directly filtered to obtain compound WX-038. 1H NMR (400 MHz, CDCl3) δ: 11.97 (br s, 1H), 9.39 (br s, 1H), 9.27 (s, 1H), 8.39 (d, J=8.8 Hz, 1H), 8.27-8.16 (m, 2H), 7.97 (dd, J=1.4, 7.9 Hz, 1H), 7.62 (d, J=6.8 Hz, 1H), 7.44 (t, J=7.9 Hz, 1H), 4.19-4.09 (m, 1H), 3.55 (s, 3H), 3.06 (d, J=4.3 Hz, 3H), 2.09-1.97 (m, 1H), 1.48 (d, J=7.0 Hz, 6H), 1.05-0.96 (m, 2H), 0.94-0.85 (m, 2H); LCMS m/z=525.1 [M+1]+.
Under a nitrogen atmosphere, compound 37-1 (532 mg, 1.94 mmol, 1 eq) and A-12 (397.56 mg, 1.94 mmol, 1 eq) were sequentially added to tetrahydrofuran (20 mL), and the mixture was stirred until dissolved. LiHMDS (1 M, 4.85 mL, 2.5 eq) was slowly added dropwise thereto at 0° C., and the mixture was stirred at 25° C. for another 2 hours. After the reaction was completed, the reaction mixture was added with water (50 mL) and extracted with ethyl acetate (50 mL*2). The organic phases were combined, washed with saturated brine (100 mL*2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (dichloromethane:methanol=100:3) to obtain compound 39-1. LCMS m/z=443.0 [M+1]+.
Compound 39-1 (786 mg, 1.77 mmol, 1 eq) and cyclopropanecarboxamide (1.51 g, 17.74 mmol, 10 eq) were dissolved in dioxane (20 mL) and NMP (0.5 mL). Cesium carbonate (1.73 g, 5.32 mmol, 3 eq) and Xantphos (154.01 mg, 266.16 μmol, 0.15 eq) were then added thereto. After replacing with nitrogen three times, Pd2(dba)3 (243.73 mg, 266.16 μmol, 0.15 eq) was added. The mixture was stirred at 130° C. for 16 hours. After the reaction was completed, the reaction mixture was cooled to room temperature, then added with water (20 mL), and extracted with ethyl acetate (20 mL*2). The organic phase was washed with saturated brine (20 mL*2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (dichloromethane:methanol=10:1) to obtain compound 38-2. LCMS m/z=492.2 [M+1]+.
Compound 39-2 (200 mg, 406.83 μmol, 1 eq) was added to a mixed solution of ethanol (10 mL) and water (5 mL), and potassium hydrogenperoxomonosulphate (375.16 mg, 610.25 mol, 1.5 eq) was added thereto. The reaction was carried out at 25° C. for 2 hours. After the reaction was completed, the reaction mixture was added with saturated sodium bisulfite aqueous solution (10 mL), and extracted with ethyl acetate (20 mL*2). The organic phase was washed with saturated brine (10 mL*2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (DCM:MeOH=10:1) to obtain compound WX-039. 1H NMR (400 MHz, CDCl3) δ: 10.58 (s, 1H), 9.10-8.90 (m, 1H), 8.35 (s, 2H), 8.28-8.21 (m, 1H), 8.20-8.14 (m, 2H), 7.62 (dd, J=1.3, 8.0 Hz, 1H), 7.33 (t, J=7.9 Hz, 1H), 7.16 (dd, J=1.5, 7.8 Hz, 1H), 6.35 (br s, 1H), 3.89-3.79 (m, 1H), 3.49 (s, 3H), 3.05 (d, J=4.8 Hz, 3H), 1.61-1.53 (m, 1H), 1.40 (d, J=7.0 Hz, 6H), 1.15-1.07 (m, 2H), 0.96-0.88 (m, 2H); LCMS m/z=524.1 [M+1]+.
In this experiment, the inhibitory effect of compounds on TYK2 JH2 pseudokinase was tested using the method of time-resolved fluorescence resonance energy transfer (TR-FRET). In the experiment, TYK2 JH2 or JAK1 JH2 pseudokinase can simultaneously bind to fluorescently labeled Tracer and Tb antibody to form a “sandwich structure”. Tb antibody, functioning as a fluorescent donor, produces fluorescence at a wavelength of 495 nm under the excitation of light at a specific wavelength. Tracer, functioning as a fluorescent acceptor, can receive fluorescence at a wavelength of 495 nm and thus produce fluorescence at a wavelength of 520 nm, i.e., a time-resolved fluorescence resonance energy transfer (TR-FRET) signal, only when it is within the “sandwich structure”, i.e., when it is sufficiently close to Tb antibody. When the compound is added to compete with Tracer for binding to the pseudokinase, the binding of Tracer decreases, leading to a weakened TR-FRET signal. The inhibitory activity of the compound binding to the pseudokinase can be reflected by the ratio of signal values at 520 nm/495 nm.
The working solution consists of the following components: HEPES (pH 7.5) at a final concentration of 20 mM; MgCl2 at a final concentration of 10 mM; Brij-35 at a final concentration of 0.015%; DTT at a final concentration of 2 mM; and BSA at a final concentration of 50 μg/mL.
Data analysis was performed using the XL-Fit software to determine the IC50 of the compounds. The results are shown in Table 11:
Conclusion: The compounds of the present disclosure exhibit good inhibitory effect on TYK2 JH2 pseudokinase.
Adenosine triphosphate (ATP) serves as a universal energy carrier in various life processes, representing the smallest unit of energy storage and transfer. The CellTiter-Glo™ Luminescent Cell Viability Assay Kit utilizes luciferase as a detection agent. During the luminescent process, luciferase requires the participation of ATP. By adding CellTiter-Glo™ reagent to the cell culture medium and measuring the luminescence, the light signal was directly proportional to the amount of ATP in the system, and ATP was positively correlated with the number of viable cells. Therefore, cell proliferation can be detected by measuring ATP content using the CellTiter-Glo kit. In this test, the cell lines used were Ba/F3-FL-TYK2-E957D and Ba/F3-TEL-TYK2. Ba/F3-FL-TYK2-E957D cells can stably express the exogenously introduced human TYK2-E957D gene, and the TYK2-E957D gene sequence contains both JH1 and JH2 domains. On the other hand, Ba/F3-TEL-TYK2 cells can stably express the exogenously introduced human TEL-TYK2 gene, and the TEL-TYK2 gene sequence contains only the JH1 domain of TYK2.
The cell lines were cultured in an incubator at 37° C. with 5% CO2. Regular passaging was performed, and cells in the logarithmic growth phase were selected for plating.
The following steps were conducted in accordance with the instructions of the Promega CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega-G7573).
Data analysis was conducted using GraphPad Prism 5.0 software. Nonlinear S-curve regression was employed to fit the data and generate dose-response curves. From these curves, the IC50 values were calculated. The data are shown in Table 12.
Conclusion: The compounds of the present disclosure exhibit strong inhibitory activity against the proliferation of Ba/F3 cells transfected with the human TYK2-E957D gene (containing both JH1 and JH2 domains of TYK2). However, they have no inhibitory activity against the proliferation of Ba/F3 cells transfected with the human TEL-TYK2 gene (which contains only the JH1 domain of TYK2). This suggests that the compounds of the present disclosure are highly selective allosteric inhibitors of TYK2 JH12.
The purpose of this experiment was to detect the inhibitory effect of the compounds on cytokine-activated JAK-STAT signaling pathways in human peripheral blood mononuclear cells (PBMC).
Flow cytometer: Brand: BD, model: Fortessa
Culture medium: 1640 culture medium+10% fetal bovine serum+1% double antibiotic+1% non-essential amino acids (all percentages are volume ratios)
Data analysis was performed using Flowjo software to determine the IC50 of the compounds. The results are shown in Table 14:
Conclusion: The compound of the present disclosure exhibits high inhibitory activity against the TYK2 signaling pathway activated by IFN-α stimulation in human PBMC cells. Meanwhile, it exhibits weak inhibitory activity against the JAK1/2 signaling pathway activated by IL-6 stimulation, the JAK2/2 signaling pathway activated by GM-CSF stimulation, and the JAK1/3 signaling pathway activated by IL-2 stimulation, thereby showing high selectivity.
The objective was to study the pharmacokinetic behavior of the compound of the present disclosure in mice and evaluate the pharmacokinetic characteristics, using male CD-1 mice aged 7 to 9 weeks as test animals. The drug concentrations in plasma at different time points after a single intravenous injection (IV) and oral administration (PO) of the compound were determined using LC/MS/MS.
The pharmacokinetic characteristics of the compound in rodents following intravenous injection and oral administration were tested using a standard protocol. Test animals were fasted for 10 to 14 hours before administration and allowed to feed 4 hours post-administration. The compound was prepared into a clear solution with the appropriate solvent for both IV (intravenous injection) and PO (oral gavage) administration. The solvent was 10% DMSO+10% solutol+80% (10% HP-β-CD aqueous solution). Whole blood samples were collected within 24 hours, centrifuged at 6000 g for 3 minutes, and the supernatant was separated to obtain plasma samples. Protein precipitation was performed by adding four times the volume of acetonitrile solution containing internal standard to the plasma samples. After centrifugation, the supernatant was added with an equal volume of water and then centrifuged before being subjected to LC-MS/MS analysis for quantifying plasma drug concentration. Pharmacokinetic parameters such as peak concentration, time to peak concentration, clearance, half-life, area under the drug-time curve, and bioavailability were calculated.
The results of the pharmacokinetic parameters are shown in Table 15.
Conclusion: The compounds of the present disclosure exhibit excellent pharmacokinetic properties in mice.
The objective was to study the pharmacokinetic behavior of the compound of the present disclosure in rats and evaluate the pharmacokinetic characteristics, using male SD rats aged 7 to 9 weeks as test animals. The drug concentrations in plasma at different time points after a single intravenous injection (IV) and oral administration (P0) of the compound were determined using LC/MS/MS.
The pharmacokinetic characteristics of the compound in rodents following intravenous injection and oral administration were tested using a standard protocol. Test animals were fasted for 10 to 14 hours before administration and allowed to feed 4 hours post-administration. The compound was prepared into a clear solution with the appropriate solvent for both IV (intravenous injection) and PO (oral gavage) administration. The solvent was 1000 DMSO+10% solutol+80% (10% HP-β-CD aqueous solution). Whole blood samples were collected within 24 hours, centrifuged at 6000 g for 3 minutes, and the supernatant was separated to obtain plasma samples. Protein precipitation was performed by adding four times the volume of acetonitrile solution containing internal standard to the plasma samples. After centrifugation, the supernatant was added with an equal volume of water and then centrifuged before being subjected to LC-MS/MS analysis for quantifying plasma drug concentration. Pharmacokinetic parameters such as peak concentration, time to peak concentration, clearance, half-life, area under the drug-time curve, and bioavailability were calculated.
The results of the pharmacokinetic parameters are shown in Table 16.
Conclusion: The compounds of the present disclosure exhibit excellent pharmacokinetic properties in rats.
The purpose of this experiment was to detect the inhibitory effect of the compound on the JAK-STAT signaling pathway activated by IFN-α in mouse whole blood. Fresh mouse whole blood was collected and placed in a 96-well plate. The test compound was added and incubated for 1 hour, followed by stimulation with IFN-α. The corresponding STAT1 phosphorylation level in the CD3+ cell population was analyzed using flow cytometer by means of surface antibody staining and intracellular phosphorylated antibody staining. The IFN-α-induced STAT1 phosphorylation in mouse whole blood is dependent on TYK2 activity. By detecting the inhibitory activity of the compound on downstream STAT5 phosphorylation, the half maximal inhibitory concentration (IC50) of the compound on TYK2 signaling pathway activity can be determined.
96-well V-bottom microplate, Greiner; 96 square-well deep well plate, Thermo; 96-well flat-bottom microplate, Corning
CO2 incubator: MCO-15AC (Thermo);
Single-channel pipettes: 0.2 to 10 μL, 20 to 200 μL, 200 to 1000 μL (Thermo);
Multi-channel pipettes: 0.2 to 10 μL, 5 to 50 μL, 20 to 300 μL (Raining);
Centrifuges: Thermo Centrifuge ST 40R; Thermo LEGEND Micro 21R;
Water purification system: Millipore Milli-Q Reference system;
Vortex mixer: EARTH REQUIRED;
Shaker: QI LIN BEI ER; MH-2;
Flow cytometer: Beckman CytoFlex.
Data were analyzed using FlowJo software. Curve fitting was performed and the IC50 was calculated using GraphPad Prism 8 software. The results are shown in Table 18.
Conclusion: In mouse whole blood, the compound of the present disclosure exhibits high inhibitory activity against the TYK2 signaling pathway activated by IFNα stimulation.
The purpose of this experiment was to detect the inhibitory effect of the compound on the JAK-STAT signaling pathway activated by cytokines in human whole blood or platelet-rich plasma. Fresh human whole blood/platelets were placed in a 96-well plate. The test compound was added and incubated for 1 hour, followed by stimulation with various cytokines. The corresponding STAT phosphorylation levels in different cell populations were analyzed using flow cytometer by means of surface antibody staining and intracellular phosphorylated antibody staining. The experiments using the cytokines IFN-α, TL-6, and TL-2 as stimulants were conducted in human whole blood, whereas the experiment using the cytokine TPO as a stimulant was conducted in platelet-rich plasma.
Data were analyzed using FlowJo software. Curve fitting was performed and the IC50 was calculated using GraphPad Prism 8 software. Data were presented as mean and standard deviation (SD). The inhibition rate of the compound was defined as follows: Inhibition %=(1−(A−B)/(C−B))*100. Where: A is the MFI (mean fluorescence intensity) for experimental wells containing both the compound and the cytokine; B is the MFI (minimum mean fluorescence intensity) for control wells without the cytokine; C is the MFI (maximum mean fluorescence intensity) for control wells containing only the cytokine. The results are shown in Table 20.
Conclusion: In human whole blood or platelets, the compound of the present disclosure exhibits high inhibitory activity against the TYK2 signaling pathway activated by IFNα stimulation. Meanwhile, it exhibits weak inhibitory activity against the JAK1/2 signaling pathway activated by IL-6 stimulation, the JAK2/2 signaling pathway activated by TPO stimulation, and the JAK1/3 signaling pathway activated by IL-2 stimulation, thereby showing high selectivity.
The purpose of this experiment was to evaluate the inhibitory effect of the compound on IL-12/IL-18-induced IFN-γ secretion by immune cells in mice. The IFN-γ protein level in serum samples of mice treated with the compound was detected using the enzyme-linked immunosorbent assay (ELISA) method.
C57BL/6J mice, aged 9 to 10 weeks, weighing 17.82 to 21.42 g, female, supplied by Shanghai Lingchang Biotechnology Co., Ltd.
This experiment was conducted twice. The specific grouping of experimental animals and administration regimen are shown in Tables 22 and 23, respectively.
The compounds were all prepared into a clear solution with 10% DMSO+10% solutol+80% (10% HP-β-CD) as a solvent for PO (oral gavage) administration. The frequency of administration was once daily (QD).
Preparation of recombinant mouse IL-12 protein: An appropriate amount of antibody was transferred to a centrifuge tube, and a certain volume of PBS buffer was added to prepare a solution with a concentration of 0.1 μg/mL. The solution was gently shaken to mix and used immediately.
Preparation of recombinant mouse IL-18/IL-1F4 protein: An appropriate amount of antibody was transferred to a centrifuge tube, and a certain volume of PBS buffer was added to prepare a solution with a concentration of 10 μg/mL. The solution was gently shaken to mix and used immediately.
For the Normal group, the administration consisted of PO (oral gavage) with blank solvent and intraperitoneal injection of blank PBS solution. For the blank control group, the administration consisted of PO (oral gavage) with blank solvent and intraperitoneal injection of inducer IL-12/IL-18.
The plate was covered with a sealing film and incubated at 37° C. for 30 minutes.
The expression level of each sample was calculated based on the standard curve, and statistical analysis of the results for each group was performed, including mean and standard error of the mean (SEM). The signal values for each group were normalized against the blank control group. Statistical analysis was then conducted to assess differences between groups based on this data. A T-test was used for analysis, and for comparisons among three or more groups, one-way ANOVA was employed. All data were analyzed using GraphPad Prism 6.02. The inhibition rate of serum IFN-γ following a single administration of the compound is shown in Table 24.
Conclusion: The compound of the present disclosure exhibits a significant dose-dependent inhibitory effect on IL-12/IL-18-induced IFNγ release in mice.
The purpose of this experiment was to evaluate the alleviating effect of the compound on colitis in a mouse model induced by CD40 antibody.
CB-17 SCID mice, 8 weeks old, weighing 18 to 20 g, female, supplied by Beijing Vital River Laboratory Animal Technology Co., Ltd.
CB-17 SCID mice were randomly divided into groups. Each group consisted of 6 mice, which received an intraperitoneal injection (IP) of CD40 antibody (80 μg per mouse) on Day 0. The positive control drug IL-12 P40 antibody and the test compound were administered from Day-1 to Day 4, with Day 5 being the endpoint of the experiment. The specific grouping of animals is shown in Table 26. The positive control drug IL-12 P40 antibody was administered via intraperitoneal injection (IP) once every three days; the test compound was administered by gavage (PO) twice a day (BID).
The compounds were all prepared into a clear solution with 10% DMSO+10% solutol+80% (10% HP-3-CD) as a solvent for PO (oral gavage) administration. Administration volume parameters: 10 mL/kg based on the body weight of the mice.
Preparation of IL-12 P40 antibody: An appropriate amount of IL-12 P40 antibody stock solution was transferred to a 15 mL centrifuge tube, and a certain volume of DPBS was added to prepare a working solution with a concentration of 2 mg/mL. The solution was gently shaken to mix and used immediately.
IL-12 P40 antibody and the test compound were administered from Day-1 to Day 4. On Day 0, 30 minutes after administration, 200 μL of CD40 antibody (4 mg/kg) was intraperitoneally injected, while mice in the blank control group were intraperitoneally injected with 200 μL of DPBS. Body weight was recorded and feces were scored during the experiment.
Fecal scoring involved placing each mouse individually in a specially designed cage for about 10 minutes for scoring. The fecal scoring criteria were as per Table 27.
If blood is observed in the feces or around the anus, occult blood testing is not conducted. For the remaining mice without visible bloody stools, feces are collected for occult blood testing. Occult blood scoring is based on the assumption that the daily test results for the mice in the Normal group are 0. Each day, the fecal samples of four mice from the blank control group are observed for the time taken to develop color on the occult blood test strips. The shortest time required for color development in the feces of these four mice is set as the daily threshold. If the feces develop color before this time point and the color intensifies within 1 to 2 minutes, a score of 2 is assigned. If no visible color or only a weak color is seen within this threshold time, and later a color develops but is significantly less intense than that of the feces scoring 2, a score of 1 is assigned.
The body weight and Disease Activity Index (DAI) scores of the animals were recorded daily to assess the disease progression in each group and the effect of the test compound on the disease. The daily Disease Activity Index (DAI) score consists of three parts, with specific criteria referenced in Table 27. The results are shown in Table 28. DAI scores are shown in Table 29.
Note: Mean refers to the average value.
Conclusion: In the mouse model of colitis induced by CD40 antibody, the compound of the present disclosure significantly outperformed the blank control group in controlling weight loss and reducing DAI scores at various doses. The compound of the present disclosure exhibits a significant alleviating effect on mouse colitis induced by CD40 antibody.
The purpose of this experiment was to evaluate the prophylactic and therapeutic effect of the test substance on IL-23-induced psoriasis-like skin lesions in mice by intradermal injection of IL-23 into the ear pinnae in a psoriasis-like skin lesion model.
C57BL/6 mice, SPF grade, weighing 19±2 g, female, sourced from the Laboratory Animal Business Department of Shanghai Institute of Planned Parenthood Research. The acclimatization period is 5 to 7 days.
Female C57BL/6 mice of qualified body weight were randomly divided into groups, with 6 mice per group. The grouping and administration information was as follows. The groups were: blank control group, model group, positive control Ustekinumab (5 mg/kg) group, WX-011A hydrochloride (30, 50 mg/kg) group, and WX-020 sulfate (30, 50 mg/kg) group. Except for the blank control group, the animals in the other groups received an intradermal injection of IL-23 (3 μg/10 μL/mouse/day, QD) in the right ear for 8 consecutive days (Day 0 to Day 7). The blank control group received an intradermal injection of an equal volume of saline solution (10 μL/mouse/day, QD) in the right ear for 8 consecutive days (Day 0 to Day 7). Concurrent with model induction, the solvent or test drug was orally administered twice a day (Bid) at 6-hour intervals for 8 consecutive days (Day 0 to Day 7). The positive control was subcutaneously injected on Day 0 and Day 3 (a total of 2 doses). During the administration period, the body weight of the animals was monitored every two days. On Day 0, Day 2, Day 4, Day 6 (all before IL-23 injection), and Day 8, the thickness of the right ear of the mice was measured, and the appearance of the ear pinnae was observed and scored. Animal grouping and administration regimen are shown in Table 31.
The compounds were all prepared into a clear solution with 10% DMSO+1000 solutol+80% (1000 HP-β-CD) as a solvent for PO (oral gavage) administration. Administration volume parameters: 10 mL/kg based on the body weight of the mice.
For the preparation of inducer IL-23, 3 vials were taken. 1.667 mL of sterile water for injection was added to each vial (500 μg), shaken and mixed, then transferred to a 10 mL EP tube, and shaken and mixed to obtain IL-23 solution at a concentration of 0.3 mg/mL. The solution was divided into 8 portions, aliquoted, and stored in a −80° C. freezer.
Ustekinumab (anti-IL-12/IL-23) preparation: 0.15 mL of Ustekinumab (anti-IL-12/IL-23) with a concentration of 5 mg/mL was pipetted and diluted with 1.35 mL of PBS (pH 7.2) solution to obtain Ustekinumab (anti-IL-12/IL-23) solution at a concentration of 0.5 mg/mL.
After grouping, IL-23 (3 μg/10 μL/mouse/day, QD) was injected intradermally into the right ears of the mice for 8 consecutive days (Day 0 to Day 7). Measurements of the thickness of the right ear and observations and scoring of the appearance of the ear pinnae were performed on Day 0, Day 2, Day 4, Day 6 (all before IL-23 injection), and Day 8. At the end of the experiment (Day 8), the left ear tissues of the mice from groups G4 to G10 (3 animals per group at each time point) were collected. The right ear, which had been modeled, was excised, an 8 mm ear punch was taken along the edge of the pinna, and its weight was measured. The excised right ear tissues were fixed in formalin for histopathological examination (H&E staining).
Concurrent with model induction, corresponding drug treatment was given for administration. Except for the positive control drug (Ustekinumab), which was subcutaneously injected on Day 0 and Day 3 (once a day), control and test drugs for each dose group were orally administered by gavage twice a day at 6-hour intervals for 8 consecutive days (Day 0 to Day 7).
The pathological scoring of mouse ear tissue sections was conducted according to the criteria in Table 33.
The experimental data are represented as Mean±SD; statistical analysis was performed using IBM SPSS Statistics 21, with p<0.05 considered to indicate a statistically significant difference between the two groups. The effect of the test substance on the thickness of mouse ears in the IL-23 induced mouse pinna epidermal hyperplasia model is shown in Table 34. The effect of the test substance on the total score and AUC for TL-23 induced mouse pinna epidermal hyperplasia is shown in Table 35. The effect of the test substance on the pathological scoring of right ear tissue sections stained with H&E is shown in Table 36.
##P < 0.01 vs. blank control group
##P < 0.01 vs. blank control group
##P < 0.01 vs. blank control group
Conclusion: Compared to the model group, the compound of the present disclosure, at various doses, exhibits a significant alleviating effect on the IL-23 induced mouse psoriasis-like condition. This was evidenced in the increase thickness of the modeled side ear, the total score of the modeled side ear, and the comprehensive pathological score of the ear tissue sections, demonstrating significant pharmacological efficacy.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202110801875.1 | Jul 2021 | CN | national |
| 202110875486.3 | Jul 2021 | CN | national |
| 202111162655.5 | Sep 2021 | CN | national |
| 202111273381.7 | Oct 2021 | CN | national |
| 202210039225.2 | Jan 2022 | CN | national |
| 202210260637.9 | Mar 2022 | CN | national |
| 202210731484.1 | Jun 2022 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/106053 | 7/15/2022 | WO |
