CN112020357B - Salt of indazolyl-containing tricyclic derivative and crystal form thereof - Google Patents
Salt of indazolyl-containing tricyclic derivative and crystal form thereof Download PDFInfo
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- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
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- A61K31/4965—Non-condensed pyrazines
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- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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- A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
- A61K31/5365—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines ortho- or peri-condensed with heterocyclic ring systems
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- A61K31/5375—1,4-Oxazines, e.g. morpholine
- A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
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- A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
- A61K31/553—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having at least one nitrogen and one oxygen as ring hetero atoms, e.g. loxapine, staurosporine
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- C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
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- C07D471/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains three hetero rings
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- C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
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Abstract
The invention relates to a salt of an indazolyl-containing tricyclic derivative and a crystal form thereof. In particular to a crystal form of a compound with a general formula (I), a preparation method, a pharmaceutical composition containing the crystal form with a therapeutically effective amount and application in preparing medicines for treating ERK-mediated related diseases.
Description
Technical Field
The invention belongs to the field of medicine synthesis, and in particular relates to a salt of an indazolyl-containing tricyclic derivative, and a preparation method and application of a crystal form of the salt.
Background
Members of the ERK signaling pathway, RAS, BRAF, etc., are often mutated in tumors, with approximately 1/3 of human tumor expression being constantly activated mutant RAS,8% of tumor expression being activated BRAF. The mutations associated with ERK signaling pathways and probability statistics in malignancy are shown in table 1. It is counted that RAS mutations are present in 90% of pancreatic cancer, 50% of colorectal cancer and 30% of lung cancer; 50% of melanoma, 50% of thyroid cancer and 15% of colorectal cancer all have BRAF mutations.
TABLE 1 ERK Signal pathway-related mutation types and probabilities in different cancers
Vemurafenib (Vemurafenib) is the first BRAF inhibitor approved by FDA for the treatment of advanced melanoma, but the efficacy can be maintained for 8-9 months, and drug resistance is likely to occur. Studies have demonstrated that reactivation of ERK signaling pathways mediates resistance of melanoma to vemurafenib. Yet another BRAF inhibitor, dabrafenib, is also very susceptible to developing resistance. In addition, vemurafenib also failed to show significant clinical activity in colorectal cancer patients carrying BRAF mutations, with an overall response rate of only 5%. In addition to BRAF inhibitors, MEK inhibitors, which are currently marketed, also exhibit varying degrees of resistance in clinical applications. MEK inhibitors have low tumor response to RAS mutations and melanoma response to BRAF mutations is only 22%. The BRAF inhibitor and the EGFR inhibitor are combined for reversing the drug resistance in clinic, and patients have multi-drug resistance after a few months.
There are a number of preclinical studies to date demonstrating that different classes of ERK upstream target inhibitor resistance can reverse BRAF inhibitor and MEK inhibitor resistance by inhibiting ERK activity, and many pharmaceutical companies are conducting studies such as: genentech, merck, lilly, but no BRAF and MEK inhibitors have been marketed in the local research of china. Patent applications for selective inhibition of ERK1/2 that have been disclosed include WO2012088314, WO2014134776, WO2014179154, WO2014137728, WO2015051314, and the like. However, the existing clinical candidate medicines such as KO-947 have the problem of poor solubility, are clinically administered by intravenous administration, can only exist in a suspension state in a conventional solvent, are unfavorable for patent medicine, and are more difficult to administer orally.
PCT patent (application number: PCT/CN 2018/110795) discloses a series of structures of pyrazolo-containing tricyclic derivative inhibitors, and in subsequent research and development, in order to facilitate processing, filtering and drying of the product, suitable characteristics of convenient storage, long-term stability of the product and the like are sought.
Disclosure of Invention
The content of PCT patent (application number: PCT/CN 2018/110795) is incorporated into the present application as part of the present application.
The invention aims to provide a crystal form of a compound shown in a general formula (I), and the structure of the crystal form is shown in the general formula (I):
wherein:
w is selected from N or CH;
R 1 selected from hydrogen atom, halogen, amino, nitro, hydroxy, cyano, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, C 1-6 Hydroxyalkyl, C 3-8 Cycloalkyl, - (CH) 2 ) n R 3 、-(CH 2 ) n OR 3 、-(CH 2 ) n C(O)R 3 、-(CH 2 ) n NR 3 R 4 Or- (CH) 2 ) n C(O)NR 3 R 4 ；
R 2 Selected from hydrogen atom, halogen, amino, nitro, hydroxy, cyano, C 1-6 Alkyl, C 1-6 Deuterated alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, C 3-8 Cycloalkyl or 3-8 membered heterocyclyl;
R 3 and R is 4 Identical or different and are each independently selected from the group consisting of hydrogen, deuterium, halogen, cyano, hydroxy, amino, nitro, C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-8 Cycloalkyl or 3-8 membered heterocyclyl, wherein said C 1-6 Alkyl, C 1-6 Haloalkyl, amino, C 3-8 Cycloalkyl and 3-8 membered heterocyclyl are optionally further selected from deuterium atoms, C 1-6 Alkyl, C 1-6 Haloalkyl, halogen, hydroxy, amino, nitro, cyano, C 1-6 Alkoxy, C 1-6 Hydroxyalkyl, C 3-8 Cycloalkyl and 3-6 membered heterocyclyl;
m is an inorganic acid or an organic acid, wherein the inorganic acid is selected from hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid or phosphoric acid; the organic acid is selected from 2, 5-dihydroxybenzoic acid, 1-hydroxy-2-naphthoic acid, acetic acid, dichloroacetic acid, trichloroacetic acid, acetohydroxamic acid, adipic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, 4-aminobenzoic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclohexane sulfamic acid, camphorsulfonic acid, aspartic acid, camphoric acid, gluconic acid, glucuronic acid, glutamic acid, isoascorbic acid, lactic acid, malic acid, mandelic acid, pyroglutamic acid, tartaric acid, dodecylsulfuric acid, dibenzoyltartaric acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactonic acid, gentisic acid, glutaric acid, 2-ketoglutaric acid, glycolic acid, hippuric acid, isethionic acid, lactonic acid, ascorbic acid, aspartic acid, lauric acid, camphoric acid, maleic acid, malonic acid, methanesulfonic acid, 1, 5-naphthalenedisulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, thiocyanic acid, undecylenic acid, trifluoroacetic acid, benzenesulfonic acid, p-toluenesulfonic acid or L-malic acid;
x is an integer selected from 0, 1, 2 or 3;
n is an integer of 0, 1, 2 or 3; and is also provided with
t is an integer of 0, 1, 2, 3, 4, 5 or 6.
In a preferred embodiment of the present invention, the crystal form of the compound of formula (I) has a structure as shown in formula (II):
wherein:
W、R 1 、R 2 m, x and t are as defined for formula (I).
In a preferred embodiment of the present invention, the compound of formula (I) has the following structure:
in a preferred embodiment of the present invention, the crystalline form of a compound of formula (I), having the structure shown in formula (III):
x and M are defined as general formula (I).
In a further preferred embodiment of the invention, it is a free base crystalline form of the compound of formula (III). Preferably, it is anhydrous, further comprising water, which is pipeline water or crystallization water, preferably containing 0.5-8 water molecules; more preferably, the hydrate contains 0.5 to 4 water molecules; further preferably 0.5 to 2.5 water molecules; still more preferably contains 1 water molecule. For example containing 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8 water.
In a further preferred embodiment of the invention, the compound of formula (III) is in crystalline form, wherein x is 1-3; still more preferably 1.
In a further preferred embodiment of the present invention, the crystalline form of the compound of formula (III) is a hydrate or anhydrate.
In a further preferred embodiment of the present invention, the crystalline form of the compound of formula (III) contains from 0.5 to 8 water molecules; preferably, the hydrate contains 0.5-4 water molecules; further preferably 0.5 to 2.5 water molecules; still more preferably contains 1 water molecule. For example containing 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8 water.
In a preferred embodiment of the invention, M is selected from the group consisting of maleic acid, p-toluenesulfonic acid, hydrochloric acid, nitric acid, sulfuric acid, salicylic acid, methanesulfonic acid, benzenesulfonic acid or 1, 5-naphthalenedisulfonic acid; and x is selected from 0.5, 1, 1.5 or 2, preferably 1.
In a preferred embodiment of the present invention, the crystalline form of the compound of formula (I) has a structure as shown in formula (IV):
x and M are defined as general formula (I).
Another object of the present invention is to provide a compound represented by the general formula (Ia), which has a structure represented by the general formula (Ia):
wherein:
w is selected from N or CH;
R 1 selected from hydrogen atom, halogen, amino, nitro, hydroxy, cyano, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, C 1-6 Hydroxyalkyl, C 3-8 Cycloalkyl, - (CH) 2 ) n R 3 、-(CH 2 ) n OR 3 、-(CH 2 ) n C(O)R 3 、-(CH 2 ) n NR 3 R 4 Or- (CH) 2 ) n C(O)NR 3 R 4 ；
R 2 Selected from hydrogen atom, halogen, amino, nitro, hydroxy, cyano, C 1-6 Alkyl, C 1-6 Deuterated alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, C 3-8 Cycloalkyl or 3-8 membered heterocyclyl;
R 3 and R is 4 Identical or different and are each independently selected from the group consisting of hydrogen, deuterium, halogen, cyano, hydroxy, amino, nitro, C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-8 Cycloalkyl or 3-8 membered heterocyclyl, wherein said C 1-6 Alkyl, C 1-6 Haloalkyl, amino, C 3-8 Cycloalkyl and 3-8 membered heterocyclyl are optionally further selected from deuterium atoms, C 1-6 Alkyl, C 1-6 Haloalkyl, halogen, hydroxy, amino, nitro, cyano, C 1-6 Alkoxy, C 1-6 Hydroxyalkyl, C 3-8 Cycloalkyl and 3-6 membered heterocyclylOne or more substituents;
m is an inorganic acid or an organic acid, wherein the inorganic acid is selected from hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid or phosphoric acid; the organic acid is selected from 2, 5-dihydroxybenzoic acid, 1-hydroxy-2-naphthoic acid, acetic acid, dichloroacetic acid, trichloroacetic acid, acetohydroxamic acid, adipic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, 4-aminobenzoic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclohexane sulfamic acid, camphorsulfonic acid, aspartic acid, camphoric acid, gluconic acid, glucuronic acid, glutamic acid, isoascorbic acid, lactic acid, malic acid, mandelic acid, pyroglutamic acid, tartaric acid, dodecylsulfuric acid, dibenzoyltartaric acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactonic acid, gentisic acid, glutaric acid, 2-ketoglutaric acid, glycolic acid, hippuric acid, isethionic acid, lactonic acid, ascorbic acid, aspartic acid, lauric acid, camphoric acid, maleic acid, malonic acid, methanesulfonic acid, 1, 5-naphthalenedisulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, thiocyanic acid, undecylenic acid, trifluoroacetic acid, benzenesulfonic acid, p-toluenesulfonic acid or L-malic acid;
y is an integer of 1, 2 or 3;
n is an integer of 0, 1, 2 or 3; and is also provided with
t is an integer of 0, 1, 2, 3, 4, 5 or 6.
In a preferred embodiment of the present invention, the compound of formula (Ia) has a structure represented by formula (IIa):
R 1 、R 2 w, M, t, x are defined as general formula (I).
In a preferred embodiment of the present invention, the compound of formula (Ia) has the following structure:
in a preferred embodiment of the present invention, the compound of formula (Ia) has a structure as shown in formula (IIIa):
m, y is defined as formula (Ia).
In a further preferred embodiment of the present invention, the compound of formula (IIIa) is water anhydrate, further comprising water, which is pipeline water or crystallization water.
In a further preferred embodiment of the present invention, the compound of formula (IIIa) contains 0.5 to 8 water molecules; preferably 0.5 to 4 water molecules; further preferably 0.5 to 2.5 water molecules; even more preferably 1 water molecule; for example containing 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8 water.
In a further preferred embodiment of the present invention, the compound of formula (Ia) has a structure as shown in formula (IVa):
M, y is as defined for formula (Ia), preferably M is selected from maleic acid and y is 1.
The invention also aims to provide a preparation method of the compound crystal form shown in the general formula (I) or the compound shown in the general formula (Ia), which specifically comprises the following steps:
1) Weighing a proper amount of free alkali, and dissolving with benign solvent;
2) Optionally, weighing a proper amount of counter-ionic acid, and dissolving the counter-ionic acid with an organic solvent; preferably, the amount of the counter-ionic acid is 1.2 equivalents;
3) Optionally, mixing the two solutions, stirring, dripping poor solvent until turbidity appears if no precipitate is separated out, stirring, and crystallizing to obtain a target product;
wherein:
the benign solvent is selected from methanol, tetrahydrofuran, N-dimethylformamide, N-dimethylacetamide or N-methylpyrrolidone; preferably N, N-dimethylformamide and N-methylpyrrolidone;
the organic solvent is selected from methanol, ethanol, ethyl acetate, methylene dichloride, acetone, normal hexane, petroleum ether, benzene, toluene, chloroform, acetonitrile, carbon tetrachloride, dichloroethane, tetrahydrofuran, 2-butanone, 3-pentanone, heptane, methyl tertiary butyl ether, isopropyl ether, 1, 4-dioxane, tertiary butanol or N, N-dimethylformamide; methanol and ethanol are preferred; the benign solvent and the organic solution need to be mutually soluble when in use;
The poor solvent is selected from heptane, water, methyl tertiary butyl ether, toluene, isopropyl ether, ethyl acetate, acetone or acetonitrile; ethyl acetate, methyl tertiary butyl ether, isopropyl ether and acetonitrile are preferred;
the counter-ionic acid is selected from hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, phosphoric acid, 2, 5-dihydroxybenzoic acid, 1-hydroxy-2-naphthoic acid, acetic acid, dichloroacetic acid, trichloroacetic acid, acetohydroxamic acid, adipic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, 4-aminobenzoic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclohexane sulfamic acid, camphorsulfonic acid, aspartic acid, camphoric acid, gluconic acid, glucuronic acid, glutamic acid, isoascorbic acid, lactic acid, malic acid, mandelic acid, pyroglutamic acid, tartaric acid, dodecylsulfuric acid, sulfuric acid, dibenzoyltartaric acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactonic acid, gentisic acid, glutaric acid, 2-ketoglutaric acid, glycolic acid, hippuric acid, hydroxyethanesulfonic acid, lactobionic acid, ascorbic acid, aspartic acid, lauric acid, camphoric acid, maleic acid, malonic acid, methanesulfonic acid, 1, 5-naphthalenedisulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, thiocyanic acid, undecylenic acid, trifluoroacetic acid, benzenesulfonic acid, p-toluenesulfonic acid or L-malic acid; preferably methanesulfonic acid, sulfuric acid, hydrochloric acid, nitric acid, benzenesulfonic acid, maleic acid, adipic acid, p-toluenesulfonic acid, citric acid, malonic acid and L-malic acid; maleic acid is more preferred.
The invention also aims to provide a crystal form of the compound shown in the general formula (I) or a preparation method of the compound shown in the general formula (Ia), which specifically comprises the following steps:
1) Weighing a proper amount of free alkali, and suspending with an unfavorable solvent;
2) Optionally, weighing a proper amount of counter-ionic acid, and dissolving the counter-ionic acid with an organic solvent; preferably, the amount of the counter-ionic acid is 1.2 equivalents;
3) Optionally, adding the solution in the step 2) into the suspension in the step 1), stirring, and crystallizing to obtain a target product;
wherein:
the poor solvent is selected from acetone, ethyl acetate, acetonitrile, ethanol, 88% acetone, tetrahydrofuran, 1, 4-dioxane, benzene, toluene, isopropanol, N-butanol, isobutanol, N-dimethylformamide, N-dimethylacetamide, N-propanol, tertiary butanol or 2-butanone; preferably methanol, ethanol, tetrahydrofuran, ethyl acetate, acetonitrile or acetone; preferably methanol, ethanol, tetrahydrofuran, ethyl acetate, acetonitrile and acetone;
the organic solvent is selected from methanol, ethanol, ethyl acetate, methylene dichloride, acetone, normal hexane, petroleum ether, benzene, toluene, chloroform, acetonitrile, carbon tetrachloride, dichloroethane, tetrahydrofuran, 2-butanone, 3-pentanone, heptane, methyl tertiary butyl ether, isopropyl ether, 1, 4-dioxane, tertiary butanol or N, N-dimethylformamide; preferably methanol or ethanol; the benign solvent and the organic solution need to be mutually soluble when in use;
The counter-ionic acid is selected from hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, phosphoric acid, 2, 5-dihydroxybenzoic acid, 1-hydroxy-2-naphthoic acid, acetic acid, dichloroacetic acid, trichloroacetic acid, acetohydroxamic acid, adipic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, 4-aminobenzoic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclohexane sulfamic acid, camphorsulfonic acid, aspartic acid, camphoric acid, gluconic acid, glucuronic acid, glutamic acid, isoascorbic acid, lactic acid, malic acid, mandelic acid, pyroglutamic acid, tartaric acid, dodecylsulfuric acid, sulfuric acid, dibenzoyltartaric acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactonic acid, gentisic acid, glutaric acid, 2-ketoglutaric acid, glycolic acid, hippuric acid, hydroxyethanesulfonic acid, lactobionic acid, ascorbic acid, aspartic acid, lauric acid, camphoric acid, maleic acid, malonic acid, methanesulfonic acid, 1, 5-naphthalenedisulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, thiocyanic acid, undecylenic acid, trifluoroacetic acid, benzenesulfonic acid, p-toluenesulfonic acid or L-malic acid; maleic acid is preferred.
The invention also aims to provide a crystal form of the compound shown in the general formula (I) or a preparation method of the compound shown in the general formula (Ia), which specifically comprises the following steps:
1) Weighing a proper amount of free alkali, adding a benign solvent, and heating until the benign solvent is dissolved;
2) Optionally, weighing a proper amount of counter-ionic acid, and dissolving the counter-ionic acid with an organic solvent; preferably, the amount of the counter-ionic acid is 1.2 equivalents;
3) Stirring, cooling and crystallizing to obtain a target product;
wherein:
the benign solvent is selected from methanol, tetrahydrofuran, N-dimethylformamide, N-dimethylacetamide or N-methylpyrrolidone; preferably N, N-dimethylformamide and N-methylpyrrolidone;
the organic solvent is selected from methanol, ethanol, ethyl acetate, methylene dichloride, acetone, normal hexane, petroleum ether, benzene, toluene, chloroform, acetonitrile, carbon tetrachloride, dichloroethane, tetrahydrofuran, 2-butanone, 3-pentanone, heptane, methyl tertiary butyl ether, isopropyl ether, 1, 4-dioxane, tertiary butanol or N, N-dimethylformamide; methanol and ethanol are preferred; the benign solvent and the organic solution need to be mutually soluble when in use;
the counter-ionic acid is selected from hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, phosphoric acid, 2, 5-dihydroxybenzoic acid, 1-hydroxy-2-naphthoic acid, acetic acid, dichloroacetic acid, trichloroacetic acid, acetohydroxamic acid, adipic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, 4-aminobenzoic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclohexane sulfamic acid, camphorsulfonic acid, aspartic acid, camphoric acid, gluconic acid, glucuronic acid, glutamic acid, isoascorbic acid, lactic acid, malic acid, mandelic acid, pyroglutamic acid, tartaric acid, dodecylsulfuric acid, sulfuric acid, dibenzoyltartaric acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactonic acid, gentisic acid, glutaric acid, 2-ketoglutaric acid, glycolic acid, hippuric acid, hydroxyethanesulfonic acid, lactobionic acid, ascorbic acid, aspartic acid, lauric acid, camphoric acid, maleic acid, malonic acid, methanesulfonic acid, 1, 5-naphthalenedisulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, thiocyanic acid, undecylenic acid, trifluoroacetic acid, benzenesulfonic acid, p-toluenesulfonic acid or L-malic acid; preferably methanesulfonic acid, sulfuric acid, hydrochloric acid, nitric acid, benzenesulfonic acid, maleic acid, adipic acid, p-toluenesulfonic acid, citric acid, malonic acid or L-malic acid; maleic acid is more preferred.
In a preferred embodiment of the present invention, the compound of formula (IV) is in a crystalline form wherein M is selected from the group consisting of maleic acid, p-toluenesulfonic acid, hydrochloric acid, nitric acid, salicylic acid, methanesulfonic acid, benzenesulfonic acid or 1, 5-naphthalenedisulfonic acid; maleic acid is preferred, and x is more preferred to be 1.
In a preferred embodiment of the present invention, the crystalline form of the compound of formula (IV), its free base crystalline form I, X-ray powder diffraction pattern has characteristic peaks at 2θ of 6.4 and 26.7 (2θ±0.2°), preferably further comprises characteristic peaks at 2θ of 7.2, 13.1, 16.8, 17.7, 18.9, 20.2, 21.2 and 28.2 (2θ±0.2°).
The crystal form of the compound shown in the general formula (IV) is a free alkali compound crystal form, and X-ray characteristic diffraction peaks expressed in terms of 2 theta angles and d values of interplanar spacings are shown in table 1 by using Cu-K alpha radiation.
TABLE 1
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The crystal form of the compound shown in the general formula (IV) is a crystal form I of a free alkali compound, and the X-ray powder diffraction pattern of the crystal form I is basically shown in figure 1.
The crystal form of the compound shown in the general formula (IV) is a crystal form I of a free alkali compound, and the TGA diagram is basically shown in figure 2.
The crystal form of the compound shown in the general formula (IV) is a crystal form I of a free alkali compound, and the DSC chart of the crystal form I is basically shown in figure 3.
In a preferred embodiment of the present invention, the crystalline form of the compound of formula (IV), its free base crystalline form II, has an X-ray powder diffraction pattern having characteristic peaks at 2θ of 8.5, 13.4 and 17.0 (2θ±0.2°), and further comprises characteristic peaks at 2θ of 10.2, 10.9, 12.8, 13.1, 16.1, 17.8, 18.8, 19.5, 23.0, 23.9, 24.4, 25.5, 26.1 and 27.6 (2θ±0.2°).
The crystal form of the compound shown in the general formula (IV) is a free alkali compound crystal form, and X-ray characteristic diffraction peaks expressed in terms of 2 theta angles and d values of interplanar spacings are shown in table 2 by using Cu-K alpha radiation.
TABLE 2
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The crystal form of the compound shown in the general formula (IV) is a crystal form II of a free alkali compound, and the X-ray powder diffraction pattern of the crystal form II is basically shown in figure 4.
The crystal form of the compound shown in the general formula (IV) is a crystal form II of a free alkali compound, and the TGA diagram is basically shown in figure 5.
The crystal form of the compound shown in the general formula (IV) is a crystal form II of a free alkali compound, and the DSC chart of the crystal form II is basically shown in figure 6.
In a preferred embodiment of the present invention, the crystalline form of the compound of formula (IV) wherein M is maleic acid and X is 1, the maleate salt form I thereof, the X-ray powder diffraction pattern having characteristic peaks at 2θ of 5.5 and 16.3 (2θ±0.2°); further comprising characteristic peaks at 2θ of 10.8, 15.3, 17.7, 26.1, 26.4 and 27.0 (2θ±0.2°); further having characteristic peaks at 2θ of 13.0, 14.9, 20.0 and 20.8 (2θ±0.2°);
The crystal form of the compound shown in the general formula (IV) is characterized in that M is maleic acid, X is 1, the maleate crystal form I uses Cu-K alpha radiation, and X-ray characteristic diffraction peaks expressed in terms of 2 theta angles and d values of interplanar spacings are shown in table 3.
TABLE 3 Table 3
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In a preferred embodiment of the present invention, the crystalline form of the compound of formula (IV), wherein M is maleic acid and X is 1, its maleate salt form I, has an X-ray powder diffraction pattern substantially as shown in figure 7.
In a preferred embodiment of the present invention, the crystalline form of the compound of formula (IV), wherein M is maleic acid and x is 1, its maleate salt form I, has a TGA profile substantially as shown in figure 8.
In a preferred embodiment of the present invention, the compound of formula (IV) is in crystalline form, wherein M is maleic acid and x is 1, in maleate form I, and the DSC profile is substantially as shown in figure 9.
In a preferred embodiment of the present invention, the crystalline form of the compound of formula (IV), wherein M is maleic acid and X is 1, is maleate form II, having an X-ray powder diffraction pattern with characteristic peaks at 2θ of 5.5, 10.8, 16.3 and 17.6 (2θ±0.2°); further comprising characteristic peaks at 14.0, 26.4, 26.6 and 27.2 in 2θ; still further included are characteristic peaks at 2θ±0.2° of 8.8, 12.9, 18.4, 18.9, 20.6, 21.5, 22.4 and 22.8.
The crystal form of the compound shown in the general formula (IV) is characterized in that M is maleic acid, X is 1, and the maleate crystal form II uses Cu-K alpha radiation, and X-ray characteristic diffraction peaks expressed in terms of 2 theta angle and d value of interplanar spacing are shown in table 4.
TABLE 4 Table 4
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In a preferred embodiment of the present invention, the crystalline form of the compound of formula (IV) wherein M is maleic acid and X is 1, the maleate salt thereof, form II, and the X-ray powder diffraction pattern thereof is substantially as shown in figure 10.
In a preferred embodiment of the present invention, the compound of formula (IV) is in crystalline form, wherein M is maleic acid and x is 1, and the maleate salt is in crystalline form II, and the DSC profile is substantially as shown in figure 11.
In a preferred embodiment of the present invention, the crystalline form of the compound of formula (IV), wherein M is maleic acid and X is 1, is maleate form III, has an X-ray powder diffraction pattern with characteristic peaks at 2θ of 4.9, 15.1, 17.2, 17.5, 26.5 and 26.9 (2θ±0.2°); further comprising characteristic peaks at 2θ of 9.8, 14.7, 18.4, and 19.8 (2θ±0.2°); still further included are peaks characterized by 2θ of 12.3, 13.0, 14.0, and 27.5 (2θ±0.2°).
The compound shown in the general formula (IV) is characterized in that M is maleic acid, X is 1, and the maleate crystal form III uses Cu-K alpha radiation, and X-ray characteristic diffraction peaks expressed in terms of 2 theta angle and interplanar spacing d are shown in table 5.
TABLE 5
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In a preferred embodiment of the present invention, the crystalline form of the compound of formula (IV), wherein M is maleic acid and X is 1, the maleate salt thereof is form III, and the X-ray powder diffraction pattern thereof is substantially as shown in fig. 12.
In a preferred embodiment of the present invention, the compound of formula (IV) is in crystalline form, wherein M is maleic acid and x is 1, and in maleate form III, and the DSC profile is substantially as shown in figure 13.
In a preferred embodiment of the present invention, the compound of formula (IV) is crystalline form wherein M is maleic acid and X is 1, and the maleate salt form IV has an X-ray powder diffraction pattern having characteristic peaks at 2θ of 4.8, 16.7; further comprising characteristic peaks at 2θ of 9.6, 11.9, and 14.9 (2θ±0.2°); still further included are those having characteristic peaks at 2θ±0.2° of 7.2, 12.9, 14.3, 19.1, 19.3, 21.5 and 23.9.
The compound shown in the formula (IV) is characterized in that M is maleic acid, X is 1, and the maleate crystal form IV uses Cu-K alpha radiation, and X-ray characteristic diffraction peaks expressed in terms of 2 theta angle and interplanar spacing d are shown in table 6.
TABLE 6
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In a preferred embodiment of the present invention, the crystalline form of the compound of formula (IV), wherein M is maleic acid and X is 1, its maleate salt form IV, has an X-ray powder diffraction pattern substantially as shown in figure 14.
In a preferred embodiment of the present invention, the compound of formula (IV) is in crystalline form, wherein M is maleic acid and x is 1, in maleate form IV, and the DSC profile is substantially as shown in figure 15.
In a preferred embodiment of the present invention, the crystalline form of the compound of formula (IV), wherein M is hydrochloric acid, its hydrochloride salt form, has an X-ray powder diffraction pattern with characteristic peaks at 2θ of 5.8 and 17.2 (2θ±0.2°); further comprising characteristic peaks at 2θ of 12.0, 13.6, 16.4, 21.7, 23.0, 26.1, 26.3 and 27.1 (2θ±0.2°); still further included are characteristic peaks at 2θ of 8.9, 28.8, and 30.1 (2θ±0.2°).
The compound shown in the formula (IV) in the invention, wherein M is hydrochloric acid, the hydrochloride crystal form of the compound is Cu-K alpha radiation, and X-ray characteristic diffraction peaks expressed in terms of 2 theta angle and d value of interplanar spacing are shown in table 7.
TABLE 7
In a preferred embodiment of the present invention, the compound of formula (IV) is in crystalline form, wherein M is hydrochloric acid, in hydrochloride form, and in X-ray powder diffraction pattern substantially as shown in figure 16.
In a preferred embodiment of the present invention, the crystalline form of the compound of formula (IV) wherein M is p-toluenesulfonic acid, which has a characteristic peak at 2θ of 6.1, 11.3, 17.0, 17.5 and 18.3 (2θ±0.2°) for the p-toluenesulfonate crystalline form I; further included are characteristic peaks at 2θ of 12.7, 19.1, 19.9, 20.6, and 22.2 (2θ±0.2°).
The compound shown in the formula (IV) is p-toluenesulfonic acid, wherein p-toluenesulfonate crystal form I is p-toluenesulfonate, cu-K alpha radiation is used, and X-ray characteristic diffraction peaks expressed in terms of 2 theta angle and interplanar spacing d value are shown in table 8.
TABLE 8
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In a preferred embodiment of the present invention, the crystalline form of the compound of formula (IV), wherein M is p-toluenesulfonic acid, which is p-toluenesulfonate salt form I, has an X-ray powder diffraction pattern substantially as shown in figure 17.
In a preferred embodiment of the invention, the crystalline form of the compound of formula (IV) wherein M is p-toluenesulfonic acid, which has a characteristic peak at 2θ of 5.4, 8.6, 11.4, 16.8, 18.2, 19.7, 20.4 and 21.9 (2θ±0.2°) for the p-toluenesulfonate form II; further comprising characteristic peaks at 2θ of 13.5, 13.7, 15.7, 17.2, 23.7, 25.5 and 27.6 (2θ±0.2°); still further included are peaks characterized by 2 theta of 16.0, 21.4, 25.9 and 28.7 (2 theta + 0.2 deg.).
The compound shown in the formula (IV) is p-toluenesulfonic acid, p-toluenesulfonate crystal form II is p-toluenesulfonate, cu-K alpha radiation is used, and X-ray characteristic diffraction peaks expressed in terms of 2 theta angle and interplanar spacing d value are shown in table 9.
TABLE 9
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In a preferred embodiment of the present invention, the crystalline form of the compound of formula (IV), wherein M is p-toluenesulfonic acid, which is p-toluenesulfonate salt form II, has an X-ray powder diffraction pattern substantially as shown in figure 18.
In a preferred embodiment of the present invention, the crystalline form of the compound of formula (IV) wherein M is p-toluenesulfonic acid, which has a characteristic peak at 2θ of 4.9, 8.6, 13.2, 18.9, 20.6 and 25.2 (2θ±0.2°) for p-toluenesulfonate form III; further comprising characteristic peaks at 2θ of 9.6, 10.9, 12.6, 15.0, 15.6, 17.0, 22.6, 25.8 and 27.5 (2θ±0.2°); still further included are those having characteristic peaks at 2θ of 10.5, 13.9, 16.5, 17.7, 21.7, 26.1, 26.6, 26.9, 27.8, 29.8, and 32.2 (2θ±0.2°).
The compound shown in the formula (IV) is p-toluenesulfonic acid, p-toluenesulfonate crystal form III is p-toluenesulfonate, cu-K alpha radiation is used, and X-ray characteristic diffraction peaks expressed in terms of 2 theta angle and interplanar spacing d value are shown in table 10.
Table 10
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In a preferred embodiment of the present invention, the crystalline form of the compound of formula (IV) wherein M is p-toluenesulfonic acid, which is p-toluenesulfonate salt form III, has an X-ray powder diffraction pattern substantially as shown in figure 19.
In a preferred embodiment of the present invention, the crystalline form of the compound of formula (IV) wherein M is p-toluenesulfonic acid, has a characteristic peak at 2θ of 5.5, 11.9, 16.3, 19.4 and 25.5 (2θ±0.2°) for the crystal form IV of the p-toluenesulfonate salt; further comprising characteristic peaks at 2θ of 13.8, 18.1, 18.6, 20.1, 21.3, 22.9 and 26.5 (2θ±0.2°); still further comprising characteristic peaks at 8.7, 9.1, 10.2, 10.8, 12.9 and 20.4 (2θ±0.2°).
The compound shown in the formula (IV) is p-toluenesulfonic acid, the p-toluenesulfonate crystal form IV is p-toluenesulfonic acid, cu-K alpha radiation is used, and X-ray characteristic diffraction peaks expressed in terms of 2 theta angle and interplanar spacing d values are shown in table 11.
TABLE 11
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In a preferred embodiment of the present invention, the crystalline form of the compound of formula (IV) wherein M is p-toluenesulfonic acid, which is p-toluenesulfonate salt form IV, has an X-ray powder diffraction pattern substantially as shown in figure 20.
In a preferred embodiment of the present invention, the crystalline form of the compound of formula (IV) wherein M is nitric acid, the nitrate crystalline form thereof, has an X-ray powder diffraction pattern with characteristic peaks at 2θ of 5.0, 16.3, 16.7 and 28.0 (2θ±0.2°); further comprising characteristic peaks at 2θ of 8.2, 8.5, 11.8, 13.2, and 29.1 (2θ±0.2°); still further included are peaks characterized by 2 theta of 19.7, 20.4, 21.0 and 24.5 (2 theta + 0.2 deg.).
The X-ray characteristic diffraction peaks expressed in terms of 2 theta angles and d values of interplanar spacings using Cu-K alpha radiation are shown in Table 12.
Table 12
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In a preferred embodiment of the present invention, the compound of formula (IV) is in the form of a crystal, wherein M is nitric acid, in the form of a nitrate salt, and in the form of an X-ray powder diffraction pattern substantially as shown in figure 21.
The invention also aims at providing a pharmaceutical composition which contains a therapeutically effective amount of the crystal form of the compound shown in the general formula (I) or the compound shown in the general formula (Ia) and one or more pharmaceutically acceptable carriers.
The invention also aims to provide the crystal form of the compound shown in the formula (I) or the application of the compound shown in the formula (Ia) and the pharmaceutical composition in the preparation of medicines for treating and/or preventing ERK-mediated cancers or tumor-related diseases.
The invention also aims to provide the application of the crystal form of the compound shown in the formula (I) or the compound shown in the formula (Ia) and the pharmaceutical composition thereof in preparing medicines for treating cancers, inflammations, chronic liver diseases, diabetes, cardiovascular diseases or AIDS.
The present invention also aims to provide a method for the treatment and/or prophylaxis of diseases which are characterized by ERK mediated pathologies, which comprises administering to the patient a therapeutically effective dose of crystals of the compound of formula (I) or of the compound of formula (Ia) and pharmaceutical compositions thereof. Diseases in which ERK mediated pathology is characterized include cancer, inflammation, chronic liver disease, diabetes, cardiovascular disease or AIDS.
The use as described above, wherein the disease is selected from the group consisting of cancer, bone disease, inflammatory disease, immune disease, neurological disease, metabolic disease, respiratory disease or cardiac disease; wherein the cancer is selected from breast cancer, pancreatic cancer, non-small cell lung cancer, thyroid cancer, seminoma, melanoma, bladder cancer, liver cancer, kidney cancer, myelodysplastic syndrome, acute myelogenous leukemia, or colorectal cancer.
Detailed description of the invention
Unless stated to the contrary, the terms used in the specification and claims have the following meanings.
The term "alkyl" refers to a saturated aliphatic hydrocarbon group which is a straight or branched chain group containing from 1 to 20 carbon atoms, preferably an alkyl group containing from 1 to 8 carbon atoms, more preferably an alkyl group containing from 1 to 6 carbon atoms, and most preferably an alkyl group containing from 1 to 3 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl, n-heptyl, 2-methylhexyl 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-dimethylpentyl, 2, 4-dimethylpentyl, 2-dimethylpentyl, 3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2, 3-dimethylhexyl, 2, 4-dimethylhexyl, 2, 5-dimethylhexyl, 2-dimethylhexyl, 3-dimethylhexyl, 4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2, 2-diethylpentyl group, n-decyl group, 3-diethylhexyl group, 2-diethylhexyl group, various branched isomers thereof, and the like. The alkyl group may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available point of attachment, preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxy or carboxylate, with methyl, ethyl, isopropyl, t-butyl, haloalkyl, deuteroalkyl, alkoxy-substituted alkyl and hydroxy-substituted alkyl being preferred.
The term "alkylene" means that one hydrogen atom of the alkyl group is further substituted, for example: "methylene" means-CH 2 - "ethylene" means- (CH) 2 ) 2 - "propylene" means- (CH) 2 ) 3 "butylene" means- (CH) 2 ) 4 -and the like, the above substituents may be linked to different carbon atoms to form a carbon chain, or may be linked to one carbon atom to form a cycloalkyl group. The term "alkenyl" refers to an alkyl group as defined above consisting of at least two carbon atoms and at least one carbon-carbon double bond, such as vinyl, 1-propenyl, 2-propenyl, 1-, 2-, or 3-butenyl, and the like. Alkenyl groups may be substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxyHeterocyclic alkoxy, cycloalkylthio, heterocyclic alkylthio.
The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring containing from 3 to 20 carbon atoms, preferably from 3 to 8 carbon atoms, more preferably from 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like; polycyclic cycloalkyl groups include spiro, fused and bridged cycloalkyl groups, preferably cyclopropyl, cyclobutyl, cyclohexyl, cyclopentyl and cycloheptyl.
The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing from 3 to 20 ring atoms in which one or more ring atoms are selected from nitrogen, oxygen or S (O) m (wherein m is an integer from 0 to 2), but does not include a ring moiety of-O-O-, -O-S-, or-S-S-, and the remaining ring atoms are carbon. Preferably containing 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably 3 to 8 ring atoms; most preferably containing 3 to 8 ring atoms. Non-limiting examples of monocyclic heterocyclyl groups include pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothienyl, dihydroimidazolyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, pyranyl, and the like, with tetrahydrofuranyl, pyrazolidinyl, morpholinyl, piperazinyl, and pyranyl being preferred. Polycyclic heterocyclyl groups include spiro, fused and bridged heterocyclic groups; wherein the heterocyclic groups of the spiro ring, the condensed ring and the bridged ring are optionally connected with other groups through single bonds, or are further connected with other cycloalkyl groups, heterocyclic groups, aryl groups and heteroaryl groups through any two or more atoms on the ring in a parallel ring manner; the heterocyclic group may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo Carboxyl or carboxylate groups.
The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing from 3 to 20 ring atoms in which one or more ring atoms are selected from nitrogen, oxygen or S (O) m (wherein m is an integer from 0 to 2), but does not include a ring moiety of-O-O-, -O-S-, or-S-S-, and the remaining ring atoms are carbon. Preferably containing 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably 3 to 8 ring atoms; most preferably containing 3 to 8 ring atoms. Non-limiting examples of monocyclic heterocyclyl groups include oxetanyl, pyrrolidinyl, pyrrolidinonyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothienyl, dihydroimidazolyl, dihydrofuranyl, dihydropyrazolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, pyranyl, and the like, with oxetanyl, pyrrolidinonyl, tetrahydrofuranyl, pyrazolidinyl, morpholinyl, piperazinyl, and pyranyl being preferred. Polycyclic heterocyclyl groups include spiro, fused and bridged heterocyclic groups; the heterocyclic groups of the spiro ring, the condensed ring and the bridged ring are optionally connected with other groups through single bonds, or are further connected with other cycloalkyl groups, heterocyclic groups, aryl groups and heteroaryl groups through any two or more atoms on the ring in a parallel ring mode.
The term "alkoxy" refers to-O- (alkyl) and-O- (unsubstituted cycloalkyl), wherein alkyl is as defined above. Alkoxy groups having 1 to 8 carbon atoms are preferred, alkoxy groups having 1 to 6 carbon atoms are more preferred, and alkoxy groups having 1 to 3 carbon atoms are most preferred. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, cyclohexyloxy. The alkoxy groups may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl, or carboxylate groups.
"haloalkyl" refers to an alkyl group substituted with one or more halogens, where alkyl is as defined above.
"haloalkoxy" refers to an alkoxy group substituted with one or more halogens, wherein the alkoxy group is as defined above.
"hydroxyalkyl" refers to an alkyl group substituted with a hydroxy group, wherein alkyl is as defined above.
"alkenyl" refers to alkenyl, also known as alkenyl, preferably alkenyl containing 2 to 8 carbon atoms, more preferably alkenyl containing 2 to 6 carbon atoms, and most preferably alkenyl containing 2 to 3 carbon atoms; wherein said alkenyl group may be further substituted with other related groups, such as: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate groups.
"alkynyl" refers to (CH≡C-), preferably alkynyl containing 2 to 8 carbon atoms, more preferably alkynyl of 2 to 6 carbon atoms, most preferably alkynyl of 2 to 3 carbon atoms. Wherein said alkynyl group may be further substituted with other related groups such as: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate groups.
"hydroxy" refers to an-OH group.
"halogen" means fluorine, chlorine, bromine or iodine.
"amino" means-NH 2 。
"cyano" refers to-CN.
"nitro" means-NO 2 。
"carboxy" means-C (O) OH.
The terms "X is selected from A, B or C", "X is selected from A, B and C", "X is A, B or C", "X is A, B and C", etc. all express the same meaning, that is, X may be any one or several of A, B, C.
"optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not.
"substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms in the group are independently substituted with a corresponding number of substituents. It goes without saying that substituents are only in their possible chemical positions, and that the person skilled in the art is able to determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, amino or hydroxyl groups having free hydrogen may be unstable when bound to carbon atoms having unsaturated (e.g., olefinic) bonds.
"stereoisomers" include three classes of geometric (cis-trans) isomerism, optical isomerism, conformational isomerism.
The hydrogen atoms of the invention can be replaced by the isotope deuterium thereof, and any hydrogen atom in the compound of the embodiment of the invention can be replaced by deuterium atoms.
"pharmaceutical composition" means a mixture comprising one or more of the compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients and thus exert biological activity.
By "pharmaceutically acceptable salts" is meant salts of the compounds of the present invention which are safe and effective when used in a mammal, and which possess the desired biological activity.
"TGA" refers to thermogravimetric analysis (TGA) experiments.
"DSC" refers to Differential Scanning Calorimetry (DSC) experiments.
"DVS" refers to a dynamic moisture sorption (DVS) experiment.
"XRPD" refers to X-ray powder diffraction (XRPD) experiments.
"HPLC" refers to High Performance Liquid Chromatography (HPLC) experiments.
"PK" refers to Pharmacokinetic (PK) experiments.
The invention is further described below in connection with examples, which are not intended to limit the scope of the invention.
Drawings
Fig. 1 is an XRPD pattern of free base form I.
Figure 2 is a TGA profile of the free base form I.
Fig. 3 is a DSC profile of the free base form I.
Fig. 4 is an XRPD pattern of free base form II.
Figure 5 is a TGA profile of form II of the free base.
Fig. 6 is a DSC profile of form II of the free base.
Fig. 7 is an XRPD pattern of maleate salt form I.
Fig. 8 is a TGA profile of maleate salt form I.
Fig. 9 is a DSC profile of maleate form I.
Fig. 10 is an XRPD pattern of maleate salt form II.
Fig. 11 is a DSC profile of maleate form II.
Fig. 12 is an XRPD pattern of maleate salt form III.
Fig. 13 is a DSC profile of maleate form III.
Fig. 14 is an XRPD pattern of maleate form IV.
Fig. 15 is a DSC profile of maleate form IV.
Fig. 16 is an XRPD illustration of the hydrochloride salt.
Fig. 17 is an XRPD pattern of p-toluenesulfonate form I.
Fig. 18 is an XRPD pattern of p-toluenesulfonate form II.
Fig. 19 is an XRPD pattern of p-toluenesulfonate form III.
Fig. 20 is an XRPD pattern of p-toluenesulfonate form IV.
Fig. 21 is an XRPD illustration of the nitrate salt.
FIG. 22 is a graphical representation of rat PK experiments.
Detailed Description
Experimental instrument
Some parameters of the physicochemical detecting instrument
Conditions for liquid phase analysis
Apparatus and device
| Instrument name | Model number |
| Analytical balance | Sartorius BSA224S-CW |
| Water purifier | Milli-Q Plus，Millipore |
| High performance liquid chromatograph | Agilent1260 |
| Pump with a pump body | Agilent G1311B |
| Sample injector | G1329B |
| Column temperature box | G1316A |
| Detector for detecting a target object | G1315D |
Chromatographic conditions
Chromatographic column: XBIdge TM (C18，3.5μm，4.6*150mm)
Flow rate: 1mL/min
Column temperature: 50 DEG C
Detection wavelength: 255nm
Sample injection volume: 5.0 mu L
Run time: 35min
A diluent: NMP-water (v/v, 1:4)
Mobile phase: a: water (0.05% trifluoroacetic acid); b: acetonitrile (0.05% trifluoroacetic acid)
Preparation of free base
Example 1
Preparation of (R) -N- (1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydropyrrolo [3,2-f ] indazole-6-carboxamide
The first step: preparation of methyl 3- (6-nitro-3- (pyridin-4-yl) -1-trityl-1H-indazol-5-yl) acrylate
6-nitro-3- (pyridin-4-yl) -1-trityl-1H-indazole-5-carbaldehyde (350 mg,0.68 mmol) and methoxyformyl methylene triphenylphosphine (344 mg,1.03 mmol) were dissolved in toluene (20 mL), heated to 105℃and stirred for 2 hours. After the reaction, the reaction solution is concentrated and subjected to column chromatography [ eluent: petroleum ether-Petroleum ether/ethyl acetate (60:40) ] to afford the pale cyan solid product, methyl 3- (6-nitro-3- (pyridin-4-yl) -1-trityl-1H-indazol-5-yl) acrylate (200 mg, 51% yield).
MS m/z(ESI)：567.1[M+H] + .
And a second step of: preparation of 3- (6-nitro-3- (pyridin-4-yl) -1-trityl-1H-indazol-5-yl) acrylic acid
3- (6-Nitro-3- (pyridin-4-yl) -1-trityl-1H-indazol-5-yl) acrylic acid methyl ester (150 mg,0.26 mmol) and lithium hydroxide monohydrate (22 mg,0.53 mmol) were dissolved in tetrahydrofuran (8 mL) and water (2 mL) and stirred at room temperature for 4 hours. After completion of the reaction, 0.5N aqueous acetic acid (30 mL) was quenched, extracted with ethyl acetate (30 mL x 2), the organic phase was washed with aqueous sodium chloride (20 mL), dried over sodium sulfate and concentrated to give 3- (6-nitro-3- (pyridin-4-yl) -1-trityl-1H-indazol-5-yl) acrylic acid (150 mg, 100% yield) as an orange-yellow solid.
MS m/z(ESI)：553.1[M+H] + 。
And a third step of: preparation of (R) -3- (6-nitro-3- (pyridin-4-yl) -1-trityl-1H-indazol-5-yl) -N- (1-phenylethyl) acryloylamide
To a solution of 3- (6-nitro-3- (pyridin-4-yl) -1-trityl-1H-indazol-5-yl) acrylic acid (150 mg,0.27 mmol), (R) -1-phenylethan-1-amine (49 mg,0.40 mmol) and O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate (207 mg,0.54 mmol) in tetrahydrofuran (20 mL) was added triethylamine (816 mg,0.81 mmol) and the mixture was stirred at room temperature for 16 hours. Ethyl acetate (60 mL) was added to the reaction mixture, followed by washing with 0.5N acetic acid (30 mL. Times.2), aqueous sodium bicarbonate (20 mL), aqueous sodium chloride (20 mL), drying over sodium sulfate, and concentration gave (R) -3- (6-nitro-3- (pyridin-4-yl) -1-trityl-1H-indazol-5-yl) -N- (1-phenylethyl) acryloylamide (150 mg, 84% yield) as a yellow solid.
MS m/z(ESI)：656.2[M+H] + .
Fourth step: preparation of (R) -N- (1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydropyrrolo [3,2-f ] indazole-6-carboxamide
(R) -3- (6-nitro-3- (pyridin-4-yl) -1-trityl-1H-indazol-5-yl) -N- (1-phenylethyl) acryloylamide (130 mg,0.19 mmol) was microwaved in triethyl phosphite (3 mL) at 140℃for 45 minutes. Concentrating the reaction solution, and purifying by column chromatography [ eluent: petroleum Ether-Petroleum ether/ethyl acetate (40:60) ] gives the product (R) -N- (1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydropyrrolo [3,2-f ] indazole-6-carboxamide (50 mg, 40% yield) as a yellow oil.
MS m/z(ESI)：624.2[M+H] + .
Fifth step: preparation of (R) -N- (1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydropyrrolo [3,2-f ] indazole-6-carboxamide
To (R) -N- (1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydropyrrolo [3,2-f]To a solution of indazole-6-carboxamide (50 mg,0.08 mmol) and triethylsilane (19 mg,0.16 mmol) in dichloromethane (2 mL) was added trifluoroacetic acid (4 mL) and the mixture was stirred at room temperature for 2 hours. Spin-drying the reaction solution, dissolving in methanol, adjusting to alkaline with ammonia water, and performing thin layer chromatography (developing agent: CH) 2 Cl 2 Meoh=10/1) to give the product (R) -N- (1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydropyrrolo [3, 2-f) as a yellow solid]Indazole-6-carboxamide (6 mg, 20% yield).
1 H NMR(400MHz，MeOD)δ8.66(d，J＝4Hz，2H)，8.41(s，1H)，8.12(d，J＝4Hz，2H)，7.53(s，1H)，7.44(d，J＝8Hz，2H)，7.39-7.30(m，3H)，7.26-7.22(m，1H)，5.29(q，J＝8Hz，1H)，1.61(d，J＝8Hz，3H).
MS m/z(ESI)：382.1[M+H] + .
Example 2
Preparation of (R) -N- (1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
The first step: preparation of 3-bromo-5, 6-dinitro-1-trityl-1H-indazole
3-bromo-5, 6-dinitro-1H-indazole (5.0 g,17.42 mmol), cesium carbonate (8.51 g,26.12 mmol) and DMF (50 mL) were stirred, triphenylchloromethane (5.83 g,20.91 mmol) was added in portions and stirred overnight at room temperature. 250mL of water was added, stirred for 1H, filtered and washed to give the solid product 3-bromo-5, 6-dinitro-1-trityl-1H-indazole (11.47 g, wet weight). Is directly used for the next reaction without drying.
And a second step of: preparation of 5, 6-dinitro-3- (pyridin-4-yl) -1-trityl-1H-indazole
3-bromo-5, 6-dinitro-1-trityl-1H-indazole (11.47 g,21.66 mmol), 4-pyridineboronic acid (5.33 g,43.36 mmol), pd (dppf) Cl 2 (1.59 g,4.33 mmol), potassium carbonate (6.0 g,14 mmol) and dioxane/water (125 mL/25 mL) are stirred for 5 hours at 80 ℃ under the protection of nitrogen, cooled to room temperature, added with water, extracted with ethyl acetate, and purified by column chromatography to obtain a solid product of 5, 6-dinitro-3- (pyridine)-4-yl) -1-trityl-1H-indazole (7.50 g, 81% yield in two steps).
MS m/z(ESI)：528.1[M+H] + .
And a third step of: preparation of 3- (pyridin-4-yl) -1-trityl-1H-indazole-5, 6-diamine
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5, 6-dinitro-3- (pyridin-4-yl) -1-trityl-1H-indazole (2.5 g,4.74 mmol), 10% Pd/C (200 mg), THF (150 mL) were hydrogenated overnight at room temperature, filtered and concentrated to dryness to give the product 3- (pyridin-4-yl) -1-trityl-1H-indazole-5, 6-diamine as a brown solid which was used directly in the next reaction.
MS m/z(ESI)：468.2[M+H] + .
Fourth step: preparation of 3- (pyridin-4-yl) -6- (trichloromethyl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole
3- (pyridin-4-yl) -1-trityl-1H-indazole-5, 6-diamine (obtained in the previous step), methyl 2, 2-trichloroacetimidate (920 mg,5.22 mmol), acetic acid (25 mL) were stirred overnight at room temperature, 100mL of water was added, stirring was performed for 1 hour, filtration was performed, and washing was performed to obtain a brown solid product 3- (pyridin-4-yl) -6- (trichloromethyl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole (4.32 g, wet weight). Directly used in the next reaction.
MS m/z(ESI)：594.1，596.1[M+H] + .
Fifth step: preparation of methyl 3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxylate
3- (pyridin-4-yl) -6- (trichloromethyl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole (4.32 g), methanol (50 mL) were refluxed overnight and concentrated to dryness. Isopropyl ether was added and stirred for 30min, filtered, and the isopropyl ether was washed to give crude 3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxylic acid methyl ester (2.44 g) as a brown solid.
MS m/z(ESI)：536.2[M+H] + .
Sixth step: (R) -N- (1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
Methyl 3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxylate (100 mg,0.19 mmol) is placed in a microwave reaction tube and (R) -1-phenylethane-1-amine (1.0 mL) is added and heated to 150℃for 60min. After cooling to room temperature, 20mL of ethyl acetate was added to the reaction solution, and the ethyl acetate layer was washed with a saturated ammonium chloride solution and a saturated sodium chloride solution, followed by drying over anhydrous sodium sulfate and column chromatography to give a crude product (80 mg) which was directly used for the next reaction.
MS m/z(ESI)：625.1[M+H] + .
Seventh step: preparation of (R) -N- (1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
The crude (R) -N- (1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (80 mg) obtained in the above step was dissolved in 4mL of methylene chloride, 4mL of trifluoroacetic acid was added thereto, and after reacting at room temperature for 3 hours, the reaction solution was dried by spinning. The obtained crude product was dissolved in a mixed solution of ethyl acetate and tetrahydrofuran, washed with a saturated sodium hydrogencarbonate solution and a saturated sodium chloride solution, and then dried over anhydrous sodium sulfate. The resulting organic solvent was dried by spin-drying and purified by preparative chromatography to give the product (R) -N- (1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (15 mg, 21% in two steps) as a yellow solid.
1 H NMR(400MHz，DMSO-d 6 )δ：13.45-13.39(m，1H)，13.21-13.08(m，1H)，9.43-9.33(m，1H)，8.72(s，2H)，8.44-8.04(m，3H)，7.99-7.87(m，1H)，7.58(m，2H)，7.36-7.32(m，2H)，7.26-7.22(m，1H)，5.25-5.21(m，1H)，1.57(d，J＝7.0Hz，3H).
MS m/z(ESI)：383.1[M+H] + .
Example 3
Preparation of N- (3, 4-dimethoxybenzyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
N- (3, 4-dimethoxybenzyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide is obtained from 3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxylic acid methyl ester and (3, 4-dimethoxyphenyl) methylamine as starting materials in reference example 2, step six and step seven.
1 H NMR(400MHz，DMSO-d 6 )δ13.48(s，1H)，9.37(d，J＝8.6Hz，1H)，8.71-8.64(m，2H)，8.13(s，1H)，7.99(s，1H)，7.96-7.89(m，2H)，7.72(t，J＝10.2Hz，1H)，6.93-6.84(m，2H)，6.89-6.78(m，1H)，4.72(dt，J＝10.0，1.0Hz，2H)，3.82(d，J＝8.5Hz，6H).
MS m/z(ESI)：429.1[M+H] + .
Example 4
Preparation of N- (3-chloro-4-fluorobenzyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
N- (3-chloro-4-fluorobenzyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide is obtained from 3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxylic acid methyl ester and (3-chloro-4-fluorophenyl) methylamine as starting materials in step six and step seven of reference example 2.
1 H NMR(400MHz，DMSO-d 6 )δ13.47(s，1H)，9.38(d，J＝8.6Hz，1H)，8.13(s，1H)，7.99(s，1H)，7.96-7.89(m，2H)，7.72(t，J＝10.2Hz，1H)，7.38(dd，J＝7.4，4.9Hz，1H)，7.14(ddq，J＝9.1，7.9，1.2Hz，2H)，4.73(dt，J＝10.1，1.0Hz，2H).
MS m/z(ESI)：421.2[M+H] + .
Example 5
Preparation of (R) -3- (pyridin-4-yl) -N- (1- (p-benzyl) ethyl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
The first step: preparation of (R) -3- (pyridin-4-yl) -N- (1- (p-benzyl) ethyl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
Methyl 3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxylate (100 mg,0.19 mmol) is placed in a microwave reaction tube and (R) -1- (p-benzyl) ethane-1-amine (1.0 mL) is added and heated to 150℃for 60min. After cooling to room temperature, 20mL of ethyl acetate was added to the reaction solution, and the ethyl acetate layer was washed with a saturated ammonium chloride solution and a saturated sodium chloride solution, followed by drying over anhydrous sodium sulfate and column chromatography to give a crude product (80 mg, yield 67%) which was directly used for the next reaction.
MS m/z(ESI)：639.3[M+H] + .
And a second step of: preparation of (R) -3- (pyridin-4-yl) -N- (1- (p-benzyl) ethyl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
Crude (R) -3- (pyridin-4-yl) -N- (1- (p-benzyl) ethyl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (80 mg) was dissolved in 4mL of methylene chloride, 4mL of trifluoroacetic acid was added thereto, and the reaction mixture was allowed to react at room temperature for 3 hours, and then dried by spin-drying. The obtained crude product was dissolved in a mixed solution of ethyl acetate and tetrahydrofuran, washed with a saturated sodium hydrogencarbonate solution and a saturated sodium chloride solution, and then dried over anhydrous sodium sulfate. The resulting organic solvent was dried by spin-drying and purified by preparative chromatography to give the product (R) -3- (pyridin-4-yl) -N- (1- (p-benzyl) ethyl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (10 mg, 20% yield) as a yellow solid.
1 H NMR(400MHz，MeOD)δ8.57(d，J＝4Hz，2H)，8.37(s，0.6H)，8.15(s，0.4H)，8.02(d，J＝4Hz，2H)，7.79(s，0.4H)，7.56(s，0.6H)，7.25(d，J＝8Hz，2H)，7.08(d，J＝8Hz，2H)，5.12-5.16(m，1H)，2.22(s，3H)，1.52(d，J＝8Hz，3H).
MS m/z(ESI)：397.1[M+H] + .
Example 6
Preparation of (R) -N- (1- (4-fluorophenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
The first step: preparation of (R) -N- (1- (4-fluorophenyl) ethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
Methyl 3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxylate (1.5 g,2.80 mmol), (R) -1- (4-fluorophenyl) ethane-1-amine (2.5 mL) was stirred at 150℃for 1h with microwaves, cooled, concentrated to dryness, and purified by column chromatography to give crude viscous material, which was used directly in the next reaction.
MS m/z(ESI)：643.2[M+H] + .
And a second step of: preparation of (R) -N- (1- (4-fluorophenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
The product of the previous step is dissolved in dichloromethane (5 mL), trifluoroacetic acid (5 mL) is added, stirred for 2h at room temperature, concentrated to dryness, and purified by column chromatography to obtain yellow solid (R) -N- (1- (4-fluorophenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4, 5-f)]Indazole-6-carboxamide (603 mg, 54% two-step yield). 1 H NMR(400MHz，MeOD)δ8.89(s，1H)，8.55(s，1H)，8.18(s，1H)，7.85(s，1H)，7.28(dd，J＝15.0，8.3Hz，1H)，6.80(ddd，J＝20.8，17.5，10.7Hz，3H)，6.28(dd，J＝16.7，1.8Hz，1H)，5.80(dd，J＝10.6，1.7Hz，1H)，4.56(m，1H)，3.98-3.81(m，3H).
MS m/z(ESI)：401.1[M+H] + .
Example 7
Preparation of N- (1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
The first step: preparation of N- (1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
Methyl 3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxylate (100 mg,0.18 mmol) and 1-phenylethane-1-amine (1.5 mL) are stirred for 1h at 150℃with microwaves, cooled, concentrated to dryness and purified by column chromatography to give crude N- (1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (27 mg).
MS m/z(ESI)：625.2[M+H] + .
And a second step of: preparation of N- (1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
Crude N- (1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (27 mg) was dissolved in dichloromethane (2 mL), trifluoroacetic acid (2 mL) was added, stirred at room temperature for 2h, concentrated to dryness, and purified by column chromatography to give N- (1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (5 mg, two-step yield 7%) as a yellow solid.
1 H NMR(400MHz，DMSO-d 6 )δ13.48(s，1H)，9.37(d，J＝8.6Hz，1H)，8.71-8.64(m，2H)，8.13(s，1H)，7.99(s，1H)，7.96-7.89(m，2H)，7.36-7.27(m，2H)，7.31-7.20(m，3H)，6.75(d，J＝9.3Hz，1H)，5.20(dqt，J＝8.7，6.8，0.9Hz，1H)，1.57(d，J＝6.7Hz，3H).
MS m/z(ESI)：383.1[M+H] + .
Example 8
Preparation of (S) -N- (2-methoxy-1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
(S) -N- (2-methoxy-1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide was obtained from 3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxylic acid methyl ester and (S) -2-methoxy-1-phenylethane-1-amine as starting materials in step six and step seven of reference example 2.
1 H NMR(400MHz，DMSO-d 6 )δ13.48(s，1H)，9.37(d，J＝8.6Hz，1H)，8.72(d，J＝5.2Hz，2H)，8.34(s，2H)，8.04(d，J＝5.1Hz，2H)，7.82-7.60(m，1H)，7.50(d，J＝7.4Hz，2H)，7.36(t，J＝7.5Hz，2H)，7.28(t，J＝7.3Hz，1H)，5.31(td，J＝8.4，4.8Hz，1H)，3.87(t，J＝9.2Hz，2H)，3.64(d，J＝5.0Hz，3H).
MS m/z(ESI)：413.1[M+H] + .
Example 9
Preparation of (R) -N- (1- (4-nitrophenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
(R) -N- (1- (4-nitrophenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide was obtained from 3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxylic acid methyl ester and (R) -1- (4-nitrophenyl) ethyl-1-amine as starting materials in step six and step seven of reference example 2.
1 H NMR(400MHz，DMSO-d 6 )δ13.44-13.39(m，1H)，13.20-13.08(m，1H)，9.43-9.33(m，1H)，8.68-8.62(m，2H)，8.22-8.14(m，2H)，8.00-7.94(m，2H)，7.70(s，2H)，7.62-7.54(m，2H)，5.27-5.22(m，1H)，1.56(d，J＝6.4Hz，3H).
MS m/z(ESI)：428.1[M+H] + .
Example 10
Preparation of (R) -N- (1- (4-bromophenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
(R) -N- (1- (4-bromophenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide was obtained from 3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxylic acid methyl ester and (R) -1- (4-bromophenyl) ethyl-1-amine as starting materials in step six and step seven of reference example 2.
1 H NMR(400MHz，DMSO-d 6 )δ13.47(s，1H)，9.48(d，J＝7.9Hz，1H)，8.71-8.64(m，2H)，8.13(s，1H)，7.99(s，1H)，7.96-7.89(m，2H)，7.50-7.42(m，2H)，7.26-7.19(m，2H)，6.75(d，J＝9.3Hz，1H)，5.22(dqt，J＝8.8，6.8，1.0Hz，1H)，1.57(d，J＝6.7Hz，3H).
MS m/z(ESI)：461.1，463.0[M+H] + .
Example 11
Preparation of (R) -3- (pyridin-4-yl) -N- (1- (3- (trifluoromethyl) phenyl) ethyl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
The first step: preparation of (R) -3- (pyridin-4-yl) -N- (1- (4- (trifluoromethyl) phenyl) ethyl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
Methyl 3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxylate (100 mg,0.19 mmol) is placed in a microwave reaction tube and (R) -1- (4- (trifluoromethyl) phenyl) ethane-1-amine (10 mL) is added and heated to 150℃for 60min. After cooling to room temperature, 20mL of ethyl acetate was added to the reaction solution, and the ethyl acetate layer was washed with a saturated ammonium chloride solution and a saturated sodium chloride solution, followed by drying over anhydrous sodium sulfate and column chromatography to give a crude product (80 mg, yield 67%) which was directly used for the next reaction.
MS m/z(ESI)：693.3[M+H] + .
And a second step of: preparation of (R) -3- (pyridin-4-yl) -N- (1- (3- (trifluoromethyl) phenyl) ethyl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
Crude (R) -3- (pyridin-4-yl) -N- (1- (4- (trifluoromethyl) phenyl) ethyl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (80 mg) was dissolved in 4mL of methylene chloride, 4mL of trifluoroacetic acid was added thereto, and the reaction mixture was allowed to react at room temperature for 3 hours, and then the reaction mixture was dried by spinning. The obtained crude product was dissolved in a mixed solution of ethyl acetate and tetrahydrofuran, washed with a saturated sodium hydrogencarbonate solution and a saturated sodium chloride solution, and then dried over anhydrous sodium sulfate. The resulting organic solvent was dried by spin-drying and purified by preparative chromatography to give the product (R) -3- (pyridin-4-yl) -N- (1- (3- (trifluoromethyl) phenyl) ethyl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (10 mg, 19% yield) as a yellow solid.
1 H NMR(400MHz，MeOD)δ8.59(d，J＝4Hz，2H)，8.39(s，0.6H)，8.15(s，0.4H)，8.06(d，J＝4Hz，2H)，7.79(s，0.4H)，7.56(s，0.6H)，7.49(d，J＝8Hz，2H)，7.21(d，J＝8Hz，2H)，5.12-5.16(m，1H)，1.52(d，J＝8Hz，3H).
MS m/z(ESI)：451.1[M+H] + .
Example 12
Preparation of (R) -N- (1-phenylpropyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
(R) -N- (1-phenylpropyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide is obtained from 3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxylic acid methyl ester and (R) -1-phenylpropan-1-ylamine as starting materials in step six and step seven of reference example 2.
1 H NMR(400MHz，DMSO-d 6 )δ13.56-13.03(m，2H)，9.25-9.14(m，1H)，8.72(d，J＝4Hz，2H)，8.50(s，0.6H)，8.06-7.90(m，2.7H)，7.57(s，0.6H)，7.45(d，J＝8Hz，2H)，7.35(t，J＝8Hz，2H)，7.28-7.23(m，1H)，5.14-5.09(m，1H)，1.75-1.70(m，2H)，0.95-0.88(m，3H).
MS m/z(ESI)：397.1[M+H] + .
Example 13
Preparation of (R) -N- (1- (3-bromophenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
The first step: preparation of (R) -N- (1- (3-bromophenyl) ethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
Methyl 3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxylate (100 mg,0.18 mmol), (R) -1- (3-bromophenyl) ethane-1-amine (1 mL) was stirred for 1h at 150 ℃ with microwaves, cooled, concentrated to dryness, and the crude (R) -N- (1- (3-bromophenyl) ethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (120 mg) was purified by column chromatography.
MS m/z(ESI)：703.1705.2[M+H] + .
And a second step of: preparation of (R) -N- (1- (3-bromophenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
(R) -N- (1- (3-bromophenyl) ethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (120 mg) was dissolved in dichloromethane (2 mL), trifluoroacetic acid (2 mL) was added, stirred at room temperature for 2h, concentrated to dryness, and purified by column chromatography to give (R) -N- (1- (3-bromophenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (26 mg, 30% yield in two steps) as a yellow solid.
1 H NMR(400MHz，DMSO-d 6 )δ13.47(s，1H)，9.48(d，J＝7.9Hz，1H)，8.71-8.64(m，2H)，8.13(s，1H)，7.99(s，1H)，7.96-7.89(m，2H)，7.56-7.47(m，2H)，7.31(dq，J＝7.5，1.4Hz，1H)，7.16(t，J＝7.5Hz，1H)，6.97(d，J＝9.3Hz，1H)，5.20(dqt，J＝8.8，6.7，0.9Hz，1H)，1.59(d，J＝6.9Hz，3H).
MS m/z(ESI)：461.1，463.0[M+H] + .
Example 14
Preparation of (R) -N- (1- (3-methoxyphenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
The first step: preparation of (R) -N- (1- (3-methoxyphenyl) ethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
3- (pyridin-4-yl) -6- (trichloromethyl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole (100 mg,0.17 mmol) was dissolved in a mixed solvent of 12mL of acetonitrile and water (acetonitrile: water=3:1), sodium hydrogencarbonate (141 mg,1.70 mmol) and (R) - (+) -1- (3-methoxyphenyl) ethylamine (28 mg,0.19 mmol) were added and reacted at 60℃under nitrogen atmosphere for 2 hours. After concentrating the reaction solution, 20mL of water was added, the aqueous layer was extracted with ethyl acetate (20 mL x 3), the ethyl acetate layer was washed with saturated sodium chloride solution and dried over anhydrous sodium sulfate, and then column chromatography (eluent: petroleum ether: ethyl acetate=1:1 to ethyl acetate) was performed to obtain a brown oily crude product (50 mg) which was directly used for the next reaction.
MS m/z(ESI)：655.1[M+H] + .
And a second step of: preparation of (R) -N- (1- (3-methoxyphenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
(R) -N- (1- (3-methoxyphenyl) ethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (50 mg) was dissolved in 4mL of methylene chloride, 4mL of trifluoroacetic acid was added thereto, and the reaction mixture was allowed to react at room temperature for 3 hours, and then the reaction mixture was dried by spin-drying. The obtained crude product was dissolved in a mixed solution of ethyl acetate and tetrahydrofuran, washed with a saturated sodium hydrogencarbonate solution and a saturated sodium chloride solution, and then dried over anhydrous sodium sulfate. The resulting organic solvent was dried by spin-drying and purified by thin layer chromatography (developer: dichloromethane: methanol=10:1) to give the product (R) -N- (1- (3-methoxyphenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (7.1 mg, 10% yield) as a yellow solid.
1 H NMR(400MHz，DMSO-d 6 )δ13.45-13.39(m，1H)，13.21-13.08(m，1H)，9.43-9.33(m，1H)，8.68-8.62(m，2H)，8.00-7.94(m，2H)，7.70(s，2H)，7.35-7.27(m，2H)，6.90-6.82(m，2H)，5.25-5.21(m，1H)，3.81(s，3H)，1.47(d，J＝6.8Hz，3H).
MS m/z(ESI)：413.1[M+H] + .
Example 15
Preparation of (S) -N- (2-hydroxy-1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
The first step: preparation of (S) -N- (2-hydroxy-1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f]Indazole-6-carboxylic acid methyl ester (100 mg,0.18 mmol) and (S) -2-amino-2-phenylethan-1-ol (102 mg,0.75 mmol) were dissolved in dioxane (3 mL) and reacted by microwave at 150℃for 3 hours. Post-concentration column chromatography [ eluent: CH (CH) 2 Cl 2 ～CH 2 Cl 2 /MeOH(97∶3)]Obtaining the product (S) -N- (2-hydroxy-1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f]Indazole-6-carboxamide (80 mg, 67% yield).
MS m/z(ESI)：641.2[M+H] + .
And a second step of: preparation of (S) -N- (2-hydroxy-1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
To (S) -N- (2-hydroxy-1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f]To a solution of indazole-6-carboxamide (80 mg,0.13 mmol) in dichloromethane (2 mL) was added trifluoroacetic acid (2 mL) and the mixture was stirred at room temperature for 3 hours. Spin-drying the reaction mixture, dissolving in methanol (5 mL), adjusting pH with ammonia water, concentrating, and performing thin layer chromatography [ CH ] 2 Cl 2 /MeOH(10∶1)～CH 2 Cl 2 /MeOH(8∶1)]Obtaining the product (S) -N- (2-hydroxy-1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f]Indazole-6-carboxamide (5.4 mg, 11% yield).
1 H NMR(400MHz，DMSO)δ13.55-13.01(m，2H)，9.25-9.14(m，1H)，8.72(d，J＝4Hz，2H)，8.50(s，0.6H)，8.07-7.91(m，2.7H)，7.59(s，0.6H)，7.45(d，J＝8Hz，2H)，7.35(t，J＝8Hz，2H)，7.28-7.23(m，1H)，5.14-5.09(m，2H)，3.84-3.77(m，2H).
MS m/z(ESI)：399.1[M+H] + .
Example 16
Preparation of (R) -3- (pyridin-4-yl) -N- (1- (m-benzyl) ethyl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
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The first step: preparation of (S) -N- (2-hydroxy-1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f]Indazole-6-carboxylic acid methyl ester (100 mg,0.19 mmol) and (R) -1- (m-benzyl) ethane-1-amine (101 mg,0.75 mmol) were stirred in N-methylpyrrolidone (3 mL) for 3 hours at 150℃with microwaves. Column chromatography after concentration (eluent: CH) 2 Cl 2 ～CH 2 Cl 2 MeOH (97:3)) to give the product (R) -3- (pyridin-4-yl) -N- (1- (m-benzyl) ethyl) -1-trityl-1, 7-dihydroimidazo [4, 5-f)]Indazole-6-carboxamide (80 mg, 67% yield).
MS m/z(ESI)：639.4[M+H] + .
And a second step of: preparation of (R) -3- (pyridin-4-yl) -N- (1- (m-benzyl) ethyl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
To (R) -3- (pyridin-4-yl) -N- (1- (m-benzyl) ethyl) -1-trityl-1, 7-dihydroimidazo [4,5-f]To a solution of indazole-6-carboxamide (80 mg,0.13 mmol) in dichloromethane (2 mL) was added trifluoroacetic acid (2 mL) and the mixture was stirred at room temperature for 3 hours. Spin-drying the reaction mixture, dissolving in methanol (5 mL), adjusting pH with ammonia water, concentrating, and performing thin layer Chromatography (CH) 2 Cl 2 MeOH (8:1)) to give the product (R) -3- (pyridin-4-yl) -N- (1- (m-benzyl) ethyl) -1, 7-dihydroimidazo [4,5-f]indazole-6-carboxamide (10 mg, 20% yield).
1 H NMR(400MHz，MeOD)δ8.57(d，J＝4Hz，2H)，8.37(s，0.6H)，8.15(s，0.4H)，8.02(d，J＝4Hz，2H)，7.79(s，0.4H)，7.56(s，0.6H)，7.42-7.30(m，3H)，7.12-08(m，1H)，5.12-5.16(m，1H)，2.22(s，3H)，1.52(d，J＝8Hz，3H).
MS m/z(ESI)：397.2[M+H] + .
Example 17
Preparation of (R) -N- (1- (3-chlorophenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
The first step: preparation of (R) -N- (1- (3-chlorophenyl) ethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
3- (pyridin-4-yl) -6- (trichloromethyl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole (2.0 g,3.36 mmol) was dissolved in 40mL of a mixed solvent of tetrahydrofuran and water (tetrahydrofuran: water=3:1), sodium hydrogencarbonate (2.82 g,33.6 mmol) and (R) -1- (3-chlorophenyl) ethylamine (356 mg,3.70 mmol) were added, and the mixture was reacted at room temperature under nitrogen for 4 hours. After concentrating the reaction solution, 40mL of water was added, the aqueous layer was extracted with ethyl acetate (40 mL x 3), the ethyl acetate layer was washed with saturated sodium chloride solution and dried over anhydrous sodium sulfate, and then column chromatography (eluent: petroleum ether: ethyl acetate=1:1 to ethyl acetate) was carried out to obtain a brown solid crude product (540 mg) which was directly used for the next reaction.
MS m/z(ESI)：659.1[M+H] + .
And a second step of: preparation of (R) -N- (1- (3-chlorophenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
(R) -N- (1- (3-chlorophenyl) ethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (540 mg) was dissolved in 5mL of methylene chloride, 5mL of trifluoroacetic acid was added thereto, and after reacting at room temperature for 3 hours, the reaction solution was dried by spin-drying. The obtained crude product was dissolved in a mixed solution of ethyl acetate and tetrahydrofuran, washed with a saturated sodium hydrogencarbonate solution and a saturated sodium chloride solution, and then dried over anhydrous sodium sulfate. The resulting organic solvent was dried by spin-drying and purified by column chromatography (eluent: dichloromethane: methanol=10:1) to give the product (R) -N- (1- (3-chlorophenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (300 mg, 21% over 2 steps) as a pale yellow solid.
1 H NMR(400MHz，DMSO)δ13.48(s，0.4H)，13.41(s，0.6H)，13.25(s，0.4H)，13.11(s，0.6H)，9.60(d，J＝8.5Hz，0.4H)，9.53(d，J＝8.5Hz，0.6H)，8.78-8.67(m，2H)，8.46(s，0.6H)，8.13(s，0.4H)，8.06(d，J＝5.9Hz，1.2H)，7.99(d，J＝5.9Hz，0.8H)，7.90(s，0.4H)，7.59(s，1.6H)，7.46-7.31(m，3H)，5.24(p，J＝7.0Hz，1H)，1.57(d，J＝7.0Hz，3H).
MS m/z(ESI)：417.1[M+H] + .
Example 18
Preparation of (S) -N- (1- (3-chlorophenyl) -2-hydroxyethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
The first step: preparation of (S) -N- (1- (3-chlorophenyl) -2-hydroxyethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
Methyl 3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxylate (100 mg,0.19 mmol) is placed in a microwave reaction tube, and (S) -2-amino-2- (3-chlorophenyl) ethanol (2.0 mL) is added and heated to 150℃for 60min. After cooling to room temperature, 20mL of ethyl acetate was added to the reaction solution, and the ethyl acetate layer was washed with a saturated ammonium chloride solution and a saturated sodium chloride solution, followed by drying over anhydrous sodium sulfate and purification by column chromatography (eluent: dichloromethane to dichloromethane: methanol=20:1) to give a crude product (90 mg) which was directly used for the next reaction.
MS m/z(ESI)：675.1[M+H] + .
And a second step of: preparation of (S) -N- (1- (3-chlorophenyl) -2-hydroxyethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
(S) -N- (1- (3-chlorophenyl) -2-hydroxyethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (90 mg) was dissolved in 4mL of methylene chloride, 4mL of trifluoroacetic acid was added thereto, and after 3 hours at room temperature, the reaction solution was dried by spin-drying. The obtained crude product was dissolved in a mixed solution of ethyl acetate and tetrahydrofuran, washed with a saturated sodium hydrogencarbonate solution and a saturated sodium chloride solution, and then dried over anhydrous sodium sulfate. The resulting organic solvent was dried by spin-drying and purified by thin layer chromatography (developer: dichloromethane: methanol=10:1) to give the product (S) -N- (1- (3-chlorophenyl) -2-hydroxyethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (22 mg, 26% over 2 steps) as a yellow solid.
1 H NMR(400MHz，DMSO-d 6 )δ13.49(s，0.4H)，13.41(s，0.6H)，13.27(s，0.4H)，13.14(s，0.6H)，9.36(d，J＝8.5Hz，0.4H)，9.27(d，J＝8.5Hz，0.6H)，8.74-8.71(m，2H)，8.50(s，0.6H)，8.14(s，0.4H)，8.07(d，J＝6.1Hz，1.2H)，7.99(d，J＝6.1Hz，0.8H)，7.91(s，0.4H)，7.60(s，0.6H)，7.56(s，1H)，7.47-7.28(m，3H)，5.19-5.07(m，2H)，3.89-3.71(m，2H).
MS m/z(ESI)：433.1[M+H] + .
Example 19
Preparation of (R) -N- (1- (3, 5-dimethoxyphenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
(R) -N- (1- (3, 5-dimethoxyphenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide is obtained from 3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxylic acid methyl ester and (R) -1- (3, 5-dimethoxyphenyl) ethyl-1-amine as raw materials in step six and step seven of reference example 2.
1 H NMR(400MHz，CDCl 3 )δ8.75(s，2H)，8.27(s，1H)，7.90(d，J＝8.3Hz，3H)，7.65(s，1H)，6.65(s，2H)，6.36(s，1H)，4.97(s，1H)，3.79(s，6H)，1.48(s，3H).
MS m/z(ESI)：443.1[M+H] + .
Example 20
Preparation of (S) -N- (2-hydroxy-1- (m-benzyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
The first step: preparation of (S) -N- (2-hydroxy-1- (m-benzyl) ethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
Methyl 3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxylate (100 mg,0.19 mmol) is placed in a microwave reaction tube, and (S) -2-amino-2- (m-tolyl) ethanol (2.0 mL) is added and heated to 150℃for 60min. After cooling to room temperature, 20mL of ethyl acetate was added to the reaction solution, and the ethyl acetate layer was washed with a saturated ammonium chloride solution and a saturated sodium chloride solution, followed by drying over anhydrous sodium sulfate and purification by column chromatography (eluent: dichloromethane to dichloromethane: methanol=20:1) to give a crude product (75 mg) which was directly used for the next reaction.
MS m/z(ESI)：655.2[M+H] + .
And a second step of: preparation of (S) -N- (2-hydroxy-1- (m-benzyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
(S) -N- (2-hydroxy-1- (m-benzyl) ethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (75 mg) was dissolved in 4mL of methylene chloride, 4mL of trifluoroacetic acid was added thereto, and after 3 hours at room temperature, the reaction solution was dried by spinning. The obtained crude product was dissolved in a mixed solution of ethyl acetate and tetrahydrofuran, washed with a saturated sodium hydrogencarbonate solution and a saturated sodium chloride solution, and then dried over anhydrous sodium sulfate. The resulting organic solvent was dried by spin-drying and purified by thin layer chromatography (developer: dichloromethane: methanol=10:1) to give the product (S) -N- (1- (3-chlorophenyl) -2-hydroxyethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide as a yellow solid (12 mg, 15% yield over 2 steps).
1 H NMR(400MHz，DMSO)δ13.49(s，0.4H)，13.42(s，0.6H)，13.26(s，0.4H)，13.13(s，0.6H)，9.26-9.08(m，1H)，8.73(d，J＝5.5Hz，2H)，8.57-7.54(m，4H)，7.27-7.21(m，3H)，7.08(d，J＝5.7Hz，1H)，5.11-5.06(m，2H)，3.88-3.79(m，1H)，3.78-3.69(m，1H)，2.31(s，3H).
MS m/z(ESI)：413.1[M+H] + .
Example 21
Preparation of (R) -N- (1- (3-cyanophenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
(R) -N- (1- (3-cyanophenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide was obtained from 3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxylic acid methyl ester and (R) -3- (1-aminoethyl) benzonitrile as starting materials in step six and step seven of reference example 2.
1 H NMR(400MHz，DMSO-d 6 )δ13.47(s，1H)，9.48(d，J＝7.9Hz，1H)，8.71-8.64(m，2H)，8.13(s，1H)，7.99(s，1H)，7.96-7.89(m，2H)，7.78(q，J＝1.4Hz，1H)，7.58(ddq，J＝6.8，3.9，1.5Hz，2H)，7.43(t，J＝7.5Hz，1H)，6.97(d，J＝9.3Hz，1H)，5.20(dddd，J＝9.3，7.9，6.8，5.8Hz，1H)，1.59(d，J＝6.8Hz，3H).
MS m/z(ESI)：408.2[M+H] + .
Example 22
Preparation of N- (2-hydroxy-1- (3-methoxyphenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
N- (2-hydroxy-1- (3-methoxyphenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide is obtained from 3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxylic acid methyl ester and 2-amino-2- (3-methoxyphenyl) ethan-1-ol as raw materials in step six and step seven of reference example 2.
MS m/z(ESI)：429.1[M+H] + .
Example 23
Preparation of (S) -N- (2-amino-2-carbonyl-1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
Reference example 2 step six and step seven were performed using 3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxylic acid methyl ester and (S) -2-amino-2-phenylacetamide as starting materials to obtain (S) -N- (2-amino-2-carbonyl-1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide.
1 H NMR(400MHz，DMSO-d 6 )δ13.48(s，1H)，12.72(s，1H)，8.71-8.64(m，2H)，8.13(s，1H)，7.99(s，1H)，7.96-7.89(m，2H)，7.67(d，J＝10.8Hz，1H)，7.54-7.45(m，2H)，7.42-7.32(m，3H)，6.97(s，2H)，5.67(dt，J＝11.0，1.0Hz，1H).
MS m/z(ESI)：412.1[M+H] + .
Example 24
Preparation of (S) -N- (cyano (phenyl) methyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
(S) -N- (cyano (phenyl) methyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide is obtained from 3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxylic acid methyl ester and (S) -2-amino-2-phenylacetonitrile as raw materials in step six and step seven of reference example 2.
1 H NMR(400MHz，DMSO-d 6 )δ13.47-13.11(m，2H)，8.85-8.71(m，3H)，8.45(s，0.7H)，8.15(s，0.3H)，8.08-7.98(m，2H)，7.85(s，0.3H)，7.51(s，0.7H)，7.42-7.33(m，5H)，5.88(m，1H).
MS m/z(ESI)：394.1[M+H] + .
Example 25
Preparation of (R) -N- (1- (4-aminophenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
(R) -N- (1- (4-aminophenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide was obtained from 3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxylic acid methyl ester and (R) -4- (1-aminoethyl) aniline as starting materials in step six and step seven of reference example 2.
1 H NMR(400MHz，DMSO-d 6 )δ13.43-13.37(m，1H)，13.18-13.03(m，1H)，9.43-9.33(m，1H)，8.68-8.62(m，2H)，8.00-7.94(m，2H)，7.70(s，2H)，7.19-7.11(m，2H)，6.53-6.48(m，2H)，5.25-5.21(m，1H)，4.33(s，2H)，1.55(d，J＝7.0Hz，3H).
MS m/z(ESI)：398.1[M+H] + .
Example 26
Preparation of (R) -N- (1- (3, 5-dichlorophenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
Reference example 2 step six and step seven were performed using 3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxylic acid methyl ester and (R) -1- (3, 5-dichlorophenyl) ethan-1-amine as starting materials to give (R) -N- (1- (3, 5-dichlorophenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide according to example 2.
1 H NMR(400MHz，CDCl 3 )δ8.75(s，2H)，8.29(s，1H)，7.89(d，J＝0.8Hz，3H)，7.56(s，1H)，7.34(s，2H)，7.28(s，1H)，4.97(s，1H)，1.48(s，3H).
MS m/z(ESI)：451.1，453.1[M+H] + .
Example 27
Preparation of (R) -N- (1- (3-fluorophenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
The first step: preparation of (R) -N- (1- (3-fluorophenyl) ethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
Methyl 3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxylate (200 mg,0.37 mmol) was placed in a microwave reaction tube, and (R) -1- (3-fluorophenyl) ethane-1-amine (1.0 mL) was added and heated to 150℃for reaction for 60min. After cooling to room temperature, 20mL of ethyl acetate was added to the reaction solution, and the ethyl acetate layer was washed with a saturated ammonium chloride solution and a saturated sodium chloride solution, followed by column chromatography after drying over anhydrous sodium sulfate to give the crude product (R) -N- (1- (3-fluorophenyl) ethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (160 mg, yield 67%) which was directly used for the next reaction.
MS m/z(ESI)：643.3[M+H] + .
And a second step of: preparation of (R) -N- (1- (3-fluorophenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
Crude (R) -N- (1- (3-fluorophenyl) ethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (160 mg) was dissolved in 4mL of methylene chloride, 4mL of trifluoroacetic acid was added thereto, and after reacting at room temperature for 3 hours, the reaction solution was dried by spin-drying. The obtained crude product was dissolved in a mixed solution of ethyl acetate and tetrahydrofuran, washed with a saturated sodium hydrogencarbonate solution and a saturated sodium chloride solution, and then dried over anhydrous sodium sulfate. The resulting organic solvent was dried by spin-drying and purified by preparative chromatography to give the product (R) -N- (1- (3-fluorophenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (30 mg, yield 27%) as a yellow solid.
1 H NMR(400MHz，DMSO-d 6 )δ13.46(s，1H)，9.50(d，J＝8Hz，1H)，8.72(d，J＝8Hz，2H)，8.36(s，2H)，8.02(d，J＝4Hz，2H)，7.62-7.73(m，1H)，7.42-7.30(m，2H)，7.14-7.06(m，2H)，5.27-5.23(m，1H)，1.56(d，J＝8Hz，3H).
MS m/z(ESI)：401.2[M+H] + .
Example 28
Preparation of (R) -N- (1- (2-chlorophenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
(R) -N- (1- (2-chlorophenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide was obtained from 3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxylic acid methyl ester and (R) -1- (2-chlorophenyl) ethyl-1-amine as starting materials in step six and step seven of reference example 2.
1 H NMR(400MHz，DMSO-d 6 )δ13.47(s，1H)，9.48(d，J＝7.9Hz，1H)，8.71-8.64(m，2H)，8.13(s，1H)，7.99(s，1H)，7.96-7.89(m，2H)，7.44-7.36(m，1H)，7.36-7.28(m，1H)，7.24-7.13(m，2H)，6.83(d，J＝9.5Hz，1H)，5.28(dqd，J＝9.5，6.8，1.0Hz，1H)，1.68(d，J＝6.9Hz，3H).
MS m/z(ESI)：417.1，419.1[M+H] + .
Example 29
Preparation of (S) -N- (2- (methylamino) -1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
The first step: preparation of (S) -N- (2-hydroxy-1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxylic acid methyl ester (50 mg,0.93 mmol), (S) -2-amino-2-phenylethane-1-ol (640 mg,4.66 mmol) and NMP (10 mL) were added to 150mL of microwave with stirring for 2h, cooled, concentrated to dryness, and purified by column chromatography to give the brown solid product (S) -N- (2-hydroxy-1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (340 mg, 56% yield).
MS m/z(ESI)：641.2[M-H] + .
And a second step of: preparation of (S) -N- (2-carbonyl-1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
(S) -N- (2-hydroxy-1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (220 mg,0.34 mmol) was dissolved in 20mL of DMSO, IBX (769 mg,2.74 mmol) was added and stirred overnight at room temperature. Concentrated to dryness and purified by column chromatography to give the crude brown solid product (S) -N- (2-carbonyl-1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (300 mg).
MS m/z(ESI)：639.2[M-H] + .
And a third step of: preparation of (S) -N- (2- (methylamino) -1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
(S) -N- (2-carbonyl-1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (100 mg), 30% methylamine in methanol solution (3 mL) and glacial acetic acid (2 mL) were dissolved in dichloromethane/methanol (10 mL/10 mL), stirred at room temperature for 30min, sodium cyanoborohydride (49 mg,0.78 mmol) was added and stirred at room temperature overnight. Concentrating to dryness, adding water, extracting with dichloromethane, drying, filtering, and concentrating to dryness. Column chromatography purification gave crude (S) -N- (2- (methylamino) -1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (100 mg) as a thick matter.
MS m/z(ESI)：654.2[M-H] + .
Fourth step: preparation of (S) -N- (2- (methylamino) -1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
The viscous (S) -N- (2- (methylamino) -1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (crude product, 100 mg) obtained in the above third step was dissolved in dichloromethane (1 mL), trifluoroacetic acid (2 mL) was added, and stirred at room temperature for 2h. Concentrated to dryness and purified by column chromatography to give the solid product (S) -N- (2- (methylamino) -1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (3.6 mg, three steps yield 7.6%).
MS m/z(ESI)：412.1[M+H] + .
Example 30
Preparation of (S) -N- (2- (cyclopropylamino) -1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
The first step: preparation of (S) -N- (2-hydroxy-1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxylic acid methyl ester (2 g,3.73 mmol), (S) -2-amino-2-phenylethan-1-ol (2.56 g,18.66 mmol) at 140℃under microwave stirring for 1h, water, ethyl acetate extraction, drying and concentration to dryness, column separation and purification to obtain (S) -N- (2-hydroxy-1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (1.46 g, yield 61%).
MS m/z(ESI)：641.2[M+H] + .
And a second step of: preparation of (S) -N- (2-carbonyl-1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
(S) -N- (2-hydroxy-1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (1.46 g,2.28 mmol) was dissolved in DMSO (10 mL), IBX (5.1 g,18.21 mmol) was added and stirred at room temperature under nitrogen for 3h. Dichloromethane and sodium carbonate solution are added for washing, drying and concentration are carried out until the mixture is dried, and oily crude products (2.52 g) are obtained after column separation and purification.
MS m/z(ESI)：639.2[M+H] + .
And a third step of: preparation of (S) -N- (2- (cyclopropylamino) -1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
(S) -N- (2-carbonyl-1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (20 mg,0.031 mmol) and cyclopropylamine (18 mg,0.31 mmol) were stirred in dichloromethane/methanol (10 mL/10 mL), 1mL of acetic acid was added and stirred for 30min. Sodium cyanoborohydride (10 mg,0.16 mmol) was added, stirred overnight, extracted with water, dried and concentrated to dryness, and the oil was purified by column separation and used directly in the next reaction.
MS m/z(ESI)：704.2[M+H] + .
Fourth step: preparation of (S) -N- (2- ((2, 2-difluoroethyl) amino) -1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
The oil obtained in the previous step was dissolved in dichloromethane (2 mL), trifluoroacetic acid (2 mL) was added and stirred at room temperature for 2h. Concentrated to dryness, and purified by column separation to give (S) -N- (2- ((2, 2-difluoroethyl) amino) -1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide as an off-white solid (3.5 mg, 25% yield in two steps).
1 H NMR(400MHz，DMSO-d 6 )δ13.47(s，1H)，9.48(d，J＝7.9Hz，1H)，8.71-8.64(m，2H)，8.13(s，1H)，7.99(s，1H)，7.96-7.89(m，2H)，7.40-7.23(m，5H)，7.10(d，J＝10.3Hz，1H)，5.12(dtt，J＝10.2，6.9，1.0Hz，1H)，3.22(dt，J＝7.7，6.1Hz，1H)，3.11(ddd，J＝12.8，7.0，6.0Hz，1H)，3.00(ddd，J＝12.5，7.0，6.1Hz，1H)，2.54-2.41(m，1H)，1.05-0.77(m，4H).
MS m/z(ESI)：438.2[M+H] + .
Example 31
Preparation of N- (2-fluoro-1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
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N- (2-fluoro-1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide is obtained from 3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxylic acid methyl ester and 2-fluoro-1-phenylethane-1-amine as starting materials in step six and step seven of reference example 2.
1 H NMR(400MHz，DMSO-d 6 )δ13.47(s，1H)，9.48(d，J＝7.9Hz，1H)，8.71-8.64(m，2H)，8.13(s，1H)，7.99(s，1H)，7.96-7.89(m，2H)，7.65(d，J＝11.0Hz，1H)，7.41-7.32(m，4H)，7.32-7.23(m，1H)，5.55-5.38(m，1H)，4.99(d，J＝7.0Hz，1H)，4.87(d，J＝7.1Hz，1H).
MS m/z(ESI)：401.1[M+H] + .
Example 32
Preparation of 3- (pyridin-4-yl) -N- (2, 2-trifluoro-1-phenylethyl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
3- (pyridin-4-yl) -N- (2, 2-trifluoro-1-phenylethyl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide was obtained from 3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxylic acid methyl ester and 2, 2-trifluoro-1-phenylethane-1-amine as starting materials in step six and step seven of reference example 2.
1 H NMR(400MHz，DMSO-d 6 )δ：13.56(s，1H)，13.32(s，1H)，9.39(d，J＝7.5Hz，1H)，8.68-8.62(m，2H)，8.00-7.94(m，2H)，7.70(s，2H)，7.41-7.30(m，4H)，7.30-7.26(m，1H)，7.05-6.91(m，1H).
MS m/z(ESI)：437.1[M+H] + .
Example 33
Preparation of (R) -N- (1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-b ] pyrazolo [4,3-e ] pyridine-6-carboxamide
The first step: preparation of (2, 6-difluoropyridin-3-yl) (pyridin-4-yl) methanol
2, 6-difluoropyridine (3.45 g,30 mmol) was dissolved in 100mL of anhydrous tetrahydrofuran, cooled to-78deg.C, 2.0M LDA in THF/n-heptane solution (15.8 mL,31.5 mmol) was added dropwise under nitrogen followed by injection of 4-pyridinecarbaldehyde (3.86 g,36 mmol). The reaction was gradually warmed from-78 ℃ to room temperature, 1.8mL of acetic acid was added to the reaction solution after the completion of the reaction, and the reaction solution was stirred at room temperature for 30 minutes. Silica gel was directly added to the reaction solution, followed by column chromatography after spin-drying to give (R) -N- (1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-b ] pyrazolo [4,3-e ] pyridine-6-carboxamide as a pale yellow solid (5.0 g, yield 75%).
MS m/z(ESI)：223.1[M+H] + .
And a second step of: preparation of (2, 6-difluoropyridin-3-yl) (pyridin-4-yl) methanone
(2, 6-difluoropyridin-3-yl) (pyridin-4-yl) methanol (5.0 g,22.5 mmol) was dissolved in 250mL DMF and PDC (25.4 g,67.5 mmol) was added in portions and reacted at room temperature for 4 hours. After the reaction mixture was concentrated, 200mL of water was added thereto, and the mixture was extracted with ethyl acetate. The ethyl acetate layer was washed with saturated NaCl solution, dried over anhydrous sodium sulfate and then subjected to column chromatography to give (2, 6-difluoropyridin-3-yl) (pyridin-4-yl) methanone (3.6 g, 73% yield) as a white solid.
MS m/z(ESI)：221.1[M+H] + .
And a third step of: preparation of 6-fluoro-3- (pyridin-4-yl) -1H-pyrazolo [3,4-b ] pyridine
(2, 6-difluoropyridin-3-yl) (pyridin-4-yl) methanone (3.6 g,16.4 mmol) was dissolved in 30mL 1, 4-dioxane and 85% hydrazine hydrate solution (1.2 g,19.7 mmol) was added and reacted at room temperature for 3 hours. Silica gel was added to the reaction solution, followed by column chromatography after direct spin-drying to give 6-fluoro-3- (pyridin-4-yl) -1H-pyrazolo [3,4-b ] pyridine (2.6 g, yield 74%) as a white solid.
MS m/z(ESI)：215.1[M+H] + .
Fourth step: preparation of 3- (pyridin-4-yl) -1H-pyrazolo [3,4-b ] pyridin-6-amine
6-fluoro-3- (pyridin-4-yl) -1H-pyrazolo [3,4-b ] pyridine (0.5 g,2.33 mmol) is placed in a microwave reaction tube and 4mL DMSO and 8mL concentrated ammonia are added. The reaction was carried out for 2 hours with microwave heating to 110 ℃. After the reaction solution was cooled, it was poured into 100mL of water and stirred, the precipitated solid was suction-filtered, and the cake was washed with water and dried in vacuo to give the crude product 3- (pyridin-4-yl) -1H-pyrazolo [3,4-b ] pyridin-6-amine (450 mg).
MS m/z(ESI)：212.1[M+H] + .
Fifth step: preparation of 5-nitro-3- (pyridin-4-yl) -1H-pyrazolo [3,4-b ] pyridin-6-amine
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3- (pyridin-4-yl) -1H-pyrazolo [3,4-b ] pyridin-6-amine (450 mg,2.13 mmol) was dissolved in 7.5mL of concentrated sulfuric acid and placed under an ice bath and concentrated nitric acid (310 mg,3.20 mmol) was added dropwise. The reaction was then heated to 55℃and allowed to react for 6 hours. After the reaction solution was cooled, it was poured into 50mL of ice water and stirred, and the pH was adjusted to neutral with 4N NaOH solution. The precipitated solid was suction-filtered, and the filter cake was washed with water and dried in vacuo to give 5-nitro-3- (pyridin-4-yl) -1H-pyrazolo [3,4-b ] pyridin-6-amine (200 mg, 36% yield) as a yellow solid.
MS m/z(ESI)：257.1[M+H] + .
Sixth step: preparation of 3- (pyridin-4-yl) -1H-pyrazolo [3,4-b ] pyridine-5, 6-diamine
5-nitro-3- (pyridin-4-yl) -1H-pyrazolo [3,4-b]Pyridin-6-amine (200 mg,0.78 mmol) was dissolved in a mixed solvent of DMF and THF (25 mL: 25 mL) and 50mg of 20% Pd (OH) was added 2 After replacing C (about 50% water) with hydrogen balloon several times, the reaction was carried out overnight at room temperature. The reaction solution was filtered to remove Pd (OH) 2 After spin-drying, the crude product 3- (pyridin-4-yl) -1H-pyrazolo [3,4-b is obtained]Pyridine-5, 6-diamine (200 mg) was used directly in the next reaction.
MS m/z(ESI)：227.1[M+H] + 。
Seventh step: preparation of methyl 3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-b ] pyrazolo [4,3-e ] pyridine-6-carboxylate
3- (pyridin-4-yl) -1H-pyrazolo [3,4-b ] pyridine-5, 6-diamine (150 mg,0.66 mmol) was dissolved in 10mL of methanol, placed in a microwave reaction tube, 2mL of triethylamine and 1.5mL of methyl dichloromethoxyacetate were added, and the mixture was heated to 100℃for reaction for 1.5 hours. After the reaction solution was cooled to room temperature, silica gel was added thereto for spin drying, and the crude product methyl 3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-b ] pyrazolo [4,3-e ] pyridine-6-carboxylate (120 mg) was obtained by column chromatography.
MS m/z(ESI)：295.1[M+H] + .
Eighth step: preparation of (R) -N- (1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-b ] pyrazolo [4,3-e ] pyridine-6-carboxamide
Methyl 3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-b]Pyrazolo [4,3-e]Pyridine-6-carboxylate (130 mg,0.44 mmol) was placed in a microwave reaction tube, 2mL (R) - (+) -1-phenylethanamine was added, and the reaction was carried out by microwave heating to 140℃for 1 hour. After the reaction solution is cooled, 20mL of 2-methyltetrahydrofuran is added, and the organic layer is successively saturated with NH 4 Cl solution and saturated NaCl solution washing, anhydrous sodium sulfate drying, through preparative HPLC to obtain light yellow solid (R) -N- (1-phenyl ethyl) -3- (pyridine-4-yl) -1, 7-dihydro imidazo [4, 5-b)]Pyrazolo [4,3-e]Pyridine-6-carboxamide (6.8 mg, 4% yield).
1 H NMR(400MHz，DMSO-d 6 )δ13.35-113.13(m，2H)，9.45-9.34(m，1H)，8.72(d，J＝8Hz，2H)，8.02(d，J＝8Hz，2H)，7.86(s，0.4H)，7.57(s，0.6H)，7.46-7.23(m，5H)，5.24-5.21(m，1H)，1.56(d，J＝8Hz，3H).
MS m/z(ESI)：384.1[M+H] + .
Example 34
Preparation of (R) -N- (1- (2-fluorophenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
(R) -N- (1- (2-fluorophenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide is obtained from 3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxylic acid methyl ester and (R) -1- (2-fluorophenyl) ethyl-1-amine as raw materials in step six and step seven of reference example 2.
1 H NMR(400MHz，DMSO-d 6 )δ13.49(s，1H)，9.47(d，J＝7.9Hz，1H)，8.72(d，J＝5.2Hz，2H)，8.35(s，4H)，8.03(d，J＝5.1Hz，2H)，7.72(s，1H)，7.61(t，J＝7.2Hz，1H)，7.32(dd，J＝13.2，5.8Hz，1H)，7.26-7.14(m，2H)，5.57-5.43(m，1H)，1.56(d，J＝7.0Hz，3H).
MS m/z(ESI)：401.1[M+H] + .
Example 35
Preparation of (R) -N- (1- (3-chloro-4-fluorophenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
(R) -N- (1- (3-chloro-4-fluorophenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide is obtained from 3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxylic acid methyl ester and (R) -1- (3-chloro-4-fluorophenyl) ethyl-1-amine as raw materials in step six and step seven of reference example 2.
1 H NMR(400MHz，DMSO-d 6 )δ13.42(s，1H)，9.46(d，J＝8.4Hz，1H)，8.65(d，J＝4.9Hz，2H)，8.27(s，5H)，7.96(d，J＝5.3Hz，2H)，7.66(dd，J＝7.2，1.9Hz，2H)，7.48-7.38(m，1H)，7.29(ddd，J＝25.1，12.7，7.0Hz，2H)，5.23-5.11(m，1H)，1.49(d，J＝7.0Hz，3H).
MS m/z(ESI)：435.1，437.1[M+H] + .
Example 36
Preparation of N- (3-hydroxy-1-phenylpropyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
N- (3-hydroxy-1-phenylpropyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide is obtained from 3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxylic acid methyl ester and 3-amino-3-phenylpropan-1-ol as starting materials in step six and step seven of reference example 2.
1 H NMR(400MHz，DMSO-d 6 )δ13.56-13.03(m，2H)，9.25-9.14(m，1H)，8.70(d，J＝4Hz，2H)，8.49(s，0.6H)，8.06-7.90(m，2.7H)，7.57(s，0.6H)，7.46(d，J＝8Hz，2H)，7.35(t，J＝8Hz，2H)，7.28-7.23(m，1H)，5.14-5.09(m，1H)，4.50(s，1H)，3.85-3.80(m，2H)，2.14-2.03(m，2H).
MS m/z(ESI)：413.1[M+H] + .
Example 37
(R) -N- (1- (3-cyclopropylphenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
The first step: preparation of tert-butyl (R) - (1- (3-bromophenyl) ethyl) carbamate
To a solution of (R) -1- (3-bromophenyl) ethylamine (500 mg,2.5 mmol) in methylene chloride (20 mL) was added triethylamine (500 mg,5 mmol), and a solution of di-t-butyl dicarbonate (640 mg,3 mmol) in methylene chloride (5 mL) was added dropwise at 0℃and the mixture was stirred for 3 hours. Dichloromethane (50 mL) was added to the reaction, washed with saturated aqueous citric acid (30 mL x 2), then saturated aqueous sodium bicarbonate (30 mL) and the organic phase was concentrated to give tert-butyl (R) - (1- (3-bromophenyl) ethyl) carbamate (750 mg, 100% yield) as a colorless transparent oil.
And a second step of: preparation of tert-butyl (R) - (1- (3-cyclopropylphenyl) ethyl) carbamate
(R) - (1- (3-bromophenyl) ethyl) carbamic acid tert-butyl ester (720 mg,2.4 mmol), cyclopropylboronic acid (413 mg,4.8 mmol), [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride dichloromethane complex (98 mg,0.12 mmol) and potassium carbonate (994 mg,7.2 mmol) were reacted in dioxane (10 mL) and water (1 mL) for 1.5 hours at microwave 120 ℃. After the reaction is finished, the reaction solution is concentrated and then purified by column chromatography [ eluent: petroleum ether-Petroleum ether/ethyl acetate (95/5) ] gives the product (R) - (1- (3-cyclopropylphenyl) ethyl) carbamic acid tert-butyl ester (430 mg, 69% yield) as a colourless oil.
And a third step of: preparation of (R) -1- (3-cyclopropylphenyl) ethane-1-amine hydrochloride
Tert-butyl (R) - (1- (3-cyclopropylphenyl) ethyl) carbamate (430 mg,1.64 mmol) was stirred in ethyl acetate hydrochloride (10 mL,4 mol/L) for 16 hours. The reaction solution was concentrated to give the product (R) -1- (3-cyclopropylphenyl) ethane-1-amine hydrochloride as a white solid (300 mg, yield 92%).
MS m/z(ESI)：162.2[M+H] + .
Fourth step: preparation of (R) -N- (1- (3-cyclopropylphenyl) ethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
3- (pyridin-4-yl) -6- (trichloromethyl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole (100 mg,0.17 mmol), (R) -1- (3-cyclopropylphenyl) ethane-1-amine hydrochloride (37 mg,0.18 mmol) and sodium carbonate (212 mg,2.52 mmol) are stirred in acetonitrile (10 mL) and water (5 mL) at 60℃for 1 hour. Tetrahydrofuran (10 mL) was added to the reaction mixture, and the mixture was stirred at 60℃for 2 hours. After the reaction was completed, water (30 mL) was added, extracted with ethyl acetate (30 mL x 2), and the organic phase was concentrated and purified by column chromatography [ eluent: petroleum ether-Petroleum ether/ethyl acetate (30:70) ] gives crude (75 mg), which is purified by thin layer chromatography (developer: petroleum ether/ethyl acetate=1/1) to give the product (R) -N- (1- (3-cyclopropylphenyl) ethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (40 mg, 36% yield) as a yellow solid.
MS m/z(ESI)：665.3[M+H] + .
Fifth step: preparation of (R) -N- (1- (3-cyclopropylphenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
To (R) -N- (1- (3-cyclopropylphenyl) ethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f]To a solution of indazole-6-carboxamide (80 mg,0.12 mmol) in dichloromethane (2 mL) was added triethylsilane (28 mg,0.24 mmol), trifluoroacetic acid (4 mL), and the mixture was stirred at room temperature for 1 hour. Spin-drying the reaction solution, dissolving in methanol, adjusting to alkaline with ammonia water, concentrating, and purifying by thin layer chromatography (developing solvent: CH) 2 Cl 2 Meoh=10/1) to give the yellow solid product (R) -N- (1- (3-cyclopropylphenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f]Indazole-6-carboxamide (26 mg, 51% yield).
1 H NMR(400MHz，DMSO-d 6 )δ13.46-13.39(m，1H)，13.12(s，1H)，9.40-9.31(m，1H)，8.72(s，2H)，8.45(s，0.6H)，8.12-7.87(m，3H)，7.58(s，0.6H)，7.23-7.18(m，3H)，6.93(d，J＝8Hz，1H)，5.20-5.16(m，1H)，1.90-1.89(m，1H)，1.54(d，J＝8Hz，3H)，0.94-0.93(m，2H)，0.67-0.66(m，2H).
MS m/z(ESI)：423.1[M+H]+.
Example 38
Preparation of (S) -N- (2- ((2, 2-difluoroethyl) amino) -1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
The first step: preparation of (S) -N- (2- ((2, 2-difluoroethyl) amino) -1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
(S) -N- (2-carbonyl-1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (200 mg,0.31 mmol) and 2, 2-difluoroethylamine (127 mg,1.56 mmol) were stirred in dichloromethane/methanol (10 mL/5 mL), 3mL of acetic acid was added and stirred for 30min. Sodium cyanoborohydride (98 mg,1.56 mmol) was added, stirred overnight, extracted with water, dichloromethane, dried and concentrated to dryness, and the crude (S) -N- (2- ((2, 2-difluoroethyl) amino) -1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (38 mg) was obtained by column separation and purification.
MS m/z(ESI)：704.2[M+H] + .
And a second step of: preparation of (S) -N- (2- ((2, 2-difluoroethyl) amino) -1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
(S) -N- (2- ((2, 2-difluoroethyl) amino) -1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (38 mg) was dissolved in dichloromethane (2 mL), trifluoroacetic acid (2 mL) was added, and stirred at room temperature for 2h. Concentrated to dryness, and purified by column separation to give (S) -N- (2- ((2, 2-difluoroethyl) amino) -1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide as an off-white solid (9 mg, 6% yield in two steps).
1 H NMR(400MHz，DMSO-d 6 )δ13.40(s，1H)，13.10(s，1H)，9.18(s，1H)，8.71-8.64(m，2H)，8.13(s，1H)，7.99(s，1H)，7.96-7.89(m，2H)，7.40-7.23(m，5H)，7.03(d，J＝10.1Hz，1H)，5.71(t，J＝7.0Hz，1H)，5.12(dtt，J＝10.4，7.1，1.0Hz，1H)，3.28(dt，J＝12.5，7.3Hz，1H)，3.17(dt，J＝12.2，7.2Hz，1H)，3.12-2.81(m，2H)，1.51(p，J＝7.2Hz，1H).
MS m/z(ESI)：462.2[M+H] + .
Example 39
Preparation of (S) -N- (2- (dimethylamino) -1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
The first step: preparation of (S) -N- (2-hydroxy-1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxylic acid methyl ester (2.0 g,3.73 mmol), (S) -2-amino-2-phenylethan-1-ol (2.56 g,18.66 mmol) at 140℃under microwave stirring for 1h, water, ethyl acetate extraction, drying concentration to dryness, column separation purification to obtain (S) -N- (2-hydroxy-1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (1.46 g, 61% yield).
MS m/z(ESI)：641.2[M+H] + .
And a second step of: preparation of (S) -N- (2-carbonyl-1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
(S) -N- (2-hydroxy-1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (1.46 g,2.28 mmol) was dissolved in DMSO (10 mL), IBX (5.1 g,18.21 mmol) was added and stirred at room temperature under nitrogen for 3h. Dichloromethane and sodium carbonate solution are added for washing, drying and concentration are carried out until the mixture is dried, and 2.52g oily crude product is obtained after column separation and purification and is directly used for the next reaction.
MS m/z(ESI)：639.2[M+H] + .
And a third step of: preparation of (S) -N- (2- (dimethylamino) -1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
(S) -N- (2-carbonyl-1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (200 mg,0.31 mmol), a 2M solution of dimethylamine in methanol (1.57 mL,3.13 mmol) was stirred in dichloromethane/methanol (10 mL/5 mL), acetic acid 0.5mL was added and stirred for 30min. Sodium cyanoborohydride (98 mg,1.56 mmol) was added, stirred overnight, extracted with water, dried and concentrated to dryness, and the oil (66 mg) was purified by column separation and used directly in the next reaction.
MS m/z(ESI)：668.3[M+H] + .
Fourth step: preparation of (S) -N- (2- (dimethylamino) -1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
(S) -N- (2- (dimethylamino) -1-phenylethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (66 mg) was dissolved in dichloromethane (5 mL), trifluoroacetic acid (2 mL) was added, and the mixture was stirred at room temperature for 1h. Concentrated to dryness, and purified by column separation to give an off-white solid (10 mg, 8% yield in two steps).
1 H NMR(400MHz，DMSO-d 6 )δ13.40(s，1H)，13.10(s，1H)，9.18(s，1H)，8.72(d，J＝5.7Hz，2H)，8.50(s，0.5H)，8.29-7.81(m，3H)，7.58(s，0.5H)，7.48(d，J＝7.4Hz，2H)，7.34(t，J＝7.5Hz，2H)，7.26(t，J＝7.3Hz，1H)，5.17(s，1H)，2.96(s，1H)，2.43(dd，J＝12.6，5.2Hz，1H)，2.24(s，6H).
MS m/z(ESI)：426.2[M+H] + .
Example 40
(R) -N- (1- (3-isopropylphenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
The first step: preparation of (R) -N- (1- (3-bromophenyl) ethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
3- (pyridin-4-yl) -6- (trichloromethyl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole (2 g,3.35 mmol), (R) -1- (3-bromophenyl) ethylamine (737 mg,3.69 mmol) and sodium bicarbonate (4.22 g,50.25 mmol) in acetonitrile (50 mL) and water (25 mL) are stirred at 60℃for 1 hour. Tetrahydrofuran (50 mL) was added to the reaction mixture, and the mixture was stirred at 60℃for 2 hours. After completion of the reaction, water (60 mL) was added, extracted with ethyl acetate (50 mL x 2), and the organic phase was concentrated and then chromatographed [ eluent: petroleum ether/ethyl acetate (30:70) ] gives the brown solid product (R) -N- (1- (3-bromophenyl) ethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (700 mg, 30% yield).
MS m/z(ESI)：703.1[M+H] + .
And a second step of: preparation of (R) -N- (1- (3- (prop-1-en-2-yl) phenyl) ethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
(R) -N- (1- (3-bromophenyl) ethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (200 mg,0.28 mmol), 4, 5-tetramethyl-2- (prop-1-en-2-yl) -1,3, 2-dioxaborolan (191 mg,1.14 mmol), a [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride dichloromethane complex (23 mg, 0.328 mmol) and potassium carbonate (118 mg,0.85 mmol) were reacted in dioxane (6 mL) and water (0.6 mL) at microwave 130℃for 1.5 hours. Concentrating the reaction solution, and purifying by column chromatography [ eluent: petroleum Ether-Petroleum ether/ethyl acetate (30:70) ] gives the product (R) -N- (1- (3- (prop-1-en-2-yl) phenyl) ethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (100 mg, 53%) as a brown oil.
MS m/z(ESI)：665.2[M+H] + .
And a third step of: preparation of (R) -N- (1- (3-isopropylphenyl) ethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
(R) -N- (1- (3- (prop-1-en-2-yl) phenyl) ethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ]Indazole-6-carboxamide (100 mg,0.15 mmol) and Pd (OH) 2 C (100 mg) in tetrahydrofuran (6 mL), under a hydrogen balloon, at room temperature for 16 hours. The reaction solution was filtered, and the filtrate was concentrated to give (R) -N- (1- (3-isopropylphenyl) ethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f ] as a yellow-black solid product]Indazole-6-carboxamide (100 mg, 100% yield).
MS m/z(ESI)：667.3[M+H]+.
Fourth step: preparation of (R) -N- (1- (3-isopropylphenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
To (R) -N- (1- (3-isopropylphenyl) ethyl) -3- (pyridin-4-yl) -1-trityl-1, 7-dihydroimidazo [4,5-f]To a solution of indazole-6-carboxamide (100 mg,0.15 mmol) in dichloromethane (2 mL) was added triethylsilane (35 mg,0.30 mmol), trifluoroacetic acid (4 mL), and the mixture was stirred at room temperature for 1 hour. Spin-drying the reaction solution, dissolving in methanol, adjusting to alkaline with ammonia water, concentrating, and performing thin-layer chromatography (developing solvent: CH) 2 Cl 2 Meoh=10/1) to give (R) -N- (1- (3-isopropylphenyl) ethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f as a yellow solid]Indazole-6-carboxamide (28 mg,yield 44%).
1 H NMR(400MHz，DMSO-d 6 )δ13.41(d，J＝28Hz，1H)，13.21-13.07(m，1H)，9.40-9.29(m，1H)，8.73-8.70(m，2H)，8.44(s，0.6H)，8.12(s，0.4H)，8.05-7.97(m，2H)，7.87(s，0.4H)，7.58(s，0.6H)，7.35(s，1H)，7.30-7.24(m，2H)，7.14-7.12(m，1H)，5.23-5.20(m，1H)，2.91-2.84(m，1H)，1.56(d，J＝8Hz，3H)，1.20(d，J＝4Hz，6H).
MS m/z(ESI)：425.2[M+H] + .
Example 41
Preparation of N-benzyl-3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide
Methyl 3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxylate (7.0 mg,0.0239 mmol) and benzylamine (1 mL) were stirred at 100deg.C under microwave for 1h, concentrated to dryness, and purified by column separation to give N-benzyl-3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide (0.8 mg, 9% yield).
1 H NMR(400MHz，DMSO-d 6 )δ13.40-13.35(m，1H)，13.20-13.08(m，1H)，8.71-8.64(m，2H)，8.13(s，1H)，7.99(s，1H)，7.96-7.89(m，2H)，7.51(s，1H)，7.37-7.23(m，5H)，4.64(dd，J＝10.0，1.0Hz，2H).
MS m/z(ESI)：369.1[M+H] + .
Compound biological assay evaluation
The invention is further illustrated below in conjunction with test examples, which do not limit the scope of the invention.
1. Testing of enzymology experiments
1.1 determination of the inhibitory effect of the Compounds of the invention on ERK-1 kinase Activity
The purpose of this test example was to measure the inhibition of ERK-1 kinase activity by a compound. Using LANCE Ultra (Perkin Elmer) method in vitro ERK-1 kinase assay. By plating in 384 well plates (Perkin Elmer OPTIPLATE TM ) To 2.5. Mu.L of test compound/DMSO (final 4%, V/V, diluted to 10 concentrations (400 nM to 0.02 nM) using a 1:3 dilution scheme) was added ERK1 enzyme (Invitrogen, # PV3311, final concentration) prepared with kinase buffer (50mM Hepes pH 7.4, 10mM MgCl2,1mM EGTA,0.01%Triton X-100,2mM DTT): 0.4 nM) and the substrate Ulight-MBP peptide (Perkin Elmer, # TRF0109-M, final concentration: 0.5. Mu.M) in a mixed solution of 5. Mu.L, and 2.5. Mu.L of ATP (Invitrogen, #PV3227, final concentration of 38.15. Mu.M) prepared from the same buffer solution as described above was added thereto, followed by mixing and starting the reaction (reaction system 10. Mu.L). The reaction mixture was incubated at room temperature for 60 minutes. The reaction was stopped by adding 10. Mu.L/well of stop buffer (1X LANCE Detection buffer (Perkinelmer#CR 97-100), 10mM EDTA (Invitrogen# 15575038), 1nM Eu-anti-p-MBP (Perkinelmer#TRF 0201-M) antibody the plate was incubated at room temperature for 60 minutes, excitation readings were made on a Synergy H1 Hybird Reader, H1MFD (Biotek) microplate Reader with excitation wavelength 320 nM. TR-FRET ratio was calculated by dividing acceptor emission signal (at 665nM signal value) by Eu donor emission signal (at 615nM signal value.) percent inhibition of wells treated with the compound relative to wells of Max signal (DMSO control) and Min signal (no enzyme added) controls on the plate. TR-FRET ratio data {% inhibition = 100- [ (test compound-Min average) ]/(Max average-Min average) ×100). The concentration of the compound was diluted 4 times in the reaction system to 10 concentrations of 100nM to 0.005nM. The abs_ic50 value was calculated using GraphPad prism fit percent inhibition and ten point concentration data to a 4 parameter nonlinear logic formula.
1.2 determination of the inhibitory effect of the Compounds of the invention on ERK2 kinase Activity
The purpose of this test case was to measure the inhibition of ERK-2 kinase activity by the compounds. In vitro ERK-2 kinase assays were performed using the LANCE Ultra (Perkin Elmer) method. By plating in 384 well plates (Perkin Elmer OPTIPLATE TM ) To this, 2.5. Mu.L of test compound/DMSO (final 4%, V/V, diluted to 10 concentrations (400 nM to 0.02 nM) using a 1:3 dilution scheme) was added, followed by addition of a kinase buffer (50mM Hepes pH 7.4,10mM MgCl 2 1mM EGTA,0.01%Triton X-100,2mM DTT) ERK-2 enzyme (Invitrogen, #PV3313, final concentration: 0.08 nM) and the substrate Ulight-MBP peptide (Perkin Elmer, # TRF0109-M, final concentration: 0.5. Mu.M) in a mixed solution of 5. Mu.L, and 2.5. Mu.L of ATP (Invitrogen, #PV3227, final concentration of 38.15. Mu.M) prepared from the same buffer solution as described above was added thereto, followed by mixing and starting the reaction (reaction system 10. Mu.L). The reaction mixture was incubated at room temperature for 60 minutes. The reaction was stopped by adding 10. Mu.L/well of stop buffer (1X LANCE Detection buffer (Perkinelmer#CR 97-100), 10mM EDTA (Invitrogen# 15575038), 1nM Eu-anti-p-MBP (Perkinelmer#TRF 0201-M) antibody the plate was incubated at room temperature for 60 minutes, excitation readings were made on a Synergy H1 Hybird Reader, H1MFD (Biotek) microplate Reader with excitation wavelength 320nM, TR-FRET ratios were calculated by dividing the acceptor emission signal (at 665nM signal value) by the Eu donor emission signal (at 615nM signal value), the percent inhibition TR-FRET ratio data {% inhibition = 100- [ (test compound-Min mean) for wells treated with the compound was calculated relative to wells of Max signal (DMSO control) and Min signal (no enzyme added) controls on the plate ]/(Max average-Min average) ×100). The concentration of the compound was diluted 4 times in the reaction system to 10 concentrations of 100nM to 0.005nM. Calculation of abs_ic using graphpadrism fit percentage inhibition and ten point concentration data to 4 parameter nonlinear logic formula 50 Values.
The method is characterized by comprising the following steps: the compounds of the present invention show a response in ERK inhibition assays of about 0.01nM to 100nM (IC 50 ) Is a biological activity of (a).
In some embodiments, the compounds of the invention are IC for ERK-1 and/or ERK-2 50 Less than about 100nM, preferably less than about 10nM, further preferably less than about 5nM, more preferably less than about 1nM, most preferably less than 0.1nM of the compounds listed in the present invention. In some embodiments, the compounds of the invention are IC to ERK 50 Less than about 100nM, preferably less than about 10nM, further preferably less than about 5nM, more preferably less than about 1nM, most preferably less than 0.1nM of the listed compounds. In some other embodiments, preferred compounds among the listed compounds of the inventionExhibits dual binding specificity and is capable of IC at less than about 100nM, less than about 10nM, less than about 5nM, less than about 1nM, less than 0.1nM 50 Values inhibit ERK kinases (e.g., ERK-1 kinase, ERK-2 kinase, etc.) and protein kinases (e.g., ras, raf, her-2, MEK1, etc.).
Specifically, the test data of the examples are shown in table 13:
table 13: inhibition of ERK-1, ERK-2 kinase activity by compounds versus IC 50 Value of
1.3 determination of the proliferation inhibitory Activity of the Compounds of the invention on tumor cells
The purpose of this test example is to determine the inhibition of tumor cell proliferation activity by the compounds of the present invention. The inhibition activity of the compound on the proliferation of tumor cells is measured by a CellTiter-Glo method, and half inhibition concentration IC of the inhibition activity of the compound on the proliferation of the cells is obtained 50 . Inoculating 50-100 mu L of tumor cell suspension in 96-hole cell culture plate with the density of 1-5 x 10 4 Cells/ml, the culture plate is cultured in an incubator for 16 to 24 hours (37 ℃,5 percent CO) 2 ). Adding gradient diluted solutions of compounds to be tested with different concentrations into cells of a culture plate, and incubating the culture plate in an incubator for 3-7 days (37 ℃ C., 5% CO) 2 ). 50-100 mu L CellTiter-Glo reagent is added to each well, and the mixture is shaken for 10 minutes and allowed to stand at room temperature for 10 minutes. The enzyme-labeled instrument measures chemiluminescent signal values. The inhibition rate was calculated by chemiluminescent signal values. IC of compounds by curve fitting based on inhibition ratios at different concentrations 50 。
The compound of the invention is used for testing the proliferation activity of pancreatic cancer tumor cells Mia Paca 2, and the tested IC 50 The values are shown in Table 14.
Table 14: compound pair pancreasCancer tumor cell Mia Paca 2 proliferation activity inhibition relative to IC 50 Value of
Conclusion: the compound has obvious inhibition effect on the proliferation activity of tumor cells.
2. Mouse pharmacokinetic assay
2.1 Experimental purposes:
the pharmacokinetic behavior of the compounds example 1, example 2, and example 6 in the Mouse body (plasma) was studied using Balb/c Mouse (male, available from Shanghai Jieshijie laboratory animal Co., ltd.) as a test animal, animal production license number (SCXK (Shanghai) 2013-0006 N0.311620400001794).
2.2 experimental protocol:
170g PEG400 and 20g Solutol HS-15 were weighed into a 250mL glass bottle and 10mL of NMP was added. Ultrasonic for 10 minutes, and uniformly mixing to obtain a clear solution.
11.2mg of example 1, 10.8mg of example 2,9.8mg of example 6 were weighed into 4mL glass bottles, 1.940mL,2.160mL and 1.525mL of the solution were added, and the solution was sonicated for 10 minutes to give colorless clear solutions at a concentration of 5mg/mL. PO was administered at a dose of 50mg/kg and a dosing volume of 10mL/kg after one night of fasting.
2.3 experimental results:
the PK experimental results for the free base compounds are shown in table 15 below:
table 15: mouse pharmacokinetic parameters of free base compounds
As can be seen from Table 15, the exposure of example 1, example 2 and example 6 to mouse plasma was good at a dose of 50 mg/kg.
3. Experiment of tumor inhibition on MiaPaca 2 transplantation tumor model
3.1 Experimental purposes:
BALB/c nude mice are used as test animals, and human pancreatic cancer cells MiaPaca 2 xenograft tumor (CDX) model is adopted to carry out in vivo efficacy experiments, so as to evaluate the anti-tumor effect of the test compounds.
3.2 laboratory instruments and reagents:
3.2.1 instruments:
ultra clean bench (BSC-1300 II A2, shanghai Bo Xie medical equipment factory)
CO 2 Incubator (Thermo-311, thermo)
Centrifuge (Centrifuge 5720R, eppendorf)
Full-automatic cell counter (Countess II, life Technologies)
Pipettor (10-20. Mu.L, eppendorf)
Microscope (Ts 2, nikang)
Vernier caliper (CD-6' AX, sanfeng Japan)
Cell culture bottle (T25/T75/T225, corning)
Constant temperature flume (HWS 12, shanghai Yihengzheng science)
3.2.2 reagents:
DMEM(11995-065，Gibco)
fetal Bovine Serum (FBS) (10091-148, gibco)
0.25% trypsin (25200-056, gibco)
Green streptomycin double antibody (P/S) (SV 30010, GE)
Phosphate Buffered Saline (PBS) (10010-023, gibco)
Matrigel(356234，Corning)
Gln(25030-081，Gibco)
3.3 experimental procedure:
MiaPaca 2 cells were removed from the cell bank and, after resuscitation, DMEM medium (10% FBS, 1% Glu, 1% P/S) was added to CO 2 Culturing in incubator (temperature of incubator is 37deg.C, CO) 2 Concentration 5%). After the cells are spread at 80-90% of the bottom of the culture flask, the cells are passaged and continuously placed in CO after the passaged 2 Culturing in an incubator. The process is repeated until the number of cells meets the requirement of in vivo pharmacodynamic inoculation, and the cells in logarithmic growth phase are collectedCounting by a dynamic cell counter, and resuspending cells with PBS and Matrigel (volume ratio of 1:1) according to the counting result to obtain cell suspension (density 8 suspension cells) 7 /ml), placed in an ice bin for use.
Animals used were BALB/c nude mice, females, 6-8 weeks old, weighing approximately 18-22 grams. Mice were kept in an environment free of specific pathogens and 5 mice per cage were housed in a single ventilated cage. All cages, pads and water were sterilized prior to use and all animals were free to obtain a standard certified commercial laboratory diet. Nude mice were marked with a universal ear tag for disposable rats and mice before the start of the experiment, the skin at the inoculation site was sterilized with 75% medical alcohol before inoculation, and each mouse was subcutaneously inoculated with 0.1ml (containing 8×10) 6 Individual cells) MiaPaca2 tumor cells. When the average tumor volume reaches 100-200mm 3 The administration of the packets was started at that time. The test compounds were administered orally and gastrally daily, and the dose, frequency, and efficacy of each group at the end of the experiment are shown in tables 16 and 17. Tumor volume (mm) was measured twice weekly with vernier calipers 3 ) The calculation formula is as follows: v=0.5 x D, wherein D and D are the major and minor diameters of the tumor, respectively. Antitumor efficacy was determined by dividing the mean tumor increase volume of compound-treated animals by the mean tumor increase volume of untreated animals. The calculation formula of the tumor inhibition rate is as follows: TGI (%) =1- [ (Vt-V0) dosing group/(Vt-V0) solvent control group]*100%. All animals were euthanized after the end of the experiment.
Table 16: drug efficacy parameters of compound for transplanted tumor mice
The results show that the example 2 can obviously inhibit the growth of MiaPaca 2 nude mice transplanted tumor under the two administration conditions of 100mg/kg QD and 50mg/kg BID after 21 days of oral continuous administration, and the drug effect is good; example 6 also significantly inhibited tumor growth at 100mg/kg QD dosing.
Table 17: drug efficacy parameters of compound for transplanted tumor mice
The results show that the oral administration of example 1 can significantly inhibit the growth of MiaPaca 2 nude mice transplanted tumor under the condition of 50mg/kg QD administration after 16 days of oral continuous administration, and the drug effect is good.
4. hERG potassium channel inhibition Activity assay
4.1 cell preparation
4.1.1 CHO-hERG cells were cultured in 175cm2 flasks, and after cell density had grown to 60-80%, the culture was removed, washed once with 7mL PBS, and then digested with 3mL Detachin.
4.1.2 after digestion is complete, 7mL of culture solution is added for neutralization, then centrifugation is carried out, supernatant is sucked away, and 5mL of culture solution is added for resuspension so as to ensure that the cell density is 2-5 for resuspension, so that fineness is ensured.
4.2 preparation of solutions
Table 18: composition of intracellular and extracellular fluids
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4.3 electrophysiological recording procedure
The single cell high impedance sealing and whole cell mode formation process is all completed automatically by Qpatch instrument, after obtaining whole cell record mode, the cell is clamped at-80 millivolts, before a depolarization stimulus of +40 millivolts for 5 seconds is given, a pre-voltage of-50 millivolts is given for 50 milliseconds, then repolarization is carried out to-50 millivolts for 5 seconds, and then return to-80 millivolts is carried out. This voltage stimulus was applied every 15 seconds, 2 minutes later with an extracellular fluid record for 5 minutes, and then the dosing process was started with compound concentrations starting from the lowest test concentration, each of which was administered for 2.5 minutes. At least 3 cells were tested for each concentration.
4.4 preparation of Compounds
4.4.1 dilution of 20mM Compound stock with extracellular fluid, adding 2495uL extracellular fluid to 5uL 20mM Compound stock, 500-fold dilution to 40uM, and then sequentially 3-fold serial dilutions in extracellular fluid containing 0.2% DMSO to obtain the final concentration to be tested.
The highest test concentration of 4.4.2 was 40uM, and 6 concentrations of 40, 13.33,4.44,1.48,0.49, and 0.16uM, respectively, were found in this order.
4.4.3 the DMSO content in the final test concentration was no more than 0.2%, and this concentration of DMSO had no effect on the hERG potassium channel.
4.5 data analysis
Experimental data were analyzed by XLFit software.
4.6 quality control
Environment: humidity is 20-50% and temperature is 22-25%. To be measured
Reagent: the experimental reagents used were purchased from Sigma company and had a purity of > 98%
Experimental data in the report must meet the following criteria:
whole cell sealing impedance > 100M must be full
Tail current amplitude > 400pA
Pharmacological parameters:
the inhibition effect of multiple concentrations of Cisapride on hERG channel was set as positive control
4.7 experimental results
Example inhibition of hERG current at multiple concentrations:
table 19: example inhibition results on hERG current at multiple concentrations
| Examples numbering | hERG(uM) |
| Example 2 | ＞40 |
Inhibition of the cardiac hERG potassium channel by drugs is the primary cause of QT prolongation syndrome by drugs. From the experimental results, it can be seen that example 2, without inhibition on cardiac hERG potassium ion channel, can avoid toxic side effects of heart at high doses.
In summary, the present invention provides a series of highly active, highly selective ERK1/2 kinase inhibitors having novel structures. The drug has good pharmacokinetic properties on a rat model and a mouse model, also has good drug effects on a Miapaca tumor-bearing mouse model, and has great potential to be developed into drugs for tumor diseases.
Crystal form study of compound free alkali and salt thereof
1. EXAMPLE 2 preparation of different crystalline forms of the free base
1.1 example 2 preparation of free base form I
4.7g of crude (R) -N- (1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide and 47mL of dichloromethane are added to a 100mL reaction flask and the temperature is reduced in an ice-water bath. 2.4mL of triethylsilane and 13.8mL of trifluoroacetic acid were added dropwise to the reaction mixture, the reaction mixture was warmed to room temperature, and the reaction mixture was stirred for 5 hours. Sampling, detecting the reaction completion, concentrating the reaction solution and removing the solvent. Purification by column chromatography gave 0.43g of a yellow solid, to which was added 8.6mL of methanol, heated to reflux for 1 hour, cooled and stirred for 3 hours, filtered, and the filter cake was washed with ethyl acetate, methyl tert-butyl ether and dried to give 0.23g of (R) -N- (1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide as a yellow solid with a purity of 99.3%. It was detected and analyzed as form I of the free base having an XRPD pattern as shown in figure 1, a TGA pattern as shown in figure 2 and a DSC pattern as shown in figure 3.
1.2 example 2 preparation of free base form II
18g of crude (R) -N- (1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide and 180mL of methylene chloride are added to a 250mL reaction flask, the ice water bath is cooled, 9.2mL of triethylsilane and 55.2mL of trifluoroacetic acid are added dropwise, the ice water bath is removed, the reaction solution is warmed to room temperature and stirred for 17 hours. Sampling, detecting that the reaction is complete, concentrating the reaction solution, removing the solvent, adding 72mL of methanol into the concentrated residue, cooling the reaction solution by using an ice water bath, dropwise adding 20mL of 4M hydrogen chloride-ethyl acetate solution, dropwise adding 100mL of ethyl acetate into the reaction solution after the dropwise adding is finished, stirring at room temperature for 2 hours, filtering, washing a filter cake by using ethyl acetate, and drying to obtain 12.1g of a earthy yellow solid. 180mL of 2-methyltetrahydrofuran and 180mL of 2N aqueous sodium hydroxide solution were added to the solid, the layers were stirred and separated, the aqueous layer was extracted with 180mL of 2-methyltetrahydrofuran again, the organic phases were combined, dried, and the solvent was concentrated to give 8.6g of a yellow solid. Then 50mL of methanol and 20mL of tetrahydrofuran solution were added to the solid, followed by 10mL of 30% hydrogen chloride-methanol solution, then 50mL of TBE was added dropwise, stirring was performed at room temperature for 1 hour, filtration was performed, 80mL of 2-methyltetrahydrofuran and 80mL of 2N aqueous sodium hydroxide solution were added to the filter cake (wet product), the mixture was stirred and separated, the aqueous layer was extracted with 80mL of 2-methyltetrahydrofuran again, the organic phases were combined, dried, the solvent was removed by concentration, 40mL of MTBE was added to the concentrated residue, stirring was performed at room temperature for 1 hour, filtration was performed, and drying was performed to obtain 5.0g of pale yellow solid (R) -N- (1-phenylethyl) -3- (pyridin-4-yl) -1, 7-dihydroimidazo [4,5-f ] indazole-6-carboxamide with a purity of 99.4%. The free base form II, as analyzed by the assay, had an XRPD pattern as shown in figure 4, a TGA pattern as shown in figure 5, and a DSC pattern as shown in figure 6.
2. Compound salt type crystal form screening
2.1 Experimental purposes:
different counter-ionic acids are selected and different salts with crystalline forms are prepared by suitable crystallization methods.
2.2 laboratory apparatus and equipment:
2.3 experimental procedure:
2.3.1 screening of salt forms of example 2
(1) Preparation of maleate salt form I
500mg of the free base of example 2 was weighed, 5mL of N, N-dimethylformamide was added and the system was heated at 50℃until complete dissolution, and 183mg of maleic acid was added to 1mL of methanol and the solution was shaken at room temperature to complete dissolution. Adding a methanol solution of maleic acid into a solution of alkali at 50 ℃, quickly separating out a precipitate, stirring for 0.5h, adding 10mL of methyl tertiary butyl ether into the system, continuously stirring for 1.5h, closing heating, cooling to room temperature, finally filtering, leaching a filter cake with 20mL of methyl tertiary butyl ether, and putting the solid into a vacuum drying oven at 40 ℃ to be dried to constant weight, thus obtaining the maleate crystal form I. A detected analysis, having an XRPD pattern as shown in figure 7, a TGA pattern as shown in figure 8, and a DSC pattern as shown in figure 9.
(2) Preparation of maleate salt form II
498.37mg of the free base of example 2 are weighed, 10mL of methanol are added, and the mixture is insoluble under stirring at 50 ℃ and is an off-white suspension. Additional 185.05mg of maleic acid was weighed and 1mL of methanol was added to the sonicated solution. A methanol solution of maleic acid was added to a free base suspension at 50 ℃ and the suspension immediately turned from off-white to yellow. After stirring the reaction at 50 ℃ for 2h, the heating was turned off, the room temperature was allowed to cool, finally filtered and the filter cake was rinsed with 10mL methyl tert-butyl ether. And (5) putting the solid into a vacuum drying oven at 40 ℃ to be dried to constant weight, and obtaining the maleate crystal form II. A detected analysis, having an XRPD pattern as shown in figure 10 and a DSC pattern as shown in figure 11.
(3) Preparation of maleate salt form III
Weighing 30mg of maleate, adding 200uL of tetrahydrofuran, pulping for 4d at 50 ℃, finally centrifuging the solid, removing supernatant, and putting the solid into a vacuum drying oven at 40 ℃ for drying until the weight is constant, thus obtaining the maleate crystal form III. A detected analysis, having an XRPD pattern as shown in figure 12 and a DSC pattern as shown in figure 13.
(4) Preparation of maleate salt form IV
Weighing 50.23mg of maleate, adding 0.75mL of N-methylpyrrolidone, completely dissolving at 50 ℃, adding 1mL of isopropyl ether into the system, quickly separating out yellow solid, reacting at 50 ℃ for 2 hours, continuously adding 2mL of isopropyl ether, turning off heating to room temperature, finally centrifuging the solid, removing supernatant, and drying the solid in a vacuum drying oven at 40 ℃ until the weight is constant, thus obtaining the maleate crystal form IV. A detected analysis, having an XRPD pattern as shown in figure 14 and a DSC pattern as shown in figure 15.
(5) Preparation of hydrochloride salt
Weighing 30mg of compound example 2 free alkali, adding 1mL of tetrahydrofuran, heating to 40 ℃ and stirring for insolubility, adding 94uL of 1M ethanol solution of hydrochloric acid into the suspension, heating the system to 50 ℃ without obvious phenomenon after the addition, adding 0.5mL of methanol, reacting for 2 hours at 50 ℃, closing the heating, stirring for 16 hours at room temperature, centrifuging, oscillating with 2mL of acetone for two times, removing supernatant, drying the solid in a vacuum drying oven at 40 ℃ until the weight is constant, and obtaining hydrochloride crystals. It has an XRPD pattern as shown in figure 16, analyzed by detection.
(6) Preparation of p-Benzenesulfonate Crystal form I
30mg of the compound example 2 free base was weighed, 1mL of methanol was added, stirring was not performed at 40℃and 94uL of 1.18M ethanol solution of p-toluenesulfonic acid was added to the suspension, and the solution was completely dissolved by ultrasonic vibration and stirred at 40℃for 1 hour to remain dissolved. The heating is turned off, a large amount of yellow solid is precipitated after stirring for 1h, the mixture is centrifuged after stirring for 48h at room temperature, 1.5mL of methyl tertiary butyl ether is used for oscillating and centrifuging twice, and the solid is dried to constant weight after removing the supernatant in a vacuum drying oven at 40 ℃. And obtaining the p-toluenesulfonate crystal form I. It has an XRPD pattern as shown in figure 17, as analyzed by detection.
(7) Preparation of p-Benzenesulfonate Crystal form II
Weighing 30mg of p-toluenesulfonate, adding 200uL of ethanol, pulping for 4d at 50 ℃, finally centrifuging the solid, removing supernatant, and drying the solid in a vacuum drying oven at 40 ℃ until the weight is constant to obtain p-toluenesulfonate crystal form II. It has an XRPD pattern as shown in figure 18, as analyzed by detection.
(8) Preparation of p-Benzenesulfonate form III
Weighing 30mg of p-toluenesulfonate, adding 200uL of 88% acetone, pulping for 4d at 50 ℃, finally centrifuging the solid, removing supernatant, and drying the solid in a vacuum drying oven at 40 ℃ until the weight is constant to obtain p-toluenesulfonate crystal form III. It has an XRPD pattern as shown in figure 19, as analyzed by detection.
(9) Preparation of p-Benzenesulfonate Crystal form IV
200mg of p-toluenesulfonate was dried at 80℃for 4h to give p-toluenesulfonate form IV, which was examined and analyzed and had an XRPD pattern as shown in FIG. 20.
Preparation of nitrate salts
50mg of the compound example 2 free base is weighed, 1mL of methanol is added, stirring is carried out at room temperature, dissolution is carried out, 157uL of 1M nitric acid aqueous solution and 1mL of ethanol mixed solution are added to the suspension, stirring is carried out at room temperature for 12 hours, filtration is carried out, filter cake is rinsed by 2mL of acetone, and finally solid is put into a vacuum drying oven at 40 ℃ and dried to constant weight, thus obtaining nitrate crystal. It has an XRPD pattern as shown in figure 21, as analyzed by detection.
3. Stability test
3.1 Experimental purposes:
and (3) examining the physicochemical stability of the candidate compound under the acceleration condition or the influence factor condition of free alkali or different salt forms of the candidate compound, and providing a basis for salt form screening and compound salt storage.
3.2 experimental protocol:
the compound example 2, free base form II and salt were weighed separately at about 2mg, sealed in a 60 ℃ oven and left open at room temperature RH95% (saturated KNO) 3 Aqueous solution) and an illumination box (5000 lx.+ -. 500 lx), the content of salt is measured by HPLC and external standard method for 5 days and 10 days, and the change of the related substances of the salt is calculated by adopting chromatographic peak area normalization method.
3.3 experimental results:
3.3.1 compound example 2 physicochemical stability of free base form II and salts the results are shown in table 20 below, the following "-" indicates undetected:
table 20: compound example 2 results of stability experiments on free base and salt
The above data show: the maleate is more stable than the free alkali crystal form II of the compound example 2 under the conditions of high temperature and high humidity, and no obvious impurity is generated.
4. Moisture permeability test
4.1 experimental purposes:
the hygroscopicity of the compound free alkali and salt under different relative humidity conditions is inspected, and a basis is provided for screening and storing the compound salt.
4.2 experimental protocol:
the compound salt is placed in saturated water vapor with different relative humidity, so that the compound and the water vapor reach dynamic balance, and the percentage of the moisture absorption and weight gain of the compound after the balance is calculated.
4.3 experimental results:
4.3.1 Compounds example 2 hygroscopicity of free base form II and salts thereof
1) Compound example 2 free base form II has a hygroscopic weight gain of about 2.23% at 80% RH and hygroscopicity.
2) The maleate crystal form I has moisture absorption and weight gain of 1.36% under the RH80% condition and slightly moisture absorption; the XRPD pattern of maleate form I was unchanged, i.e. there was no form change, by 1 wet absorption and desorption cycles at 0-95% relative humidity.
5. Solubility experiment
5.1 Experimental purposes:
the solubility of the compound free alkali and salt in water, artificial Simulated Gastric Fluid (SGF), fasted artificial simulated intestinal fluid (FaSSIF), non-fasted artificial simulated intestinal fluid (FeSSIF) and other mediums is compared, and a basis is provided for salt patentability evaluation.
5.2 experimental protocol:
about 2mg of the compound was suspended in various media for 24 hours and the thermodynamic solubility of the compound at room temperature was determined by HPLC, external standard method.
5.3 experimental results:
5.3.1 Compound example 2 solubility results for free base form II, maleate form I and p-Benzenesulfonate form IV are shown in Table 21 below:
table 21 compound example 2 results of crystal form solubility experiments of free base and salt
From the results of the solubility of the compound example 2, form II of the free base and maleate in four media, it can be seen that the compound remains insoluble in water after salt formation, but the solubility in FeSSIF medium is improved after maleate formation; while p-toluenesulfonate increases solubility in the medium FaSSIF but decreases solubility in SGF.
6. Polycrystalline screening experiments
6.1 experimental purposes:
through polycrystalline screening, a relatively stable crystal form is found.
6.2 experimental protocol:
The method comprises the steps of selecting an organic solvent with certain solubility and water, suspending the compound in a solvent system, stirring and pulping for 1 week at room temperature, centrifuging, discarding supernatant, and measuring XRPD of the solid after the solid is dried in vacuum (-0.1 Mpa) at 40 ℃ overnight, and comparing with XRPD of a salt of a raw material compound.
6.3 experimental procedure:
1) Compound example 2 preparation of various crystalline forms of maleate salt:
(1) preparation of maleate salt form I
500mg of the compound of example 2, form II of free base, 5mL of N, N-dimethylformamide was added and the system was heated at 50℃until complete dissolution, and 183mg of maleic acid was added to 1mL of methanol and shaken at room temperature to complete dissolution. Adding a methanol solution of maleic acid into a solution of alkali at 50 ℃, quickly separating out a precipitate, stirring for 0.5h, adding 10mL of methyl tertiary butyl ether into the system, continuously stirring for 1.5h, closing heating, cooling to room temperature, finally filtering, leaching a filter cake with 20mL of methyl tertiary butyl ether, and putting the solid into a vacuum drying oven at 40 ℃ to be dried to constant weight, thus obtaining the maleate crystal form I.
(2) Preparation of maleate salt form II
498.37mg of the compound example 2, form II, is weighed out, 10mL of methanol is added and stirred at 50℃for insolubilization, as an off-white suspension. Additional 185.05mg of maleic acid was weighed and 1mL of methanol was added to the sonicated solution. A methanol solution of maleic acid was added to a free base suspension at 50 ℃ and the suspension immediately turned from off-white to yellow. After stirring the reaction at 50 ℃ for 2h, the heating was turned off, the room temperature was allowed to cool, finally filtered and the filter cake was rinsed with 10mL methyl tert-butyl ether. And (5) putting the solid into a vacuum drying oven at 40 ℃ to be dried to constant weight, and obtaining the maleate crystal form II.
(3) Preparation of maleate salt form III
Weighing 30mg of maleate crystal form I, adding 200uL of tetrahydrofuran, pulping for 4d at 50 ℃, finally centrifuging the solid, removing supernatant, and drying the solid in a vacuum drying oven at 40 ℃ until the weight is constant to obtain maleate crystal form III.
(4) Preparation of maleate salt form IV
Weighing 50.23mg of maleate crystal form I, adding 0.75mL of N-methylpyrrolidone, completely dissolving at 50 ℃, adding 1mL of isopropyl ether into the system, quickly separating out yellow solid, reacting at 50 ℃ for 2 hours, continuously adding 2mL of isopropyl ether, turning off heating to room temperature, centrifuging the solid, removing supernatant, and drying the solid in a vacuum drying oven at 40 ℃ until the weight is constant, thus obtaining maleate crystal form IV.
6.4 experimental results:
6.4.1 Competition relationship between forms of compound example 2 maleate:
1) The maleate form II is very stable in methanol.
2) The maleate salt form III is stable in THF.
3) The maleate crystal form I is relatively stable in acetone, acetonitrile, ethanol, ethyl acetate, isopropanol and water.
PK experiment
7.1 Experimental purposes:
the pharmacokinetic behavior of the compounds example 2, form II of free base, form I of maleate and form IV of p-toluenesulfonate orally administered in rats (plasma) was studied in SD rats, male as test animals.
7.2 experimental protocol:
the compound example 2, form II of the free base, form I of the maleate salt and form IV of the p-toluenesulfonate salt, were uniformly suspended in an aqueous solution containing 0.5% of HPMC K4M and administered to rats by gavage, three rats in parallel, at a dose of 100mg/kg, and the amounts of the compounds were all converted to the amounts of the same compound example 2, free base.
7.3 experimental results:
compound example 2 free base and different salts crystalline form PK experimental results are shown in table 22 below:
table 22: compound example 2 results of crystal form SD rat PK experiments with free base and salt
As can be seen from table 22:
1) Comparative compound example 2 the exposure to free base form II, maleate form I and p-benzoate form IV were increased.
2) Comparative compound example 2T of free base form II max T of maleate salt form I max And is reduced.
3) Compound example 2 changes in the concentration of drug in rat plasma over 24h in form II of free base, form I of maleate and form IV of p-toluenesulfonate are shown in figure 22.
Claims (53)
1. A compound of the general formula (Ia) has the structure:
wherein:
w is selected from-N-or-CH-;
R 1 selected from hydrogen atom, halogen, cyano, C 1-3 Alkyl, C 1-3 Halogenated compoundsAlkyl, C 1-3 Hydroxyalkyl, - (CH) 2 ) n OR 3 、-(CH 2 ) n NR 3 R 4 Or- (CH) 2 ) n C(O)NR 3 R 4 ；
R 2 Selected from hydrogen atom, halogen, amino, nitro, C 1-3 Alkyl, C 1-3 Deuterated alkyl, C 1-3 Haloalkyl, C 1-3 Alkoxy or C 3-6 Cycloalkyl;
R 3 and R is 4 Identical or different and are each independently selected from hydrogen atoms, deuterium atoms, C 1-3 Alkyl, C 1-3 Haloalkyl or C 3-6 Cycloalkyl;
m is an inorganic acid or an organic acid, wherein the inorganic acid is selected from hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid or phosphoric acid; the organic acid is selected from 2, 5-dihydroxybenzoic acid, 1-hydroxy-2-naphthoic acid, acetic acid, dichloroacetic acid, trichloroacetic acid, acetohydroxamic acid, adipic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, 4-aminobenzoic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclohexane sulfamic acid, camphorsulfonic acid, aspartic acid, camphoric acid, gluconic acid, glucuronic acid, glutamic acid, isoascorbic acid, lactic acid, malic acid, mandelic acid, pyroglutamic acid, tartaric acid, dodecylsulfuric acid, dibenzoyltartaric acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactonic acid, gentisic acid, glutaric acid, 2-ketoglutaric acid, glycolic acid, hippuric acid, isethionic acid, lactonic acid, ascorbic acid, aspartic acid, lauric acid, camphoric acid, maleic acid, malonic acid, methanesulfonic acid, 1, 5-naphthalenedisulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, thiocyanic acid, undecylenic acid, trifluoroacetic acid, benzenesulfonic acid, p-toluenesulfonic acid or L-malic acid;
y is an integer of 1, 2 or 3;
n is an integer of 0, 1, 2 or 3; and is also provided with
t is an integer of 0, 1, 2 or 3.
2. The compound of formula (Ia) according to claim 1, wherein the compound has the structure of formula (IIa):
3. the compound of formula (Ia) according to claim 1, wherein the compound has the structure:
4. the compound of formula (Ia) according to claim 1, wherein the compound has the structure of formula (IIIa):
5. the compound of formula (Ia) according to claim 4, wherein said compound of formula (IIIa) is anhydrous and further comprises water, which is pipeline water or crystallization water.
6. The compound of formula (Ia) according to claim 5, wherein said compound of formula (IIIa) is anhydrous and further comprises 0.5 to 8 water molecules.
7. The compound of formula (Ia) according to claim 6, wherein said compound of formula (IIIa) is anhydrous and further comprises 0.5 to 4 water molecules.
8. The compound of formula (Ia) according to claim 7, wherein said compound of formula (IIIa) is anhydrous and further comprises 0.5 to 2.5 water molecules.
9. The compound of formula (Ia) according to claim 8, wherein said compound of formula (IIIa) is anhydrous and further comprises 1 water molecule.
10. The compound of formula (Ia) according to claim 4, wherein the compound of formula (IIIa) is amorphous.
11. The compound of formula (Ia) according to claim 1, wherein the compound has the structure of formula (IVa):
12. a crystal form of a compound shown in a general formula (I) has a structure shown in the formula (I):
wherein:
w is selected from N or CH;
R 1 selected from hydrogen atom, halogen, cyano, C 1-3 Alkyl, C 1-3 Haloalkyl, C 1-3 Hydroxyalkyl, - (CH) 2 ) n OR 3 、-(CH 2 ) n NR 3 R 4 Or- (CH) 2 ) n C(O)NR 3 R 4 ；
R 2 Selected from hydrogen atom, halogen, amino, nitro, C 1-3 Alkyl, C 1-3 Deuterated alkyl, C 1-3 Haloalkyl, C 1-3 Alkoxy or C 3-6 Cycloalkyl;
R 3 and R is 4 Identical or different and are each independently selected from hydrogen atoms, deuterium atoms, C 1-3 Alkyl, C 1-3 Haloalkyl or C 3-6 Cycloalkyl;
m is an inorganic acid or an organic acid, wherein the inorganic acid is selected from hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid or phosphoric acid; the organic acid is selected from 2, 5-dihydroxybenzoic acid, 1-hydroxy-2-naphthoic acid, acetic acid, dichloroacetic acid, trichloroacetic acid, acetohydroxamic acid, adipic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, 4-aminobenzoic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclohexane sulfamic acid, camphorsulfonic acid, aspartic acid, camphoric acid, gluconic acid, glucuronic acid, glutamic acid, isoascorbic acid, lactic acid, malic acid, mandelic acid, pyroglutamic acid, tartaric acid, dodecylsulfuric acid, dibenzoyltartaric acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactonic acid, gentisic acid, glutaric acid, 2-ketoglutaric acid, glycolic acid, hippuric acid, isethionic acid, lactonic acid, ascorbic acid, aspartic acid, lauric acid, camphoric acid, maleic acid, malonic acid, methanesulfonic acid, 1, 5-naphthalenedisulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, thiocyanic acid, undecylenic acid, trifluoroacetic acid, benzenesulfonic acid, p-toluenesulfonic acid or L-malic acid;
x is an integer selected from 0, 1, 2 or 3;
n is an integer of 0, 1, 2 or 3; and is also provided with
t is an integer of 0, 1, 2 or 3.
13. The crystalline form of a compound of formula (I) according to claim 12, wherein the compound has the structure of formula (II):
14. the crystalline form of a compound of formula (I) according to claim 12, having the structure:
15. the crystalline form of a compound of formula (I) according to claim 12, wherein the compound has the structure of formula (III):
16. the crystalline form of the compound of formula (I) according to claim 15, wherein x is 0 and the compound of formula (III) is in the form of a free base;
or when x is selected from 1, 2 or 3, the compound of the formula (III) is in a salt crystal form.
17. The crystalline form of the compound of formula (I) according to claim 16, wherein x is 1 when the compound of formula (III) is in the form of a salt.
18. The crystalline form of the compound of formula (I) according to claim 15, wherein the compound of formula (III) is anhydrous and further comprises water, which is pipeline water or crystallization water.
19. The crystalline form of the compound of formula (I) according to claim 18, wherein the compound of formula (III) is anhydrous and further comprises 0.5 to 8 water molecules.
20. The crystalline form of the compound of formula (I) according to claim 19, wherein the compound of formula (III) is anhydrous and further comprises 0.5 to 4 water molecules.
21. The crystalline form of the compound of formula (I) according to claim 20, wherein the compound of formula (III) is anhydrous and further comprises 0.5 to 2.5 water molecules.
22. The crystalline form of the compound of formula (I) according to claim 21, wherein the compound of formula (III) is anhydrous and further comprises 1 water molecule.
23. The crystalline form of a compound of formula (I) according to claim 12, wherein the compound has the structure of formula (IV):
24. a process for preparing a compound according to any one of claims 1 to 11 or a crystalline form of a compound according to any one of claims 12 to 23, comprising in particular the steps of:
1) Weighing a proper amount of free alkali, and dissolving with benign solvent;
2) Optionally, weighing a proper amount of counter-ionic acid, and dissolving the counter-ionic acid with an organic solvent;
3) Optionally, combining the two solutions, stirring, and dripping a poor solvent until turbidity appears if no precipitate is separated out;
4) Stirring and crystallizing to obtain a target product;
Wherein:
the benign solvent is selected from methanol, tetrahydrofuran, N-dimethylformamide, N-dimethylacetamide or N-methylpyrrolidone;
the organic solvent is selected from methanol, ethanol, ethyl acetate, methylene dichloride, acetone, normal hexane, petroleum ether, benzene, toluene, chloroform, acetonitrile, carbon tetrachloride, dichloroethane, tetrahydrofuran, 2-butanone, 3-pentanone, heptane, methyl tertiary butyl ether, isopropyl ether, 1, 4-dioxane, tertiary butanol or N, N-dimethylformamide; the benign solvent and the organic solution need to be mutually soluble when in use;
the poor solvent is selected from heptane, water, methyl tertiary butyl ether, toluene, isopropyl ether, ethyl acetate, acetone or acetonitrile;
the counter-ionic acid is selected from hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, phosphoric acid, 2, 5-dihydroxybenzoic acid, 1-hydroxy-2-naphthoic acid, acetic acid, dichloroacetic acid, trichloroacetic acid, acetohydroxamic acid, adipic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, 4-aminobenzoic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclohexane sulfamic acid, camphorsulfonic acid, aspartic acid, camphoric acid, gluconic acid, glucuronic acid, glutamic acid, isoascorbic acid, lactic acid, malic acid, mandelic acid, pyroglutamic acid, tartaric acid, dodecylsulfuric acid, sulfuric acid, dibenzoyltartaric acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactonic acid, gentisic acid, glutaric acid, 2-ketoglutaric acid, glycolic acid, hippuric acid, hydroxyethanesulfonic acid, lactobionic acid, ascorbic acid, aspartic acid, lauric acid, camphoric acid, maleic acid, malonic acid, methanesulfonic acid, 1, 5-naphthalenedisulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, thiocyanic acid, undecylenic acid, trifluoroacetic acid, benzenesulfonic acid, p-toluenesulfonic acid or L-malic acid.
25. The process according to claim 24, wherein,
the benign solvent is selected from N, N-dimethylformamide and N-methylpyrrolidone;
the organic solvent is selected from methanol and ethanol;
the poor solvent is selected from ethyl acetate, methyl tertiary butyl ether, isopropyl ether and acetonitrile;
the counter ion acid is selected from methanesulfonic acid, sulfuric acid, hydrochloric acid, nitric acid, benzenesulfonic acid, maleic acid, adipic acid, p-toluenesulfonic acid, citric acid, malonic acid and L-malic acid;
the amount of the counter-ionic acid was 1.2 equivalents.
26. The process according to claim 25, wherein,
the counter-ionic acid is selected from maleic acid.
27. A process for preparing a compound according to any one of claims 1 to 11 or a crystalline form of a compound according to any one of claims 12 to 23, comprising in particular the steps of:
1) Weighing a proper amount of free alkali, and suspending with an unfavorable solvent;
2) Optionally, weighing a proper amount of counter-ionic acid, and dissolving the counter-ionic acid with an organic solvent;
3) Optionally, adding the solution of 2) to the suspension of 1);
4) Stirring and crystallizing to obtain a target product;
wherein:
the poor solvent is selected from acetone, ethyl acetate, acetonitrile, ethanol, 88% acetone, tetrahydrofuran, 1, 4-dioxane, benzene, toluene, isopropanol, N-butanol, isobutanol, N-dimethylformamide, N-dimethylacetamide, N-propanol, tertiary butanol or 2-butanone;
The organic solvent is selected from methanol, ethanol, ethyl acetate, methylene dichloride, acetone, normal hexane, petroleum ether, benzene, toluene, chloroform, acetonitrile, carbon tetrachloride, dichloroethane, tetrahydrofuran, 2-butanone, 3-pentanone, heptane, methyl tertiary butyl ether, isopropyl ether, 1, 4-dioxane, tertiary butanol or N, N-dimethylformamide; the benign solvent and the organic solution need to be mutually soluble when in use;
the counter-ionic acid is selected from hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, phosphoric acid, 2, 5-dihydroxybenzoic acid, 1-hydroxy-2-naphthoic acid, acetic acid, dichloroacetic acid, trichloroacetic acid, acetohydroxamic acid, adipic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, 4-aminobenzoic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclohexane sulfamic acid, camphorsulfonic acid, aspartic acid, camphoric acid, gluconic acid, glucuronic acid, glutamic acid, isoascorbic acid, lactic acid, malic acid, mandelic acid, pyroglutamic acid, tartaric acid, dodecylsulfuric acid, sulfuric acid, dibenzoyltartaric acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactonic acid, gentisic acid, glutaric acid, 2-ketoglutaric acid, glycolic acid, hippuric acid, hydroxyethanesulfonic acid, lactobionic acid, ascorbic acid, aspartic acid, lauric acid, camphoric acid, maleic acid, malonic acid, methanesulfonic acid, 1, 5-naphthalenedisulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, thiocyanic acid, undecylenic acid, trifluoroacetic acid, benzenesulfonic acid, p-toluenesulfonic acid or L-malic acid.
28. The process according to claim 27, wherein,
the poor solvent is selected from methanol, ethanol, tetrahydrofuran, ethyl acetate, acetonitrile and acetone;
the organic solvent is selected from methanol and ethanol;
the counter-ionic acid is selected from maleic acid;
the amount of the counter-ionic acid was 1.2 equivalents.
29. A process for the preparation of a compound according to any one of claims 1 to 11 or a crystalline form of a compound according to any one of claims 12 to 23, comprising in particular the steps of:
1) Weighing a proper amount of free alkali, adding a benign solvent, and heating until the benign solvent is dissolved;
2) Optionally, weighing a proper amount of counter-ionic acid, and dissolving the counter-ionic acid with an organic solvent;
3) Optionally, adding the solution of 2) to the solution of 1);
4) Stirring, cooling and crystallizing to obtain a target product;
wherein:
the benign solvent is selected from methanol, tetrahydrofuran, N-dimethylformamide, N-dimethylacetamide or N-methylpyrrolidone;
the organic solvent is selected from methanol, ethanol, ethyl acetate, methylene dichloride, acetone, normal hexane, petroleum ether, benzene, toluene, chloroform, acetonitrile, carbon tetrachloride, dichloroethane, tetrahydrofuran, 2-butanone, 3-pentanone, heptane, methyl tertiary butyl ether, isopropyl ether, 1, 4-dioxane, tertiary butanol or N, N-dimethylformamide; the benign solvent and the organic solution need to be mutually soluble when in use;
The counter-ionic acid is selected from hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, phosphoric acid, 2, 5-dihydroxybenzoic acid, 1-hydroxy-2-naphthoic acid, acetic acid, dichloroacetic acid, trichloroacetic acid, acetohydroxamic acid, adipic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, 4-aminobenzoic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclohexane sulfamic acid, camphorsulfonic acid, aspartic acid, camphoric acid, gluconic acid, glucuronic acid, glutamic acid, isoascorbic acid, lactic acid, malic acid, mandelic acid, pyroglutamic acid, tartaric acid, dodecylsulfuric acid, sulfuric acid, dibenzoyltartaric acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactonic acid, gentisic acid, glutaric acid, 2-ketoglutaric acid, glycolic acid, hippuric acid, hydroxyethanesulfonic acid, lactobionic acid, ascorbic acid, aspartic acid, lauric acid, camphoric acid, maleic acid, malonic acid, methanesulfonic acid, 1, 5-naphthalenedisulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, thiocyanic acid, undecylenic acid, trifluoroacetic acid, benzenesulfonic acid, p-toluenesulfonic acid or L-malic acid.
30. The process according to claim 29, wherein,
the benign solvent is selected from N, N-dimethylformamide and N-methylpyrrolidone;
the organic solvent is selected from methanol and ethanol;
the counter ion acid is selected from methanesulfonic acid, sulfuric acid, hydrochloric acid, nitric acid, benzenesulfonic acid, maleic acid, adipic acid, p-toluenesulfonic acid, citric acid, malonic acid and L-malic acid;
the amount of the counter-ionic acid was 1.2 equivalents.
31. The process according to claim 30, wherein,
the counter-ionic acid is selected from maleic acid.
32. A compound of formula (Ia) according to any one of claims 1 to 11, characterised in that: m is selected from maleic acid, p-toluenesulfonic acid, hydrochloric acid, nitric acid, salicylic acid, methanesulfonic acid, benzenesulfonic acid or 1, 5-naphthalenedisulfonic acid;
y is 1.
33. A compound of formula (Ia) according to any one of claims 1 to 11, characterised in that: m is selected from maleic acid.
34. The crystalline form of the compound of formula (I) according to any one of claims 12 to 23,
m is selected from maleic acid, p-toluenesulfonic acid, hydrochloric acid, nitric acid, salicylic acid, methanesulfonic acid, benzenesulfonic acid or 1, 5-naphthalenedisulfonic acid;
x is 1.
35. The crystalline form of the compound of formula (I) according to any one of claims 12 to 23,
M is selected from maleic acid.
36. A crystalline form of the compound of formula (IV) as claimed in claim 23, which is free base crystalline form I, X-ray powder diffraction pattern having characteristic peaks at 2Θ of 6.4 and 26.7 (2Θ ± 0.2 °);
its free base crystalline form II, X-ray powder diffraction pattern has characteristic peaks at 2Θ of 8.5, 13.4, and 17.0.
37. The crystalline form of the compound of formula (IV) according to claim 36, wherein the free base crystalline form I, X-ray powder diffraction pattern has characteristic peaks at 2Θ of 7.2, 13.1, 16.8, 17.7, 18.9, 20.2, 21.2, and 28.2 (2Θ ± 0.2 °);
the free base crystalline form II, X-ray powder diffraction pattern, has characteristic peaks (2θ±0.2°) at 2θ of 10.2, 10.9, 12.8, 13.1, 16.1, 17.8, 18.8, 19.5, 23.0, 23.9, 24.4, 25.5, 26.1 and 27.6.
38. A crystalline form of the compound of formula (IV) as described in claim 23, which is a maleate salt form, and x is 1,
the maleate crystal form I has characteristic peaks at 2 theta of 5.5 and 16.3 (2 theta plus or minus 0.2 DEG) of an X-ray powder diffraction pattern;
the maleate crystal form II has characteristic peaks at 2 theta of 5.5, 10.8, 16.3 and 17.6 (2 theta plus or minus 0.2 DEG);
The maleate crystal form III has characteristic peaks at 2 theta of 4.9, 15.1, 17.2, 17.5, 26.5 and 26.9 (2 theta plus or minus 0.2 DEG);
the maleate crystal form IV has an X-ray powder diffraction pattern with characteristic peaks of 4.8 and 16.7 at 2 theta.
39. The crystalline form of the compound of formula (IV) according to claim 38, wherein the maleate salt form I, X-ray powder diffraction pattern has characteristic peaks at 2Θ of 10.8, 15.3, 17.7, 26.1, 26.4, and 27.0 (2Θ ± 0.2 °);
the maleate salt form II, X-ray powder diffraction pattern having characteristic peaks at 14.0, 26.4, 26.6 and 27.2 in 2Θ;
the maleate salt crystal form III has characteristic peaks at 2 theta of 9.8, 14.7, 18.4 and 19.8 (2 theta plus or minus 0.2 DEG);
the maleate salt form IV, X-ray powder diffraction pattern, has characteristic peaks at 2θ of 9.6, 11.9, and 14.9 (2θ±0.2°).
40. The crystalline form of compound of formula (IV) according to claim 39, wherein the maleate salt form I, X-ray powder diffraction pattern has characteristic peaks at 2Θ of 13.0, 14.9, 20.0, and 20.8 (2Θ ± 0.2 °);
the maleate salt form II, X-ray powder diffraction pattern has characteristic peaks at 2θ of 8.8, 12.9, 18.4, 18.9, 20.6, 21.5, 22.4 and 22.8 (2θ±0.2°);
The maleate salt crystal form III has characteristic peaks at 2 theta of 12.3, 13.0, 14.0 and 27.5 (2 theta plus or minus 0.2 DEG);
the maleate salt form IV, X-ray powder diffraction pattern has characteristic peaks at 2θ of 7.2, 12.9, 14.3, 19.1, 19.3, 21.5 and 23.9 (2θ±0.2°).
41. A crystalline form of the compound of formula (IV) as claimed in claim 23, which is a hydrochloride salt crystalline form, and x is 1, the x-ray powder diffraction pattern has characteristic peaks at 2Θ of 5.8 and 17.2 (2Θ±0.2°).
42. The crystalline form of the compound of formula (IV) of claim 41, wherein the hydrochloride crystalline form has a characteristic peak at 2Θ (2Θ ± 0.2 °) of 12.0, 13.6, 16.4, 21.7, 23.0, 26.1, 26.3, and 27.1.
43. The crystalline form of the compound of formula (IV) of claim 42, wherein the hydrochloride crystalline form has an X-ray powder diffraction pattern with characteristic peaks at 2Θ at 8.9, 28.8, and 30.1 (2Θ ± 0.2 °).
44. A crystalline form of the compound of formula (IV) as claimed in claim 23 which is a crystalline form of p-toluenesulfonate salt and X is 1, which has a characteristic peak at 2Θ (2Θ±0.2°) of 6.1, 11.3, 17.0, 17.5 and 18.3 for the p-toluenesulfonate salt crystalline form I;
The p-toluenesulfonate crystal form II has characteristic peaks at 2 theta of 5.4, 8.6, 11.4, 16.8, 18.2, 19.7, 20.4 and 21.9 (2 theta plus or minus 0.2 DEG);
the p-toluenesulfonate crystal form III has characteristic peaks at 2 theta of 4.9, 8.6, 13.2, 18.9, 20.6 and 25.2 (2 theta plus or minus 0.2 DEG);
the p-toluenesulfonate crystal form IV has characteristic peaks at 2 theta of 5.5, 11.9, 16.3, 19.4 and 25.5 (2 theta +/-0.2 DEG) in X-ray powder diffraction pattern.
45. The crystalline form of the compound of formula (IV) according to claim 44, wherein the p-benzosulfonate crystalline form I, X-ray powder diffraction pattern has characteristic peaks at 2Θ of 12.7, 19.1, 19.9, 20.6, and 22.2 (2Θ ± 0.2 °);
the p-toluenesulfonate crystal form II has an X-ray powder diffraction pattern with characteristic peaks at 2θ of 13.5, 13.7, 15.7, 17.2, 23.7, 25.5 and 27.6 (2θ±0.2°);
the p-toluenesulfonate crystal form III, X-ray powder diffraction pattern has characteristic peaks at 2θ of 9.6, 10.9, 12.6, 15.0, 15.6, 17.0, 22.6, 25.8 and 27.5 (2θ±0.2°);
the p-toluenesulfonate crystal form IV, X-ray powder diffraction pattern has characteristic peaks at 2θ of 13.8, 18.1, 18.6, 20.1, 21.3, 22.9 and 26.5 (2θ.+ -. 0.2 ℃).
46. The crystalline form of compound of formula (IV) according to claim 45, wherein the p-benzosulfonate crystalline form II, X-ray powder diffraction pattern has characteristic peaks at 2Θ of 16.0, 21.4, 25.9, and 28.7 (2Θ ± 0.2 °);
the p-toluenesulfonate crystal form III, X-ray powder diffraction patterns have characteristic peaks at 2θ of 10.5, 13.9, 16.5, 17.7, 21.7, 26.1, 26.6, 26.9, 27.8, 29.8 and 32.2 (2θ±0.2°);
the p-toluenesulfonate crystal form IV, X-ray powder diffraction pattern has characteristic peaks at 2θ of 8.7, 9.1, 10.2, 10.8, 12.9 and 20.4 (2θ±0.2°).
47. A crystalline form of the compound of formula (IV) as claimed in claim 23, which is a nitrate crystalline form and x is 1, the x-ray powder diffraction pattern having characteristic peaks at 2Θ of 5.0, 16.3, 16.7 and 28.0 (2Θ±0.2°).
48. The crystalline form of compound of formula (IV) of claim 47, wherein the nitrate crystalline form has an X-ray powder diffraction pattern with characteristic peaks at 2Θ (2Θ ± 0.2 °) of 8.2, 8.5, 11.8, 13.2, and 29.1.
49. The crystalline form of compound of formula (IV) of claim 48, wherein the nitrate crystalline form has an X-ray powder diffraction pattern with characteristic peaks at 2Θ at 19.7, 20.4, 21.0, and 24.5 (2Θ ± 0.2 °).
50. A pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (Ia) according to any one of claims 1 to 11, together with one or more pharmaceutically acceptable carriers.
51. A pharmaceutical composition comprising a therapeutically effective amount of a crystalline form of a compound of formula (I) according to any one of claims 12 to 23, together with one or more pharmaceutically acceptable carriers.
52. A pharmaceutical composition comprising a therapeutically effective amount of a crystalline form of a compound of formula (IV) according to any one of claims 36 to 47, together with one or more pharmaceutically acceptable carriers.
53. The use of a compound of general formula (Ia) according to any one of claims 1 to 11 and a pharmaceutical composition according to claim 50 for the manufacture of a medicament for the treatment of ERK mediated related diseases selected from cancer, bone diseases, inflammatory diseases, immune diseases, neurological diseases, metabolic diseases, respiratory diseases or heart diseases; wherein the cancer is selected from breast cancer, pancreatic cancer, non-small cell lung cancer, thyroid cancer, seminoma, melanoma, bladder cancer, liver cancer, kidney cancer, myelodysplastic syndrome, acute myelogenous leukemia, or colorectal cancer.
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| CN201910260428 | 2019-04-02 | ||
| CN2019102604282 | 2019-04-02 | ||
| PCT/CN2020/082042 WO2020200161A1 (en) | 2019-04-02 | 2020-03-30 | Salt of indazolyl-containing tricyclic derivative and crystal form thereof |
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Citations (4)
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| US5476851A (en) * | 1994-09-08 | 1995-12-19 | Rhone-Poulenc Rorer Pharmaceuticals, Inc. | Pyrazolo[3,4-g]quinoxaline compounds which inhibit PDGF receptor protein tyrosine kinase |
| WO2015051341A1 (en) * | 2013-10-03 | 2015-04-09 | Araxes Pharma Llc | Inhibitors of erk and methods of use |
| CN104829613A (en) * | 2015-04-13 | 2015-08-12 | 上海创诺医药集团有限公司 | Diaryl-substituted pyrazolo ring derivative and preparation method thereof, and applications of diaryl-substituted pyrazolo ring derivative in medicine field |
| WO2017222958A1 (en) * | 2016-06-20 | 2017-12-28 | Kura Oncology, Inc. | Treatment of squamous cell carcinomas with inhibitors of erk |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004203804A (en) * | 2002-12-26 | 2004-07-22 | Taiho Yakuhin Kogyo Kk | New indazole derivative or salt thereof and pharmaceutical with the same as active ingredient |
| WO2014179154A2 (en) * | 2013-04-30 | 2014-11-06 | Merck Sharp & Dohme Corp. | Novel compounds that are erk inhibitors |
| CN104447765A (en) * | 2014-12-31 | 2015-03-25 | 深圳铂立健医药有限公司 | Tricyclic compound and pharmaceutical compositions thereof and application thereof |
| WO2019076336A1 (en) * | 2017-10-19 | 2019-04-25 | 江苏豪森药业集团有限公司 | Pyrazolyl-containing tricyclic derivative, preparation method therefor and use thereof |
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- 2020-03-30 WO PCT/CN2020/082042 patent/WO2020200161A1/en active Application Filing
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5476851A (en) * | 1994-09-08 | 1995-12-19 | Rhone-Poulenc Rorer Pharmaceuticals, Inc. | Pyrazolo[3,4-g]quinoxaline compounds which inhibit PDGF receptor protein tyrosine kinase |
| WO2015051341A1 (en) * | 2013-10-03 | 2015-04-09 | Araxes Pharma Llc | Inhibitors of erk and methods of use |
| CN104829613A (en) * | 2015-04-13 | 2015-08-12 | 上海创诺医药集团有限公司 | Diaryl-substituted pyrazolo ring derivative and preparation method thereof, and applications of diaryl-substituted pyrazolo ring derivative in medicine field |
| WO2017222958A1 (en) * | 2016-06-20 | 2017-12-28 | Kura Oncology, Inc. | Treatment of squamous cell carcinomas with inhibitors of erk |
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| WO2020200161A1 (en) | 2020-10-08 |
| TW202102507A (en) | 2021-01-16 |
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