Patent Application
20240325394
This application claims the priorities of Chinese patent application 2020113542899 filed on Nov. 26, 2020 and Chinese patent application 2021113892168 filed on Nov. 22, 2021. The contents of the Chinese patent applications are incorporated herein by reference in their entireties.
The present disclosure belongs to the field of biomedicine, specifically related to a salt and crystal form of nitrogen-containing heterocyclic derivative, a preparation method therefor and an application thereof.
Rat sarcomas (RAS), encoded by proto-oncogenes HRAS, NRAS, and KRAS, is calssified as 4 proteins, HRAS, NRAS, KRAS4A and KRAS4B, and is a GTP (guanosine triphosphate) binding protein. RAS is located on the inner surface of a cell membrane, upstream of which is receptor tyrosine kinase (RTK), after activation, RAS regulates downstream PI3K, RAF and other signaling pathways, thereby regulating cell growth, survival, migration, differentiation, and other functions.
RAS has two main states in the body: an inactivated state combined with GDP (guanosine diphosphate) and an activated state combined with GTP. Its activity is regulated by two proteins, guanine nucleotide exchange factor (GEF) promotes the release of GDP from the RAS protein, allowing GTP to bind to activate RAS; GTPase activating protein (GAP) activates the GTPase activity of RAS protein, hydrolyzes the GTP bound to RAS protein into GDP, and inactivates the RAS. Under normal circumstances, the RAS protein is in a non-activated state, the conformation changes after mutation, and the RAS is in a continuously activated state, and downstream signaling pathways are also continuously activated, leading to the occurrence of various cancers.
As the first identified oncogene, RAS is the oncogene with the highest mutation rate, accounting for an average of 25% of human cancers. The most common oncegenic mutation in the RAS family is KRAS (85%), while NRAS (12%) and HRAS (3%) are relatively rare. KRAS mutations mainly occur in a series of cancers such as pancreatic cancer (95%), colorectal cancer (52%) and lung cancer (31%), etc. The most common mutation mode of KRAS is point mutation, which mostly occurs in G12, G13 in p-loop (aa 10 to 17) and Q61 in Switch II region (aa 59 to 76), where G12 mutation is the most common (83%). KRAS G12C is one of the most common mutations in non-small cell lung cancer (NSCLC) and colorectal cancer.
Although there are great clinical demands, no drugs that directly target KRAS have been marketed so far, and currently, patients with KRAS mutations in clinical treatment can only be treated with chemotherapy. The difficulty in the development of KRAS inhibitors is mainly due to two factors: first, the structure of RAS protein is smooth, and small molecules are difficult to bind to the protein surface; secondly, the affinity of RAS GTPase for GTP is as high as picomolar (pM) level, and the level of endogenous GTP is high, small molecule drugs are difficult to block the combination of the two. Recent studies have found that after the mutation of Glycine (Gly) at 12-position of KRAS to Cysteine (Cys), the conformation changes and a new pocket is formed for covalent binding of small molecules, which irreversibly locks KRAS G12C in binding to GDP in a non-activated state. Therefore, KRAS G12C inhibitors are expected to be the first drug directly targeting KRAS.
At present, many KRAS G12C inhibitors have entered the clinical research stage, such as AMG 510 developed by Amgen, ARS-3248 developed by Wellspring Biosciences and MTRX849 developed by Mirati, all of which are currently in the clinical Phase I research stage, but none of them have been developed and marketed as KRAS G12C inhibitors yet.
There is no specific target drug for KRAS G12C, and there is a large clinical demand. The KRAS G12C inhibitors with higher selectivity, better activity and better safety have the potential to treat a variety of cancers, and have broad market prospects.
The patent application of Jiangsu Hanson Pharmaceutical Group Co., Ltd. (application No.: PCT/CN2020/093285) disclosed the structure of a series of pyridazine derivative inhibitors. In the subsequent research and development, in order to make the product easy to process, filter, dry, convenient for storage, long-term stability of the product, and high bioavailability, the present disclosure has carried out a comprehensive study on the salt and crystal form of the above substances, and is committed to obtaining the most suitable crystal form.
All the contents involved in the patent application PCT/CN2020/093285 are incorporated in the present disclosure by citation.
The object of the present disclosure is to provide an acid salt of a compound represented by general formula (I):
In a preferred embodiment of the present disclosure, in the acid salt of the compound represented by general formula (I), Ra is each independently selected from hydrogen, deuterium, halogen, amino, hydroxyl, sulfhydryl, cyano, nitro, C1-6 alkyl, C1-6 deuterated alkyl, C1-6 haloalkyl, C1-6 alkoxy, —SRaa, —C(O)Raa, —NRaaRbb, C1-6 haloalkoxy or C1-6 hydroxyalkyl;
R5 is selected from hydrogen, deuterium, halogen, amino, hydroxyl, sulfhydryl, cyano, nitro, C1-6 alkyl, C1-6 deuterated alkyl, C1-6 haloalkyl, C1-6 alkoxy, —SRaa, —C(O)Raa, —NRaaRbb or C1-6 hydroxyalkyl;
In a preferred embodiment of the present disclosure, for the acid salt of the compound, the compound is shown in general formula (II):
In a preferred embodiment of the present disclosure, for the acid salt of the compound, the compound is further shown in general formula (II-A) or (II-B):
In a preferred embodiment of the present disclosure, for the acid salt of the compound, wherein the compound is selected from:
In a more preferred embodiment of the present disclosure, for the acid salt of the compound, wherein the compound is selected from:
Acid in the acid salt is selected from hydroxyethyl sulfonic acid, sulfuric acid, 1,5-naphthalene disulfonic acid, methanesulfonic acid, hydrobromic acid, phosphoric acid, benzenesulfonic acid, oxalic acid, maleate acid, adipic acid, hydrochloric acid, citric acid, malonic acid, L-malic acid, pamoic acid, p-toluenesulfonic acid or fumaric acid, preferably hydroxyethyl sulfonic acid or sulfuric acid.
In a further preferred embodiment of the present disclosure, the number of acid is 0.2-3; preferably 0.2, 0.5, 1, 1.5, 2, 2.5 or 3; more preferably 0.5, 1, 2 or 3.
In a further preferred embodiment of the present disclosure, the acid salt is a hydrate or an anhydrate, and when the acid salt is the hydrate, the number of water is 0.2-3; preferably 0.2, 0.5, 1, 1.5, 2, 2.5 or 3; more preferably 0.5, 1, 2 or 3.
In the most preferred embodiment of the present disclosure, an acid salt of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one is provided, wherein the acid in the acid salt is selected from hydroxyethyl sulfonic acid, sulfuric acid, 1,5-naphthalene disulfonic acid, methylsulfonic acid, hydrobromic acid, phosphoric acid, benzenesulfonic acid, oxalic acid, maleate acid, adipic acid, hydrochloric acid, citric acid, malonic acid, L-malic acid, pamoic acid, p-toluenesulfonic acid or fumaric acid, wherein structure of the acid salt of the compound is as follows:
In a preferred embodiment of the present disclosure, the acid salt is in a crystal form; preferably a crystal form of the acid salt of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one;
In a preferred embodiment of the present disclosure, the crystal form of the acid salt of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one is provided.
In a more preferred embodiment of the present disclosure, the acid salt of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one is in a crystal form; preferably the crystal form of hydroxyethyl sulfonate, the crystal form of sulfate, the crystal form of 1,5-naphthalene disulfonate, the crystal form of methanesulfonate, the crystal form of hydrobromate, the crystal form of phosphate, the crystal form of benzenesulfonate, the crystal form of oxalate, the crystal form of maleate, the crystal form of adipate, the crystal form of hydrochloride, the crystal form of citrate, the crystal form of malonate, the crystal form of L-malate, the crystal form of pamoate, the crystal form of p-toluenesulfonate or the crystal form of fumarate.
In a preferred embodiment of the present disclosure, the acid salt is in a crystal form; wherein the number of acid is 0.2-3; preferably 0.2, 0.5, 1, 1.5, 2, 2.5 or 3; more preferably 0.5, 1, 2 or 3.
In a preferred embodiment of the present disclosure, crystal forms I-III of hydroxyethyl sulfonate and crystal forms I-IV of sulfate of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one are provided:
In a further preferred embodiment of the present disclosure, crystal forms I-III of hydroxyethyl sulfonate and crystal forms I-IV of sulfate of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one are provided:
In a further preferred embodiment of the present disclosure, crystal forms I-III of hydroxyethyl sulfonate and crystal forms I-IV of sulfate of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one are provided:
In a further preferred embodiment of the present disclosure, crystal forms I-III of hydroxyethyl sulfonate and crystal forms I-IV of sulfate of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one are provided:
In a further preferred embodiment of the present disclosure, for the crystal form I of hydroxyethyl sulfonate of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one, measured by Cu-Kα radiation, X-ray characteristic diffraction peaks represented by 20 angles and interplanar spacing d values are shown in Table 1.
The crystal form I of hydroxyethyl sulfonate of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one shown in embodiment 13-1 described in the present disclosure has an X-ray powder diffraction pattern basically as shown in
In a further preferred embodiment of the present disclosure, for the crystal form II of hydroxyethyl sulfonate of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one shown in embodiment 13-1, measured by Cu-Kα radiation, X-ray characteristic diffraction peaks represented by 20 angles and interplanar spacing d values are shown in Table 2.
The crystal form II of hydroxyethyl sulfonate of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one shown in embodiment 13-1 described in the present disclosure has an X-ray powder diffraction pattern basically as shown in
In a further preferred embodiment of the present disclosure, for the crystal form III of hydroxyethyl sulfonate of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one shown in embodiment 13-1, measured by Cu-Kα radiation, X-ray characteristic diffraction peaks represented by 20 angles and interplanar spacing d values are shown in Table 3.
The crystal form III of hydroxyethyl sulfonate of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one shown in embodiment 13-1 described in the present disclosure has an X-ray powder diffraction pattern basically as shown in
In a further preferred embodiment of the present disclosure, for the crystal form I of sulfate of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one shown in embodiment 13-1, measured by Cu-Kα radiation, X-ray characteristic diffraction peaks represented by 20 angles and interplanar spacing d values are shown in Table 4.
The crystal form I of sulfate of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1l-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one shown in embodiment 13-1 described in the present disclosure has an X-ray powder diffraction pattern basically as shown in
In a further preferred embodiment of the present disclosure, for the crystal form II of sulfate of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one shown in embodiment 13-1, measured by Cu-Kα radiation, X-ray characteristic diffraction peaks represented by 20 angles and interplanar spacing d values are shown in Table 5.
The crystal form II of sulfate of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1l-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one shown in embodiment 13-1 described in the present disclosure has an X-ray powder diffraction pattern basically as shown in
In a further preferred embodiment of the present disclosure, for the crystal form III of sulfate of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one shown in embodiment 13-1, measured by Cu-Kα radiation, X-ray characteristic diffraction peaks represented by 20 angles and interplanar spacing d values are shown in Table 6.
The crystal form III of sulfate of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1l-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one shown in embodiment 13-1 described in the present disclosure has an X-ray powder diffraction pattern basically as shown in
In a further preferred embodiment of the present disclosure, for the crystal form IV of sulfate of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one shown in embodiment 13-1, measured by Cu-Kα radiation, the X-ray characteristic diffraction peaks represented by 20 angles and interplanar spacing d values are shown in Table 7.
The crystal form IV of sulfate of compound P-4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one shown in embodiment 13-1 described in the present disclosure has an X-ray powder diffraction pattern basically as shown in
In a further preferred embodiment of the present disclosure, positions of diffraction peaks with relative peak intensity of top ten in the X-ray powder diffraction pattern of the crystal form I of hydroxyethyl sulfonate and diffraction peaks at the corresponding positions in
In a further preferred embodiment of the present disclosure, for the acid salt of the compound, the crystal form of the acid salt is a hydrate or an anhydrate, when the crystal form of the acid salt is the hydrate, the number of water is 0.2 to 3, preferably 0.2, 0.5, 1, 1.5, 2, 2.5 or 3, more preferably 0.5, 1, 2 or 3; further, the water in the hydrate is pipeline water, or crystal water, or a combination of both.
In a further preferred embodiment of the present disclosure, a method for preparing an acid salt comprises the following steps:
In a further preferred embodiment of the present disclosure, a method for preparing the acid salt of the compound and a crystal form thereof comprises the following steps:
In a further preferred embodiment of the present disclosure, a method for preparing a crystal form of the acid salt of the compound comprises the following steps:
In a further preferred embodiment of the present disclosure, a method for preparing the acid salt of the compound or the crystal form thereof comprises the following steps:
The reaction solvent used in step 1) is an organic solvent, preferably at least one of ethanol, propanol, isopropanol, 2-methyltetrahydrofuran, n-heptane, methyl tert-butyl ether, toluene, isopropyl acetate, tert-butanol, n-butanol, tetrahydrofuran, acetone, 2-butanone, ethyl acetate or 1,4-dioxane;
The organic solvent in step 2) is selected from one or more of alcohol, ether, ketone or ester solvents, preferably at least one of ethanol, propanol, isopropanol, 2-methyltetrahydrofuran, n-heptane, methyl-tert-butyl ether, toluene, isopropyl acetate, tert-butanol, n-butanol, tetrahydrofuran, acetone, 2-butanone, ethyl acetate or 1,4-dioxane;
The present disclosure also provides a preferred embodiment, and relates to a pharmaceutical composition comprising a therapeutically effective amount of the acid salt of the compound represented by general formula (I) or the crystal form thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.
The present disclosure further relates to a use of any one of the acid salts of the compound represented by general formula (I) or the crystal forms thereof, or the pharmaceutical composition in the manufacture of a medicament of a KRAS inhibitor; preferably a use in the manufacture of a medicament of a KRAS G12C mutation inhibitor.
In some embodiments, the pharmaceutically acceptable salt of the compound and the crystal form thereof, or the composition of the present disclosure is used for treating Noonan syndrome, leopard syndrome, leukemia, neuroblastoma, melanoma, esophagus cancer, head and neck tumor, breast cancer, lung cancer and colon cancer; preferably non-small cell lung cancer, colon cancer, esophagus cancer, and head and neck tumor.
Unless otherwise stated, the terms used in the description 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 1 to 20 carbon atoms, preferably alkyl containing 1 to 8 carbon atoms, more preferably alkyl containing 1 to 6 carbon atoms, most preferably alkyl containing 1 to 3 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-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, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-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, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched isomers. More preferably lower alkyl containing 1 to 6 carbon atoms, non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. The alkyl may be substituted or unsubstituted, when substituted, the substituents may be substituted at any available attachment point, the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxyl, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl, or carboxylate, preferably alkyl substituted by methyl, ethyl, isopropyl, tert-butyl, haloalkyl, deuterated alkyl, alkoxy-substituted alkyl and hydroxyl-substituted alkyl.
The term “alkylene” refers to that one hydrogen atom of alkyl is further substituted, for example: “methylene” refers to —CH2—, “ethylene” refers to —(CH2)2—, and “propylene” refers to —(CH2)3—, “butylene” refers to —(CH2)4—, etc. The term “alkenyl” refers to alkyl as defined above containing 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 etc. The alkenyl may be substituted or unsubstituted, when substituted, the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.
The term “cycloalkyl” refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctanyl, etc.; polycylic cycloalkyl includes spiro, fused and bridged cycloalkyl, preferably cyclopropyl, cyclobutyl, cyclohexyl, cyclopentyl and cycloheptyl.
The term “spirocycloalkyl” refers to polycyclyl that shares one carbon atom (called a spiro atom) between 5- to 20-membered monocyclic rings, which may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. Preferably 6-14-membered, more preferably 7-10-membered. According to the number of shared spiro atoms between the rings, the spirocycloalkyl is classified into monospirocycloalkyl, bispirocycloalkyl or polyspirocycloalkyl, preferably monospirocycloalkyl and bispirocycloalkyl. More preferably, 3-membered/6-membered, 3-membered/5-membered, 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered, or 5-membered/6-membered monospirocycloalkyl. Non-limiting examples of spirocycloalkyl include:
The term “fused cycloalkyl” refers to a 5-20-membered all-carbon polycyclic group in which each ring in the system shares an adjacent pair of carbon atoms with other rings in the system, wherein one or more of the rings may comprise one or multiple double bonds, but none of the rings has a fully conjugated π-electron system. Preferably 6-14-membered, more preferably 7-10-membered. According to the number of constituent rings, it can be classified into bicyclic, tricyclic, tetracyclic or polycyclic fused cycloalkyl, preferably bicyclic or tricyclic, and more preferably 5-membered/5-membered or 5-membered/6-membered bicyclic alkyl. Non-limiting examples of fused cycloalkyls include;
The term “bridged cycloalkyl” refers to 5 to 20-membered all-carbon polycyclic group, in which any two rings share two carbon atoms that are not directly connected, it may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. Preferably 6-14-membered, more preferably 7-10-membered. According to the number of constituent rings, it can be classified into bicyclic, tricyclic, tetracyclic or polycyclic bridged cycloalkyl, preferably bicyclic, tricyclic, or tetracyclic, and more preferably bicyclic or tricyclic. Non-limiting examples of bridged cycloalkyl include:
The cycloalkyl ring may be fused to an aryl, heteroaryl or heterocycloalkyl ring, wherein the ring connected to the parent structure is cycloalkyl, non-limiting examples include indanyl, tetrahydronaphthyl, benzocycloheptanyl, etc. The cycloalkyl may be substituted or unsubstituted, when substituted, the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl or carboylate.
The term “heterocyclyl” refers to saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, wherein one or more of the ring atoms are heteroatoms selected from nitrogen, oxygen or S(O)m (wherein m is an integer of 0 to 2), but not including the ring part of —O—O—, —O—S— or —S—S—, and the remaining ring atoms are carbon. It preferably contains 3 to 12 ring atoms, wherein 1 to 4 ring atoms are heteroatoms; more preferably contains 3 to 8 ring atoms; most preferably contains 3 to 8 ring atoms; further preferably 3-8-membered heterocyclyl containing 1 to 3 nitrogen atoms, optionally substituted by 1 to 2 oxygen atoms, sulfur atoms or oxo, including nitrogen-containing monocyclic heterocyclyl, nitrogen-containing spiro heterocyclyl or nitrogen-containing fused heterocyclyl.
Non-limiting examples of monocyclic heterocyclyl include pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothienyl, dihydroimidazolyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, azepyl, 1,4-diazepanyl, pyranyl, etc., preferably pyrrolidinyl, morpholinyl, piperidinyl, azepanyl, 1,4-diazepanyl and piperazinyl. Polycyclic heterocyclyl include spiro-, fused- and bridged heterocyclyl; the spiro-, fused- and bridged heterocyclyl are optionally connected to other groups through a single bond, or to connect to other cycloalkyl, heterocyclyl, aryl and heteroaryl through any two or more of ring atoms.
The term “spiroheterocyclyl” refers to polycyclic heterocyclyl sharing one atom (called a spiro atom) between 5-20-membered monocyclic ring, wherein one or more ring atoms are selected from nitrogen, oxygen or S(O)m (wherein m is an integer of 0 to 2) heteroatoms, and the remaining ring atoms are carbon. It may contain one or more double bonds, but none of the rings has fully conjugated π-electron system. Preferably 6-14-membered, more preferably 7-10-membered. According to the number of spiro atoms shared between the rings, the spiro heterocyclyl is classified into monospiroheterocyclyl, dispiroheterocyclyl or polyspiroheterocyclyl, preferably monospiroheterocyclyl and dispiroheterocyclyl. More preferably, 3-membered/5-membered, 3-membered/6-membered, 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered, or 5-membered/6-membered monospiroheterocyclyl. Non-limiting examples of spiroheterocyclyl include:
The term “fused heterocyclyl” refers to a 5-20-membered polycyclic heterocylic group in which each ring in the system shares an adjacent pair of atoms with other rings in the system, one or more of the rings may comprise one or multiple double bonds, but none of the rings has a fully conjugated π-electron system, wherein one or more of the ring atoms are heteroatoms selected from nitrogen, oxygen or S(O)m (wherein m is an integer of 0 to 2), the rest of the ring atoms are carbon. Preferably 6-14-membered, more preferably 7-10-membered. According to the number of constituent rings, it can be classified into bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclyl, preferably bicyclic or tricyclic, and more preferably 5-membered/5-membered or 5-membered/6-membered bicyclic fused heterocylyl. Non-limiting examples of fused heterocylyl include:
The term “bridged heterocyclyl” refers to polycyclic heterocylic group in which any two rings share two atoms that are not directly connected, it may contain one or multiple double bonds, but none of the rings has a fully conjugated π-electron system, wherein one or more of the ring atoms are heteroatoms selected from nitrogen, oxygen or S(O)m (wherein m is an integer of 0 to 2), the rest of the ring atoms are carbon. Preferably 6-14-membered, more preferably 7-10-membered. According to the number of constituent rings, it can be classified into bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocyclyl, preferably bicyclic, tricyclic, or tetracyclic, and more preferably bicyclic or tricyclic. Non-limiting examples of bridged heterocylyl include:
The heterocyclic ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring connected to the parent structure is heterocyclyl, and non-limiting examples of heterocyclyl include:
The heterocyclyl may be substituted or unsubstituted, when substituted, the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl or carboylate.
The term “aryl” refers to a 6-14-membered all-carbon monocyclic or fused polycyclic (that is, rings sharing adjacent pairs of carbon atoms) with conjugated π-electron system, preferably 6-12-membered, such as phenyl and naphthyl. More preferably phenyl. The aryl ring may be fused on a heteroaryl, heterocyclyl or cycloalkyl ring, including benzo 5-10-membered heteroaryl, benzo 3-8-membered cycloalkyl and benzo 3-8-membered heteroalkyl, preferably benzo 5-6-membered heteroaryl, benzo 3-6-membered cycloalkyl and benzo 3-6-membered heteroalkyl, wherein the heterocyclyl is heterocyclyl containing 1 to 3 nitrogen atoms, oxygen atoms and sulfur atoms; or a 3-membered nitrogen-containing fused ring containing a benzene ring.
Herein, the ring connected to the parent structure is an aryl ring, and non-limiting examples of aryl include:
The aryl may be substituted or unsubstituted, when substituted, the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalky, heterocycloalky, aryl, heteroaryl, cycloalkoxyl, heterocycloalkoxyl, cycloalkylthio, heterocycloalkylthio, carboxyl or carboylate.
The term “heteroaryl” refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen. The heteroaryl is preferably 5-12-membered, more preferably 5- or 6-membered, such as imidazole, furanyl, thiophenyl, thiazolyl, pyrazolyl, oxazolyl, pyrrolyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl, thiadiazole, pyrazinyl, etc., preferably triazolyl, thiophenyl, imidazolyl, pyrazolyl, oxazolyl, pyrimidinyl or thiazolyl; more preferably pyrazolyl, pyrrolyl and oxazolyl.
The heteroaryl ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring connected to the parent structure is the heteroaryl ring, and non-limiting examples of heteroaryl include:
The heteroaryl may be optionally substituted or unsubstituted, when substituted, the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalky, heterocycloalky, aryl, heteroaryl, cycloalkoxyl, heterocycloalkoxyl, cycloalkylthio, heterocycloalkylthio, carboxyl or carboylate.
The term “alkoxy” refers to —O-(alkyl) and —O-(unsubstituted cycloalkyl), wherein the definition of alkyl is as described above, preferably alkyl containing 1 to 8 carbon atoms, more preferably alkyl containing 1 to 6 carbon atoms, most preferably alkyl containing 1 to 3 carbon atoms. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy. The alkoxy may be optionally substituted or unsubstituted, when substituted, the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboylate.
The term “alkylthio” refers to —S-(alkyl) and —S-(unsubstituted cycloalkyl), wherein the definition of alkyl is as described above. Preferably alkyl containing 1 to 8 carbon atoms, more preferably alkyl containing 1 to 6 carbon atoms, most preferably alkyl containing 1 to 3 carbon atoms. Non-limiting examples of alkylthio include: methylthio, ethylthio, propylthio, butylthio, cyclopropylthio, cyclobutylthio, cyclopentylthio, cyclohexylthio. The alkylthio may be optionally substituted or unsubstituted, when substituted, the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboylate.
“Alkylthio-alkyl” refers to alkylthio attached to alkyl, wherein the alkyl and the alkylthio are as defined above.
“Alkylaminocarbonyl” refers to (alkyl)-N—C(O)—, wherein the alkyl is as defined above.
“Haloalkyl” refers to alkyl substituted by one or more halogens, wherein the alkyl is as defined above.
“Haloalkoxy” refers to alkoxy substituted by one or more halogens, wherein the alkoxy is as defined above.
“Haloalkylthio” refers to alkylthio substituted by one or more halogens, wherein the alkylthio is as defined above.
“Hydroxyalkyl” refers to alkyl substituted by one or more hydroxyl, wherein the alkyl is as defined above.
“Alkenyl” refers to chain alkenyl, also known as alkylene, preferably alkyl containing 2 to 8 carbon atoms, more preferably alkyl containing 2 to 6 carbon atoms, most preferably alkyl containing 2 to 3 carbon atoms. Herein, the alkenyl may be further substituted with other related groups, such as: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalky, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate.
“Alknyl” refers to (CH≡C—), preferably alkyl containing 2 to 8 carbon atoms, more preferably alkyl containing 2 to 6 carbon atoms, most preferably alkyl containing 2 to 3 carbon atoms. Herein, the alknyl may be further substituted by other related groups, for example: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboylate.
The term “alkenylcarbonyl” refers to —C(O)-(alkenyl), wherein the alkenyl is as defined above. Non-limiting examples of alkenylcarbonyl include: vinylcarbonyl, propenylcarbonyl, butenylcarbonyl. The alkenylcarbonyl may be optionally substituted or unsubstituted, when substituted, the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboylate.
“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” and other terms all express the same meaning, which means that X can be any one or more of A, B, and C.
The hydrogen atom described in the present disclosure may be replaced by its isotope deuterium, and any hydrogen atom in the compounds according to the embodiments of the present disclosure may also be replaced by a deuterium atom.
“Optional” or “optionally” refers to that the event or environment described later may, but not necessarily, occur, and the description includes occasions where the event or environment occurs or does not occur. For example, “heterocyclic group optionally substituted by alkyl” refers to that alkyl may, but not necessarily, be present, and the description includes the case where the heterocyclic group is substituted by alkyl and the case where the heterocyclic group is not substituted by alkyl.
“Substituted” refers to one or more hydrogen atoms in the group, preferably up to 5, more preferably 1 to 3 hydrogen atoms, independently substituted by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine possible or impossible substitutions (by experiment or theory) without too much effort. For example, amino or hydroxyl having free hydrogen may be unstable when combined with a carbon atom having an unsaturated (e.g., olefinic) bond.
“Pharmaceutical composition” refers to a mixture containing one or more of the compounds described herein or the physiologically/pharmaceutically acceptable salt or prodrug thereof and other chemical components, and the other component is, for example, physiological/pharmaceutically acceptable carrier and excipient. The purpose of the pharmaceutical composition is to promote the administration to an organism, facilitate the absorption of an active ingredient and then exert the biological activity.
“Pharmaceutically acceptable salt” refers to the salt of the compound of the present disclosure, which is safe and effective when used in mammals, and has due biological activity.
The following embodiments will further describe the present disclosure, but these embodiments do not limit the scope of the present disclosure.
The structures of the compounds of the present disclosure were determined by nuclear magnetic resonance (NMR) or/and liquid chromatography-mass spectrometry (LC-MS). NMR chemical shift (δ) was given in units of parts per million (ppm). NMR was determined using a Bruker AVANCE-400 NMR instrument with deuterated dimethyl sulfoxide (DMSO-d6), deuterated methanol (CD3OD) and deuterated chloroform (CDCl3) as solvents and tetramethylsilane (TMS) as internal standard.
Liquid chromatography-mass spectrometry LC-MS was determined with an Agilent 1200 Infinity Series mass spectrometer. HPLC determinations were performed using an Agilent 1200DAD high pressure liquid chromatograph (Sunfire C18 150×4.6 mm column) and a Waters 2695-2996 high pressure liquid chromatograph (Gimini C18 150×4.6 mm column).
Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate was used as a thin-layer chromatography silica gel plate, the specification of TLC was 0.15 mm to 0.20 mm, and the specification of thin-layer chromatography separation and purification products was 0.4 mm to 0.5 mm. Generally, Yantai Huanghai silica gel 200 to 300 mesh silica gel was used as a carrier for column chromatography.
The starting materials in the embodiments of the present disclosure are known and commercially available, or can be synthesized by using or following methods known in the art.
Unless otherwise specified, all reactions of the present disclosure were carried out under continuous magnetic stirring under dry nitrogen or argon atmosphere, the solvent was a dry solvent, and the unit of the reaction temperature was degrees Celsius.
2,4-Dichloropyridin-3-amine (4.5 g, 27.78 mmol), 4,4,5,5-tretramethyl-2-(prop-1-en-2-yl)-1,3,2-dioxaborolane (5.13 g, 30.56 mmol), potassium carbonate (11.5 g, 83.34 mmol), Pd(PPh3)4 were added to dioxane (120 mL), and the reaction mixture was uniformly mixed and then stirred overnight in an oil bath at 100° C. The mixture was concentrated under reduced pressure, and the resulting crude product was purified by rapid silica gel column chromatography to obtain the target compound 4-chloro-2-(prop-1-en-2-yl)pyridin-3-amine as a colorless oily liquid (4.5 g, yield: 96%).
MS m/z (ESI): 169.1 [M+H]+.
4-Chloro-2-(prop-1-en-2-yl)pyridin-3-amine (2 g, 11.9 mmol) and sodium thiomethoxide (10 mL, 20% aqueous solution) were added to dioxane (3 mL). The reaction mixture was uniformly mixed, then reacted at 100° C. for 2 days, cooled to room temperature, and concentrated under reduced pressure, and the resulting crude product was purified by rapid silica gel column chromatography to obtain the compound 4-(methylthio)-2-(prop-1-en-2-yl)pyridin-3-amine as a pale yellow liquid (1.7 g, yield: 79%).
MS m/z (ESI): 181.2 [M+H]+.
Methanol (50 mL) was added to 4-(methylthio)-2-(prop-1-en-2-yl)pyridin-3-amine (2 g, 11.11 mmol) and Pd/C (4 g), the reaction mixture was uniformly mixed, then reacted overnight at room temperature and concentrated under reduced pressure. The resulting crude product was added to a mixed solution of methanol (5 mL), N,N-diisopropylethylamine (0.5 mL) and acrylonitrile (1 mL), and the reaction was carried out at room temperature for 2 hours. The mixture was concentrated under reduced pressure and purified by rapid silica gel column chromatography to obtain the compound 2-isopropyl-4-(methylthio)pyridin-3-amine as a colorless liquid (500 mg, yield: 25%).
MS m/z (ESI): 183.2 [M+H]+.
THF (10 mL) was added to 2,6-dichloro-5-fluoronicotinamide (500 mg, 2.44 mmol) and oxalyl chloride (1.32 mL, 2.54 mmol), and the reaction mixture was uniformly mixed and then the reaction was carried out at 60° C. for 3 hours, the reaction temperature was reduced to room temperature, and triethylamine (680 mg, 6.6 mmol) and 2-isopropyl-4-(methylthio)pyridin-3-amine (400 mg, 2.2 mmol) were added thereto, and the reaction was carried out at room temperature for 1 hour. The mixture was concentrated under reduced pressure, and the resulting crude product was purified by rapid silica gel column chromatography to obtain the compound 2,6-dichloro-5-fluoro-N-((2-isopropyl-4-(methylthio)pyridin-3-yl)carbamoyl)nicotinamide as a white solid (800 mg, yield: 87%).
MS m/z (ESI): 417.1 [M+H]+.
2,6-Dichloro-5-fluoro-N-((2-isopropyl-4-(methylthio)pyridin-3-yl)carbamoyl)nicotinamide (800 mg, 1.92 mmol) was added to THF (20 mL), and after the reaction mixture was uniformly mixed, KHMDS (4.8 mL, 4.8 mmol) was slowly added thereto at 0° C., and the reaction was carried out at room temperature for 1 hour. The mixture was concentrated under reduced pressure, and the resulting crude product was purified by rapid silica gel column chromatography to obtain the compound 7-chloro-6-fluoro-4-hydroxy-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one as a white solid (600 mg, yield: 82%).
MS m/z (ESI): 381.1 [M+H]+.
7-Chloro-6-fluoro-4-hydroxy-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (300 mg, 0.79 mmol), phosphorus oxychloride (600 mg, 3.95 mmol), DIPEA (1 g. 7.9 mmol) were added to THF (40 mL), the reaction mixture was uniformly mixed, and then the reaction was carried out at 80° C. for 1 hour, the reaction temperature was reduced to room temperature, and tert-butyl (S)-3-methylpiperazine-1-carboxylate (240 mg. 1.19 mmol) was slowly added to the reaction solution, then the reaction was carried out at room temperature for 1 hour. The mixture was concentrated under reduced pressure, and the resulting crude product was purified by rapid silica gel column chromatography to obtain compound tert-butyl (S)-4-(7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)-2-carbonyl-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate as a white solid (400 mg, yield: 90%).
MS m/z (ESI): 563.1 [M+H]+.
tert-Butyl (S)-4-(7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)-2-carbonyl-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate (400 mg, 0.71 mmol), TFA (2 mL) were added to CH2Cl2 (30 mL), the reaction mixture was uniformly mixed, and then the reaction was carried out at room temperature for 1 hour, then the mixture was concentrated under reduced pressure. CH2Cl2 (20 mL) and DIPEA (0.3 mL) were added to the resulting crude product, the reaction temperature was reduced to 0° C., acryloyl chloride (0.1 mL) was slowly added to the reaction solution, and the reaction was carried out at room temperature for 1 hour, the mixture was then concentrated under reduced pressure. The resulting crude product was purified by rapid silica gel column chromatography to obtain the compound (S)-4-(4-acryloyl-2-methylpiperazin-1-yl)-7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one as a yellow solid (200 mg, yield: 55%).
MS m/z (ESI): 517.1 [M+H]+.
(S)-4-(4-Acryloyl-2-methylpiperazin-1-yl)-7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (50 mg, 0.1 mmol), (2-fluoro-6-hydroxyphenyl) boric acid (30 mg. 0.2 mmol), Pd(dppf)Cl2 (16 mg. 0.02 mmol) and cesium carbonate (100 mg. 0.3 mmol) were added to dixoane (1.5 mL), the reaction mixture was uniformly mixed, and then the reaction was carried out at 100° C. under microwave irradiation for 1 hour, then the mixture was concentrated under reduced pressure. CH2Cl2 (20 mL) and DIPEA (0.3 mL) were added into the resulting crude product, and the reaction temperature was reduced to 0° C., acryloyl chloride (0.1 mL) was slowly added to the reaction solution, and the reaction was carried out at room temperature for 1 hour, then the mixture was concentrated under reduced pressure. The resulting crude product was purified by Pre-HPLC to obtain the compound 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one as a white solid (14 mg, yield: 24%).
MS m/z (ESI): 593.1 [M+H]+.
1H NMR (400 MHZ, MeOD-d4) δ 8.40 (d, J=5.6 Hz, 1H), 8.22-8.27 (m, 1H), 7.21-7.27 (m, 2H), 6.79-6.88 (m, 1H), 6.58-6.66 (m, 2H), 6.28-6.34 (m, 1H), 5.84 (d, J=12.0 Hz, 1H), 5.06 (s, 1H), 4.43-4.59 (m, 2H), 4.07-4.23 (s, 1H), 3.57-3.85 (m, 2H), 3.20-3.48 (m, 1H), 2.79-2.85 (m, 1H), 2.41 (s, 3H), 1.47 (d, J=4.8 Hz, 3H), 1.20 (d, J=6.4 Hz, 3H), 1.06 (d, J=6.8 Hz, 3H).
Embodiment 1 was resolved by SFC to obtain two axial chiral isomers, embodiment 1-1 and embodiment 1-2, SFC: chiral preparation conditions:
tR=1.92 min
MS m/z (ESI): 593.1 [M+H]+.
1H NMR (400 MHZ, MeOD-d4) δ 8.40 (d, J=5.6 Hz, 1H), 8.22-8.27 (m, 1H), 7.21-7.27 (m, 2H), 6.79-6.88 (m, 1H), 6.58-6.66 (m, 2H), 6.28-6.34 (m, 1H), 5.84 (d, J=12.0 Hz, 1H), 5.06 (s, 1H), 4.43-4.59 (m, 2H), 4.07-4.23 (s, 1H), 3.57-3.85 (m, 2H), 3.20-3.48 (m, 1H), 2.79-2.85 (m, 1H), 2.41 (s, 3H), 1.47 (d, J=4.8 Hz, 3H), 1.20 (d, J=6.4 Hz, 3H), 1.06 (d, J=6.8 Hz, 3H).
tR=2.43 min
MS m/z (ESI): 593.1 [M+H]+.
1HNMR (400 MHZ, MeOD-d4) δ 8.40 (d, J=5.6 Hz, 1H), 8.25 (t, J=10.8 Hz, 1H), 7.21-7.27 (m, 2H), 6.79-6.90 (m, 1H), 6.58-6.66 (m, 2H), 6.28-6.34 (m, 1H), 5.83 (dd, J=10.8 Hz, 2.0 Hz, 1H), 5.05-5.10 (m, 1H), 4.41-4.57 (m, 2H), 4.07-4.21 (m, 1H), 3.61-3.87 (m, 2H), 3.24-3.36 (m, 1H), 2.77-2.83 (m, 1H), 2.41 (s, 3H), 1.46-1.49 (m, 3H), 1.19 (d, J=6.8 Hz, 3H), 1.06 (d, J=6.8 Hz, 3H).
N,N-Diisopropylethylamine (407 mg, 3.16 mmol) was added to a solution of 7-chloro-6-fluoro-4-hydroxy-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (200 mg, 0.526 mmol) in acetonitrile (10 mL) at room temperature, then phosphorus oxychloride (242 mg, 1.58 mmol) was added thereto and the mixture was stirred at 80° C. for 1 hour. The mixture was cooled to room temperature and directly used in the next reaction.
Step 2: Preparation of tert-butyl (2R,5S)-4-(7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)-2-carbonyl-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-2,5-dimethylpiperazine-1-carboxylate
N,N-Diisopropylethylamine (678 mg, 5.26 mmol) and tert-butyl (2R,5S)-2,5-dimethylpiperazine-1-carboxylate (224 mg, 1.005 mmol) were added to the reaction mixture of the previous step and stirred for 1 hour at room temperature after the addition. Water (60 mL) was added thereto and the mixture was extracted with ethyl acetate (40 mL×3), the organic phase was washed with ammonium chloride aqueous solution (40 mL) and then washed with sodium chloride aqueous solution (30 mL), concentrated and then subjected to column chromatography [eluent: dichloromethane to methanol/dichloromethane from 0% to 2.2%] to obtain tert-butyl (2R,5S)-4-(7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)-2-carbonyl-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-2,5-dimethylpiperazine-1-carboxylate (200 mg, two-step yield: 66%) as a yellow solid.
MS m/z (ESI): 577.2 [M+H]+, 579.2 [M+H+2]+.
Trifluoroacetic acid (1.2 mL) was added to a solution of tert-butyl (2R,5S)-4-(7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)-2-carbonyl-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-2,5-dimethylpiperazine-1-carboxylate (200 mg, 0.347 mmol) in dichloromethane (6 mL), and the mixture was stirred at room temperature for 1.5 hours after the addition. Then the reaction mixture was concentrated at low temperature to obtain 7-chloro-4-((2S,5R)-2,5-dimethylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one trifluoroacetate (200 mg) as a red oil, which was used rapidly in the next reaction.
MS m/z (ESI): 477.2 [M+H]+, 479.2 [M+H+2]+.
N,N-Diisopropylethylamine (447 mg, 3.47 mmol) was added to a solution of 7-chloro-4-((2S,5R)-2,5-dimethylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one trifluoroacetate (200 mg, 0.347 mmol) in dichloromethane (15 mL), then acryloyl chloride (63 mg, 0.694 mmol) was added dropwise thereto at 0° C., and the mixture was stirred for 1 hour after the addition. The reaction mixture was quenched with ammonium chloride aqueous solution (30 mL), extracted with dichloromethane (30 mL×3), the dichloromethane phase was washed with saturated NaCl aqueous solution (20 mL), dried over anhydrous sodium sulfate, concentrated and then subjected to column chromatography [eluent: dichloromethane to methanol/dichloromethane from 0% to 2.5%] to obtain 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (130 mg, two-step yield: 71%) as a yellow solid.
MS m/z (ESI): 530.2 [M+H]+, 532.2 [M+H+2]+.
4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (130 mg, 0.246 mmol), (2-fluoro-6-hydroxyphenyl) boric acid (77 mg, 0.491 mmol), [1,1′-bis(diphenylphosphino)ferrocene]palladium(II)dichloride dichloromethane complex (40 mg, 0.0491 mmol) and cesium carbonate (240 mg, 0.738 mmol) were added to dioxane (4 mL) and water (1 mL), the mixture was replaced with nitrogen, and stirred at 100° C. under microwave irradiation for 1 hour. The reaction mixture was concentrated, then subjected to column chromatography [eluent: dichloromethane to methanol/dichloromethane from 0% to 2.5%] to obtain 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (90 mg, yield: 60%) as a yellow solid.
MS m/z (ESI): 606.2 [M+H]+.
1H NMR (400 MHZ, MeOD-d4) δ 8.40 (d, J=8 Hz, 1H), 8.29-8.18 (m, 1H), 7.30-7.18 (m, 2H), 6.93-6.73 (m, 1H), 6.70-6.56 (m, 2H), 6.36-6.20 (m, 1H), 5.89-5.75 (m, 1H), 5.15-4.98 (m, 1H), 4.63-4.22 (m, 2H), 4.11-3.82 (m, 2H), 3.68-3.40 (m, 1H), 2.88-2.65 (m, 1H), 2.40 (d, J=4 Hz, 3H), 1.53-1.43 (m, 3H), 1.36 (t, J=8 Hz, 1H), 1.28 (t, J=8 Hz, 2H), 1.23-1.16 (m, 3H), 1.10-1.01 (m, 3H).
Embodiment 2 was resolved by SFC to obtain two axial chiral isomers, embodiment 2-1 and embodiment 2-2, SFC: chiral preparation conditions:
tR=1.99 min
MS m/z (ESI): 606.2 [M+H]+.
1H NMR (400 MHZ, MeOD-d4) δ 8.40 (d, J=8 Hz, 1H), 8.29-8.18 (m, 1H), 7.30-7.18 (m, 2H), 6.93-6.73 (m, 1H), 6.70-6.56 (m, 2H), 6.36-6.20 (m, 1H), 5.89-5.75 (m, 1H), 5.15-4.98 (m, 1H), 4.63-4.22 (m, 2H), 4.11-3.82 (m, 2H), 3.68-3.40 (m, 1H), 2.88-2.65 (m, 1H), 2.40 (d, J=4 Hz, 3H), 1.53-1.43 (m, 3H), 1.36 (t, J=8 Hz, 1H), 1.28 (t, J=8 Hz, 2H), 1.23-1.16 (m, 3H), 1.10-1.01 (m, 3H).
tR=2.87 min
MS m/z (ESI): 606.2 [M+H]+.
1H NMR (400 MHZ, MeOD-d4) δ 8.40 (d, J=8 Hz, 1H), 8.27-8.18 (m, 1H), 7.30-7.19 (m, 2H), 6.94-6.74 (m, 1H), 6.70-6.56 (m, 2H), 6.36-6.20 (d, J=16 Hz, 1H), 5.90-5.75 (m, 1H), 5.14-4.98 (m, 1H), 4.63-4.22 (m, 2H), 4.12-3.82 (m, 2H), 3.68-3.41 (m, 1H), 2.87-2.65 (m, 1H), 2.40 (d, J=4 Hz, 3H), 1.53-1.42 (m, 3H), 1.36 (t, J=8 Hz, 1H), 1.28 (t, J=8 Hz, 2H), 1.23-1.16 (d, J=4 Hz, 3H), 1.10-1.01 (d, J=4 Hz, 3H).
4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one was prepared with reference to embodiment 1.
MS m/z (ESI): 609.1 [M+H]+.
4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-(methylthio)phenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one was prepared with reference to embodiment 1.
MS m/z (ESI): 622.8 [M+H]+.
4-Chloro-3-fluoroaniline (1.45 g. 0.01 mol) was dissolved in THF (150 mL), Na2CO3 (3.18 g, 0.03 mol) was added thereto, the mixture was cooled to 0° C. under nitrogen atmosphere, trifluoroacetic anhydride (4.2 mL, 0.03 mol) was added dropwise thereto, and the mixture was then stirred at room temperature for 10 hours after the addition. The reaction mixture was added to water (150 mL). The mixture was then extracted three times with ethyl acetate (100 mL). The organic phases were combined, dried over anhydrous sodium sulfate, concentrated to obtain a crude product, and the crude product was purified by column chromatography (PE/EA=5:1) to obtain a white solid target product N-(4-chloro-3-fluorophenyl)-2,2,2-trifluoroacetamide (2.3 g. yield: 95%).
1H NMR (400 MHZ, MeOD-d4) δ 7.70 (dd, J=11.1, 2.0 Hz, 1H), 7.49-7.40 (m, 2H);
19F NMR (376 MHZ, MeOD-d4) δ−77.17 (s);
MS m/z (ESI): 242.1 [M+H]+.
N-(4-Chloro-3-fluorophenyl)-2,2,2-trifluoroacetamide (2.3 g. 9.5 mmol) was dissolved in THF (40 mL), the mixture was cooled to −78° C. under nitrogen atmosphere, and n-BuLi (7.9 mL, 19.0 mmol, 2.4 M) was added dropwise thereto, then the mixture was stirred at −50° C. for 50 minutes after the addition. The reaction mixture was cooled to −78° C., triisopropyl borate (2.3 g. 9.5 mmol) (4.8 mL, 20.9 mmol) was added dropwise thereto, the mixture was stirred at the same temperature for 20 minutes after the addition, the dry ice bath was removed, and the mixture was stirred at room temperature for 2 hours. Then, the reaction mixture was cooled to 0° C., dilute hydrochloric acid (19 mL, 1M) was added dropwise thereto, the temperature was raised to 40° C., and the mixture was stirred for 1 hour. The mixture was then extracted three times with ethyl acetate (100 mL). The organic phases were combined, dried over anhydrous sodium sulfate, concentrated to obtain a crude product, and the crude product was purified by column chromatography (PE/EA=4:1) to obtain a gray solid target product (6-amino-3-chloro-2-fluorophenyl) boric acid (1.1 g. yield: 56%).
MS m/z (ESI): 190.0 [M+H]+.
(6-Amino-3-chloro-2-fluorophenyl) boric acid (100 mg, 0.53 mmol) was dissolved in MeOH (20 mL), Pd/C (20 mg) was added thereto, the mixture was replaced with hydrogen for three times, then stirred and reacted for 2 hours at 15 psi, and the complete reaction was detected by TLC (PE/EA 1:1). The mixture was filtered, and the filtrate was concentrated to obtain a yellow solid target product (2-amino-6-fluorophenyl) boric acid (80 mg, yield: 97%), which was used directly in the next reaction without purification.
MS m/z (ESI): 156.0 [M+H]+.
(S)-4-(4-Acryloyl-2-methylpiperazin-1-yl)-7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (26 mg, 0.05 mmol), (6-amino-3-chloro-2-fluorophenyl) boric acid (23.2 mg, 0.15 mmol) and cesium carbonate (48.87 mg, 0.15 mmol) were dissolved in dioxane/H2O (1.5 mL/0.3 mL). The mixture was replaced with nitrogen for 1 minute, and the reaction was carried out at 100° C. under microwave irradiation for 1 hour. When the reaction was completed, the reaction mixture was concentrated, purified by column chromatography (CH2Cl2/MeOH=20:1) to obtain a crude product, and then the crude product was purified by preparative HPLC to obtain a yellow solid target product 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-6-fluorophenyl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (7.0 mg, yield: 24%).
1H NMR (400 MHZ, MeOD-d4) δ 8.46 (d, J=5.4 Hz, 1H), 8.25 (dd, J=21.2, 12.0 Hz, 1H), 7.27 (d, J=5.5 Hz, 1H), 7.11 (dd, J=14.7, 8.2 Hz, 1H), 6.84 (d, J=14.2 Hz, 1H), 6.49 (d, J=8.3 Hz, 1H), 6.41-6.27 (m, 2H), 5.83 (dd, J=10.6, 1.6 Hz, 1H), 4.48 (dd, J=52.4, 11.6 Hz, 2H), 4.30-3.83 (m, 2H), 3.74 (d, J=9.7 Hz, 2H), 3.22 (s, 1H), 2.98-2.80 (m, 1H), 2.43 (d, J=0.7 Hz, 3H), 1.56-1.40 (m, 3H), 1.22 (d, J=6.6 Hz, 3H), 1.01 (d, J=6.6 Hz, 3H).
19F NMR (376 MHZ, MeOD−d4) δ −114.58-−114.95 (m), −114.95-−115.34 (m), −125.12-−126.48 (m).
MS m/z (ESI): 592.2 [M+H]+.
2-(4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-7-yl)-3-fluorobenzamide was prepared with reference to embodiment 1.
MS m/z (ESI): 619.7 [M+H]+.
4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(2-(dimethylamino)-6-fluorophenyl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one was prepared with reference to embodiment 1.
MS m/z (ESI): 619.7 [M+H]+.
4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluoro-6-(methylamino)phenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyridinyl[2,3-d]pyrimidin-2(1H)-one was prepared with reference to embodiment 1.
MS m/z (ESI): 605.7 [M+H]+.
(S)-4-(4-Acryloyl-2-methylpiperazin-1-yl)-7-chloro-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (26.7 mg, 0.05 mmol), (6-amino-3-chloro-2-fluorophenyl) boric acid (28.4 mg, 0.15 mmol) and potassium acetate (15.0 mg, 0.15 mmol) were dissolved in dioxane/H2O (1.5 mL/0.3 mL). The mixture was replaced with nitrogen for 1 minute, and the reaction was carried out at 100° C. under microwave irradiation for 1 hour. When the reaction was completed, the reaction mixture was concentrated, purified by column chromatography (CH2Cl2/MeOH=20:1) to obtain a crude product, and the crude product was then purified by preparative HPLC to obtain a yellow solid target product 4-((S)-4-acryloyl-2-methylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (3.7 mg, yield: 14%).
MS m/z (ESI): 642.1 [M+H]+.
1H NMR (400 MHZ, MeOD-d4) δ 8.56-8.39 (m, 2H), 7.24 (t, J=5.3 Hz, 1H), 7.15 (dd, J=15.4, 6.9 Hz, 1H), 6.84 (d, J=9.9 Hz, 1H), 6.53-6.46 (m, 1H), 6.32 (d, J=15.9 Hz, 1H), 5.84 (d, J=12.2 Hz, 1H), 4.68-4.36 (m, 3H), 4.10 (dd, J=45.7, 31.6 Hz, 2H), 3.76 (s, 1H), 2.94 (s, 2H), 2.42 (d, J=6.2 Hz, 3H), 1.57-1.43 (m, 3H), 1.22 (d, J=6.7 Hz, 3H), 1.06 (dd, J=42.4, 6.7 Hz, 3H). 19F NMR (376 MHz, MeOD) δ −117.04-−117.24 (m), −117.24-−117.51 (m).
Embodiment 9 was resolved by SFC to obtain two axial chiral isomers, embodiment 9-1 and embodiment 9-2, SFC: chiral preparation conditions:
tR=1.74 min
MS m/z (ESI): 642.1 [M+H]+.
1H NMR (400 MHZ, MeOD-d4) δ 8.56-8.39 (m, 2H), 7.24 (t, J=5.3 Hz, 1H), 7.15 (dd, J=15.4, 6.9 Hz, 1H), 6.84 (d, J=9.9 Hz, 1H), 6.53-6.46 (m, 1H), 6.32 (d, J=15.9 Hz, 1H), 5.84 (d, J=12.2 Hz, 1H), 4.68-4.36 (m, 3H), 4.10 (dd, J=45.7, 31.6 Hz, 2H), 3.76 (s, 1H), 2.94 (s, 2H), 2.42 (d, J=6.2 Hz, 3H), 1.57-1.43 (m, 3H), 1.22 (d, J=6.7 Hz, 3H), 1.06 (dd, J=42.4, 6.7 Hz, 3H).
19F NMR (376 MHZ, MeOD-d4) δ −117.04-−117.24 (m), −117.24-−117.51 (m).
tR=2.49 min
MS m/z (ESI): 642.1 [M+H]+.
1H NMR (400 MHZ, MeOD-d4) δ 8.56-8.39 (m, 2H), 7.27-7.10 (m, 2H), 6.84 (dd, J=28.3, 17.7 Hz, 1H), 6.50 (d, J=8.8 Hz, 1H), 6.32 (d, J=16.9 Hz, 1H), 5.83 (d, J=11.7 Hz, 1H), 4.63-4.41 (m, 2H), 4.23-4.02 (m, 1H), 3.79-3.57 (m, 2H), 3.36 (s, 2H), 2.99-2.86 (m, 1H), 2.41 (d, J=7.6 Hz, 3H), 1.51 (d, J=25.9 Hz, 3H), 1.21 (d, J=6.6 Hz, 3H), 1.05 (dd, J=44.8, 6.7 Hz, 3H).
(S)-4-(4-Acryloyl-2-methylpiperazin-1-yl)-7-chloro-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (26 mg, 0.05 mmol), (6-amino-3-chloro-2-fluorophenyl) boric acid (28.4 mg, 0.15 mmol) and cesium carbonate (48.8 mg, 0.15 mmol) were dissolved in dioxane/H2O (1.5 mL/0.3 mL). The mixture was replaced with nitrogen for 1 minute, and the reaction was carried out at 100° C. under microwave irradiation for 1 hour. When the reaction was completed, the reaction mixture was evaporated to dryness by rotary evaporation, purified by column chromatography (CH2Cl2/MeOH=20:1) to obtain a crude product, and then the crude product was purified by preparative HPLC to obtain a yellow solid target product 4-(S)-4-acryloyl-2-methylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (4.4 mg, yield: 14%).
1H NMR (400 MHZ, MeOD-d4) δ 8.47 (d, J=5.4 Hz, 1H), 8.38-8.24 (m, 1H), 7.27 (d, J=5.4 Hz, 1H), 7.17 (t, J=8.6 Hz, 1H), 6.85 (d, J=14.9 Hz, 1H), 6.49 (d, J=8.9 Hz, 1H), 6.32 (d, J=16.3 Hz, 1H), 5.84 (d, J=10.5 Hz, 1H), 4.57 (d, J=23.5 Hz, 2H), 4.42 (s, 1H), 4.24-3.89 (m, 2H), 3.73 (dd, J=14.4, 7.9 Hz, 1H), 2.92 (s, 1H), 2.43 (s, 3H), 1.54-1.40 (m, 3H), 1.22 (d, J=6.7 Hz, 3H), 1.01 (d, J=6.6 Hz, 3H).
19F NMR (376 MHZ, MeOD-d4) δ−116.46-−116.73 (m), −116.87 (dd, J=39.0, 8.4 Hz), −126.18 (dd, J=24.9, 15.2 Hz).
MS m/z (ESI): 626.1 [M+H]+.
4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(2,3-difluoro-6-hydroxyphenyl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one was prepared with reference to embodiment 2.
MS m/z (ESI): 611.1 [M+H]+.
1H NMR (400 MHZ, MeOD-d4) δ 8.41 (d, J=5.6 Hz, 1H), 8.32-8.25 (m, 1H), 7.25 (d, J=5.6 Hz, 1H), 7.20-7.13 (m, 1H), 6.92-6.82 (m, 1H), 6.62-6.58 (m, 1H), 6.34-6.28 (m, 1H), 5.83 (d, J=10.4 Hz, 1H), 5.14-5.04 (m, 1H), 4.64-4.42 (m, 2H), 4.25-4.07 (m, 1H), 3.89-3.61 (m, 3H), 2.88-2.77 (m, 1H), 2.42 (s, 3H), 1.52-1.46 (m, 3H), 1.20 (d, J=6.4 Hz, 3H), 1.05 (d, J=6.4 Hz, 3H).
(S)-4-(4-Acryloyl-2-methylpiperazin-1-yl)-7-(2,6-difluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one was prepared with reference to embodiment 2.
MS m/z (ESI): 611.1 [M+H]+.
Under N2 atmosphere, 2,5,6-trichloronicotinamide (6.2 g, 27.7 mmol) was dissolved in THF (60 mL), oxalyl chloride (15.2 mL, 31.5 mmol) (2 M/L dichloromethane solution) was added dropwise thereto at −78° C., and the mixture was stirred at −78° C. for 10 minutes, then the mixture was stirred at 60° C. for 3 hours, then the reaction mixture was cooled to 0° C., triethylamine (18 mL, 111 mmol) was added dropwise thereto. A solution of 2-isopropyl-4-(methylthio)pyridin-3-amine (5 g, 27.7 mmol) in THF was added dropwise thereto, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was quenched with brine, extracted with water and ethyl acetate (3*100 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, then the crude product was purified by column chromatography (DCM/MeOH=100:1 to 70:1) to obtain the target product 2,5,6-trichloro-N-((2-isopropyl-4-(methylthio)pyridin-3-yl)carbamoyl)nicotinamide (8.6 g, yield: 72%).
MS m/z (ESI): 433.1 [M+H]+, 435.1 [M+H+2]+.
2,5,6-Trichloro-N-((2-isopropyl-4-(methylthio)pyridin-3-yl)carbamoyl)nicotinamide (10.4 g, 24.1 mmol) was dissolved in anhydrous THF (80 mL), cooled to 0° C. under nitrogen atmosphere, KHMDS (48 mL, 48.2 mmol) was added dropwise thereto, and the mixture was stirred for 0.5 hours. The reaction mixture was then quenched with saturated ammonium chloride aqueous solution, then extracted with water and ethyl acetate (3*100 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated to obtain a crude product, and the crude product was slurried with ethyl acetate and purified to obtain the target product 6,7-dichloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2,4(1H,3H)-dione (8 g, yield: 84%).
MS m/z (ESI): 397.1 [M+H]+, 399.1 [M+H+2]+.
6,7-Dichloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2,4(1H,3H)-dione (5.2 g, 13.1 mmol) was dissolved in ACN (50 mL); DIEA (23 mL, 66 mmol) and POC3 (3 mL, 19.7 mmol) were added thereto, and the mixture was stirred at 80° C. for 0.5 hours. The resulting product was directly used in the next reaction.
MS m/z (ESI): 415.1 [M+H]+, 417.1 [M+H+2]+.
DIEA (23 mL, 66 mmol) and tert-butyl (2R,5S)-2,5-dimethylpiperazine-1-carboxylate (6.2 g, 26.2 mmol) were added to a solution of 4,6,7-trichloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one in acetonitrile (50 mL), and the mixture was stirred at room temperature for 1 hour. The reaction mixture was then quenched with water, then extracted with water and ethyl acetate (3*100 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated to obtain a crude product, and the crude product was purified by column chromatography (CH2Cl2/MeOH=30:1) to obtain the target (2R,5S)-4-(6,7-dichloro-1-(2-isopropyl-4-product tert-butyl (methylthio)pyridin-3-yl)-2-carbonyl-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-2,5-dimethylpiperazine-1-carboxylate (6.1 g, yield: 77%).
MS m/z (ESI): 593.1 [M+H]+, 595.1 [M+H+2]+.
tert-Butyl (2R,5S)-4-(6,7-dichloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)-2-carbonyl-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-2,5-dimethylpiperazine-1-carboxylate (6.1 g, 10.3 mmol) was dissolved in dichloromethane (20 mL), TFA (20 mL) was added thereto, and the mixture was stirred at room temperature for 1 hour. The mixture was concentrated to obtain the crude target product 6,7-dichloro-4-((2S,5R)-2,5-dimethylpiperazin-1-yl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (6.1 g, yield: 100%).
MS m/z (ESI): 493.1 [M+H]+, 495.1 [M+H+2]+.
6,7-Dichloro-4-((2S,5R)-2,5-dimethylpiperazin-1-yl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (6 g, 12.2 mmol) was dissolved in dichloromethane (30 mL); DIEA (30 mL, 131 mmol) and acryloyl chloride (1.08 mL, 13.13 mmol) were added thereto, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was then quenched with water, then extracted with water and ethyl acetate (3*100 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated to obtain a crude product, and the crude product was purified by column chromatography (CH2Cl2/MeOH=20:1) to obtain the target product 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-6,7-dichloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (1.6 g. yield: 22%).
MS m/z (ESI): 547.1 [M+H]+, 549.1 [M+H+2]+.
Under N2 atmosphere, 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-6,7-dichloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (700 mg. 1.3 mmol), (6-amino-3-chloro-2-fluorophenyl) boric acid (380 mg. 2.6 mmol) was dissolved in a mixture of 1,4-dioxane and water (6 mL:0.3 mL); and Pd(dppf)Cl2.DCM (100 mg. 0.1 mmol) and KOAc (400 mg. 4 mmol) were added thereto, and the reaction was carried out at 100° C. for 1 hour under microwave irradiation. The reaction mixture was then quenched with water, then extracted with water and ethyl acetate (3*50 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated to obtain a crude product, and the crude product was purified by column chromatography (CH2Cl2/MeOH=200:1 to 80:1) to obtain the target product 4-((2S,5R)-4-acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-3-chloro-2-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (400 mg, yield: 48%).
MS m/z (ESI): 656.1 [M+H]+, 658.1 [M+H+2]+.
1H NMR (400 MHZ, Methanol-d4) δ 8.47-8.34 (m, 2H), 7.24-7.20 (m, 1H), 7.10-7.14 (m, 1H), 6.79-6.68 (m, 1H), 6.42-6.40 (d, J=8.0 Hz, 1H), 6.24-6.17 (m, 1H), 5.75-5.71 (m, 1H), 5.01-4.94 (m, 2H), 4.46-4.40 (m, 1H), 4.26-4.17 (m, 1H), 4.03-3.99 (m, 1H), 3.84-3.79 (m, 1H), 2.86-2.77 (m, 1H), 2.36 (s, 3H), 1.26-1.19 (m, 9H), 1.14-1.11 (m, 3H).
Embodiment 13 was resolved by SFC to obtain two axial chiral isomers, embodiment 13-1 and embodiment 13-2, SFC: chiral preparation conditions:
tR=1.74 min
MS m/z (ESI): 656.1 [M+H]+, 658.1 [M+H+2]+.
1H NMR (400 MHZ, MeOD-d4) δ 8.47-8.34 (m, 2H), 7.24-7.20 (m, 1H), 7.10-7.14 (m, 1H), 6.79-6.68 (m, 1H), 6.42-6.40 (d, J=8.0 Hz, 1H), 6.24-6.17 (m, 1H), 5.75-5.71 (m, 1H), 5.01-4.94 (m, 2H), 4.46-4.40 (m, 1H), 4.26-4.17 (m, 1H), 4.03-3.99 (m, 1H), 3.84-3.79 (m, 1H), 2.86-2.77 (m, 1H), 2.36 (s, 3H), 1.26-1.19 (m, 9H), 1.14-1.11 (m, 3H).
tR=2.49 min
MS m/z (ESI): 656.1 [M+H]+, 658.1 [M+H+2]+.
1H NMR (400 MHZ, DMSO-d6) δ 8.55-8.38 (m, 2H), 7.25-7.20 (m, 1H), 7.18-7.11 (m, 1H), 6.88-6.76 (m, 1H), 6.51-6.47 (d, J=8.0 Hz, 1H), 6.33-6.27 (m, 1H), 5.84-5.80 (m. 1H), 5.12-5.10 (m, 2H), 4.46-4.23 (m, 2H), 4.15-3.89 (m, 2H), 3.64-3.50 (m, 1H), 2.89-2.82 (m, 1H), 2.43 (s, 3H), 1.51-0.99 (m. 12H).
4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one was prepared with reference to embodiment 13.
MS m/z (ESI): 623.1 [M+H]+, 625.1 [M+H+2]+.
1H NMR (400 MHZ, Methanol-d4) δ 8.47-8.34 (m, 2H), 7.21-7.20 (m, 2H), 6.89-6.77 (m, 1H), 6.64-6.55 (m, 2H), 6.32-6.26 (m, 1H), 5.84-5.80 (m, 1H), 5.08-5.03 (m, 2H), 4.56-4.49 (m, 1H), 4.34-4.26 (m, 1H), 4.13-4.04 (m, 1H), 3.92-3.88 (m, 1H), 2.79-2.72 (m, 1H), 2.40 (s, 3H), 1.55-1.43 (m, 3H), 1.35-1.27 (m, 3H), 1.20-1.17 (m, 3H), 1.08-1.05 (t, J=8.0 Hz, 3H).
Embodiment 14 was resolved by SFC to obtain two axial chiral isomers, embodiment 14-1 and embodiment 14-2, SFC: chiral preparation conditions:
tR=2.46 min
MS m/z (ESI): 623.1 [M+H]+, 625.1 [M+H+2]+.
1H NMR (400 MHZ, Methanol-d4) δ 8.47-8.34 (m, 2H), 7.21-7.20 (m, 2H), 6.89-6.77 (m, 1H), 6.64-6.55 (m, 2H), 6.32-6.26 (m, 1H), 5.84-5.80 (m, 1H), 5.08-5.03 (m, 2H), 4.56-4.49 (m, 1H), 4.34-4.26 (m, 1H), 4.13-4.04 (m, 1H), 3.92-3.88 (m, 1H), 2.79-2.72 (m, 1H), 2.40 (s, 3H), 1.55-1.43 (m, 3H), 1.35-1.27 (m, 3H), 1.20-1.17 (m, 3H), 1.08-1.05 (t, J=8.0 Hz, 3H).
tR=3.08 min
MS m/z (ESI): 623.1 [M+H]+, 625.1 [M+H+2]+.
1H NMR (400 MHZ, Methanol-d4) δ 8.48-8.34 (m, 2H), 7.23-7.21 (m, 2H), 6.90-6.78 (m, 1H), 6.66-6.58 (m, 2H), 6.33-6.28 (m, 1H), 5.85-5.82 (m, 1H), 5.10-5.06 (m, 2H), 4.58-4.50 (m, 1H), 4.34-4.27 (m, 1H), 4.13-4.06 (m, 1H), 3.93-3.88 (m, 1H), 2.79-2.71 (m, 1H), 2.41 (s, 3H), 1.56-1.46 (m, 3H), 1.37-1.29 (m, 3H), 1.21-1.18 (m, 3H), 1.07-1.05 (t, J=8.0 Hz, 3H).
(S)-4-(4-Acryloyl-2-methylpiperazin-1-yl)-7-(2,6-difluorophenyl)-6-fluoro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one was prepared with reference to embodiment 2.
MS m/z (ESI): 595.1 [M+H]+.
1H NMR (400 MHZ, Methanol-d4) δ 8.40-8.32 (m, 2H), 7.51 (t, J=7.6 Hz, 1H), 7.22 (d, J=5.4 Hz, 1H), 7.05 (t, J=8.4 Hz, 2H), 6.86-6.79 (m, 1H), 6.37-6.26 (m, 1H), 5.84 (d, J=10.6 Hz, 1H), 5.08 (m, 2H), 4.56-4.46 (m, 2H), 4.21-4.08 (m, 1H), 3.85-3.62 (m, 2H), 2.86-2.82 (m, 1H), 2.40 (s, 3H), 1.47 (d, J=6.6 Hz, 3H), 1.21-1.19 (d, J=6.8 Hz, 3H), 1.04 (d, J=6.8 Hz, 3H).
(S)-4-(4-Acryloyl-2-methylpiperazin-1-yl)-6-fluoro-7-(2-fluorophenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one was prepared with reference to embodiment 2.
MS m/z (ESI): 576.7 [M+H]+.
4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-7-(2-amino-3,5-dichloro-6-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one was prepared with reference to embodiment 13.
MS m/z (ESI): 690.1 [M+H]+, 692.1 [M+H+2]+.
1H NMR (400 MHZ, Methanol-d4) δ 8.46-8.34 (m, 2H), 7.25-7.21 (m, 1H), 7.11-7.14 (m, 1H), 6.44-6.42 (d, J=8.0 Hz, 1H), 6.23-6.16 (m, 1H), 5.73-5.70 (m, 1H), 5.03-4.97 (m, 2H), 4.47-4.42 (m, 1H), 4.25-4.16 (m, 1H), 4.06-4.02 (m, 1H), 3.86-3.83 (m, 1H), 2.84-2.79 (m, 1H), 2.34 (s, 3H), 1.27-1.19 (m, 9H), 1.16-1.14 (m, 3H).
4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-5-chloro-3,6-difluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one was prepared with reference to embodiment 13.
MS m/z (ESI): 660.1 [M+H]+, 662.1 [M+H+2]+.
1H NMR (400 MHZ, Methanol-d4) δ 8.58-8.38 (m, 2H), 7.53-7.36 (m, 1H), 7.23-7.15 (m, 1H), 6.97-6.79 (m, 1H), 6.22 (d, J=16 Hz, 1H), 5.77 (d, J=8 Hz, 1H), 5.45-5.40 (m, 2H), 5.07-4.82 (m, 1H), 4.50-3.98 (m, 3H), 3.92-3.49 (m, 2H), 3.17-3.02 (m, 1H), 2.93-2.63 (m, 1H), 2.44-2.26 (m, 3H), 1.43-1.27 (m, 3H), 1.08 (d, J=4 Hz, 3H), 1.04-0.86 (m, 3H).
4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-5,6-difluoro-3-methylphenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one was prepared with reference to embodiment 13.
MS m/z (ESI): 640.1 [M+H]+, 642.1 [M+H+2]+.
1H NMR (400 MHZ, Methanol-d4) δ 8.57-8.35 (m, 3H), 7.25-7.04 (m, 2H), 6.96-6.79 (m, 1H), 6.29-6.14 (m, 1H), 5.77 (d, J=12 Hz, 1H), 5.09-4.82 (m, 1H), 4.76-4.58 (m, 2H), 4.48-3.98 (m, 3H), 3.94-3.59 (m, 2H), 2.93-2.69 (m, 1H), 2.44-2.29 (m, 3H), 2.10-1.95 (m, 3H), 1.42-1.26 (m, 3H), 1.08 (d, J=4 Hz, 3H), 1.05-0.87 (m, 3H).
4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-7-(2-amino-5-chloro-3,6-difluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one was prepared with reference to embodiment 13.
MS m/z (ESI): 674.1 [M+H]+, 676.1 [M+H+2]+.
1H NMR (400 MHZ, Methanol-d4) δ 8.61-8.39 (m, 2H), 7.56-7.35 (m, 1H), 7.27-7.14 (m, 1H), 6.96-6.75 (m, 1H), 6.20 (d, J=16 Hz, 1H), 5.82-5.71 (m, 1H), 5.53-5.38 (m, 2H), 4.95-4.69 (m, 1H), 4.57-4.30 (m, 1H), 4.24-4.00 (m, 2H), 3.98-3.79 (m, 2H), 2.95-2.60 (m, 1H), 2.44-2.25 (m, 3H), 1.40-1.13 (m, 6H), 1.10-0.87 (m, 6H).
4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-7-(2-amino-3,6-difluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one was prepared with reference to embodiment 13.
MS m/z (ESI): 674.1 [M+H]+.
4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-3,6-difluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one was prepared with reference to embodiment 13.
MS m/z (ESI): 626.1 [M+H]+, 628.1 [M+H+2]+.
1H NMR (400 MHZ, Methanol-d4) δ 8.58-8.34 (m, 2H), 7.26-6.99 (m, 2H), 6.95-6.77 (m, 1H), 6.47-6.27 (m, 1H), 6.26-6.13 (m, 1H), 5.77 (d, J=16 Hz, 1H), 5.22 (s, 2H), 5.09-4.80 (m, 1H), 4.50-3.99 (m, 3H), 3.95-3.53 (m, 2H), 3.20-2.98 (m, 1H), 2.94-2.65 (m, 1H), 2.42-2.24 (m, 3H), 1.43-1.25 (m, 3H), 1.09 (d, J=4 Hz, 3H), 1.04-0.82 (m, 3H).
4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-3,5,6-trifluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one was prepared with reference to embodiment 13.
MS m/z (ESI): 644.1 [M+H]+.
4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-7-(2-amino-3,5,6-trifluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one was prepared with reference to embodiment 13.
MS m/z (ESI): 658.1 [M+H]+.
4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-7-(2-amino-5,6-difluoro-3-methylphenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one was prepared with reference to embodiment 13.
MS m/z (ESI): 654.1 [M+H]+, 656.1 [M+H+2]+.
1H NMR (400 MHZ, Methanol-d4) δ 8.58-8.31 (m, 2H), 7.25-7.03 (m, 2H), 6.94-6.73 (m, 1H), 6.19 (d, J=16 Hz, 1H), 5.81-5.69 (m, 1H), 4.96-4.59 (m, 3H), 4.55-4.38 (m, 1H), 4.29-3.96 (m, 2H), 3.93-3.72 (m, 2H), 3.00-2.60 (m, 1H), 2.45-2.25 (m, 3H), 2.07-1.94 (m, 3H), 1.43-1.13 (m, 6H), 1.12-0.82 (m, 6H).
4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-2,3,4-trifluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one was prepared with reference to embodiment 13.
MS m/z (ESI): 658.1 [M+H]+,
4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(6-amino-2,3,4-trifluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one was prepared with reference to embodiment 13.
MS m/z (ESI): 644.1 [M+H]+,
4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-7-(6-amino-2,3-difluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one was prepared with reference to embodiment 13.
MS m/z (ESI): 640.2 [M+H]+,
4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(6-amino-2,3-difluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one was prepared with reference to embodiment 13.
MS m/z (ESI): 626.1 [M+H]+,
4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-methylphenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one was prepared with reference to embodiment 13.
MS m/z (ESI): 607.1 [M+H]+, 609.1 [M+H+2]+.
1H NMR (400 MHZ, Methanol-d4) δ 8.57-8.34 (m, 2H), 7.43-7.31 (m, 1H), 7.18 (d, J=4 Hz, 1H), 7.15-7.01 (m, 2H), 6.95-6.78 (m, 1H), 6.28-6.14 (m, 1H), 5.77 (d, J=12 Hz, 1H), 5.07-4.86 (m, 1H), 4.45-4.25 (m, 2H), 4.22-3.98 (m, 1H), 3.93-3.58 (m, 2H), 3.21-3.02 (m, 1H), 2.87-2.69 (m, 1H), 2.40-2.27 (m, 3H), 1.98-1.85 (m, 3H), 1.41-1.28 (m, 3H), 1.08 (d, J=8 Hz, 3H), 1.02-0.79 (m, 3H).
4-((2S,5R)-4-Acryloyl-2-methylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-methylphenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one was prepared with reference to embodiment 13.
MS m/z (ESI): 621.2 [M+H]+,
4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-6-chloro-7-(2-chloro-6-fluorophenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one was prepared with reference to embodiment 13.
MS m/z (ESI): 627.1 [M+H]+, 629.1 [M+H+2]+.
1H NMR (400 MHZ, Methanol-d4) δ 8.56-8.30 (m, 2H), 7.58-7.36 (m, 3H), 7.19 (s, 1H), 6.87 (s, 1H), 6.24-6.19 (d, J=20.0 Hz, 1H), 5.79-5.76 (d, J=12.0 Hz, 1H), 4.97 (s, 1H), 4.32-4.04 (m, 3H), 3.80-3.49 (m, 3H), 2.72 (s, 1H), 2.35 (s, 3H), 1.34-0.91 (m, 9H).
4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-6-chloro-7-(2-chloro-6-fluorophenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one was prepared with reference to embodiment 13.
MS m/z (ESI): 641.6 [M+H]+,
(S)-4-(4-Acryloyl-2-methylpiperazin-1-yl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)-7-(o-benzyl)pyrido[2,3-d]pyrimidin-2(1H)-one was prepared with reference to embodiment 13.
MS m/z (ESI): 589.1 [M+H]+, 591.1 [M+H+2]+.
(S)-4-(4-Acryloyl-2-methylpiperazin-1-yl)-6-chloro-7-(2-chlorophenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one was prepared with reference to embodiment 13.
MS m/z (ESI): 609.6 [M+H]+,
(S)-4-(4-Acryloyl-2-methylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-(trifluoromethyl)phenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one was prepared with reference to embodiment 13.
MS m/z (ESI): 661.1 [M+H]+,
4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)-7-(o-benzyl)pyrido[2,3-d]pyrimidin-2(1H)-one was prepared with reference to embodiment 13.
MS m/z (ESI): 603.1 [M+H]+, 605.1 [M+H+2]+.
4-((2S,5R)-4-Acryloyl-2,5-dimethylpiperazin-1-yl)-6-chloro-7-(2-chlorophenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one was prepared with reference to embodiment 13.
MS m/z (ESI): 623.6 [M+H]+,
4-((2S,5R)-(4-Acryloyl-2,5-dimethylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-(trifluoromethyl)phenyl)-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one was prepared with reference to embodiment 13.
MS m/z (ESI): 675.1 [M+H]+,
4-((S)-4-Acryloyl-2-methylpiperazin-1-yl)-7-(2-amino-3,5-dichloro-6-fluorophenyl)-6-chloro-1-(2-isopropyl-4-(methylthio)pyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one was prepared with reference to embodiment 13.
MS m/z (ESI): 676.1 [M+H]+, 678.1 [M+H+2]+.
1H NMR (400 MHz, Methanol-d4) δ 8.40-8.32 (m, 2H), 7.51 (t, J=7.6 Hz, 1H), 7.22 (d, J=5.4 Hz, 1H), 7.05 (t, J=8.4 Hz, 2H), 6.86-6.79 (m, 1H), 6.37-6.26 (m, 1H), 5.84 (d, J=10.6 Hz, 1H), 5.08 (m, 2H), 4.56-4.46 (m, 2H), 4.21-4.08 (m, 1H), 3.85-3.62 (m, 2H), 2.86-2.82 (m, 1H), 2.40 (s, 3H), 1.47 (d, J=6.6 Hz, 3H), 1.21-1.19 (d, J=6.8 Hz, 3H), 1.04 (d, J=6.8 Hz, 3H).
The present disclosure is further described below in conjunction with test embodiments to explain the present disclosure, but these embodiments are not meant to limit the scope of the present disclosure.
To determine the inhibitory effect of the compounds of the embodiments on the proliferation activity of KRAS G12C mutant cell lines NCI-H358 and Mia PaCa-2 cells.
The cell culture plate was purchased from Corning Company, the article number was 3610.
When NCI-H358 or Mia PaCa-2 cells were cultured to the appropriate fusion level, the NCI-H358 or Mia PaCa-2 cells were collected, and the cells were adjusted to the appropriate cell concentration using a complete medium, and the cell suspension was spread in a 96-well plate, 90 μL per well, and placed in a 37° C., 5% CO2 incubator overnight; and compound solutions of different concentrations were prepared using DMSO and culture medium; and a solvent control was set, the compound solution was added to a 96-well plate, 10 μL per well, at 37° C. in a 5% CO2 incubator for 72 hours; CellTiter-Glo solution was added thereto and the mixture was mixed well by shaking, incubated for 10 min in the dark, and read by BioTek Synergy H1 microplate reader.
The luminescence signal values were used to calculate the inhibition rate, the concentration and the inhibition rate were fitted to a nonlinear regression curve using Graphpad Prism software, then the IC50 value was obtained.
The experimental results are shown in Table 8, IC50 values of the inhibitory activity of the compounds of the embodiments on the proliferation of NCI-H358 and Mia PaCa-2 cells.
According to the data, the compounds of the embodiments of the present disclosure have a good inhibitory effect on the proliferation of NCI-H358 and Mia PaCa-2 cells.
To determine the ability of the compound to improve the stability of KRAS G12C protein (the degree of increase in protein melting temperature can be used to characterize the compound's ability to bind to KRAS G12C protein).
In this experiment, the thermal shift method was used to test the degree of change in the melting temperature (Tm) of the KRAS G12C protein before and after the binding of the compound, in order to characterize the ability of the compound to improve the stability of the KRAS G12C protein.
The specific experiment operation was as follows:
The experimental data file of PCR instrument was imported into thermal shift software, and the melting temperature (Tm) of each treatment group was obtained, and the change value of melting temperature (ATm) was obtained by subtracting the Tm of DMSO solvent control group.
According to the above scheme, the compounds of the present disclosure show the ability to increase the melting temperature of the protein as shown in Table 9 in the experiment of improving the binding stability of KRAS G12C protein.
The above data show that the compounds of the embodiments of the present disclosure have good binding ability to KRAS G12C protein.
To determine the inhibitory activity of the compounds of the embodiments on the level of phosphorylated ERK in KRAS G12C mutant cells Mia PaCa-2.
When Mia PaCa-2 cells were cultured to the appropriate fusion level, Mia PaCa-2 cells were collected, and the cell density was adjusted to 1×106/mL using complete culture medium, the cell suspension was spread on a 96-well plate, 50 μL per well, and placed adherent to the wall in a 37° C., 5% CO2 incubator overnight, compound solutions with different concentrations were prepared using DMSO and complete culture medium, a solvent control was set, the compound solution was added to a 96-well plate, 25 μL per well, and placed in a 37° C., 5% CO2 incubator for 2 hours of continuous culture, the supernatant was discarded from the cell culture plate, 50 μL of lysis solution was added to each well, and lysing was performed for 30 minutes by shaking at room temperature, then the mixture was centrifuged at 1000 rpm for 1 minute, 15 μL of supernatant was transferred to 384 well plate, 5 μL of detection mixture (Eu-labeled anti-ERK1/2 (T202-Y204) antibody with final concentration of 0.5 nM and ULight labeled anti-ERK1/2 antibody with final concentration of 5 nM) was added to each well, centrifuged at 1000 rpm for 1 minute and mixed uniformly, the reaction was carried out overnight at room temperature, the plate was read with BioTek Synergy H1, and the signal values was detected at 620 nm and 665 nm emission wavelengths by time-resolved fluorescence program.
The ratio of the signal values at 665 nm and 620 nm emission wavelength were calculated, and the ratio was used to calculate the inhibition rate, the concentration and the inhibition rate were fitted to a nonlinear regression curve using Graphpad Prism software, then the IC50 value was obtained.
The above data show that the compounds of the embodiments of the present disclosure have a good inhibitory effect on pERK in Mia PaCa-2 cells.
To study the pharmacokinetic behavior of the compounds in mice (plasma) after oral administration using Balb/c mice as test animals.
The compound of the embodiment of the present disclosure was self-made;
Balb/c Mice, male, purchased from Shanghai Jiesijie Laboratory Animal Co., Ltd, Animal Production License No. (SCXK (Shanghai) 2013-0006 NO. 311620400001794).
5 g of Hydroxyethyl cellulose (HEC, CMC-Na, viscosity: 800-1200 Cps) was weighed, dissolved in 1000 mL of purified water, and 10 g of Tween 80 was added. The mixture was mixed well to form a clear solution.
The compounds of the embodiments were weighed and added into 4-mL glass bottles, respectively, 2.4 mL of the solution was added, and ultrasound was performed for 10 minutes to obtain a colorless clear solution with a concentration of 1 mg/mL.
Balb/C mice, males; PO, after overnight fasting, respectively, at a dose of 10 mg/kg, administered in a volume of 10 mL/kg.
Blood samples were collected before administration and 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h and 8 h after administration, the blood was placed in EDTA-2K tube, centrifuged at 4° C. 6000 rpm for 6 min to separate plasma, and stored at −80° C.; food was consumed 4 hours after drug administration.
The final determination results obtained by applying LCMS/MS method are shown in Table 11.
The above data show that the compounds of the embodiments of the present disclosure have good pharmacokinetic parameters in mice.
BALB/c nude mice were used as the test animals, and the human pancreatic cancer cell MiaPaca 2 xenograft (CDX) model was used for in vivo pharmacodynamic experiments to evaluate the antitumor effects of the test compounds.
MiaPaca 2 cells were removed from the cell bank, revived and added to DMEM medium (containing 10% FBS, 1% Glu, 1% P/S) and incubated in a CO2 incubator (incubator temperature was 37° C., CO2 concentration was 5%). After the cells were spread to 80-90% of the bottom of the culture flask, the cells were continued to be cultured in the CO2 incubator. The process was repeated until the number of cells met the in vivo pharmacological inoculation requirement, and the cells in logarithmic growth period were collected and counted with an automatic cell counter, resuspended with PBS and Matrigel (volume ratio 1:1) according to the count results, made into a cell suspension (the density was 8×107/mL), and placed in an ice box for use.
BALB/c nude mice, female, 6-8 weeks old, weighing about 18-22 g. The mice were kept in an environment free of special pathogens and in a single ventilated cage with 5 mice in each cage. All cages, bedding and water were sterilized before use, and all animals had free access to standard certified commercial laboratory diets. Nude mice were labeled with disposable universal ear tags for mice and rats before the start of the experiment, and the skin of the inoculation site was disinfected with 75% medical alcohol before inoculation, 0.1 mL (containing 8*106 cells) of MiaPaca 2 tumor cells were inoculated subcutaneously on the right back of each mouse. When the tumor volume reached 100-200 mm3, the group administration was started. The tested compounds were administered daily by oral intragastric administration, dosage/frequency (6 mg/kg QD×3w), and the efficacy of each group at the end of the experiment was shown in Table 5.
The tumor volume (mm3) was measured with vernier caliper twice a week, the calculation formula was V=0.5*D*D*D, wherein D and d were the long and short diameter of the tumor, respectively. The anti-tumor efficacy was determined by dividing the average tumor increased volume of the compound-treated animals by the average tumor increased volume of the untreated animals. The formula of tumor inhibition rate is: TGI (%)=1−[(Vt−V0) administration group/(Vt−V0) solvent control group]*100%. After the experiment, all animals were euthanized.
The above data show that after oral administration for 21 days, the compounds of the embodiments of the present disclosure can significantly inhibit the growth of transplanted tumor in MiaPaca 2 nude mice under the condition of oral administration of 6 mg/kg per day.
To evaluate the efficacy of the compound in vivo on xenograft tumor model of human lung cancer NCI-H358 cells.
BALB/c nude mice, 6-8 weeks old, female, purchased from Shanghai Xipuer-Bikai Experimental Animal Co., Ltd.
Tumor volume calculation: tumor volume (mm3)=length (mm)×width (mm)×width (mm)/2
Calculation of TGI (%) of compound tumor inhibition rate: when there was no tumor regression, TGI (%)=[(1−(mean tumor volume at the end of the administration in a treatment group-mean tumor volume at the start of administration in the treatment group))/(mean tumor volume at the end of treatment in the solvent control group-mean tumor volume at the start of treatment in the solvent control group)]×100%. When there was tumor regression, TGI (%)=[1−(mean tumor volume at the end of dosing in a treatment group-mean tumor volume at the beginning of dosing in the treatment group)/mean tumor volume at the beginning of dosing in the treatment group]×100%.
The above data show that after 15 days of continuous oral administration, the compounds of the embodiments of the present disclosure significantly inhibited the growth of the tumors of nude mouse transplanted with human lung cancer NCI-H358 cells under the condition of oral administration of 10 mg/kg per day, which was significantly better than the reference data.
7.1.1 CHO-hERG cells were cultured in a 175 cm2 flask, when the cell density reached 60-80%, the culture medium was removed, the cells were washed with 7 mL PBS, and then digested with 3 mL Detachin.
7.1.2 After complete digestion, 7 mL culture medium was added to neutralize, then the mixture was centrifuged, the supernatant was aspirated, and then 5 mL culture medium was added to re-suspend, ensuring 2-5×106/mL of cell density.
The process of single cell high impedance sealing and whole cell mode formation were all automatically completed by Qpatch instrument, after obtaining the whole cell recording mode, the cells were clamped at −80 mV, before giving a 5-second +40 mV depolarization stimulus, a 50 millisecond −50 mV prevoltage was given first, and then repolarized to −50 mV for 5 seconds, then returned to −80 mV. This voltage stimulation was applied every 15 seconds and recorded for 2 minutes before giving extracellular fluid recordings for 5 minutes, and then the administration process was started, the compound concentration was given from the lowest test concentration, each test concentration was given for 2.5 minutes, and the positive control compound 3 μM of Cisapride was given after all concentrations were continuously given. At least 3 cells (n≥3) were tested at each concentration.
7.4.1 20 mM of compound mother liquor was diluted with extracellular fluid, 5 μL of 20 mM compound mother liquor was added into 2495 μL of extracellular fluid and diluted 500-fold to 40 μM, and then the final concentration to be tested was obtained by sequential 3-fold serial dilutions in extracellular solution containing 0.2% DMSO.
7.4.2 The highest test concentration was 40 μM, in a total of 6 concentrations of 40, 13.33, 4.44, 1.48, 0.49 and 0.16 μM respectively.
7.4.3 The content of DMSO in the final test concentration was not more than 0.2%, and this concentration of DMSO had no effect on hERG potassium channel.
The experimental data were analyzed by XLFit software.
The inhibitory effect of multiple concentrations of Cisapride on hERG channel was set as positive control.
The inhibition of drugs on the cardiac hERG potassium channel was the main cause of QT prolonged syndrome caused by drugs. It can be seen from the experimental results that the embodiment compound of the present disclosure had no obvious inhibitory effect on the cardiac hERG potassium ion channel, and can avoid the toxic and side effects to the heart at a high dose.
The purpose of this experiment was to examine the stability of the compounds of the embodiments in mouse, rat, dog and human plasma.
Animal or human whole blood was collected, then the blood was put into a test tube containing anticoagulant, centrifuged at 3500 rpm for 10 min, and the upper layer of pale yellow plasma was collected.
The compound was weighed, the stock solution was prepared with DMSO and the working solution was prepared with 100 mM phosphate buffer.
2.36 mg of Propantheline was weighed and diluted to 10 mM stock solution with 1 mL of DMSO; 10 μL of 10 mM stock solution was pipetted into 1 mL of 100 mM phosphate buffer to a final concentration of 100 μM.
4.05 mg of lovastatin was weighed and diluted to 10 mM stock solution with 1 mL of DMSO; 10 μL of 10 mM stock solution was pipetted into 1 mL of 100 mM phosphate buffer to a final concentration of 100 μM.
The above data show that the plasma stability of the compounds of the embodiments in the present disclosure is high with little species difference.
Using human liver microsomal incubation system, the inhibition of CYP450 enzyme subtypes by compounds was rapidly predicted by single point method.
2.5 mM NADPH: 100 mM phosphate buffer was added to 4.165 mg of NADPH (reduced nicotinamide adenine dinucleotide phosphate) to 2 mL. 0.25 mg/mL microsome: 4 mL of 100 mM phosphate buffer was added to 50 μL of 20 mg/mL microsome and mixed well.
The embodiment compound to be tested was weighed and diluted to 10 mM with DMSO and then diluted to 100 UM with 100 mM phosphate buffer.
The above data show that the embodiment compound of the present disclosure has no strong inhibition on each CYP enzyme subtype, and the risk of DDI is small.
The purpose of this experimental method was to detect the plasma protein binding of the compounds of the embodiments in plasma.
Liquid-phase mass spectrometer, centrifuge, vortexer, pipette, continuous liquid dispenser, 96-well plate, tissue homogenizer (for tissue sample analysis), 50% methanol aqueous solution, acetonitrile solution with internal standard, blank matrix (plasma, urine or tissue homogenate, etc.)
The embodiment compound was prepared into a 1 mM solution A with DMSO.
Solution A was added to the plasma solution and prepared into a 5 μM solution B.
The above data show that the compounds of the embodiments of the present disclosure exhibit high plasma protein binding rate with little species difference.
The pharmacokinetic behavior of the compound of the embodiment 13-1 and AMG-510 compound, administered orally at a dose of 6 mg/kg, in mice (plasma, tumor tissue and intestine) was studied using MiaPaca 2 tumor-bearing mice as test animals.
Embodiment 13-1 of the present disclosure, AMG-510 compound, self-made.
24 MiaPaca 2 tumor-bearing mice, females. 3 for each time point (0 h, 1 h, 2 h, 4 h, 6 h, 8 h, 16 h, 24 h). Shanghai xipuer-bikai Laboratory Animal Co., Ltd, Animal Production License No. (SCXK (Shanghai) 2018-0006.
5 g of Hydroxymethyl cellulose was weighed, dissolved in 1000 mL of purified water, and 10 g of Tween 80 was added. The mixture was mixed well to form a clear solution.
Embodiment compound 13-1 and compound AMG-510 were weighed and dissolved in the solution, the mixture was shaken well, and ultrasound was performed for 15 minutes to obtain a uniform suspension with a concentration of 0.6 mg/mL.
MiaPaca 2 tumor-bearing mice were administered at a dose of 6 mg/kg in a volume of 10 mL/kg, respectively, based on body weight p.o. after fasting (animals were not administered at point 0 h).
Before and after administration, mice were sacrificed with CO2, 0.5 mL blood was collected from the heart and placed in EDTA-2K tube, centrifuged at 4° C. 6000 rpm for 6 min to separate plasma, and stored at −80° C.; after the tumor tissues were weighing, placed in a 2 mL centrifuge tube and stored at −80° C. The duodenum, ileum and colon tissues were cut with scissors, the contents were removed and cleaned twice with PBS, after absorbing water with absorbent paper, they were weighed, placed in a 2 mL centrifuge tube and stored at −80° C.
At a dose of 6 mg/kg, the ratio of exposure of the compound of the embodiment of the present disclosure in the tumor of the mouse to the exposure in the blood was higher than that of AMG-510, with longer T1/2 and MRT.
It is well known to those skilled in the art that when the above compounds of the embodiments are shown to have a good inhibitory effect on the proliferation of NCI-H358 and Mia PaCa-2 cells, the pharmaceutically acceptable salts may often have the same pharmacological and pharmacodynamic activities. On this basis, the inventors further study the physical and chemical properties of the salt forms and crystal forms of the corresponding compounds, but the preparation and characterization of the following specific salt forms or crystal forms described below do not limit the scope of protection of the present disclosure, and more salt forms and crystal forms of the compounds of the present disclosure can be obtained by conventional salt-forming or crystallization methods based on the present disclosure by those skilled in the art, and these salt forms and crystal forms are the schemes protected by the present disclosure. Details are as follows:
To screen the salt forms of compound.
10 mg of compound was weighed, 200 μL of solvent was added thereto, the mixture was stirred at room temperature. Different acids were added respectively thereto, the mixture was stirred overnight, dried by centrifugation or volatilization to obtain a salt of the compound.
A good solvent was selected, the acid was weighed, the good solvent was added thereto to prepare a stock solution containing the compound in the concentration of 100 mg/mL. An anti-solvent was added thereto, 100 mg of compound was weighed respectively. 1 mL of the good solvent was added, completely dissolved and then filtered. 0.2 mL of filtrate was taken, the anti-solvent was added dropwise thereto respectively (stop adding if there is a precipitate, and adding 1.8 mL of anti-solvent at most), the mixture was stirred for a period of time, and the filtrate was removed by quick centrifugation to obtain the salt of the compound.
Through the salt form screening experiment, sulfuric acid, hydroxyethyl sulfonic acid and 1,5-naphthalene disulfonic acid can form salt with the free base of the compound.
As mentioned above, more pharmaceutically acceptable salts can be obtained by those skilled in the art using conventional methods based on the present disclosure.
To determine the number of hydroxyethyl sulfonic acid in the hydroxyethyl sulfonate of the compound of embodiment 13-1
An appropriate amount of the free base of the compound of embodiment 13-1 was weighed, methanol was added thereto to prepare a series of linear solutions with the concentration of 0.05-0.30 mg/mL.
An appropriate amount of the hydroxyethyl sulfonate of the compound of embodiment 13-1 was weighed, methanol was added thereto to prepare a solution containing the hydroxyethyl sulfonate of the compound of embodiment 13-1 with the concentration of 0.25 mg/mL. The above linear solution and a sample solution were taken for injection respectively.
The results of the external standard method show that hydroxyethyl sulfonic acid and free base form a salt in the molar ratio of 1:1.
To determine the number of hydroxyethyl sulfonic acid in the hydroxyethyl sulfonate of the compound of embodiment 13-1
An appropriate amount of the hydroxyethyl sulfonic acid was weighed, methanol was added thereto to prepare a series of linear solutions containing the hydroxyethyl sulfonic acid in the concentration of 0.5-1 mg/mL.
An appropriate amount of the hydroxyethyl sulfonate of compound of embodiment 13-1 was weighed, methanol was added to prepare a solution containing the hydroxyethyl sulfonate of compound of embodiment 13-1 with the concentration of 5.0 mg/mL. The above linear solution and a sample solution were taken for injection respectively.
Sample Area mg/mL
The number of the hydroxyethyl sulfonic acid in the hydroxyethyl sulfonate of the compound of embodiment 13-1 is calculated to be 1.
To screen the salt for crystal form of compound.
10 mg of the compound of embodiment 13-1 was weighed, different reaction solvents were added thereto respectively, and then the final volume of the mixture was 200 μL. The mixture was stirred, added with acid, and stirred for 12 hours. After centrifugation and drying, the XRPD of the mixture was measured.
The good solvent was selected, the acid was weighed, the good solvent was added thereto to prepare the stock solution containing the compound in the concentration of 100 mg/mL. The anti-solvent was added thereto, 100 mg of compound was weighed respectively, 1 mL of the good solvent was added, completely dissolved and then filtered. 0.2 mL of filtrate was taken, the anti-solvent was added dropwise thereto respectively (stop adding if there is a precipitate, and adding 1.8 mL of anti-solvent at most). The mixture was stirred for a period of time, the filtrate was removed by quick centrifugation, the XRPD of a solid was measured after dying.
Through experiments on the crystal form of the salt of the compound, the resulting salt forms with crystal forms were hydroxyethyl sulfonate, sulfate, and 1,5-naphthalenedisulfonate.
To prepare the crystal forms of the compound of embodiment 13-1
500 mg of the compound of embodiment 13-1 was weighed, 9.08 mL of isopropanol was added, and the mixture was heated to 50° C. and stirred. 0.914 mL of hydroxyethyl sulfonic acid (1.0 M in MeOH) was added thereto, precipitated after dissolved clarification, stirred at room temperature for 2 hours. The solid was dried under vacuum at 50° C. after filtration to obtain the crystal form I of hydroxyethyl sulfonate, which has an XRPD pattern as shown in
Alternatively, the compound of embodiment 13-1 (100 g), isopropanol (1200 mL) were added to a 3 L three-necked flask, heated to 40 to 45° C., stirred to dissolved clarification; and the 2-hydroxyethyl sulfonic acid (28.84 g) was dispersed in 800 mL of ethanol, the ethanol solution was added dropwise to the reaction system at a controlled temperature of 39 to 42° C. for about 10 minutes. 500 mg of seed crystal was added to the above reaction mixture and a solid was precipitated rapidly. The heating was removed, the reaction mixture was cooled to 25° C. and stirred for 12 hours. The reaction mixture was filtered, and the filter cake was washed with 400 mL of isopropanol, drained to dryness and dried under vacuum at 45° C. for 16 hours to obtain 92.57 g of a pale yellow solid with a purity of 97.9%, a chiral purity of 92%, and a mass yield of 92%. The pale yellow solid has an XRPD pattern as shown in
10 mg of the compound of embodiment 13-1 was weighed, 0.2 mL of tetrahydrofuran was added, and the mixture was heated to 50° C. and stirred. 18.3 μL of hydroxyethyl sulfonic acid (1.0 M in MeOH) was added thereto, precipitated after dissolved clarification, stirred at room temperature for 2 hours. The solid was dried under vacuum at 50° C. after filtration to obtain the crystal form II of hydroxyethyl sulfonate, which has an XRPD pattern as shown in
20 mg of the crystal form I of hydroxyethyl sulfonate was weighed, 0.2 mL of methanol and 0.45 mL of methyl tert-butyl ether were added, and the mixture was heated to 50° C. and stirred overnight. The solid was dried under vacuum at 50° C. after filtration to obtain the crystal form III of hydroxyethyl sulfonate, which has an XRPD pattern as shown in
10 mg of the compound of embodiment 13-1 was weighed, 0.2 mL of ethanol was added, and the mixture was heated to 50° C. and stirred. 18.3 μL of sulfuric acid (1.0 M in MeOH) was added thereto, precipitated after dissolved clarification, stirred at room temperature overnight. The solid was dried under vacuum at 50° C. after filtration to obtain the crystal form I of sulfate, which has an XRPD pattern as shown in
100 mg of the compound of embodiment 13-1 was weighed, 1.82 mL of isopropanol was added, and the mixture was heated to 50° C. and stirred. 183 μL of sulfuric acid (1.0 M in MeOH) was added thereto, precipitated a solid after dissolved clarification, stirred at room temperature overnight. The solid was dried under vacuum at 50° C. after filtration to obtain the crystal form II of sulfate, which has an XRPD pattern as shown in
10 mg of the crystal form I of sulfate was weighed, 0.2 mL of isopropanol was added, and the mixture was heated to 50° C. and stirred for 5 days. The solid was dried under vacuum at 50° C. after filtration to obtain the crystal form III of sulfate, which has an XRPD pattern as shown in
10 mg of the crystal form I of sulfate was weighed, 0.2 mL of ethyl acetate was added, and the mixture was heated to 50° C. and stirred for 5 days. The solid was dried under vacuum at 50° C. after filtration to obtain the crystal form IV of sulfate, which has an XRPD pattern as shown in
To investigate the physical and chemical stability of crystal form of the compound under high temperature, high humidity, high temperature and high humidity, and light conditions, so as to provide a basis for screening and storage of crystal form.
4.1.3.1 An appropriate amount of crystal form I of hydroxyethyl sulfonate of the compound of embodiment 13-1 was weighed and treated under light (≥1.2×106 lux-h, 10 days), high humidity (25° C., 75%, 10 days), high humidity (25° C., 90%, 10 days), high temperature (40° C., 30 days), high temperature (60° C., 30 days) and micropowder conditions for a period of time, respectively, then the XRPD of the crystal form I of hydroxyethyl sulfonate was measured.
An appropriate amount of crystal form I of hydroxyethyl sulfonate of the compound of embodiment 13-1 was weighed and placed under light (5000±500 lux), high temperature (60° C.), high humidity (92.5% RH), and high temperature and high humidity (50° C.&75% RH) conditions for 10 days, respectively, and a solution containing free base of the embodiment 13-1 at a concentration of 0.25 mg/mL was prepared by adding diluent methanol, analysed by HPLC, and the change of related substances was calculated according to the peak area normalization method.
The above experimental results show that the crystal form I of hydroxyethyl sulfonate of compound of embodiment 13-1 is relatively stable under light, high humidity, high temperature, and micropowder conditions.
To investigate the physical and chemical stability of crystal form of the compound under high temperature, high humidity, high temperature and high humidity, and light conditions, so as to provide a basis for screening and storage of crystal form.
An appropriate amount of crystal form II of sulfate of the compound of embodiment 13-1 was weighed and placed under light (5000±500 lux), high temperature (60° C.), high humidity (92.5% RH), and high temperature and high humidity (50° C. &75% RH) conditions for 10 days, respectively, and a solution containing free base of the embodiment 13-1 at a concentration of 0.25 mg/mL was prepared by adding diluent methanol, analysed by HPLC, and the change of related substances was calculated according to the peak area normalization method.
The crystal form II of sulfate is relatively stable under light, high humidity, high temperature and high humidity conditions.
To investigate the solubility of crystal form I of hydroxyethyl sulfonate and crystal form II of sulfate in different pH media, water, artificial simulated gastric fluid (FaSSGF), fasting artificial simulated intestinal fluid (FaSSIF) and non-fasting artificial simulated intestinal fluid (FeSSIF), so as to provide a basis for the assessment of salt druggablitity.
Approximately 1 mg of different salt forms of the compound was weighed and suspended into 1 mL of artificial simulated gastric fluid (FaSSGF), fasting artificial simulated intestinal fluid (FaSSIF), non-fasting artificial simulated intestinal fluid (FeSSIF), and pure water for 24 hours, respectively, the thermodynamic solubility of the compound at 37° C. was measured by HPLC with external standard method.
To obtain the thermodynamically stable crystal form of hydroxyethyl sulfonate by screening of polycrystal forms.
10 mg of crystal form I of hydroxyethyl sulfonate was weighed, 200 μL of organic solvent was added respectively, and the mixture was slurried at room temperature and 50° C. for 5 days, centrifuged. The supernatant was discarded, and the solid was dried and the XRPD of the solid was measured.
The above results show that the crystal form I of hydroxyethyl sulfonate is a stable crystal form of hydroxyethyl sulfonate.
To obtain the thermodynamically stable crystal form of sulfate by screening of polycrystal forms.
10 mg of crystal form II of sulfate was weighed, 200 μL of organic solvent was added respectively, the mixture was slurried at 50° C. for 5 days, centrifuged. The supernatant was discarded, and the solid was dried, and the XRPD of the solid was measured.
The above results show that the crystal form II of sulfate is a stable crystal form of sulfate.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202011354289.9 | Nov 2020 | CN | national |
| 202111389216.8 | Nov 2021 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/133653 | 11/26/2021 | WO |
