CN112672994A - Heterospirocyclic compounds as LSD1 inhibitors and application thereof - Google Patents

Heterospirocyclic compounds as LSD1 inhibitors and application thereof Download PDF

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CN112672994A
CN112672994A CN201980058619.9A CN201980058619A CN112672994A CN 112672994 A CN112672994 A CN 112672994A CN 201980058619 A CN201980058619 A CN 201980058619A CN 112672994 A CN112672994 A CN 112672994A
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alkyl
hydrochloride salt
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CN112672994B (en
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吴凌云
汪秋燕
黎健
陈曙辉
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Nanchang Hongyi Technology Co Ltd
Nanchang Hongyi Pharmaceutical Co Ltd
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Medshine Discovery Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C211/00—Compounds containing amino groups bound to a carbon skeleton
    • C07C211/33—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to carbon atoms of rings other than six-membered aromatic rings
    • C07C211/34—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to carbon atoms of rings other than six-membered aromatic rings of a saturated carbon skeleton
    • C07C211/35—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to carbon atoms of rings other than six-membered aromatic rings of a saturated carbon skeleton containing only non-condensed rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C217/00—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton
    • C07C217/54—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups bound to carbon atoms of at least one six-membered aromatic ring and amino groups bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings of the same carbon skeleton
    • C07C217/74—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups bound to carbon atoms of at least one six-membered aromatic ring and amino groups bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings of the same carbon skeleton with rings other than six-membered aromatic rings being part of the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D205/00—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom
    • C07D205/02—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings
    • C07D205/04—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/14—Nitrogen atoms not forming part of a nitro radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/08—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms
    • C07D211/10—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with radicals containing only carbon and hydrogen atoms attached to ring carbon atoms
    • C07D211/14—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with radicals containing only carbon and hydrogen atoms attached to ring carbon atoms with hydrocarbon or substituted hydrocarbon radicals attached to the ring nitrogen atom

Abstract

Relates to a heterocyclic spiro compound which can be used as a lysine specific demethylase 1(LSD1) inhibitor, and also relates to application thereof in preparing medicaments for treating diseases related to LSD 1. In particular to a compound shown as a formula (I), an isomer and pharmaceutically acceptable salt thereof.

Description

Heterospirocyclic compounds as LSD1 inhibitors and application thereof
The following priority is claimed in the present application:
CN201811070310.5, application date: 2018.09.13, respectively;
CN201910099370.8, application date: 2019.01.31.
Technical Field
the invention relates to a heterocyclic spiro compound serving as a lysine specific demethylase 1(LSD1) inhibitor and application thereof in preparing a medicament for treating diseases related to LSD 1. In particular to a compound shown as a formula (I), an isomer and pharmaceutically acceptable salts thereof.
Background
Histone post-translational modification including methylation, acetylation, phosphorylation, ubiquitination and other processes is an important regulation and control means of epigenetics, and gene expression is influenced by changing chromatin structure [ Xueshun Wang, Boshi Huang, Takayoshi Suzuki et al, Epigenomics,2015, 1379-; ]. Although these modifications do not alter the underlying sequence of DNA, this epigenetic change may persist throughout the cell life cycle or through cell iterative processes through cell division [ Adrian Bird, Nature,2007,396-398 ]. Epigenetic dysfunction is therefore closely related to the pathological processes of various diseases [ James T Lynch, William J Harris & Tim C P someville, Expert opin. Ther. targets,2012,1239-1249], such as various solid tumors, hematological tumors, viral infections, neurological disorders, and the like. Therefore, epigenetics is now a research hotspot in the field of drug development. The methylation state of histones is regulated by both histone methyltransferases and histone demethylases. Lysine specific demethylase (LSD1, also known as KDM1A) is the first histone Lysine demethylase reported, and it widely participates in transcriptional regulation by regulating the methylation state of histone Lysine, affecting many physiological processes such as cell proliferation and differentiation, and pluripotency of embryonic stem cells. [ Yujiang Shi, Fei Lan, Caitlin Matson et al, Cell,2004, 941-953 ] [ Daniel P.Mould, Alison E.McGonagle, Daniel H.Wiseman et al, Medicinal Research Reviews 2015, 586-618 ]. The LSD1 structure includes three main parts: an N-terminal SWIRM domain, a C-terminal aminooxidase domain (AOL) and a central Tower domain. [ Ruchi Anand, Ronen Marmorstein, Journal of Biological Chemistry,2007, 35425-35429 ]. The C-terminal aminooxidase domain contains two active pockets, one is the site for FAD binding and the other is the site for recognition and binding to the substrate [ Pete Stavropoulos, Hunter Blbel, Andre Hoelz, Nature Structral & Molecular Biology,2006,626-632 ]. The function of the SWIRM domain has not been clearly concluded, it is not directly involved in FAD or substrate binding, but mutation or removal of this region would reduce the activity of LSD1, thus presumably affecting the action of the active region by modulating conformation. [ Yong Chen, Yuting Yang, Feng Wang et al, Biochemistry,2006, 13956-. The Tower domain is the binding domain of LSD1 with other protein factors. LSD1 can be combined with different protein factors to act on different substrates, thereby playing different roles in regulating and controlling histone and gene expression. For example, LSD1, when combined with CoREST, will preferentially act on histone H3K4, through demethylation, remove activation related histone mark, inhibit gene transcription; while binding to androgen receptor proteins, recombinant LSD1 preferentially acts on H3K9, activating androgen receptor-associated gene transcription by demethylation [ Ruchi Anand, Ronen Marmorstein, Journal of Biological Chemistry,2007, 35425-35429; eric Metzger, Melanie Wissmann, Na Yin et al, Nature,2005, 436-. In addition, LSD1 also regulates the methylation status of some non-histone substrates, including the tumor suppressor p53 and DNA methyltransferase 1(DNA methyltransferase 1, DNMT1) etc. [ Yi Chao Zheng, Jinlian Ma, Zhiru Wang, Medicinal Research Reviews,2015, 1032-1071 ].
LSD1 is an FAD-dependent amino oxidase, where proton transfer is considered to be its most likely oxidation mechanism [ Zheng Y C, Yu B, Chen Z S, et al.]. Firstly, the N-CH of the substrate is transferred by proton3The bond is converted to an imine bond, and this imine ion intermediate undergoes hydrolysis to form, on the one hand, the demethylated amine and, on the other hand, formaldehyde. During this catalytic cycle FAD is reduced to FADH2, which is subsequently oxidized back to FAD by a molecule of oxygen, producing a molecule of H2O2[ Yujiang Shi, Fei Lan, Caitlin Matson, Cell,2004, 941-]。
LSD1 is aberrantly expressed in a number of different types of tumors. LSD1 is highly expressed in Acute Myeloid Leukemia (AML) subtype, and is an important factor in maintaining Leukemic Stem Cell (LSC) potential. LSD1 is highly expressed in various solid tumors such as lung cancer, breast cancer, prostate cancer, liver cancer and pancreatic cancer, and is closely related to poor prognosis of tumors. LSD1 inhibits cadherin expression and is closely related to tumor invasion and epithelial-mesenchymal transition (EMT) [ Hosseini A, Minucci S. Epigenomics,2017,9, 1123-.
No drug is approved for the market of the LSD1 inhibitor at present, and 8 drugs are in clinical research stage and mainly used for treating diseases such as hematological tumors, small cell lung cancer, Ewing's sarcoma and the like. However, in the face of a huge unmet market, there is still a need in the art for better active, more pharmacokinetic parameters of candidate compounds to be advanced to clinical trials to meet therapeutic needs.
Disclosure of Invention
The present invention provides a compound of formula (I), an isomer thereof or a pharmaceutically acceptable salt thereof,
Figure PCTCN2019105680-APPB-000001
wherein the content of the first and second substances,
L 1is selected from- (CH)2)g-、-C(＝O)-、-S(＝O) 2-, -C (═ O) -O-, and-C (═ O) -NH-;
R 1is selected from NH2、CN、COOH、-S(＝O) 2-NH 2、C 1-6Alkyl, -C (═ O) -C1-4Alkyl-phenyl and-C1-4Alkyl-4-7 membered heterocycloalkyl wherein said C1-6Alkyl, -C (═ O) -C1-4Alkyl-phenyl and-C1-4Alkyl-4-7 membered heterocycloalkyl optionally substituted with 1,2 or 3RaSubstitution;
m is 0, 1 or 2;
n is 0, 1 or 2, and m and n cannot be 0 at the same time;
r is 0 or 1;
q is 0 or 1;
g is 0, 1,2, 3 or 4;
R aselected from F, Cl, Br, I, OH, NH2、CN、COOH、C 1-3Alkylamino and-NH-C (═ O) -C1-3Alkyl radical, wherein said C1-3Alkylamino and-NH-C (═ O) -C1-3Alkyl is optionally substituted with 1,2 or 3R;
r is selected from F, Cl, Br, I, OH and NH2And C1-3An alkyl group;
said 4-7 membered heterocycloalkyl contains 1,2, 3 or 4 heteroatoms or groups of heteroatoms independently selected from-NH-, -O-, -S-and N;
with "-" carbon atoms as chiral carbon atoms, in the form of (R) or (S) single enantiomers or enriched in one enantiomer;
the carbon atom with "#" is a chiral carbon atom and exists in the form of a single enantiomer (R) or (S) or in a form enriched in one enantiomer.
In some embodiments of the present invention, R is selected from F, Cl, Br, I, OH, NH2、CH 3and-CH2CH 3The other variables are as defined herein.
In some embodiments of the invention, R is as defined aboveaSelected from F, Cl, Br, I, OH, NH2、CN、COOH、
Figure PCTCN2019105680-APPB-000002
Figure PCTCN2019105680-APPB-000003
Wherein said
Figure PCTCN2019105680-APPB-000004
Optionally substituted with 1,2 or 3R, the other variables being as defined herein.
In some embodiments of the invention, R is as defined aboveaSelected from F, Cl, Br, I, OH, NH2、CN、COOH、
Figure PCTCN2019105680-APPB-000005
Figure PCTCN2019105680-APPB-000006
Other variables are as defined herein.
In some embodiments of the invention, R is as defined above1Is selected from NH2、CN、COOH、-S(＝O) 2-NH 2、C 1-4Alkyl, -C (═ O) -C1-3Alkyl-phenyl and-C1-3Alkyl-5-6 heterocycloalkyl wherein said C1-4Alkyl, -C (═ O) -C1-3Alkyl-phenyl and-C1-3Alkyl-5-6 heterocycloalkyl optionally substituted with 1,2 or 3RaAnd the other variables are as defined herein.
In some embodiments of the invention, R is as defined above1Is selected from NH2、CN、COOH、-S(＝O) 2-NH 2、-CH 3、-CH 2-CH 3、
Figure PCTCN2019105680-APPB-000007
Figure PCTCN2019105680-APPB-000008
Wherein said CH3、-CH 2-CH 3、
Figure PCTCN2019105680-APPB-000009
Figure PCTCN2019105680-APPB-000010
Optionally substituted by 1,2 or 3RaAnd the other variables are as defined herein.
In some embodiments of the invention, R is as defined above1Is selected from NH2、CN、COOH、
Figure PCTCN2019105680-APPB-000011
-CH 3、-CH 2-CH 3、
Figure PCTCN2019105680-APPB-000012
Figure PCTCN2019105680-APPB-000013
Other variables are as defined herein.
In some embodiments of the invention, L is1Selected from single bonds, -CH2-、-(CH 2) 2-、
Figure PCTCN2019105680-APPB-000014
-C (═ O) -, and-S (═ O)2The other variables are as defined herein.
The present invention provides a compound of formula (I), an isomer thereof or a pharmaceutically acceptable salt thereof,
Figure PCTCN2019105680-APPB-000015
wherein the content of the first and second substances,
L 1is selected from- (CH)2)g-、-C(＝O)-、-S(＝O) 2-, -C (═ O) -O-, and-C (═ O) -NH-;
R 1is selected from NH2、CN、COOH、-S(＝O) 2-NH 2、C 1-6Alkyl and-C (═ O) -C1-4Alkyl-phenyl, wherein said C1-6Alkyl and-C (═ O) -C1-4Alkyl-phenyl optionally substituted by 1,2 or 3RaSubstitution;
m is 0, 1 or 2;
n is 0, 1 or 2, and m and n cannot be 0 at the same time;
r is 0 or 1;
q is 0 or 1;
g is 0, 1,2, 3 or 4;
R aselected from F, Cl, Br, I, OH, NH2CN, COOH and C1-3Alkylamino, wherein said C1-3Alkylamino is optionally substituted with 1,2 or 3R;
r is selected from F, Cl, Br, I, OH and NH2And CH3；
With "-" carbon atoms as chiral carbon atoms, in the form of (R) or (S) single enantiomers or enriched in one enantiomer;
the carbon atom with "#" is a chiral carbon atom and exists in the form of a single enantiomer (R) or (S) or in a form enriched in one enantiomer.
In some embodiments of the invention, L is1Is selected from- (CH)2) g-is, R1Is selected from NH2、CN、COOH、-S(＝O) 2-NH 2、C 1-6Alkyl and-C (═ O) -C1-4Alkyl-phenyl, wherein said C1-6Alkyl and-C (═ O) -C1-4Alkyl-phenyl optionally substituted by 1,2 or 3RaThe other variables are as defined herein.
In some embodiments of the invention, L is1Selected from-C (═ O) -, -S (═ O)2when-C (- ═ O) -O-and-C (-O) -NH-, -, R1Is selected from NH2、CN、COOH、-S(＝O) 2-NH 2And C1-6Alkyl radical, wherein said C1-6Alkyl is optionally substituted by 1,2 or 3RaAnd the other variables are as defined herein.
In some embodiments of the invention, R is as defined aboveaSelected from F, Cl, Br, I, OH, NH2CN, COOH and
Figure PCTCN2019105680-APPB-000016
wherein said
Figure PCTCN2019105680-APPB-000017
Optionally substituted with 1,2 or 3R, the other variables being as defined herein.
In some aspects of the inventionR is as defined aboveaSelected from F, Cl, Br, I, OH, NH2CN, COOH and
Figure PCTCN2019105680-APPB-000018
other variables are as defined herein.
In some embodiments of the invention, R is as defined above1Is selected from NH2、CN、COOH、-S(＝O) 2-NH 2、C 1-4Alkyl and-C1-3Alkyl-phenyl, wherein said C1-4Alkyl and-C1-3Alkyl-phenyl optionally substituted by 1,2 or 3RaAnd the other variables are as defined herein.
In some embodiments of the invention, R is as defined above1Is selected from NH2、CN、COOH、-S(＝O) 2-NH 2、CH 3、-CH 2-CH 3、
Figure PCTCN2019105680-APPB-000019
Figure PCTCN2019105680-APPB-000020
Wherein said CH3、-CH 2-CH 3、
Figure PCTCN2019105680-APPB-000021
Optionally substituted by 1,2 or 3RaAnd the other variables are as defined herein.
In some embodiments of the invention, R is as defined above1Is selected from NH2、CN、COOH、
Figure PCTCN2019105680-APPB-000022
CH 3、
Figure PCTCN2019105680-APPB-000023
Figure PCTCN2019105680-APPB-000024
Figure PCTCN2019105680-APPB-000025
Other variables are as defined herein.
In some embodiments of the invention, L is1Selected from single bonds, -CH2-、-(CH 2) 2-、
Figure PCTCN2019105680-APPB-000026
-C (═ O) -, and-S (═ O)2The other variables are as defined herein.
The present invention provides a compound of formula (I), an isomer thereof or a pharmaceutically acceptable salt thereof,
Figure PCTCN2019105680-APPB-000027
wherein the content of the first and second substances,
L 1is selected from- (CH)2)g-、-C(＝O)-、-S(＝O) 2-, -C (═ O) -O-, and-C (═ O) -NH-;
R 1is selected from NH2、CN、COOH、-S(＝O) 2-NH 2And C1-6Alkyl radical, wherein said C1-6Alkyl is optionally substituted by 1,2 or 3RaSubstitution; m is 0, 1 or 2;
n is 0, 1 or 2, and m and n cannot be 0 at the same time;
r is 0 or 1;
q is 0 or 1;
g is 0, 1,2, 3 or 4;
R aselected from F, Cl, Br, I, OH, NH2CN and OOH, wherein said phenyl is optionally substituted with 1,2 or 3R;
r is selected from F, Cl, Br, I, OH and NH2；
With "-" carbon atoms as chiral carbon atoms, in the form of (R) or (S) single enantiomers or enriched in one enantiomer;
the carbon atom with "#" is a chiral carbon atom and exists in the form of a single enantiomer (R) or (S) or in a form enriched in one enantiomer.
In some embodiments of the invention, R is as defined aboveaSelected from F, Cl, Br, I, OH, NH2CN and COOH, other variables are as defined herein.
In some embodiments of the invention, R is as defined above1Is selected from NH2、CN、COOH、-S(＝O) 2-NH 2And C1-4Alkyl radical, wherein said C1-4Alkyl is optionally substituted by 1,2 or 3RaAnd the other variables are as defined herein.
In some embodiments of the invention, R is as defined above1Is selected from NH2、CN、COOH、-S(＝O) 2-NH 2、CH 3、-CH 2-CH 3、
Figure PCTCN2019105680-APPB-000028
Figure PCTCN2019105680-APPB-000029
Wherein said CH3、-CH 2-CH 3、
Figure PCTCN2019105680-APPB-000030
Optionally substituted by 1,2 or 3RaAnd the other variables are as defined herein.
In some embodiments of the invention, R is as defined above1Is selected from NH2、CN、COOH、
Figure PCTCN2019105680-APPB-000031
CH 3、
Figure PCTCN2019105680-APPB-000032
Figure PCTCN2019105680-APPB-000033
Other variables are as defined herein.
In some embodiments of the invention, L is1Selected from single bonds, -CH2-、-(CH 2) 2-、
Figure PCTCN2019105680-APPB-000034
-C (═ O) -, and-S (═ O)2The other variables are as defined herein.
Still other embodiments of the present invention are derived from any combination of the above variables.
In some embodiments of the invention, the above compound, isomer thereof or pharmaceutically acceptable salt thereof is selected from
Figure PCTCN2019105680-APPB-000035
Figure PCTCN2019105680-APPB-000036
Wherein the content of the first and second substances,
g and R1As defined herein.
The invention also provides a compound of the formula, an isomer thereof or a pharmaceutically acceptable salt thereof,
Figure PCTCN2019105680-APPB-000037
Figure PCTCN2019105680-APPB-000038
in some embodiments of the invention, the above-described compound, isomer thereof, or pharmaceutically acceptable salt thereof,
Figure PCTCN2019105680-APPB-000039
the invention also provides the compound, the isomer or the pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt is selected from hydrochloride.
The invention also provides application of the compound, the isomer of the compound or the pharmaceutically acceptable salt of the compound in preparing a medicament for treating LSD1 related diseases.
Technical effects
As a novel LSD1 inhibitor, the compound has remarkable inhibitory activity on LSD1, and has remarkable inhibitory activity on NCI-H1417, HL60 and MV-4-11 cell proliferation; meanwhile, the compound has good pharmacokinetic properties; and the combination of the monoclonal antibody and PD-L1 monoclonal antibody in a CT-26 mouse colon cancer transplantation tumor model and the combination of the monoclonal antibody and PD-1 monoclonal antibody in an MC38 mouse colon cancer transplantation tumor model has excellent tumor inhibition effect.
Definitions and explanations
As used herein, the following terms and phrases are intended to have the following meanings, unless otherwise indicated. A particular term or phrase, unless specifically defined, should not be considered as indefinite or unclear, but rather construed according to ordinary meaning. When a trade name appears herein, it is intended to refer to its corresponding commodity or its active ingredient.
The term "pharmaceutically acceptable" as used herein is intended to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The term "pharmaceutically acceptable salts" refers to salts of the compounds of the present invention, prepared from the compounds of the present invention found to have particular substituents, with relatively nontoxic acids or bases. When compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of a base in neat solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amines or magnesium salts or similar salts. When compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in neat solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, and the like; and salts of organic acids including acids such as acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-toluenesulfonic, citric, tartaric, methanesulfonic, and the like; also included are salts of amino acids such as arginine and the like, and salts of organic acids such as glucuronic acid and the like. Certain specific compounds of the invention contain both basic and acidic functionalities and can thus be converted to any base or acid addition salt.
The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound, which contains an acid or base, by conventional chemical methods. In general, such salts are prepared by the following method: prepared by reacting these compounds in free acid or base form with a stoichiometric amount of the appropriate base or acid, in water or an organic solvent or a mixture of the two.
The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis and trans isomers, (-) -and (+) -enantiomers, (R) -and (S) -enantiomers, diastereomers, (D) -isomers, (L) -isomers, as well as racemic and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are included within the scope of the present invention.
Unless otherwise indicated, the terms "enantiomer" or "optical isomer" refer to stereoisomers that are mirror images of each other.
Unless otherwise indicated, the term "cis-trans isomer" or "geometric isomer" results from the inability of a double bond or a single bond to rotate freely within a ring-forming carbon atom.
Unless otherwise indicated, the term "diastereomer" refers to a stereoisomer in which the molecules have two or more chiral centers and a non-mirror image relationship between the molecules.
Unless otherwise indicated, "(D)" or "(+)" means dextrorotation, "(L)" or "(-) -means levorotation," (DL) "or" (±) "means racemization.
Using solid wedge keys, unless otherwise indicated
Figure PCTCN2019105680-APPB-000040
And wedge dotted bond
Figure PCTCN2019105680-APPB-000041
Showing the absolute configuration of a solid centre, by means of straight solid keys
Figure PCTCN2019105680-APPB-000042
And straight dotted line bond
Figure PCTCN2019105680-APPB-000043
Showing the relative configuration of the centres of solids, by wavy lines
Figure PCTCN2019105680-APPB-000044
Representing solid-line keys of wedge shape
Figure PCTCN2019105680-APPB-000045
Or wedge dotted bond
Figure PCTCN2019105680-APPB-000046
Or by wavy lines
Figure PCTCN2019105680-APPB-000047
Indicating straight solid-line keys
Figure PCTCN2019105680-APPB-000048
And straight dotted line bond
Figure PCTCN2019105680-APPB-000049
Unless otherwise indicated, when a double bond structure such as a carbon-carbon double bond, a carbon-nitrogen double bond and a nitrogen-nitrogen double bond is present in a compound and two different substituents are attached to each atom on the double bond (in a double bond containing a nitrogen atom, one lone pair of electrons on the nitrogen atom is considered as one substituent to which it is attached), if a wavy line is used between the atom on the double bond and its substituent in the compound
Figure PCTCN2019105680-APPB-000050
The term "linked" means either the (Z) -isomer, the (E) -isomer, or a mixture of both isomers of the compound. For example, the following formula (A) represents that the compound exists in the form of a single isomer of formula (A-1) or formula (A-2) or in the form of a mixture of two isomers of formula (A-1) and formula (A-2); the following formula (B) represents that the compound exists in the form of a single isomer of formula (B-1) or formula (B-2) or in the form of a mixture of two isomers of formula (B-1) and formula (B-2). The following formula (C) represents that the compound exists in the form of a single isomer of formula (C-1) or formula (C-2) or a mixture of two isomers of formula (C-1) and formula (C-2).
Figure PCTCN2019105680-APPB-000051
The compounds of the invention may be present specifically. Unless otherwise indicated, the term "tautomer" or "tautomeric form" means that at room temperature, the isomers of different functional groups are in dynamic equilibrium and can be rapidly interconverted. If tautomers are possible (e.g., in solution), then the chemical equilibrium of the tautomers can be reached. For example, proton tautomers (prototropic tautomers), also known as proton transfer tautomers (prototropic tautomers), include interconversions by proton transfer, such as keto-enol isomerization and imine-enamine isomerization. Valence isomers (valencetatomer) include interconversion by recombination of some of the bonding electrons. A specific example of where keto-enol tautomerism is the interconversion between two tautomers of pentane-2, 4-dione and 4-hydroxypent-3-en-2-one.
Unless otherwise indicated, the terms "enriched in one isomer", "isomer enriched", "enantiomer enriched" or "enantiomeric enrichment" refer to a content of one isomer or enantiomer of less than 100%, and the content of the isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.
Unless otherwise indicated, the term "isomeric excess" or "enantiomeric excess" refers to the difference between the relative percentages of two isomers or enantiomers. For example, if the content of one isomer or enantiomer is 90%, and the content of the other isomer or enantiomer is 10%, the isomer or enantiomer excess (ee value) is 80%.
Optically active (R) -and (S) -isomers as well as D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one of the enantiomers of a compound of the invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (e.g., amino) or an acidic functional group (e.g., carboxyl), diastereomeric salts are formed with an appropriate optically active acid or base, followed by diastereomeric resolution by conventional methods known in the art, and the pure enantiomers are recovered. Furthermore, separation of enantiomers and diastereomers is typically accomplished by using chromatography employing a chiral stationary phase, optionally in combination with chemical derivatization (e.g., carbamate formation from amines). The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be labelled with radioactive isotopes, such as tritium (A), (B), (C3H) Iodine-125 (125I) Or C-14(14C) In that respect For example, deuterium can be used to replace hydrogen to form a deuterated drug, the bond formed by deuterium and carbon is stronger than the bond formed by common hydrogen and carbon, and compared with an undeuterated drug, the deuterated drug has the advantages of reducing toxic and side effects, increasing the stability of the drug, enhancing the curative effect, prolonging the biological half-life period of the drug and the like. All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention. "optional" or "optionally"It is meant that the subsequently described event or circumstance may, but need not, occur and that the description includes instances where said event or circumstance occurs and instances where it does not.
The term "substituted" means that any one or more hydrogen atoms on a particular atom is replaced with a substituent, and may include variations of deuterium and hydrogen, so long as the valency of the particular atom is normal and the substituted compound is stable. When the substituent is oxygen (i.e., ═ O), it means that two hydrogen atoms are substituted. Oxygen substitution does not occur on aromatic groups. The term "optionally substituted" means that it may or may not be substituted, and unless otherwise specified, the kind and number of substituents may be arbitrary on the basis of chemical realizability.
When any variable (e.g., R) occurs more than one time in the composition or structure of a compound, its definition in each case is independent. Thus, for example, if a group is substituted with 0-2R, the group may optionally be substituted with up to two R, and there are separate options for R in each case. Furthermore, combinations of substituents and/or variants thereof are permissible only if such combinations result in stable compounds.
When the number of one linking group is 0, e.g. - (CRR)0-, represents that the linking group is a single bond.
When one of the variables is selected from a single bond, it means that the two groups to which it is attached are directly connected, for example, where L represents a single bond in A-L-Z means that the structure is actually A-Z.
When a substituent is absent, it indicates that the substituent is absent, e.g., when X is absent in A-X, it indicates that the structure is actually A. When a substituent bond can be cross-linked to more than two atoms of a ring, such substituent may be bonded to any atom of the ring, e.g. a building block
Figure PCTCN2019105680-APPB-000052
Represents a substituent thereofR can be substituted at any position on cyclohexyl or cyclohexadiene. . When no atom through which a substituent is attached to a substituted group is indicated in the listed substituents, such substituents may be bonded through any atom thereof, for example, a pyridyl group as a substituent may be attached to a substituted group through any one of carbon atoms on the pyridine ring.
When the listed linking groups do not indicate their direction of attachment, the direction of attachment is arbitrary, for example,
Figure PCTCN2019105680-APPB-000053
the linking group L in (A) is-M-W-, in which case-M-W-can be formed by connecting ring A and ring B in the same direction as the reading sequence from left to right
Figure PCTCN2019105680-APPB-000054
The ring A and the ring B may be connected in the reverse direction of the reading sequence from left to right
Figure PCTCN2019105680-APPB-000055
Combinations of the linking groups, substituents, and/or variants thereof are permissible only if such combinations result in stable compounds.
Unless otherwise specified, the number of atoms on a ring is generally defined as the number of ring members, e.g., "5-7 membered ring" means a "ring" around which 5-7 atoms are arranged.
Unless otherwise specified, "3-12 membered ring" means a cycloalkyl, heterocycloalkyl, cycloalkenyl or heterocycloalkenyl group consisting of 3 to 12 ring atoms. The ring includes monocyclic ring, and also includes bicyclic or polycyclic ring systems such as spiro ring, fused ring and bridged ring. Unless otherwise specified, the ring optionally contains 1,2, or 3 heteroatoms independently selected from O, S and N. The 3-12 membered ring includes 3-10 membered, 3-9 membered, 3-8 membered, 3-7 membered, 3-6 membered, 3-5 membered, 4-10 membered, 4-9 membered, 4-8 membered, 4-7 membered, 4-6 membered, 4-5 membered, 5-10 membered, 5-9 membered, 5-8 membered, 5-7 membered, 5-6 membered, 6-10 membered, 6-9 membered, 6-8 membered, 6-7 membered ring and the like. The term "5-7 membered heterocycloalkyl" includes piperidinyl and the like, but does not include phenyl. The term "ring" also includes ring systems containing at least one ring, each of which "ring" independently conforms to the above definition.
Unless otherwise specified, a "5-6 membered ring" means a cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, cycloalkynyl, heterocycloalkynyl, aryl or heteroaryl group consisting of 5 to 6 ring atoms. The ring includes monocyclic ring, and also includes bicyclic ring systems such as spiro ring, fused ring and bridged ring. Unless otherwise specified, the ring optionally contains 1,2, or 3 heteroatoms independently selected from O, S and N. The 5-6 membered ring includes 5-membered, 6-membered rings, and the like. "5-6 membered ring" includes, for example, phenyl, pyridyl, piperidyl and the like; on the other hand, the term "5-6 membered heterocycloalkyl" includes piperidinyl and the like, but does not include phenyl. The term "ring" also includes ring systems containing at least one ring, each of which "ring" independently conforms to the above definition.
Unless otherwise specified, the term "C1-6Alkyl "is intended to mean a straight or branched saturated hydrocarbon group consisting of 1 to 6 carbon atoms. Said C is1-6The alkyl group comprising C1-5、C 1-4、C 1-3、C 1-2、C 2-6、C 2-4、C 6And C5Alkyl, etc.; it may be monovalent (e.g., methyl), divalent (e.g., methylene), or multivalent (e.g., methine). C1-6Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, s-butyl and t-butyl), pentyl (including n-pentyl, isopentyl and neopentyl), hexyl, and the like.
Unless otherwise specified, the term "C1-4Alkyl "is intended to mean a straight or branched saturated hydrocarbon group consisting of 1 to 4 carbon atoms. Said C is1-4The alkyl group comprising C1-2、C 1-3And C2-3Alkyl, etc.; it may be monovalent (e.g., methyl), divalent (e.g., methylene), or multivalent (e.g., methine). C1-4Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, s-butyl and t-butyl), and the like.
Unless otherwise specified, the term "C1-3Alkyl "is intended to mean a straight or branched saturated hydrocarbon group consisting of 1 to 3 carbon atoms. Said C is1-3The alkyl group comprising C1-2And C2-3Alkyl, etc.; it may be monovalent (e.g., methyl), divalent (e.g., methylene), or multivalent (e.g., methine). C1-3Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.
Unless otherwise specified, the term "C1-6Alkoxy "denotes those alkyl groups containing 1 to 6 carbon atoms which are attached to the rest of the molecule through an oxygen atom. Said C is1-6Alkoxy radicals comprising C1-4、C 1-3、C 1-2、C 2-6、C 2-4、C 6、C 5、C 4And C3Alkoxy, and the like. C1-6Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), butoxy (including n-butoxy, isobutoxy, s-butoxy and t-butoxy), pentyloxy (including n-pentyloxy, isopentyloxy and neopentyloxy), hexyloxy, and the like.
Unless otherwise specified, the term "C1-3Alkoxy "denotes those alkyl groups containing 1 to 3 carbon atoms which are attached to the rest of the molecule through an oxygen atom. Said C is1-3Alkoxy radicals comprising C1-2、C 2-3、C 3And C2Alkoxy, and the like. C1-3Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), and the like.
Unless otherwise specified, the term "C1-3Alkylamino "refers to those packets attached to the rest of the molecule through an amino groupAlkyl groups containing 1 to 3 carbon atoms. Said C is1-3Alkylamino radicals comprising C1-2、C 3And C2Alkylamino, and the like. C1-3Examples of alkylamino include, but are not limited to, -NHCH3、-N(CH 3) 2、-NHCH 2CH 3、-N(CH 3)CH 2CH 3、-NHCH 2CH 2CH 3、-NHCH 2(CH 3) 2And the like.
Unless otherwise specified, the term "C" in the present invention6-10Aromatic rings "and" C6-10Aryl "is used interchangeably with the term" C6-10Aromatic ring "or" C6-10Aryl "denotes a cyclic hydrocarbon group consisting of 6 to 10 carbon atoms with a conjugated pi-electron system, which may be a monocyclic, fused bicyclic or fused tricyclic ring system, wherein each ring is aromatic. It may be monovalent, divalent or polyvalent, C6-10Aryl radicals including C6-9、C 9、C 10And C6Aryl, and the like. C6-10Examples of aryl groups include, but are not limited to, phenyl, naphthyl (including 1-naphthyl and 2-naphthyl, and the like).
Unless otherwise specified, the term "4-7 membered heterocycloalkyl" by itself or in combination with other terms denotes a saturated cyclic group consisting of 4 to 7 ring atoms, 1,2, 3 or 4 of which are heteroatoms independently selected from O, S and N, the remainder being carbon atoms, wherein the nitrogen atom is optionally quaternized, and the carbon, nitrogen and sulfur heteroatoms may optionally be oxidized (i.e., C (═ O), NO and s (O))pAnd p is 1 or 2). It includes monocyclic and bicyclic ring systems, wherein bicyclic ring systems include spiro, fused and bridged rings. Furthermore, with respect to the "4-7 membered heterocycloalkyl", the heteroatom may occupy the position of the heterocycloalkyl linkage to the rest of the molecule. The 4-7 membered heterocycloalkyl group includes 5-6 membered, 4 membered, 5 membered, 6 membered and 7 membered heterocycloalkyl groups and the like. Examples of 4-7 membered heterocycloalkyl include, but are not limited to, azetidinyl, oxetanyl, thietanyl, pyrrolidinylPyrazolidinyl, imidazolidinyl, tetrahydrothienyl (including tetrahydrothien-2-yl, tetrahydrothien-3-yl, etc.), tetrahydrofuranyl (including tetrahydrofuran-2-yl, etc.), tetrahydropyranyl, piperidinyl (including 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, etc.), piperazinyl (including 1-piperazinyl, 2-piperazinyl, etc.), morpholinyl (including 3-morpholinyl, 4-morpholinyl, etc.), dioxanyl, dithianyl, isoxazolidinyl, isothiazolidinyl, 1, 2-oxazinyl, 1, 2-thiazinyl, hexahydropyridazinyl, homopiperazinyl, homopiperidinyl, or pyrrolidin-2-onyl, etc.
Unless otherwise specified, the term "5-6 membered heterocycloalkyl" by itself or in combination with other terms denotes a saturated cyclic group consisting of 5 to 6 ring atoms, 1,2, 3 or 4 of which are heteroatoms independently selected from O, S and N, the remainder being carbon atoms, wherein the nitrogen atom is optionally quaternized, and the carbon, nitrogen and sulfur heteroatoms may optionally be oxidized (i.e., C (═ O), NO and s (O))pAnd p is 1 or 2). It includes monocyclic and bicyclic ring systems, wherein bicyclic ring systems include spiro, fused and bridged rings. Furthermore, with respect to the "5-6 membered heterocycloalkyl", the heteroatom may occupy the position of the heterocycloalkyl linkage to the rest of the molecule. The 5-6 membered heterocycloalkyl group includes 5-and 6-membered heterocycloalkyl groups. Examples of 5-6 membered heterocycloalkyl include, but are not limited to, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrothienyl (including tetrahydrothien-2-yl and tetrahydrothien-3-yl, etc.), tetrahydrofuryl (including tetrahydrofuran-2-yl, etc.), tetrahydropyranyl, piperidinyl (including 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, etc.), piperazinyl (including 1-piperazinyl and 2-piperazinyl, etc.), morpholinyl (including 3-morpholinyl and 4-morpholinyl, etc.), dioxanyl, dithianyl, isoxazolidinyl, isothiazolidinyl, 1, 2-oxazinyl, 1, 2-thiazinyl, hexahydropyridazinyl, homopiperazinyl, homopiperidinyl, or pyrrolidin-2-onyl, etc.
Unless otherwise specified, Cn-n+mOr Cn-C n+mIncluding any one particular case of n to n + m carbons, e.g. C1-12Comprising C1、C 2、C 3、C 4、C 5、C 6、C 7、C 8、C 9、C 10、C 11And C12Also included are any ranges of n to n + m, e.g. C1-12Comprising C1-3、C 1-6、C 1-9、C 3-6、C 3-9、C 3-12、C 6-9、C 6-12And C9-12Etc.; similarly, n to n + m means the number of atoms on the ring is n to n + m, for example, the 3-12 membered ring includes a 3-membered ring, a 4-membered ring, a 5-membered ring, a 6-membered ring, a 7-membered ring, an 8-membered ring, a 9-membered ring, a 10-membered ring, a 11-membered ring, and a 12-membered ring, and any range of n to n + m is also included, for example, the 3-12 membered ring includes a 3-6-membered ring, a 3-9-membered ring, a 5-6-membered ring, a 5-7-membered ring, a 6-8-membered ring, and a 6-.
The term "leaving group" refers to a functional group or atom that can be substituted by another functional group or atom through a substitution reaction (e.g., an affinity substitution reaction). For example, representative leaving groups include triflate; chlorine, bromine, iodine; sulfonate groups such as methanesulfonate, toluenesulfonate, p-bromobenzenesulfonate, p-toluenesulfonate and the like; acyloxy groups such as acetoxy, trifluoroacetyloxy, and the like.
The term "protecting group" includes, but is not limited to, "amino protecting group," hydroxyl protecting group, "or" thiol protecting group. The term "amino protecting group" refers to a protecting group suitable for use in preventing side reactions at the amino nitrogen position. Representative amino protecting groups include, but are not limited to: a formyl group; acyl, for example alkanoyl (such as acetyl, trichloroacetyl or trifluoroacetyl); alkoxycarbonyl such as tert-butoxycarbonyl (Boc); arylmethoxycarbonyl groups such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl groups such as benzyl (Bn), trityl (Tr), 1-bis- (4' -methoxyphenyl) methyl; silyl groups, such as Trimethylsilyl (TMS) and t-butyldimethylsilyl (TBS), and the like. The term "hydroxy protecting group" refers to a protecting group suitable for use in preventing side reactions of a hydroxy group. Representative hydroxy protecting groups include, but are not limited to: alkyl groups such as methyl, ethyl and tert-butyl; acyl groups, such as alkanoyl (e.g., acetyl); arylmethyl groups such as benzyl (Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (benzhydryl, DPM); silyl groups, such as Trimethylsilyl (TMS) and t-butyldimethylsilyl (TBS), and the like.
The compounds of the present invention may be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combinations thereof with other chemical synthetic methods, and equivalents thereof known to those skilled in the art, with preferred embodiments including, but not limited to, examples of the present invention.
The solvent used in the present invention can be commercially available. The invention employs the following abbreviations: aq represents water; HATU represents O- (7-azabenzotriazol-1-yl) -N, N' -tetramethyluronium hexafluorophosphate; EDC stands for N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride; m-CPBA represents 3-chloroperoxybenzoic acid; eq represents equivalent, equivalent; CDI represents carbonyldiimidazole; DCM represents dichloromethane; PE represents petroleum ether; DIAD represents diisopropyl azodicarboxylate; DMF represents N, N-dimethylformamide; DMSO represents dimethyl sulfoxide; EtOAc for ethyl acetate; EtOH stands for ethanol; MeOH represents methanol; CBz represents benzyloxycarbonyl, an amine protecting group; BOC represents tert-butoxycarbonyl as an amine protecting group; HOAc represents acetic acid; NaCNBH3Represents sodium cyanoborohydride; r.t. represents room temperature; O/N stands for overnight; THF represents tetrahydrofuran; boc2O represents di-tert-butyl dicarbonate; TFA represents trifluoroacetic acid; DIPEA stands for diisopropylethylamine; the hydrochloride of the compound of the invention is added with saturated sodium bicarbonate solution to adjust the pH value to be neutral, and the free alkali of the compound is obtained by high performance liquid chromatography separation (neutral ammonium bicarbonate system).
The compounds are used according to the conventional naming principle in the field
Figure PCTCN2019105680-APPB-000056
Software nomenclature, commercial Compounds adopted supplier Catalogue namesWeighing is carried out.
Detailed Description
The present invention is described in detail below by way of examples, but is not meant to be limited to any of the disadvantages of the present invention. Having described the invention in detail and having disclosed specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.
Example 1
Figure PCTCN2019105680-APPB-000057
The synthetic route is as follows:
Figure PCTCN2019105680-APPB-000058
first step of
Sodium hydroxide (279g,6.99mol) was dissolved in water (3L), the reaction solution was cooled to 10 ℃ with an ice-water bath, and Compound 1-1(997g,3.49mol) was added to the reaction solution in portions and stirred at 10 ℃ for 2 hours. To the reaction mixture was added ethyl acetate (2L x 1) for extraction, and ethyl acetate (1.6L x 1) for extraction. The combined organic phases were washed with water (1.5L x 1), then with saturated brine (1.5L x 1), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent, thereby obtaining compound 1-2.1H NMR(400MHz,CDCl 3)δ7.18-7.14(m,2H),7.08-7.04(m,1H),6.95-6.92(m,2H),2.48-2.44(m,1H),1.80-1.76(m,1H),0.98-0.87(m,2H)。
Second step of
Compound 1-3(1.00g,3.92mmol) and compound 1-2(522mg,3.92mmol) were dissolved in anhydrous dichloromethane (20mL), and glacial acetic acid (706mg,11.8mmol) was added to the reaction solution. The reaction was stirred at 20 ℃ for 1h, sodium borohydride acetate (2.49g,11.8mmol) was added and the reaction was stirred at 20 ℃ for a further 10 h. The reaction solution was diluted with dichloromethane (80mL)After release, the mixture was washed successively with saturated aqueous sodium bicarbonate (100 mL. times.3), water (100 mL. times.2), and saturated brine (100 mL. times.1), dried over anhydrous sodium sulfate, filtered, and the resulting mother liquor was concentrated to give compound 1-4.1H NMR(400MHz,CDCl 3) δ 7.18-7.16(m,2H),7.11-7.17(m,1H),6.96-6.94(m,2H),3.92-3.89(m,1H),3.61-3.55(m,1H),3.52-3.48(m,3H),3.27-3.23(m,2H),2.24-2.21(m,1H),1.99-1.94(m,1H),1.85-1.79(m,1H),1.55-1.47(m,6H),1.38(s,9H),1.01-0.90(m, 2H). MS-ESI calculated value [ M + H%] +373, found 373.
The third step
Compound 1-4(1.10g,2.95mmol) was dissolved in anhydrous dichloromethane (20mL), and triethylamine (448mg,4.43mmol) and trifluoroacetic anhydride (930mg,4.43mmol) were added. The reaction mixture was stirred at 15 ℃ for 12 hours. Dichloromethane (50mL) was added to the reaction solution, the organic phase was washed with hydrochloric acid (1M,50mL × 1) and saturated brine (50mL × 1), dried over anhydrous sodium sulfate, filtered, the mother liquor was concentrated, and the crude product was isolated by column chromatography (5/1 dichloromethane/methanol, Rf ═ 0.38) to give compound 1-5. MS-ESI calculated value [ M-56+ H ]] +413,[M-Boc+H] +369, found 413,369.
The fourth step
Compounds 1-5(600mg,1.28mmol) were dissolved in anhydrous dichloromethane (6mL) and trifluoroacetic acid (4.62g,40.5mmol) was added at 20 ℃. The reaction solution was stirred at 20 ℃ for 2 hours, and the solvent was removed by concentration under reduced pressure, and the residue was dissolved in methylene chloride (6mL), and triethylamine (250. mu.L) was added thereto, followed by stirring at room temperature for half an hour, and the solvent was removed by concentration under reduced pressure to obtain compounds 1 to 6. MS-ESI calculated value [ M + H%] +369, found 369.
The fifth step
Compound 1-6(200mg,0.543mmol) and triethylamine (164mg,1.63mmol) were dissolved in acetonitrile (10mL), compound 1-7(157mg,0.869mmol) was added, and the reaction mixture was reacted at 50 ℃ for 12 hours. 1-7(49.1mg,0.271mmol) and triethylamine (54.9mg,0.543mmol) were further added to the reaction mixture, and the reaction mixture was reacted at 50 ℃ for 24 hours. The solvent was removed by concentration under reduced pressure, the product was dissolved in dichloromethane (50mL x 1) and then saturated with sodium bicarbonate (15mL x 1), water (15mL x 1) and saturated water in that orderAnd brine (15mL x 1), dried over anhydrous sodium sulfate, filtered, and the mother liquor concentrated. The product was isolated by thin layer chromatography (10/1 dichloromethane/methanol, Rf ═ 0.56) to afford compounds 1-8. MS-ESI calculated value [ M + H%] +469, found 469.
The sixth step
Compound 1-8(100mg,0.213mmol) was dissolved in a mixed solution of tetrahydrofuran (2mL), ethanol (2mL) and water (2mL), and sodium hydroxide (25.6mg,0.640mmol) was added. The reaction was carried out at 50 ℃ for 2 hours. The organic phase was removed by concentration under reduced pressure, the residue was dissolved in water (10mL), the pH was adjusted to 4 with hydrochloric acid (1mol/L), and the hydrochloride of Compound 1 was isolated by high performance liquid chromatography (acidic, hydrochloric acid system).1H NMR(400MHz,D 2O) δ 7.36-7.32(m,2H),7.28-7.25(m,1H),7.17-7.16(m,2H), 4.21-4.19(m,1H),4.16-4.08(m,2H),3.49-3.36(m,4H),3.17-3.11(m,2H),2.94-2.93(m,1H),2.84(t, J ═ 6.8Hz,2H),2.59-2.50(m,1H),2.45-2.39(m,1H),2.08-1.98(m,4H),1.85-1.77(m,1H),1.52-1.50(m,1H),1.44-1.39(m, 1H). MS-ESI calculated value [ M + H%] +345, found 345.
Example 2
Figure PCTCN2019105680-APPB-000059
The synthetic route is as follows:
Figure PCTCN2019105680-APPB-000060
first step of
Compound 2-2 was obtained by the fifth step of reference example 1. MS-ESI calculated value [ M + H%] +483, found 483.
Second step of
The hydrochloride of compound 2 was obtained according to the sixth step of example 1.1H NMR(400MHz,D 2O)δ7.34-7.30(m,2H),7.26-7.23(m,1H),7.16-7.14(m,2H),4.19-4.15(m,1H),4.08-4.07(m,2H),3.43(t,J＝13.2Hz,2H),3.09-3.05(m,3H),2.94-2.88(m,1H),2.53-2.49(m,1H),2.43-2.36(m,3H),2.13-1.91(m,7H),1.83-1.75(m,1H),1.53-1.48(m,1H),1.41-1.36(m, 1H). MS-ESI calculated value [ M + H%] +359, found 359.
Example 3
Figure PCTCN2019105680-APPB-000061
The synthetic route is as follows:
Figure PCTCN2019105680-APPB-000062
first step of
Compound 3-1(200mg,1.01mmol) was dissolved in anhydrous dichloromethane (5mL), triethylamine (306mg,3.03mmol) was added and cooled to 0 deg.C, Compound 3-2(231mg,2.02mmol) was added under nitrogen at 0 deg.C, reacted at 25 deg.C for 3 hours, the organic solvent was distilled off under reduced pressure, the residue and triethylamine (275mg,2.71mmol) were dissolved in acetonitrile (5mL), Compound 1-6(333mg, 0.905. mu. mol) was added, and reacted at 25 deg.C for 12 hours. The solvent was removed by concentration under reduced pressure, the residue was dissolved in dichloromethane (30mL), washed successively with saturated sodium bicarbonate (10 mL. times.2) and saturated brine (10 mL. times.1), dried over anhydrous sodium sulfate, filtered, and the mother liquor was concentrated. Separation by thin layer chromatography (1/1 petroleum ether/ethyl acetate, Rf 0.56) afforded compound 3-3. MS-ESI calculated value [ M + H%] +447, found 447.
Second step of
Compound 3-3(120mg,0.269mmol) was dissolved in a mixed solution of tetrahydrofuran (2mL), ethanol (2mL) and water (2mL), and sodium hydroxide (32.3mg,0.806mmol) was added. The reaction was carried out at 50 ℃ for 2 hours. The organic solvent was removed by concentration under reduced pressure, the residue was dissolved in water (10mL), the pH was adjusted to 4 with hydrochloric acid (1mol/L), and the hydrochloride of Compound 3 was isolated by high performance liquid chromatography (acidic, hydrochloric acid system).1H NMR(400MHz,CD 3OD)δ7.33-7.30(m,2H),7.25-7.22(m,1H),7.19-7.17(m,2H),4.19-4.02(m,3H),3.46-3.42(m,2H),3.17-3.06(m,2H),3.03-2.99(m,1H),2.83(s,3H),2.52-2.48(m,1H),2.38(m,1H),1.96-1.85(m,4H),1.71-1.63(m,1H),1.53(m,1H),1.43(m, 1H). MS-ESI calculated value [ M + H%] +351, found value 351.
Example 4
Figure PCTCN2019105680-APPB-000063
The synthetic route is as follows:
Figure PCTCN2019105680-APPB-000064
first step of
Compound 1-6(300mg,0.622mmol), compound 4-1(125mg,0.933mmol) and triethylamine (189mg,1.87mmol) were dissolved in acetonitrile (5 mL). The reaction was stirred at 50 ℃ for 10 hours, concentrated under reduced pressure to remove the solvent, the residue was dissolved in dichloromethane (50mL), the organic phase was washed successively with water (50mL x 1) and saturated brine (50mL x 1), dried over anhydrous sodium sulfate, filtered, the mother liquor was concentrated, and the crude product was isolated by thin layer chromatography (1/2 petroleum ether/ethyl acetate, Rf ═ 0.24) to give compound 4-2. MS-ESI calculated value [ M + H%] +422, measured value 422.
Second step of
Compound 4-2(100mg,0.237mmol) was dissolved in tetrahydrofuran (2mL), water (2mL) and ethanol (2mL), and sodium hydroxide (28.5mg,0.712mmol) was added. The reaction solution was stirred at 50 ℃ for 2 hours, then tetrahydrofuran and ethanol were removed by concentration under reduced pressure, the residue was dissolved in water (10mL), pH was adjusted to 4 with hydrochloric acid (1M), and the residue after concentration under reduced pressure was separated by high performance liquid chromatography (acidic, hydrochloric acid system) to give the hydrochloride of compound 4.1H NMR(400MHz,CD 3OD)δ7.23-7.20(m,2H),7.15-7.10(m,3H),4.15-4.10(m,1H),4.07-4.02(m,2H),3.48-3.36(m,3H),3.32-3.29(m,1H),3.17-3.06(m,3H),2.91-2.90(m,1H),2.73-2.71(m,1H),2.56-2.53(m,1H),2.33-2.30(m,1H),2.08-2.01(m,4H),1.90-1.87(m,1H),1.56-1.52(m,1H),1.31-1.29(m,1H). MS-ESI calculated value [ M + H%] +326, found 326.
Example 5
Figure PCTCN2019105680-APPB-000065
The synthetic route is as follows:
Figure PCTCN2019105680-APPB-000066
first step of
Dissolving compound 5-1(56.5mg,0.298mmol), O- (7-azabenzotriazol-1-yl) -N, N, N, N-tetramethyluronium hexafluorophosphate (155mg,0.407mmol) and N, N-diisopropylethylamine (70.2mg,0.543mmol) in dichloromethane (6mL), stirring the reaction solution at 30 ℃ for 0.5 h, then, compound 1-6(100mg,0.271mmol) was added to the reaction solution, and the new reaction solution was further stirred at 30 ℃ for 10 hours, this was diluted with dichloromethane (10mL), washed successively with water (20mL x 1) and saturated brine (20mL x 1), dried over anhydrous sodium sulfate, filtered, the mother liquor was concentrated, and the crude product was isolated by thin layer chromatography (2/1 petroleum ether/ethyl acetate, Rf ═ 0.3) to give compound 5-2. MS-ESI calculated value [ M + H%] +540, found 540.
Second step of
Compound 5-2(70.0mg,0.130mmol) was dissolved in methanol hydrochloride (4mol/L,5mL) at 30 ℃. The reaction mixture was stirred at 30 ℃ for 10 hours, the solvent was removed by concentration under reduced pressure, the residue was dissolved in tetrahydrofuran (2mL), water (2mL) and ethanol (2mL), and sodium hydroxide (15.6mg,0.389mmol) was added. The reaction solution was stirred at 50 ℃ for 2 hours, concentrated under reduced pressure to remove tetrahydrofuran and ethanol, the residue was diluted with water (10mL), adjusted to pH 4 with hydrochloric acid (1mol/L), and concentrated under reduced pressure, and the residue was subjected to high performance liquid chromatography (acidic, hydrochloric acid system) to give the hydrochloride of compound 5.1H NMR(400MHz,CD 3OD)δ7.35-7.31(m,2H),7.26-7.21(m,3H),4.48-4.45(m,1H),4.20-3.97(m,4H),3.71-3.49(m,2H),3.37-3.17(m,1H),3.01(t,J＝3.2Hz,1H) 2.66-2.63(m,1H),2.43-2.41(m,1H),2.08-1.99(m,1H),1.97-1.80(m,3H),1.72-1.58(m,2H),1.51-1.40(m, 4H). MS-ESI calculated value [ M + H%] +344, found 344.
Example 6
Figure PCTCN2019105680-APPB-000067
The synthetic route is as follows:
Figure PCTCN2019105680-APPB-000068
first step of
Compound 6-2 was obtained in the first step of reference example 5. MS-ESI calculated value [ M + H%] +568, found 568.
Second step of
The second step of reference example 4 gave crude compound 6-3. MS-ESI calculated value [ M + H%] +472, found 472.
The third step
Compound 6-3(70.0mg, 148. mu. mol) was dissolved in methanol hydrochloride (4M,10mL) at 25 ℃. The reaction solution is stirred and reacted for 1 hour at 25 ℃, the solvent is removed by decompression and concentration, and the residue is separated by high performance liquid chromatography (acidity, hydrochloric acid system) to obtain the hydrochloride of the compound 6.1H NMR(400MHz,CD 3OD) δ 7.35-7.31(m,2H),7.26-7.21(m,3H),4.40-4.35(m,1H),4.20-3.96(m,4H),3.81-3.64(m,1H),3.62-3.43(m,1H),3.48-3.16(m,1H),3.03-3.01(m,1H),2.68-2.62(m,1H),2.47-2.37(m,1H),2.22-2.18(m,1H),2.09-2.05(m,1H),1.97-1.81(m,3H),1.68-1.58(m,2H),1.45-1.40(m,1H),1.14-1.10(m,3H),1.05-1.02(m, 3H). MS-ESI calculated value [ M + H%] +372, measured value 372.
Example 7
Figure PCTCN2019105680-APPB-000069
The synthetic route is as follows:
Figure PCTCN2019105680-APPB-000070
first step of
Compound 7-1(80.0mg,0.639mmol) was dissolved in anhydrous tetrahydrofuran (6mL), and after cooling to 0 deg.C, N-diisopropylethylamine (165mg,1.28mmol) and methanesulfonyl chloride (110mg,0.959mmol) were added to the solution, and the reaction solution was stirred at 29 deg.C for 1 hour. The reaction solution was diluted with ethyl acetate (30mL), washed successively with a saturated sodium bicarbonate solution (30mL × 1) and a saturated sodium chloride solution (30mL × 1), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give crude compound 7-2.1H NMR(400MHz,DMSO-d 6)δ4.56(t,J＝6.4Hz,2H),3.52(t,J＝6.4Hz,2H),3.29(s,3H)。
Second step of
Compound 7-3 was obtained in the first step of reference example 4. MS-ESI calculated value [ M + H%] +476, found 476.
The third step
The second step of reference example 4 gave the hydrochloride salt of compound 7.1H NMR(400MHz,CD 3OD) δ 7.36-7.32(m,2H),7.28-7.24(m,1H),7.22-7.20(m,2H),4.25-4.21(m,1H),4.18-4.12(m,2H),3.67-3.65(m,2H),3.62-3.50(m,4H),3.32-3.26(m,2H),3.06-3.03(m,1H),2.63-2.58(m,1H),2.48-2.43(m,1H),2.27-2.06(m,4H),2.00-1.94(m,1H),1.64-1.59(m,1H),1.47-1.42(m, 1H). MS-ESI calculated value [ M + H%] +380, found 380.
Example 8
Figure PCTCN2019105680-APPB-000071
The synthetic route is as follows:
Figure PCTCN2019105680-APPB-000072
first step of
Compound 8-2 was obtained in the first step of reference example 4. MS-ESI calculated value [ M + H%] +490, found 490.
Second step of
The second step of reference example 4 gave the hydrochloride salt of compound 8.1H NMR(400MHz,D 2O) delta 7.38-7.34(m,2H),7.30-7.27(m,1H),7.20-7.18(m,2H),4.26-4.21(m,1H),4.16-4.08(m,2H),3.63-3.46(m,2H),3.33-3.25(m,4H), 3.19-3.07(m,2H),2.98-2.93(m,1H),2.64-2.53(m,1H),2.47-2.41(m,1H),2.28-2.20(m,2H),2.16-1.95(m,4H),1.89-1.79(m,1H),1.56-1.50(m,1H),1.46-1.41(m, 1H). MS-ESI calculated value [ M + H%] +394, found 394.
Example 9
Figure PCTCN2019105680-APPB-000073
The synthetic route is as follows:
Figure PCTCN2019105680-APPB-000074
first step of
Compound 1-6(150mg,0.407mmol) was dissolved in acetonitrile (2mL), compound 9-1(221mg,2.04mol) and triethylamine (206mg,2.04mmol) were added to the reaction solution, and the system was raised to 50 ℃ and stirred for reaction for 12 hours. The solvent was removed by concentration under reduced pressure, and the crude product was isolated and purified by thin layer chromatography (10/1 dichloromethane/methanol, Rf ═ 0.4) to give compound 9-2. MS-ESI calculated value [ M + H%] +441, measured value 441.
Second step of
Compound 9-2(131mg,0.291mmol) was dissolved in tetrahydrofuran (2mL) and absolute ethanol (2mL), and sodium hydroxide (33.8mg,0.845mmol) was dissolved in water (2mL) and added dropwise to the solution, followed by stirring at 50 ℃ for 2 hours. Concentrating the reaction solution under reduced pressureRemoving solvent, adjusting pH to 5 with hydrochloric acid (1mol/L), concentrating under reduced pressure, and separating and purifying the crude product by high performance liquid chromatography (acidic, hydrochloric acid system) to obtain hydrochloride of compound 9.1H NMR(400MHz,CD 3OD) δ 7.33-7.28(m,2H),7.25-7.19(m,3H),4.22-7.14(m,3H),3.68-3.54(m,2H),3.36-3.25(m,3H),3.17-3.15(m,1H),3.04-3.00(m,1H),2.66-2.60(m,1H),2.55-2.39(m,1H),2.34-2.19(m,1H),2.16-1.96(m,4H),1.67-1.60(m,1H),1.45-1.35(m, 7H). MS-ESI calculated value [ M + H%] +345, found 345.
Example 10
Figure PCTCN2019105680-APPB-000075
Synthetic route
Figure PCTCN2019105680-APPB-000076
First step of
Compound 10-1(5.00g,2.87mmol) was dissolved in ethyl acetate (5mL) and ethyl acetate hydrochloride (4mol/L,15mL), and the reaction mixture was stirred at 15 ℃ for 1 hour. Concentrating under reduced pressure to obtain crude compound 10-2.1HNMR(400MHz,DMSO-d 6)δ8.51(s,3H),1.38(s,6H)。
Second step of
Compound 10-2(200mg,1.43mmol) was dissolved in methanol (5ml), formaldehyde (465mg, 37% aqueous solution, 5.73mmol) and Pd/C (153mg, 10% wet palladium on carbon, 0.143mmol) were added, the reaction mixture was reacted under a hydrogen atmosphere (50psi) at 20 ℃ for 16 hours, and the reaction mixture was filtered and concentrated to give compound 10-3.1HNMR(400MHz,DMSO-d 6)δ2.74(s,6H),1.50(s,6H)。
The third step
Dissolving compound 10-3(100mg,0.762mmol) in N, N-dimethylformamide (5ml), and adding compound 1-6(247mg,0.610mmol), triethylamine (386mg,3.81mmol) and O- (7-azabenzotriazol-1-yl) -N, N, N, N-tetramethyluronium hexafluoro-chlorideA phosphonium salt (386mg,2.29mmol), the reaction mixture was stirred at 30 ℃ for 15 hours, ethyl acetate (20mL) and saturated brine (5mL) were added to the reaction mixture, the organic layer was washed with saturated brine (10mL × 2), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the crude product was isolated by column chromatography (20/1 dichloromethane/methanol, Rf ═ 0.51) to give compound 10-4.1H NMR(400MHz,CDCl 3) δ 7.33-7.30(m,2H),7.24-7.22(m,1H),7.07-7.05(m,2H),4.72-4.49(m,1H),4.10-3.97(m,2H),3.03-3.01(m,1H),2.96(s,1H),2.88(s,1H),2.80(s,6H),2.41-2.08(m,6H),1.96-1.66(m,4H),1.61-1.45(m,4H),1.26-1.24(m, 3H). MS-ESI calculated value [ M + H%] +482, found 482.
The fourth step
Dissolving compound 10-4(143mg,0.173mmol) in ethanol (3mL), tetrahydrofuran (3mL) and water (3mL), adding sodium hydroxide (20.8mg), stirring the reaction solution at 50 ℃ for 2 hours, concentrating the reaction solution under reduced pressure, and separating the crude product by high performance liquid chromatography (acidic, hydrochloric acid system) to obtain the hydrochloride of compound 10.1H NMR(400MHz,CD 3OD) δ 7.32-7.28(m,2H),7.24-7.18(m,3H),4.07-3.99(m,5H),3.58-3.33(m,2H),3.02-2.99(m,1H),2.89-2.65(m,6H),2.63-2.58(m,1H),2.43-2.37(m,1H), 2.06-1.99(m,1H),1.93-1.81(m,3H),1.67(s,6H),1.64-1.57(m,2H),1.43-1.38(m, 1H). MS-ESI calculated value [ M + H%] +386, found 386.
Example 11
Figure PCTCN2019105680-APPB-000077
The synthetic route is as follows:
Figure PCTCN2019105680-APPB-000078
first step of
Mixing compound 11-1(64.9mg,0.298mmol), O- (7-azabenzotriazole-1-yl) -N, N, N, N-tetramethyluronium hexafluorophosphate (124mg,0.326mmol) and N, N-diisopropylethylamine(105mg,0.814mmol) was dissolved in N, N-dimethylformamide (2mL), the reaction was stirred at 25 ℃ for 1 hour, then Compound 1-6(100mg,0.271mmol) was added to the reaction, and the reaction was stirred at 25 ℃ for 12 hours. This was diluted with water (30mL), extracted with ethyl acetate (10mL × 3), washed with saturated brine (30mL × 1), dried over anhydrous sodium sulfate, filtered, the mother liquor was concentrated, and the crude product was isolated by thin layer chromatography (1:1 petroleum ether/ethyl acetate, Rf ═ 0.8) to give compound 11-2.1H NMR(400MHz,CDCl 3) δ 7.34-7.31(m,2H),7.25-7.23(m,1H),7.11-7.06(m,2H),5.45-5.44(m,1H),4.64-4.58(m,1H),4.10-4.05(m,1H),4.00-3.91(m,3H),3.50-3.32(m,2H),3.24-3.21(m,2H),2.41-2.33(m,1H),2.20-2.07(m,2H),1.76-1.73(m,2H),1.54-1.43(m,11H),1.28-1.25(m, 8H). MS-ESI calculated value [ M + H%] +568, found 568.
Second step of
Compound 11-2(150mg,0.264mmol) was dissolved in tetrahydrofuran (2mL) and absolute ethanol (2mL), and sodium hydroxide (21.1mg,0.528mmol) was dissolved in water (2mL) and added dropwise to the solution, followed by stirring at 20 ℃ for 2 hours. Adjusting pH to 5 with hydrochloric acid (1mol/L), and concentrating under reduced pressure to obtain crude compound 11-3. MS-ESI calculated value [ M + H%] +472, found 472.
The third step
Compound 11-3(150mg,0.318mmol) was dissolved in dichloromethane (2mL), and trifluoroacetic acid (109mg, 0.954mmol) was added to the reaction mixture at 0 ℃ to raise the temperature to 25 ℃ naturally, followed by stirring and reaction for 1 hour. Concentrating under reduced pressure to remove solvent, and separating the residue by high performance liquid chromatography (acidic, hydrochloric acid system) to obtain hydrochloride of compound 11.1H NMR(400MHz,CD 3OD) δ 7.34-7.30(m,2H),7.26-7.19(m,3H),4.22-4.07(m,3H),4.00-3.97(m,2H),3.43-3.40(m,2H),3.05-3.01(m,3H),2.62-2.58(m,1H),2.44-2.37(m,1H),2.04-1.98(m,1H),1.87-1.75(m,3H),1.64-1.55(m,2H),1.45-1.40(m, 7H). MS-ESI calculated value [ M + H%] +372, measured value 372.
Example 12
Figure PCTCN2019105680-APPB-000079
The synthetic route is as follows:
Figure PCTCN2019105680-APPB-000080
first step of
Compound 12-2 was obtained in the first step of reference example 11.1H NMR(400MHz,CDCl 3) δ 7.34-7.31(m,2H),7.25-7.23(m,1H),7.11-7.06(m,2H),5.36-5.32(m,1H),4.64-4.61(m,1H),4.51-4.44(m,1H),4.09-4.04(m,1H),3.99-3.95(m,1H),3.75-3.40(m,2H),3.26-3.17(m,1H),2.40-2.33(m,1H),2.20-2.07(m,2H),1.97-1.83(m,2H),1.76-1.69(m,2H),1.60-1.58(m,2H),1.51-1.43(m,11H),0.99-1.86(m, 6H). MS-ESI calculated value [ M + H%] +568, found 568.
Second step of
The second step of reference example 11 gave compound 12-3. MS-ESI calculated value [ M + H%] +472, found 472.
The third step
The third step of reference example 11 gave the hydrochloride salt of compound 12.1H NMR(400MHz,CD 3OD) δ 7.34-7.30(m,2H),7.26-7.19(m,3H),4.38-4.32(m,1H),4.24-3.97(m,4H),3.79-3.16(m,3H),3.02-3.00(m,1H),2.63-2.57(m,1H),2.46-2.35(m,1H),2.22-2.14(m,1H),2.07-1.98(m,1H),1.91-1.75(m,3H),1.70-1.53(m,2H),1.44-1.39(m,1H),1.12-1.08(m,3H),1.03-1.00(m, 3H). MS-ESI calculated value [ M + H%] +372, measured value 372.
Example 13
Figure PCTCN2019105680-APPB-000081
The synthetic route is as follows:
Figure PCTCN2019105680-APPB-000082
first step of
Compound 13-2 was obtained in the first step of reference example 11. MS-ESI calculated value [ M + H%] +540, found 540.
Second step of
The second step of reference example 11 gave compound 13-3. MS-ESI calculated value [ M + H%] +444, found value 444.
The third step
The third step of reference example 11 gave the hydrochloride salt of compound 13.1H NMR(400MHz,CD 3OD) δ 7.33-7.29(m,2H),7.25-7.18(m,3H),4.47-4.42(m,1H),4.17-3.98(m,4H),3.69-3.43(m,2H),3.27-3.15(m,1H),3.02-2.99(m,1H),2.61-2.56(m,1H),2.45-2.35(m,1H),2.06-1.96(m,1H),1.93-1.72(m,3H),1.69-1.51(m,2H),1.48-1.39(m, 4H). MS-ESI calculated value [ M + H%] +344, found 344.
Example 14
Figure PCTCN2019105680-APPB-000083
The synthetic route is as follows:
Figure PCTCN2019105680-APPB-000084
first step of
Compound 14-2 was obtained in the first step of reference example 11.1H NMR(400MHz,CDCl 3) δ 7.34-7.30(m,2H),7.27-7.23(m,1H),7.10-7.05(m,2H),5.25-5.24(m,1H),4.69-4.57(m,1H),4.19-4.04(m,1H),4.01-3.94(m,1H),3.59-3.37(m,2H),3.29-3.14(m,1H),2.80(s,2H),2.47-2.32(m,3H),2.21-2.04(m,2H),1.76-1.69(m,2H),1.60-1.42(m,3H)1.29-1.24(m, 6H). MS-ESI calculated value [ M + H%] +469, found 469.
Second step of
The third step of reference example 11 gave compound 14 as hydrochloric acidAnd (3) salt.1H NMR(400MHz,CD 3OD) delta 7.33-7.29(M,2H),7.25-7.18(M,3H),4.16-4.06(M,4H),3.84-3.77(M,1H),3.51-3.46(M,1H),3.27-3.19(M,1H),3.04-2.97(M,1H),2.63-2.52(M,3H),2.40-2.35(M,1H),1.99-1.96(M,1H),1.86-1.77(M,3H),1.65-1.51(M,2H),1.44-1.39(M,1H),1.28(s,6H) MS-ESI calculation [ M + H] +373, found 373.
Example 15
Figure PCTCN2019105680-APPB-000085
The synthetic route is as follows:
Figure PCTCN2019105680-APPB-000086
first step of
Compound 15-2 was obtained in the first step of reference example 11. MS-ESI calculated value [ M + H%] +526, measured value 526.
Second step of
The second step of reference example 11 gave compound 15-3. MS-ESI calculated value [ M + H%] +430, measured value 430.
The third step
The third step of reference example 11 gave compound 15 as the hydrochloride salt.1H NMR(400MHz,CD 3OD) δ 7.33-7.29(m,2H),7.24-7.18(m,3H),4.17-3.91(m,6H),3.54-3.41(m,2H),3.30-3.22(m,1H),3.02-3.00(m,1H),2.64-2.60(m,1H),2.42-2.36(m,1H),2.06-1.98(m,1H),1.89-1.73(m,3H),1.69-1.52(m,2H),1.43-1.38(m, 1H). MS-ESI calculated value [ M + H%] +330, measured value 330.
Example 16
Figure PCTCN2019105680-APPB-000087
The synthetic route is as follows:
Figure PCTCN2019105680-APPB-000088
first step of
Compound 16-2 was obtained in the first step of reference example 11. MS-ESI calculated value [ M + H%] +554, found 554.
Second step of
The second step of reference example 11 gave compound 16-3. MS-ESI calculated value [ M + H%] +458, measured value 458.
The third step
The third step of reference example 11 gave compound 16 as the hydrochloride salt.1H NMR(400MHz,CD 3OD) δ 7.33-7.29(m,2H),7.24-7.18(m,3H),4.17-4.08(m,3H),4.05-3.85(m,2H),3.52-3.33(m,2H),3.02-3.00(m,1H),2.63-2.56(m,1H),2.43-2.37(m,1H),2.04-1.98(m,1H),1.86-1.80(m,3H),1.68-1.57(m,8H),1.44-1.39(m, 1H). MS-ESI calculated value [ M + H%] +358, found 358.
Example 17
Figure PCTCN2019105680-APPB-000089
The synthetic route is as follows:
Figure PCTCN2019105680-APPB-000090
first step of
Compound 17-1(109mg,0.503mmol), O- (7-azobenzotriazol) -N, N, N, N, -tetramethyluronium hexafluorophosphate (225mg,0.593mmol), N, N-diisopropylethylamine (95.8mg,0.741mmol) were dissolved in N, N-dimethylformamide (3mL) and stirred at 25 ℃ for 0.5 hour. Compounds 1-6(200mg,0.494mmol) were added to the reaction solution. The reaction mixture was stirred at 25 ℃ for 11.5 hours, poured into saturated aqueous sodium bicarbonate (10mL), and then treated with acetic acidExtraction with ethyl ester (20mL × 3), combination of organic phases, washing of the organic phase with saturated aqueous sodium chloride solution (20mL × 1), drying over anhydrous sodium sulfate, filtration, and column chromatography of the crude product (2/1 petroleum ether/ethyl acetate, Rf ═ 0.15) gave compound 17-2. MS-ESI calculated value [ M + H%] +568, found 568.
Second step of
Compound 17-2(204mg,0.359mmol) and sodium hydroxide (43.1mg,0.450mmol) were dissolved in tetrahydrofuran (1mL), ethanol (1mL) and water (1mL), and the reaction mixture was stirred at 20 ℃ for 2 hours and concentrated under reduced pressure to give compound 17-3. MS-ESI calculated value [ M + H%] +472, found 472.
The third step
Compound 17-3(216mg,0.458mmol) was dissolved in methanol hydrochloride (5 mL). The reaction solution is stirred and reacted for 1 hour at the temperature of 25 ℃ under the condition of nitrogen, the residue is separated by high performance liquid chromatography (acidity, hydrochloric acid system) after the reaction solution is decompressed and concentrated, and the hydrochloride of the compound 17 is obtained.1H NMR(400MHz,CD 3OD) δ 7.33-7.19(m,5H),4.17-4.10(m,4H),3.53-3.49(m,1H),3.48-3.46(m,1H),3.42-3.31(m,1H),3.02-3.00(m,1H),2.82-2.73(m,2H),2.62(s,1H),2.41-2.37(m,1H),2.01-2.03(m,1H),1.88-1.73(m,3H),1.63-1.50(m,2H),1.42(s, 7H). MS-ESI calculated value [ M + H%] +372, measured value 372.
Example 18
Figure PCTCN2019105680-APPB-000091
The synthetic route is as follows:
Figure PCTCN2019105680-APPB-000092
first step of
Dissolving the compound 18-1(56.5mg,0.299mmol) in anhydrous N, N-dimethylformamide (2mL), and adding N, N-diisopropylethylamine (70.2mg,0.543mmol) and O- (7-azabenzotriazole-1-yl) -N, N-diisopropylethylamine (N, N-diisopropylethylamine) and O- (7-azabenzotriazole-1-yl)N, N-tetramethyluronium hexafluorophosphate (155mg,0.407 mmol). The reaction solution was stirred at 30 ℃ for 1 hour. Then, compound 1-6(100mg,0.271mmol) was added to the reaction mixture. The reaction solution was stirred at 30 ℃ for 4 hours. Water (10mL) was added to the reaction solution, extracted with ethyl acetate (10mL x 3), the organic phases were combined, washed with saturated sodium chloride solution (20mL x 1), the organic phase was dried over anhydrous sodium sulfate, filtered, and the crude product was separated by thin layer chromatography (1/1 petroleum ether/ethyl acetate, Rf ═ 0.41) to give compound 18-2. MS-ESI calculated value [ M + H%] +540, found 540.
Second step of
Compound 18-2(130mg,0.238mmol) was dissolved in tetrahydrofuran (2mL) and water (2mL), and sodium hydroxide (19.1mg,0.477mmol) was added to the reaction mixture. The reaction mixture was stirred at 50 ℃ for 3 hours, concentrated under reduced pressure to remove the solvent, the residue was diluted with water (10mL), extracted with ethyl acetate (10 mL. times.3), the organic phases were combined, washed with saturated sodium chloride (30 mL. times.1), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give compound 18-3. MS-ESI calculated value [ M + H%] +444, found value 444.
The third step
Compound 18-3(100mg,0.225mmol) was dissolved in ethyl acetate (42mL), and ethyl acetate hydrochloride (4mol/L,0.563mL,2.25mmol) was added to the reaction mixture. The reaction mixture was stirred at 25 ℃ for 2 hours, and the solvent was removed by concentration under reduced pressure. The hydrochloride of compound 18 is prepared by high performance liquid chromatography (acidic, hydrochloric acid system).1H NMR(400MHz,CD 3OD) δ 7.35-7.31(m,2H),7.25-7.21(m,3H),4.19-4.11(m,4H),3.68-3.61(m,1H),3.52-3.46(m,1H),3.24-3.21(m,3H),3.04-3.02(m,1H),2.89-2.83(m,2H),2.65-2.64(m,1H),2.42-2.40(m,1H),2.07-2.02(m,1H),1.91-1.73(m,3H),1.72-1.53(m,2H),1.45-1.42(m, 1H). MS-ESI calculated value [ M + H%] +344, found 344.
Example 19
Figure PCTCN2019105680-APPB-000093
The synthetic route is as follows:
Figure PCTCN2019105680-APPB-000094
first step of
Compound 19-1(429mg,4.12mmol) was dissolved in anhydrous N, N-dimethylformamide (10mL), and N, N-diisopropylethylamine (665mg,5.15mmol), 1-hydroxybenzotriazole (556mg,4.12mmol) and 1- (3-dimethylaminopropyl) -3-acetaldehyde hydrochloride (789mg,4.12mmol) were added to the reaction solution. The reaction solution was stirred at 30 ℃ for 1 hour. Then, compound 1-6(800mg,2.06mmol) was added to the reaction solution. The reaction solution was stirred at 30 ℃ for 12 hours. Water (20mL) was added to the reaction solution, extracted with ethyl acetate (10mL x 3), the organic phases were combined, washed with saturated sodium chloride (20mL x 1), the organic phase was dried over anhydrous sodium sulfate, filtered, and the crude product was separated by thin layer chromatography (1/1 petroleum ether/ethyl acetate, Rf ═ 0.51) to give compound 19-2. MS-ESI calculated value [ M + H%] +455, found value 455.
Second step of
Compound 19-2(700mg,1.54mmol) was dissolved in tetrahydrofuran (4mL) and water (4mL), and sodium hydroxide (123mg,3.08mmol) was added to the reaction solution. The reaction mixture was stirred at 50 ℃ for 3 hours, the solvent was removed by concentration under reduced pressure, and the residue was diluted with water (10mL) and adjusted to pH 4 with an aqueous hydrochloric acid solution (1 mol/L). The hydrochloride salt of compound 19 was prepared by high performance liquid chromatography (acidic, hydrochloric acid system).1H NMR(400MHz,CD 3OD) δ 7.36-7.32(m,2H),7.28-7.26(m,1H),7.17-7.16(m,2H),4.22-4.20(m,1H),4.16-4.12(m,1H),4.07-4.05(m,2H),3.84-3.56(m,2H),3.49-3.20(m,1H),2.95-2.91(m,1H),2.53-2.49(m,1H),2.46-2.40(m,1H),1.97-1.92(m,1H),1.84-1.69(m,3H),1.65-1.58(m,1H),1.53-1.49(m,1H),1.45-1.41(m,1H),1.39(s, 6H). MS-ESI calculated value [ M + H%] +359, found 359.
Example 20
Figure PCTCN2019105680-APPB-000095
The synthetic route is as follows:
Figure PCTCN2019105680-APPB-000096
first step of
Compound 20-1(53.8mg,0.407mmol) was dissolved in anhydrous N, N-dimethylformamide (2mL), and N, N-diisopropylethylamine (87.7mg,0.679mmol), O- (7-azabenzotriazol-1-yl) -N, N, N, N-tetramethyluronium hexafluorophosphate salt (206mg,0.543mmol) was added to the reaction mixture. The reaction solution was stirred at 30 ℃ for 1 hour. Then, compound 1-6(100mg,0.271mmol) was added to the reaction mixture. The reaction solution was stirred at 30 ℃ for 4 hours. Water (10mL) was added to the reaction solution, extracted with ethyl acetate (10mL x 3), the organic phases were combined, washed with saturated sodium chloride (20mL x 1), the organic phase was dried over anhydrous sodium sulfate, filtered, and the crude product was separated by thin layer chromatography (1/1 petroleum ether/ethyl acetate, Rf ═ 0.40) to give compound 20-2. MS-ESI calculated value [ M + H%] +483, found 483.
Second step of
Compound 20-2(100mg,0.207mmol) was dissolved in tetrahydrofuran (1mL) and water (1mL), and sodium hydroxide (24.9mg,0.622mmol) was added to the reaction solution. The reaction mixture was stirred at 60 ℃ for 3 hours, the solvent was removed by concentration under reduced pressure, the residue was diluted with water (10mL), and the pH was adjusted to about 7 with an aqueous hydrochloric acid solution (1 mol/L). Compound 20 was prepared by high performance liquid chromatography (neutral system).1H NMR(400MHz,CD 3OD) δ 7.27-7.23(m,2H),7.16-7.12(m,1H),7.08-7.06(m,2H),3.96-3.92(m,1H),3.73-3.64(m,3H),3.56-3.50(m,1H),3.30-3.23(m,1H),2.67-2.64(m,2H),2.53-2.50(m,2H),2.36-2.32(m,1H),2.18-2.13(m,1H),1.99-1.92(m,1H),1.81-1.63(m,6H),1.11-1.04(m, 2H). MS-ESI calculated value [ M + H%] +373, found 373.
Example 21
Figure PCTCN2019105680-APPB-000097
The synthetic route is as follows:
Figure PCTCN2019105680-APPB-000098
first step of
Compound 1-6(100mg,0.271mmol) and compound 21-1(50.0mg,0.299mmol) were dissolved in acetonitrile (6mL), and triethylamine (54.9mg,0.543mmol) was added to the reaction mixture. The reaction solution was stirred at 50 ℃ for 12 hours. The reaction was directly concentrated, and the crude product was isolated by thin layer chromatography (10/1 dichloromethane/methanol, Rf ═ 0.50) to give compound 21-2. MS-ESI calculated value [ M + H%] +455, found value 455.
Second step of
Compound 21-2(120mg,0.264mmol) was dissolved in tetrahydrofuran (2mL) and water (2mL), and sodium hydroxide (31.7mg,0.792mmol) was added to the reaction solution. The reaction mixture was stirred at 60 ℃ for 3 hours, the solvent was removed by concentration under reduced pressure, the residue was diluted with water (10mL), and the pH was adjusted to about 5 with an aqueous hydrochloric acid solution (1 mol/L). The hydrochloride salt of compound 21 was prepared by high performance liquid chromatography (acidic, hydrochloric acid system).1H NMR(400MHz,CD 3OD) δ 7.35-7.31(m,2H),7.26-7.21(m,3H),4.24-4.14(m,3H),3.56-3.49(m,2H),3.33-3.26(m,4H),3.05-3.03(m,1H),2.72-2.67(m,1H),2.46-2.43(m,1H),2.19-2.09(m,4H),1.98-1.94(m,3H),1.71-1.66(m,1H),1.43-1.41(m,1H),1.28(s, 6H). MS-ESI calculated value [ M + H%] +359, found 359.
Example 22
Figure PCTCN2019105680-APPB-000099
The synthetic route is as follows:
Figure PCTCN2019105680-APPB-000100
first step of
Compound 22-2 was obtained in the first step of reference example 11. MS-ESI calculated value [ M + H%] +602, measured value 602.
Second step of
The second step of reference example 11 gave compound 22-3. MS-ESI calculated value [ M + H%] +506, measured value 506.
The third step
The third step of reference example 11 gave compound 22 as the hydrochloride salt.1H NMR(400MHz,CD 3OD) δ 7.60-7.43(m,5H),7.30-7.13(m,5H),5.55-5.51(m,1H),4.21-4.01(m,4H),3.48-3.37(m,2H),3.24-3.05(m,1H),2.98-2.94(m,1H),2.56-2.55(m,1H),2.41-2.17(m,1H),2.08-1.94(m,1H),1.86-1.68(m,2H),1.57-1.35(m, 4H). MS-ESI calculated value [ M + H%] +406, measured value 406.
Example 23
Figure PCTCN2019105680-APPB-000101
The synthetic route is as follows:
Figure PCTCN2019105680-APPB-000102
first step of
The compound 1-6(200mg,0.494mmol) and the compound 23-1(61.1mg,0.593mmol) were dissolved in N, N-dimethylformamide (4mL), and O- (7-azabenzotriazol-1-yl) -N, N, N, N-tetramethyluronium hexafluorophosphate salt (282mg,0.741mmol), N, N-diisopropylethylamine (128mg,0.988mmol) and the reaction mixture were added. The reaction solution was stirred at 25 ℃ for 12 hours. Water (10mL) was added, extracted with ethyl acetate (10mL x 3), and the organic phase was washed with saturated sodium chloride (20mL x 1), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was isolated by thin layer chromatography (1:1 petroleum ether/ethyl acetate, Rf ═ 0.40) to afford compound 23-2. MS-ESI calculated value [ M + H%] +454, found 454.
Second step of
Reacting the compound 23-2(203mg,0.439mmol) was dissolved in tetrahydrofuran (2mL) and water (2mL), and sodium hydroxide (17.5mg,0.439mmol) was added to the reaction solution. The reaction mixture was stirred at 50 ℃ for 12 hours, concentrated under reduced pressure to remove the organic solvent, and adjusted to pH 5 with hydrochloric acid (1 mol/L). The hydrochloride salt of compound 23 was prepared by high performance liquid chromatography (acidic, hydrochloric acid system).1H NMR(400MHz,CD 3OD) δ 7.35-7.31(m,2H),7.26-7.21(m,3H),4.37-4.29(m,2H),4.23-4.08(m,4H),3.50-3.42(m,2H),3.30-3.23(m,1H),3.06-3.02(m,1H),2.99-2.98(m,6H),2.68-2.64(m,1H),2.44-2.39(m,1H),2.08-2.03(m,1H),1.95-1.78(m,3H),1.73-1.59(m,2H),1.46-1.40(m, 1H). MS-ESI calculated value [ M + H%] +358, found 358.
Example 24
Figure PCTCN2019105680-APPB-000103
The synthetic route is as follows:
Figure PCTCN2019105680-APPB-000104
first step of
The compound 1-6(200mg,0.494mmol) and the compound 24-1(76.6mg,0.593mmol) were dissolved in N, N-dimethylformamide (4mL), and O- (7-azabenzotriazol-1-yl) -N, N, N, N-tetramethyluronium hexafluorophosphate (282mg,0.741mmol), N, N-diisopropylethylamine (128mg,0.988mmol) and the reaction mixture were added. The reaction solution was stirred at 25 ℃ for 12 hours. Water (10mL) was added, extraction was performed with ethyl acetate (10mL x 3), the organic phase was washed with saturated sodium chloride (20mL x 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was isolated by thin layer chromatography (1:1 petroleum ether/ethyl acetate, Rf ═ 0.42) to afford compound 24-2. MS-ESI calculated value [ M + H%] +480, found 480.
Second step of
Compound 24-2(201mg,0.412mmol) was dissolved in tetrahydrofuran (2mL) and water (2mL), and sodium hydroxide (16.5mg,0.412mmol) was added to the reaction solution.The reaction mixture was stirred at 50 ℃ for 12 hours, concentrated under reduced pressure to remove the organic solvent, and then adjusted to pH 5 with hydrochloric acid (1 mol/L). The hydrochloride salt of compound 24 was prepared by high performance liquid chromatography (acidic, hydrochloric acid system).1H NMR(400MHz, CD 3OD) δ 7.35-7.31(m,2H),7.26-7.21(m,3H),4.48-4.37(m,2H),4.20-4.13(m,4H),3.77-3.76(m,2H),3.51-3.47(m,2H),3.30-3.20(m,3H),3.04-3.02(m,1H),2.68-2.64(m,1H),2.44-2.39(m,1H),2.20-2.17(m,2H),2.10-2.06(m,3H),1.94-1.80(m,3H),1.75-1.59(m,2H),1.45-1.40(m, 1H). MS-ESI calculated value [ M + H%] +384, found 384.
Example 25
Figure PCTCN2019105680-APPB-000105
The synthetic route is as follows:
Figure PCTCN2019105680-APPB-000106
first step of
The compound 1-6(200mg,0.494mmol) and the compound 25-1(86.1mg,0.593mmol) were dissolved in N, N-dimethylformamide (4mL), and O- (7-azabenzotriazol-1-yl) -N, N, N, N-tetramethyluronium hexafluorophosphate (282mg,0.741mmol), N, N-diisopropylethylamine (128mg,0.998mmol) and the reaction mixture were added. The reaction solution was stirred at 25 ℃ for 12 hours. Water (10mL) was added, extraction was performed with ethyl acetate (10mL x 3), the organic phase was washed with saturated sodium chloride (20mL x 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was isolated by thin layer chromatography (1:1 petroleum ether/ethyl acetate, Rf ═ 0.42) to afford compound 25-2. MS-ESI calculated value [ M + H%] +496, found 496.
Second step of
Compound 25-2(205mg,0.411mmol) was dissolved in tetrahydrofuran (2mL) and water (2mL), and sodium hydroxide (16.4mg,0.411mmol) was added to the reaction solution. The reaction solution is stirred and reacted for 12 hours at 50 ℃, the organic solvent is removed by concentration under reduced pressure, and hydrochloric acid (1 mol-L) adjusting the pH to 5. The hydrochloride salt of compound 25 was prepared by high performance liquid chromatography (acidic, hydrochloric acid system).1H NMR(400MHz,CD 3OD) δ 7.35-7.31(m,2H),7.26-7.21(m,3H),4.46-4.35(m,2H),4.20-4.05(m,6H),3.94-3.88(m,2H),3.61-3.58(m,2H),3.53-3.49(m,2H),3.33-3.22(m,3H),3.04-3.03(m,1H),2.66-2.65(m,1H),2.44-2.39(m,1H),2.09-2.03(m,1H),1.95-1.80(m,3H),1.75-1.59(m,2H),1.45-1.40(m, 1H). MS-ESI calculated value [ M + H%] +400, found value 400.
Example 26
Figure PCTCN2019105680-APPB-000107
The synthetic route is as follows:
Figure PCTCN2019105680-APPB-000108
first step of
The compound 1-6(200mg,0.494mmol) and the compound 26-1(84.9mg,0.593mmol) were dissolved in N, N-dimethylformamide (4mL), and O- (7-azabenzotriazol-1-yl) -N, N, N, N-tetramethyluronium hexafluorophosphate (282mg,0.741mmol), N, N-diisopropylethylamine (128mg,0.998mmol) and the reaction mixture were added. The reaction solution was stirred at 25 ℃ for 12 hours. Water (10mL) was added, extraction was performed with ethyl acetate (10mL x 3), the organic phase was washed with saturated sodium chloride (20mL x 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was isolated by thin layer chromatography (1:1 petroleum ether/ethyl acetate, Rf ═ 0.42) to afford compound 26-2. MS-ESI calculated value [ M + H%] +494, found 494.
Second step of
Compound 26-2(200mg,0.402mmol) was dissolved in tetrahydrofuran (2mL) and water (2mL), and sodium hydroxide (16.1mg,0.402mmol) was added to the reaction solution. The reaction mixture was stirred at 50 ℃ for 12 hours, concentrated under reduced pressure to remove the organic solvent, and then adjusted to pH 5 with hydrochloric acid (1 mol/L). Preparation of the salt of compound 26 by high performance liquid chromatography (acidic, hydrochloric acid system)An acid salt.1H NMR(400MHz,CD 3OD) δ 7.35-7.31(m,2H),7.26-7.21(m,3H),4.36-4.26(m,2H),4.20-4.05(m,4H),3.59-3.43(m,4H),3.30-3.23(m,1H),3.13-3.11(m,2H),3.04-2.99(m,1H),2.71-2.67(m,1H),2.43-2.40(m,1H),2.09-2.05(m,1H),1.93-1.83(m,8H),1.71-1.67(m,2H),1.61-1.56(m,1H),1.42-1.39(m, 1H). MS-ESI calculated value [ M + H%] +398, found 398.
Example 27
Figure PCTCN2019105680-APPB-000109
The synthetic route is as follows:
Figure PCTCN2019105680-APPB-000110
first step of
The compound 1-6(200mg,0.494mmol) and the compound 27-1(77.8mg,0.593mmol) were dissolved in N, N-dimethylformamide (4mL), and O- (7-azabenzotriazol-1-yl) -N, N, N, N-tetramethyluronium hexafluorophosphate salt (282mg,0.741mmol), N, N-diisopropylethylamine (128mg,0.998mmol) and the reaction mixture were added. The reaction solution was stirred at 25 ℃ for 12 hours. Water (10mL) was added, extraction was performed with ethyl acetate (10mL x 3), the organic phase was washed with saturated sodium chloride (20mL x 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was isolated by thin layer chromatography (1:1 petroleum ether/ethyl acetate, Rf ═ 0.47) to afford compound 27-2. MS-ESI calculated value [ M + H%] +482, found 482.
Second step of
Compound 27-2(209mg,0.428mmol) was dissolved in tetrahydrofuran (2mL) and water (2mL), and sodium hydroxide (17.1mg,0.428mmol) was added to the reaction solution. The reaction mixture was stirred at 50 ℃ for 12 hours, concentrated under reduced pressure to remove the organic solvent, and then adjusted to pH 5 with hydrochloric acid (1 mol/L). The hydrochloride salt of compound 27 was prepared by high performance liquid chromatography (acidic, hydrochloric acid system).1H NMR(400MHz,CD 3OD)δ7.35-7.31(m,2H),7.26-7.21(m,3H),4.35-4.25(m,2H),4.20-4.11(m,4H),3.57-3.46(m,2H),3.32-3.25(m,5H),3.04-3.02(m,1H),2.70-2.62(m,1H),2.44-2.40(m,1H),2.10-2.03(m,1H),1.95-1.81(m,3H),1.72-1.57(m,2H),1.45-1.40(m,1H),1.38-1.33(m, 6H). MS-ESI calculated value [ M + H%] +386, found 386.
Example 28
Figure PCTCN2019105680-APPB-000111
The synthetic route is as follows:
Figure PCTCN2019105680-APPB-000112
first step of
The compound 1-6(200mg,0.494mmol) and the compound 28-1(69.4mg,0.593mmol) were dissolved in N, N-dimethylformamide (4mL), and O- (7-azabenzotriazol-1-yl) -N, N, N, N-tetramethyluronium hexafluorophosphate (282mg,0.741mmol), N, N-diisopropylethylamine (128mg,0.998mmol) and the reaction mixture were added. The reaction solution was stirred at 25 ℃ for 12 hours. Water (10mL) was added, extraction was performed with ethyl acetate (10mL x 3), the organic phase was washed with saturated sodium chloride (20mL x 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was isolated by thin layer chromatography (1:1 petroleum ether/ethyl acetate, Rf ═ 0.43) to afford compound 28-2. MS-ESI calculated value [ M + H%] +468, measured value 468.
Second step of
Compound 28-2(206mg,0.437mmol) was dissolved in tetrahydrofuran (2mL) and water (2mL), and sodium hydroxide (17.5mg,0.437mmol) was added to the reaction mixture. The reaction mixture was stirred at 50 ℃ for 12 hours, concentrated under reduced pressure to remove the organic solvent, and then adjusted to pH 5 with hydrochloric acid (1 mol/L). The hydrochloride salt of compound 28 was prepared by high performance liquid chromatography (acidic, hydrochloric acid system).1H NMR(400MHz,CD 3OD)δ7.14-7.10(m,2H),7.05-7.00(m,3H),4.06-3.92(m,5H),3.83-3.81(m,1H),3.43-3.37(m,1H),3.30-.3.21(m,1H),3.10-3.00(m,1H),2.84-2.81(m,1H),2.48-2.44(m,1H),2.22-2.18(m,1H),1.99(s,3H),1.89-1.82(m,1H),1.70-1.56(m,3H),1.49-1.35(m,2H),1.24-1.18(m, 1H). MS-ESI calculated value [ M + H%] +372, measured value 372.
Example 29
Figure PCTCN2019105680-APPB-000113
The synthetic route is as follows:
Figure PCTCN2019105680-APPB-000114
first step of
The compound 1-6(200mg,0.494mmol) and the compound 29-1(86.1mg,0.593mmol) were dissolved in N, N-dimethylformamide (4mL), and O- (7-azabenzotriazol-1-yl) -N, N, N, N-tetramethyluronium hexafluorophosphate (282mg,0.741mmol), N, N-diisopropylethylamine (128mg,0.998mmol) and the reaction mixture were added. The reaction solution was stirred at 25 ℃ for 12 hours. Water (10mL) was added, extraction was performed with ethyl acetate (10mL x 3), the organic phase was washed with saturated sodium chloride (20mL x 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was isolated by thin layer chromatography (1:1 petroleum ether/ethyl acetate, Rf ═ 0.52) to afford compound 29-2. MS-ESI calculated value [ M + H%] +496, found 496.
Second step of
Compound 29-2(208mg,0.383mmol) was dissolved in tetrahydrofuran (2mL) and water (2mL), and sodium hydroxide (15.3mg,0.383mmol) was added to the reaction solution. The reaction mixture was stirred at 50 ℃ for 12 hours, concentrated under reduced pressure to remove the organic solvent, and then adjusted to pH 5 with hydrochloric acid (1 mol/L). The hydrochloride salt of compound 29 was prepared by high performance liquid chromatography (acidic, hydrochloric acid system).1H NMR(400MHz,CD 3OD)δ7.35-7.31(m,2H),7.26-7.21(m,3H),4.22-4.05(m,6H),3.64-3.60(m,1H),3.49-3.45(m,1H),3.30-3.23(m,1H),3.06-3.02(m,1H),2.67-2.56(m,2H),2.43-2.37(m,1H),2.05-2.02(m,1H),1.89-1.77(m,3H),1.68-1.56(m,2H),1.45-1.40(m,1H),1.18-1.15(d,J＝6.8, 6H). MS-ESI calculated value [ M + H%] +400, found value 400.
Example 30
Figure PCTCN2019105680-APPB-000115
The synthetic route is as follows:
Figure PCTCN2019105680-APPB-000116
first step of
The compound 1-6(200mg,0.494mmol) and the compound 30-1(84.9mg,0.593mmol) were dissolved in N, N-dimethylformamide (4mL), and O- (7-azabenzotriazol-1-yl) -N, N, N, N-tetramethyluronium hexafluorophosphate (282mg,0.741mmol), N, N-diisopropylethylamine (128mg,0.998mmol) and the reaction mixture were added. The reaction solution was stirred at 25 ℃ for 12 hours. Water (10mL) was added, extraction was performed with ethyl acetate (10mL x 3), the organic phase was washed with saturated sodium chloride (20mL x 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was isolated by thin layer chromatography (1:1 petroleum ether/ethyl acetate, Rf ═ 0.43) to afford compound 30-2. MS-ESI calculated value [ M + H%] +494, found 494.
Second step of
Compound 30-2(201mg,401umol) was dissolved in tetrahydrofuran (2mL) and water (2mL), and sodium hydroxide (16.1mg,402umol) was added to the reaction mixture. The reaction mixture was stirred at 50 ℃ for 12 hours, concentrated under reduced pressure to remove the organic solvent, and then adjusted to pH 5 with hydrochloric acid (1 mol/L). The hydrochloride salt of compound 30 was prepared by high performance liquid chromatography (acidic, hydrochloric acid system).1H NMR(400MHz,CD 3OD) δ 7.35-7.31(m,2H),7.26-7.21(m,3H),4.27-4.19(m,5H),4.16-4.12(m,1H),3.57-3.53(m,4H),3.25-3.20(m,1H),3.04-3.03(m,1H),2.67-2.66(m,1H),2.52-2.50(m,2H),2.49-2.48(m,1H),2.14-2.05(m,3H),1.78-1.68(m,3H),1.66-1.50(m,2H),1.43-1.41(m, 1H). MS-ESI calculated value [ M + H%] +398, found 398.
Example 31
Figure PCTCN2019105680-APPB-000117
The synthetic route is as follows:
Figure PCTCN2019105680-APPB-000118
first step of
The compound 1-6(200mg,0.494mmol) and the compound 31-1(86.1mg,0.593mmol) were dissolved in N, N-dimethylformamide (4mL), and O- (7-azabenzotriazol-1-yl) -N, N, N, N-tetramethyluronium hexafluorophosphate (282mg,0.741mmol), N, N-diisopropylethylamine (128mg,0.998mmol) and the reaction mixture were added. The reaction solution was stirred at 25 ℃ for 12 hours. Water (10mL) was added, extraction was performed with ethyl acetate (10mL x 3), the organic phase was washed with saturated sodium chloride (20mL x 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was isolated by thin layer chromatography (1:1 petroleum ether/ethyl acetate, Rf ═ 0.34) to afford compound 31-2. MS-ESI calculated value [ M + H%] +496, found 496.
Second step of
Compound 31-2(209mg,0.352mmol) was dissolved in tetrahydrofuran (2mL) and water (2mL), and sodium hydroxide (14.1mg,0.352mmol) was added to the reaction solution. The reaction mixture was stirred at 50 ℃ for 12 hours, concentrated under reduced pressure to remove the organic solvent, and then adjusted to pH 5 with hydrochloric acid (1 mol/L). The hydrochloride salt of compound 31 was prepared by high performance liquid chromatography (acidic, hydrochloric acid system).1H NMR(400MHz,CD 3OD) δ 7.35-7.31(m,2H),7.27-7.21(m,3H),4.21-4.15(m,4H),3.69-3.66(m,1H),3.56-3.48(m,1H),3.45-3.42(m,2H),3.31-3.26(m,4H),3.05-3.02(m,2H),2.99-2.97(m,3H),2.70-2.60(m,1H),2.44-2.38(m,1H),2.13-2.01(m,1H),1.93-1.77(m,3H),1.69-1.62(m,2H),1.38-1.35(m, 6H). MS-ESI calculated value [ M + H%] +400, found value 400.
Example 32
Figure PCTCN2019105680-APPB-000119
The synthetic route is as follows:
Figure PCTCN2019105680-APPB-000120
first step of
The compound 1-6(200mg,0.494mmol) and the compound 32-1(94.4mg,0.593mmol) were dissolved in N, N-dimethylformamide (4mL), and O- (7-azabenzotriazol-1-yl) -N, N, N, N-tetramethyluronium hexafluorophosphate (282mg,0.741mmol), N, N-diisopropylethylamine (128mg,0.998mmol) and the reaction mixture were added. The reaction solution was stirred at 25 ℃ for 12 hours. Water (10mL) was added, extraction was performed with ethyl acetate (10mL x 3), the organic phase was washed with saturated sodium chloride (20mL x 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was isolated by thin layer chromatography (1:1 petroleum ether/ethyl acetate, Rf ═ 0.32) to afford compound 32-2. MS-ESI calculated value [ M + H%] +510, measured value 510.
Second step of
Compound 32-2(101mg, 67.0. mu. mol) was dissolved in tetrahydrofuran (2mL) and water (2mL), and sodium hydroxide (2.68mg, 67.0. mu. mol) was added to the reaction mixture. The reaction mixture was stirred at 50 ℃ for 12 hours, concentrated under reduced pressure to remove the organic solvent, and then adjusted to pH 5 with hydrochloric acid (1 mol/L). The hydrochloride salt of compound 32 was prepared by high performance liquid chromatography (acidic, hydrochloric acid system).1H NMR(400MHz,CD 3OD) δ 7.35-7.32(m,2H),7.25-7.20(m,3H),4.20-4.05(m,6H),3.88-3.82(m,2H),3.73-3.66(m,1H),3.59-3.46(m,5H),3.27-3.17(m,3H),3.05-3.00(m,3H),2.62-2.59(m,1H),2.44-2.38(m,1H),2.06-1.97(m,1H),1.94-1.78(m,3H),1.63-1.59(m,2H),1.47-1.41(m, 1H). MS-ESI calculated value [ M + H%] +414, measured value 414.
Example 33
Figure PCTCN2019105680-APPB-000121
The synthetic route is as follows:
Figure PCTCN2019105680-APPB-000122
first step of
The compound 1-6(200mg,0.494mmol) and the compound 33-1(86.6mg,0.593mmol) were dissolved in N, N-dimethylformamide (4mL), and O- (7-azabenzotriazol-1-yl) -N, N, N, N-tetramethyluronium hexafluorophosphate (282mg,0.741mmol), N, N-diisopropylethylamine (128mg,0.998mmol) and the reaction mixture were added. The reaction solution was stirred at 25 ℃ for 12 hours. Water (10mL) was added, extraction was performed with ethyl acetate (10mL x 3), the organic phase was washed with saturated sodium chloride (20mL x 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was isolated by thin layer chromatography (1:1 petroleum ether/ethyl acetate, Rf ═ 0.34) to afford compound 33-2. MS-ESI calculated value [ M + H%] +497, found 497.
Second step of
Compound 33-2(186mg,0.334mmol) was dissolved in tetrahydrofuran (2mL) and water (2mL), and sodium hydroxide (13.4mg,0.334mmol) was added to the reaction solution. The reaction mixture was stirred at 50 ℃ for 12 hours, concentrated under reduced pressure to remove the organic solvent, and then adjusted to pH 5 with hydrochloric acid (1 mol/L). The hydrochloride salt of compound 33 was prepared by high performance liquid chromatography (acidic, hydrochloric acid system).1H NMR(400MHz,CD 3OD) δ 7.35-7.31(m,2H),7.27-7.21(m,3H),4.25-4.20(m,1H),4.18-4.16(m,2H),3.30-3.25(m,4H),3.06-3.04(m,1H),2.67-2.63(m,1H),2.59(s,2H),2.50-2.43(m,1H),2.15-2.02(m,4H),1.90-1.83(m,1H),1.67-1.64(m,1H),1.46-1.42(m,1H),1.28-1.26(m, 6H). MS-ESI calculated value [ M + H%] +401, found value 401.
Example 34
Figure PCTCN2019105680-APPB-000123
The synthetic route is as follows:
Figure PCTCN2019105680-APPB-000124
first step of
Compound 34-1(200mg,0.127mmol) was dissolved in dichloromethane (2mL), and oxalyl chloride (194mg,0.153mmol) and N, N-dimethylformamide (9.30mg,0.127mol) were added to the reaction solution at 0 ℃. The reaction solution was stirred at 0-15 ℃ for 1 hour. The reaction solution was directly concentrated to obtain a crude compound 34-2.
Second step of
Compound 34-2(200mg,1.14mmol) and compound 1-6(210mg,0.569mmol) were dissolved in dichloromethane (5mL), and triethylamine (230mg,2.28mmol) was added to the reaction solution. The reaction solution was stirred at 15 ℃ for 3 hours. Water (10mL) was added, extraction was performed with dichloromethane (10mL x 3), the organic phase was washed with saturated sodium chloride (20mL x 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was isolated by thin layer chromatography (1:1 petroleum ether/ethyl acetate, Rf ═ 0.31) to afford compound 34-3. MS-ESI calculated value [ M + H%] +508, found 508.
The third step
Compound 34-3(144mg,0.282mmol) was dissolved in tetrahydrofuran (2mL) and water (2mL), and sodium hydroxide (22.6mg,0.564mmol) was added to the reaction solution. The reaction mixture was stirred at 50 ℃ for 3 hours, concentrated under reduced pressure to remove the organic solvent, and then adjusted to pH 5 with hydrochloric acid (1 mol/L). The hydrochloride salt of compound 34 was prepared by high performance liquid chromatography (acidic, hydrochloric acid system).1H NMR(400MHz,CD 3OD) δ 7.33-7.31(m,2H),7.27-7.21(m,3H),4.23-4.15(m,4H),3.60-3.59(m,1H),3.57-3.56(m,2H),3.42-3.41(m,1H),3.40-3.39(m,2H),3.38-3.37(m,1H),3.05-2.99(m,5H),2.65-2.63(m,1H),2.48-2.47(m,1H),2.02-2.01(m,1H),1.94-1.80(m,8H),1.67-1.57(m,3H),1.43-1.42(m, 1H). MS-ESI calculated value [ M + H%] +412, found 412.
And (3) biochemical detection:
experimental example 1: evaluation of enzyme Activity
The aim of this assay was to detect the body of compound on LSD1Exo-inhibitory activity. The enzyme adopted in the test is humanized LSD1, the standard substrate is histone H3K4me peptide (20 mu M), and the enzyme fluorescence coupling method is adopted to jointly detect H generated after LSD1 reaction through Horseradish Peroxidase (HPR) and a fluorescent reagent Amplex Red2O 2The method of (3) determining the activity of the compound. Starting from 10. mu.M, 3-fold dilutions were made and the IC was determined at 10 concentrations of compound50The value is obtained. The compounds were incubated for 30 minutes with enzyme and substrate before the reaction was initiated by addition of substrate. A fluorescence detector: EnVision, excitation wavelength: Ex/Em 530/590 nM.
The compounds tested had inhibitory activity against LSD1, with the results shown in table 1.
Table 1: results of in vitro enzyme Activity screening test for Compounds of the invention
Compound numbering IC 50(nM) Compound numbering IC 50(nM)
Hydrochloride salt of Compound 1 116.5 Hydrochloride salt of compound 18 8.54
Hydrochloride salt of Compound 2 2289 Hydrochloride salt of Compound 19 317.8
Hydrochloride salt of Compound 3 388.5 Hydrochloride salt of Compound 20 935.2
Hydrochloride salt of Compound 4 14.62 Hydrochloride salt of Compound 21 34.06
Hydrochloride salt of Compound 5 46.42 Hydrochloride salt of Compound 22 29.29
Hydrochloride salt of Compound 6 86.24 Hydrochloride salt of compound 23 15.72
Hydrochloride salt of compound 7 640 Hydrochloride salt of compound 24 13.19
Hydrochloride salt of Compound 8 12.21 Hydrochloride salt of Compound 25 68.14
Hydrochloride salt of Compound 9 60.42 Hydrochloride salt of compound 26 18.83
Hydrochloride salt of Compound 10 36.58 Hydrochloride salt of compound 27 8.74
Hydrochloride salt of Compound 11 11.36 Hydrochloride salt of Compound 28 41.1
Hydrochloride salt of Compound 12 37.35 Hydrochloride salt of compound 29 66.24
Hydrochloride salt of Compound 13 7.05 Hydrochloride salt of compound 30 84.8
Hydrochloride salt of Compound 14 44.76 Hydrochloride salt of compound 31 35.53
Hydrochloride salt of Compound 15 12.89 Hydrochloride salt of Compound 32 27.99
Hydrochloride salt of Compound 16 3.7 Hydrochloride salt of compound 34 42.15
Hydrochloride salt of Compound 17 12.89
And (4) conclusion: the compound of the invention has obvious inhibitory activity on LSD 1.
Experimental example 2: evaluation of NCI-H1417 cell proliferation inhibitory Activity:
purpose of the experiment: detecting the inhibition activity of the test compound on the H1417 cell proliferation.
Experimental materials: RPMI 1640 medium, fetal bovine serum, Promega CellTiter-Glo reagent. The NCI-H1417 cell line was purchased from ATCC. Envision multi-label analyzer (PerkinElmer).
The experimental method comprises the following steps: compounds were dissolved to 10mM, compounds were diluted 5-fold in DMSO in compound plates, compounds were initially 2mM, three-fold diluted with Bravo, 10 concentrations, and 250nL in Echo transfer plates to upper and lower duplicate wells of blank 384 cell plates, and 250nL of DMSO/compound was added to the top of the cell suspension per well/1000 cells/50 μ L, compounds were diluted 200-fold, i.e., the initial concentration was 10 μ M. The cell plates were placed in a carbon dioxide incubator for 10 days. To the cell plate, 25. mu.L of Promega CellTiter-Glo reagent per well was added, and the luminescence signal was stabilized by shaking at room temperature for 10 minutes. Readings were taken using a PerkinElmer Envision multi-label analyzer.
And (3) data analysis: the original data was converted to the inhibition rate, IC, using the equation (Max-Ratio)/(Max-Min) × 100%50The values of (a) can be obtained by curve fitting of four parameters (mode 205 in XLFIT5, idss).
The test compounds had inhibitory activity against NCI-H1417 cell proliferation, and the results are shown in Table 2.
Table 2: results of the experiment for inhibiting NCI-H1417 cell proliferation by the Compound of the present invention
Compound numbering IC 50(nM) Compound numbering IC 50(nM)
Hydrochloride salt of Compound 1 32.49 Hydrochloride salt of Compound 16 0.90
Hydrochloride salt of Compound 5 1.28 Hydrochloride salt of Compound 17 3.95
Hydrochloride salt of Compound 6 1.74 Hydrochloride salt of compound 18 2.84
Hydrochloride salt of Compound 9 2.28 Hydrochloride salt of Compound 19 10.25
Hydrochloride salt of Compound 10 8.17 Hydrochloride salt of Compound 21 3.04
Hydrochloride salt of Compound 11 2.79 Hydrochloride salt of Compound 22 2.26
Hydrochloride salt of Compound 12 7.20 Hydrochloride salt of compound 23 6.95
Hydrochloride salt of Compound 13 1.83 Hydrochloride salt of Compound 25 22.38
Hydrochloride salt of Compound 14 24.05 Hydrochloride salt of compound 30 19.09
Hydrochloride salt of Compound 15 2.18 Hydrochloride salt of Compound 32 10.39
And (4) conclusion: the compound has obvious inhibitory activity on NCI-H1417 cell proliferation.
Experimental example 3: evaluation of HL60 cell proliferation inhibitory activity:
purpose of the experiment: and detecting the inhibitory activity of the test compound on HL60 cell proliferation.
Experimental materials: RPMI-1640 medium, fetal bovine serum, penicillin/streptomycin antibiotics were purchased from Vitrexate. CellTiter-Glo (cell viability chemiluminescence detection reagent) reagent was purchased from Promega. The HL60 cell line was purchased from Nanjing Kebai Life technologies, Inc. Nivo multi-label analyzer (PerkinElmer).
The experimental method comprises the following steps: HL60 cells were seeded in white 384-well plates containing 600 HL60 cells in 40 μ L cell suspension per well. The cell plates were placed in a carbon dioxide incubator overnight. The test compounds were diluted 5-fold with a calandria to the 10 th concentration, i.e. from 2mM to 1.024nM, setting up a double-well experiment. Add 78. mu.L of medium to the intermediate plate, transfer 2. mu.L of each well of the gradient dilution compound to the intermediate plate according to the corresponding position, mix well and transfer 10. mu.L of each well to the cell plate. The cell plates were placed in a carbon dioxide incubator for 6 days. A separate cell plate was prepared, and the signal values were read on the day of drug addition as maximum values (Max values in the following equation) for data analysis. To each well of this cell plate, 20. mu.L of a cell viability chemiluminescence detection reagent was added, and the plate was incubated at room temperature for 10 minutes to stabilize the luminescence signal. Reading with a multi-label analyzer.
And (3) data analysis: the original data was converted to inhibition rate, IC, using the equation (Sample-Min)/(Max-Min) × 100%50The values of (A) can be obtained by curve fitting of four parameters (obtained in the GraphPad Prism "log (inhibitor)" vs. response- -Variable slope "mode).
The test compounds had inhibitory activity against HL60 cell proliferation, and the results are shown in table 3.
Table 3: results of HL60 cell proliferation inhibition assay using the compounds of the present invention
Compound numbering IC 50(nM) Compound numbering IC50(nM)
Hydrochloride salt of Compound 1 15.29 Hydrochloride salt of Compound 17 0.6
Hydrochloride salt of Compound 5 0.73 Hydrochloride salt of compound 18 1.05
Hydrochloride salt of Compound 6 1.9 Hydrochloride salt of Compound 19 3.14
Hydrochloride salt of Compound 10 7.97 Hydrochloride salt of Compound 21 4.51
Hydrochloride salt of Compound 11 0.46 Hydrochloride salt of Compound 22 0.59
Hydrochloride salt of Compound 12 <0.005 Hydrochloride salt of compound 23 3.52
Hydrochloride salt of Compound 13 0.79 Hydrochloride salt of Compound 25 14.32
Hydrochloride salt of Compound 14 6.77 Hydrochloride salt of compound 30 7.46
Hydrochloride salt of Compound 15 2.30 Hydrochloride salt of Compound 32 2.89
Hydrochloride salt of Compound 16 0.32
And (4) conclusion: the compound has obvious inhibitory activity on HL60 cell proliferation.
Experimental example 4: evaluation of MV-4-11 cell proliferation inhibitory Activity:
purpose of the experiment: and detecting the inhibition activity of the test compound on the MV-4-11 cell proliferation.
Experimental materials: IMDM medium, fetal bovine serum, penicillin/streptomycin antibiotics were purchased from vison. CellTiter-Glo (cell viability chemiluminescence detection reagent) reagent was purchased from Promega. MV-4-11 cell line was purchased from Nanjing Kebai Life technologies, Inc. Nivo multi-label analyzer (PerkinElmer).
The experimental method comprises the following steps: MV-4-11 cells were seeded in white 96-well plates in 80. mu.L cell suspension per well, containing 6000 MV-4-11 cells. The cell plates were placed in a carbon dioxide incubator overnight.
The test compounds were diluted 5-fold with a calandria to 8 th concentration, i.e. from 2mM to 25.6nM, setting up a duplicate well experiment. Add 78. mu.L of medium to the intermediate plate, transfer 2. mu.L of each well of the gradient dilution compound to the intermediate plate according to the corresponding position, mix well and transfer 20. mu.L of each well to the cell plate. The cell plates were placed in a carbon dioxide incubator for 6 days. A separate cell plate was prepared, and the signal values were read on the day of drug addition as maximum values (Max values in the following equation) for data analysis. To each well of this cell plate, 25. mu.L of a cell viability chemiluminescence detection reagent was added, and the plate was incubated at room temperature for 10 minutes to stabilize the luminescence signal. Reading with a multi-label analyzer.
And (3) data analysis: the original data was converted to inhibition rate, IC, using the equation (Sample-Min)/(Max-Min) × 100%50The values of (A) can be obtained by curve fitting of four parameters (obtained in the GraphPad Prism "log (inhibitor)" vs. response- -Variable slope "mode).
The test compounds had MV-4-11 cell proliferation inhibitory activity, and the results are shown in Table 4.
Table 4: results of MV-4-11 cell proliferation inhibition assay by the Compounds of the present invention
Compound numbering IC 50(nM) Compound numbering IC 50(nM)
Hydrochloride salt of Compound 1 26.67 Hydrochloride salt of Compound 17 2.41
Hydrochloride salt of Compound 5 0.4 Hydrochloride salt of compound 18 0.56
Hydrochloride salt of Compound 6 1.16 Hydrochloride salt of Compound 19 3.46
Hydrochloride salt of Compound 10 2.63 Hydrochloride salt of Compound 21 1.74
Hydrochloride salt of Compound 11 1.18 Hydrochloride salt of Compound 22 1.16
Hydrochloride salt of Compound 12 0.99 Hydrochloride salt of compound 23 1.93
Hydrochloride salt of Compound 13 1.01 Salt of compound 25Acid salts 3.74
Hydrochloride salt of Compound 14 7.22 Hydrochloride salt of compound 30 3.56
Hydrochloride salt of Compound 15 1.32 Hydrochloride salt of Compound 32 1.7
Hydrochloride salt of Compound 16 <0.128
And (4) conclusion: the compound has obvious inhibition activity on MV-4-11 cell proliferation.
Experimental example 5: compound pharmacokinetic evaluation
Purpose of the experiment: testing the pharmacokinetics of Compounds in CD-1 mice
Experimental materials:
CD-1 mouse (Male, 7 ~ 9 weeks old, Shanghai Si Laike)
And (3) experimental operation:
rodent pharmacokinetic characteristics of the compound after intravenous injection and oral administration are tested by a standard scheme, and a candidate compound is prepared into a clear solution in an experiment and is given to a mouse for single intravenous injection and oral administration. The intravenous injection and oral administration solvent is a mixed solvent prepared from 10% of dimethyl sulfoxide and 90% of 10% of hydroxypropyl beta cyclodextrin. Four male CD-1 mice were used for this project, two mice were administered intravenously at a dose of 1mg/kg, plasma samples were collected at 0h (before administration) and 0.0833,0.25,0.5,1,2,4,8,24h after administration, two other mice were administered orally at a dose of 2mg/kg by gavage, and 0h (given to a patient) was collectedPre-drug) and 0.25,0.5,1,2,4,8,24h after drug administration, collecting whole blood sample within 24h, centrifuging for 15 min at 3000g, separating supernatant to obtain plasma sample, adding 4 times volume of acetonitrile solution containing internal standard to precipitate protein, centrifuging to obtain supernatant, adding equal volume of water, centrifuging to obtain supernatant, sampling, quantitatively analyzing blood concentration by LC-MS/MS analysis method, and calculating drug substitution parameters such as peak concentration (C)max) Clearance (CL), half-life (T)1/2) Tissue distribution (Vdss), area under the time curve (AUC)0-last) Bioavailability (F), etc.
The results of the experiment are shown in table 5:
TABLE 5 pharmacokinetic testing results
Figure PCTCN2019105680-APPB-000125
And (4) conclusion: the compound of the invention has good pharmacokinetic properties, including good oral bioavailability, oral exposure, half-life, clearance rate and the like.
Experimental example 6: in vivo pharmacodynamic study of compound on CT-26 mouse colon cancer transplantation tumor model
3.1 purpose of experiment:
the purpose of the experiment is to research the evaluation of the in vivo efficacy of the compound on a CT-26 mouse colon cancer transplantation tumor model.
3.2 Experimental animals:
the species are as follows: mouse
Strain: BALB/c mice
Week age and body weight: 7 weeks old, weight 18-23 g
Sex: female
The supplier: shanghai Bikai laboratory animals Co., Ltd
3.3 Experimental methods and procedures
3.3.1 cell culture
Name: CT-26 (mouse colon cancer cell)
Cell source: ATCC (American type culture Collection)
Cell culture: the culture medium is 1640 medium containing 10% fetal calf serum, and the culture condition is 37 deg.C and 5% carbon dioxide. The passage ratio is 1: 2-1: 3, and the passage is performed for 2-3 times per week.
3.3.2 tumor cell inoculation
0.1mL (3X 10)5Individual) cells were inoculated subcutaneously into the right dorsal back of each mouse. Animals were randomly grouped by weight on the same day.
3.3.3 preparation of test substances
The experimental solvent is 0.5% methylcellulose solution, and the preparation method comprises weighing 5g methylcellulose, dissolving in 800mL ultrapure water, stirring, and diluting to 1000mL with ultrapure water. Dissolving the test substance with solvent to obtain a uniform solution with a certain concentration, and storing at 4 deg.C.
3.3.4 tumor measurement and Experimental indices
The experimental index is to investigate whether the tumor growth is inhibited, delayed or cured. Tumor diameters were measured twice weekly using a vernier caliper. The formula for tumor volume is: v is 0.5a × b2And a and b represent the major and minor diameters of the tumor, respectively.
The tumor-inhibiting therapeutic effect of the compound was evaluated relative to the tumor proliferation rate T/C (%). Relative tumor proliferation rate T/C (%): the calculation formula is as follows: T/C%RTV/C RTV×100％(T RTV: treatment group RTV; cRTV: negative control group RTV). Calculating Relative Tumor Volume (RTV) according to the tumor measurement result, wherein the calculation formula is that RTV is Vt/V 0In which V is0When administered in groups (i.e. d)0) Measurement of the mean tumor volume, VtMean tumor volume at a certain measurement, TRTVAnd CRTVThe same day data was taken.
3.4 the results of the experiment are shown in Table 6:
TABLE 6 evaluation of the tumor-inhibiting efficacy of test compounds on CT-26 mouse colon carcinoma transplantable tumor model (calculated based on tumor volume on day 32 after grouping)
Figure PCTCN2019105680-APPB-000126
PD-L1 monoclonal antibody source: BioXcell.
And (4) conclusion: the compound has excellent tumor inhibiting effect on a colon cancer transplantation tumor model of a CT-26 mouse when being combined with PD-L1 monoclonal antibody.
Experimental example 7: in vivo pharmacodynamic study of compound on MC38 mouse colon cancer transplantation tumor model
7.1 purpose of the experiment:
the purpose of the experiment is to study the evaluation of the drug effect of the compound of the invention on the MC38 mouse colon cancer transplantation tumor model in vivo.
7.2 Experimental animals:
the species are as follows: mouse
Strain: c57BL/6 mice
The week age is as follows: 6-8 weeks old
Sex: female
The supplier: shanghai Slek laboratory animals Co., Ltd
7.3 Experimental methods and procedures
7.3.1 cell culture
Name: MC38 (mouse colon cancer cell)
Cell source: ATCC (American type culture Collection)
Cell culture: the culture medium is 1640 medium containing 10% fetal calf serum, and the culture condition is 37 deg.C and 5% carbon dioxide. The passage ratio is 1: 2-1: 3, and the passage is performed for 2-3 times per week.
7.3.2 tumor cell inoculation
0.1mL (2X 10)5Individual) cells were inoculated subcutaneously into the right dorsal back of each mouse. Animals were randomly grouped by weight on the same day.
7.3.3 preparation of test substances
The experimental solvent is 0.5% methylcellulose solution, and the preparation method comprises weighing 5g methylcellulose, dissolving in 800mL ultrapure water, stirring, and diluting to 1000mL with ultrapure water. Dissolving the test substance with solvent to obtain a uniform solution with a certain concentration, and storing at 4 deg.C.
7.3.4 tumor measurement and Experimental indices
The experimental index is to investigate whether the tumor growth is inhibited, delayed or cured. Tumor diameters were measured twice weekly using a vernier caliper. The formula for tumor volume is: v is 0.5a × b2And a and b represent the major and minor diameters of the tumor, respectively.
The tumor-inhibiting therapeutic effect of the compound was evaluated relative to the tumor proliferation rate T/C (%). Relative tumor proliferation rate T/C (%): the calculation formula is as follows: T/C%RTV/C RTV×100％(T RTV: treatment group RTV; cRTV: negative control group RTV). Calculating Relative Tumor Volume (RTV) according to the tumor measurement result, wherein the calculation formula is that RTV is Vt/V 0In which V is0When administered in groups (i.e. d)0) Measurement of the mean tumor volume, VtMean tumor volume at a certain measurement, TRTVAnd CRTVThe same day data was taken.
7.4 the results of the experiment are shown in Table 7:
TABLE 7 evaluation of the tumor-inhibiting efficacy of test compounds on MC38 mouse colon carcinoma graft tumor model (calculated based on tumor volume on day 27 after grouping)
Figure PCTCN2019105680-APPB-000127
PD-1 monoclonal antibody source: BioXcell.
And (4) conclusion: the compound has excellent tumor inhibition effect on a colon cancer transplantation tumor model of an MC38 mouse when being combined with PD-1 monoclonal antibody.

Claims (13)

  1. A compound of formula (I), an isomer thereof or a pharmaceutically acceptable salt thereof,
    Figure PCTCN2019105680-APPB-100001
    wherein the content of the first and second substances,
    L 1is selected from- (CH)2)g-、-C(＝O)-、-S(＝O) 2-, -C (═ O) -O-, and-C (═ O) -NH-;
    R 1is selected from NH2、CN、COOH、-S(＝O) 2-NH 2、C 1-6Alkyl, -C (═ O) -C1-4Alkyl-phenyl and-C1-4Alkyl-4-7 membered heterocycloalkyl,
    wherein said C1-6Alkyl, -C (═ O) -C1-4Alkyl-phenyl and-C1-4Alkyl-4-7 membered heterocycloalkyl optionally substituted with 1,2 or 3RaSubstitution;
    m is 0, 1 or 2;
    n is 0, 1 or 2, and m and n cannot be 0 at the same time;
    r is 0 or 1;
    q is 0 or 1;
    g is 0, 1,2, 3 or 4;
    R aselected from F, Cl, Br, I, OH, NH2、CN、COOH、C 1-3Alkylamino and-NH-C (═ O) -C1-3Alkyl radical, wherein said C1-3Alkylamino and-NH-C (═ O) -C1-3Alkyl is optionally substituted with 1,2 or 3R;
    r is selected from F, Cl, Br, I, OH and NH2And C1-3An alkyl group;
    said 4-7 membered heterocycloalkyl contains 1,2, 3 or 4 heteroatoms or groups of heteroatoms independently selected from-NH-, -O-, -S-and N;
    with "-" carbon atoms as chiral carbon atoms, in the form of (R) or (S) single enantiomers or enriched in one enantiomer;
    the carbon atom with "#" is a chiral carbon atom and exists in the form of a single enantiomer (R) or (S) or in a form enriched in one enantiomer.
  2. A compound, isomer, or pharmaceutically acceptable salt thereof according to claim 1, wherein R is selected from F, Cl, Br, I, OH, NH2、CH 3and-CH2CH 3。
  3. The compound, an isomer thereof, or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein RaSelected from F, Cl, Br, I, OH, NH2、CN、COOH、
    Figure PCTCN2019105680-APPB-100002
    Wherein said
    Figure PCTCN2019105680-APPB-100003
    Figure PCTCN2019105680-APPB-100004
    Optionally substituted with 1,2 or 3R.
  4. A compound, isomer, or pharmaceutically acceptable salt thereof according to claim 3, wherein RaSelected from F, Cl, Br, I, OH, NH2、CN、COOH、
    Figure PCTCN2019105680-APPB-100005
  5. A compound, isomer, or pharmaceutically acceptable salt thereof according to claim 1, wherein R1Is selected from NH2、CN、COOH、 -S(＝O) 2-NH 2、C 1-4Alkyl, -C (═ O) -C1-3Alkyl-phenyl and-C1-3Alkyl-5-6 heterocycloalkyl wherein said C1-4Alkyl, -C (═ O) -C1-3Alkyl-phenyl and-C1-3Alkyl-5-6 heterocycloalkyl optionally substituted with 1,2 or 3RaAnd (4) substitution.
  6. A compound, isomer, or pharmaceutically acceptable salt thereof according to claim 5, wherein R1Is selected from NH2、CN、COOH、-S(＝O) 2-NH 2、-CH 3、-CH 2-CH 3、
    Figure PCTCN2019105680-APPB-100006
    Figure PCTCN2019105680-APPB-100007
    Wherein said CH3、-CH 2-CH 3、
    Figure PCTCN2019105680-APPB-100008
    Figure PCTCN2019105680-APPB-100009
    Optionally substituted by 1,2 or 3RaAnd (4) substitution.
  7. A compound, isomer, or pharmaceutically acceptable salt thereof according to claim 6, wherein R1Is selected from NH2、CN、COOH、
    Figure PCTCN2019105680-APPB-100010
    -CH 3、-CH 2-CH 3、
    Figure PCTCN2019105680-APPB-100011
    Figure PCTCN2019105680-APPB-100012
  8. A compound, isomer, or pharmaceutically acceptable salt thereof according to claim 1, wherein L1Selected from single bonds, -CH2-、-(CH 2) 2-、
    Figure PCTCN2019105680-APPB-100013
    -C (═ O) -, and-S (═ O)2-。
  9. The compound according to any one of claims 1 to 7, an isomer thereof or a pharmaceutically acceptable salt thereof, which is selected from the group consisting of
    Figure PCTCN2019105680-APPB-100014
    Figure PCTCN2019105680-APPB-100015
    Wherein the content of the first and second substances,
    g is as defined in claim 1;
    R 1as defined in any one of claims 1 to 6.
  10. A compound of the formula, an isomer thereof or a pharmaceutically acceptable salt thereof,
    Figure PCTCN2019105680-APPB-100016
    Figure PCTCN2019105680-APPB-100017
  11. the compound according to claim 10, an isomer thereof, or a pharmaceutically acceptable salt thereof,
    Figure PCTCN2019105680-APPB-100018
  12. the compound, an isomer thereof, or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 11, wherein the pharmaceutically acceptable salt is selected from hydrochloride salts.
  13. Use of a compound according to any one of claims 1-12, an isomer thereof, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a disorder associated with LSD 1.
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