Novel tricyclic aromatic heterocyclic compound, and preparation method, pharmaceutical composition and application thereof
阅读说明：本技术 新型三环芳香杂环化合物，及其制备方法、药物组合物和应用 (Novel tricyclic aromatic heterocyclic compound, and preparation method, pharmaceutical composition and application thereof ) 是由 王喆 白海云 潜安然 李德亮 于 2021-05-24 设计创作，主要内容包括：本发明提供了一种新型三环芳香杂环化合物,及其制备方法、药物组合物和应用,具体地,本发明提供了一种如下式I所示的化合物,或其光学异构体、水合物、溶剂合物,或其药学上可接受的盐；其中,各基团的定义如说明书中所述。所述的式I化合物可以用于治疗与PD-1/PD-L1信号通路有关的疾病。(The invention provides a novel tricyclic aromatic heterocyclic compound, a preparation method, a pharmaceutical composition and application thereof, and particularly provides a compound shown as the following formula I, or an optical isomer, a hydrate, a solvate or a pharmaceutically acceptable salt thereof; wherein, the definition of each group is described in the specification. The compounds of formula I are useful for treating diseases associated with the PD-1/PD-L1 signaling pathway.)
1. A compound of formula I, or an optical isomer, hydrate, solvate, or pharmaceutically acceptable salt thereof:
wherein n is 0, 1,2 or 3;
m, p, q and t are each independently selected from 0, 1,2,3 or 4;
X1、X2、X3、X4、X5and X6Each independently selected from the group consisting of: n, O, S, SO2、C(R)2、NR；
Y1、Y2、Y3、Y4、Y5And Y6Each independently selected from the group consisting of: n, CH, C;
selected from the group consisting of: substituted or unsubstituted C1-C15 aryl, or substituted or unsubstituted 4-12 membered (preferably 5-7 membered) heteroaryl having 1-4 heteroatoms, substituted or unsubstituted 4-12 membered heterocyclyl, substituted or unsubstituted 4-12 membered C5-C12 cycloalkyl;
selected from the group consisting of: substituted or unsubstituted C1-C15 arylene, or substituted or unsubstituted 4-12 membered (preferably 5-7 membered) heteroarylene having 1-4 heteroatoms, substituted or unsubstituted 4-12 membered heterocyclylene, substituted or unsubstituted 4-12 membered C5-C12 cycloalkylene;
R、R1、R2、R3、R4and R5Each independently selected from the group consisting of: H. -CN, trifluoromethyl, sulfonamido, nitro, hydroxy, halogen, -S-R8、-S(O)-R8、-S(O)2-R8Substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C3-C8 cycloalkyl, oxo (i.e., ═ O), ═ NRf, -CN, hydroxy, NRdRe (e.g., amino), substituted or unsubstituted C1-C6 amine, substituted or unsubstituted- (C1-C6 alkylene) -NH- (C1-C6 alkylene), carboxy, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-12 membered heteroaryl having 1-3 heteroatoms, substituted or unsubstituted 3-12 membered heterocyclyl having 1-4 heteroatoms, substituted or unsubstituted C6-C10 arylSubstituted or unsubstitutedOr (L)1a)r-(L2a)s-(L3a)s-；-C0-8-O-R8,-C0-8-C(O)OR8,-C0-8-OC(O)OR8,-C0-8-NR8R9,-C0-8-N(R8)C(O)R9,-C0-8-C(O)NR8R9；
Or R1And R2Together with the ring atoms to which they are attached form a 5, 6 or 7 membered carbocyclic ring or a 5, 6 or 7 membered heterocyclic ring, wherein said heterocyclic ring contains 1,2 or 3 ring members selected from N, S or O; the carbocycle or heterocycle can be an aromatic ring conjugated with the B ring, and can also be a non-aromatic ring containing unsaturated bonds or a saturated ring;
R8and R9Each independently selected from the group consisting of: H. hydroxy, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C3-C8 cycloalkyl, oxo (i.e., ═ O), ═ NRf, -CN, hydroxy, NRdRe (e.g., amino), substituted or unsubstituted C1-C6 amine, substituted or unsubstituted- (C1-C6 alkylene) -NH- (C1-C6 alkylene), carboxy, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-to 12-membered heteroaryl having 1 to 3 heteroatoms, substituted or unsubstituted 3-to 12-membered heterocyclyl having 1 to 4 heteroatoms, substituted or unsubstituted C6-C10 arylSubstituted or unsubstitutedOr (L)1a)r-(L2a)s-(L3a)s-；
Each L1aEach independently is a group selected from the group consisting of; chemical bond, substituted or unsubstituted C1-C7Alkylene, substituted or unsubstituted C2-C4 alkenylene, substituted or unsubstituted C2-C4 alkynylene, -S-, -O-, substituted or unsubstituted-NH-, -S (O) -, or-S (O)2-；
L2aSelected from the group consisting of: substituted or unsubstituted C6-C12 arylene, substituted or unsubstituted 5-12 membered heteroarylene having 1-3 heteroatoms, substituted or unsubstitutedSubstituted C3-C8 cycloalkylene, substituted or unsubstituted 5-10 membered heterocyclylene having 1-3 heteroatoms;
L3aselected from the group consisting of: substituted or unsubstituted C1-C10 alkyl, C1-C10 aryl, -CN, hydroxy, amino, carboxy, -CO-NH-SO2-Rg、-NH-SO2-Rg、-SO2-NH-CO-Rg；
r is 1,2,3, 4, 5, 6;
s is 0, 1,2 respectively;
rd, Re and Rg are each independently selected from the group consisting of: H. substituted or unsubstituted C1-C6Alkyl, substituted or unsubstituted C3-C10Cycloalkyl, substituted or unsubstituted C6-C10An aryl group; or Rd and Re together form a substituted or unsubstituted 5-10 membered heterocyclyl having 1-3 heteroatoms selected from N, S and O;
said Rf is selected from the group consisting of: H. substituted or unsubstituted C1-C6Alkyl, substituted or unsubstituted C6-C10Aryl, substituted or unsubstituted C6-C10Heteroaryl, cyano, -C (═ O) -NRdRe, -C (═ O) -substituted or unsubstituted C1-C6Alkoxy, -C (═ O) -substituted or unsubstituted C1-C6Alkyl, -C (═ O) -substituted or unsubstituted C3-C10Cycloalkyl, -C (═ O) -substituted or unsubstituted C2-C6Alkenyl, -C (═ O) -substituted or unsubstituted C2-C6An alkynyl group;
unless otherwise specified, "substituted" means substituted with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: halogen: including but not limited to-F, Cl, Br, -CH2Cl、-CHCl2、-CCl3、-CH2F、-CHF2、-CF3Oxo, -CN, hydroxy, amino, C1-C6 alkylamino, carboxy, -NHAc, a group selected from the group consisting of unsubstituted or substituted with one or more substituents selected from the group consisting of: C1-C6 alkyl, C1-C6 alkoxy, C6-C10 aryl, C3-C8 cycloalkyl, halogenated C6-C10 aryl, 5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and OA 5-10 membered heterocyclyl having 1-3 heteroatoms selected from N, S and O; the substituents are selected from the following group: halogen, hydroxy, carboxy, cyano, C1-C6 alkoxy, C1-C6 alkylamino;
in the formulae above, any of the heteroatoms is selected from the group consisting of: B. p, N, S and O.
2. The compound of claim 1, or an isomer, optical isomer, hydrate, solvate, or pharmaceutically acceptable salt thereofIs a ring-forming divalent group selected from the group consisting of: wherein, the bonding position of the ring can be N or C.
3. The compound of claim 1, wherein said compound isIs a structure selected from the group consisting of:
and said R is1And R2Each independently selected from the group consisting of: H. halogen, substituted or unsubstituted C1-C10 alkyl.
4. The compound of claim 1, wherein said compound isIs a structure selected from the group consisting of:
5. the compound of claim 1, wherein said compound isIs a structure selected from the group consisting of:
6. the compound of claim 1, or an optical isomer, hydrate, solvate, or pharmaceutically acceptable salt thereof, selected from the group consisting of;
7. a process for the preparation of a compound of formula I according to claim 1, comprising a step selected from the group consisting of schemes 1 and 2:
synthesis scheme 1:
(a) taking the intermediates 1 and 2 as raw materials, and obtaining a target product I-1 through a Sonogashira coupling reaction catalyzed by a palladium catalyst;
preferably, the preparation method of the intermediate 1 is as follows:
taking a compound 1-1 as a raw material, and carrying out a coupling reaction under the conditions of a palladium catalyst and a ligand to form an intermediate 1-2;
(b) taking 1-2 as a raw material, carrying out a glycidyl reaction with primary and secondary amines under the catalysis of Lewis acid, adding a proper reducing agent, and further reducing to form an intermediate 1-3; or direct reductive amination reaction in the presence of a reducing agent to form intermediates 1-3;
(c) taking 1-3 as a raw material, and carrying out a halogenation reaction under the catalysis of Lewis acid to obtain an intermediate 1;
synthesis scheme 2:
(a) the compound 3-1 is used as raw material to react with nitrating agent (such as concentrated sulfuric acid/NaNO)3Concentrated sulfuric acid/fuming nitric acid, etc.) to form intermediate 3-2;
(b) using 3-2 as raw material, under the condition of reduction (Pd-C/H)2(ii) a Zinc powder/ammonium chloride; iron powder/acetic acid, etc.), and a reduction reaction occurs to form an intermediate 3-3;
(c) taking 3-3 and 3-4 as raw materials, carrying out condensation reaction under the condition of a water reducing agent, adding a proper oxidant (such as DDQ and the like) to carry out oxidation reaction, and obtaining an intermediate 3-5;
(d) taking 3-5 and 3-6 as raw materials, and carrying out coupling reaction under the conditions of a catalyst and a ligand to form an intermediate I-2.
8. A pharmaceutical composition comprising (1) a compound of claim 1 or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof; (2) a pharmaceutically acceptable carrier.
9. Use of a compound according to claim 1 or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition according to claim 7, for the preparation of a pharmaceutical composition for the prophylaxis and/or treatment of diseases associated with the activity or expression of PD-1/PD-L1.
10. A PD-1/PD-L1 modulator, wherein said modulator comprises a compound of claim 1, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof.
Technical Field
The invention relates to the field of small molecule medicines, and particularly provides a small molecule compound which can be used for treating diseases related to a PD-1/PD-L1 signal pathway.
Background
The immune system plays a crucial role in controlling and curing many diseases, such as various cancers, diseases caused by viruses, and the like. Cancer cells often multiply rapidly by evading or suppressing the immune system in some way. One such way is to alter the expression of activating and inhibitory molecules on immune cells. Blocking the suppressive immune checkpoint, like PD-1, has proven to be a very effective method of suppressing cancer cells.
PD-1 is programmed cell death protein-1, also known as CD 279. It is expressed mainly in activated T cells and B cells and functions to inhibit the activation of cells, which is a normal homeostatic mechanism of the immune system, and PD-1 is a protective wall of our human body because excessive T/B cell activation causes autoimmune diseases. PD-1 is a type I transmembrane glycoprotein composed of 268 amino acids, and its structure mainly includes an outer immunoglobulin variable region, a hydrophobic transmembrane region, and an intracellular region. The intracellular region contains two phosphorylation sites, located in the immunoreceptor tyrosine inhibitory motif and immunoreceptor tyrosine switch motif, which also demonstrates that PD-1 is capable of back-regulating T cell receptor-mediated signaling. PD-1 has two ligands, PD-L1 and PD-L2, which differ in the manner of expression. PD-L1, which is up-regulated in many tumor cells, binds to PD-1 on T cells, inhibits T cell proliferation and activation, leaves T cells in an inactive state, and ultimately induces immune escape.
PD-1/PD-L1 exerts a reverse immunomodulatory effect. When PD-1 binds to PD-L1, tyrosine in the tyrosine-converting motif domain of the immune receptor of T cells can be phosphorylated, and the phosphorylated tyrosine can bind to phosphatase protein tyrosinase 2 and protein tyrosinase 1. This can block activation of extracellular signal-regulated kinase, and can block activation of phosphoinositide 3-kinase (PI3K) and serine-threonine protein kinase (Akt), thereby inhibiting T lymphocyte proliferation and secretion of related cytokines. While the PD-1/PD-L1 signal inhibits the activation and proliferation of T cells, the secretion of cytokines interleukin 2, interferon gamma and IL-10 can be realized. In addition, PD-1/PD-L1 signal has similar immune function to B cell, and when PD-1 binds to B cell antigen receptor, PD-1 cytoplasm region acts with tyrosinase containing protein tyrosinase 2 binding site, thereby preventing B cell activation.
PD-1/PD-L1-based immunotherapy is a new generation of immunotherapy that is of great interest. In recent years, a series of surprising research results prove that the PD-1/PD-L1 inhibitor has strong antitumor activity on various tumors. PD-1/PD-L1 antibody modulators currently on the market are Ninolumab from BMS, Lamboluzumab from Merck, and Atezolizumab from Roche. In addition to this, there are many PD-1/PD-L1 antibody modulators in the research, including Cure Tech's Pidilizumab, GSK's AMP-224, and AstraZeneca's MEDI-4736.
Although tumor immunotherapy is considered as a new generation of revolution for targeted post-treatment cancer therapy. However, currently marketed and researched PD-1 single drug has its own defects, including injection administration, oral administration, instability in vivo, easy protease decomposition, easy immune cross reaction, difficult purification, high production cost, etc. Therefore, small molecule modulators of the PD-1/PD-L1 interaction are a better choice for tumor immunotherapy.
In view of the foregoing, there is a pressing need in the art to develop novel small molecule modulators of the PD-1/PD-L1 interaction.
Disclosure of Invention
The invention aims to provide a novel small molecule regulator of PD-1/PD-L1 interaction.
In a first aspect of the present invention, there is provided a compound represented by the following formula I, or an optical isomer, hydrate, solvate, or a pharmaceutically acceptable salt thereof:
wherein n is 0, 1,2 or 3;
m, p, q and t are each independently selected from 0, 1,2,3 or 4;
X1、X2、X3、X4、X5and X6Each independently selected from the group consisting of: n, O, S, SO2、C(R)2、NR；
Y1、Y2、Y3、Y4、Y5And Y6Each independently selected from the group consisting of: n, CH, C;
selected from the group consisting of: substituted or unsubstituted C1-C15 aryl, or substituted or unsubstituted 4-12 membered (preferably 5-7 membered) heteroaryl having 1-4 heteroatoms, substituted or unsubstituted 4-12 membered heterocyclyl, substituted or unsubstituted 4-12 membered C5-C12 cycloalkyl;
selected from the group consisting of: substituted or unsubstituted C1-C15 arylene, or substituted or unsubstituted 4-12 membered (preferably 5-7 membered) heteroarylene having 1-4 heteroatoms, substituted or unsubstituted 4-12 membered heterocyclylene, substituted or unsubstituted 4-12 membered C5-C12 cycloalkylene;
R、R1、R2、R3、R4and R5Each independently selected from the group consisting of: H. -CN, trifluoromethyl, sulfonamido, nitro, hydroxy, halogen, -S-R8、-S(O)-R8、-S(O)2-R8Substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C3-C8 cycloalkyl, oxo (i.e., ═ O), ═ NRf, -CN, hydroxy, NRdRe (e.g., amino), substituted or unsubstituted C1-C6 amine, substituted or unsubstituted- (C1-C6 alkylene) -NH- (C1-C6 alkylene), carboxy, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-12 membered heteroaryl having 1-3 heteroatoms, substituted or unsubstituted 3-12 membered heterocyclyl having 1-4 heteroatoms, substituted or unsubstituted C6-C10 arylSubstituted or unsubstitutedOr (L)1a)r-(L2a)s-(L3a)s-；-C0-8-O-R8,-C0-8-C(O)OR8,-C0-8-OC(O)OR8,-C0-8-NR8R9,-C0-8-N(R8)C(O)R9,-C0-8-C(O)NR8R9；
Or R1And R2Together with the ring atoms to which they are attached form a 5, 6 or 7 membered carbocyclic ring or a 5, 6 or 7 membered heterocyclic ring, wherein said heterocyclic ring contains 1,2 or 3 ring members selected from N, S or O; the carbocycle or heterocycle can be an aromatic ring conjugated with the B ring, and can also be a non-aromatic ring containing unsaturated bonds or a saturated ring;
R8and R9Each independently selected from the group consisting of: H. hydroxy, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C3-C8 cycloalkyl, oxo (i.e., ═ O), ═ NRf, -CN, hydroxy, NRdRe (e.g., amino), substituted or unsubstituted C1-C6 amine, substituted or unsubstituted- (C1-C6 alkylene) -NH- (C1-C6 alkylene), carboxy, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-to 12-membered heteroaryl having 1 to 3 heteroatoms, substituted or unsubstituted 3-to 12-membered heterocyclyl having 1 to 4 heteroatoms, substituted or unsubstituted C6-C10 arylSubstituted or unsubstitutedOr (L)1a)r-(L2a)s-(L3a)s-；
Each L1aEach independently is a group selected from the group consisting of; chemical bond, substitution orUnsubstituted C1-C7Alkylene, substituted or unsubstituted C2-C4 alkenylene, substituted or unsubstituted C2-C4 alkynylene, -S-, -O-, substituted or unsubstituted-NH-, -S (O) -, or-S (O)2-；
L2aSelected from the group consisting of: substituted or unsubstituted C6-C12 arylene, substituted or unsubstituted 5-12 membered heteroarylene having 1-3 heteroatoms, substituted or unsubstituted C3-C8 cycloalkylene, substituted or unsubstituted 5-10 membered heterocyclylene having 1-3 heteroatoms;
L3aselected from the group consisting of: substituted or unsubstituted C1-C10 alkyl, C1-C10 aryl, -CN, hydroxy, amino, carboxy, -CO-NH-SO2-Rg、-NH-SO2-Rg、-SO2-NH-CO-Rg；
r is 1,2,3, 4, 5, 6;
s is 0, 1,2 respectively;
rd, Re and Rg are each independently selected from the group consisting of: H. substituted or unsubstituted C1-C6Alkyl, substituted or unsubstituted C3-C10Cycloalkyl, substituted or unsubstituted C6-C10An aryl group; or Rd and Re together form a substituted or unsubstituted 5-10 membered heterocyclyl having 1-3 heteroatoms selected from N, S and O;
said Rf is selected from the group consisting of: H. substituted or unsubstituted C1-C6Alkyl, substituted or unsubstituted C6-C10Aryl, substituted or unsubstituted C6-C10Heteroaryl, cyano, -C (═ O) -NRdRe, -C (═ O) -substituted or unsubstituted C1-C6Alkoxy, -C (═ O) -substituted or unsubstituted C1-C6Alkyl, -C (═ O) -substituted or unsubstituted C3-C10Cycloalkyl, -C (═ O) -substituted or unsubstituted C2-C6Alkenyl, -C (═ O) -substituted or unsubstituted C2-C6An alkynyl group;
unless otherwise specified, "substituted" means substituted with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: halogen: including but not limited to-F, Cl, Br, -CH2Cl、-CHCl2、-CCl3、-CH2F、-CHF2、-CF3Oxo, -CN, hydroxy, amino, C1-C6 alkylamino, carboxy, -NHAc, a group selected from the group consisting of unsubstituted or substituted with one or more substituents selected from the group consisting of: C1-C6 alkyl, C1-C6 alkoxy, C6-C10 aryl, C3-C8 cycloalkyl, halogenated C6-C10 aryl, 5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O, 5-10 membered heterocyclyl having 1-3 heteroatoms selected from N, S and O; the substituents are selected from the following group: halogen, hydroxy, carboxy, cyano, C1-C6 alkoxy, C1-C6 alkylamino;
in the formulae above, any of the heteroatoms is selected from the group consisting of: B. p, N, S and O.
In another preferred embodiment, R4Is composed ofWherein R isbAnd Rc RdEach independently selected from the group consisting of: H. substituted or unsubstituted C1-C8An alkyl group; or said RbAnd RcTogether with the adjacent N atom, form a substituted or unsubstituted 4-10 membered heterocyclic group having 1-3 heteroatoms selected from N, S and O.
In another preferred embodiment, theIs a ring-forming divalent group selected from the group consisting of: saidIs a ring-forming divalent group selected from the group consisting of:
wherein, the bonding position of the ring can be N or C.
In another advantageIn the alternative, R is3Selected from the group consisting of: methyl, -F, Cl, Br, -CH2Cl、-CHCl2、-CCl3、-CH2F、-CHF2、-CF3CN, -hydroxyl and amino.
In another preferred embodiment, theIs a structure selected from the group consisting of:
and said R is1And R2Each independently selected from the group consisting of: H. halogen, substituted or unsubstituted C1-C10 alkyl.
In another preferred embodiment, theIs a structure selected from the group consisting of:
in another preferred embodiment, theThe structure is as follows:
in another preferred embodiment, theIs a structure selected from the group consisting of:
in another preferred embodiment, the compound is selected from the following compounds;
in a second aspect of the invention, there is provided a process for the preparation of a compound of formula I according to the first aspect of the invention, comprising a step selected from the group consisting of those depicted in schemes 1 and 2:
synthesis scheme 1:
(a) taking the intermediates 1 and 2 as raw materials, and obtaining a target product I-1 through a Sonogashira coupling reaction catalyzed by a palladium catalyst;
preferably, the preparation method of the intermediate 1 is as follows:
taking a compound 1-1 as a raw material, and carrying out a coupling reaction under the conditions of a palladium catalyst and a ligand to form an intermediate 1-2;
(b) taking 1-2 as a raw material, carrying out a glycidyl reaction with primary and secondary amines under the catalysis of Lewis acid, adding a proper reducing agent, and further reducing to form an intermediate 1-3; or direct reductive amination reaction in the presence of a reducing agent to form intermediates 1-3;
(c) taking 1-3 as a raw material, and carrying out a halogenation reaction under the catalysis of Lewis acid to obtain an intermediate 1;
synthesis scheme 2:
(a) the compound 3-1 is used as raw material to react with nitrating agent (such as concentrated sulfuric acid/NaNO)3Concentrated sulfuric acid/fuming nitric acid, etc.) to form intermediate 3-2;
(b) using 3-2 as raw material, under the condition of reduction (Pd-C/H)2(ii) a Zinc powder/ammonium chloride; iron powder/acetic acid, etc.), and a reduction reaction occurs to form an intermediate 3-3;
(c) taking 3-3 and 3-4 as raw materials, carrying out condensation reaction under the condition of a water reducing agent, adding a proper oxidant (such as DDQ and the like) to carry out oxidation reaction, and obtaining an intermediate 3-5;
(d) taking 3-5 and 3-6 as raw materials, and carrying out coupling reaction under the conditions of a catalyst and a ligand to form an intermediate I-2.
In a third aspect of the present invention, there is provided a pharmaceutical composition comprising (1) a compound according to the first aspect of the present invention, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof; (2) a pharmaceutically acceptable carrier.
In a fourth aspect of the present invention, there is provided a use of a compound according to the first aspect of the present invention or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition according to the third aspect of the present invention, for the preparation of a pharmaceutical composition for the prevention and/or treatment of a disease associated with an activity or an expression amount of PD-1/PD-L1.
In a fifth aspect of the invention, there is provided a PD-1/PD-L1 modulator comprising a compound of the first aspect of the invention, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof.
In another preferred embodiment, the pharmaceutical composition is used for treating a disease selected from the group consisting of: cancer, infectious disease, autoimmune disease.
In another preferred embodiment, the cancer is selected from the group consisting of: pancreatic cancer, bladder cancer, colorectal cancer, breast cancer, prostate cancer, kidney cancer, hepatocellular cancer, lung cancer, ovarian cancer, cervical cancer, gastric cancer, esophageal cancer, melanoma, neuroendocrine cancer, central nervous system cancer, brain cancer, bone cancer, soft tissue sarcoma, non-small cell lung cancer, small cell lung cancer or colon cancer, skin cancer, lung cancer, urologic tumors, hematologic tumors, glioma, digestive system tumors, reproductive system tumors, lymphoma, nervous system tumors, brain tumors, head and neck cancer.
In another preferred embodiment, the cancer is selected from the group consisting of: acute Lymphocytic Leukemia (ALL), Acute Myelogenous Leukemia (AML), Chronic Lymphocytic Leukemia (CLL), Small Lymphocytic Lymphoma (SLL), myelodysplastic syndrome (MDS), myeloproliferative disorder (MPD), Chronic Myelogenous Leukemia (CML), Multiple Myeloma (MM), non-hodgkin's lymphoma (NHL), Mantle Cell Lymphoma (MCL), follicular lymphoma, Waldestrom Macroglobulinemia (WM), T-cell lymphoma, B-cell lymphoma or diffuse large B-cell lymphoma (DLBCL).
In another preferred embodiment, the infectious disease is selected from bacterial infection and viral infection.
In another preferred embodiment, the autoimmune disease is selected from organ-specific autoimmune disease, systemic autoimmune disease.
In another preferred embodiment, the organ-specific autoimmune disease comprises chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, goodpasture's syndrome, primary biliary cirrhosis, multiple sclerosis, acute idiopathic polyneuritis.
In another preferred embodiment, the systemic autoimmune disease comprises rheumatoid arthritis, systemic lupus erythematosus, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia.
In another preferred embodiment, the pharmaceutical composition is also used for improving the T cell function of Chronic Hepatitis B (CHB) patients.
In another preferred embodiment, the inhibitor further comprises at least one therapeutic agent selected from the group consisting of: nivolumab, pembrolizumab, atezolizumab, or ipilimumab.
In a sixth aspect of the invention, there is provided a method of modulating the PD-1/PD-L1 interaction in vivo, comprising the steps of: contacting a compound according to the first aspect of the invention, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, with PD-L1 protein.
Detailed Description
The present inventors have extensively and intensively studied to find a class of PD-1/PD-L1 interaction modulators having an excellent modulating effect. On this basis, the inventors have completed the present invention.
Definition of
As used herein, the term "alkyl" includes straight or branched chain alkyl groups. E.g. C1-C8Alkyl represents a straight or branched chain alkyl group having 1 to 8 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, and the like.
As used herein, the term "alkenyl" includes straight or branched chain alkenyl groups. E.g. C2-C6Alkenyl means straight-chain or branched alkenyl having 2 to 6 carbon atoms, e.g. vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl2-butenyl, or the like.
As used herein, the term "alkynyl" includes straight or branched chain alkynyl groups. E.g. C2-C6Alkynyl means straight or branched chain alkynyl having 2 to 6 carbon atoms, such as ethynyl, propynyl, butynyl, or the like.
As used herein, the term "C3-C10Cycloalkyl "refers to cycloalkyl groups having 3 to 10 carbon atoms. It may be a single ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or the like. It may also be in the form of a double ring, for example a bridged or spiro ring.
As used herein, the term "C1-C8Alkylamino "is defined as being substituted by C1-C8The amino group substituted by the alkyl can be mono-substituted or di-substituted; for example, methylamino, ethylamino, propylamino, isopropylamino, butylamino, isobutylamino, tert-butylamino, dimethylamino, diethylamino, dipropylamino, diisopropylamino, dibutylamino, diisobutylamino, di-tert-butylamino and the like.
As used herein, the term "C1-C8Alkoxy "means a straight or branched chain alkoxy group having 1 to 8 carbon atoms; for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy and the like.
As used herein, the term "3-10 membered heterocycloalkyl having 1-3 heteroatoms selected from the group consisting of N, S and O" refers to a saturated or partially saturated cyclic group having 3-10 atoms and wherein 1-3 atoms are heteroatoms selected from the group consisting of N, S and O. It may be monocyclic or may be in the form of a double ring, for example a bridged or spiro ring. Specific examples may be oxetane, azetidine, tetrahydro-2H-pyranyl, piperidinyl, tetrahydrofuranyl, morpholinyl, pyrrolidinyl, and the like.
As used herein, the term "C6-C10Aryl "means an aryl group having 6 to 10 carbon atoms, for example, phenyl or naphthyl and the like.
As used herein, the term "5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S and O" refers to a cyclic aromatic group having 5-10 atoms and wherein 1-3 atoms are heteroatoms selected from the group consisting of N, S and O. It may be a single ring or a condensed ring form. Specific examples may be pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrrolyl, pyrazolyl, imidazolyl, (1,2,3) -triazolyl and (1,2,4) -triazolyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl and the like.
Unless specifically stated to be "substituted or unsubstituted", the groups of the present invention may be substituted with a substituent selected from the group consisting of: halogen, nitrile group, nitro group, hydroxyl group, amino group, C1-C6Alkyl-amino, C1-C6Alkyl radical, C2-C6Alkenyl radical, C2-C6Alkynyl, C1-C6Alkoxy, halo C1-C6Alkyl, halo C2-C6Alkenyl, halo C2-C6Alkynyl, halo C1-C6Alkoxy, allyl, benzyl, C6-C12Aryl radical, C1-C6alkoxy-C1-C6Alkyl radical, C1-C6Alkoxy-carbonyl, phenoxycarbonyl, C2-C6Alkynyl-carbonyl, C2-C6Alkenyl-carbonyl, C3-C6Cycloalkyl-carbonyl, C1-C6Alkyl-sulfonyl, and the like.
As used herein, "halogen" or "halogen atom" refers to F, Cl, Br, and I. More preferably, the halogen or halogen atom is selected from F, Cl and Br. "halogenated" means substituted with an atom selected from F, Cl, Br, and I.
Unless otherwise specified, the structural formulae depicted herein are intended to include all isomeric forms (e.g., enantiomers, diastereomers and geometric isomers (or conformational isomers)): for example, R, S configuration containing an asymmetric center, (Z), (E) isomers of double bonds, and the like. Thus, individual stereochemical isomers of the compounds of the present invention or mixtures of enantiomers, diastereomers or geometric isomers (or conformers) thereof are within the scope of the present invention.
As used herein, the term "tautomer" means that structural isomers having different energies may exceed the low energy barrier, thereby converting with each other. For example, proton tautomers (i.e., proton transmutations) include interconversion by proton shift, such as 1H-indazoles and 2H-indazoles. Valence tautomers include interconversion by recombination of some of the bonding electrons.
As used herein, the term "solvate" refers to a complex of a compound of the present invention coordinated to solvent molecules in a specific ratio.
As used herein, the term "hydrate" refers to a complex formed by the coordination of a compound of the present invention with water.
Active ingredient
As used herein, "compounds of the invention" refers to compounds of formula I, and also includes various crystalline forms, pharmaceutically acceptable salts, hydrates, or solvates of the compounds of formula I.
Preferred compounds of the present invention include compounds 1-360 (including various classes of R configuration and/or S configuration stereoisomers of each compound, and/or E-/Z-cis-trans isomers).
In another preferred embodiment, the pharmaceutically acceptable salts include salts formed in combination with inorganic acids, organic acids, alkali metal ions, alkaline earth metal ions, or organic bases capable of providing physiologically acceptable cations, as well as ammonium salts.
In another preferred embodiment, the inorganic acid is selected from hydrochloric acid, hydrobromic acid, phosphoric acid or sulfuric acid; the organic acid is selected from methanesulfonic acid, p-toluenesulfonic acid, trifluoroacetic acid, lycic acid, tartaric acid maleate, fumaric acid, citric acid or lactic acid; the alkali metal ions are selected from lithium ions, sodium ions and potassium ions; the alkaline earth metal ions are selected from calcium ions and magnesium ions; the organic base capable of providing a physiologically acceptable cation is selected from methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris (2-hydroxyethyl) amine.
All such salts within the scope of the present invention may be prepared by conventional methods. During the preparation of the compounds of formula I and solvates and salts thereof, different crystallization conditions may occur as polycrystals or co-crystals.
Preparation of Compounds of formula I
For the preparation of the compounds of the general formula I according to the invention, the preparation of the compounds of the general formula I according to the invention can be obtained by the following synthetic route, depending on the structure of the general formula I.
Equation 1:
(a) taking the intermediates 1 and 2 as raw materials, and obtaining a target product I-1 through coupling reaction catalyzed by a proper palladium catalyst;
the preparation of intermediate 1 is exemplified as follows:
(a) taking a compound 1-1 as a raw material (X is Cl, Br or I), and carrying out coupling reaction under the conditions of a proper palladium catalyst and a ligand to form an intermediate 1-2;
(b) taking 1-2 as a raw material, carrying out a glycidyl reaction with primary and secondary amines under the catalysis of proper Lewis acid, adding a proper reducing agent, and further reducing to form an intermediate 1-3; or direct reductive amination in the presence of a suitable reducing agent to form intermediates 1-3;
(c) taking 1-3 as a raw material, and carrying out a halogenation reaction under the catalysis of a proper Lewis acid to obtain an intermediate 1;
the preparation method of the intermediate 2 comprises the following steps:
(a) taking compounds 2-1 and 2-2 as raw materials, and carrying out coupling reaction (such as Suzuki, Buchwald and the like) under the condition of a proper palladium catalyst and a ligand to obtain an intermediate 2-3;
(b) starting with 2-2, the silicon-based protecting group is removed using a suitable reagent to give intermediate 2.
In addition, the starting materials and intermediates in the above reactions are readily available, and the reactions in each step can be readily synthesized according to reported literature or by conventional methods in organic synthesis to those skilled in the art. The compounds of formula I may exist in the form of solvates or non-solvates, and crystallization using different solvents may give different solvates.
Equation 2:
(a) the compound 3-1 is used as raw material to react with a proper nitrating agent (such as concentrated sulfuric acid/NaNO)3Concentrated sulfuric acid/fuming nitric acid, etc.) to form intermediate 3-2;
(b) using 3-2 as raw material, under proper reducing condition (Pd-C/H)2(ii) a Zinc powder/ammonium chloride; iron powder/acetic acid, etc.), and a reduction reaction occurs to form an intermediate 3-3;
(c) taking 3-3 and 3-4 as raw materials, carrying out condensation reaction under the condition of a proper water reducing agent, adding a proper oxidant (such as DDQ and the like) to carry out oxidation reaction to obtain an intermediate 3-5;
(d) taking 3-5 and 2-1 as raw materials, and carrying out coupling reaction under the condition of a proper catalyst and a ligand to form an intermediate I-2;
in addition, the starting materials and intermediates in the above reactions are readily available, and the reactions in each step can be readily synthesized according to reported literature or by conventional methods in organic synthesis to those skilled in the art. The compounds of formula I may exist in the form of solvates or non-solvates, and crystallization using different solvents may give different solvates.
Pharmaceutical compositions and methods of administration
The compound of the present invention and various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates thereof, and pharmaceutical compositions containing the compound of the present invention as a main active ingredient can be used for preventing and/or treating (stabilizing, alleviating or curing) diseases (e.g., cancer, infectious diseases, autoimmune diseases) associated with the PD-1/PD-L1 interaction, because the compound of the present invention has excellent activity of regulating the PD-1/PD-L1 interaction.
The pharmaceutical compositions of the present invention comprise a safe and effective amount of a compound of the present invention in combination with a pharmaceutically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000mg of a compound of the invention per dose, more preferably, 10-200mg of a compound of the invention per dose. Preferably, said "dose" is a capsule or tablet.
"pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the components of the composition are capable of intermixing with and with the compounds of the present invention without significantly diminishing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g. sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g. stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g. soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g. propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (e.g. tween, etc.)) Wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.
The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, parenteral (intravenous, intramuscular or subcutaneous).
Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) disintegrating agents, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.
Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be delayed in release in a certain part of the digestive tract. Examples of embedding components which can be used are polymeric substances and wax-like substances. If desired, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.
Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly employed in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of such materials and the like.
In addition to these inert diluents, the compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.
The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds (e.g., other anti-cancer agents).
When administered in combination, the pharmaceutical composition further comprises one or more (2, 3, 4, or more) other pharmaceutically acceptable compounds. One or more (2, 3, 4, or more) of the other pharmaceutically acceptable compounds may be used simultaneously, separately or sequentially with the compounds of the invention for the prevention and/or treatment of a disease associated with the PD-1/PD-L1 interaction.
When the pharmaceutical composition is used, a safe and effective amount of the compound of the present invention is suitable for mammals (such as human beings) to be treated, wherein the administration dose is a pharmaceutically-considered effective administration dose, and for a human body with a weight of 60kg, the daily administration dose is usually 1 to 2000mg, preferably 20 to 500 mg. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.
The main advantages of the invention include:
(1) the compound has high regulation activity on the interaction of PD-1/PD-L1, has strong binding capacity with PD-L1 protein, and has the capacity of relieving the inhibition of IFN gamma by PD-L1.
(2) The compound of the invention has better solubility; the toxicity to normal cells is very low and can therefore be applied to the treated subject over a wide dose range.
(3) Compared with the prior art, the compound of the invention has better solubility and good drug forming property, and the compound of the invention has good bioavailability in vivo experiments, and in addition, compared with the prior art, the compound of the invention can be easily prepared into pharmaceutically acceptable salts, thereby being beneficial to further preparation.
(4) The in vivo efficacy research shows that the compound can obviously inhibit the growth of subcutaneous tumors no matter on the tumor volume or the tumor weight, and can obviously increase the number of lymphocytes in the blood and the spleen of a mouse.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are percentages and parts by weight.
The test materials and reagents used in the following examples are commercially available without specific reference.
General materials and test methods:
the instruments and materials involved in the examples are described below:
the NMR spectrum was obtained by analysis with a Bruker AV-400(400MHz) NMR spectrometer.
Chemical shifts are reported in ppm units using tetramethylsilane as an internal standard (CDC 1)3Delta 7.26 ppm). The data reported are the chemical shifts and their split and coupling constants (s: singlet; d: doublet; t: triplet; q: quartet; br: broad; m: multiplet).
Mass spectrometry data were analyzed using a liquid mass spectrometer from the Finnigen advanced LCQ company (Finnigan LCQ Advantage), all reactions operated under anhydrous and oxygen-free conditions under dry argon protection, except for other requirements. The solid organometallic compound was stored in an argon protected dry box.
The tetrahydrofuran and the diethyl ether are obtained by distillation, and metal sodium and benzophenone are added into the tetrahydrofuran and the diethyl ether during the distillation. Methylene chloride, pentane and hexane were treated with calcium hydride.
The special raw materials and intermediates involved in the present invention are provided by custom-made processing of Tianjin Changsen pharmaceutical Co., Ltd, and all other chemical reagents are purchased from reagent suppliers such as Shanghai chemical reagent company, Aldrich company (Aldrich), Acros company (Acros). If the intermediates or products needed by the reaction in the synthesis process are not enough for the next experiment, the synthesis is repeated for a plurality of times until the intermediates or products are enough.
The raw materials and reagents related to the invention can be obtained by commercial or customized processing and purchase except for special instructions.
The compounds of the invention may contain one or more asymmetric centres and so the series of compounds may be in racemic or single enantiomeric form. The compound prepared by the invention is a heterocyclic compound with the purity higher than 95 percent, and the structural representation of each final product is respectively represented by MS or/and hydrogen spectrum nuclear magnetic resonance (1H NMR) analysis. The synthesis of the various compounds and intermediates of the invention is illustrated by the examples below.
EXAMPLE 1 Synthesis of Compound 001
Step 1-1:
taking 1-1(100g,1.0eq) of raw materials and 27g,1.0eq of sodium hydroxide, adding the raw materials into water (500ml) and methanol (2000ml), keeping the brown color slightly turbid, cooling to about-25 ℃ of an internal bath, adding NIS (151g,1.0eq) at one time, naturally heating for reaction, increasing the solid, then gradually dissolving to the dark brown color, clearing, reacting for 2 hours, adding 1000ml of water, adding 2M hydrochloric acid (400ml), performing weak acidity on the system, filtering, rinsing with MTBE for 2 times to obtain the solid: 161g, yield: 87 percent. MS-APCI 275[ M + H ]]+.
Step 1-2:
adding DMF (1000ml, 2BV) into a reaction bottle, adding 1-2(500g,1.0eq) and cuprous cyanide (172g,1.05eq) into the reaction bottle under the protection of nitrogen, replacing the raw materials with nitrogen, heating to 115 ℃ in an internal bath, reacting for 6 hours, and tracking by LCMS; and (3) post-treatment: adding THF for dilution, filtering, concentrating the filtrate, adding a sodium hydroxide aqueous solution (1.1eq, 6BV) at room temperature, and adjusting the pH value to 8-9; filtering, adjusting the pH of the filtrate to 7 by using 2M sulfuric acid, adding ferrous sulfate and activated carbon, stirring at room temperature for 1 hour, filtering, adjusting the pH of the filtrate to 2-3 by adding 2M sulfuric acid (400ml), filtering the solid, pulping isopropanol (200ml) for 30 minutes, filtering, and drying to obtain 211g of brown solid. MS-APCI:174[ M + H ]]+.
Step 1-3:
4.5 g of compound 1-3 are dissolved in 200ml of DCM (in a 500ml single-neck flask) and 6.3 g of NIS are added under argon. The reaction was carried out at room temperature overnight. TLC spot plate 1-2 reaction was complete. The reaction mixture was added with 100mL of 2M HCl, separated, and dried. Purification by spin-drying over column (Hep-DCM 1:1, then DCM) gave 6g of product as a pale yellow solid. MS-APCI of 300[ M + H ]]+.
Step 1-4:
in a 50 ml three-necked flask 945 mg of compound 1-4,636 mg of compound 1-5,25 mg of CuI and 25mg of Pd (PPh)3)2Cl2Under the protection of argon, 20ml of DMF and 1.2 ml of DIPEA were added and reacted at 55 ℃ for 3 hours. TLC spot plate material disappeared. The reaction solution was added to 300 ml of ice water, extracted twice with 300 ml of ethyl acetate, washed once with 2M NaOH, dried and spun dry. Purification by column (DCM-MeOH 10:1) afforded 120mg of a pale yellow solid. MS-APCI:364[ M + H]+.
Step 1-5:
215 mg of the compound 1-6,248 mg of azetidine-3-carboxylic acid methyl ester hydrochloride, 125mg of Na (CN) BH3Dissolved in 10 ml of THF under argon protection, 167 mg of triethylamine are added and the reaction is carried out at 70 ℃ for 24 hours. The reaction solution was added to 100ml of ice water, extracted twice with 100ml of EA, and dried. Purification by column chromatography (DCM-MeOH100,50:1,10:1,5:1) afforded 53mg of a pale yellow solid 1-7, MS-APCI:463[ M + H ]]+.
Step 1-6:
53mg of starting material 1-7 was dissolved in 5ml of MeOH, 2 ml of 2M NaOH was added, and the reaction was carried out at 26 ℃ for 5 hours. The reaction solution was spun dry at 40 ℃ and the residual solid was adjusted to pH7 with 50 mL of water and 2M HCl, and the solid was filtered off, washed with water, washed with THF, dried and further purified to give 5mg of a yellow solid. MS-APCI 449[ M + H ]]+
Using the procedure for the synthesis of compound 001, the following compounds in the list were also synthesized:
EXAMPLE 2 Synthesis of Compound 026
Step 2-1:
adding 1-3(110g, 1.0eq) of raw materials into acetic acid (660ml,6BV), carrying out turbidity, heating to 40 ℃ of an internal bath, slowly dropwise adding fuming nitric acid (84g,2.1eq) to obtain turbidity, carrying out heat preservation reaction for about 1 hour, carrying out LCMS tracking, and carrying out aftertreatment after 3.5% of raw materials remain: cooling to 10 ℃, stirring for 30 minutes, filtering, HEP rinsing, and airing to obtain 117g of solid, wherein the yield is as follows: 84 percent of purity 88 percent and 8 percent of raw material, adding THF (600ml) into an external bath, pulping for 1 hour at 50 ℃, and filtering at room temperature to obtain 106g of solid, wherein the yield is as follows: 76 percent. MS-APCI 219[ M + H ]]+.
Step 2-2:
taking 2-4(20g, 1.0eq) raw materials, adding TFA (12g,1.1eq) into THF (400ml,20BV), carrying out turbidity, adding 5% platinum carbon (10-20%), carrying out hydrogen replacement, carrying out room temperature reaction, carrying out LCMS tracking, (the reaction speed is influenced by multiple factors), and carrying out aftertreatment: filtered, rinsed with 3BVTHF and directly dosed to the next step. MS-APCI:189[ M + H ]]+.
Step 2-3:
2.02 g of the compound 2-3 is dissolved in 50 ml of DMAC, 1.98 g of the compound 2-4 is added, and the mixture is reacted for 3 hours at room temperature under the protection of argon. 3g of DDQ was added and reacted at room temperature overnight. The reaction was poured into 150 ml of water, extracted twice with DCM (100 ml) and dried. Purification by spin-drying over column (DCM-MeOH100,50:1, 30:1, 20:1,10:1, 5:1) afforded 100mg of productA pale yellow solid. MS-APCI 387[ M + H ]]+。
Step 2-4:
dissolve intermediate 2-7(20.7g, 3.3eq) in THF (143mL, 10V/W), add intermediate 2-8(14.3g, 1.0eq), add NaBH3CN (5.0eq, 11.6g) in place of nitrogen. Stir in an oil bath at 70 ℃ overnight. And (4) detecting by LC-MS, stopping the reaction when about 15% of 2-7 is remained, cooling to room temperature, combining with another batch of 1g of the reaction, adding water, extracting twice by EA, combining organic phases, washing by using salt water, drying and concentrating. The sample was stirred through the column with MeOH in DCM (2%) to give 17.2g of product and 5.0g of cross-over point as an off-white solid. Pulping with DCM (1: 1100 mL) and DCM (20 mL) respectively, stirring at room temperature for 10 minutes, filtering, rinsing with Hep, collecting filter cakes, drying to obtain 10.3g and 1.0g respectively, detecting by LCMS (liquid chromatography S) that no obvious impurity exists, and combining to obtain 11.1g of off-white solid, wherein the HPLC yield is 51.8%. The chiral resolution is carried out to obtain 14.98 g of peak and 25.12 g of peak.
Step 2-5:
tert-butyl ester 2-9(1g,1.0eq) was dissolved in TFA (10v/w) and stirred at room temperature for 1 hour. LCMS check reaction complete, concentrate directly to give yellow to tan oil. Adding water (10v/w) to the concentrated residue to precipitate viscous semi-oil semi-solid, adjusting pH to 6-7 with saturated NaHCO3 solution, and aggregating a large amount of viscous semi-oil semi-solid into mass(1). The coarse filtration is carried out once, a filter cake (or pasty) is collected, water (20v/w) is added, ultrasonic treatment is carried out for 5-10 minutes, and the viscous substance is gradually solidified and partially dispersed. Stirring at room temperature for 2 hr, basically dispersing, filtering, washing with water, collecting filter cake, and drying to obtain 500mg of product MS-APCI:486[ M + H ]]+。
Step 2-6:
the compound borate ester raw materials 2-11 (commerical, 37mg), 2-10(100mg), potassium phosphate (88mg) and RuPhos-Pd-G3(5mg) are sequentially added into a single-neck bottle filled with 1,4-Dioxane/H2O (4:1, 5mL), protected by nitrogen, stirred for 1 hour at 90 ℃ in an oil bath, and the reaction is detected by LC-MS. After the reaction is completed, filtering, spin-drying solvent, Prep-HPLC to obtain 10mg of compound 026 (white solid), ESI (APCI) 542[ M + H ]]+.
1H NMR(400MHz,DMSO-d6)δ12.94(s,1H),10.34(s,1H),8.47(s,1H),8.08(dd,J＝6.9,2.6Hz,1H),7.77–7.39(m,2H),7.10–6.64(m,3H),5.12(s,1H),4.28(s,4H),3.67–3.4(m,3H),3.24–3.15(m,3H),2.37–2.10(m,2H)，2.00–1.93(m,1H).
Using the procedure for the synthesis of compound 002, the following compounds in the list were synthesized:
example 3 synthesis of compound 025 step 3-1:
compound 3-1(1125mg, 2.32mmol), cyclopropylsulfonamide (310mg, 2.56mmol), DMAP (567mg, 4.64mmol) and EDCI (889mg, 4.64mmol) were added sequentially to a single vial containing DCM (15mL), stirred at room temperature for 1 hour and checked by TLC (weak UV, dot plate after washing with 0.5M dilute hydrochloric acid). After the reaction is completed, washing with 0.5M dilute hydrochloric acid and brine, drying with anhydrous sodium sulfate, and performing column chromatography to obtain 548mg of white solid. ESI (APCI) 589[ M-H ] -.
Step 3-2:
compound borate ester starting materials 2-11 (commerical, 28mg), 2-10(90mg), potassium phosphate (88mg) and Xantphos-PdCl2(13mg) was added to the flask in the same order with 1,4-Dioxane/H2O (4:1, 5mL) in a single-neck flask, under nitrogen, stirred for 2 hours at 90 ℃ in an oil bath, and the reaction was checked by LC-MS. After the reaction is completed, filtering, spin-drying solvent, Prep-HPLC to obtain 10mg of compound 025 (white solid), ESI (APCI) 645[ M + H ]]+.
1H NMR(400MHz,DMSO-d6)δ8.50(s,1H),8.11(dd,J＝6.8,2.6Hz,1H),7.72–7.61(m,2H),6.99(dd,J＝5.2,3.1Hz,2H),6.94(dd,J＝8.3,2.1Hz,1H),5.17(d,J＝7.0Hz,1H),4.31(s,4H),3.49(d,J＝8.2Hz,2H),3.32(s,2H),3.24(ddd,J＝17.2,8.6,3.1Hz,2H),2.96(dq,J＝12.8,6.6,5.9Hz,2H),2.69–2.54(m,2H).
Biological assay
Example A: PD-1/PD-L1 homogeneous time-resolved fluorescence (HTRF) binding assay
The assay was performed in standard black 384 well polystyrene plates with a final volume of 20. mu.L. Inhibitors were first serially diluted in DMSO and added to the plate wells, followed by the addition of the other reaction components. The final concentration of DMSO in the assay was 1%. The assay was performed in PBS buffer (pH7.4) containing 0.05% Tween-20 and 0.1% BSA at 25 ℃. Recombinant human PD-L1 protein (19-238) with a His tag at the C-terminus was purchased from Acro biosystems (PD 1-H5229). Recombinant human PD-1 protein (25-167) with an Fc marker at the C-terminus was also purchased from Acrobiosystems (PD 1-H5257). PD-L1 and PD-1 protein were diluted in assay buffer and 0.1. mu.l of the solution was extracted and added to the plate well. The plates were centrifuged and the proteins were preincubated with inhibitors for 40 minutes. After incubation, 0.1. mu.l of HTRF detection buffer containing europium-blocked labeled anti-human IgG (Perkinelmer-AD0212) Fc specific and anti-HisAllophycocyanin (APC, Perkinelmer-AD0059H) conjugated antibody. After centrifugation, the plates were incubated at 25 ℃ for 60 minutes. The data were read (665nm/620nm ratio) in a PHERAStar FS plate reader. The final concentrations in the assay were-3 nM PD1, 10nM PD-L1, 1nM europium anti-human IgG and 20nM anti-His-allophycocyanin. IC of inhibitor was derived using GraphPad prism5.0 software to fit the activity data50The value is obtained.
Compounds illustrated in the examples IC50The values are expressed in the following manner: IC (integrated circuit)50：+：≤100nM；++：100nM～1000nM；+++：>1000nM。
Data for the example compounds obtained using the PD-1/PD-L1 Homogeneous Time Resolved Fluorescence (HTRF) binding assay described in example a are provided in table 1.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
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