CN116589464A - Pyrimidine ring compound, preparation method and application thereof - Google Patents

Pyrimidine ring compound, preparation method and application thereof Download PDF

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CN116589464A
CN116589464A CN202310075494.9A CN202310075494A CN116589464A CN 116589464 A CN116589464 A CN 116589464A CN 202310075494 A CN202310075494 A CN 202310075494A CN 116589464 A CN116589464 A CN 116589464A
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amino
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程建军
谢成英
蒋华良
张金凤
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Shanghai Institute of Materia Medica of CAS
ShanghaiTech University
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Abstract

The invention discloses a pyrimido ring compound, a preparation method and application thereof. The invention provides a compound shown as a formula I, pharmaceutically acceptable salt thereof, tautomer thereof or solvate thereof. The compounds of formula I of the invention are usefulAs adenosine A2A receptor antagonists or histone deacetylase HDAC inhibitors. Further, the compounds of the present invention may have both adenosine A2A receptor antagonistic activity and histone deacetylase HDAC inhibitory activity. The compounds of formula I of the present invention may be used for the treatment and/or prophylaxis of diseases associated with the adenosine A2A receptor and/or histone deacetylase HDAC, such as tumors and central nervous system diseases.

Description

Pyrimidine ring compound, preparation method and application thereof
Technical Field
The invention relates to a pyrimido ring compound, a preparation method and application thereof.
Background
Adenosine, an important endogenous signaling molecule, is involved in the regulation of a variety of important physiological functions (Ohta, gorelik et al, proceedings of the National Academy of Sciences,2006,103 (35), 13132-13137). As a purine nucleoside, adenosine acts on adenosine receptors on cell membranes to perform downstream signal transduction. Adenosine receptors belong to the G protein-coupled receptor (GPCR) and are divided into four subtypes: a is that 1 ,A 2A ,A 2B And A 3 (Antonioli, fornai et al Pharmacology and Therapeutics,2008,120 (3), 233-532), wherein A 2A The receptor is coupled to Gs protein, activating downstream adenylate cyclase, which increases the concentration of cAMP (Markus Klinger, cellular Signalling,2002,14,99-108). Adenosine A 2A Receptors are densely distributed in the central nervous system and are closely related to the pathogenesis of various degenerative central nervous system diseases such as parkinson's disease, alzheimer's disease, huntington's disease, etc. (Gomes et al, biochimica et Biophysica Acta,2011,1808,1380-1399). In Parkinson's disease, A 2A The receptor is highly expressed in the nigrostriatal body and can co-localize with the dopamine D2 receptor and form heterodimers, a 2A Activation of the receptor inhibits dopamine D2 receptor signaling (Shook, jackson, ACS Chemical Neuroscience,2011,2,555-567). Accordingly, A 2A Receptor antagonists are capable of releasing this inhibition, enhancing the downstream signaling of the D2 receptor, and thus enhancing dopaminergic activity, and are therefore useful as medicaments for the treatment of parkinson's disease. A is that 2A Receptor antagonists as therapeutic agents for parkinson's disease can also alleviate the dyskinesia side-effects induced by L-DOPA (L-DOPA). Multiple A 2A Small molecule antagonists of the receptor have entered clinical research as drugs for the treatment of parkinson's disease (Aren van waade et al Medicinal Research Reviews,2018,38,5-56), wherein itratheophylline (itradefyline, KW-6002) is marketed in japan as a drug for the treatment of parkinson's disease in 2013 as an adjunct to parkinson's disease, and is approved by the united states drug food administration (FDA) for use in the treatment of parkinson's disease in 8 months of 2019. The compound pre-dant (MK-3814), tozadant (SYN-115), etc. have also entered clinical studies. Although most of A 2A Clinical trials of receptor antagonists as single agents for the treatment of parkinson's disease have poor efficacy, but a 2A The potential of receptor antagonists for the treatment of related degenerative central nervous system diseases has been preliminarily demonstrated.
In recent years, A 2A The receptor is a potential tumor immunotherapy target and is of great interest. Although current tumor immunotherapy has achieved very good results for the treatment of specific cancer types, a number of drugs such as PD-1, PD-L1 antibody Keystuda, opdivoAnd tecantriq, CTLA4 antibody Yervoy et al have been marketed in the united states (Hoos, nature Reviews Drug Discovery 2016,15,235-247), but the effectiveness of these drugs is still low due to the variety of immunosuppressive mechanisms present in the tumor microenvironment, and the development of new tumor immunity drugs is still urgent. Adenosine is an important immunosuppressive factor in the tumor microenvironment, involved in immunosuppression (Antonioli, blandizi et al Nature Reviews Cancer,2013,13 (12), 842-857). The hypoxic microenvironment of the tumor limits energy utilization and induces aggregation of extracellular ATP, which can be hydrolyzed by the nucleases CD39 and CD73 to adenosine, resulting in a significant increase in adenosine concentration at the periphery of the tumor, thus forming an "adenosine protection ring" that helps the cancer resist attack by the immune system of the body (Linden, american Journal of Physiology Cell Physiology,2006,291 (3), 405-406). Adenosine and adenosine a 1 、A 2A 、A 2B And A 3 Receptor binding activates the receptor and thus performs a different regulatory function. Wherein A is 2A Receptors act as immune checkpoint proteins on the surface of T cells, mainly by inhibiting the action of effector T cells to exert immunosuppressive functions (Jacobson, gao, nature Reviews Drug Discovery,2006,5 (3), 247-264). Ohta and Sitkovsky et al, as early as 2001, found blocking A 2A The receptor may reverse immunosuppression (Sitkovsky, ohta, nature,2001,414,916-919). A on adenosine activated T cells 2A After the receptor, intracellular cAMP is raised, thereby inhibiting the immune function of T cells. And add A 2A Receptor antagonists, blocking A 2A The receptor, which reduces the concentration of cAMP, reverses this immunosuppression (Antonioli, blandizi et al, nature Review Cancer,2013,13 (12), 842-857). Many studies have shown that A 2A Receptors are a promising target for the development of tumor immunotherapy. Several known A2A receptor antagonists such as vipadent, CPI-444, PBF-509 and AZD4635 have been put into clinical study as drugs for tumor immunotherapy. But A is 2A Receptor antagonists have no direct killing effect on tumor cells, and A is clinically present 2A Receptor antagonists are used in combination with other anti-cancer drugs to treat tumors (Vecchio, white et al, pharmacology and Therapeutics,2019,198,20-33). How to make it anti-tumor The effect is stronger, and new breakthrough needs to be made.
Histone deacetylases (histone deacetylases, HDACs) are another drug target that is closely related to both tumor and degenerative central nervous system diseases. HDACs and histone acetyltransferases (histone acetyltransferases, HATs) are two key enzymes regulating epigenetic science, capable of reversibly acetylating histone lysine residues, and act in opposition (Kazantsev and Thompson, nature Reviews Drug Discovery,2008,7,854-868). HATs catalyze the acetylation of lysine residues at the N-terminus of histones to place chromatin in a relatively loosely open state, so that transcription factors are accessible to DNA to promote gene expression, a transcriptional coactivator; the function of HDACs is to catalyze the removal of acetyl groups from lysine residues of the histone, placing chromatin in a compact conformation to block DNA transcription and gene expression, a transcriptional co-inhibitor (Ellmeier and Seiser, nature Reviews Immunology,2018,18 (10), 617-634). The presently found humanized HDACs are 18 subtypes and can be divided into four subfamilies of Class I-IV. Class I includes HDACs 1, 2, 3, and 8; class II is further divided into class IIa (HDAC 4, 5, 7 and 9) and class IIb (HDAC 6 and 10); class IV has only one member HDAC 11. All three subfamilies are Zn 2+ Dependent HDACs are also known as classical HDACs. Class III, also known as sirtuins, includes SIRT 1-7, depends on NAD + Exerting catalytic activity (Wilfried Ellmeier and Christian Seiser, nature Reviews Immunology,2018,18 (10), 617-634).
Currently, HDAC inhibitors have a wide range of applications in anti-tumor. Over-expression of HDAC inhibits expression of a range of oncogenes and thus promotes growth of tumor cells, e.g., functional abnormalities of HDAC cause deacetylation of p53 protein to block its binding to DNA and thus block transcription of apoptotic genes; it also leads to decreased expression of the cell cycle inhibitor p21, which leads to cell cycle arrest; in addition, HDAC is also involved in vascularization of tumor tissue, regulation of immune cell function, etc. (Falkenberg and Johnstone, nature Reviews Drug Discovery,2014,13,673-691). In view of the great potential of HDAC inhibitors in inhibiting tumor proliferation, there is a great deal of interest in their research and application as antitumor agents, and four HDAC inhibitors (vorinostat/SAHA, romidepsin/FK228, belinostat/PDX-101, panobinostat/LBH-589) are currently approved by the us FDA for marketing: SAHA was developed by Merck corporation to treat refractory Cutaneous T Cell Lymphomas (CTCL); romidepsin was developed by Celgene pharmaceutical company for the treatment of CTCL and Peripheral T Cell Lymphoma (PTCL); panobinostat developed by nowa is an oral combination with bortezomib and dexamethasone for the treatment of multiple myeloma; belinostat developed by Spectrum pharmaceutical Co., ltd.) is used for the treatment of PTCL. The chidamide is subtype selective HDAC inhibitor developed and marketed by Shenzhen micro-core organisms, is approved by CFDA in 12 months 2014, and is suitable for patients with recurrent or refractory peripheral T cell lymphomas which have received at least one systemic chemotherapy in the past. Other HDAC inhibitors such as abexinostat/PCI024781, givinostat/ITF2375, mocetinostat/MGCD-0103, etc. are in clinical studies at different stages.
In addition to their use in anti-tumor applications, genetic evidence suggests that HDACs and HATs are involved in maintaining central nervous system homeostasis, and that these are associated with neurological disorders such as Rubinstein-Taybi syndrome and Rett syndrome (Kazantsev and Thompson, nature Reviews Drug Discovery,2008,7 (10), 854-868). Therefore, HDAC inhibitors are also receiving increasing attention in the areas of neurological diseases such as Alzheimer's disease, parkinson's disease, huntington's chorea, etc. (Falkenberg and Johnstone, nature Reviews Drug Discovery,2014,13,673-691). For example, HDAC6 can modulate the level of phosphorylation of tau protein, thereby affecting the progression of tau-driven neurological disease (Selenica et al, alzheimer's Research & Therapy,2014,6,12). HDAC6 can also regulate the degradation of misfolded proteins, which are pathological features of various neurological diseases such as alzheimer's disease, parkinson's disease, huntington's disease, etc., by modulating protein aggregation and HSP90 function. As another example, HDAC2 has been demonstrated to regulate brain function and neurological development and deterioration in various models; HDAC2 overexpression can down-regulate synaptic plasticity and number and dendritic spine density, which in turn leads to degradation of learning cognitive function (Guan et al Nature,2009,459,55-60). It has been demonstrated in the literature that HDAC inhibitors can have therapeutic effects on neurological diseases, such as panobinostat can reverse symptoms of Huntington's chorea in animal models by inhibiting HDAC function (Siebzehnreubl et al Proceedings of the National Academy of Sciences,2018,115 (37), E8765-8774), SAHA can significantly improve cognition in animal models (Guan et al Nature,2009,459,55-60). However, it is difficult to obtain an antitumor therapeutic effect sufficiently potent when HDAC inhibitors are used alone, and the range of indications as drugs is also narrow. Currently, HDAC inhibitors are used clinically in combination with other antitumor agents (Bots and Johnstone, clinical Cancer Research,2009,15 (12), 3970-7 2009).
For diseases with complex pathogenesis, such as tumors and neurodegenerative diseases, the design and development of single drug molecules with multiple pharmacological activities is a more advantageous and effective strategy. In view of A 2A The receptor and HDAC have similar effects with tumors and various central nervous system diseases, and the synergistic use of the receptor and the HDAC is very likely to exert stronger therapeutic effects in the treatment of related diseases.
Chinese patent applications CN201911152880.3, CN201911153069.7, CN202010168088.3 report that the core structure has a 2A Compounds that act in both receptor antagonism and HDAC inhibition. Yan et al disclose a hybrid having a double or triple ring parent nucleus with a 2A The compound with double functions of receptor antagonism and HDAC inhibition has good in vivo anti-tumor activity (J Med Chem 2021,64,16573-16597). However, the above-mentioned compounds are limited in kind and structure, and more kinds of compounds are obtained, which have A 2A Compounds that act both as receptor antagonists and HDAC inhibitors are a major problem to be solved by those skilled in the art.
Disclosure of Invention
The invention aims to solve the problems of needlesThe technology provides a pyrimidyl ring compound based on a double-target small molecule structure of an A2A receptor antagonist and an HDAC inhibitor, a preparation method and application thereof. The pyrimido-cyclic compound of the invention can be used as adenosine A 2A Receptor antagonists or histone deacetylase HDAC inhibitors with adenosine a 2A Receptor antagonistic activity and histone deacetylase HDAC inhibitory activity, useful for the treatment and/or prevention of adenosine a 2A Receptor and/or histone deacetylase HDAC related diseases, such as tumors and neurodegenerative diseases.
The invention provides a compound shown in a formula I, pharmaceutically acceptable salt, tautomer or solvate thereof:
wherein A and B are independently CH or N;
R 1 and R is 2 Independently H or C 1 -C 6 An alkyl group;
R 3 is a 5-10 membered heteroaryl or is substituted with one or more R 3-1 Substituted 5-10 membered heteroaryl; in the heteroaryl, the number of the heteroatoms is 1, 2 or 3, and the heteroatoms are selected from one, two or three of N, O and S;
l is-C 1 -C 10 Alkylene-, -C 1 -C 10 alkylene-C 6 -C 10 Arylene-, -C 1 -C 10 alkylene-C 6 -C 10 arylene-C 2 -C 4 alkenylene-C 1 -C 10 alkylene-O-C 6 -C 10 Arylene-, -C 1 -C 10 alkylene-C 6 -C 10 arylene-O-C 1 -C 10 Alkylene radical,or-C 1 -C 10 Alkylene-5-10 membered heteroarylene-O-C 1 -C 10 Alkylene-;in the 5-10 membered heteroarylene, the number of heteroatoms is 1, 2 or 3, and the heteroatoms are selected from one, two or three of N, O and S; wherein the band "# end is connected with the ZBG;
ZBG is
R 3-1 Independently cyano or C 1 -C 6 An alkyl group;
L 1 、L 2 independently-C 1 -C 10 Alkylene or-O-C 1 -C 10 An alkylene group;
R 6 independently hydrogen or halogen;
m is 1, 2, 3 or 4.
In certain embodiments, in the compounds of formula I described above or pharmaceutically acceptable salts thereof, certain groups have the following definitions, and the non-mentioned groups are as described in any of the remaining schemes (hereinafter referred to in this paragraph as "in a scheme"):
R 3 wherein the heteroaryl is independently preferably a 5-6 membered heteroaryl; the heteroatoms in the heteroaryl group are independently preferably O, and the number of heteroatoms is independently preferably 1 or 2. The heteroaryl groups are independently preferably furyl groups, and further independently preferably furyl groups
In one embodiment, R 1 、R 2 And R is 3-1 In (C) 1 -C 6 Alkyl is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl.
In one embodiment L, R 4 And R is 5 In (C) 1 -C 10 Alkylene is independently C 1 -C 7 Alkylene, more preferably methylene, ethylene, propylene, butylene, pentylene, hexylene or heptylene, preferably methylene, n-ethylene, n-propylene, n-butylene,N-pentylene, n-hexylene or n-heptylene.
In one embodiment, L is the same as C 6 -C 10 Arylene is independently phenylene, more preferably
In one embodiment, L is the same as C 2 -C 4 Alkenylene is independently vinylidene, more preferably
In one embodiment, L, the 5-10 membered heteroarylene is a 5-6 membered heteroarylene; the hetero atom in the 5-10 membered heteroarylene and the 5-6 membered heteroarylene is preferably N, and the number of the hetero atom is preferably 1 or 2. The 5-to 6-membered heteroarylene group is preferably a pyridyl group, more preferably
In one embodiment, R 6 Wherein the halogen is fluorine, chlorine, bromine or iodine.
In one aspect, in ZBG, theIs->
In one embodiment, L is
In one embodiment, R 1 And R is 2 H.
In one embodiment, R 3 Is that
In a certain scheme, the compound shown in the formula I is a substituted pyrimidine compound shown in the formula I-1, a substituted pyrimidine compound shown in the formula I-2, a substituted pyrimidine compound shown in the formula I-3 or a substituted pyrimidine compound shown in the formula I-4;
in one embodiment, the compound shown in formula I is any one of the following compounds:
the invention also provides a pharmaceutical composition which comprises the compound shown in the formula I or pharmaceutically acceptable salt thereof and at least one pharmaceutical excipient.
The invention also provides application of the compound shown in the formula I or pharmaceutically acceptable salt thereof or the pharmaceutical composition in preparation of an adenosine A2A receptor antagonist and/or a histone deacetylase HDAC inhibitor.
In such applications, the adenosine A2A receptor antagonist and/or histone deacetylase HDAC inhibitor may be used in mammalian organisms; it is also useful in vitro, mainly as an experimental use, for example: the kit can be used as a standard sample or a control sample for comparison or prepared according to a conventional method in the field to provide rapid detection for the antagonism effect of the adenosine A2A receptor and/or the HDAC inhibition effect of the histone deacetylase.
The invention also provides application of the compound shown in the formula I or pharmaceutically acceptable salt thereof or the pharmaceutical composition in preparation of medicines for treating and/or preventing diseases.
The dosage of the compound shown as the formula I or the pharmaceutically acceptable salt thereof or the pharmaceutical composition can be a therapeutically effective amount.
The disease may be cancer or a central nervous system disease.
In certain embodiments, when ZBG in the compound of formula I is In the meantime, the histone deacetylase HDAC is histone deacetylase HDAC1 or histone deacetylase HDAC6; when ZBG in the compound shown in formula I is +.>In this case, the histone deacetylase HDAC is histone deacetylase HDAC1.
The cancer may be a cancer of the head and neck (e.g. thyroid, nasopharyngeal, meningeal or intracranial metastasis), a cancer of the respiratory system (e.g. small cell lung or non-small cell lung), a cancer of the digestive system (e.g. liver, stomach, oesophageal, rectal, colon or pancreas), a cancer of the urinary system (e.g. renal, bladder, prostate or testicular), bone, gynaecological (e.g. breast, cervical or ovarian), a cancer of the blood system (e.g. leukaemia, lymphoma or myeloma) or other types of cancer (e.g. melanoma, glioma or skin cancer).
The central nervous system disorder may be Parkinson's disease, alzheimer's disease or Huntington's disease.
The compounds of formula I or pharmaceutically acceptable salts thereof, as described above, or the pharmaceutical compositions described, may also be used in any disease process characterized by abnormal proliferation of cells, such as benign prostatic hyperplasia, neurofibromatosis, atherosclerosis, pulmonary fibrosis, arthritis, psoriasis, glomerulonephritis, restenosis following angioplasty or vascular surgery, inflammatory bowel disease, transplant rejection, endotoxic shock and fungal infections.
The invention also provides application of the compound shown in the formula I or pharmaceutically acceptable salt thereof or the pharmaceutical composition in preparation of a preparation for regulating the activity of an adenosine A2A receptor and/or histone deacetylase HDAC.
In certain embodiments, the histone deacetylase HDAC is histone deacetylase HDAC1.
The choice of the pharmaceutical excipients is varied depending on the route of administration and the nature of the action, and may generally be fillers, diluents, binders, wetting agents, disintegrants, lubricants, emulsifiers, suspending agents, etc. which are conventional in the art.
The pharmaceutical compositions may be administered by oral, injectable (intravenous, intramuscular, subcutaneous and intracoronary), sublingual, buccal, rectal, transurethral, vaginal, nasal, inhaled or topical routes, the preferred route being oral.
The invention also provides a preparation method of the compound shown in the formula I, which comprises the following steps of carrying out the following reaction on the compound shown in the formula II in an organic solvent to obtain the compound shown in the formula I,
therein, A, B, R 1 、R 2 、R 3 L and ZBG are as defined above; r is R a Is H or C 1 -C 6 An alkyl group.
The reaction conditions in the preparation method of the compound shown in the formula I are conventional in the art.
The invention also provides a compound shown as a formula II,
therein, A, B, R 1 、R 2 、R 3 L and R a Is defined as above.
In one embodiment of the present invention, the compound represented by formula II is the following compound:
in the present invention, the following terms appearing in the specification and claims of the present invention have the following meanings, unless otherwise indicated:
in the present invention, the term "substitution" or "substituent" means that one or more hydrogen atoms are replaced with the specified groups. When no substitution positions are indicated, substitution may be at any position, but only formation of a stable or chemically feasible chemical is allowed.
In the present invention, the term "optional" or "optionally" means 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. For example, the term "optionally substituted" means that the substituents may or may not be substituted, and the types and numbers of substituents may be arbitrary on the basis that they can be chemically achieved unless otherwise specified.
When any variable (e.g. R 3-1 ) Where the composition or structure of a compound occurs more than once, its definition is independent in each case. Thus, for example, if a group is substituted with 1 to 2R 3-1 Substituted, the radicals may optionally be substituted by up to two R's, and R's in each case have an independent option. Furthermore, combinations of substituents and/or variants thereof are only permissible if such combinations result in stable compounds.
In the present invention, the term "alkyl" refers to a saturated straight or branched monovalent hydrocarbon group having the indicated number of carbon atoms, e.g., C 1 -C 10 Alkyl refers to straight or branched chain alkyl groups having 1 to 10 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, isobutyl, s-butyl, t-butyl), and pentyl (e.g., n-pentyl, isopentyl, neopentyl).
In the present invention, the term "alkylene" refers to a saturated straight or branched divalent hydrocarbon group having the indicated number of carbon atoms. Thereby C 1 Alkylene (i.e., methylene) refers to-CH 2 -，C 2 Alkylene (i.e., ethylene) refers to-CH 2 -CH 2 -，C 3 Alkylene means-CH 2 -CH 2 -CH 2 -。
In the present invention, the term "alkenylene" refers to a straight-chain divalent hydrocarbon radical having the indicated number of carbon atoms and at least one carbon-carbon double bond, where the carbon-carbon double bond may be located anywhere within the alkenylene radical. Thereby C 2 Alkenylene (i.e., vinylidene) means-ch=ch-, C 3 Alkenylene means-CH 2 -ch=ch-and-CH 2 ＝CH-CH 2 -，C 4 Alkenylene groupfinger-CH 2 -CH＝CH-CH 2 -、-CH 2 ＝CH-CH 2 -CH 2 -and-CH 2 -CH-CH 2 ＝CH 2 -。
In the present invention, the term "aryl" refers to any stable mono-or polycyclic (e.g., bi-or tricyclic) carbocycle of up to 6-10 atoms in each ring, wherein at least one ring is aromatic. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, 2, 3-indanyl, biphenyl, phenanthryl, anthracenyl, or acenaphthyl (acenaphthyl). It will be appreciated that where the aryl substituent is a bicyclic substituent and one of the rings is a non-aromatic ring, the connection is through an aromatic ring.
In the present invention, the term "arylene" refers to a divalent aryl group. 1, 4-phenylene radical, i.e.Or 1, 3-phenylene, i.e.)>
In the present invention, the term "heteroaryl" refers to a stable mono-or polycyclic (e.g., bi-or tricyclic) carbocycle of up to 6-10 atoms in each ring, wherein at least one ring is aromatic and contains at least one heteroatom selected from O, N and S. Heteroaryl groups may be attached to other moieties in the molecule through heteroatoms or carbon atoms therein. Examples of heteroaryl groups include, but are not limited to, acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrazolyl, indolyl, benzotriazole, furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl (e.g., pyrrol-1-yl, i.e.) Pyrrol-2-yl->) Tetrahydroquinolinyl. Can be managedIn the case where the heteroaryl substituent is a bicyclic substituent and one of the rings is a non-aromatic ring, the connection is through an aromatic ring.
In the present invention, the term "heteroarylene" refers to a divalent heteroaryl group. Examples of 1,4- (6 membered heteroarylene) in which 1 and 4 do not refer to the original numbering of the ring atoms in the 6 membered heteroarylene, but rather to the relative positions of the two attachment sites of the 6 membered heteroarylene, are para, and 1,4- (6 membered heteroarylene) include, but are not limited to
Examples of 1,3- (5 membered heteroarylene) include, but are not limited to, as in the case of 1,3- (5 membered heteroarylene) above
When the linking group exemplified in the present invention does not indicate its linking direction, the linking direction is linked in the same direction as the reading order from left to right, for example,medium linking group L 1 is-C-D-, in which case-C-D-connects ring A and ring B in the same direction as the reading sequence from left to right to form +.>But not form->When L is +.>When (I)>The formed structure isBut not +.>
Combinations of such linking groups, substituents and/or variants thereof are permissible only if such combinations result in stable compounds.
In the present invention, unless otherwise indicated, the term "halogen" means F, cl, br, I.
In the present invention, the term "pharmaceutically acceptable salt" means a salt formed from a suitable non-toxic organic acid, inorganic acid, organic base or inorganic base with a compound as shown in formula I, which retains the biological activity of the compound as shown in formula I. The organic acid may be various organic acids capable of forming salts, which are conventional in the art, preferably one or more of methanesulfonic acid, p-toluenesulfonic acid, maleic acid, fumaric acid, citric acid, tartaric acid, malic acid, lactic acid, formic acid, acetic acid, propionic acid, trifluoroacetic acid, oxalic acid, succinic acid, benzoic acid, isethionic acid, naphthalenesulfonic acid and salicylic acid. The mineral acid may be any of a variety of mineral acids conventionally known in the art capable of salt formation, preferably one or more of hydrochloric acid, sulfuric acid and phosphoric acid. The organic base can be various organic bases capable of forming salts, which are conventional in the art, and preferably one or more of pyridines, imidazoles, pyrazines, indoles, purines, tertiary amines and anilines. The tertiary amine organic base is preferably triethylamine and/or N, N-diisopropylethylamine. The aniline organic base is preferably N, N-dimethylaniline. The pyridine organic base is preferably one or more of pyridine, picoline, 4-dimethylaminopyridine and 2-methyl-5-ethylpyridine. The inorganic base may be various inorganic bases capable of forming salts, which are conventional in the art, preferably one or more of alkali metal hydrides, alkali metal hydroxides, alkali metal alkoxides, potassium carbonate, sodium carbonate, lithium carbonate, cesium carbonate, potassium bicarbonate and sodium bicarbonate. The alkali metal hydride is preferably sodium hydride and/or potassium hydride. The alkali metal hydroxide is preferably one or more of sodium hydroxide, potassium hydroxide and lithium hydroxide. The alkali metal alkoxide is preferably one or more of sodium methoxide, sodium ethoxide, potassium tert-butoxide and sodium tert-butoxide. In some embodiments of the invention, the pharmaceutically acceptable salt is the hydrochloride salt.
The term "solvate" refers to a substance formed from a compound of formula I with a suitable solvent. The solvent is preferably water or an organic solvent.
The compounds of the invention and their structures are also meant to include all isomeric (e.g., enantiomers, diastereomers, geometric isomers and conformational isomers) forms which may be defined as (R) -/(S) -or (D) -/(L) -or (R, R) -/(R, S) -/(S, S) -, depending on the absolute stereochemistry for the amino acid. The present invention includes all such possible isomers, as well as their racemic, enantiomerically enriched, and optionally pure forms. The optically active (+) and (-), (R) -and (S) -and (R, R) -/(R, S) -/(S, S) -or (D) -and (L) -isomers can be prepared using chiral synthesis, chiral resolution, or can be resolved using conventional techniques such as, but not limited to, high performance liquid phase (HPLC) using chiral columns. When a compound described herein contains an alkenyl double bond or other geometric asymmetric center, unless otherwise specified, the compound includes both E and Z geometric isomers. Likewise, all tautomeric forms are also included.
In the present invention, the term "tautomer" refers to the movement of a proton from one atom of a molecule from the original position to another position of the same molecule. The present invention includes tautomers of any of the compounds.
The term "therapeutically effective amount" with respect to a drug or pharmacologically active agent refers to a sufficient amount of the drug or agent that is non-toxic but achieves the intended effect. For the purposes of the present oral dosage form, a "therapeutically effective amount" of one active agent in a composition refers to that amount which is required to achieve the desired effect when combined with another active agent in the composition. Determination of an effective amount varies from person to person, depending on the age and general condition of the recipient, and also on the particular active substance, a suitable effective amount in an individual case can be determined by one skilled in the art according to routine experimentation.
On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.
The reagents and materials used in the present invention are commercially available.
The invention has the positive progress effects that: provides a thiazolo-ring compound, a preparation method, an intermediate and application thereof, and the thiazolo-ring compound can be used as an adenosine A2A receptor antagonist or a histone deacetylase HDAC inhibitor. Further, the thiazolo cyclic compound of the present invention may have both adenosine A2A receptor antagonistic activity and histone deacetylase HDAC inhibitory activity. The thiazolo cyclic compounds of the present invention may be used for the treatment and/or prevention of diseases associated with adenosine A2A receptors and/or histone deacetylase HDAC, such as tumors and central nervous system diseases.
Drawings
FIG. 1 is a graph showing the relative tumor growth rate of the intragastric administration of I-14.
FIG. 2 shows the relative tumor growth rate of intraperitoneal administration of Compound I-14.
Detailed Description
The invention will be further illustrated in the following examples. These examples are only intended to illustrate the invention and do not limit the scope of the invention in any way. All parameters and the remaining description in the examples are in terms of mass (grams) unless otherwise indicated.
Example 1: preparation of 5- (6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -N-hydroxypentanamide (compound I-1)
Step 1: preparation of methyl 5- (6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) pentanoate (intermediate Int-1)
The starting material compound A1 is synthesized by reference to the literature (Gillespie, cliffe et al, bioorg Med Chem Lett.,2008,18,2924-2929). Methyl 5-Bromovalerate (218 mg,1.12 mmol), K 2 CO 3 (206 mg,1.49 mmol) and Compound C-1 (150 mg,0.745 mmol) were dissolved in DMF (15 mL) and stirred overnight at 100deg.C. The reaction solution was diluted with water, extracted three times with ethyl acetate, and the organic phases were combined with anhydrous Na 2 SO 4 Drying once, evaporating the solvent under reduced pressure, and separating and purifying the residual solid by silica gel column chromatography to obtain yellow oily intermediate Int-1 (84 mg, yield 34%). 1 H NMR(600MHz,DMSO-d 6 )δ8.23(s,1H),8.05(dd,J＝1.7,0.8Hz,1H),7.42(dd,J＝3.5,0.8Hz,1H),6.87(brs,2H),6.78(dd,J＝3.5,1.7Hz,1H),4.19(t,J＝6.9Hz,2H),3.55(s,3H),2.33(t,J＝7.4Hz,2H),1.84–1.78(m,2H),1.52–1.45(m,2H).HRMS(ESI)C 15 H 18 N 5 O 3 + [M+H] + Calculated 316.1404, found 316.1406.
Step 2: preparation of 5- (6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -N-hydroxypentanamide (compound I-1)
A solution of potassium hydroxide (1.29 g,23 mmol) in anhydrous methanol (3.22 mL) was added dropwise to a solution of hydroxylamine hydrochloride (1.07 g,15.4 mmol) in anhydrous methanol (5.52 mL) at 0deg.C, and the mixture was stirred at room temperature slowly for 0.5 hours, and the solid was removed by filtration to give a methanol solution of hydroxylamine. 5- (6-amino-4- (furan-2-yl) -1H-pyrazolo [3, 4-d)]Methyl pyrimidin-1-yl) valerate (intermediate Int-1) (0.076 g,0.241 mmol) was dissolved in a methanol solution of the above hydroxylamine and stirred at room temperature for 1 hour. The reaction solution was neutralized to pH7.4 with a 1, 4-dioxane solution (4M) of hydrogen chloride, the solvent was evaporated under reduced pressure, and water was added to the solid residue, which was stirred at room temperature for 1 hour, followed by suction filtration to give yellow solid (I-1) (0.030 g, yield 38%). 1 H NMR(800MHz,DMSO-d 6 )δ10.33(s,1H),8.66(s,1H),8.23(s,1H),8.05(d,J＝1.7Hz,1H),7.42(d,J＝3.4Hz,1H),6.88(brs,2H),6.78(dd,J＝3.5,1.7Hz,1H),4.18(t,J＝7.1Hz,2H),1.98(t,J＝7.4Hz,2H),1.78–1.73(m,2H),1.49–1.44(m,2H).HRMS(ESI)C 14 H 17 N 6 O 3 + [M+H] + Calculated 317.1357, found 317.1360.
Example 2: preparation of 6- (6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -N-hydroxyhexanamide (compound I-2)
The 5-bromopentanoate in step 1 of example 1 was replaced with 6-methylbromoheptanoate, and the remaining necessary raw materials, reagents and production method were the same as those of example 1 to give compound (I-2) (0.116 g, yield 84%) as a white solid. 1 H NMR(600MHz,DMSO-d 6 )δ10.31(s,1H),8.65(d,J＝1.7Hz,1H),8.22(s,1H),8.05(dd,J＝1.8,0.8Hz,1H),7.42(dd,J＝3.6,0.8Hz,1H),6.87(brs,2H),6.78(dd,J＝3.5,1.7Hz,1H),4.17(t,J＝7.1Hz,2H),1.92(t,J＝7.4Hz,2H),1.79(p,J＝7.4Hz,2H),1.51(p,J＝7.5Hz,2H),1.26–1.19(m,2H).HRMS(ESI)C 15 H 19 N 6 O 3 + [M+H] + Calculated 331.1513, found 331.1515.
Example 3: preparation of 7- (6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -N-hydroxyheptanamide (compound I-3)
The 5-bromopentanoate in step 1 of example 1 was replaced with 7-methylbromoheptanoate, and the remaining necessary raw materials, reagents and production method were the same as those of example 1 to give compound (I-3) (0.190 g, yield 88%) as a white solid. 1 H NMR(800MHz,DMSO-d 6 )δ10.31(s,1H),8.64(s,1H),8.22(s,1H),8.05(d,J＝1.6Hz,1H),7.42(dd,J＝3.5,0.7Hz,1H),6.86(brs,2H),6.78(dd,J＝3.5,1.7Hz,1H),4.17(t,J＝7.1Hz,2H),1.91(t,J＝7.4Hz,2H),1.78(p,J＝7.2Hz,2H),1.45(p,J＝7.4Hz,2H),1.28–1.21(m,4H).HRMS(ESI)C 16 H 21 N 6 O 3 + [M+H] + Calculated 345.1670, found 345.1678.
Example 4: preparation of 5- (6-amino-4- (5-methylfuran-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -N-hydroxyvaleramide (compound I-4)
Step 1: preparation of methyl 5- (6-amino-4- (5-methylfuran-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) pentanoate (intermediate Int-2)
The starting material compound A2 is synthesized by reference to the literature (Gillespie, cliffe et al, bioorg Med Chem Lett.,2008,18,2924-2929). The compound C-1 in step 1 of example 1 was replaced with compound C-2, and the remaining desired starting materials, reagents and preparation method were the same as in step 1 of example 1 to give intermediate Int-2 (42 mg, yield 18%) as a yellow oil. 1 H NMR(600MHz,DMSO-d 6 )δ8.23(s,1H),7.35(d,J＝3.4Hz,1H),6.82(brs,2H),6.41(dd,J＝3.3,1.2Hz,1H),4.18(t,J＝6.9Hz,2H),3.55(s,3H),2.45(s,3H),2.33(t,J＝7.4Hz,2H),1.84–1.76(m,2H),1.52–1.45(m,2H).HRMS(ESI)C 16 H 20 N 5 O 3 + [M+H] + Calculated 330.1561, found 330.1562.
Step 2: preparation of 5- (6-amino-4- (5-methylfuran-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -N-hydroxyvaleramide (compound I-4)
The reagent and preparation method were the same as in example 1, step 2, to give compound (I-4) (0.048 g, yield 67%) as a white solid. 1 H NMR(800MHz,DMSO-d 6 )δ10.33(s,1H),8.66(s,1H),8.23(s,1H),7.35(d,J＝3.4Hz,1H),6.82(brs,2H),6.41(dd,J＝3.4,1.2Hz,1H),4.17(t,J＝7.1Hz,2H),2.45(s,3H),1.97(t,J＝7.4Hz,2H),1.78–1.73(m,2H),1.49–1.43(m,2H).HRMS(ESI)
C 15 H 19 N 6 O 3 + [M+H] + Calculated 331.1513, found 331.1515.
Example 5: preparation of 6- (6-amino-4- (5-methylfuran-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -N-hydroxyhexanamide (compound I-5)
The 5-bromopentanoate in step 1 of example 1 was replaced with 6-methylbromoheptanoate, and the remaining necessary raw materials, reagents and production method were the same as those of example 4 to give compound (I-5) (0.186 g, yield 81%) as a white solid. 1 H NMR(600MHz,DMSO-d 6 )δ10.31(s,1H),8.65(d,J＝1.8Hz,1H),8.22(s,1H),7.34(d,J＝3.4Hz,1H),6.82(brs,2H),6.41(dd,J＝3.4,1.1Hz,1H),4.16(t,J＝7.1Hz,2H),2.45(s,3H),1.92(t,J＝7.4Hz,2H),1.78(p,J＝7.3Hz,2H),1.51(p,J＝7.5Hz,2H),1.27–1.18(m,2H).HRMS(ESI)C 16 H 21 N 6 O 3 + [M+H] + Calculated 345.1670, found 345.1679.
Example 6: preparation of 7- (6-amino-4- (5-methylfuran-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -N-hydroxyheptanamide (compound I-6)
The 5-bromopentanoate in step 1 of example 4 was replaced with 7-methylbromoheptanoate, and the remaining necessary raw materials, reagents and production method were the same as those of example 4 to give compound (I-6) (0.105 g, yield 85%) as a white solid. 1 H NMR(800MHz,DMSO-d 6 )δ10.32(s,1H),8.65(s,1H),8.22(s,1H),7.34(d,J＝3.3Hz,1H),6.81(brs,2H),6.40(dd,J＝3.4,1.1Hz,1H),4.16(t,J＝7.1Hz,2H),2.45(s,3H),1.91(t,J＝7.4Hz,2H),1.77(p,J＝7.2Hz,2H),1.45(p,J＝7.4Hz,2H),1.28–1.21(m,4H).HRMS(ESI)C 17 H 23 N 6 O 3 + [M+H] + Calculated 359.1826, found 359.1822.
Example 7: preparation of 7- (2-amino-6- (furan-2-yl) -9H-purin-9-yl) -N-hydroxyheptanamide (Compound I-7)
Step 1: preparation of methyl 7- (2-amino-6- (furan-2-yl) -9H-purin-9-yl) heptanoate (intermediate Int-3)
The starting material compound A3 is synthesized by reference to the literature (Gillespie, cliffe et al, bioorg Med Chem Lett.,2008,18,2924-2929). Methyl 7-Bromoheptanoate (133 mg,0.596 mmol), K 2 CO 3 (137 mg,0.994 mmol) and Compound C-3 (100 mg,0.497 mmol) were dissolved in DMF (15 mL) and stirred at room temperature overnight. The reaction solution was diluted with water, extracted three times with ethyl acetate, and the organic phases were combined with anhydrous Na 2 SO 4 Drying once, evaporating the solvent under reduced pressure, and separating and purifying the residual solid by silica gel column chromatography to obtain yellow oily intermediate Int-3 (35 mg, yield 21%). 1 H NMR(800MHz,DMSO-d 6 )δ8.12(s,1H),7.95–7.94(m,1H),7.72(d,J＝3.3Hz,1H),6.74(dd,J＝3.4,1.7Hz,1H),6.54(brs,2H),4.04(t,J＝7.2Hz,2H),3.56(s,3H),2.27(t,J＝7.4Hz,2H),1.77(p,J＝7.4Hz,2H),1.49(p,J＝7.4Hz,2H),1.31–1.26(m,2H),1.27–1.22(m,2H).HRMS(ESI)C 17 H 22 N 5 O 3 + [M+H] + Calculated 344.1717, found 344.1715.
Step 2: preparation of 7- (2-amino-6- (furan-2-yl) -9H-purin-9-yl) -N-hydroxyheptanamide (Compound I-7)
The intermediate Int-1 in step 2 of example 1 was replaced with intermediate Int-3, and the remaining desired starting materials, reagents and preparation method were the same as in step 2 of example 1 to give compound (I-7) (0.039 g, yield 57%) as a white solid. 1 H NMR(800MHz,DMSO-d 6 )δ10.32(s,1H),8.65(s,1H),8.12(s,1H),7.96–7.94(m,1H),7.72(d,J＝3.4Hz,1H),6.74(dd,J＝3.4,1.7Hz,1H),6.55(brs,2H),4.04(t,J＝7.2Hz,2H),1.92(t,J＝7.4Hz,2H),1.77(p,J＝7.3Hz,2H),1.47(p,J＝7.3Hz,2H),1.29–1.22(m,4H).HRMS(ESI)C 16 H 21 N 6 O 3 + [M+H] + Calculated 345.1670, found 345.1669.
The compounds listed in examples 8-10 can be prepared in the same manner by changing the corresponding starting materials according to the methods listed in examples 1-7, as detailed in Table 1.
TABLE 1
Example 11: preparation of 4- ((6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) -N- (2-aminophenyl) benzamide (compound I-11)
Step 1: preparation of methyl 4- ((6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) benzoate (intermediate Int-4)
The 5-bromopentanoate in step 1 of example 1 was replaced with methyl 4- (bromomethyl) benzoate, the reaction conditions were changed to room temperature at 100℃and the remaining necessary starting materials, reagents and preparation method were the same as in step 1 of example 1 to give intermediate (Int-4) as a yellow solid (0.152 g, yield 26%). 1 H NMR(800MHz,Chloroform-d)δ8.28(s,1H),7.98–7.95(m,2H),7.72(dd,J＝1.7,0.8Hz,1H),7.41(d,J＝3.6Hz,1H),7.34–7.31(m,2H),6.62(dd,J＝3.5,1.7Hz,1H),5.51(s,2H),5.36(brs,2H),3.88(s,3H)。HRMS(ESI)C 18 H 16 N 5 O 3 + [M+H] + Calculated 350.1248, found 350.1245.
Step 2: preparation of 4- ((6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) benzoic acid (intermediate Int-5)
To a solution of ester intermediate Int-4 (0.096 g,0.274 mmol) in a mixture of tetrahydrofuran (12 mL) and water (3 mL), lithium hydroxide (0.036 g,0.151 mmol) was added and stirred overnight at room temperature. The reaction solution is neutralized to pH7.4 by 1, 4-dioxane solution (4M) of hydrogen chloride, and the solvent is evaporated to dryness under reduced pressure to obtain a crude product of a carboxylic acid intermediate Int-5, which is directly used for the next reaction. HRMS (ESI) C 17 H 14 N 5 O 3 + ([M+H] + ) Calculated 336.1091, found 336.2810.
Step 3: preparation of 4- ((6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) -N- (2-aminophenyl) benzamide (compound I-11)
A mixture of all intermediate Int-5 obtained in step 2 and lithium chloride was dissolved in anhydrous DMF (15 mL), HATU (0.209 g, 0.268 mmol) was added, stirred at room temperature for 20 minutes, then o-phenylenediamine (0.119 g,1.10 mmol) and DIPEA (0.106 g,0.823 mmol) were added and stirred at room temperature overnight. The reaction solution was diluted with water, extracted three times with ethyl acetate, the organic phases were combined, the solvent was distilled off under reduced pressure, and the remaining solid was separated and purified by silica gel column chromatography to give Compound I-11 (0.063 g, yield 54%). 1 H NMR(800MHz,DMSO-d6)δ9.60(s,1H),8.31(s,1H),8.07–
8.06(m,1H),7.92(d,J＝7.9Hz,2H),7.46(dd,J＝3.5,0.8Hz,1H),7.30(d,J＝8.1Hz,2H),7.17–7.13(m,1H),6.98–6.94(m,3H),6.80(dd,J＝3.5,1.8Hz,1H),6.76(dd,J＝8.0,1.4Hz,1H),6.58(t,J＝7.5Hz,1H),5.51(s,2H),4.89(brs,2H)。HRMS(ESI)
C 23 H 20 N 7 O 2 + ([M+H] + ) Calculation of426.1673, 426.1670.
Example 12: preparation of 4- ((6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) -N-hydroxybenzoamide (compound I-12)
The intermediate Int-1 in step 2 of example 1 was replaced with intermediate Int-4, and the other desired raw materials, reagents and preparation method were the same as those in step 2 of example 1 to give an off-white solid compound (I-12) (0.053 g, yield 97%). 1 H NMR(800MHz,DMSO-d6)δ11.16(s,1H),8.95(brs,1H),8.30(s,1H),8.06(d,J＝1.6Hz,1H),7.68(d,J＝8.2Hz,2H),7.46(d,J＝3.5Hz,1H),7.23(d,J＝8.1Hz,2H),6.95(brs,2H),6.79(dd,J＝3.5,1.8Hz,1H),5.47(s,2H)。HRMS(ESI)C 17 H 15 N 6 O 3 + [M+H] + Calculated 351.1200, found 351.1187.
Example 13: preparation of 4- ((6-amino-4- (5-methylfuran-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) -N- (2-aminophenyl) benzamide (compound I-13)
Step 1: preparation of methyl 4- ((6-amino-4- (5-methylfuran-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) benzoate (intermediate Int-6)
The procedure of example 4, step 1, was followed except that methyl 5-bromopentanoate was replaced with methyl 4- (bromomethyl) benzoate and the reaction conditions were changed to room temperature at 100℃to give a white solid intermediate (Int-6) (0.050 g, yield 14%). 1 H NMR(800MHz,DMSO-d 6 )δ8.32(s,1H),7.90(d,J＝8.2Hz,2H),7.38(d,J＝3.4Hz,1H),7.27(d,J＝8.1Hz,2H),6.91(brs,2H),6.42(dd,J＝3.4,1.2Hz,1H),5.51(s,2H),3.82(s,3H),2.46(s,3H)。HRMS(ESI)C 19 H 18 N 5 O 3 + [M+H] + Calculated 364.1404, found 364.1387.
Step 2-3: preparation of 4- ((6-amino-4- (5-methylfuran-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) -N- (2-aminophenyl) benzamide (compound I-13)
The intermediate Int-4 in step 2 of example 11 was replaced with intermediate Int-6, and the other desired materials, reagents and preparation method were the same as in step 2 of example 11 to give the product as an off-white solid compound (I-13) (0.073 g, yield 55%) in step 3 of example 11. 1 H NMR(800MHz,DMSO-d 6 )δ9.60(s,1H),8.32(s,1H),7.92(d,J＝7.9Hz,2H),7.38(d,J＝3.4Hz,1H),7.28(d,J＝8.0Hz,2H),7.15(d,J＝8.4Hz,1H),6.96(td,J＝7.6,1.5Hz,1H),6.90(brs,2H),6.76(dd,J＝8.0,1.4Hz,1H),6.58(td,J＝7.6,1.4Hz,1H),6.43(dd,J＝3.4,1.1Hz,1H),5.50(s,2H),4.89(s,2H),2.46(s,3H)。HRMS(ESI)C 24 H 22 N 7 O 2 + [M+H] + Calculated 440.1829, found 440.1811.
Example 14: preparation of 4- (2- (6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) ethyl) -N- (2-aminophenyl) benzamide (compound I-14)
Step 1: preparation of methyl 4- (2- (6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) ethyl) benzoate (intermediate Int-7)
The 5-bromopentanoate methyl ester in step 1 of example 1 was replaced with methyl 4- (2-bromoethyl) benzoate, the reaction conditions were changed to room temperature at 100℃and the remaining necessary raw materials, reagents and preparation method were the same as in step 1 of example 1 to give intermediate (Int-7) as a yellow solid (0.096 g, yield 35%). 1 H NMR(800MHz,Chloroform-d)δ8.23(s,1H),7.92–7.90(m,2H),7.72(dd,J＝1.7,0.8Hz,1H),7.42(s,1H),7.26–7.23(m,2H),6.62(dd,J＝
3.5,1.7Hz,1H),5.31(brs,2H),4.53(t,J＝7.6Hz,2H),3.87(s,3H),3.27(d,J＝7.6Hz,2H)。HRMS(ESI)C 19 H 18 N 5 O 3 + [M+H] + Calculated 364.1404, found 364.1413.
Step 2-3: preparation of 4- (2- (6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) ethyl) -N- (2-aminophenyl) benzamide (compound I-14)
The intermediate Int-4 in step 2 of example 11 was replaced with intermediate Int-7, and the remaining desired starting materials, reagents and preparation method were the same as in step 2 of example 11 to give the product as a tan solid compound (I-14) (0.039 g, yield 70%) as in step 3 of example 11. 1 H NMR(800MHz,DMSO-d6)δ9.57(s,1H),8.24(s,1H),8.05(d,J＝1.7Hz,1H),7.86(d,J＝7.7Hz,2H),7.42(d,J＝3.5Hz,1H),7.30(d,J＝7.8Hz,2H),7.14(d,J＝7.8Hz,1H),6.95(t,J＝7.5Hz,1H),6.88(s,2H),6.78(dd,J＝3.5,1.7Hz,1H),6.77(d,J＝8.8Hz,1H),6.58(t,J＝7.5Hz,1H),4.87(brs,2H),4.48(t,J＝7.2Hz,2H),3.26(t,J＝7.2Hz,2H)。HRMS(ESI)C 24 H 22 N 7 O 2 + [M+H] + Calculated 440.1829, found 440.1826.
Example 15: preparation of 4- (2- (6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) ethyl) -N-hydroxybenzoamide (compound I-15)
The intermediate Int-1 in step 2 of example 1 was replaced with intermediate Int-7, and the remaining desired starting materials, reagents and preparation method were the same as in step 2 of example 1, further purified by preparative HPLC to give compound (I-15) as a yellow solid (0.070 g, yield 80%). 1 H NMR(800MHz,DMSO-d 6 )δ11.11(s,1H),8.95(brs,1H),8.24(s,1H),8.05(d,J＝1.7Hz,1H),7.63(d,J＝8.0Hz,2H),7.43(d,J＝3.5Hz,1H),7.23(d,J＝7.8Hz,2H),6.88(brs,2H),6.78(dd,J＝3.5,1.7Hz,1H),4.45(t,J＝7.3Hz,2H),3.21(t,J＝7.3Hz,2H)。HRMS(ESI)C 18 H 17 N 6 O 3 + [M+H] + Calculated 365.1357, found 365.1355.
Example 16: preparation of 4- (2- (6-amino-4- (5-methylfuran-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) ethyl) -N- (2-aminophenyl) benzamide (compound I-16)
Step 1: preparation of methyl 4- (2- (6-amino-4- (5-methylfuran-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) ethyl) benzoate (intermediate Int-8)
The 5-bromopentanoate methyl ester in step 1 of example 4 was replaced with methyl 4- (2-bromoethyl) benzoate, the reaction conditions were changed to room temperature at 100℃and the remaining necessary raw materials, reagents and preparation method were the same as those in step 1 of example 4 to give an off-white solid intermediate (Int-8) (0.083 g, yield 32%). 1 H NMR(800MHz,DMSO-d 6 )δ8.22(s,1H),7.83–7.80(m,2H),7.33(d,J＝3.4Hz,1H),7.30–7.27(m,2H),6.80(brs,2H),6.41(dd,J＝3.4,1.1Hz,1H),4.45(t,J＝7.2Hz,2H),3.81(s,3H),3.23(t,J＝7.2Hz,2H),2.45(s,3H)。HRMS(ESI)C 20 H 20 N 5 O 3 + [M+H] + Calculated 378.1561, found 378.1557.
Step 2-3: preparation of 4- (2- (6-amino-4- (5-methylfuran-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) ethyl) -N- (2-aminophenyl) benzamide (compound I-16)
The intermediate Int-4 in step 2 of example 11 was replaced with intermediate Int-8, and the remaining desired starting materials, reagents and preparation method were the same as in step 2 of example 11, and the obtained product was obtained as a yellow solid compound (I-16) (0.056 g) in step 3 of example 11Yield 55%). 1 H NMR(800MHz,DMSO-d 6 )δ9.59(s,1H),8.24(s,1H),7.86(d,J＝7.9Hz,2H),7.34(d,J＝3.4Hz,1H),7.29(d,J＝8.0Hz,2H),7.15(d,J＝7.8Hz,1H),6.96(td,J＝7.6,1.6Hz,1H),6.82(brs,2H),6.78(dd,J＝8.0,1.5Hz,1H),6.60(t,J＝7.5Hz,1H),6.41(dd,J＝3.3,1.1Hz,1H),4.98(s,2H),4.47(t,J＝7.2Hz,2H),3.25(t,J＝7.2Hz,2H),2.45(s,3H)。HRMS(ESI)C 25 H 24 N 7 O 2 + [M+H] + Calculated 454.1986, found 454.1980.
Example 17: preparation of 4- (2- (6-amino-4- (5-methylfuran-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) ethyl) -N-hydroxybenzoamide (compound I-17)
The intermediate Int-1 in step 2 of example 1 was replaced with intermediate Int-8, and the remaining desired starting materials, reagents and preparation method were the same as in step 2 of example 1, further purified by preparative HPLC to give compound (I-17) (0.041 g, yield 56%) as a yellow solid. 1 H NMR(800MHz,DMSO-d 6 )δ11.11(s,1H),8.95(s,1H),8.24(s,1H),7.62(d,J＝8.2Hz,2H),7.35(d,J＝3.4Hz,1H),7.23(d,J＝8.3Hz,2H),6.83(brs,2H),6.42(dd,J＝3.3,1.2Hz,1H),4.44(t,J＝7.2Hz,2H),3.20(t,J＝7.2Hz,2H),2.45(s,3H)。HRMS(ESI)C 19 H 19 N 6 O 3 + [M+H] + Calculated 379.1513, found 379.1501.
Example 18: preparation of 4- (3- (6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) propyl) -N- (2-aminophenyl) benzamide (compound I-18)
Step 1-4: preparation of methyl 4- (3- (6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) propyl) benzoate (intermediate Int-9)
Compound D-1 is as defined hereinSynthetic methods of donation (Yan et al, J Med chem.,2021,64,16573-16597). Compound D-1 (803 mg,1.18 mmol), K 2 CO 3 (260 mg,1.57 mmol) and Compound C-1 (189 mg,0.79 mmol) were dissolved in DMF (20 mL) and stirred overnight at 100deg.C. The reaction solution was diluted with water, extracted three times with ethyl acetate, and the organic phases were combined with anhydrous Na 2 SO 4 Drying once, evaporating the solvent under reduced pressure, and separating and purifying the remaining solid by silica gel column chromatography to obtain white solid Int-9 (0.105 g, yield 35%). 1 H NMR(800MHz,DMSO-d 6 )δ8.23(s,1H),8.05(dd,J＝1.7,0.8Hz,1H),7.87–7.85(m,2H),7.42(dd,J＝3.5,0.8Hz,1H),7.37–7.34(m,2H),6.88(s,2H),6.78(dd,J＝3.5,1.7Hz,1H),4.21(t,J＝6.9Hz,2H),3.82(s,3H),2.66(t,J＝7.6Hz,2H),2.18–2.12(m,2H)。
Step 5-6: preparation of 4- (3- (6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) propyl) -N- (2-aminophenyl) benzamide (compound I-18)
The intermediate Int-4 in step 2 of example 11 was replaced with intermediate Int-9, and the remaining desired starting materials, reagents and preparation method were the same as in step 2 of example 11 to give the product as an off-white solid compound (I-18) (0.104 g, yield 69%) in step 3 of example 11. 1 H NMR(800MHz,DMSO-d 6 )δ9.60(s,1H),8.27(s,1H),8.06(dd,J＝1.8,0.8Hz,1H),7.91(d,J＝7.8Hz,2H),7.43(dd,J＝3.5,0.7Hz,1H),7.36(d,J＝8.1Hz,2H),7.16(d,J＝7.8Hz,1H),6.96(td,J＝7.6,1.6Hz,1H),6.89(brs,2H),6.79–6.78(m,1H),6.77(d,J＝1.5Hz,1H),6.59(td,J＝7.5,1.4Hz,1H),4.88(brs,2H),4.23(t,J＝7.0Hz,2H),2.67(t,J＝7.6Hz,2H),2.16(p,J＝7.3Hz,2H)。HRMS(ESI)C 25 H 24 N 7 O 2 + [M+H] + Calculated 454.1986, found 454.1988.
Example 19: preparation of 4- ((2-amino-4- (furan-2-yl) -7H-pyrrolo [2,3-d ] pyrimidin-7-yl) methyl) -N- (2-aminophenyl) benzamide (compound I-19)
Step 1: preparation of methyl 4- ((2-amino-4- (furan-2-yl) -7H-pyrrolo [2,3-d ] pyrimidin-7-yl) methyl) benzoate (intermediate Int-10)
The starting material compound C-4 is synthesized by reference (Gillespie, cliffe et al, bioorg Med Chem Lett.,2008,18,2924-2929). Methyl 4- (bromomethyl) benzoate (433 mg,1.90 mmol), K 2 CO 3 (349 mg,2.53 mmol) and Compound C-4 (255 mg,1.26 mmol) were dissolved in DMF (15 mL) and stirred at room temperature overnight. The reaction solution was diluted with water, extracted three times with ethyl acetate, and the organic phases were combined with anhydrous Na 2 SO 4 Drying once, evaporating the solvent under reduced pressure, and separating and purifying the residual solid by silica gel column chromatography to obtain an off-white solid intermediate Int-10 (207 mg, yield 47%). 1 H NMR(600MHz,DMSO-d 6 )δ7.97(dd,J＝1.8,0.8Hz,1H),7.93–
7.89(m,2H),7.29–7.26(m,3H),7.22(d,J＝3.6Hz,1H),6.77(d,J＝3.6Hz,1H),6.72(dd,J＝3.5,1.7Hz,1H),6.28(brs,2H),5.38(s,2H),3.82(s,3H)。HRMS(ESI)C 19 H 17 N 4 O 3 +
[M+H] + Calculated 349.1295, found 349.1278.
Step 2: preparation of 4- ((2-amino-4- (furan-2-yl) -7H-pyrrolo [2,3-d ] pyrimidin-7-yl) methyl) -N- (2-aminophenyl) benzamide (compound I-19)
The intermediate Int-4 in step 2 of example 11 was replaced with intermediate Int-10, and the remaining desired starting materials, reagents and preparation method were the same as in step 2 of example 11 to give the product as a yellow solid compound (I-19) (0.050 g, yield 41%) in step 3 of example 11. 1 H NMR(800MHz,DMSO-d 6 )δ9.61(s,1H),7.96(d,J＝1.8Hz,1H),7.92(d,J＝7.9Hz,2H),7.30(d,J＝8.0Hz,2H),7.28(dd,J＝3.5,0.8Hz,1H),7.24(d,J＝3.6Hz,1H),7.15(d,J＝7.8Hz,1H),6.96(td,J＝7.6,1.6Hz,1H),6.78(d,J＝3.6Hz,1H),6.77(dd,J＝8.1,1.6Hz,1H),6.72(dd,J＝3.4,1.7Hz,1H),6.58(td,J＝7.5,1.4Hz,1H),6.27(s,2H),5.37(s,2H),4.87(s,2H)。HRMS(ESI)C 24 H 21 N 6 O 2 + [M+H] + Calculated 425.1721, found 425.1737.
Example 20: preparation of 4- ((2-amino-6- (furan-2-yl) -9H-purin-9-yl) methyl) -N- (2-aminophenyl) benzamide (Compound I-20)
Step 1: preparation of methyl 4- ((2-amino-6- (furan-2-yl) -9H-purin-9-yl) methyl) benzoate (intermediate Int-11)
The procedure of example 7, step 1 was followed except that methyl 7-bromoheptanoate was replaced with methyl 4- (bromomethyl) benzoate, the reaction condition was changed to room temperature at 100℃and the remaining necessary raw materials, reagents and preparation method were the same as in example 7, step 1, to give intermediate (Int-11) as a pale yellow solid (0.289 g, yield 88%). 1 H NMR(800MHz,DMSO-d 6 )δ8.24(s,1H),7.96(dd,J＝1.7,0.8Hz,1H),7.94–7.92(m,2H),7.74(dd,J＝3.4,0.9Hz,1H),7.37–7.34(m,2H),6.75(dd,J＝3.4,1.8Hz,1H),6.59(brs,2H),5.42(s,2H),3.83(s,3H)。HRMS(ESI)
C 18 H 16 N 5 O 3 + [M+H] + Calculated 350.1248, found 350.1253.
Step 2: preparation of 4- ((2-amino-6- (furan-2-yl) -9H-purin-9-yl) methyl) -N- (2-aminophenyl) benzamide (Compound I-20)
The intermediate Int-4 in step 2 of example 11 was replaced with intermediate Int-11, and the remaining desired starting materials, reagents and preparation methods were the same as in step 2 of example 11, to give the product as a pale yellow solid compound (I-20) (0) in step 3 of example 11.080g, yield 63%). 1 H NMR(800MHz,DMSO-d 6 )δ9.61(s,1H),8.26(s,1H),7.96(dd,J＝1.8,0.8Hz,1H),7.94(d,J＝8.0Hz,2H),7.74(dd,J＝3.4,0.8Hz,1H),7.38(d,J＝8.0Hz,2H),7.15(d,J＝7.8Hz,1H),6.96(ddd,J＝8.4,7.3,1.6Hz,1H),6.77(d,J＝1.4Hz,1H),6.76–6.75(m,1H),6.60(s,2H),6.58(dd,J＝7.5,1.4Hz,1H),5.41(s,2H),4.88(s,2H)。HRMS(ESI)C 23 H 20 N 7 O 2 + [M+H] + Calculated 426.1673, found 426.1654.
Example 21: preparation of 4- (2- (2-amino-6- (furan-2-yl) -9H-purin-9-yl) ethyl) -N- (2-aminophenyl) benzamide (Compound I-21)
Step 1: preparation of methyl 4- (2- (2-amino-6- (furan-2-yl) -9H-purin-9-yl) ethyl) benzoate (intermediate Int-12)
The procedure of example 7, step 1 was repeated except for substituting methyl 7-bromoheptanoate with methyl 4- (2-bromoethyl) benzoate and then substituting the other desired starting materials, reagents and preparation method for the same procedures as in example 7, step 1 to give intermediate (Int-12) as a pale yellow solid (0.187 g, yield 34%). 1 H NMR(800MHz,DMSO-d 6 )δ7.94(dd,J＝1.7,0.8Hz,1H),7.90(s,1H),7.88–7.85(m,2H),7.68(dd,J＝3.4,0.8Hz,1H),7.34–7.31(m,2H),6.73(dd,J＝3.4,1.7Hz,1H),6.58(brs,2H),4.36(t,J＝7.2Hz,2H),3.82(s,3H),3.23(t,J＝7.2Hz,2H)。HRMS(ESI)C 19 H 18 N 5 O 3 + [M+H] + Calculated 364.1404, found 364.1402.
Step 2: preparation of 4- (2- (2-amino-6- (furan-2-yl) -9H-purin-9-yl) ethyl) -N- (2-aminophenyl) benzamide (Compound I-21)
Intermediate Int-4 in step 2 of example 11 was replaced with intermediate Int-12, the remaining starting materials required,the reagent and preparation method were the same as in example 11, step 2, and the obtained product was a pale yellow solid compound (I-21) (0.060 g, yield 49%) according to example 11, step 3. 1 H NMR(800MHz,DMSO-d 6 )δ9.60(s,1H),7.95(dd,J＝1.7,0.8Hz,1H),7.93(s,1H),7.90(d,J＝7.9Hz,2H),7.69(dd,J＝3.4,0.8Hz,1H),7.32(d,J＝8.0Hz,2H),7.14(d,J＝7.8Hz,1H),6.96(ddd,J＝8.6,7.3,1.6Hz,1H),6.77(dd,J＝8.0,1.5Hz,1H),6.73(dd,J＝3.4,1.7Hz,1H),6.59(brs,2H),6.58(d,J＝8.1Hz,1H),4.87(s,2H),4.38(t,J＝7.2Hz,2H),3.24(t,J＝7.2Hz,2H)。HRMS(ESI)C 24 H 22 N 7 O 2 + [M+H] + Calculated 440.1829, found 440.1814.
Example 22: preparation of 4- ((2-amino-6- (5-methylfuran-2-yl) -9H-purin-9-yl) methyl) -N- (2-aminophenyl) benzamide (Compound I-22)
Step 1: preparation of methyl 4- ((2-amino-6- (5-methylfuran-2-yl) -9H-purin-9-yl) methyl) benzoate (intermediate Int-13)
The starting material compound C-5 is synthesized by reference (Gillespie, cliffe et al, bioorg Med Chem Lett.,2008,18,2924-2929). Methyl 4- (bromomethyl) benzoate (153 mg,0.669 mmol), K 2 CO 3 (123 mg,0.892 mmol) and Compound C-5 (96 mg, 0.4476 mmol) were dissolved in DMF (15 mL) and stirred at room temperature overnight. The reaction solution was diluted with water, extracted three times with ethyl acetate, and the organic phases were combined with anhydrous Na 2 SO 4 Drying once, evaporating the solvent under reduced pressure, and separating and purifying the residual solid by silica gel column chromatography to obtain white solid intermediate Int-13 (85 mg, yield 52%). 1 H NMR(600MHz,DMSO-d 6 )δ8.20(s,1H),7.95–7.91(m,2H),7.68(d,J＝3.3Hz,1H),7.35(d,J＝8.4Hz,2H),6.57(brs,2H),6.38(dd,J＝3.3,1.1Hz,1H),5.40(s,2H),3.83(s,3H),2.40(s,3H)。HRMS(ESI)C 19 H 18 N 5 O 3 + [M+H] + Calculated 364.1404, found 364.1400.
Step 2: preparation of 4- ((2-amino-6- (5-methylfuran-2-yl) -9H-purin-9-yl) methyl) -N- (2-aminophenyl) benzamide (Compound I-22)
The intermediate Int-4 in step 2 of example 11 was replaced with intermediate Int-13, and the remaining desired starting materials, reagents and preparation method were the same as in step 2 of example 11 to give the product as a yellow solid compound (I-22) (0.039 g, 40% yield) as in step 3 of example 11. 1 H NMR(800MHz,DMSO-d 6 )δ9.62(s,1H),8.22(s,1H),7.94(d,J＝7.9Hz,2H),7.68(d,J＝3.3Hz,1H),7.37(d,J＝8.0Hz,2H),7.15(d,J＝7.8Hz,1H),6.96(td,J＝7.6,1.6Hz,1H),6.77(dd,J＝8.0,1.4Hz,1H),6.59(d,J＝8.1Hz,1H),6.57(brs,2H),6.39(dd,J＝3.4,0.9Hz,1H),5.39(s,2H),4.93(s,2H),2.41(s,3H)。HRMS(ESI)C 24 H 22 N 7 O 2 + [M+H] + Calculated 440.1829, found 440.1822.
Example 23: preparation of 4- ((2-amino-6- (5-methylfuran-2-yl) -9H-purin-9-yl) methyl) -N-hydroxybenzoamide (Compound I-23)
The intermediate Int-1 in step 2 of example 1 was replaced with intermediate Int-13, and the remaining required raw materials, reagents and preparation method were the same as in step 2 of example 1 to give compound (I-23) (0.077 g, yield 87%) as a yellow solid. 1 H NMR(800MHz,DMSO-d 6 )δ11.18(s,1H),9.02(s,1H),8.20(s,1H),7.72–7.69(m,2H),7.67(d,J＝3.3Hz,1H),7.30(d,J＝8.2Hz,2H),6.57(brs,2H),6.38(dd,J＝3.4,1.2Hz,1H),5.35(s,2H),2.40(s,3H)。HRMS(ESI)C 18 H 17 N 6 O 3 + [M+H] + Calculated 365.1357, found 365.1334.
Example 24: preparation of 4- ((5-amino-7- (furan-2-yl) -3H- [1,2,3] triazolo [4,5-d ] pyrimidin-3-yl) methyl) -N- (2-aminophenyl)) benzamide (Compound I-24)
Step 1: preparation of methyl 4- ((5-amino-7- (furan-2-yl) -3H- [1,2,3] triazolo [4,5-d ] pyrimidin-3-yl) methyl) benzoate (intermediate Int-14)
The starting material compound C-6 is synthesized by reference (Gillespie, cliffe et al, bioorg Med Chem Lett.,2008,18,2924-2929). Methyl 4- (bromomethyl) benzoate (293 mg,1.25 mmol), cs 2 CO 3 (651 mg,2.00 mmol) and Compound C-6 (165 mg,0.816 mmol) in CH 3 CN and DMF (1:1 v/v each 10 mL) was stirred overnight at room temperature. The reaction solution was diluted with water, extracted three times with ethyl acetate, and the organic phases were combined with anhydrous Na 2 SO 4 Drying once, evaporating the solvent under reduced pressure, and separating and purifying the residual solid by silica gel column chromatography to obtain yellow solid intermediate Int-14 (23 mg, yield 8%). 1 H NMR(800MHz,DMSO)δ8.16–8.12(m,1H),7.98–7.95(m,2H),7.75(d,J＝3.5Hz,1H),7.49(d,J＝8.5Hz,2H),7.15(s,2H),6.85(dd,1H),6.00(s,2H),3.84(s,3H)。HRMS(ESI)C 17 H 15 N 6 O 3 + [M+H] + Calculated 351.1200, found 351.1201.
Step 2: preparation of 4- ((5-amino-7- (furan-2-yl) -3H- [1,2,3] triazolo [4,5-d ] pyrimidin-3-yl) methyl) -N- (2-aminophenyl)) benzamide (Compound I-24)
The intermediate Int-4 in step 2 of example 11 was replaced with intermediate Int-14, and the remaining desired starting materials, reagents and preparation method were the same as in step 2 of example 11 to give the product as a yellow solid compound (I-24) (0.015 g, yield 12%) in step 3 of example 11. 1 H NMR(800MHz,DMSO-d 6 )δ9.65(s,1H),8.13(dd,J＝1.7,0.8Hz,1H),7.98(d,J＝7.9Hz,2H),7.74(dd,J＝3.5,0.8Hz,1H),7.51(d,J＝8.1Hz,2H),7.15(dd,1H),7.05(s,2H),6.96(m,1H),6.85(dd,J＝3.5,1.7Hz,1H),6.76(dd,J＝8.0,1.5Hz,1H),6.58(m,1H),5.98(s,2H),4.88(s,2H)。HRMS(ESI)C 22 H 19 N 8 O 2 + [M+H] + Calculated 427.1625, found 427.1624.
Example 25: preparation of 4- ((5-amino-7- (5-methylfuran-2-yl) -3H- [1,2,3] triazolo [4,5-d ] pyrimidin-3-yl) methyl) -N-hydroxybenzoamide (compound I-25)
Step 1: preparation of 4- ((5-amino-7- (5-methylfuran-2-yl) -3H- [1,2,3] triazolo [4,5-d ] pyrimidin-3-yl) methyl) benzoate (intermediate Int-15)
The starting material compound C-7 was synthesized by reference to the literature (Gillespie, cliffe et al, bioorg Med Chem Lett.,2008,18,2924-2929). Methyl 4- (bromomethyl) benzoate (427 mg,1.87 mmol), cs 2 CO 3 (967 mg,2.97 mmol) and 7- (5-methylfuran-2-yl) -3H- [1,2,3]Triazolo [4,5-d ]]Pyrimidine-5-amine (262 mg,1.21 mmol) in CH 3 CN and DMF (1:1 v/v each 10 mL) was stirred overnight at room temperature. The reaction solution was diluted with water, extracted three times with ethyl acetate, and the organic phases were combined with anhydrous Na 2 SO 4 Drying once, evaporating the solvent under reduced pressure, and separating and purifying the residual solid by silica gel column chromatography to obtain yellow solid intermediate Int-14 (21 mg, yield 7%). 1 HNMR(800MHz,DMSO-d 6 )δ7.94(m,2H),7.88(d,J＝3.3Hz,1H),7.37(m,2H),7.34–
7.19(s,2H),6.51(dd,J＝3.4,1.1Hz,1H),5.75(s,2H),3.83(s,3H),2.45(s,3H)。HRMS(ESI)C 18 H 17 N 6 O 3 + [M+H] + Calculated 365.1357, found 365.1403.
Step 2: preparation of 4- ((5-amino-7- (5-methylfuran-2-yl) -3H- [1,2,3] triazolo [4,5-d ] pyrimidin-3-yl) methyl) -N-hydroxybenzoamide (compound I-25)
The intermediate Int-1 in step 2 of example 1 was replaced with intermediate Int-15, and the remaining desired starting materials, reagents and preparation method were the same as in step 2 of example 1 to give compound (I-25) (0.018 g, 69% yield) as a yellow solid. 1 H NMR(800MHz,DMSO-d 6 )δ11.17 9.03(s,1H),9.03(s,1H),7.87(d,J＝3.4Hz,1H),7.73–7.69(m,2H),7.40(s,2H),7.32–7.29(m,2H),6.51(dd,J＝3.4,1.1Hz,1H),5.70(s,2H),2.45(s,3H)。HRMS(ESI)C 17 H 16 N 7 O 3 + [M+H] + Calculated 366.1309, found 366.1329.
Example 26: (E) Preparation of-3- (4- ((6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenyl) -N- (2-aminophenyl) acrylamide (compound I-26)
Step 1: preparation of methyl (E) -3- (4- ((6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenyl) acrylate (intermediate Int-16)
The procedure of example 1, step 1, was followed except that methyl 5-bromopentanoate was replaced with methyl (E) -3- (4- (bromomethyl) phenyl) acrylate, the reaction conditions were changed to room temperature at 100℃and the remaining desired starting materials, reagents and preparation were the same, to give intermediate (Int-7) (0.081 g, yield 29%) as a yellow solid. 1 H NMR(800MHz,DMSO-d 6 )δ8.29(s,1H),8.06(dd,J＝1.7,0.8Hz,1H),7.67(d,J＝7.7Hz,2H),7.62(d,J＝16.0Hz,1H),7.45(dd,J＝3.5,0.8Hz,1H),7.21(d,J＝7.2Hz,2H),6.95(brs,2H),6.79(dd,J＝3.5,1.7Hz,1H),6.60(d,J＝16.0Hz,1H),5.45(s,2H),3.71(s,3H)。HRMS(ESI)C 20 H 18 N 5 O 3 +
[M+H] + Calculated 376.1404, found 376.1405.
Step 2: (E) Preparation of-3- (4- ((6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenyl) -N- (2-aminophenyl) acrylamide (compound I-26)
The intermediate Int-4 in step 2 of example 11 was replaced with intermediate Int-16, and the remaining desired starting materials, reagents and preparation method were the same as in step 2 of example 11 to give the product as a yellow solid compound (I-26) (0.047 g, yield 55%) as in step 3 of example 11. 1 H NMR(800MHz,DMSO-d 6 )δ9.38(s,1H),8.30(s,1H),8.07(dd,J＝1.7,0.8Hz,1H),7.57(d,J＝8.0Hz,2H),7.51(d,J＝15.7Hz,1H),7.46(dd,J＝3.5,0.8Hz,1H),7.32(dd,J＝8.0,1.6Hz,1H),7.24(d,J＝8.0Hz,2H),6.96(brs,2H),6.91(dd,J＝7.6,1.5Hz,1H),6.85(d,J＝15.7Hz,1H),6.80(dd,J＝3.5,1.7Hz,1H),6.74(dd,J＝7.9,1.5Hz,1H),6.57(td,J＝7.5,1.4Hz,1H),5.46(s,2H),4.93(s,2H)。HRMS(ESI)C 25 H 22 N 7 O 2 + [M+H] + Calculated 452.1829, found 452.1851.
Example 27: (E) Preparation of-3- (4- ((6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenyl) -N-hydroxyacrylamide (compound I-27)
The intermediate Int-1 in step 2 of example 1 was replaced with intermediate Int-16, and the remaining desired starting materials, reagents and preparation methods were the same as in step 2 of example 1, further purified by preparative HPLC to give compound (I-27) as a yellow solid (0.026 g, 38% yield). 1 H NMR(800MHz,DMSO-d 6 )δ10.74(s,1H),9.02(s,1H),8.29(s,1H),8.06(dd,J＝1.7,0.8Hz,1H),7.51(d,J＝8.0Hz,2H),7.45(dd,J＝3.5,0.8Hz,1H),7.41(d,J＝15.8Hz,1H),7.21(d,J＝8.0Hz,2H),6.95(brs,2H),6.79(dd,J＝3.5,1.7Hz,1H),6.41(d,J＝15.8Hz,1H),5.44(s,2H)。HRMS(ESI)C 19 H 17 N 6 O 3 + [M+H] + Calculated 377.1357, found 377.1359.
Example 28: (E) Preparation of-3- (4- ((5-amino-7- (furan-2-yl) -3H- [1,2,3] triazolo [4,5-d ] pyrimidin-3-yl) methyl) phenyl) -N- (2-aminophenyl) acrylamide (Compound I-28)
Step 1: preparation of methyl (E) -3- (4- ((5-amino-7- (furan-2-yl) -3H- [1,2,3] triazolo [4,5-d ] pyrimidin-3-yl) methyl)) phenyl) acrylate (intermediate Int-17)
The procedure for preparation and preparation was as in example 24, step 1, substituting methyl 4- (bromomethyl) benzoate for methyl (E) -3- (4- (bromomethyl) phenyl) acrylate to give intermediate (Int-7) (0.083 g, 11% yield) as a yellow solid. 1 H NMR(800MHz,DMSO)δ8.12(d,J＝0.9Hz,1H),7.91(d,J＝3.3Hz,1H),7.71(d,J＝8.3Hz,2H),7.63(d,J＝16.0Hz,1H),7.36(s,2H),7.31(d,J＝8.3Hz,2H),6.86(dd,J＝3.5,1.7Hz,1H),6.62(d,J＝16.0Hz,1H),5.70(s,2H),3.71(s,3H)。HRMS(ESI)C 19 H 17 N 6 O 3 + [M+H] + Calculated 377.1357, found 377.1361.
Step 2: (E) Preparation of-3- (4- ((5-amino-7- (furan-2-yl) -3H- [1,2,3] triazolo [4,5-d ] pyrimidin-3-yl) methyl) phenyl) -N- (2-aminophenyl) acrylamide (Compound I-28)
The intermediate Int-4 in step 2 of example 11 was replaced with intermediate Int-17, and the remaining desired starting materials, reagents and preparation were the same as in step 2 of example 11 to give the product as a tan solid compound (I-28) (0.006 g, yield 4%) as in step 3 of example 11. 1 H NMR(800MHz,DMSO-d 6 )δ9.40(s,1H),8.13(dd,J＝1.8,0.8Hz,1H),7.91(dd,J＝3.5,0.8Hz,1H),7.60(d,J＝8.2Hz,2H),7.53-1.51(d,J＝15.7Hz,1H),7.38(s,2H),7.33(d,J＝8.3Hz,2H),7.32(d,J＝1.5Hz,1H),6.91(m,1H),6.88-6.86(d,J＝15.8,1H),6.88–6.86(d,J＝15.1Hz,1H),6.74(dd,J＝8.0,1.5Hz,1H),6.57(td,J＝7.5,1.4Hz,1H),5.71(s,2H),4.94(s,2H)。HRMS(ESI)C 24 H 21 N 8 O 2 + [M+H] + Calculated 453.1782, found 453.1721.
Example 29: (E) Preparation of-3- (3- ((6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenyl) -N- (2-aminophenyl) acrylamide (compound I-29)
Step 1: preparation of methyl (E) -3- (3- ((6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenyl) acrylate (intermediate Int-18)
3- (bromomethyl) benzaldehyde (0.284 g,1.43 mmol), K 2 CO 3 (275 mg,1.99 mmol) and 4- (furan-2-yl) -1H-pyrazolo [3,4-d]Pyrimidine-6-amine (160 mg,0.795 mmol) was dissolved in DMF (15 mL) and stirred overnight at room temperature. The reaction solution was diluted with water, extracted three times with ethyl acetate, and the organic phases were combined with anhydrous Na 2 SO 4 Drying once, evaporating the solvent under reduced pressure, and separating and purifying the remaining solid by silica gel column chromatography to obtain white solid intermediate Int-18a (0.105 g, yield 41%). 1 H NMR(800MHz,DMSO-d 6 )δ9.96(s,1H),8.31(s,1H),8.07(dd,J＝1.7,0.8Hz,1H),7.83(dt,J＝7.5,1.5Hz,1H),7.68(q,J＝1.3,0.9Hz,1H),7.58(t,J＝7.6Hz,1H),7.54(dt,J＝7.9,1.5Hz,1H),7.45(dd,J＝3.5,0.8Hz,1H),6.96(brs,2H),6.79(dd,J＝3.5,1.7Hz,1H),5.53(s,2H)。HRMS(ESI)C 17 H 14 N 5 O 2 + [M+H] + Calculated 320.1142, found 320.1144. Methyl 2- (dimethoxyphosphoryl) acetate (111 mg,0.608 mmol) and C 4 H 9 OK (55 mg,0.498 mmol) was dissolved in DMF (15 mL) and stirred at 0deg.C for 15 min, and then the solution of Int-18a in DMF was added dropwise and stirred at RT for 2 h. The reaction solution was diluted with water, extracted three times with ethyl acetate, and the organic phases were combined with anhydrous Na 2 SO 4 Drying once, evaporating solvent under reduced pressure, and separating residual solid with silica gel column Chromatography separation and purification gave intermediate Int-18 (0.092 g, 75% yield) as a white solid. 1 H NMR(800MHz,DMSO-d 6 )δ8.29(s,1H),8.06(dd,J＝1.7,0.8Hz,1H),7.65–7.63(m,1H),7.62(d,J＝16.1Hz,1H),7.59–7.58(m,1H),7.45(dd,J＝3.5,0.8Hz,1H),7.37(t,J＝7.7Hz,1H),7.18(d,J＝7.8Hz,1H),6.95(brs,2H),6.79(dd,J＝3.5,1.7Hz,1H),6.59(d,J＝16.1Hz,1H),5.45(s,2H),3.71(s,3H)。HRMS(ESI)C 20 H 17 N 5 O 3 + [M+H] + Calculated 376.1404, found 376.1397.
Step 2: (E) Preparation of-3- (3- ((6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenyl) -N- (2-aminophenyl) acrylamide (compound I-29)
The intermediate Int-4 in step 2 of example 11 was replaced with intermediate Int-18, and the remaining desired starting materials, reagents and preparation were the same as in step 2 of example 11 to give the product as a yellow solid compound (I-29) (0.028 g, yield 26%) in step 3 of example 11. 1 H NMR(800MHz,DMSO-d 6 )δ9.38(s,1H),8.30(s,1H),8.06(d,J＝1.7Hz,1H),7.52(d,J＝8.9Hz,1H),7.51(d,J＝15.9Hz,2H),7.45(dd,J＝3.5,0.8Hz,1H),7.40(t,J＝7.7Hz,1H),7.32(dd,J＝7.8,1.6Hz,1H),7.21(d,J＝7.7Hz,1H),6.95(brs,2H),6.91(td,J＝7.8,1.8Hz,1H),6.85(d,J＝15.8Hz,1H),6.79(dd,J＝3.5,1.8Hz,1H),6.74(dd,J＝8.0,1.5Hz,1H),6.57(td,J＝7.5,1.4Hz,1H),5.47(s,2H),4.93(brs,2H)。HRMS(ESI)C 25 H 22 N 7 O 2 + [M+H] + Calculated 452.1829, found 452.1830.
Example 30: (E) Preparation of-3- (3- ((6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenyl) -N-hydroxyacrylamide (compound I-30)
The intermediate Int-1 in step 2 of example 1 was replaced with intermediate Int-18, and the remaining desired starting materials, reagents and preparation methods were the same as in step 2 of example 1, further purified by preparative HPLC to give compound (I-30) as a white solid (0.060 g, 84% yield). 1 H NMR(800MHz,DMSO-d 6 )δ10.76(s,1H),9.04(s,1H),8.29(s,1H),8.06(d,J＝1.7Hz,1H),7.46(d,J＝7.9Hz,1H),7.45(d,J＝3.5Hz,1H),7.43(s,1H),7.40(d,J＝15.8Hz,1H),7.36(t,J＝7.7Hz,1H),7.17(d,J＝7.7Hz,1H),6.94(brs,2H),6.79(dd,J＝3.6,1.7Hz,1H),6.41(d,J＝15.8Hz,1H),5.44(s,2H)。HRMS(ESI)C 19 H 17 N 6 O 3 + [M+H] + Calculated 377.1357, found 377.1354.
Example 31: (E) Preparation of-3- (3- ((6-amino-4- (5-methylfuran-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenyl) -N- (2-aminophenyl) acrylamide (compound I-31)
Step 1: (E) Preparation of methyl-3- (3- ((6-amino-4- (5-methylfuran-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenyl) acrylate (intermediate Int-19)
4- (furan-2-yl) -1H-pyrazolo [3,4-d ] as in example 29, step 1]Substitution of pyrimidin-6-amine with 4- (5-methylfuran-2-yl) -1H-pyrazolo [3,4-d]Pyrimidine-6-amine, the remaining required raw materials, reagents and preparation method were the same as in example 29, step 1, to give compound (I-31) (0.313 g, yield 57%) as a pale yellow solid. 1 H NMR(600MHz,DMSO-d 6 )δ8.30(s,1H),7.64–7.60(m,2H),7.57(t,J＝1.8Hz,1H),7.39–7.34(m,2H),7.17(dt,J＝7.8,1.3Hz,1H),6.90(brs,2H),6.58(d,J＝16.0Hz,1H),6.42(dd,J＝3.4,1.1Hz,1H),5.44(s,2H),3.71(s,3H),2.45(s,3H)。HRMS(ESI)C 21 H 20 N 5 O 3 + [M+H] + Calculated 390.1561, found 390.1563.
Step 2: (E) Preparation of-3- (3- ((6-amino-4- (5-methylfuran-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenyl) -N- (2-aminophenyl) acrylamide (compound I-31)
The intermediate Int-4 in step 2 of example 11 was replaced with intermediate Int-19, and the remaining desired starting materials, reagents and preparation method were the same as in step 2 of example 11 to give the product as a yellow solid compound (I-31) (0.064 g, yield 49%) in step 3 of example 11. 1 H NMR(600MHz,DMSO-d 6 )δ9.38(s,1H),8.31(s,1H),7.54–7.47(m,3H),7.42–7.36(m,2H),7.32(dd,J＝7.9,1.5Hz,1H),7.20(d,J＝7.6Hz,1H),6.93–6.88(m,3H),6.84(d,J＝15.7Hz,1H),6.74(dd,J＝8.0,1.5Hz,1H),6.57(td,J＝7.5,1.4Hz,1H),6.43(dd,J＝3.3,1.2Hz,1H),5.46(s,2H),4.93(s,2H),2.46(s,3H)。HRMS(ESI)C 26 H 24 N 7 O 2 + [M+H] + Calculated 466.1986, found 466.1982.
Example 32: preparation of 4- (2- (6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) ethoxy) -N- (2-aminophenyl) benzamide (compound I-32)
Step 1: preparation of methyl 4- (2- (6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) ethoxy) benzoate (intermediate Int-20)
Compound D-2 was synthesized by literature methods (Yan et al, J Med chem.,2021,64,16573-16597). Compound C-1 (0.081 g,0.40 mmol), compound D-2 (0.125 g,0.48 mmol) and K 2 CO 3 (111 mg,0.80 mmol) was dissolved in DMF (15 mL) and stirred overnight at 100deg.C. The reaction solution was diluted with water, extracted three times with ethyl acetate, and the organic phases were combined with anhydrous Na 2 SO 4 Drying once, evaporating the solvent under reduced pressure, and separating and purifying the remaining solid by silica gel column chromatography to obtain white solid intermediate Int-20 (0.099 g, yield 65%). 1 H NMR(800MHz,Chloroform-d)δ8.27(s,1H),7.96–7.92(m,2H),7.74(dd,J＝1.7,0.8Hz,1H),7.51–7.41(m,1H),6.89–6.87(m,2H),6.64(dd,J＝3.5,1.7Hz,1H),5.28(brs,2H),4.70(t,J＝5.9Hz,2H),4.49(t,J＝5.9Hz,2H),3.86(s,3H)。HRMS(ESI)C 19 H 18 N 5 O 4 + [M+H] + Calculated 380.1353, found 380.1351.
Step 2: preparation of 4- (2- (6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) ethoxy) -N- (2-aminophenyl) benzamide (compound I-32)
The intermediate Int-4 in step 2 of example 11 was replaced with intermediate Int-20, and the remaining desired starting materials, reagents and preparation were the same as in step 2 of example 11 to give the product as a yellow solid compound (I-32) (0.025 g, yield 42%) in step 3 of example 11. 1 H NMR(800MHz,DMSO-d6)δ9.53(s,1H),8.28(s,1H),8.05(d,J＝1.7Hz,1H),7.93(d,J＝8.5Hz,2H),7.44(d,J＝3.5Hz,1H),7.13(d,J＝7.8Hz,1H),7.00(d,J＝8.8Hz,2H),6.96(dd,J＝7.7,1.6Hz,1H),6.94(brs,2H),6.78(dd,J＝3.5,1.7Hz,1H),6.77(dd,J＝8.1,1.5Hz,1H),6.59(t,J＝7.5Hz,1H),4.85(brs,2H),4.61(t,J＝5.5Hz,2H),4.53(t,J＝5.5Hz,2H)。HRMS(ESI)C 24 H 22 N 7 O 3 + [M+H] + Calculated 456.1779, found 456.1773.
Example 33: preparation of 4- (2- (6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) ethoxy) -N-hydroxybenzoamide (compound I-33)
The intermediate Int-1 in step 2 of example 1 was replaced with intermediate Int-20, and the remaining desired starting materials, reagents and preparation methods were the same as in step 2 of example 1, further purified by preparative HPLC to give compound (I-33) as an off-white solid (0.066 g, yield 49%). 1 H NMR(800MHz,DMSO-d6)δ11.03(s,1H),8.86(brs,1H),8.26(s,1H),8.04(d,J＝1.7Hz,1H),7.68(d,J＝8.9Hz,2H),7.43(d,J＝3.5Hz,1H),6.94(d,J＝8.8Hz,2H),6.91(brs,2H),6.78(dd,J＝3.5,1.7Hz,1H),4.58(t,J＝5.5Hz,2H),4.49(t,J＝5.5Hz,2H)。HRMS(ESI)C 18 H 17 N 6 O 4 + [M+H] + Calculated 381.1306, found 381.1317.
Example 34: preparation of 4- (3- (6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) propoxy) -N- (2-aminophenyl) benzamide (compound I-34)
Step 1: preparation of methyl 4- (3- (6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) propoxy) benzoate (intermediate Int-21)
The 1, 2-dibromoethane in step 1 of example 32 was replaced with 1, 3-dibromopropane, and the other required raw materials, reagents and preparation method were the same as those in step 1 of example 32 to obtain a white solid intermediate Int-21 (0.060 g, yield 28%). 1 H NMR(800MHz,DMSO-d 6 )δ8.24(s,1H),8.05(dd,J＝1.7,0.8Hz,1H),7.89–7.87(m,2H),7.42(dd,J＝3.5,0.8Hz,1H),7.01–6.98(m,2H),6.86(brs,2H),6.78(dd,J＝3.5,1.7Hz,1H),4.38(t,J＝6.9Hz,2H),4.09(t,J＝6.1Hz,2H),3.80(s,3H),2.29(p,J＝6.5Hz,2H)。
HRMS(ESI)C 20 H 20 N 5 O 4 + [M+H] + Calculated 394.1510, found 394.1515.
Step 2: preparation of 4- (3- (6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) propoxy) -N- (2-aminophenyl) benzamide (compound I-34)
The intermediate Int-4 in step 2 of example 11 is replaced by intermediate Int-21, and the rest of the required raw materials, reagents and preparation method are the same as those in step 2 of example 11, and the obtained product is obtained according to the actual stateExample 11 step 3 gave compound (I-34) as a yellow solid (0.032 g, 32% yield). 1 H NMR(800MHz,DMSO-d 6 )δ9.53(s,1H),8.25(s,1H),8.05(d,J＝1.6Hz,1H),7.94(d,J＝8.3Hz,2H),7.43(d,J＝3.5Hz,1H),7.14(d,J＝7.5Hz,1H),7.00(d,J＝8.9Hz,2H),6.96(td,J＝7.6,1.5Hz,1H),6.88(s,2H),6.80–6.77(m,2H),6.60(t,J＝7.5Hz,1H),4.93(s,2H),4.40(t,J＝6.9Hz,2H),4.10(t,J＝6.1Hz,2H),2.30(p,J＝6.5Hz,2H)。HRMS(ESI)C 25 H 24 N 7 O 3 + [M+H] + Calculated 470.1935, found 470.1982.
The compounds listed in examples 35-44 can be prepared in the same manner by changing the corresponding starting materials according to the methods listed in examples 11-34, as detailed in Table 2.
TABLE 2
Example 45: preparation of 5- (3- ((6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenoxy) -N-hydroxypentanamide (compound I-45)
Step 1: preparation of methyl 5- (3- ((6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenoxy) pentanoate (intermediate Int-22)
M-cresol (1 g,9.25 mmol), K 2 CO 3 (2.58 g,18.49 mmol) and methyl 5-bromopentanoate (2.18 g,11.10 mmol) were dissolved in DMF (15 mL) and stirred overnight at room temperature. The reaction solution was diluted with water, extracted three times with ethyl acetate, and the organic phases were combined with anhydrous Na 2 SO 4 Drying once, evaporating the solvent under reduced pressure, and separating and purifying the residual solid by silica gel column chromatography to obtain colorless liquid intermediate Int-22a (1.559 g, yield 76%). HRMS (ESI) C 13 H 19 O 3 + [M+H] + Calculated 223.1441, found 223.1466. Int-22a (1.559 g,7.01 mmol), 1-bromopyrrolidine-2, 5-dione (1.5 g,8.42 mmol) and benzoic acid peroxy anhydride (85 mg,0.350 mmol) were dissolved in CCl 4 (30 mL) and refluxed overnight. The reaction solution was diluted with water, extracted three times with ethyl acetate, and the organic phases were combined with anhydrous Na 2 SO 4 Drying once, evaporating the solvent under reduced pressure, and separating and purifying the residual solid by silica gel column chromatography to obtain a pale yellow oily intermediate Int-22b (1.461 g, yield 69%). HRMS (ESI) C 13 H 18 BrO 3 + [M+H] + Calculated 301.0434, found 301.0389. Int-22b (1.460 g,4.85 mmol), K 2 CO 3 (463 mg,3.37 mmol) and 4- (furan-2-yl) -1H-pyrazolo [3, 4-d)]Pyrimidine-6-amine (399 mg,1.68 mmol) was dissolved in DMF (15 mL) and stirred overnight at room temperature. The reaction solution was diluted with water, extracted three times with ethyl acetate, and the organic phases were combined with anhydrous Na 2 SO 4 Drying once, evaporating the solvent under reduced pressure, and separating and purifying the remaining solid by silica gel column chromatography to obtain yellow oily intermediate Int-22 (0.140 g, yield 20%). 1 H NMR(800MHz,DMSO-d 6 )δ8.28(s,1H),8.06(dd,J＝1.8,0.8Hz,1H),7.44(dd,J＝3.5,0.8Hz,1H),7.21(t,J＝7.9Hz,1H),6.93(brs,2H),6.83–6.80(m,1H),6.79(dd,J＝3.5,1.7Hz,1H),6.74(t,J＝2.0Hz,1H),6.73–6.70(m,1H),5.38(s,2H),3.90(t,J＝6.1Hz,2H),3.57(s,3H),2.35(t,J＝7.2Hz,2H),1.70–1.66(m,2H),1.66–1.62(m,2H)。HRMS(ESI)C 22 H 24 N 5 O 4 + [M+H] + Calculated 422.1823, found 422.1829.
Step 2: preparation of 5- (3- ((6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenoxy) -N-hydroxypentanamide (compound I-45)
The intermediate Int-1 in step 2 of example 1 was replaced with intermediate Int-22, and the other desired raw materials, reagents and preparation method were the same as in step 2 of example 1 to give the compound (I-45) (0.075 g, yield 59%) as an off-white solid. 1 H NMR(800MHz,DMSO-d 6 )δ10.35(s,1H),8.67(d,J＝1.7Hz,1H),8.28(s,1H),8.06(dd,J＝1.7,0.8Hz,1H),7.44(dd,J＝3.5,0.8Hz,1H),7.21(t,J＝7.9Hz,1H),6.94(brs,2H),6.83–6.81(m,1H),6.79(dd,J＝3.5,1.7Hz,1H),6.76(t,J＝2.0Hz,1H),6.71(d,J＝7.7Hz,1H),5.38(s,2H),3.90(t,J＝6.2Hz,2H),1.99(t,J＝7.2Hz,2H),1.68–1.63(m,2H),1.63–1.58(m,2H)。HRMS(ESI)C 21 H 23 N 6 O 4 + [M+H] + Calculated 423.1775, found 423.1787.
Example 46: preparation of 6- (3- ((6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenoxy) -N-hydroxyhexanamide (compound I-46)
Step 1: preparation of 6- (3- ((6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenoxy) -N-hydroxyhexanamide (intermediate Int-23)
The 5-bromopentanoate in step 1 of example 45 was replaced with methyl 6-bromohexanoate, and the remaining desired starting materials, reagents and preparation were the same as in step 1 of example 45 to give intermediate Int-23 (0.173 g, 40% yield) as a yellow oil. 1 H NMR(800MHz,DMSO-d 6 )δ8.28(s,1H),8.06(dd,J＝1.8,0.7Hz,1H),7.44(dd,J＝3.5,0.8Hz,1H),7.20(t,J＝7.9Hz,1H),6.94(brs,2H),6.81(ddd,J＝8.2,2.6,0.9Hz,1H),6.79(dd,J＝3.5,1.7Hz,1H),6.74–6.70(m,2H),5.38(s,2H),3.89(t,J＝6.4Hz,2H),3.56(s,3H),2.30(t,J＝7.4Hz,2H),1.69–1.64(m,2H),1.58–1.52(m,2H),1.40–1.35(m,2H)。HRMS(ESI)C 23 H 26 N 5 O 4 + [M+H] + Calculated 436.1979, found 436.1980.
Step 2: preparation of 6- (3- ((6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenoxy) -N-hydroxyhexanamide (compound I-46)
The intermediate Int-1 in step 2 of example 1 was replaced with intermediate Int-23, and the remaining desired starting materials, reagents and preparation method were the same as in step 2 of example 1 to give compound (I-46) (0.130 g, yield 81%) as a yellow solid. 1 H NMR(800MHz,DMSO-d 6 )δ10.33(s,1H),8.65(s,1H),8.28(s,1H),8.06(d,J＝1.7Hz,1H),7.44(dd,J＝3.5,0.8Hz,1H),7.20(t,J＝7.9Hz,1H),6.94(brs,2H),6.81(dd,J＝8.2,2.6Hz,1H),6.79(dd,J＝3.5,1.7Hz,1H),6.74(t,J＝2.1Hz,1H),6.71(d,J＝7.6Hz,1H),5.38(s,2H),3.88(t,J＝6.4Hz,2H),1.95(t,J＝7.4Hz,2H),1.66(p,J＝6.6Hz,2H),1.52(p,J＝7.5Hz,2H),1.37–1.32(m,2H)。HRMS(ESI)C 22 H 25 N 6 O 4 + [M+H] + Calculated 437.1932, found 437.1930.
Example 47: preparation of 7- (3- ((6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenoxy) -N-hydroxyheptanamide (compound I-47)
Step 1: preparation of methyl 7- (3- ((6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenoxy) heptanoate (intermediate Int-24)
The 5-bromopentanoate in step 1 of example 45 was replaced with 7-methylbromoheptanoate, and the remaining necessary raw materials, reagents and preparation method were the same as those in step 1 of example 45 to give Int-24 (0.067 g, yield 50%) as a yellow oily intermediate. HNMR (800 MHz, DMSO-d) 6 )δ8.28(s,1H),8.06(dd,J＝1.8,0.8Hz,1H),7.44(dd,J＝3.5,0.8Hz,1H),7.20(t,J＝7.9Hz,1H),6.94(brs,2H),6.81(ddd,J＝8.2,2.6,0.9Hz,1H),6.79(dd,J＝3.5,1.7Hz,1H),6.73–6.70(m,2H),5.38(s,2H),3.88(t,J＝6.5Hz,2H),3.57(s,3H),2.28(t,J＝7.4Hz,2H),1.67–1.62(m,2H),1.54–1.49(m,2H),1.38–1.33(m,2H),1.30–1.26(m,2H)。HRMS(ESI)C 24 H 28 N 5 O 3 + [M+H] + Calculated 450.2136, found 450.2136.
Step 2: preparation of 7- (3- ((6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenoxy) -N-hydroxyheptanamide (compound I-47)
The intermediate Int-1 in step 2 of example 1 was replaced with intermediate Int-24, and the other desired raw materials, reagents and preparation method were the same as in step 2 of example 1 to give the compound (I-47) as an off-white solid (0.076 g, yield 52%). 1 H NMR(800MHz,DMSO-d 6 )δ10.32(s,1H),8.64(brs,1H),8.28(s,1H),8.06(d,J＝1.7Hz,1H),7.46–7.43(m,1H),7.20(t,J＝7.9Hz,1H),6.94(brs,2H),6.81(dd,J＝8.2,2.5Hz,1H),6.79(dd,J＝3.5,1.7Hz,1H),6.74(t,J＝2.0Hz,1H),6.71(d,J＝7.6Hz,1H),5.38(s,2H),3.88(t,J＝6.5Hz,2H),1.93(t,J＝7.4Hz,2H),1.64(p,J＝6.7Hz,2H),1.48(p,J＝7.5Hz,2H),1.35(tt,J＝9.4,6.6Hz,2H),1.29–1.22(m,2H)。HRMS(ESI)C 23 H 27 N 6 O 4 + [M+H] + Calculated 451.2088, found 451.2082.
Example 48: preparation of 5- (3- ((6-amino-4- (5-methylfuran-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenoxy) -N-hydroxypentanamide (compound I-48)
Step 1: preparation of methyl 5- (3- ((6-amino-4- (5-methylfuran-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenoxy) pentanoate (intermediate Int-25)
4- (furan-2-yl) -1H-pyrazolo [3,4-d ] as in example 45 step 1]Substitution of pyrimidin-6-amine with 4- (5-methylfuran-2-yl) -1H-pyrazolo [3,4-d]Pyrimidine-6-amine, the remaining required raw materials, reagents and preparation method were the same as in example 45, step 1, to afford intermediate Int-25 (0.070 g, yield 24%) as a yellow oil. 1 H NMR(800MHz,DMSO-d 6 )δ8.29(s,1H),7.37(d,J＝3.3Hz,1H),7.20(t,J＝7.9Hz,1H),6.88(brs,2H),6.81(ddd,J＝8.2,2.6,0.9Hz,1H),6.72(t,J＝2.0Hz,1H),6.70(dt,J＝7.7,1.3Hz,1H),6.42(dd,J＝3.4,1.1Hz,1H),5.37(s,2H),3.90(t,J＝6.1Hz,2H),3.57(s,3H),2.46(s,3H),2.35(t,J＝7.2Hz,2H),1.70–1.62(m,4H)。HRMS(ESI)C 23 H 26 N 5 O 4 + [M+H] + Calculated 436.1979, found 436.1973.
Step 2: preparation of 5- (3- ((6-amino-4- (5-methylfuran-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenoxy) -N-hydroxypentanamide (compound I-48)
The intermediate Int-1 in step 2 of example 1 was replaced with intermediate Int-25, and the remaining desired starting materials, reagents and preparation method were the same as in step 2 of example 1 to give compound (I-48) (0.027 g, 39% yield) as a yellow solid. 1 H NMR(600MHz,DMSO-d 6 )δ10.35(s,1H),8.68(d,J＝1.9Hz,1H),8.29(s,1H),7.37(d,J＝3.4Hz,1H),7.20(t,J＝7.9Hz,1H),6.88(brs,2H),6.82(dd,J＝8.2,2.5Hz,1H),6.74(t,J＝2.0Hz,1H),6.70(d,J＝7.6Hz,1H),6.42(d,J＝3.1Hz,1H),5.37(s,2H),3.90(t,J＝6.1Hz,2H),2.46(s,3H),1.99(t,J＝7.1Hz,2H),1.71–1.58(m,4H)。HRMS(ESI)C 22 H 25 N 6 O 4 + [M+H] + Calculated 436.1979, found 436.1973.
Example 49: preparation of 7- (3- ((6-amino-4- (5-methylfuran-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenoxy) -N-hydroxyheptanamide (compound I-49)
Step 1: preparation of methyl 7- (3- ((6-amino-4- (5-methylfuran-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenoxy) heptanoate (intermediate Int-26)
4- (furan-2-yl) -1H-pyrazolo [3,4-d ] as in example 47, step 1]Substitution of pyrimidin-6-amine with 4- (5-methylfuran-2-yl) -1H-pyrazolo [3,4-d]Pyrimidine-6-amine, the remaining desired starting materials, reagents and preparation were the same as in example 47, step 1, to afford intermediate Int-26 (0.043 g, 30% yield) as a yellow oil. 1 H NMR(800MHz,DMSO-d 6 )δ8.28(s,1H),7.36(d,J＝3.4Hz,1H),7.19(d,J＝8.0Hz,1H),6.89(brs,2H),6.81–6.79(m,1H),6.72–6.70(m,2H),6.41(dd,J＝3.4,1.1Hz,1H),5.38(s,2H),3.87(t,J＝6.5Hz,2H),3.56(s,3H),2.45(s,3H),2.27(t,J＝7.4Hz,2H),1.63(p,J＝6.6Hz,2H),1.50(p,J＝7.4Hz,2H),1.38–1.32(m,2H),1.30–1.23(m,2H)。HRMS(ESI)C 25 H 30 N 5 O 4 + [M+H] + Calculated 464.2292, found 464.2282.
Step 2: preparation of 7- (3- ((6-amino-4- (5-methylfuran-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenoxy) -N-hydroxyheptanamide (compound I-49)
The intermediate Int-1 of step 2 of example 1 was replaced with intermediate Int-26, and the remaining desired starting materials, reagents and preparation methods were the same as in step 2 of example 1, further purified by preparative HPLC to give the white solid compound (I-49) (0.036 g, yield 55%). 1 H NMR(800MHz,DMSO-d 6 )δ10.31(s,1H),8.64(d,J＝1.8Hz,1H),8.29(s,1H),7.37(d,J＝3.4Hz,1H),7.20(t,J＝7.9Hz,1H),6.88(brs,2H),6.82–6.80(m,1H),6.72(s,1H),6.70(d,J＝7.7Hz,1H),6.42(dd,J＝3.4,1.1Hz,1H),5.37(s,2H),3.88(t,J＝6.5Hz,2H),2.46(s,3H),1.93(t,J＝7.4Hz,2H),1.64(p,J＝6.6Hz,2H),1.48(p,J＝7.5Hz,2H),1.35(p,J＝7.1Hz,2H),1.29–1.24(m,2H)。HRMS(ESI)C 24 H 29 N 6 O 4 + [M+H] + Calculated 465.2245, found 465.2242.
Example 50: preparation of 7- (3- ((6-amino-4- (3-cyanophenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenoxy) -N-hydroxyheptanamide (compound I-50)
Step 1: preparation of 7- (3- ((6-amino-4- (3-cyanophenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenoxy) heptanoate (intermediate Int-27)
The starting material compound C-8 is synthesized by reference (Gillespie, cliffe et al, bioorg Med Chem Lett.,2008,18,2924-2929). Methyl 7- (3- (bromomethyl) phenoxy) heptanoate (218 mg,1.12 mmol), K 2 CO 3 (206 mg,1.49 mmol) and Compound C-8 (150 mg,0.745 mmol) were dissolved in DMF (15 mL) and stirred at room temperature overnight. The reaction solution was diluted with water, extracted three times with ethyl acetate, and the organic phases were combined with anhydrous Na 2 SO 4 Drying once, evaporating the solvent under reduced pressure, and separating and purifying the residual solid by silica gel column chromatography to obtain white solid intermediate Int-27 (28 mg, yield 34%). 1 H NMR(800MHz,DMSO-d 6 )δ8.53(t,J＝1.7Hz,1H),8.49(dt,J＝8.0,1.4Hz,1H),8.42(s,1H),8.06(dt,J＝7.7,1.4Hz,1H),7.80(t,J＝7.8Hz,1H),7.21(t,J＝7.9Hz,1H),7.10(brs,2H),6.83–6.80(m,1H),6.76(t,J＝2.1Hz,1H),6.74(d,J＝7.9Hz,1H),5.42(s,2H),3.89(t,J＝6.4Hz,2H),3.56(s,3H),2.27(t,J＝7.4Hz,2H),1.67–1.62(m,2H),1.51(p,J＝7.5Hz,2H),1.38–1.33(m,2H),1.31–1.26(m,2H)。HRMS(ESI)C 27 H 29 N 6 O 3 + [M+H] + Calculated 485.2296, found 485.2291.
Step 2: preparation of 7- (3- ((6-amino-4- (3-cyanophenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) phenoxy) -N-hydroxyheptanamide (compound I-50)
The intermediate Int-1 in step 2 of example 1 was replaced with intermediate Int-27, and the remaining desired starting materials, reagents and preparation methods were the same as in step 2 of example 1, further purified by preparative HPLC to give the compound (I-50) as a yellow-white solid (0.007 g, 31% yield). 1 H NMR(800MHz,DMSO-d 6 )δ10.33(s,1H),9.20(s,1H),8.50(s,1H),8.46(dt,J＝7.9,1.4Hz,1H),8.40(t,J＝1.8Hz,1H),7.91(dt,J＝7.8,1.4Hz,1H),7.82(t,J＝7.8Hz,1H),7.22(t,J＝7.9Hz,1H),7.10(brs,2H),6.83(dd,J＝8.3,2.5Hz,1H),6.77(t,J＝2.1Hz,1H),6.74(d,J＝7.6Hz,1H),5.43(s,2H),3.90(t,J＝6.5Hz,2H),1.93(t,J＝7.4Hz,2H),1.65(p,J＝6.7Hz,2H),1.48(p,J＝7.5Hz,2H),1.39–1.33(m,2H),1.29–1.24(m,2H)。HRMS(ESI)C 26 H 28 N 7 O 3 + [M+H] + Calculated 485.2296, found 485.2291.
Example 51: preparation of 7- ((6- ((6-amino-4- (3-cyanophenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) pyridin-2-yl) oxy) -N-hydroxyheptanamide (compound I-51)
Step 1: preparation of methyl 7- ((6- ((6-amino-4- (3-cyanophenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) pyridin-2-yl) oxy) heptanoate (intermediate Int-28)
6-methylpyridin-2-ol (0.96 g,8.80 mmol), K 2 CO 3 (2.43 g,17.59 mmol), methyl 7-bromoheptanoate (2.36 g,10.56 mmol) and sodium iodide (200 mg,0.15 mmol) were dissolved in DMF (15 mL) and stirred at room temperature overnight. The reaction solution was diluted with water, extracted three times with ethyl acetate, and the organic phases were combined with anhydrous Na 2 SO 4 Drying once, evaporating the solvent under reduced pressure, and separating and purifying the residual solid by silica gel column chromatography to obtain colorless liquid intermediate Int-28a (1.317 g, yield 60%). 1 H NMR(800MHz,Chloroform-d)δ7.44(dd,J＝8.2,7.2Hz,1H),6.69(d,J＝7.2Hz,1H),6.50(d,J＝8.2Hz,1H),4.24(t,J＝6.6Hz,2H),3.66(s,3H),2.43(s,3H),2.32(t,J＝7.5Hz,2H),1.81–1.74(m,2H),1.66(p,J＝7.6Hz,2H),1.51–1.44(m,2H),1.42–1.37(m,2H)。HRMS(ESI)C 14 H 22 O 3 + [M+H] + Calculated 252.1594, found 252.1645. Int-28a (1.317 g,5.24 mmol), 1-bromopyrrolidine-2, 5-dione (1.165 g,6.29 mmol) and benzoic acid peroxy anhydride (66 mg,0.262 mmol) were dissolved in CCl 4 (30 mL) and refluxed overnight. The reaction solution was diluted with water, extracted three times with ethyl acetate, and the organic phases were combined with anhydrous Na 2 SO 4 Drying once, evaporating the solvent under reduced pressure, and separating and purifying the remaining solid by silica gel column chromatography to obtain colorless oily intermediate Int-28b (0.715 g, yield 33%). HRMS (ESI) C 14 H 21 BrO 3 + [M+H] + Calculated 330.0699, found 330.0700. Int-28b (0.470 g,1.44 mmol), K 2 CO 3 (351 mg,2.54 mmol) and 3- (6-amino-1H-pyrazolo [3, 4-d)]Pyrimidine-4 yl) benzonitrile (200 mg,0.846 mmol) was dissolved in DMF (15 mL) and stirred overnight at room temperature. The reaction solution was diluted with water, extracted three times with ethyl acetate, and the organic phases were combined with anhydrous Na 2 SO 4 Drying once, evaporating the solvent under reduced pressure, and separating and purifying the residual solid by silica gel column chromatography to obtain an off-white solid intermediate Int-28 (0.045 g, yield 11%). 1 H NMR(800MHz,DMSO-d 6 )δ8.55(t,J＝1.8Hz,1H),8.51(dt,J＝7.9,1.4Hz,1H),8.46(s,1H),8.07(dt,J＝7.7,1.4Hz,1H),7.80(t,J＝7.8Hz,1H),7.60(dd,J＝8.2,7.4Hz,1H),7.06(brs,2H),6.64(d,J＝8.2Hz,1H),6.56(d,J＝7.3Hz,1H),5.47(brs,2H),4.02(t,J＝6.8Hz,2H),3.57(s,3H),2.21(t,J＝7.4Hz,2H),1.52–1.48(m,2H),1.45–1.39(m,2H),1.21–1.16(m,4H)。HRMS(ESI)C 26 H 28 N 7 O 3 + [M+H] + Calculated 486.2248, found 486.2247.
Step 2: preparation of 7- ((6- ((6-amino-4- (3-cyanophenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) pyridin-2-yl) oxy) -N-hydroxyheptanamide (compound I-51)
The intermediate Int-1 in step 2 of example 1 was replaced with intermediate Int-28, and the remaining desired starting materials, reagents and preparation methods were the same as in step 2 of example 1, further purified by preparative HPLC to give the yellow-white solid compound (I-51) (0.003 g, yield 10%). 1 H NMR(800MHz,DMSO-d 6 )δ10.35(brs,1H),9.20(brs,1H),8.54(s,1H),8.47(dt,J＝7.8,1.4Hz,1H),8.42(t,J＝1.8Hz,1H),7.92(dt,J＝7.9,1.4Hz,1H),7.83(t,J＝7.8Hz,1H),7.61(dd,J＝8.2,7.4Hz,1H),7.09(brs,2H),6.66(d,J＝8.2Hz,1H),6.51(d,J＝7.4Hz,1H),5.47(s,2H),4.06(t,J＝6.7Hz,2H),1.91(t,J＝7.4Hz,2H),1.58–1.53(m,2H),1.46–1.40(m,2H),1.27–1.19(m,4H)。HRMS(ESI)C 25 H 27 N 8 O 3 + [M+H] + Calculated 487.2201, found 487.2199.
Example 52: preparation of 5- ((6- ((6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) pyridin-2-yl) oxy) -N-hydroxypentanamide (compound I-52)
Step 1: preparation of methyl 5- ((6- ((6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) pyridin-2-yl) oxy) pentanoate (intermediate Int-29)
The procedure of example 45, step 1, was followed except for substituting methyl 5-bromopentanoate for methyl 5- ((6- (bromomethyl) pyridin-2-yl) oxy) pentanoate to give intermediate Int-29 as a white solid (0.108 g, 30% yield). 1 H NMR(800MHz,DMSO-d 6 )δ8.31(s,1H),8.06(dd,J＝1.8,0.8Hz,1H),7.59(t,J＝7.8Hz,1H),7.45(dd,J＝3.5,0.8Hz,1H),6.91(brs,2H),6.79(dd,J＝3.5,1.7Hz,1H),6.65(d,J＝8.2Hz,1H),6.48(d,J＝7.4Hz,1H),5.43(s,2H),4.09(t,J＝6.4Hz,2H),3.56(s,3H),2.28(t,J＝7.3Hz,2H),1.60–1.56(m,2H),1.55–1.51(m,2H)。HRMS(ESI)C 21 H 23 N 6 O 4 + [M+H] + Calculated 423.1775, found 423.1779.
Step 2: preparation of 5- ((6- ((6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) pyridin-2-yl) oxy) -N-hydroxypentanamide (compound I-52)
The intermediate Int-1 in step 2 of example 1 was replaced with intermediate Int-29, and the remaining desired starting materials, reagents and preparation method were the same as in step 2 of example 1, further purified by preparative HPLC to give the compound (I-52) as a yellow-white solid (0.088 g, 88% yield). 1 H NMR(600MHz,DMSO-d 6 )δ10.35(s,1H),8.68(d,J＝1.7Hz,1H),8.32(s,1H),8.07(d,J＝1.8Hz,1H),7.59(dd,J＝8.3,7.4Hz,1H),7.45(dd,J＝3.6,0.7Hz,1H),6.92(brs,2H),6.80(dd,J＝3.5,1.7Hz,1H),6.66(d,J＝8.2Hz,1H),6.43(d,J＝7.4Hz,1H),5.42(s,2H),4.11(t,J＝6.3Hz,2H),1.96(t,J＝7.1Hz,2H),1.62–1.51(m,4H)。HRMS(ESI)C 20 H 22 N 7 O 4 + [M+H] + Calculated 424.1728, found 424.1723.
Example 53: preparation of 7- ((6- ((6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) pyridin-2-yl) oxy) -N-hydroxyheptanamide (compound I-53)
Step 1: preparation of methyl 7- ((6- ((6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) pyridin-2-yl) oxy) heptanoate (intermediate Int-30)
The procedure for preparation was as in example 45 step 1 except substituting methyl 5-bromopentanoate for methyl 7- ((6- (bromomethyl) pyridin-2-yl) oxy) heptanoate to give intermediate Int-30 (0.058 g, 26% yield) as a yellow solid. 1 H NMR(800MHz,DMSO-d 6 )δ8.30(s,1H),8.06(dd,J＝1.7,0.8Hz,1H),7.60(dd,J＝8.2,7.4Hz,1H),7.44(dd,J＝3.5,0.8Hz,1H),6.90(brs,2H),6.79(dd,J＝3.5,1.7Hz,1H),6.64(dd,J＝8.2,0.8Hz,1H),6.52(dd,J＝7.3,0.8Hz,1H),5.42(s,2H),4.04(t,J＝6.8Hz,2H),3.57(s,3H),2.24(t,J＝7.4Hz,2H),1.51(p,J＝7.3Hz,2H),1.45(p,J＝7.3Hz,2H),1.23–1.17(m,4H)。HRMS(ESI)C 23 H 27 N 6 O 4 + [M+H] + Calculated 451.2088, found 451.2086.
Step 2: preparation of 7- ((6- ((6-amino-4- (furan-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) methyl) pyridin-2-yl) oxy) -N-hydroxyheptanamide (compound I-53)
The intermediate Int-1 in step 2 of example 1 was replaced with intermediate Int-30, and the other desired raw materials, reagents and preparation method were the same as in step 2 of example 1 to give an off-white solid compound (I-53) (0.026 g, yield 52%). 1 H NMR(800MHz,DMSO-d 6 )δ10.32(s,1H),8.65(s,1H),8.31(s,1H),8.08–8.05(m,1H),7.59(t,J＝8.7Hz,1H),7.45(dd,J＝3.5,0.8Hz,1H),6.90(brs,2H),6.79(dd,J＝3.5,1.8Hz,1H),6.64(d,J＝8.2Hz,1H),6.49(d,J＝7.4Hz,1H),5.43(s,2H),4.04(t,J＝6.8Hz,2H),1.91(t,J＝7.4Hz,2H),1.53(p,J＝6.8Hz,2H),1.43(p,J＝7.4Hz,2H),1.26–1.17(m,4H)。HRMS(ESI)C 22 H 26 N 7 O 4 + [M+H] + Calculated 452.2041, found 452.2038.
Example 54: preparation of 7- (3- ((2-amino-6- (furan-2-yl) -9H-purin-9-yl) methyl) phenoxy) -N-hydroxyheptanamide (Compound I-54)
Step 1: preparation of methyl 7- (3- ((2-amino-6- (furan-2-yl) -9H-purin-9-yl) methyl) phenoxy) heptanoate (intermediate Int-31)
The test was performed by substituting methyl 7-bromoheptanoate in step 1 of example 7 with methyl 7- (3- (bromomethyl) phenoxy) heptanoate, the remaining required starting materialsThe preparation and preparation method were the same as in example 7, step 1, to give intermediate Int-31 (0.073 g, yield 28%) as a white solid. 1 H NMR(800MHz,DMSO-d 6 )δ8.21(s,1H),7.96(dd,J＝1.7,0.8Hz,1H),7.73(dd,J＝3.4,0.8Hz,1H),7.26–7.22(m,1H),6.84(tdd,J＝3.8,2.5,0.9Hz,2H),6.81(dt,J＝7.8,1.1Hz,1H),6.75(dd,J＝3.4,1.7Hz,1H),6.60(brs,2H),5.27(s,2H),3.91(t,J＝6.5Hz,2H),3.57(s,3H),2.29(t,J＝7.4Hz,2H),1.70–1.63(m,2H),1.53(p,J＝7.5Hz,2H),1.40–1.34(m,2H),1.32–1.27(m,2H)。HRMS(ESI)C 24 H 28 N 5 O 3 + [M+H] + Calculated 450.2136, found 450.2198.
Step 2: preparation of 7- (3- ((2-amino-6- (furan-2-yl) -9H-purin-9-yl) methyl) phenoxy) -N-hydroxyheptanamide (Compound I-54)
The intermediate Int-1 in step 2 of example 1 was replaced with intermediate Int-31, and the remaining required raw materials, reagents and preparation method were the same as in step 2 of example 1 to give compound (I-54) (0.050 g, yield 88%) as a white solid. 1 H NMR(800MHz,DMSO-d 6 )δ10.32(d,J＝1.8Hz,1H),8.65(d,J＝1.8Hz,1H),8.21(s,1H),7.96(d,J＝1.6Hz，1H)，7.72(d，J＝3.3Hz，1H)，7.27–7.20(m，1H)，6.84(d，J＝6.5Hz，2H)，6.80(d，J＝7.6Hz,1H),6.75(dd,J＝3.4,1.7Hz,1H),6.59(brs,2H),5.26(s,2H),3.91(t,J＝6.5Hz,2H),1.93(t,J＝7.4Hz,2H),1.66(p,J＝6.7Hz,2H),1.49(p,J＝7.5Hz,2H),1.40–1.33(m,2H),1.30–1.24(m,2H)。HRMS(ESI)C 23 H 27 N 6 O 4 + [M+H] + Calculated 451.2088, found 451.2082.
Example 55: preparation of 7- ((6- ((2-amino-6- (furan-2-yl) -9H-purin-9-yl) methyl) pyridin-2-yl) oxy) -N-hydroxyheptanamide (Compound I-55)
Step 1: preparation of methyl 7- ((6- ((2-amino-6- (furan-2-yl) -9H-purin-9-yl) methyl) pyridin-2-yl) oxy) heptanoate (intermediate Int-32)
The procedure for preparation and use of example 54, step 1, was repeated except for substituting methyl 7- (3- (bromomethyl) phenoxy) heptanoate for methyl 7- ((6- (bromomethyl) pyridin-2-yl) oxy) heptanoate to give intermediate Int-32 (0.129 g, 37% yield) as a yellow oil. 1 H NMR(800MHz,DMSO-d 6 )δ8.19(s,1H),7.96–7.92(m,1H),7.74(dd,J＝3.4,0.9Hz,1H),7.64(dd,J＝8.2,7.3Hz,1H),6.74(dd,J＝3.4,1.7Hz,1H),6.73–6.71(m,1H),6.66(d,J＝8.2Hz,1H),6.52(brs,2H),5.33(s,2H),4.04(t,J＝6.8Hz,2H),3.57(s,3H),2.23(t,J＝7.4Hz,2H),1.49(p,J＝6.9Hz,2H),1.44(p,J＝7.4Hz,2H),1.21–1.17(m,4H)。HRMS(ESI)C 23 H 27 N 6 O 4 + [M+H] + Calculated 451.2088, found 451.2088.
Step 2: preparation of 7- ((6- ((2-amino-6- (furan-2-yl) -9H-purin-9-yl) methyl) pyridin-2-yl) oxy) -N-hydroxyheptanamide (Compound I-55)
The intermediate Int-1 in step 2 of example 1 was replaced with intermediate Int-32, and the remaining desired starting materials, reagents and preparation method were the same as in step 2 of example 1, further purified by preparative HPLC to give compound (I-55) as a white solid (0.051 g, yield 41%). 1 H NMR(800MHz,DMSO-d 6 )δ10.34–10.30(m,1H),8.66(d,J＝1.8Hz,1H),8.20(s,1H),7.95(t,J＝1.2Hz,1H),7.74(d,J＝3.6Hz,1H),7.64(d,J＝7.9Hz,1H),6.75(dd,J＝3.4,1.7Hz,1H),6.70(d,J＝7.4Hz,1H),6.67(d,J＝8.2Hz,1H),6.52(brs,2H),5.33(s,2H),4.04(t,J＝6.8Hz,2H),1.90(t,J＝7.4Hz,2H),1.51(p,J＝6.9Hz,2H),1.42(p,J＝7.4Hz,2H),1.25–1.20(m,2H),1.20–1.16(m,2H)。HRMS(ESI)C 22 H 26 N 7 O 4 + [M+H] + Calculated 452.2041, found 452.2037.
Example 56: preparation of 7- (3- ((2-amino-6- (5-methylfuran-2-yl) -9H-purin-9-yl) methyl) phenoxy) -N-hydroxyheptanamide (Compound I-56)
Step 1: preparation of methyl 7- (3- ((2-amino-6- (5-methylfuran-2-yl) -9H-purin-9-yl) methyl) phenoxy) heptanoate (intermediate Int-33)
The procedure of example 22, step 1, was followed except for substituting methyl 7- (4- (bromomethyl) benzoate with methyl 7- (3- (bromomethyl) phenoxy) heptanoate and then substituting the other necessary materials, reagents and preparation method to obtain the yellow-white solid intermediate Int-33 (0.147 g, yield 46%). 1 H NMR(800MHz,DMSO-d 6 )δ8.17(s,1H),7.67(d,J＝3.3Hz,1H),7.24–7.21(m,1H),6.84–6.82(m,2H),6.80(dt,J＝7.8,1.2Hz,1H),6.57(brs,2H),6.37(dd,J＝3.3,1.0Hz,1H),5.25(s,2H),3.90(t,J＝6.5Hz,2H),3.57(s,3H),2.40(s,3H),2.28(t,J＝7.4Hz,2H),1.68–1.62(m,2H),1.54–1.49(m,2H),1.39–1.33(m,2H),1.31–1.26(m,2H)。HRMS(ESI)C 25 H 30 N 5 O 4 + [M+H] + Calculated 464.2292, found 464.2290.
Step 2: preparation of 7- (3- ((2-amino-6- (5-methylfuran-2-yl) -9H-purin-9-yl) methyl) phenoxy) -N-hydroxyheptanamide (Compound I-56)
The intermediate Int-1 in step 2 of example 1 was replaced with intermediate Int-33, and the remaining desired starting materials, reagents and preparation method were the same as in step 2 of example 1 to give compound (I-56) (0.129 g, yield 96%) as a white solid. 1 H NMR(800MHz,DMSO-d 6 )δ10.32(s,1H),8.65(s,1H),8.17(s,1H),7.66(d,J＝3.3Hz,1H),7.25–7.22(m,1H),6.85–6.82(m,2H),6.79(d,J＝7.6Hz,1H),6.56(brs,2H),6.38(dd,J＝3.3,1.2Hz,1H),5.25(s,2H),3.90(t,J＝6.5Hz,2H),2.40(s,3H),1.93(t,J＝7.4Hz,2H),1.65(p,J＝6.7Hz,2H),1.49(p,J＝7.5Hz,2H),1.39–1.34(m,2H),1.30–1.24(m,2H)。HRMS(ESI)C 24 H 29 N 6 O 4 + [M+H] + Calculated 465.2245, found 465.2249.
Example 57: preparation of 7- ((6- ((2-amino-6- (5-methylfuran-2-yl) -9H-purin-9-yl) methyl) pyridin-2-yl) oxy) -N-hydroxyheptanamide (Compound I-57)
Step 1: preparation of methyl 7- ((6- ((2-amino-6- (5-methylfuran-2-yl) -9H-purin-9-yl) methyl) pyridin-2-yl) oxy) heptanoate (intermediate Int-34)
The procedure for preparation and use of example 56, step 1, was repeated except for substituting methyl 7- (3- (bromomethyl) phenoxy) heptanoate for methyl 7- ((6- (bromomethyl) pyridin-2-yl) oxy) heptanoate to give Int-34 as an off-white solid intermediate (0.127 g, 42% yield). 1 H NMR(800MHz,DMSO-d 6 )δ8.16(s,1H),7.68(d,J＝3.3Hz,1H),7.64(t,J＝7.7Hz,1H),6.72(d,J＝7.3Hz,1H),6.66(d,J＝8.2Hz,1H),6.51(brs,2H),6.37(dd,J＝3.3,1.2Hz,1H),5.32(s,2H),4.04(t,J＝6.8Hz,2H),3.57(s,3H),2.40(s,3H),2.22(t,J＝7.5Hz,2H),1.49(p,J＝6.9Hz,2H),1.44(p,J＝7.4Hz,2H),1.23–1.15(m,4H)。HRMS(ESI)C 24 H 29 N 6 O 4 + [M+H] + Calculated 465.2245, found 465.2267.
Step 2: preparation of 7- ((6- ((2-amino-6- (5-methylfuran-2-yl) -9H-purin-9-yl) methyl) pyridin-2-yl) oxy) -N-hydroxyheptanamide (Compound I-57)
The intermediate Int-1 in the step 2 of the example 1 is replaced by the intermediate Int-34, and the rest required raw materials, reagents and preparation methods are the same as those in the step 2 of the example 1, so that yellow is obtainedSolid compound (I-57) (0.105 g, yield 80%). 1 H NMR(800MHz,DMSO-d 6 )δ10.32(s,1H),8.66(s,1H),8.17(s,1H),7.68(d,J＝3.2Hz,1H),7.64(t,J＝7.8Hz,1H),6.69(d,J＝7.3Hz,1H),6.67(d,J＝8.2Hz,1H),6.51(brs,2H),6.38(dd,J＝3.3,1.2Hz,1H),5.32(s,2H),4.05(t,J＝6.8Hz,2H),2.40(s,3H),1.91(t,J＝7.4Hz,2H),1.52(p,J＝6.9Hz,2H),1.43(p,J＝7.4Hz,2H),1.25–1.17(m,4H)。HRMS(ESI)C 23 H 28 N 7 O 4 + [M+H] + Calculated 466.2197, found 466.2183.
Example 58: preparation of 7- (3- ((2-amino-4- (furan-2-yl) -7H-pyrrolo [2,3-d ] pyrimidin-7-yl) methyl) phenoxy) -N-hydroxyheptanamide (compound I-58)
Step 1: preparation of methyl 7- (3- ((2-amino-4- (furan-2-yl) -7H-pyrrolo [2,3-d ] pyrimidin-7-yl) methyl) phenoxy) heptanoate (intermediate Int-35)
The procedure of example 19, step 1, was followed except for substituting methyl 4- (bromomethyl) benzoate with methyl 7- (3- (bromomethyl) phenoxy) heptanoate and then substituting the other desired starting materials, reagents and preparation method to give Int-35 (0.061 g, yield 18%) as a yellow solid. 1 H NMR(800MHz,DMSO-d 6 )δ7.96(d,J＝1.8Hz,1H),7.26(d,J＝3.4Hz,1H),7.22–7.19(m,2H),6.80(dd,J＝8.3,2.3Hz,1H),6.75–6.72(m,3H),6.72(dd,J＝3.4,1.7Hz,1H),6.27(brs,2H),5.23(s,2H),3.88(t,J＝6.5Hz,2H),3.56(s,3H),2.28(t,J＝7.4Hz,2H),1.64(p,J＝6.6Hz,2H),1.51(p,J＝7.4Hz,2H),1.38–1.33(m,2H),1.30–1.26(m,2H)。HRMS(ESI)C 25 H 29 N 4 O 4 + [M+H] + Calculated 449.2183, found 449.2185.
Step 2: preparation of 7- (3- ((2-amino-4- (furan-2-yl) -7H-pyrrolo [2,3-d ] pyrimidin-7-yl) methyl) phenoxy) -N-hydroxyheptanamide (compound I-58)
The intermediate Int-1 in step 2 of example 1 was replaced with intermediate Int-35, and the remaining desired starting materials, reagents and preparation method were the same as in step 2 of example 1 to give compound (I-58) (0.034 g, 53% yield) as a yellow solid. 1 H NMR(800MHz,DMSO-d 6 )δ10.32(s,1H),8.65(s,1H),7.96(d,J＝1.7Hz,1H),7.26(d,J＝3.4Hz,1H),7.22–7.19(m,2H),6.81(dd,J＝8.1,2.5Hz,1H),6.76–6.70(m,4H),6.27(brs,2H),5.23(s,2H),3.88(t,J＝6.5Hz,2H),1.93(t,J＝7.4Hz,2H),1.67–1.62(m,2H),1.51–1.46(m,2H),1.38–1.33(m,2H),1.29–1.23(m,2H)。HRMS(ESI)C 24 H 28 N 5 O 4 + [M+H] + Calculated 450.2136, found 450.2134.
The compounds listed in examples 59-66 can be prepared in the same manner by changing the corresponding starting materials according to the methods listed in examples 45-58, as detailed in Table 3.
TABLE 3 Table 3
Biological test example 1: assay of histone deacetylase HDAC inhibitory activity by compounds. The specific operation method is as follows:
(1) Preparing a buffer solution (50mM Tris PH,0.01% Tween-20,50mM NaCl) for experiments;
(2) Preparing a compound to be tested into a corresponding DMSO solution with the concentration of 10mM, diluting the solution to 1mM by using DMSO, and carrying out gradient dilution by 3 times, wherein the concentration points are 10;
(3) Different concentrations of test compounds were transferred to 384 well plates (Perkin Elmer, cat.no. 6007279) with Echo, 250nL per well (final DMSO content of 1%);
(4) Preparing a solution of histone deacetylase with the buffer in step (1) at a final concentration of 4nM for HDAC1 (BPS bioscience, cat. No. 50051) and 5nM for HDAC6 (BPS bioscience, cat. No. 50056);
(5) Preparing a mixed solution of a substrate (LGK (Ac) -AMC, trypsin) with the buffer in step (1), for the determination of HDAC1 activity: LGK (Ac) -AMC (gil biochemistry) concentration was 8 μm, trypsin concentration was 0.05 μm, for the determination of HDAC6 activity: LGK (Ac) -AMC final concentration 11 μm, trypsin final concentration 0.01 μm;
(6) Add 15. Mu.L of the enzyme solution prepared in step (4) to each well in the 384 well plate tested, add 15. Mu.L of the buffer solution in step (1) to the low control group, centrifuge at 1000rmp for 1 min, then incubate at room temperature for 15 min;
(7) 10. Mu.L of the enzyme solution prepared in step (5) was added to each well of the 384-well plate for testing, centrifuged at 1000rmp for 1 minute, and then incubated at room temperature for 60 minutes;
(8) Reading the value with Synergy MX (maximum excitation light: 355nm, maximum emission light 460 nm);
(9) Data were processed with GraphPad Prism5 to calculate IC 50 Values, results are shown in table 4.
The inhibitory activity of the compounds of table 4 on HDAC 1.
Biological test example 2: determination of A2A receptor binding Activity of the compounds of the invention. The compounds were tested for binding activity to human A2A receptor using a radioisotope ligand-based competitive binding assay. The specific operation method is as follows:
(1) Test compounds were prepared as corresponding 10mM DMSO solutions. Then diluting to 10 mu M by using a buffer solution, and then carrying out gradient dilution by using the buffer solution by 3 times, wherein the concentration points are 10;
(2) Different concentrations of the test compound were transferred to 384 well plates with Echo, 50nL per well;
(3) A suspension of human A2A receptor cell membranes (RBHA 2AM400UA; perkin Elmer) and wheat germ lectin coated yttrium silicate SPA beads (RPNQ 0023; perkin Elmer) was prepared: (0.0334 mg/mL A2a cell membrane, 3.33mg/mL SPA beads, 0.02mg/mL ADA, assay buffer containing 1 XDPBS, 10mM MgCl) 2 1% dmso), room temperature for 20 minutes;
(4) Add 20. Mu.L to each well 3 Solutions of H SCH58261 (ART 2128; ARC) (15 nM SCH58261, assay buffer containing 1 XDBS, 10mM MgCl) 2 1% DMSO), 1000rmp for 1 min;
(5) Add 30. Mu. L A2A cell membrane/SPA bead suspension to each well, centrifuge for 1 min at 1000rmp, seal the plate and incubate at room temperature with continued shaking for 60 min;
(6) CPM values were read with Microbeta 2 (Perkin Elmer);
(7) Data were processed with GraphPad Prism 5 to calculate IC 50 The value is given to K i The results of the values are shown in Table 5.
TABLE 5 results of the competitive binding strength of example compounds to radioligands for the A2A receptor
Biological test example 3: test of tumor cell proliferation inhibition activity of the compounds of the present invention. The activity of the compound for inhibiting tumor cell proliferation is determined by using MC-38 and CT-26 cells.
(1) Cell plating
a. Preparing a complete culture medium, and fully and uniformly mixing.
b. Selecting a cell line with good growth state.
c. The cell culture flask was removed from the incubator, and the cell name, medium type and cell number marked on the flask were checked.
d. The cell suspension was pipetted into a centrifuge tube and centrifuged at 800-1000rmp for 3-5 minutes.
e. The cell supernatant in the centrifuge tube is sucked and removed, a proper volume of culture medium is added into the centrifuge tube, and the cells are gently beaten to be resuspended uniformly.
f. The Cell suspension was brought to the appropriate concentration using a Vi-Cell XR cytometer.
g. The cell suspension was added to a bottom wall white 384 well plate at 36 μl/well. Labeling detailed information such as cell name, plate density, date, etc., placing the culture plate in CO 2 The incubator was left overnight.
(2) Cell experiment:
a. test compounds were made 200× with DMSO, and compounds were diluted 3-fold with DMSO to give 10 concentration gradients of compounds.
b. After 24 hours of cell plating, 1. Mu.L of the compound was added to 19. Mu.L of the medium to prepare a 10X intermediate plate, and then 4. Mu.L of the 10X corresponding compound was added to each well, followed by incubation in an incubator at 37℃for 72 hours.
c. Cell morphology was observed under an inverted microscope.
d. The cell culture plates were left to equilibrate at room temperature for 30 minutes, 25 μl of CTG was added to each well, and then mixed on a plate shaker for 10 minutes to induce cell lysis.
e. The 384 well plate was left at room temperature for 10 minutes to stabilize the luminescence signal, and then a white bottom film was attached to the bottom of the plate using a Flexstation 3 plate (related settings: luminescence, integration time 500 ms).
f. The results of the experiment obtained by the analysis were recorded and are shown in table 6.
Experimental results of inhibition of tumor cell proliferation by the compounds of table 6
Biological test example 4: pharmacokinetic property test of the Compounds of the invention administered to C57 Male mice in a single pass by gastric lavage and intravenous injection
(1) Purpose of experiment
After the compound is administrated in a single dose in a C57 male mouse, blood samples are collected at different time points, the concentration of the compound in the plasma of the mouse is measured by LC-MS/MS, relevant pharmacokinetic parameters are calculated, and the pharmacokinetic condition of the compound in the mouse is examined.
(2) Design of experiment
Male C57 mice, 3 in each group, were supplied by Suzhou Zhaoyan laboratory animals, inc., and the experiments were performed according to the dosing regimen of Table 3 below.
TABLE 3 dosing regimen of compounds
(3) Sample collection
Each animal was anticoagulated with 0.030mL of blood per orbital procedure, EDTA-K2, at the time point of collection: 0,5,15,30min,1,2,4,6,8,24h after administration of the test agent. Blood samples were collected and placed on ice and the plasma was centrifuged within 30 minutes (centrifugation conditions: 5000 rpm, 10 minutes, 4 ℃). Stored at-80 ℃ prior to analysis.
(4) Data processing
The data acquisition and control system software is Analyst1.5.1 software (Applied Biosystem). The peak integration mode of the map sample is automatic integration; regression was performed using the ratio of the peak area of the sample to the peak area of the internal standard as an index, and the concentration of the sample. Regression mode: linear regression, weight coefficient 1/X2. Pharmacokinetic parameters were analyzed using a non-compartmental model using WinNonlin Professional v 6.3.6.3 (Pharsight, USA). Cmax is the measured maximum blood concentration, the area under the blood concentration-time curve AUC (0- > t) is calculated by a trapezoidal method, and Tmax is the peak time of the blood concentration after administration. Experimental data are expressed as "Mean ± standard deviation" (Mean ± ICR, n.
(5) Experimental results
The pharmacokinetic results of the compounds of the invention are shown in the following table. From this, it can be seen that the compound has good pharmacokinetic properties in C57 male mice. See in particular tables 4 and 5
TABLE 4 oral parameters for Compounds I-14
Remarks: "-" means not applicable.
Biological test example 4: inhibitory Activity of Compound I-14 on growth of mouse MC38 colon cancer subcutaneous transplantation tumor
(1) Experimental animal
C57BL/6 mice, 6-8 weeks, male, purchased from Fukang Biotech Co., ltd. The use and welfare of the experimental animals were carried out in compliance with the regulations of the international commission on assessment and approval of experimental animals (AAALAC). The health condition and death of animals are monitored every day, and routine examination includes observing the influence of test substances and medicines on the daily behavior of animals, such as behavior activity, weight change, appearance signs and the like.
(2) Experimental procedure
Mouse MC38 colon cancer cells were cultured at 5.0X10 5 Cells/mice were inoculated subcutaneously into the right armpit of the mice, respectively. Mice were monitored daily and vernier caliper measurements were started when tumors became visible. The width (W) and length (L) of each tumor were measured by calipers using the equation v= (l×w2)/2. When the average tumor volume is up to 100mm 3 After that, mice were randomly grouped. Compound I-14 (30 mg/kg, bid;60mg/kg, bid) was intraperitoneally administered as I-14 (90 mg/kg, bid;120mg/kg, qd), respectively. The control group was given an equal amount of solvent daily for intraperitoneal injection or lavage. Tumor volumes and mouse body weights were recorded every two days. Relative tumor volume rtv=vt/V0, vt refers to the tumor volume on the day of measurement, V0 refers to the initial tumor volume prior to administration. The evaluation index of tumor inhibition is relative tumor growth rate: T/C (%) = (average tumor growth volume of treated group/average tumor growth volume of control group) ×100%.
(3) Experimental results
The compound I-14 of the embodiment of the invention has an inhibition effect on the growth of mouse MC38 colon cancer subcutaneous transplantation tumor. As shown in FIG. 1, the relative tumor growth rates T/C (%) of I-14 (90 mg/kg, bid;120mg/kg, qd) given by gastric lavage were 56% and 74% (P < 0.05), respectively, and there was no significant difference in body weight gain among the groups of mice; in fig. 1, P <0.05. As shown in FIG. 2, the relative tumor growth rates T/C (%) of the intraperitoneally administered compound I-14 (30 mg/kg, bid;60mg/kg, bid) were 32% and 15% (P < 0.01), respectively, and are shown as P <0.01 in FIG. 2.

Claims (11)

1. A compound of formula I, a pharmaceutically acceptable salt thereof, a tautomer thereof, or a solvate thereof:
Wherein A and B are independently CH or N;
R 1 and R is 2 Independently H or C 1 -C 6 An alkyl group;
R 3 is a 5-10 membered heteroaryl or is substituted with one or more R 3-1 Substituted 5-10 membered heteroaryl; in the heteroaryl, the number of the heteroatoms is 1, 2 or 3, and the heteroatoms are selected from one, two or three of N, O and S;
l is-C 1 -C 10 Alkylene-, -C 1 -C 10 alkylene-C 6 -C 10 Arylene-, -C 1 -C 10 alkylene-C 6 -C 10 arylene-C 2 -C 4 alkenylene-C 1 -C 10 alkylene-O-C 6 -C 10 Arylene-, -C 1 -C 10 alkylene-C 6 -C 10 arylene-O-C 1 -C 10 Alkylene radical,or-C 1 -C 10 Alkylene-5-10 membered heteroarylene-O-C 1 -C 10 Alkylene-; the number of hetero atoms in the 5-10 membered heteroarylene is 1, 2 or 3, and the hetero atoms are selected from N, O and SOne, two or three of the following materials; wherein the band "# end is connected with the ZBG;
ZBG is
R 3-1 Independently cyano or C 1 -C 6 An alkyl group;
L 1 、L 2 independently-C 1 -C 10 Alkylene or-O-C 1 -C 10 An alkylene group;
R 6 independently hydrogen or halogen;
m is 1, 2, 3 or 4.
2. A compound of formula I, a pharmaceutically acceptable salt thereof, a tautomer thereof, or a solvate thereof, according to claim 1, which satisfies one or more of the following conditions:
(1)R 3 wherein the heteroaryl is independently a 5-6 membered heteroaryl; the hetero atom in the heteroaryl is independently preferably O, and the number of the hetero atom is independently preferably 1 or 2; the heteroaryl groups are independently preferably furyl groups, and further independently preferably furyl groups
(2)R 1 、R 2 And R is 3-1 In (C) 1 -C 6 Alkyl is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl;
(3)L、R 4 and R is 5 In (C) 1 -C 10 Alkylene is independently C 1 -C 7 Alkylene, preferably methylene, ethylene, propylene, butylene, pentylene, hexylene or heptylene, further preferably methylene, n-ethylene, n-propylene, n-butylene, n-pentylene, n-hexylene or n-heptylene;
(4) In L, said C 6 -C 10 Arylene groupIndependently phenylene, preferably(5) In L, said C 2 -C 4 Alkenylene is independently vinylidene, preferably +.>
(6) In L, the 5-10 membered heteroarylene is a 5-6 membered heteroarylene; the hetero atom in the heteroarylene group is preferably N, and the number of the hetero atom is preferably 1 or 2; the 5-to 6-membered heteroarylene group is preferably a pyridyl group, more preferably
(7)R 6 Wherein the halogen is fluorine, chlorine, bromine or iodine.
3. A compound of formula I, a pharmaceutically acceptable salt thereof, a tautomer thereof, or a solvate thereof, according to claim 2, which satisfies one or more of the following conditions:
(1) L is
(2)R 1 And R is 2 Is H;
(3)R 3 is that
(4) The compound shown in the formula I is a substituted pyrimidine compound shown in the formula I-1, a substituted pyrimidine compound shown in the formula I-2, a substituted pyrimidine compound shown in the formula I-3 or a substituted pyrimidine compound shown in the formula I-4;
(5) In ZBG, theIs->
4. A compound of formula I, a pharmaceutically acceptable salt thereof, a tautomer thereof, or a solvate thereof, as claimed in claim 3, wherein the compound of formula I is any one of the following:
5. a pharmaceutical composition comprising a compound of formula I, a pharmaceutically acceptable salt thereof, a tautomer thereof, or a solvate thereof, as claimed in any one of claims 1 to 4, and at least one pharmaceutically acceptable adjuvant.
6. Use of a compound according to any one of claims 1 to 4, a pharmaceutically acceptable salt thereof, a tautomer thereof or a solvate thereof according to formula I or a pharmaceutical composition according to claim 5 for the preparation of an adenosine A2A receptor antagonist and/or a histone deacetylase HDAC inhibitor.
7. Use of a compound of formula I, a pharmaceutically acceptable salt thereof, a tautomer thereof or a solvate thereof, according to any one of claims 1 to 4, or a pharmaceutical composition according to claim 5, for the manufacture of a medicament for the treatment and/or prophylaxis of a disease.
8. The use according to claim 7, wherein the disease is cancer or a central nervous system disorder; the cancer can be head and neck cancer, respiratory system cancer, digestive system cancer, urinary system cancer, bone cancer, gynecological cancer, blood system cancer, melanoma, glioma or skin cancer; the central nervous system disorder may be Parkinson's disease, alzheimer's disease or Huntington's disease.
9. A preparation method of a compound shown in a formula I comprises the following steps of carrying out the following reaction on a compound shown in a formula II in an organic solvent to obtain the compound shown in the formula I,
therein, A, B, R 1 、R 2 、R 3 L and ZBG are as described in any one of claims 1-4; r is R a Is H or C 1 -C 6 An alkyl group.
10. A compound of the formula (II),
therein, A, B, R 1 、R 2 、R 3 L and R a Is defined as set forth in claim 9.
11. The compound of claim 10, wherein the compound of formula II is the following compound:
CN202310075494.9A 2022-02-14 2023-01-18 Pyrimidine ring compound, preparation method and application thereof Pending CN116589464A (en)

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