Patent Application
20220378816
The present application is based on and claims the benefit of priority from Chinese application No. 201910542124.5, filed on Jun. 21, 2019, the disclosures of which are incorporated herein by reference in its entirety.
The present application belongs to the technical field of medicine, specifically relates to a 2-benzylidene hydrazinoadenosine compound having A2A adenosine receptor agonistic activity and a pharmaceutical composition containing the same. These compounds and compositions can be used as a medicament.
For drugs for the treatment of central nervous system diseases, one of the main reasons for the failure of their development and related research lies in the obstruction of the blood-brain barrier (BBB), which prevents a drug from being delivered to the central nervous system and accumulating in the brain to reach an effective dose to produce a corresponding therapeutic effect. Therefore, a key factor for the successful development of drugs that target the center nervous system is to overcome the blood-brain barrier. Research on methods of opening the blood-brain barrier has become a hot spot for intracerebral drug delivery. Drug delivery across the blood-brain barrier has been a challenging research field in the past few decades. Researchers have made considerable efforts to develop various drug delivery systems, and a series of strategic studies have revealed that the delivery of drugs and contrast agents across the blood-brain barrier are very difficult. However, for the treatment of central nervous system diseases, such as brain tumors, strokes, trauma and neurodegenerative diseases, as well as nerve agent poisoning, the demand for effective therapeutic drugs has increased dramatically, and the design of drugs that can penetrate the blood-brain barrier is particularly important. Therefore, it is necessary to develop an efficient blood-brain barrier disruption (BBBD) strategy, which has less nerve damage, can deliver drugs of larger molecular weight, and has better pharmacokinetic characteristics.
The BBB restricts the entry of molecules into the brain through two main structural features. First of all, the tight junctions (TJs) structure seals endothelial cells, resulting in low permeability of blood molecules through the BBB. On the other hand, compared with peripheral vascular endothelial cells, there are few transport pathways between cerebral vascular endothelial cells, but the expression level of active efflux transporters, such as P glycoprotein (P-gp) on brain capillary endothelial cells (BCECs), is very high. Considering the key role of TJs in restricting the entry of molecules into the brain (HUBER J D et al., Trends Neurosci, 2001, 24(12): 719-25), reversibly changing the tightness of TJs may be a feasible way to up-regulate the permeability of BBB. Temporary opening of TJs is a feasible way to deliver drugs into the brain because of the high passing efficiency and less molecular weight restriction for therapeutic drugs. Bynoe et al. recently demonstrated that the specific activation of A2A adenosine receptor (A2AAR) on BCECs of mouse can promote the absorption of drug in the brain (CARMAN AJ et al., J Neurosci, 2011, 31(37): 13272-80). Further studies have shown that activation of A2A adenosine receptor can up-regulate BBB permeability and temporarily increase the intercellular space of brain capillary endothelial cells. Studies have shown that the A2AAR signaling pathway modulates cytoskeletal elements by regulating intracellular actin, resulting in cell morphology contraction, destruction of TJs integrity, and increase of barrier permeability (SOHAIL MA et al., Hepatology, 2009, 49(1): 185-94). Therefore, these studies have greatly expanded the potential application fields and development space of A2AAR agonists. The development of efficient A2AAR agonists is of great significance to the study of strategies for the blood-brain barrier disruption (patent application: CN200980117596.0).
At the same time, due to the widespread distribution of A2AAR in the human body, A2AAR agonists are recommended for the treatment of various pathological diseases. Adenosine mediates A2AAR to produce potential immunosuppressive and blood pressure lowering effects. One of the main potential therapeutic effects of A2AAR agonists is anti-inflammatory and immunosuppressive effects by regulating the activity of neutrophils, macrophages and T lymphocytes (DE LERA RUIZ M et al., J Med Chem, 2014, 57(9): 3623-50; VARANI K et al., FASEB J, 2010, 24(4): 1192-204). From the perspective of cell signaling pathways, the activation of A2A adenosine receptors reduces the NF-kB pathway, reduces inflammatory cytokines such as tumor necrosis factor α (TNF-α) and Interleukin-1 Beta (IL-1β), IL-8, IL-6, and inhibits the release of matrix metalloproteinase-1 (MMP-1) and MMP-3 (HASKO G, etc., Nat Rev Drug Discov, 2008, 7(9): 759-70). Therefore, selective agonists have been developed to treat related diseases, such as allergic rhinitis, asthma, and chronic obstructive pulmonary disease. However, the systematic use of A2AAR agonist for anti-inflammatory drugs is limited, because when the activation of A2AAR produces anti-inflammatory effects, it stimulates the heart and blood vessels to cause potent hypotensive activity. On the other hand, A2AAR agonists are powerful vasodilators and have been used as diagnostic reagents for cardiac pharmacologic stress tests (patent application: CN200580033215.2). Although A2AAR agonists as powerful vasodilators can produce systemic side effects, it is reported that low doses may not produce significant cardiovascular side effects. In addition, further potential therapeutic applications of A2AAR agonists are the treatment of psychosis and Huntington's disease (AKKARI R et al., Curr Top Med Chem, 2006, 6(13): 1375-99; BOSCH MP et al., J Med Chem, 2004, 47(16): 4041-53). A2AAR agonists have been shown to have neuroprotective effects on neurodegenerative disease models by reducing the release of excitatory neurotransmitters, apoptosis and inflammation (MULLER C E et al., Biochim Biophys Acta, 2011, 1808(5): 1290-308; RIVERA-OLIVER M, etc., Life Sci, 2014, 101(1-2): 1-9).
Although the aforementioned A2AAR agonists have been increasingly developed, only one receptor agonist, Regadenoson (an adenosine analog), is approved as a coronary vasodilator in the United States. Regadenoson is a selective A2A adenosine receptor agonist jointly developed by CV Therapeutics and Astellas, which has been marketed in the United States and Europe. It is mainly used as a coronary vasodilator for myocardial perfusion imaging. Therefore, there is still a need in the art for novel, effective A2A receptor agonists that optionally have one or more physiological and/or physicochemical advantages, and it is important to further synthesize and test other A2A receptor agonists in order to develop new and improved therapeutic agents.
The purpose of the present application is to find and develop a new type of small molecule agonist acting on A2A adenosine receptor, which can agonize A2A adenosine receptor, so that on the one hand, the purpose for the prevention or treatment of a human pathological condition or symptom is achieved, in which the prevention or treatment of a human pathological condition or symptom involves the activity of A2A adenosine receptor and the activation of this activity is required; on the other hand, the purpose of increasing the permeability of blood-brain barrier of the subject receiving the therapeutic drug is also achieved. In the present application, it has been found through research that the compound with the following general Formula I can act on A2A adenosine receptor and is an A2A adenosine receptor agonist, so it can be used for the above two purposes. The present application is completed based on the above findings.
Therefore, the first aspect of the present application provides a compound represented by the general Formula (I), a stereoisomer, a pharmaceutically acceptable salt, a pharmaceutically acceptable hydrate or solvate, or a pharmaceutically acceptable ester thereof,
wherein,
n is 1, 2, 3, 4 or 5;
R represents a substituent attached to the benzene ring, and each R is independently selected from the group consisting of hydrogen, halogen, cyano, benzyloxy, halogenated benzyloxy, C1-6 alkyl, halogenated C1- 6 alkyl, C1-6 alkoxy, hydroxyl, C1-6 alkylamino, di(C1-6 alkyl)amino, anilino, diphenylamino, phenylamino,-NHC(O)R10, aryl, heteroaryl, cycloalkyl, and heterocycloalkyl, where R10 is C1-6 alkyl.
The second aspect of the present application provides a method for preparing the compound represented by the general Formula (I), a stereoisomer, a pharmaceutically acceptable salt, a pharmaceutically acceptable hydrate or solvate, or a pharmaceutically acceptable ester as described in the first aspect of the present application, comprising:
In some embodiments, in the method described in the second aspect of the present application, the compound represented by Formula V reacts with the substituted benzaldehyde represented by Formula VI in a methanol solution under microwave heating at 70° C. to 90° C.
In some embodiments, in the method described in the second aspect of the present application, the compound represented by Formula V is produced by the hydrazinolysis of a compound represented by Formula IV with hydrazine hydrate at 40° C. to 60° C.
In some embodiments, in the method described in the second aspect of the present application, the compound represented by Formula IV is produced by the ammonolysis of a compound represented by Formula III in a solution of ammonia in methanol at 90° C. to 110° C.
In some embodiments, in the method described in the second aspect of the present application, the compound represented by Formula III is produced by a substitution reaction of a compound represented by Formula VII with a compound represented by Formula II in the presence of tin tetrachloride as a catalyst at 110° C. to 130° C.
The third aspect of the present application provides a pharmaceutical composition, which comprises at least one of the compound, a stereoisomer, a pharmaceutically acceptable salt, a pharmaceutically acceptable hydrate or solvate, or a pharmaceutically acceptable ester thereof as described in the first aspect of the present application, and one or more pharmaceutically acceptable carriers or excipients.
The fourth aspect of the present application provides use of the compound, a stereoisomer, a pharmaceutically acceptable salt, a pharmaceutically acceptable hydrate or solvate, or a pharmaceutically acceptable ester thereof as described in the first aspect of the present application, or the pharmaceutical composition as described in the third aspect of the present application in the manufacture of a medicament as an A2A adenosine receptor agonist, or in the manufacture of a medicament for the prevention and/or treatment of a human pathological condition or symptom, wherein the human pathological condition or symptom is related to the activity of A2A adenosine receptor, and the prevention or treatment of the human pathological condition or symptom requires the activation of A2A adenosine receptor.
According to some embodiments of the present application, the human pathological condition or symptom described in the present application is selected from the group consisting of: autoimmune irritation, inflammation, allergic disease, skin disease, infectious disease, wasting disease, neuropathic pain, open trauma, adverse reaction caused by drug therapy, cardiovascular disease, ischemia-reperfusion injury, gout, chemical trauma, thermal trauma, diabetic nephropathy, sickle cell disease, laminitis, foundrymen's disease, glaucoma, ocular hypertension, spinal cord injury, myocardial infarction, and acute myocardial infarction.
The fifth aspect of the present application provides use of the compound, a stereoisomer, a pharmaceutically acceptable salt, a pharmaceutically acceptable hydrate or solvate, or a pharmaceutically acceptable ester thereof as described in the first aspect of the present application, or the pharmaceutical composition as described in the third aspect of the present application in the manufacture of a medicament for diagnosing a human abnormal myocardial perfusion, or in manufacture of a medicament as a coronary vasodilator.
The sixth aspect of the present application provides use of the compound, a stereoisomer, a pharmaceutically acceptable salt, a pharmaceutically acceptable hydrate or solvate, or a pharmaceutically acceptable ester thereof as described in the first aspect of the present application, or the pharmaceutical composition as described in the third aspect of the present application in the manufacture of a medicament for increasing the permeability of blood-brain barrier of a subject receiving a therapeutic drug, wherein the subject benefits from the increased permeability of blood-brain barrier for delivering the therapeutic drug across the blood-brain barrier.
According to some embodiments of the present application, in the use described in the sixth aspect of the present application, the therapeutic drug is selected from the group consisting of: drug for treating disease or disorder of central nervous system, antidote to nerve agent, and drug for treating glioma.
The seventh aspect of the present application provides a pharmaceutical composition, which comprises:
at least one of the compound, a stereoisomer, a pharmaceutically acceptable salt, or a pharmaceutically acceptable hydrate or solvate as described in the first aspect of the present application, and
a drug that needs to cross blood-brain barrier, which is selected from drug for treating disease or disorder of central nervous system, antidote to nerve agent, drug for treating glioma; and
one or more pharmaceutically acceptable carriers or excipients.
The eighth aspect of the present application provides a method for preventing and/or treating a human pathological condition or symptom, comprising administering to a patient in need thereof a prophylactically and/or therapeutically effective amount of the compound , a stereoisomer, a pharmaceutically acceptable salt, a pharmaceutically acceptable hydrate or solvate, or a pharmaceutically acceptable ester thereof as described in the first aspect of the present application, or the pharmaceutical composition as described in the third aspect of the present application, wherein the human pathological condition or symptom is related to the activity of A2A adenosine receptor, and the prevention or treatment of the human pathological condition or symptom requires the activation of A2A adenosine receptor.
The ninth aspect of the present application provides the compound represented by the general Formula (I), a stereoisomer, a pharmaceutically acceptable salt, a pharmaceutically acceptable hydrate or solvate, or a pharmaceutically acceptable ester thereof as described in the first aspect of the present application, for use in the prevention and/or treatment of a human pathological condition or symptom, wherein the human pathological condition or symptom is related to the activity of A2A adenosine receptor, and the prevention or treatment of the human pathological or symptom requires the activation of A2A adenosine receptor.
The tenth aspect of the present application provides the compound represented by the general Formula (I), a stereoisomer, a pharmaceutically acceptable salt, a pharmaceutically acceptable hydrate or solvate, or a pharmaceutically acceptable ester thereof as described in the first aspect of the present application, for use as an A2A adenosine receptor agonist or coronary vasodilator, or
for use in diagnosing a human abnormal myocardial perfusion, or
for use in increasing the permeability of blood-brain barrier of a subject receiving a therapeutic drug, and the subject benefits from the increased permeability of blood-brain barrier for delivering the therapeutic drug across the blood-brain barrier,
Preferably, the therapeutic drug is selected from the group consisting of: drug for treating disease or disorder of central nervous system, antidote to nerve agent, and drug for treating glioma.
The eleventh aspect of the present application also provides a method for diagnosing human abnormal myocardial perfusion, comprising administering a patient in need thereof a diagnostically effective amount of the compound, a stereoisomer, a pharmaceutically acceptable salt, a pharmaceutically acceptable hydrate or solvate, or a pharmaceutically acceptable ester thereof as described in the first aspect of the present application, or the pharmaceutical composition as described in the third aspect of the present application.
The twelfth aspect of the present application also provides a method for increasing the permeability of blood-brain barrier of a subject receiving a therapeutic drug, wherein the method comprises administering to the subject an effective amount of the compound, a stereoisomer, a pharmaceutically acceptable salt, a pharmaceutically acceptable hydrate or solvate, or a pharmaceutically acceptable ester thereof as described in the first aspect of the present application, or the pharmaceutical composition as described in the third aspect of the present application, wherein the subject benefits from the increased permeability of blood-brain barrier for delivering the therapeutic drug cross the blood-brain barrier.
According to some embodiments of the present application, in the method described in the twelfth aspect of the present application, the therapeutic drug is selected from the group consisting of: drug for treating disease or disorder of central nervous system, antidote to nerve agent, and drug for treating glioma.
According to some embodiments of the present application, the human pathological condition or symptom described in the present application is selected from the group consisting of: autoimmune irritation, inflammation, allergic disease, skin disease, infectious disease, wasting disease, neuropathic pain, open trauma, adverse reaction caused by drug therapy, cardiovascular disease, ischemia-reperfusion injury, gout, chemical trauma, thermal trauma, diabetic nephropathy, sickle cell disease, laminitis, foundrymen's disease, glaucoma, ocular hypertension, spinal cord injury, myocardial infarction, and acute myocardial infarction.
According to some embodiments of the present application, n is 1, 2 or 3 in the general Formula (I).
According to some embodiments of the present application, n is 1 in the general Formula (I).
According to some embodiments of the present application, n is 2 in the general Formula (I).
According to some embodiments of the present application, n is 3 in the general Formula (I).
According to some embodiments of the present application, each R in the general Formula (I) is independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, cyano, benzyloxy, fluorobenzyloxy, C1-4 alkyl, halogenated C1-4 alkyl, C1-4 alkoxy, hydroxyl, C1-4 alkylamino, di(C1-4 alkyl)amino, phenylamino, diphenylamino, -NHC(O)R10, phenyl, pyridyl, pyrrolidinyl, cyclopentyl, cyclohexyl, morpholinyl, imidazolyl, wherein R10 is C1-4 alkyl.
According to some embodiments of the present application, each R in the general Formula (I) is independently hydrogen, di(C1-6 alkyl)amino, C1-6 alkylamino, benzyloxy, halogenated benzyloxy, phenyl, halophenyl or cyano.
According to some embodiments of the present application, each R in the general Formula (I) is independently hydrogen, di(C1-4 alkyl)amino, C1-4 alkylamino, benzyloxy, halogenated benzyloxy, phenyl, halophenyl or cyano.
According to some embodiments of the present application, each R in the general Formula (I) is independently hydrogen, benzyloxy, phenyl, 4-fluorobenzyl, diethylamino, or cyano.
According to some embodiments of the present application, each R in the general Formula (I) is independently di(C1-6 alkyl)amino.
According to some embodiments of the present application, each R in the general Formula (I) is independently C1-6 alkylamino.
According to some embodiments of the present application, each R in the general Formula (I) is independently selected from the group consisting of: hydrogen, fluorine, chlorine, bromine, iodine, cyano, benzyloxy, fluorobenzyloxy, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, tert-pentyl, neopentyl, hexyl, trifluoromethyl, difluoromethyl, fluoromethyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentyloxy, n-hexyloxy, hydroxyl, methylamino, ethylamino, propylamino, butylamino, dimethylamino, diethylamino, dipropylamino, dibutylamino, phenylamino, diphenylamino, acetamido, formylamino, propionamido, phenyl, pyridyl, pyrrolidinyl, cyclopentyl, cyclohexyl, morpholinyl, and imidazolyl.
According to some embodiments of the present application, each R in the general Formula (I) is independently selected from the group consisting of: hydrogen, methoxy, ethoxy, acetamido, benzyloxy, trifluoromethyl, diphenylamino, 4-fluorobenzyloxy, chlorine, pyridin-2-yl, phenyl, pyrrolidin-1-yl, 1H-imidazol-1-yl, propoxy, diethylamino, hydroxyl, morpholin-4-yl, and cyano.
According to some embodiments of the present application, the compound represented by the general Formula (I) of the present application has the structure represented by the Formula (I-1),
wherein, R1, R2, R3, R4 are each independently selected from the group consisting of: hydrogen, halogen, cyano, benzyloxy, halogenated benzyloxy, C1-6 alkyl, halogenated C1-6 alkyl, C1-6 alkoxy, hydroxyl, C1-6 alkylamino, di(C1-6 alkyl)amino, anilino, diphenylamino, phenylamino,-NHC(O)R10, aryl, heteroaryl, cycloalkyl, and heterocycloalkyl, wherein R10 is C1-6 alkyl.
According to some embodiments of the present application, R1, R2, R3, and R4 in the Formula (I-1) are each independently selected from the group consisting of: hydrogen, fluorine, chlorine, bromine, iodine, cyano, benzyloxy, fluorobenzyloxy, C1-4 alkyl, halogenated C1-4 alkyl, C1-4 alkoxy, hydroxyl, C1-4 alkylamino, di(C1-4 alkyl)amino, phenylamino, diphenylamino,-NHC(O)R10, phenyl, pyridyl, pyrrolidinyl, cyclopentyl, cyclohexyl, morpholinyl, imidazolyl, wherein R10 is C1-4 alkyl.
According to some embodiments of the present application, R1, R2, R3, and R4 in the Formula (I-1) are each independently selected from the group consisting of: hydrogen, fluorine, chlorine, bromine, iodine, cyano, benzyloxy, fluorobenzyloxy, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, tert-pentyl, neopentyl, hexyl, trifluoromethyl, difluoromethyl, fluoromethyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentyloxy, n-hexyloxy, hydroxyl, methylamino, ethylamino, propylamino, butylamino, dimethylamino, diethylamino, dipropylamino, dibutylamino, phenylamino, diphenylamino, acetamido, formylamino, propionamido, phenyl, pyridyl, pyrrolidinyl, cyclopentyl, cyclohexyl, morpholinyl, and imidazolyl.
According to some embodiments of the present application, R1, R2, R3, and R4 in the Formula (I-1) are each independently selected from the group consisting of: hydrogen, methoxy, ethoxy, acetyl, acetamido, benzyloxy, trifluoromethyl, diphenylamino, 4-fluorobenzyloxy, chlorine, pyridin-2-yl, phenyl, pyrrolidin-1-yl, 1H-imidazol-1-yl, propoxy, diethylamino, hydroxyl, morpholin-4-yl, and cyano.
According to some embodiments of the present application, R1 in the Formula (I-1) is hydrogen or methoxy.
According to some embodiments of the present application, R1 in the Formula (I-1) is hydrogen.
According to some embodiments of the present application, R2 in the Formula (I-1) is di(C1-6 alkyl)amino.
According to some embodiments of the present application, R2 in the Formula (I-1) is C1-6 alkylamino.
According to some embodiments of the present application, R2 in the Formula (I-1) is di(C1-4 alkyl)amino.
According to some embodiments of the present application, R2 in the Formula (I-1) is C1-4 alkylamino.
According to some embodiments of the present application, R2 in the Formula (I-1) is benzyloxy or halogenated benzyloxy,
According to some embodiments of the present application, R2 in the Formula (I) is phenyl. According to some embodiments of the present application, R2 in the Formula (I) is halogenated phenyl.
According to some embodiments of the present application, R2 in the Formula (I-1) is hydrogen, methoxy, acetyl, benzyloxy, trifluoromethyl, diphenylamino, 4-fluorobenzyloxy, chlorine, pyridin-2-yl, phenyl, pyrrolidin-1-yl, 1H-imidazol-1-yl, propoxy, diethylamino, hydroxyl, morpholin-4-yl, or cyano.
According to some embodiments of the present application, R3 in the Formula (I-1) is hydrogen, benzyloxy, trifluoromethyl, ethoxy, or methoxy.
According to some embodiments of the present application, R3 in the Formula (I-1) is hydrogen or benzyloxy.
According to some embodiments of the present application, R3 in the Formula (I-1) is benzyloxy.
According to some embodiments of the present application, R4 in the Formula (I-1) is hydrogen, trifluoromethyl or cyano.
According to some embodiments of the present application, R4 in the Formula (I-1) is hydrogen.
According to some embodiments of the present application, R1, R3, and R4 in the Formula (I-1) are each independently hydrogen; R2 is di(C1-6 alkyl)amino, C1-6 alkylamino, benzyloxy, halogenated benzyloxy, phenyl, halogenated phenyl or cyano.
According to some embodiments of the present application, R1 and R4 in the Formula (I-1) are each independently hydrogen; R2 is di(C1-6 alkyl)amino, C1-6 alkylamino, benzyloxy, halogenated benzyloxy, phenyl, halogenated phenyl or cyano; R3 is hydrogen or benzyloxy.
According to some embodiments of the present application, in the Formula (I-1), R1, and R4 are each independently hydrogen; R2 is benzyloxy, phenyl, 4-fluorobenzyl, diethylamino, or cyano; R3 is hydrogen or benzyloxy.
According to some embodiments of the present application, the compound represented by the general Formula (I) described in the first aspect of the present application is selected from the group consisting of:
N-{4-{(E)-{2-{6-amino-9-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran -2-yl]-9H-purin-2-yl}hydrazono}methyl}phenyl}acetamide;
(2R,3R,4S,5R)-2-{6-amino-2-{2-[(E)-3,4-bis(benzyloxy)benzylidene]hydrazino}-9H-pruin-9-yl}-5-(hydroxymethyl)tetrahydrofuran-3,4-diol;
(2R,3R,4S,5R)-2-{6-amino-2-{2-[(E)-2,4-bis(trifluoromethyl)benzylidene]hydrazino}-9H-pruin-9-yl}-5-(hydroxymethyl)tetrahydrofuran-3,4-diol;
(2R,3R,4S,5R)-2-{6-amino-2-{2-[(E)-4-(diphenylamino)benzylidene]hydrazino}-9H-pruin-9-yl}-5-(hydroxymethyl)tetrahydrofuran-3,4-diol;
(2R,3R,4S,5R)-2-{6-amino-2-{2-{(E)-4-[(4-fluorobenzyl)oxy]benzylidene}hydrazino}-9H-purin-9-yl}-5-(hydroxymethyl)tetrahydrofuran-3,4-diol;
(2R,3R,4S,5R)-2-{6-amino-2-{2-[(E)-3-(benzyloxy)benzylidene]hydrazino}-9H-purin-9-yl}-5-(hydroxymethyl)tetrahydrofuran-3,4-diol;
(2R,3R,4S,5R)-2-{6-amino-2-{2-[(E)-4-chloro-3-(trifluoromethyl)benzylidene]hydrazino}-9H-purin-9-yl}-5-(hydroxymethyl)tetrahydrofuran-3,4-diol;
(2R,3R,4S,5R)-2-{6-amino-2-{2-[(E)-4-(pyridin-2-yl)benzylidene]hydrazino)-9H-purin-9-yl}-5-(hydroxymethyl)tetrahydrofuran-3,4-diol;
(2R,3R,4S,5R)-2-{2-{2-[(E)-[1,1′-biphenyl]-4-yl-methylene]hydrazino}-6-amino-9H-purin-9-yl}-5-(hydroxymethyl)tetrahydrofuran-3,4-diol;
(2R,3R,4S,5R)-2-{6-amino-2-{2-[(E)-4-(pyrrolidin-1-yl)benzylidene]hydrazino)-9H-pruin-9-yl}-5-(hydroxymethyl)tetrahydrofuran-3,4-diol;
(2R,3R,4S,5R)-2-{6-amino-2-{2-[(E)-4-(trifluoromethyl)benzylidene]hydrazino}-9H-purin-9-yl}-5-(hydroxymethyl)tetrahydrofuran-3,4-diol;
(2R,3R,4S,5R)-2-{2-{2-[(E)-4-(1H-imidazol-1-yl)benzylidene]hydrazino}-6-amino-9H-pruin-9-yl}-5-(hydroxymethyl)tetrahydrofuran-3,4-diol;
(2R,3R,4S,5R)-2-{6-amino-2-{2-[(E)-4-propoxybenzylidene]hydrazino}-9H-purin-9-yl}-5-(hydroxymethyl)tetrahydrofuran-3,4-diol;
2-{(E)-{2-{6-amino-9-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-9H-purinylpyridin-2-yl}hydrazono}methyl}benzonitrile;
(2R,3R,4S,5R)-2-{6-amino-2-{2-[(E)-4-(diethylamino)benzylidene]hydrazino}-9H-purin-9-yl}-5-(hydroxymethyl)tetrahydrofuran-3,4-diol;
(2R,3R,4S,5R)-2-{6-amino-2-{2-[(E)-3-ethoxy-4-hydroxybenzylidene]hydrazino}-9H-purin-9-yl}-5-(hydroxymethyl)tetrahydrofuran-3,4-diol;
(2R,3R,4S,5R)-2-{6-amino-2-{2-[(E)-4-morpholinobenzylidene]hydrazino}-9H-purin-9-yl}-5-(hydroxymethyl)tetrahydrofuran-3,4-diol;
(2R,3R,4S,5R)-2-{6-amino-2-{2-[(E)-3,4,5-trimethoxybenzylidene]hydrazino}-9H-purin-9-yl}-5-(hydroxymethyl)tetrahydrofuran-3,4-diol;
(2R,3R,4S,5R)-2-{6-amino-2-{2-[(E)-4 -(benzyloxy)-3-methoxybenzylidene]hydrazino}-9H-purin-9-yl}-5-(hydroxymethyl)tetrahydrofuran-3,4-diol; 4-{E-{2-{6-amino-9-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-9H-purinylpyridin-2-yl}hydrazono}methyl}benzonitrile.
According to some embodiments of the present application, the method described in the second aspect of the present application has a synthesis reaction process as follows:
Ribofuranose tetraacetate (Compound of Formula VII) as starting material is subjected to a substitution reaction with 2,6-dichloropurine (Compound of formula II) in the presence of tin tetrachloride as catalyst at 110° C. to 130° C. to produce 2,6-dichloro-2′,3′,5′-triacetylpurine nucleoside (Compound of Formula III); the obtained 2,6-dichloro-2′,3′,5′-triacetylpurine nucleoside (Compound of formula III) is subjected to a ammonolysis in a method solution of ammonia under sealing condition to obtain 2-chloroadenosine (Compound of Formula IV); 2-chloroadenosine (Compound of Formula IV) is subjected to hydrazinolysis with hydrazine hydrate at 40° C. to 60° C. to produce 2-hydrazinoadenosine (Compound of Formula V); finally, 2-hydrazinoadenosine (Compound of Formula V) reacts with a substituted benzaldehyde (Compound of Formula VI) in a methanol solution at 70° C. to 90° C. under microwaves to obtain a 2-benzylidenehydrazinoadenosine compound (Compounds of Formula I), wherein the definitions of the substituent R and n are the same as those described in the first aspect of the present application, and can be selected as required.
Definition of Substituent
The term “alkyl” as used herein refers to a saturated linear or branched monovalent hydrocarbonyl preferably having 1 to 6, 1 to 4 or 1 to 3 carbon atoms. For example, “C1-6 alkyl” refers to a saturated linear or branched monovalent hydrocarbonyl having 1 to 6 carbon atoms. Typical examples of “alkyl” include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, tert-pentyl, neopentyl, hexyl.
The term “hydroxyl” as used herein refers to —OH.
The term “halogen” as used herein refers to fluorine, chlorine, bromine or iodine. The preferred halogen group is fluorine, chlorine or bromine.
The term “halogenated C1-6 alkyl” as used herein refers to C1-6 alkyl mono- or poly-substituted by halogen such as fluorine, chlorine, bromine or iodine. The preferred halogenated alkyl groups include chloromethyl, chloroethyl, dichloroethyl, trifluoromethyl, difluoromethyl, monofluoromethyl and the like.
The term “C1-6 alkylamino” as used herein refers to an amino group substituted with one C1-6 alkyl. Typical examples of “C1-6 alkylamino” include but are not limited to methylamino, ethylamino, propylamino, butylamino and the like.
The term “di(C1-6 alkyl)amino” as used herein refers to an amino group substituted with two C1-6 alkyl groups. Typical examples of “di(C1-6 alkyl)amino” include, but are not limited to, dimethylamino, diethylamino, dipropylamino, dibutylamino and the like.
The term “cycloalkyl” as used herein refers to a saturated cyclic hydrocarbonyl having 3 to 12 carbon atoms and having monocyclic or bicyclic or multiple rings (including fused and bridged ring systems), preferably having 3 to 10, 3 to 8, 5 to 8, 3 to 6 or 5 to 6 carbon atoms. Typical examples of “cycloalkyl” include, but are not limited to, monocyclic structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and so on, bicyclic structures such as bicyclo[2.2.1]heptyl, and polycyclic structures such as adamantyl, etc.
The term “heterocycloalkyl” as used herein refers to a cycloalkyl as defined herein containing one, two or more heteroatoms independently selected from N, O and S. Typical examples of “heterocycloalkyl” include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperazinyl, thiazinyl, piperidinyl, morpholinyl and the like.
The term “aryl” as used herein refers to an unsaturated aromatic carbocyclic group having 5 to 14 carbon atoms and having a monocyclic ring or fused ring of two or more rings. The aryl preferably has 5 to 10, 5 to 8 or 5 to 6 carbon atoms. Typical examples of “aryl” include, but are not limited to, phenyl, naphthyl, anthryl and the like.
The term “heteroaryl” as used herein refers to a heteroaromatic cyclic group having 5 to 14 ring members, including monocyclic heteroaromatic ring and polycyclic aromatic ring, in which the monocyclic aromatic ring is fused with one or more other aromatic rings. The heteroaryl has one or two or more heteroatoms selected from O, S or N. The term “heteroaryl” as used herein also includes groups in which an aromatic ring is fused with one or more non-aromatic (carbocyclic or heterocyclic) rings, wherein the linking group or point is located on the aromatic ring or non-aromatic ring. The heteroaryl preferably has 5 to 10 ring members, more preferably 5 to 6 ring members. Typical examples of “heteroaryl” include, but are not limited to, furyl, imidazolyl, triazolyl, indolyl, tetrazolyl, pyridyl, pteridyl, pyrimidinyl, triazolyl, quinolinyl, isoquinolinyl, quinazolinyl, quinoxalinyl and the like.
The term “C1-6 alkoxy” as used herein refers to —OR11, wherein R11 is C1-6 alkyl as defined herein. Typical examples of “C1-6 alkoxy” include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentyloxy, n-hexyloxy, 1,2-dimethylbutoxy, etc.
When the name of compound used herein is inconsistent with the chemical structural formula, the chemical structural formula shall prevail.
According to some embodiments of the present application, the pharmaceutically acceptable salt of the compound of general Formula (I) described in the present application includes salts formed with inorganic or organic acids, and salts formed with inorganic or organic bases. The present application relates to all forms of the above-mentioned salts, includes but not limited to: sodium salt, potassium salt, calcium salt, lithium salt, meglumine salt, hydrochloride, hydrobromide, hydriodate, nitrate, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, acetate, propionate, butyrate, oxalate, trimethylacetate, adipate, alginate, lactate, citrate, tartrate, succinate, maleate, fumarate, picrate, aspartate, gluconate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate.
According to some embodiments of the present application, the compound of general Formula (I) described in the present application can form a pharmaceutically acceptable ester with an organic or inorganic acid. The pharmaceutically acceptable ester includes phosphate, sulfate, nitrate, formate, acetate, propionate, butyrate, valerate, and caproate, which are hydrolyzable in vivo.
The carrier described in the present application includes, but is not limited to: ion exchanger, alumina, aluminum stearate, lecithin, serum protein such as human albumin, buffer substance such as phosphate, glycerol, sorbic acid, potassium sorbate, mixture of partial glyceride of saturated plant fatty acid, water, salt or electrolyte such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salt, colloidal silica, magnesium tri silicate, polyvinylpyrrolidone, cellulosic substance, polyethylene glycol, sodium carboxymethylcellulose, polyacrylate, beeswax, lanolin.
The term “excipient” as used in the present application refers to an additive other than the main active ingredient in a pharmaceutical preparation. It is stable in nature, has no incompatibility with the main active ingredient, does not produce side effects, does not affect therapeutic effect, is not prone to deform, dry, crack, mildew, be worm-eaten at room temperature, is harmless to the human body, has no physiological effect, does not produce chemical or physical effects on the function of main active ingredient, does not affect the content determination of the main active ingredient, etc. For example, binder, filler, disintegrant, lubricant in tablet; preservative, antioxidant, corrigent, flavoring agent, cosolvent, emulsifier, solubilizer, osmotic pressure regulator and coloring agent in oral liquid preparation, etc. can all be called excipients.
The pharmaceutical composition described in the present application can be administered through various routes, such as oral tablet, capsule, powder, oral liquid, injection and transdermal preparation. The above-mentioned various preparation forms can be prepared according to conventional methods in the field of pharmacy. According to conventional pharmaceutical practices, the pharmaceutically acceptable carrier includes diluent, filler, disintegrant, wetting agent, lubricant, coloring agent, flavoring agent or other conventional additives. Typical pharmaceutically acceptable carriers include, for example, microcrystalline cellulose, starch, crospovidone, povidone, polyvinylpyrrolidone, maltitol, citric acid, sodium laurylsulfonate or magnesium stearate, etc.
According to the present application, the pharmaceutical composition can be administered in any of the following routes: oral administration, spray inhalation, rectal administration, nasal administration, buccal administration, vaginal administration, topical administration, parenteral administration such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal and intracranial injection or infusion, or administration with the aid of an explanted reservoir.
As described herein, “effective amount” refers to an amount that is sufficient to treat or prevent or diagnose a patient's disease but is sufficiently low to avoid serious side effects (at a reasonable benefit/risk ratio) within the scope of reasonable medical judgment. The therapeutically or prophylactically or diagnostically effective amount of the compound will vary according on the factors such as the specific compound selected (for example, considering the potency, effectiveness and half-life of the compound), the route of administration selected, the disease to be treated or prevented or diagnosed, the severity of the disease to be treated or prevented or diagnosed, the age, size, weight and physical disease of the patient being treated, the medical history of the patient to be treated, the duration of treatment or prevention or diagnosis, the nature of concurrent therapy, the desired effects of the treatment or prevention or diagnosis and so on, but it can still be routinely determined by those skilled in the art.
In addition, it should be noted that the specific dosage and usage of the compound of general Formula (I) described in the present application for different patients depends on many factors, including the patient's age, weight, gender, natural health status, nutritional status, and the active strength of the compound, the administration time, metabolic rate, severity of disease and the subjective judgment of physician. Herein it is preferable to use a dose of 0.001 to 1000 mg/kg body weight/day.
Beneficial Technical Effects of the Present Application
The compound represented by general Formula (I), a stereoisomer, a pharmaceutically acceptable salt or hydrate as provided in the present application can agonize A2A adenosine receptor, so that it can be used to prevent or treat a human pathological condition or symptom, in which the human pathological condition or symptom can be improved by agonizing the activity of A2A adenosine receptor.
The present application can be further described through the following examples and test examples. However, the scope of the present application is not limited to the following examples or test examples. Those skilled in the art can understand that various changes and modifications can be made to the present application without departing from the spirit and scope of the present application. The present application provides a general and/or specific description of the materials and test methods used in the test. Although many materials and operating methods used to achieve the purpose of the present application are well known in the art, the present application is still described here in as much detail as possible.
For all the following examples, standard operations and purification methods known to those skilled in the art could be used. Unless otherwise stated, all temperatures were expressed in ° C. (Celsius). The structure of the compound was determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). The compound's melting point m.p. was determined by RY-1 melting point meter, in which the thermometer had not been corrected, and the m.p. was given in ° C. 1H NMR was measured by JEOL JNM-ECA-400 NMR spectrometer. The mass spectrum was measured by API3000 (ESI) instrument. All solvents in reaction that were not specified were subject to standardized pretreatment.
21 g (0.066 mol) of (2S,3R,4R,5R)-5-(acetoxymethyl)tetrahydrofuran-2,3,4-triyl triacetate (Compound of Formula VII) was heated to 90° C. until it became clear, and then 12 g (0.063 mol) of 2,6-dichloropurine (Compound of Formula II) and 0.3 g of tin tetrachloride were added and stirred. The reaction solution was further heated to 120° C. and stirred for 15 minutes. Then the solvent was evaporated in vacuum and the residue was cooled. Methanol (50 ml) was added to the residue, and the crude solid product was separated by filtration. The crude product was recrystallized in ethanol to obtain 12 g of pale yellow powder product (2R,3R,4R,5R)-2-(acetoxymethyl)-5-(2,6-dichloro-9H-purin-9-yl)tetrahydrofuran-3,4-diyl diacetate (Compound of Formula III), which was directly used in the next reaction.
10 g (0.022 mol) of (2R,3R,4R,5R)-2-(acetoxymethyl)-5-(2,6-dichloro-9H-purin-9-yl) tetrahydrofuran-3,4-diyl diacetate (Compound of Formula III) was heated to 100° C. in 200 ml of solution of ammonia in methanol and kept for 24 hours in an autoclave. The solution was further stirred for 24 hours to room temperature, and then the solution was evaporated to dryness under reduced pressure to remove ammonia. The residue was purified by flash chromatography, in which a mixed solvent of CH2Cl2 and MeOH (CH2C2:MeOH=10:1 (v/v)) was used as the eluent, and the product was dried below 50 ° C. to obtain 4.5 g of light yellow powder (2R,3R,4S,5R)-2-(6-amino-2-chloro-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (Compound of Formula IV), which was directly used in the next reaction.
5 g (0.017 mol) of (2R,3R,4S,5R)-2-(6-amino-2-chloro-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (Compound of Formula IV) was added to 25 ml of hydrazine hydrate (65 wt % aqueous solution), the resulting mixture was heated to 50° C. while stirring, continuously heated for 4 hours until the reactant (Compound of Formula IV) disappeared, in which the progress of the reaction was monitored by TLC (CH2Cl2:MeOH=3:1 (v/v)). Then the reaction mixture was heated to 25° C., and 2-propanol (50 ml) was added for dilution, then stirred overnight. The separated precipitate was filtered to obtain 4.4 g of yellow solid (2R,3R,4S,5R)-2-(6-amino-2-hydrazino-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (Compound of Formula V), which was directly used in the next reaction.
0.5 g (0.0017 mol) of (2R,3R,4S,5R)-2-(6-amino-2-hydrazino-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (Compound of Formula V) and 0.31 g (0.0019 mol) of 4-acetamidobenzaldehyde (1.1 equivalent) were mixed in methanol (30 ml) and heated by microwave at 80° C. for 30 minutes. The crude product was precipitated from methanol. After filtration, the crude product was further purified on a C18 reverse phase column using preparative medium pressure chromatography to obtain 171 mg of white solid (Compound 1). m.p. 126° C.; 1H NMR (DMSO-d6):δ(ppm) 10.54 (s, 1H), 10.03 (s, 1H), 8.02 (s, 2H), 7.66-7.59 (m, 4H), 7.03 (br, 2H), 5.79 (d, 1H, J=6.4 Hz), 5.44 (d, 1H, J=6.0Hz), 5.24-5.21 (m, 1H), 5.11 (d, 1H, J=4.4), 4.67 (dd, 1H, J=5.6 Hz,5.6 Hz), 4.24-4.21 (m, 1H), 3.98-3.96 (m, 1H), 3.75-3.57 (m, 2H), 2.06 (s, 3H); HRMS (ESI+) m/z [M+H]+calculated for C19H22N8O5: 443.1786; found: 443.1786.
Compounds 2 to 20 could be prepared by referring to the method of Example 1, using different reactants (various substituted benzaldehydes represented by Formula VI) instead of 4-acetamidobenzaldehyde in step 1.4.
By referring to the method of step 1.4 in Example 1, and replacing 4-acetamidobenzaldehyde with 3,4-dibenzyloxybenzaldehyde, the title compound was obtained as 553 mg of white solid (Compound 2). m.p. 220° C.; 1H NMR (DMSO-d6):δ (ppm) 10.55 (s, 1H), 8.00(s, 1H), 7.98 (s, 1H), 7.96 (s, 1H), 7.52-7.30 (m, 10H), 7.09-7.05 (m, 4H), 5.76 (d, 1H, J=7.2 Hz), 5.49-5.47 (m, 2H), 5.19-5.14 (m, 5H), 4.89-4.84 (m, 1H), 4.20-4.18 (m, 1H), 4.00 (s, 1H), 3.76-3.54 (m, 2H); HRMS (ESI+) m/z [+H]+calculated for C31H31N7O6: 598.2409; found: 598.2408.
By referring to the method of step 1.4 in Example 1, and replacing 4-acetamidobenzaldehyde with 2,4-bis(trifluoromethyl)benzaldehyde, the title compound was obtained as 474 mg of white solid (Compound 3). m.p. 239° C.; 1H NMR (DMSO-d6):δ (ppm) 11.44 (s, 1H), 8.70 (d, 1H, J=8.4 Hz),8.47 (s, 1H), 8.12-8.01 (m, 3H), 7.31 (br, 2H), 5.81 (d, 1H, J=6.4 Hz), 5.47 (br, 1H), 5.30 (br, 1H), 5.19 (br, 1H), 4.73-4.71 (m, 1H), 4.25-4.24 (m, 1H), 4.00-3.98 (m, 1H), 3.77-3.62 (m, 2H); HRMS (ESI+) m/z [M+H]+calculated for C19H17F6N7O4: 522.1319; found: 522.1319.
By referring to the method of step 1.4 in Example 1, and replacing 4-acetamidobenzaldehyde with 4-(N,N-diphenylamino)benzaldehyde, the title compound was obtained as 390 mg of yellow solid (Compound 4). m.p. 184° C.; 1H NMR (DMSO-d6): δ (ppm) 10.56 (s, 1H), 8.01 (s, 2H), 7.63 (d, 2H,J=8.4 Hz), 7.35-6.94 (m, 14H), 5.77 (d, 1H,J=6.4 Hz), 5.45 (d, 1H,J=6.0 Hz), 5.27-5.24 (m, 1H), 5.11 (d, 1H,J=4.4 Hz), 4.70-4.66 (m, 1H), 4.21-4.18 (m, 1H), 3.93 (d, 1H,J=2.4 Hz), 3.73-3.53 (m, 2H); HRMS (ESI+) m/z [M'H]+calculated for C29H28N8O4:553.2306; found: 553.2306.
By referring to the method of step 1.4 in Example 1, and replacing 4-acetamidobenzaldehyde with 4-(4-fluorobenzyloxy)benzaldehyde, the title compound was obtained as 291 mg of white solid (Compound 5). m.p. 144° C.; 1-H NMR (DMSO-d6):δ (ppm) 10.51 (s, 1H), 8.02 (s, 2H), 7.68 (d, 2H, J=8.8 Hz), 7.54-7.50 (m, 2H), 7.26-7.22 (m, 2H), 7.04 (d, 4H, J=8.8 Hz), 5.80 (d, 1H, J=6.8 Hz), 5.47 (d, 1H, J=6.4 Hz), 5.31-5.28 (m, 1H), 5.16 (d, 1H, J=4.0 Hz), 5.12 (s, 2H), 4.70-4.66 (m, 1H), 4.22-4.19 (m, 1H), 3.98-3.96 (m, 1H), 3.74-3.57 (m, 2H); HRMS (ESI+) m/z [M+H]+calculated for C24H24FN7O5:510.1896; found: 510.1895.
By referring to the method of step 1.4 in Example 1, and replacing 4-acetamidobenzaldehyde with 3-benzyloxybenzaldehyde, the title compound was obtained as 355 mg of white solid (Compound 6). m.p. 148° C.; 1H NMR (DMSO-d6): δ (ppm) 10.73 (s, 1H), 8.05 (s, 2H), 7.62-6.95 (m, 11H), 5.79 (d, 1H, J=6.8 Hz), 5.48 (d, 1H, J=6.0 Hz), 5.38 (s, 1H), 5.16 (s, 3H), 4.79-4.75 (m, 1H), 4.19 (s, 1H), 3.98 (s, 1H), 3.74-3.55 (m, 2H); HRMS (ESI+) m/z [M+H]+calculated for C24H25N7O5: 492.1990; found: 492.1990.
By referring to the method of step 1.4 in Example 1, and replacing 4-acetamidobenzaldehyde with 4-chloro-3-(trifluoromethyl)benzaldehyde, the title compound was obtained as 443 mg of white solid (Compound 7). m.p. 242° C.; 1H NMR (DMSO-d6):δ (ppm) 10.94 (s, 1H), 8.46 (s, 1H), 8.07 (s, 1H), 8.01 (s, 1H), 7.89 (d, 1H, J=8.0 Hz), 7.68 (d, 1H, J=8.4 Hz), 7.13 (br, 2H), 5.73 (d, 1H, J=7.2 Hz), 5.46 (d, 1H, J=5.2 Hz), 5.39 (d, 1H, J=6.4 Hz), 5.14 (s, 1H), 4.79-4.77 (m, 1H), 4.12 (s, 1H), 3.96 (s, 1H), 3.68-3.51 (m, 2H); HRMS (ESI+) m/z [M+H]+calculated for C18H17C1F3N7O4: 488.1055; found: 488.1055.
By referring to the method of step 1.4 in Example 1, and replacing 4-acetamidobenzaldehyde with 4-(2-pyridyl)benzaldehyde, the title compound was obtained as 537 mg of white solid (Compound 8). m.p. 164° C.; 1-H NMR (DMSO-d6): δ (ppm) 10.83 (s, 1H), 8.69 (s, 1H), 8.15-7.85 (m, 8H), 7.36-7.35 (m, 1H), 7.18 (br, 2H), 5.83 (d, 1H, J=6.4 Hz), 5.50 (s, 1H), 5.28 (s, 1H), 5.20 (s, 1H), 4.71 (s, 1H), 4.25 (s, 1H), 4.00 (s, 1H), 3.77-3.62 (m, 2H); HRMS (ESI+) m/z [M+H]+calculated for C22H22N8O4: 463.1837; found: 463.1838.
By referring to the method of step 1.4 in Example 1, and replacing 4-acetamidobenzaldehyde with 4-biphenyldehyde, the title compound was obtained as 467 mg of white solid (Compound 9). m.p. 234° C.; 1H NMR (DMSO-d6): δ (ppm) 10.78 (s, 1H), 8.12 (s, 1H),8.07 (s, 1H), 7.83 (d, 2H, J=8.4 Hz), 7.31-7.71 (m, 4H), 7.48 (t, 2H, J=7.2 Hz), 7.38 (t, J=7.2Hz,1H), 7.16 (br, 2H), 5.82 (d, 1H, J=6.8 Hz), 5.49 (d, 1H, J=6.0 Hz), 5.28 (s, 1H), 5.19 (d, 1H, J=4.4 Hz), 4.69-4.67 (m, 1H), 4.22 (d, 1H, J=2.4 Hz), 3.98 (d, 1H, J=2.4 Hz), 3.75-3.60 (m, 2H); HRMS (ESI+) m/z [M+H]+calculated for C23H23N704: 462.1884; found: 462.1884.
By referring to the method of step 1.4 in Example 1, and replacing 4-acetamidobenzaldehyde with 4-(1-pyrrolidinyl)benzaldehyde, the title compound was obtain as 352 mg of white solid (Compound 10). m.p. 174° C.; 1H NMR (DMSO-d6): δ (ppm) 10.23 (s, 1H), 7.98 (s, 1H), 7.94 (s, 1H), 7.54 (d, 2H, J=8.8 Hz), 6.95 (br, 2H), 6.54 (d, 2H, J=8.8 Hz), 5.78 (d, 1H, J=6.8 Hz), 5.44 (d, 1H, J=6.4 Hz), 5.27-5.24 (m, 1H), 5.11 (d, 1H, J=4.0 Hz), 4.69-4.65 (m, 1H), 4.20 (s, 1H), 3.97 (s, 1H), 3.73-3.57 (m, 2H), 3.26 (s, 4H), 1.96 (s, 4H); HRMS (ESI+) m/z [M+H]+ calculated for C21H26N8O4: 455.2150; found: 455.2150.
By referring to the method of step 1.4 in Example 1, and replacing 4-acetamidobenzaldehyde with 4-(trifluoromethyl)benzaldehyde, the title compound was obtained as 404 mg of white solid (Compound 11). m.p. 260° C.; 1H NMR (DMSO-d6): δ (ppm) 10.97 (s, 1H), 8.14 (s, 1H), 8.08 (s, 1H), 7.96 (d, 2H, J=8.4 Hz), 7.74 (d, 2H, J=8.4Hz), 7.18 (br, 2H), 5.82 (d, 1H, J=6.4 Hz), 5.47 (d, 1H, J=5.6 Hz), 5.29 (s, 1H), 5.18 (d, 1H, J=3.2 Hz), 4.71-4.69(m, 1H), 4.23 (s, 1H), 3.98 (d, 1H, J=2 Hz), 3.76-3.60 (m, 2H); HRMS (ESI+) m/z [M+H]30 calculated for C18H18F3N7O4: 454.1445; found: 454.1446.
By referring to the method of step 1.4 in Example 1, and replacing 4-acetamidobenzaldehyde with 4-(1H-imidazol-1-yl)benzaldehyde, the title compound was obtained as 615 mg of white solid (Compound 12). m.p. 276° C.; 1H NMR (DMSO-d6): δ (ppm) 10.77 (s, 1H), 8.33 (s, 1H), 8.11 (s, 1H), 8.05 (s, 1H), 7.89 (d, 2H, J=8.4 Hz), 7.81 (s, 1H), 7.70 (d, 2H, J=8.8 Hz), 7.13 (s, 1H), 7.11 (br, 2H), 5.81 (d, 1H, J=6.8 Hz), 5.48 (d, 1H, J=6.4 Hz), 5.35-5.32 (m, 1H), 5.19 (d, 1H, J=4.0 Hz), 4.74-4.70 (m, 1H), 4.22-4.20 (m, 1H), 3.99 (s, 1H), 3.76-3.59 (m, 1H); HRMS (ESI+) m/z [M+H]+calculated for C20H21N9O4: 452.1789; found: 452.1789.
By referring to the method of step 1.4 in Example 1, and replacing 4-acetamidobenzaldehyde with 4-propoxybenzaldehyde, the title compound was obtained as 306 mg of white solid (Compound 13). m.p. 218° C.; 1H NMR (DMSO-d6): δ (ppm) 10.55 (s, 1H), 8.04 (s, 1H), 8.02 (s, 1H), 7.67 (d, 2H, J=8.4 Hz), 7.11 (br, 2H), 6.95 (d, 1H, J=8.8 Hz), 5.80 (d, 1H, J=6.4 Hz), 5.47 (d, 1H, J=5.2 Hz), 5.28 (s, 1H), 5.17 (s, 1H), 4.68 (s, 1H), 4.20 (s, 1H), 3.97-3.94 (m, 3H), 3.73-3.61 (m, 2H), 1.76-1.71 (m, 2H), 0.98 (t, 3H, J=7.6 Hz); HRMS (ESI+) m/z [M+H]+calculated for C20H25N7O5: 444.1990; found: 444.1990.
By referring to the method of step 1.4 in Example 1, and replacing 4-acetamidobenzaldehyde with 2-cyanobenzaldehyde, the title compound was obtained as 359 mg of white solid (Compound 14). m.p. 260° C.; 1H NMR (DMSO-d6): δ (ppm) 11.21 (s, 1H), 8.45 (s, 1H), 8.35 (d, 1H, J=8.0 Hz), 8.08 (s, 1H), 7.83 (d, 1H, J=7.6 Hz), 7.72 (t, 1H, J=7.6 Hz), 7.48 (t, 1H, J=7.6 Hz), 7.18 (br, 2H), 5.81 (d, 1H, J=6.4), 5.47 (d, 1H, J=5.6 Hz), 5.29-5.27 (m, 1H), 5.17 (d, 1H, J=3.6 Hz), 4.73-4.71 (m, 1H), 4.23 (s, 1H), 3.98 (s, 1H), 3.75-3.58 (m, 2H); HRMS (ESI+) m/z [M+H]+calculated for C18H18N8O4: 411.1524; found: 411.1523.
By referring to the method of step 1.4 in Example 1, and replacing 4-acetamidobenzaldehyde with 4-(N,N-diethyl)aminobenzaldehyde, the title compound was obtained as 299 mg of white solid (Compound 15). m.p. 164° C.; 1H NMR (DMSO-d6): δ (ppm) 10.27 (s, 1H), 7.99 (s, 1H), 7.92 (s, 1H), 7.51 (d, 2H, J=8.8 Hz), 6.99 (br, 2H), 6.66 (d, 2H, J=8.8 Hz), 5.78 (d, 1H, J=6.8 Hz), 5.47 (d, 1H, J=6 Hz), 5.30-5.26 (m, 1H), 5.15 (d, 1H, J=4.4 Hz), 4.69-4.65 (m, 1H), 4.21-4.18 (m, 1H), 3.97-3.95 (m, 1H), 3.73-3.56 (m, 2H), 3.39-3.34 (m, 4H), 1.10 (t, 6H, J=6.8 Hz); HRMS (ESI+) m/z [M+H]+calculated for C21 H28N8O4: 457.2306; found: 457.2305.
By referring to the method of step 1.4 in Example 1, and replacing 4-acetamidobenzaldehyde with ethyl vanillin, the title compound was obtained as 322 mg of white solid (Compound 16). m.p. 198° C.; 1H NMR (DMSO-d6): δ (ppm) 10.43 (s, 1H), 9.19 (s, 1H), 7.99 (s, 1H), 7.94 (s, 1H), 7.66 (s, 1H), 7.04 (br, 2H), 6.92 (d, 1H, J=8.0 Hz), 6.77 (d, 1H, J=8.4 Hz), 5.75 (d, 1H, J=7.2 Hz), 5.48 (d, 1H, J=6.4 Hz), 5.44-5.43 (m, 1H), 5.14 (d, 1H, J=4.0 Hz), 4.86-4.84 (m, 1H), 4.17 (s, 1H), 4.09 (q, 2H, J=6.8 Hz), 4.00 (s, 1H), 3.72-3.52 (m, 2H), 1.37 (t, 3H, J=6.8 Hz); HRMS (ESI+) m/z [M+H]+calculated for C19H23N7O6: 446.1783; found: 446.1782.
By referring to the method of step 1.4 in Example 1, and replacing 4-acetamidobenzaldehyde with 4-(morpholin-4-yl)benzaldehyde, the title compound was obtained as 348 mg of white solid (Compound 17). m.p. 186° C.; 1H NMR (DMSO-d6): δ (ppm) 10.42 (s, 1H), 8.01 (s, 1H), 7.97 (s, 1H), 7.59 (d, 2H, J=8.4 Hz), 7.02 (br, 2H), 6.95 (d, 2H, J=8.8 Hz), 5.79 (d, 1H, J=6.4 Hz), 5.46 (d, 1H, J=6.0 Hz), 5.29-5.26 (m, 1H), 5.15 (d, 1H, J=4.0 Hz), 4.69-4.65 (m, 1H), 4.21-4.18 (m, 1H), 3.97-3.95 (m, 1H), 3.74 (t, 4H, J=8.4 Hz), 3.71-3.56 (m, 2H), 3.17 (t, 4H, J=8.8 Hz); HRMS (ESI+) m/z [M+H]+calculated for C21H26N8O5: 471.2099; found: 471.2100.
By referring to the method of step 1.4 in Example 1, and replacing 4-acetamidobenzaldehyde with 3,4,5-trimethoxybenzaldehyde, the title compound was obtained as 352 mg of white solid (Compound 18). m.p. 246° C.; 1H NMR (DMSO-d6): δ (ppm) 10.68 (s, 1H), 8.01 (s, 1H), 7.98 (s, 1H), 7.16 (s, 2H), 7.09 (br, 2H), 5.75 (d, 1H, J=7.2 Hz), 5.49-5.45 (m, 2H), 5.12 (s, 1H), 4.92-4.88 (m, 1H), 4.15 (s, 1H), 3.99 (s, 1H), 3.84 (s, 6H), 3.68 (s, 3H), 3.72-3.52 (m, 2H); HRMS (ESI+) m/z [M+H]+calculated for C20H25N7O7: 476.1888; found: 476.1887.
By referring to the method of step 1.4 in Example 1, and replacing 4-acetamidobenzaldehyde with 4-(benzyloxy)-3-methoxybenzaldehyde, the title compound was obtained as 281 mg of white solid (Compound 19). m.p. 146° C.; 1H NMR (DMSO-d6): δ (ppm) 10.54 (s, 1H), 8.00 (s, 1H), 7.99 (s, 1H), 7.77 (s, 1H), 7.47-4.33 (m, 5H), 7.06 (br, 2H), 7.03 (s, 2H), 5.75 (d, 1H, J=7.2H), 5.48 (d, 1H, J=6.4 Hz), 5.46 (d, 1H, J=4.0 Hz), 5.13 (d, 1H, J=4. 0 Hz), 5.11 (s, 2H), 4.90-4.85 (m, 1H), 4.17-4.15 (m, 1H), 3.99 (s, 1H), 3.85 (s, 3H), 3.74-3.53 (m, 2H); HRMS (ESI+) m/z [M+H]+calculated for C25H27N7O6: 522.2096; found: 522.2096.
By referring to the method of step 1.4 in Example 1, and replacing 4-acetamidobenzaldehyde with 4-cyanobenzaldehyde, the title compound was obtained as 373 mg of white solid (Compound 20). m.p. 276° C.; 1H NMR (DMSO-d6): δ (ppm) 10.99 (s, 1H), 8.11 (s, 1H), 8.07 (s, 1H), 7.91 (d, 2H, J=8.4 Hz), 7.83 (d, 2H, J=8.0 Hz), 7.13 (br, 2H), 5.81 (d, 1H, J=6.4 Hz), 5.44 (d, 1H, J=6.4 Hz), 5.28-5.26 (m, 1H), 5.15 (d, 1H, J=4.8 Hz), 4.72-4.67 (m, 1H), 4.23-4.20 (m, 1H), 3.98 (d, 1H, J=2.4 Hz), 3.75-3.57 (m, 2H); HRMS (ESI+) m/z [M+H]+calculated for C18H18N8O4: 411.1524; found: 411.1525.
1) Experimental Materials
[3H]CGS21680 (2-[p-(2-carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine, [carboxy-1-ethyl-3H(N)]-;250 μCi) was purchased from PerkinElmer Research Products (Boston, Mass.).
Cell membranes stably transfected with (human) A2A adenosine receptors were prepared in HEK-293 cells. The cell membranes were obtained from PerkinElmer Research Products (Boston, Mass.).
CGS21680 (2-[p-(2-carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine) was purchased from Selleck (Shanghai, CN).
All other reagents were of analytical grade and obtained from commercial sources.
2) Experimental Method
The A2A adenosine receptors used were all expressed in the cell membranes. The compound was diluted 3 times serially with DMSO (Solarbio, D8371-250 ml) to generate compound source plates with 10 different concentrations (10 μM, 3.3 μM, 1.1 μM, 0.37 μM, 0.12 μM, 0.0412 μM, 0.0137 μM, 0.0046 μM, 0.0015 μM, 0.0005 μM), 250 nL of the compound was added to 384-well Opti-plate, sealed with parafilm. 20 U hA2A HEK-293 cell membrane was diluted with 1 mL assay buffer (50 mM Tris-HCl pH 7.4, 10 mM MgCl2, 1 mM EDTA, 1 μg/mL adenosine deaminase), 0.75 μCi [3H]CGS 21680 (final 25 nM) was added to the diluted cell membrane, and mixed well. 50 μL of the prepared dilution solution of cell membrane was transferred to the 384-well Opti-plate containing compound and incubated at 25° C. for 90 minutes. 100 μL of 0.5% Polyethyleneimine solution (PEI) was added to UNIFILTER-96 GF/B filter plate, and soaked for 90 minutes at 4° C., then 500 μL of washing buffer/well (50 mM Tris-HCl pH 7.4, 154 mM NaCl) was transferred with Cell Harvester to wash the UNIFILTER-96 GF/B filter plate twice. The mixed system in the Opti-plate was transferred to the washed UNIFILTER-96 GF/B filter plate, 500 μL of washing buffer/well (50 mM Tris-HCl pH 7.4, 154 mM NaCl) was used to wash the UNIFILTER-96 GF/B filter plate 9 times. Incubation was performed in 37° C. incubator for 3 minutes. 40 μL of ULTIMA GOLD scintillation solution (Perkin Elmer, Cat #77-16061) was added to each well, CPM (count per minute) value was read by a MicroBeta liquid scintillation counter (PerkinElmer). The specific binding percentage of [3H]CGS21680 was calculated according to the CPM value, % specific binding of [3H]CGS21680=(CPMsample−CPMLow Control) /(CPMhigh Control−CPMLow Control)*100, in which High Control was 0.5% DMSO, Low Control was 100 μM CGS21680. The IC50 value was calculated by curve fitting according to the compound concentration and the specific binding percentage of [3H]CGS21680.
3) Experimental Results
The inhibition constant (Ki) value was calculated from the IC50 value according to the Cheng and Prusoff equation, Ki=IC50/(1+[S]/Km), wherein [S] was the concentration of radioligand (25 nM) and Km was dissociation constant (22 nM) of [3H]CGS21680 binding to human A2AAR. Table 1 shows the inhibition constant Ki for Compounds 1 to 20 of the present application binding to A2A adenosine receptor.
1) Experimental Materials
Experimental reagents and consumables: DMEM/F12, G418, Penicillin-Streptomycin, Versene Solution, HEPES, Hank's Buffered Saline Solution, PBS (pH 7.4, 1×, sterile), FBS, BSA Stabilizer 7.5%, Rolipram, NECA were separately purchased from Gibico, Hyclone and Sigma. LANCE® Ultra cAMP kit (Eu-cAMP tracer, Ulight-anti-cAMP reagent, cAMP detection buffer) and hADORA2A-HEK293 cells were purchased from PerkinElmer Research Products (Boston, Mass.). All other reagents were of analytical grade and obtained from commercial sources. 384-well polypropylene microplate and 384-well solid white plate were purchased from Labcyte and Corning, respectively.
Experimental instruments: TECAN automated liquid handling workstation, Echo Acoustic Liquid Handler and EnVison multimode plate reader were purchased from TECAN, Labcyte and Envision, respectively.
2) Experimental Method
The cells stably expressing human adenosine receptor A2A (hADORA2A-HEK293 cells) were cultured in DMEM/F12 medium containing 10% FBS, 1×Penicillin-Streptomycin and 400 μg/ml G418 in a 37° C. and 5% CO2 environment. Before the experiment, the cells were digested with Versene solution, and the cells were collected by centrifugation at 200 g and room temperature for 5 minutes, and finally resuspended with assay buffer (Hank's buffered saline solution, containing 5 mM HEPES, 0.1% BSA stabilizer and 10 μM Rolipram, pH 7.4). The TECAN automated liquid handling workstation was used to dilute the compound in a 384-well polypropylene microplate with DMSO to 11 concentration points in a 3-fold gradient to prepare the compound source plate, in which the 11 concentration points of the compound were 10 mM, 3.33 mM, 1.11 mM., 0.3 7mM, 0.1 2 mM, 0.041 mM, 0.013 mM, 4.57×10−3 mM, 1.52×10−3 mM, 5×10−4 mM and 1.7×10−4 mM. The Echo Acoustic Liquid Handler (Labcyte) was used to transfer the test compound from the compound source plate to an assay plate, in which the transfer volume of the compound was 10 nl/well. The hADORA2A-HEK293 cell suspension was diluted with assay buffer to 30,000 cells/ml, and the cell suspension was transferred to the assay plate at a volume of 10 μl/well (300 cells/well). The assay plate was centrifuged at 150 g for 1 minute and pre-incubated at room temperature for 30 minutes. The Eu-cAMP tracer working solution (Eu-cAMP tracer 40 μl, cAMP detection buffer 1.96 ml) (5 μl/well) was added to the assay plate, and then the Ulight-anti-cAMP working solution (13 μl of Ulight-anti-cAMP reagent and 1.95 ml of cAMP detection buffer) (5 82 l/well) was added to the assay plate. The assay plate was rotated at 150 g for 30 seconds and incubated at room temperature for 30 minutes. The EnVision multimode plate reader (PerkinElmer) was used determine the level of cyclic adenosine monophosphate in the final solution (λex=320 nm, λem=665 nm & 615 nm). The EC50 (nM) value of the compound interacting with the A2A adenosine receptor to stimulate the production of cyclic adenosine monophosphate was calculated and the A2A receptor agonist titer of the compound was expressed as the EC50 (nM) value.
3) Experimental Results
When the test compounds interacted with A2AAR, the EC50 (nM) values for stimulating the production of cyclic AMP were shown in Table 2. The results showed that Compounds 2, 5, 9, 15 and 20 prepared in the present application were all shown as hA2AAR agonists. When Compounds 2, 5, 9, 15 and 20 interacted with A2AAR, their inhibitory constant Ki and EC50 values for stimulating cAMP production were basically in the same nanomolar range.
1) Experimental Materials
Fluorescein-labeled dextran FITC-Dextran (CAS: 60842-46-8) with molecular weight of 10,000 MW was purchased from TCI (Shanghai) Development Co., Ltd.; PBS solution and experimental animal SD rats were obtained from commercial sources.
2) Experimental Method
FITC-Dextran solutions with six concentration gradients (0.001, 0.01, 0.1, 1, 0.5, 10 μg/ml) were prepared with PBS, and standard curve of FITC-Dextran concentration was made by using microplate reader (λex=490 nm, λem=520 nm). Additionally, 10 mg/ml FITC-Dextran solution was prepared, Compound 5 was added to PBS solution to prepare 1 mg/ml solution. 1 ml of 10 mg/ml FITC-Dextran solution and 1 ml of 1 mg/ml Compound 5 in PBS solution were taken and mixed to prepare administration solvent. 1 ml of 10mg/ml FITC-Dextran solution and 1 ml of PBS solution were taken and mixed to prepare blank control solution. 6 SD rats were injected with 2 ml of the administration solvent via tail vein, respectively, while another 6 SD rats were injected with 2 ml of the blank control solution via tail vein. After 30 minutes, brain tissues were taken out from all SD rats, homogenized and centrifuged at 10,000 rpm for 15 minutes, and the supernatants were taken for later test. The solution to be tested was subjected to fluorescence detection by using the microplate reader (λex=490 nm, λem=520 nm).
3) Experimental Results
The measured fluorescence value by the microplate reader was converted into the corresponding FITC-Dextran average concentration according to the obtained standard curve of FITC-Dextran concentration. The results were shown in Table 3. The results showed that the macromolecule FITC-Dextran itself could not pass through the blood-brain barrier, while FITC-Dextran to which Compound 5 was added could enter the brain through the BBB, indicating that Compound 5 could open the blood-brain barrier.
Although the specific embodiments of the present application have been described in details, those skilled in the art will understand that according to all the teachings that have been disclosed, various modifications and substitutions can be made to those details, and these changes are all within the scope of protection of the present application. The full scope of the present application is given by the appended claims and any equivalents thereof. The publications and patent documents cited in the present application are incorporated herein by reference.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201910542124.5 | Jun 2019 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/097442 | 6/22/2020 | WO |
