Patent Application
20220380402
The present disclosure belongs to the technical field of medicine, and specifically relates to 6-hydrazinoadenosine and derivatives thereof as an A2A adenosine receptor agonist, and a pharmaceutical composition containing the same. These compounds and composition can be used as 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 cross the blood-brain barrier. Drug delivery across the blood-brain barrier has been a challenging research area in the past few decades. Researchers have made considerable efforts to develop various drug delivery systems. A series of studies of strategics have revealed that it is very difficult to transport drugs and contrast agents across the blood-brain barrier.
BBB (blood-brain barrier) restricts molecules from entering the brain through two main structural characteristics. First, there are tight junctions (TJs) between cerebral vascular endothelial cells, which seal the endothelial cells and result in low permeability of molecules in blood through the BBB. Second, compared with peripheral vascular endothelial cells, there are few transport pathways between cerebral vascular endothelial cells, but active efflux transporters, such as P-glycoprotein (P-gp) on brain capillary endothelial cells (BCECs), are at a very high expression level.
Considering the key role of TJs in restricting molecules from entering 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 BBB permeability. Temporarily opening TJs is a feasible way of brain drug delivery, and this way has a high passing efficiency for the therapeutic drugs and less limitation in molecular weight. Bynoe et al. demonstrated that the specific agonizing of A2A adenosine receptor (A2AAR) on mouse BCECs could promote brain drug absorption (CARMAN A J et al., J Neurosci, 2011, 31(37): 13272-80).
Further studies have shown that the agonizing of A2A adenosine receptor (A2AAR) 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 intracellular actin to change cytoskeletal elements, which leads to cell morphology contraction, destruction of TJs integrity, and increased barrier permeability (SOHAIL M A et al., Hepatology, 2009, 49(1): 185-94). These studies have greatly expanded the potential application fields and development space of A2AAR agonists. The development of high-efficiency A2AAR agonists is of great significance to the study of strategies for opening the blood-brain barrier (patent CN200980117596.0).
Due to the widespread distribution of A2AAR in the human body, A2AAR agonists can be used to treat various pathological diseases. Adenosine mediates A2AAR to produce potential immunosuppressive and hypotensive effects. One of the main potential therapeutic effects of A2AAR agonists is anti-inflammatory and immunosuppressive effect. It regulates 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) to achieve the above functions. From the perspective of cell signaling pathways, the agonizing of A2A reduces the NF-kB pathway, reduces inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin-1 β (IL-1β), IL-8, IL-6, and inhibits the release of matrix metalloproteinase-1 (MMP-1) and MMP-3 (HASKO G et al., 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. Furthermore, A2AAR agonists are powerful vasodilators and have been used as diagnostic reagents for cardiac pharmacological stress tests (patent CN200580033215.2). In addition, the further potential therapeutic application of A2AAR agonists is 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). It has been shown that A2AAR agonists 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 A2AAR agonists as described above 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 Pharmaceuticals and Astellas. This product 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 A2A receptor agonists that have novel structure, are effective and optionally have one or more physiological and/or physicochemical advantages, and it is important to continuously synthesize and test additional A2A receptor agonists so as to develop new and improved therapeutic agents.
The present disclosure provides a new class of small molecule agonists acting on A2A adenosine receptor, which can agonize A2A adenosine receptor, thereby achieving, on the one hand, the purpose for prevention and/or treatment of a human pathological state or symptom, in which the prevention and/or treatment of a human pathological state or symptom is related to the activity of A2A adenosine receptor, and the prevention and/or treatment of a human pathological state or symptom requires agonizing of A2A adenosine receptor; on the other hand, the purpose for increasing the permeability of blood-brain barrier of a subject receiving the therapeutic drug.
The first aspect of the present disclosure provides a compound represented by general Formula (I), or a stereoisomer thereof, or a pharmaceutically acceptable salt of the compound or stereoisomer, or a pharmaceutically acceptable hydrate or solvate of the compound or stereoisomer, or a pharmaceutically acceptable ester of the compound or stereoisomer, wherein the compound has a structure represented by the general Formula (I) as follows:
In some embodiments, R1 is selected from C6-10 aryl, 5- to 7-membered heteroaryl, 5- to 6-membered cycloalkyl, 5- to 6-membered heterocycloalkyl, C1-10 alkyl, C1-10 heteroalkyl or C2-10 alkenyl.
In some embodiments, R1 is selected from the group consisting of phenyl, pyrrolyl, imidazolyl, thiazolyl, furyl, pyridyl, cyclopentyl, cyclohexyl, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, trifluoromethyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentyloxy, n-hexyloxy, methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, tert-butylthio, sec-butylthio, n-pentylthio, n-hexylthio or C2-10 alkenyl.
In some embodiments, R1 is selected from the group consisting of phenyl, pyrrolyl, furyl, imidazolyl, thiazolyl, cyclohexyl, alkylthio, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, tert-pentyl, neopentyl, hexyl, trifluoromethyl, difluoromethyl, fluoromethyl, vinyl, or decadienyl.
In some embodiments, R1 is phenyl. R1 is optionally substituted with one or more R′, each R′ is independently selected from the group consisting of phenyl, halophenyl, amino-substituted phenyl, benzyloxy, halobenzyloxy, phenylamino, heteroaryl, cycloalkyl, heterocycloalkyl, C1-6 alkyl, halogenated C1-6 alkyl, C2-6 alkenyl, C1-6 alkoxy, C1-6 alkylthio, —NHC(O)R10, halogen or cyano, wherein R10 is C1-6 alkyl.
In some embodiments, R1 is halopyridyl, such as bromopyridyl, such as 5-bromopyridyl, such as 5-bromopyridin-2-yl.
In some embodiments, R1 is thiazolyl, such as thiazol-5-yl.
In some embodiments, R1 is cyclohexyl.
In some embodiments, R1 is selected from the following groups:
In some embodiments, each R′ is independently selected from the group consisting of phenyl, halophenyl, amino-substituted phenyl, benzyloxy, halobenzyloxy, phenylamino, imidazolyl, pyridyl, 5- to 6-membered cycloalkyl, 5- to 6-membered heterocycloalkyl, C1-6 alkyl, C1-6 haloalkyl, —NHC(O)R10, halogen or cyano, wherein R10 is C1-4 alkyl;
In some embodiments, each R′ is independently selected from the group consisting of phenyl, halophenyl, dimethylamino-substituted phenyl, benzyloxy, halobenzyloxy, diphenylamino, 1H-imidazol-1-yl, pyridin-2-yl, 1H-imidazol-1-yl, pyrrolidin-1-yl, cyclopentyl, cyclohexyl, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, trifluoromethyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentyloxy, n-hexyloxy, methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, tert-butylthio, sec-butylthio, n-pentylthio, n-hexylthio, —NH(CO)CH3, F, Cl, Br or cyano.
In some embodiments, the compound represented by general Formula (I) has the structure represented by Formula (I-1), and the compound has the structure represented by Formula I-1:
In some embodiments, each R2 is independently selected from the group consisting of C1-6 alkyl, C1-6 heteroalkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, C1-6 alkylamino, acylamino, phenyl, benzyloxy, halobenzyloxy, phenylamino, 5- to 6-membered heterocycloalkyl, —NH(CO)CH3, halogen, hydroxy, or cyano.
In some embodiments, each R2 is independently selected from the group consisting of phenyl, halophenyl, amino-substituted phenyl, benzyloxy, halobenzyloxy, phenylamino, heteroaryl, cycloalkyl, heterocycloalkyl, C1-6 alkyl, C1-6 haloalkyl, C2-10 alkenyl (such as C2-6 alkenyl), C1-6 alkoxy, —NHC(O)R10, halogen or cyano, wherein R10 is C1-4 alkyl.
In some embodiments, each R2 is independently selected from the group consisting of methyl, trifluoromethyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, C1-3 alkoxy, phenyl, diphenylamino, benzyloxy, halobenzyloxy, pyridin-2-yl, 1H-imidazol-1-yl, pyrrolidin-1-yl, —NH(CO)CH3, F, Cl, Br or cyano.
In some embodiments, each R2 is independently selected from the group consisting of —NH(CO)(R′), benzyloxy, halobenzyloxy, trifluoromethyl, pyridin-2-yl, phenyl, pyrrolidin-1-yl, 1H-imidazol-1-yl, C1-3 alkoxy, diphenylamino. Each R′ is independently selected from the group consisting of phenyl, halophenyl, amino-substituted phenyl, benzyloxy, halobenzyloxy, phenylamino, heteroaryl, cycloalkyl, heterocycloalkyl, C1-6 alkyl, halogenated C1-6 alkyl, C2-10 alkenyl (such as C2-6 alkenyl), C1-6 alkoxy, C1-6 alkylthio, —NHC(O)R10, halogen or cyano, wherein R10 is C1-6 alkyl.
In some embodiments, R′ is C1-6 alkyl, such as C1-3 alkyl, such as methyl.
In some embodiments, each R2 is independently selected from halobenzyloxy.
In some embodiments, each R2 is independently selected from 4-fluorobenzyloxy.
In some embodiments, n=1.
In some embodiments, n=2.
In some embodiments, n=3.
In some embodiments, n=4.
In some embodiments, n=5.
In some embodiments, each R2 is independently selected from benzyloxy or halobenzyloxy.
In some embodiments, R2 is halobenzyloxy, such as chlorobenzyloxy.
In some embodiments, the compound represented by general Formula (I) has a structure represented by Formula (I-2), and the compound has a structure represented by Formula 1-2:
R3 is selected from the group consisting of phenyl, halophenyl, amino-substituted phenyl, C1-4 alkylamino-substituted phenyl, di(C1-4 alkyl)amino-substituted phenyl, C1-8 alkyl or C2-8 alkenyl.
In some embodiments, R3 is dimethylamino-substituted phenyl.
In some embodiments, R3 is 1-heptenyl.
The second aspect of the present disclosure provides a method for preparing the compound of general Formula (I), or the stereoisomer thereof, or the pharmaceutically acceptable salt of the compound or stereoisomer, or the pharmaceutically acceptable hydrate or solvate of the compound or stereoisomer, or the pharmaceutically acceptable ester of the compound or stereoisomer, as described in the first aspect of the present disclosure, comprising:
In some embodiments, the compound of Formula (vii) reacts with a substituted formaldehyde (viii) in a methanol solution under microwaves at 70° C. to 90° C.;
In some embodiments, the compound of Formula (vii) is prepared by hydrazinolyzing a compound of Formula (vi) with hydrazine hydrate (N2H4.H2O) at 60˜80° C.
In some embodiments, the method of synthesizing the compound of Formula (I) is as follows:
The third aspect of the present disclosure provides a pharmaceutical composition, which comprises at least one of the compound, or the stereoisomer thereof, or the pharmaceutically acceptable salt of the compound or stereoisomer, or the pharmaceutically acceptable hydrate or solvate of the compound or stereoisomer, or the pharmaceutically acceptable ester of the compound or stereoisomer, as described in the first aspect of the present disclosure, and one or more pharmaceutically acceptable carriers or excipients.
In some embodiments, the above-mentioned pharmaceutical composition further comprises: a drug for crossing the blood-brain barrier, which is selected from the group consisting of a drug for treating a disease or disorder of the central nervous system, a neurotoxin antidote, and a drug for treating a brain glioma.
The fourth aspect of the present disclosure provides use of the compound, or the stereoisomer thereof, or the pharmaceutically acceptable salt of the compound or stereoisomer, or the pharmaceutically acceptable hydrate or solvate of the compound or stereoisomer, or the pharmaceutically acceptable ester of the compound or stereoisomer, as described in the first aspect of the present disclosure, or the pharmaceutical composition as described in the third aspect of the present disclosure in the manufacture of a medicament as an A2A adenosine receptor agonist, or
In some embodiments, the human pathological condition or symptom is selected from the following: 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, founder's disease, glaucoma, ocular hypertension, spinal cord injury, myocardial infarction, and acute myocardial infarction.
The fifth aspect of the present disclosure provides use of the compound, or the stereoisomer thereof, or the pharmaceutically acceptable salt of the compound or stereoisomer, or the pharmaceutically acceptable hydrate or solvate of the compound or stereoisomer, or the pharmaceutically acceptable ester of the compound or stereoisomer, as described in the first aspect of the present disclosure, or the pharmaceutical composition as described in the third aspect of the present disclosure in the manufacture of a medicament for diagnosing a human myocardial perfusion abnormality.
The sixth aspect of the present disclosure provides use of the compound, or the stereoisomer thereof, or the pharmaceutically acceptable salt of the compound or stereoisomer, or the pharmaceutically acceptable hydrate or solvate of the compound or stereoisomer, or the pharmaceutically acceptable ester of the compound or stereoisomer, as described in the first aspect of the present disclosure, or the pharmaceutical composition as described in the third aspect of the present disclosure in the manufacture of a medicament for increasing a blood-brain barrier permeability of a subject receiving a therapeutic drug, wherein the subject is benefited from the increased blood-brain barrier permeability for delivering the therapeutic drug across the blood-brain barrier.
In some embodiments, the therapeutic drug is selected from the following: a drug that is effective in treating a disease or disorder of the central nervous system, a neurotoxin antidote, and a drug for treating a brain glioma.
The seventh aspect of the present disclosure provides a pharmaceutical composition, which comprises:
The eighth aspect of the present disclosure provides a method for prevention and/or treatment of a human pathological condition or symptom, comprising administering to a patient in need of such treatment a therapeutically effective amount of the compound, or the stereoisomer thereof, or the pharmaceutically acceptable salt of the compound or stereoisomer, or the pharmaceutically acceptable hydrate or solvate of the compound or stereoisomer, or the pharmaceutically acceptable ester of the compound or stereoisomer, as described in the first aspect of the present disclosure, or the pharmaceutical composition according to the third aspect of the present disclosure, wherein the human's pathological condition or symptom is related to the activity of A2A adenosine receptor, and the prevention or treatment of the pathological condition or symptom of the patient requires agonizing of the A2A adenosine receptor.
The ninth aspect of the present disclosure provides the compound represented by the general Formula (I), or the stereoisomer thereof, or the pharmaceutically acceptable salt of the compound or stereoisomer, or the pharmaceutically acceptable hydrate or solvate of the compound or stereoisomer, or the pharmaceutically acceptable ester of the compound or stereoisomer, as described in the first aspect of the present disclosure, for use in 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 agonizing of the A2A adenosine receptor.
The tenth aspect of the present disclosure provides the compound represented by the general Formula (I), or the stereoisomer thereof, or the pharmaceutically acceptable salt of the compound or stereoisomer, or the pharmaceutically acceptable hydrate or solvate of the compound or stereoisomer, or the pharmaceutically acceptable ester of the compound or stereoisomer, as described in the first aspect of the present disclosure,
The eleventh aspect of the present disclosure also provides a method for diagnosing a human myocardial perfusion abnormality, comprising administering to a patient in need of such diagnosis a diagnostically effective amount of the compound represented by the general Formula (I), or the stereoisomer thereof, or the pharmaceutically acceptable salt of the compound or stereoisomer, or the pharmaceutically acceptable hydrate or solvate of the compound or stereoisomer, or the pharmaceutically acceptable ester of the compound or stereoisomer, as described in the first aspect of the present disclosure, or the pharmaceutical composition as described in the third aspect of the present disclosure.
The twelfth aspect of the present disclosure also provides a method for increasing the permeability of the blood-brain barrier of a subject receiving a therapeutic drug, the method comprising administering to the subject an effective amount of the compound represented by the general Formula (I), or the stereoisomer thereof, or the pharmaceutically acceptable salt of the compound or stereoisomer, or the pharmaceutically acceptable hydrate or solvate of the compound or stereoisomer, or the pharmaceutically acceptable ester of the compound or stereoisomer, as described in the first aspect of the present disclosure, or the pharmaceutical composition as described in the third aspect of the present disclosure, wherein the subject benefits from the increased permeability of the blood-brain barrier for delivering the therapeutic drug across the blood-brain barrier.
According to some embodiments of the present disclosure, in the method described in the twelfth aspect of the present disclosure, the therapeutic drug is selected from: a drug for treating a disease or disorder of the central nervous system, a neurotoxin antidote, and a drug for treating a brain glioma.
According to some embodiments of the present disclosure, the human pathological condition or symptom described in the present disclosure is selected from: 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, founder's disease, glaucoma, ocular hypertension, spinal cord injury, myocardial infarction, and acute myocardial infarction.
In some embodiments, the compound, or the stereoisomer thereof, or the pharmaceutically acceptable salt of the compound or stereoisomer, or the pharmaceutically acceptable hydrate or solvate of the compound or stereoisomer, or the pharmaceutically acceptable ester of the compound or stereoisomer, as described in the first aspect of the present disclosure, or the pharmaceutical composition as described in the third aspect of the present disclosure, has one or more of the following beneficial effects:
As used in this application, the term “alkyl” used alone or in combination with other terms refers to a saturated linear or branched monovalent hydrocarbon group, preferably having 1-6, 1-4 or 1-3 carbon atoms. For example, “C1-6 alkyl” refers to a saturated linear or branched monovalent hydrocarbon group 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, etc.
The term “hydroxyl” as used herein refers to —OH.
The term “halogen” as used herein refers to fluorine, chlorine, bromine or iodine. Preferred halogen is fluorine, chlorine or bromine.
The term “halo” as used herein refers to substitution by one or more halogen atoms.
The term “halogenated C1-6 alkyl” as used herein refers to a C1-6 alkyl mono- or polysubstituted by halogen such as fluorine, chlorine, bromine or iodine. Preferred haloalkyl is 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 so on.
As used in this application, the term “aryl” used alone or in combination with other terms refers to monocyclic or polycyclic (for example, having 2, 3 or 4 condensed rings) aromatic hydrocarbon group, such as but not limited to, phenyl, 1-naphthyl, 2-naphthyl, anthryl, phenanthryl, etc. In certain embodiments, the aryl is a C6-14 aryl. In certain embodiments, the aryl is a C6-10 aryl. In certain embodiments, the aryl is a naphthyl ring or phenyl ring. In certain embodiments, the aryl is phenyl.
As used in this application, the term “heteroaryl” used alone or in combination with other terms refers to a monocyclic or polycyclic (e.g., having 2, 3 or 4 condensed rings) aromatic heterocyclic moiety having one or more heteroatom ring members selected from nitrogen, sulfur and oxygen. In certain embodiments, the heteroaryl has 1, 2, 3 or 4 heteroatom ring members. In certain embodiments, the heteroaryl has 1, 2 or 3 heteroatom ring members. In certain embodiments, the heteroaryl has 1 or 2 heteroatom ring members. In certain embodiments, the heteroaryl has 1 heteroatom ring member. In certain embodiments, the heteroaryl is 5- to 10-membered or 5- to 6-membered. In certain embodiments, the heteroaryl is 5-membered. In certain embodiments, the heteroaryl is 6-membered. Examples of the heteroaryl include, but are not limited to, pyrrolyl, imidazolyl, thiazolyl, furyl or pyridyl, etc.
The term “cycloalkyl” as used herein refers to a saturated cyclic hydrocarbon group having 3 to 12 carbon atoms and having a monocyclic or bicyclic or multiple fused rings (including fused and bridged ring systems), preferably having 3-10, 3-8, 5-8, 3-6 or 5-6 carbon atoms. Typical examples of “cycloalkyl” include, but are not limited to, monocyclic structures, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc.; bicyclic structures, such as bicyclo[2.2.1]heptyl, and polycyclic structures such as adamantyl and the like.
The term “heterocycloalkyl” as used herein refers to a cycloalkyl as defined herein that contains 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.
As used in this application, the term “aralkyl” used alone or in combination with other terms refers to a lower alkyl or cycloalkyl as defined above, in which one hydrogen atom has been substituted by an aryl as defined above, or in the case of cycloalkyl, two adjacent carbon atoms are fused in benzo form to a substituted or unsubstituted phenyl to form a bicyclic group.
As used in this application, the term “heteroalkyl” used alone or in combination with other terms refers to an alkyl in which one or more carbon atoms are substituted by heteroatoms independently selected from S, O and N.
The term “C1-6 alkoxy” as used herein refers to —OR11, where R11 is a 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.
The term “C1-6 alkylthio” as used herein refers to —SR11, where R11 is a C1-6 alkyl as defined herein. Typical examples of “C1-6 alkylthio” include, but are not limited to, methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, tert-butylthio, sec-butylthio, n-pentylthio, n-hexylthio, 1,2-dimethylbutylthio, etc.
When the names of compounds used herein is inconsistent with the chemical structural formulae, the chemical structural Formulae shall prevail.
According to some embodiments of the present invention, the pharmaceutically acceptable salt of the compound of general Formula (I) described in this application includes its inorganic or organic acid salt, and inorganic or organic base salt, and this application relates to all forms of the above-mentioned salt, which includes but not limited to: sodium salt, potassium salt, calcium salt, lithium salt, meglumine salt, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, hydrogensulfate, phosphate, hydrogenphosphate, acetate, propionate, butyrate, oxalate, trimethylacetate, adipate, alginate, lactate, citrate, tartrate, succinate, maleate, fumarate, picrate, aspartate, gluconate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate, etc.
According to some embodiments of the present invention, the compound of general Formula (I) of the present invention can form a pharmaceutically acceptable ester with an organic or inorganic acid, and the pharmaceutically acceptable ester includes phosphate, sulfate, nitrate, formate, acetate, propionate, butyrate, valerate, and caproate, etc., that are hydrolyzable in vivo.
The carrier of the present invention 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, partial glyceride mixture of plant saturated fatty acid, water, salt or electrolyte, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salt, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substance, polyethylene glycol, sodium carboxymethylcellulose, polyacrylate, beeswax, lanolin.
The term “excipient” as used in the present invention refers to an additive other than the main drug in a pharmaceutical preparation. It is stable in nature, has no incompatibility with the main drug, does not produce side effects, does not affect therapeutic effect, is not easy to be deformed, dried, cracked, funked, wormed, is harmless to the human body, has no physiological effect, and does not produce chemical or physical effect with the main drug, does not affect the content determination of the main drug, etc. For example, the binder, filler, disintegrant, lubricant in tablets; the preservative, antioxidant, flavor, fragrance, cosolvent, emulsifier, solubilizer, osmotic pressure regulator, coloring agent, etc. in oral liquid preparations can all be called excipients.
The pharmaceutical composition described in this application can be administered through various routes, such as oral tablet, capsule, powder, oral liquid, injection and transdermal preparation. The above-mentioned various dosage forms of drugs can be prepared according to conventional methods in the field of pharmacy. According to conventional pharmaceutical practices, pharmaceutically acceptable carriers include 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 ways: oral, 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 stated in this article, “effective amount” refers to an amount that is sufficient to treat or prevent or diagnose a disease of a patient, but is low enough 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 to the specifically selected compound (for example, considering the potency, effectiveness and half-life of the compound), the selected route of administration, 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 being treated, the duration of treatment or prevention or diagnosis, the nature of concurrent therapy, the required treatment or prevention or diagnosis effect, etc., but it could still be routinely determined by those skilled in the art.
In addition, it should be pointed out that the specific dosage and usage of the compound of general Formula (I) described in this application for different patients are determined by many factors, including the age, weight, gender, natural health status, nutritional status of the patient, the activity strength, administration time, metabolic rate of the compound, the severity of the disease and the subjective judgment of the physician. The preferred dosage here is between 0.001 to 100 mg/kg body weight/day.
The present disclosure can be further described through the following examples and test examples. However, the scope of the present disclosure 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 disclosure without departing from the spirit and scope of the present disclosure. This disclosure provides a general and/or specific description of the materials and methods used herein. Although many materials and operating methods used to achieve the purpose of the present disclosure are well-known in the art, the present disclosure is still described herein as much detail as possible.
For all the following examples, standard operations and purification methods known to those skilled in the art can be used. Unless otherwise stated, all temperatures were expressed in ° C. (Celsius). The structure of compound was determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). The melting point m.p. of compound was determined by RY-1 melting point instrument. The thermometer had not been corrected. The m.p. was given in ° C. 1H NMR was measured by JNM-ECA-400 nuclear magnetic resonance instrument of JEOL. The mass spectrum was measured by API3000 (ESI) instrument. All reaction solvents that were not specified were subject to standardized pretreatment.
To 10 ml of hydrazine hydrate (65 wt % aqueous solution), 5 g (0.018 mol) of (2R,3R,4S,5R)-2-(6-chloro-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (vi) was added, heated to 70° C. while stirring, continued heating for 2 hours until the reactant (I) disappeared, the reaction progress was monitored by TLC (CH2Cl2:MeOH=3:1 (v/v)). Then, the reaction mixture was heated to 25° C., diluted with 2-propanol (50 ml) and stirred overnight. The separated precipitate was filtered to obtain 4.8 g of (2R,3R,4S,5R)-2-(6-hydrazino-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (vii) as white solid, which was directly used in the next reaction.
0.5 g (0.0018 mol) of (2R,3R,4S,5R)-2-(6-hydrazino-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (II) and 0.19 g (0.002 mol) of pyrrole-2-carbaldehyde (1H-pyrrole-2-carbaldehyde, 1.1 equivalent) were mixed in methanol (20 ml) and heated by microwave at 70° C. for 30 minutes.
The crude product was precipitated from methanol. After filtration, the crude product was further purified by medium pressure preparative chromatography using C18 reverse phase column, and 323 mg of white solid (Compound 1) was obtained. m.p. 160° C.; 1H NMR (DMSO-d6): δ (ppm) 11.52(s, 1H), 11.44(s, 1H), 8.50(s, 1H), 8.34(s, 1H), 8.28(s, 1H), 6.90(s, 1H), 6.43(s, 1H), 6.14(s, 1H), 5.95(d, 1H, J=6.0 Hz), 5.54(d, 1H, J=6.0 Hz), 5.40(dd, 1H, J=2.0 Hz, 4.8 Hz), 5.26(d, 1H, J=4.4 Hz), 4.65(dd, 1H, J=5.6 Hz, 5.6 Hz), 4.18(d, 1H, J=3.6 Hz), 3.99(d, 1H, J=2.8 Hz), 3.73-3.55(m, 2H); HRMS (ESI+) m/z [M+H]+ calculated for C15H17N7O4: 360.1415; found: 360.1415.
The following compounds could be prepared by referring to the method of Example 1, using different reactants (such as the above-mentioned compound of Formula viii, various substituted formaldehydes) in place of pyrrole-2-carboxaldehyde in step 1.2.
The method of step 1.2 in Example 1 was adopted, in which 3-(methylsulfanyl)propanal was used in place of pyrrole-2-carboxaldehyde to prepare Compound 2, and 424 mg of white solid (Compound 2) was obtained. m.p. 94° C.; 1H NMR (DMSO-d6): δ (ppm) 11.42(s, 1H), 8.47(s, 1H), 8.31(s, 1H), 7.72(t, 1H, J=5.2 Hz), 5.93(d, 1H, J=6.0 Hz), 5.5-(d, 1H, J=6.0 Hz), 5.34(t, 1H, J=6.0 Hz), 5.23(d, 1H, J=4.8 Hz), 4.61(dd, 1H, J=5.2 Hz, 6.0 Hz), 4.16(d, 1H, J=3.6 Hz), 3.97(d, 1H, J=3.6 Hz), 3.71-3.54(m, 2H), 2.71(t, 2H, J=7.2 Hz), 2.58(t, 2H, J=6.2 Hz), 2.10(s, 3H); HRMS (ESI+) m/z [M+H]+ calculated for C14H20N6O4S: 369.1340; found: 369.1340.
The method of step 1.2 in Example 1 was adopted, and 4-acetamidobenzaldehyde (N-(4-formylphenyl)acetamide) was used in place of pyrrole-2-carbaldehyde to prepare Compound 3, and 331 mg of white solid (Compound 3) was obtained.
m.p. 170° C.; 1H NMR (DMSO-d6): δ (ppm) 11.72(s, 1H), 10.13(s, 1H), 8.53(s, 1H), 8.38(s, 1H), 7.67(s, 4H), 5.96(d, 1H, J=5.6 Hz), 5.51(d, 1H, J=6.0 Hz), 5.33(t, 1H, J=5.6 Hz), 5.24(d, 1H, J=4.8 Hz), 4.63(d, 1H, J=5.6 Hz), 4.17(s, 1H), 3.98(s, 1H), 3.72-3.55(m, 2H), 2.07(s, 3H); HRMS (ESI+) m/z [M+H]+ calculated for C19H21N7O5: 428.1677; found: 428.1677.
The method of step 1.2 in Example 1 was adopted, in which 3,4-bis(benzyloxy)benzaldehyde was used in place of pyrrole-2-carbaldehyde to prepare Compound 4, and 880 mg of white solid (Compound 4) was obtained. m.p. 186° C.; 1H NMR (DMSO-d6): δ (ppm) 11.71(s, 1H), 8.54(s, 1H), 8.39(s, 1H), 8.27(s, 1H), 7.53-7.14(m, 13H), 5.95(s, 1H), 5.51(s, 1H), 5.34(s, 1H), 5.25(s, 1H), 5.21(s, 4H), 4.63(s, 1H), 4.17(s, 1H), 3.99(s, 1H), 3.72-3.58(m, 2H); HRMS (ESI+) m/z [M+H]+ calculated for C31H30N6O6: 583.2300; found: 583.2298.
The method of step 1.2 in Example 1 was adopted, in which 5-(4-bromophenyl)furan-2-carbaldehyde was used in place of pyrrole-2-carbaldehyde to prepare Compound 5, and 712 mg of yellow solid (Compound 5) was obtained. m.p. 162° C.; 1H NMR (DMSO-d6): δ (ppm) 11.99(br, 1H), 8.58(s, 1H), 8.44(s, 1H), 8.35(s, 1H), 7.76(d, 2H, J=8.4 Hz), 7.68(d, 2H, J=8.4 Hz), 7.22(d, 1H, J=3.6 Hz), 7.02(d, 1H, J=3.6 Hz), 5.98(d, 1H, J=6.0 Hz), 5.55-5.28(br, 3H), 4.65(s, 1H), 4.19(s, 1H), 4.01(s, 1H), 3.73-3.57(m, 2H); HRMS (ESI+) m/z [M +H]+ calculated for C21H19BrN6O5: 515.0673; found: 515.0673.
The method of step 1.2 in Example 1 was adopted, in which 2,4-bis(trifluoromethyl)benzaldehyde was used in place of pyrrole-2-carbaldehyde to prepare Compound 6, and 592 mg of white solid (Compound 6) was obtained. m.p. 200° C.; 1H NMR (DMSO-d6): δ (ppm) 12.42(s, 1H), 8.78(s, 1H), 8.65(s, 1H), 8.62(d, 1H, J=8.4 Hz), 8.50(s, 1H), 8.19(d, 1H, J=8.4 Hz), 8.08(s, 1H), 6.01(d, 1H, J=6.0 Hz), 5.57(d, 1H, J=6.0 Hz), 5.31-5.28(m, 2H), 4.65(dd, 1H, J=4.8 Hz, 6.0 Hz), 4.20(d, 1H, J=3.6 Hz), 4.01(d, 1H, J=3.2 Hz), 3.75-3.57(m, 2H); HRMS (ESI+) m/z [M+H]+ calculated for C19H16F6N6O4: 507.1210; found: 507.1209.
The method of step 1.2 in Example 1 was adopted, in which 4-(4-fluorobenzyloxy)benzaldehyde (4-[(4-fluorophenyl)methoxy]benzaldehyde) was used in place of pyrrole-2-carboxaldehyde to prepare Compound 7, and 859 mg of white Solid (Compound 7) was obtained. m.p. 206° C.; 1H NMR (DMSO-d6): δ (ppm) 11.69(s, 1H), 8.54(s, 1H), 8.38(s, 1H), 8.31(s, 1H), 7.70(d, 2H, J=8.8 Hz), 7.53(t, 2H, J=5.6 Hz), 7.24(t, 2H, J=8.8 Hz), 7.11(d, 2H, J=8.4 Hz), 5.97(d, 1H, J=6.0 Hz), 5.52(d, 1H,J=6.4 Hz), 5.34(t, 1H, J=5.20 Hz), 5.24(d, 1H, J=4.4 Hz), 5.14(s, 2H), 4.63(d, 1H, J=5.6 Hz), 4.18(s, 1H), 3.99(s, 1H), 3.72-3.56(m, 2H); HRMS (ESI+) m/z [M+H]+ calculated for C24H23FN6O5: 495.1787; found: 495.1787.
The method of step 1.2 in Example 1 was adopted, in which 3-(benzyloxy)benzaldehyde was used in place of pyrrole-2-carbaldehyde to prepare Compound 8, and 568 mg of white solid (Compound 8) was obtained. m.p. 140° C.; 1H NMR (DMSO-d6): δ (ppm) 11.80(s, 1H), 8.57(s, 1H), 8.40(s, 1H), 8.32(s, 1H), 7.51-7.32(m, 8H), 7.07-7.04(m, 1H), 5.97(d, 1H, J=5.6 Hz), 5.49(d, 1H, J=6.4 Hz), 5.29(t, 1H, J=5.20 Hz), 5.21(d, 1H, J=4.8 Hz), 5.17(s, 2H), 4.63(dd, 1H, J=5.6 Hz, 5.6 Hz), 4.18(dd, 1H, J=3.6 Hz, 4.8 Hz), 3.99(d, 1H, J=3.2 Hz), 3.73-3.55(m, 2H); HRMS (ESI+) m/z [M+H]+ calculated for C24H24N6O5: 477.1881; found: 477.1883.
The method in step 1.2 of Example 1 was adopted, in which 4-(pyridin-2-yl)benzaldehyde was used in place of pyrrole-2-carbaldehyde to prepare Compound 9, and 652 mg of white solid (Compound 9) was obtained. m.p. 236° C.; 1H NMR (DMSO-d6): δ (ppm) 11.88(s, 1H), 8.70(d, 1H, J=5.6 Hz), 8.58(s, 1H), 8.43(s, 2H), 8.20(d, 2H, J=8.4 Hz), 8.03(d, 1H,J=8.0 Hz), 7.92(d, 1H, J=9.2 Hz), 7.88(d, 2H, J=8.4 Hz), 7.40-7.37(m, 1H), 5.99(d, 1H, J=6.0 Hz), 5.51(d, 1H,J=6.4 Hz), 5.30(t, 1H, J=6.4 Hz), 5.22(d, 1H, J=4.8 Hz), 4.64(dd, 1H, J=5.6 Hz, 5.6 Hz), 4.19(dd, 1H, J=3.6 Hz, 4.4 Hz), 4.00(d, 1H, J=3.2 Hz), 3.73-3.57(m, 2H); HRMS (ESI+) m/z [M+H]+ calculated for C22H21N7O4: 448.1728; found: 448.1729.
The method of step 1.2 in Example 1 was adopted, in which 4-phenylbenzaldehyde was used in place of pyrrole-2-carboxaldehyde to prepare Compound 10, and 698 mg of white solid (Compound 10) was obtained. m.p. 170° C.; 1H NMR (DMSO-d6): δ (ppm) 11.87(s, 1H), 8.59(s, 1H), 8.43(s, 2H), 7.86 (d, 2H, J=8.0 Hz), 7.78(d, 2H, J=8.4 Hz), 7.74(d, 2H, J=7.2 Hz), 7.51(t, 2H, J=7.2 Hz), 7.41(t, 1H, J=7.6 Hz), 5.99(d, 1H, J=5.6 Hz), 5.54(d, 1H, J=6.4 Hz), 5.34(t, 1H, J=5.6 Hz), 5.26(d, 1H, J=4.8 Hz), 4.64(dd, 1H, J=5.2 Hz, 5.6 Hz), 4.19(dd, 1H, J=3.6 Hz, 4.4 Hz), 4.00(d, 1H, J=4.0 Hz), 3.74-3.57(m, 2H); HRMS (ESI+) m/z [M+H]+ calculated for C23H22N6O4: 447.1775; found: 447.1775.
The method of step 1.2 in Example 1 was adopted, in which 4-(1-pyrrolidin-1-yl)benzaldehyde was used in place of pyrrolidin-2-carbaldehyde to prepare Compound 11, and 664 mg of white solid (Compound 11) was obtained. m.p. 202° C.; 1H NMR (DMSO-d6): δ (ppm) 11.43(s, 1H), 8.48(s, 1H), 8.33(s, 1H), 8.24(s, 1H), 7.54 (d, 2H, J=8.4 Hz), 6.59(d, 2H, J=8.4 Hz), 5.95(d, 1H, J=6.4 Hz), 5.48(d, 1H, J=6.0 Hz), 5.36(t, 1H, J=4.8 Hz), 5.20(d, 1H, J=4.8 Hz), 4.63(d, 1H, J=5.2 Hz), 4.17(d, 1H, J=2.8 Hz), 3.98(d, 1H, J=4.0 Hz), 3.72-3.55(m, 2H), 3.3(t, 4H, J=6.6 Hz), 1.97(s, 4H); HRMS (ESI+) m/z [M+H]+ calculated for C21H25N7O4: 440.2041; found: 440.2039.
The method of step 1.2 in Example 1 was adopted, in which 4-(1H-imidazol-1-yl)benzaldehyde was used in place of pyrrole-2-carboxaldehyde to prepare Compound 12, and 683 mg white solid (Compound 12) was obtained. m.p. 222° C.; 1H NMR (DMSO-d6): δ (ppm) 11.87(s, 1H), 8.57(s, 1H), 8.41(s, 1H), 8.38(s, 1H), 8.35(s, 1H), 7.90 (d, 2H, J=8.4 Hz), 7.83(s, 1H), 7.76(d, 2H, J=8.8 Hz), 7.14(s, 1H), 5.98(d, 1H, J=6.0 Hz), 5.50(d, 1H, J=6.0 Hz), 5.29(t, 1H, J=6.0 Hz), 5.22(d, 1H, J=4.8 Hz), 4.63(dd, 1H, J=5.2 Hz, 5.6 Hz), 4.19(dd, 1H, J=3.6 Hz, 4.4 Hz), 3.99(d, 1H, J=3.6 Hz), 3.74-3.56(m, 2H); HRMS (ESI+) m/z [M+H]+ calculated for C20H20N8O4: 437.1680; found: 437.1714.
The method of step 1.2 in Example 1 was adopted, in which 4-propoxybenzaldehyde was used in place of pyrrole-2-carboxaldehyde to prepare Compound 13, and 686 mg of white solid (Compound 13) was obtained. m.p. 202° C.; 1H NMR (DMSO-d6): δ (ppm) 11.67(s, 1H), 8.54(s, 1H), 8.38(s, 1H), 8.31(s, 1H), 7.68 (d, 2H, J=8.4 Hz), 7.01(d, 2H, J=8.8 Hz), 5.97(d, 1H, J=5.6 Hz), 5.52(d, 1H,J=6.0 Hz), 5.35(s, 1H), 5.24(d, 1H, J=4.8 Hz), 4.63(d, 1H, J=4.8 Hz), 4.18(d, 1H, J=3.2 Hz), 3.99(s, 2H), 3.96(s, 1H), 3.72-3.57(m, 2H), 1.75(sext, 2H, J=7.2 Hz, 6.8 Hz, 6.4 Hz), 1.00(t, 3H, J=7.6 Hz); HRMS (ESI+) m/z [M+H]+ calculated for C20H24N6O5: 429.1881; found: 429.1881.
The method of step 1.2 in Example 1 was adopted, in which 4-(trifluoromethyl)benzaldehyde was used in place of pyrrole-2-carboxaldehyde to prepare Compound 14, and 639 mg of white solid was obtained. m.p. 182° C.; 1H NMR (DMSO-d6): δ (ppm) 12.06(s, 1H), 8.60(s, 1H), 8.45(s, 1H), 8.42(s, 1H), 7.97 (d, 2H, J=8.4 Hz), 7.82(d, 2H, J=8.8 Hz), 5.99(d, 1H, J=5.6 Hz), 5.54(d, 1H,J=5.6 Hz), 5.31(t, 1H, J=5.2 Hz), 5.26(d, 1H, J=4.8 Hz), 4.63(dd, 1H, J=5.2 Hz, 5.6 Hz), 4.19(dd, 1H, J=3.6 Hz, 4.4 Hz), 3.96(d, 1H, J=3.6 Hz), 3.73-3.59(m, 2H); HRMS (ESI+) m/z [M+H]+ calculated for C18H17F3N6O4: 439.1336; found: 439.1336.
The method in step 1.2 of Example 1 was adopted, in which 5-bromo-2-pyridinaldehyde (5-bromopyridine-2-carbaldehyde) was used in place of pyrrole-2-carbaldehyde to prepare Compound 15, and 590 mg of yellow solid (Compound 15) was obtained. m.p. 211° C.; 1H NMR (DMSO-d6): δ (ppm) 12.14(s, 1H), 8.72(s, 1H), 8.61(s, 1H), 8.45(s, 1H), 8.36(s, 1H), 8.15 (d, 1H, J=8.0 Hz), 8.03(d, 1H, J=8.8 Hz), 5.99(d, 1H, J=6.0 Hz), 5.50(d, 1H,J=6.0 Hz), 5.25(t, 1H, J=6.0 Hz), 5.22(d, 1H, J=4.8 Hz), 4.62(dd, 1H, J=5.2 Hz, 5.6 Hz), 4.19(dd, 1H, J=3.6 Hz, 4.8 Hz), 3.99(d, 1H, J=3.2 Hz), 3.73-3.56(m, 2H); HRMS (ESI+) m/z [M+H]+ calculated for C16H16BrN7O4: 450.0520; found: 450.0520.
The method in step 1.2 of Example 1 was obtained, in which thiazole-5-formaldehyde (1,3-thiazole-5-carbaldehyde) was used in place of pyrrole-2-carbaldehyde to prepare Compound 16, and 440 mg of white solid (Compound 16) was obtained. m.p. 224° C.; 1H NMR (DMSO-d6): δ (ppm) 12.00(s, 1H), 9.13(s, 1H), 8.65(s, 1H), 8.56(s, 1H), 8.42(s, 1H), 8.20 (s, 1H), 5.96(d, 1H, J=6.0 Hz), 5.52(d, 1H ,J=6.0 Hz), 5.30(s, 1H), 5.24(d, 1H, J=4.8 Hz), 4.62(dd, 1H, J=5.2 Hz, 5.6 Hz), 4.17(dd, 1H, J=3.6 Hz), 3.98(d, 1H, J=3.6 Hz), 3.72-3.55(m, 2H); HRMS (ESI+) m/z [M+H]+ calculated for C14H15N7O4S: 378.0979; found: 378.0978.
The method in step 1.2 of Example 1 was adopted, in which 4-dimethylamino-cinnamaldehyde ((2E)-3-[4-(dimethylamino)phenyl]prop-2-enal) was used in place of pyrrole-2-carboxaldehyde to prepare Compound 17, and 490 mg of yellow solid (Compound 17) was obtained. m.p. 172° C.; 1H NMR (DMSO-d6): δ (ppm) 11.57(br, 1H), 8.51(d, 1H, J=3.6 Hz), 8.37(d, 1H, J=6.0 Hz), 8.16(br, 1H), 7.44(d, 1H, J=8.4 Hz), 7.31(d, 1H, J=8.8 Hz), 6.83-6.70(m, 4H), 5.95(d, 1H, J=5.6 Hz), 5.52(d, 1H, J=6.0 Hz), 5.35(dd, 1H, J=5.2 Hz, 6.0 Hz), 5.24(d, 1H, J=4.4 Hz), 4.63(dd, 1H, J=4.8 Hz, 6.0 Hz), 4.17(d, 1H, J=3.6 Hz), 3.98(d, 1H, J=3.2 Hz), 3.71-3.56(m, 2H), 2.95(s, 6H); HRMS (ESI+) m/z [M+H]+ calculated for C21H25N7O4: 440.2041; found: 440.2044.
The method of step 1.2 in Example 1 was adopted, in which 4-chloro-3-(trifluoromethyl)benzaldehyde was used in place of pyrrole-2-carboxaldehyde to prepare Compound 18, and 600 mg of white solid was obtained. m.p. 196° C.; 1H NMR (DMSO-d6): δ (ppm) 12.10(s, 1H), 8.60(s, 1H), 8.45(s, 1H), 8.40(s, 1H), 8.22 (s, 1H), 8.06(d, 1H, J=8.4 Hz), 7.82(d, 1H, J=8.4 Hz), 5.99(d, 1H, J=6.0 Hz), 5.53(d, 1H,J=5.6 Hz), 5.29(t, 1H, J=5.6 Hz), 5.25(d, 1H, J=4.8 Hz), 4.63(dd, 1H, J=5.2 Hz, 6.0 Hz), 4.19(dd, 1H, J=3.6 Hz, 4.8 Hz), 3.99(d, 1H, J=3.6 Hz), 3.73-3.56(m, 2H); HRMS (ESI+) m/z [M+H]+ calculated for C18H16ClF3N6O4: 473.0946; found: 473.0945.
The method of step 1.2 in Example 1 was obtained, in which 4-(N,N-diphenylamino)benzaldehyde (4-(diphenylamino)benzaldehyde) was used in place of pyrrole-2-carbaldehyde to prepare Compound 19, and 460 mg of white solid (Compound 19) was obtained. m.p. 160° C.; 1H NMR (DMSO-d6): δ (ppm) 11.66(s, 1H), 8.51(s, 1H), 8.37(s, 1H), 8.31(s, 1H), 7.63(d, 2H, J=8.4 Hz), 7.35(t, 4H, J=8.0 Hz), 7.13-7.07(m, 6H), 6.99(d, 2H, J=8.8 Hz), 5.96(d, 1H, J=6.0 Hz), 5.48(d, 1H,J=6.0 Hz), 5.31(t, 1H, J=6.0 Hz), 5.20(d, 1H, J=4.4 Hz), 4.62(dd, 1H, J=5.2 Hz, 5.6 Hz), 4.17(dd, 1H, J=3.6 Hz, 4.8 Hz), 3.98(d, 1H, J=3.2 Hz), 3.72-3.55(m, 2H); HRMS (ESI+) m/z [M+H]+ calculated for C29H27N7O4: 538.2197; found: 538.2198.
The method in step 1.2 of Example 1 was adopted, in which 2-butyl-5-chloro-1H-imidazole-4-carbaldehyde was used in place of pyrrole-2-carbaldehyde to prepare Compound 20, and 640 mg of white solid (Compound 20) was obtained. m.p. 178° C.; 1H NMR (DMSO-d6): δ (ppm) 12.79(s, 1H), 11.78(s, 1H), 8.53(s, 1H), 8.38(s, 1H), 8.36(s, 1H), 5.96(d, 1H, J=6.0 Hz), 5.53(d, 1H, J=6.4 Hz), 5.36(s, 1H), 5.26(d, 1H, J=4.4 Hz), 4.64(dd, 1H, J=5.2 Hz, 5.6 Hz), 4.17(d, 1H, J=3.6 Hz), 3.99(d, 1H, J=2.8 Hz), 3.71-3.56(m, 2H), 2.66(t, 2H, J=7.6 Hz), 1.62(quint, 2H, J=7.6 Hz,7.2 Hz), 1.29(sext, 2H, J=7.6 Hz, 7.6 Hz, 7.2 Hz), 0.891(t, 3H, J=7.2 Hz); HRMS (ESI+) m/z [M+H]+ calculated for C18H23ClN8O4: 451.1604; found: 451.1606.
The method of step 1.2 in Example 1 was adopted, in which (2E,4E)-deca-2,4-dienal was used in place of pyrrole-2-carboxaldehyde to prepare Compound 21, and 112 mg of white solid (Compound 21) was obtained. m.p. 178° C.; 1H NMR (DMSO-d6): δ (ppm) 11.59(s, 1H), 8.50(s, 1H), 8.33(s, 1H), 8.04(d, 1H, J=9.6 Hz), 6.61-6.00(m, 3H), 5.93(d, 1H, J=5.6 Hz), 5.50(d, 1H, J=6.4 Hz), 5.32(t, 1H, J=4.8 Hz), 5.23(d, 1H, J=4.8 Hz), 4.61(dd, 1H, J=5.2 Hz, 6.0 Hz), 4.16(d, 1H, J=3.6 Hz), 3.97(d, 1H, J=3.2 Hz), 3.71-3.54(m, 2H), 2.13(dd, 1H, J=6.8 Hz, 7.2 Hz), 1.43-1.23(m, 8H), 0.88(t, 3H, J=6.8 Hz); HRMS (ESI+) m/z [M+H]+ calculated for C20H28N6O4: 417.2245; found: 417.2245.
The method of step 1.2 in Example 1 was adopted, in which cyclohexanecarbaldehyde was used in place of pyrrole-2-carbaldehyde to prepare Compound 22, and 300 mg of white solid (Compound 22) was obtained. m.p. 132° C.; 1H NMR (DMSO-d6): δ (ppm) 11.25(s, 1H), 8.46(s, 1H), 8.30(s, 1H), 7.60(d, 1H, J=4.8 Hz), 5.93(d, 1H, J=6.4 Hz), 5.50(d, 1H, J=6.0 Hz), 5.35(s, 1H), 5.23(d, 1H, J=4.4 Hz), 4.61(dd, 1H, J=5.6 Hz, 5.6 Hz), 4.16(d, 1H, J=3.2 Hz, 4.4 Hz), 3.97(dd, 1H, J=3.2 Hz, 3.6 Hz), 3.70-3.54(m, 2H), 2.27(d, 1H, J=4.8 Hz), 1.80-1.62(m, 5H), 1.35-1.18(m, 5H); HRMS (ESI+) m/z [M+H]+ calculated for C17H24N6O4: 377.1932; found: 377.1934.
[3H]CGS21680 (2-[p-(2-carboxyethyl)phenylethylamino]-5′-N-ethylformamidoadenosine, [carboxy-1-ethyl-3H(N)]; 250 μCi) was purchased from PerkinElmer Research Products (Boston, Mass.).
Cell membrane stably transfected with (human) A2A adenosine receptor was prepared in HEK-293 cells. The cell membrane was obtained from PerkinElmer Research Products (Boston, Mass.).
CGS21680 (2-[p-(2-carboxyethyl)phenylethylamino]-5′-N-ethylformamidoadenosine) was purchased from Selleck (Shanghai, CN).
All other reagents were of analytical grade and obtained from commercial sources.
The A2A adenosine receptors used were all expressed in the cell membrane. The compound was diluted 3 times serially with DMSO (Solarbio, D8371-250 ml) so as to generate a compound source plate with 10 different concentrations (10 μM, 3.3 μM, 1.1 μM, 0.0412 μM, 0.0137 μM, 0.0046 μM, 0.0015 μM, 0.0005 μM); 250 nL of the compound was added to a 384-well Opti-plate, sealed with parafilm; to 1 mL of detection buffer (50 mM Tris-HCl pH 7.4, 10 mM MgCl2, 1 mM EDTA, 1 μg/mL adenosine deaminase), 20 U of hA2A HEK-293 cell membrane was added for dilution; to the diluted cell membrane, 0.75 μCi [3H]CGS 21680 (final 25 nM) was added and mixed well; 50 μL of the prepared cell membrane diluent was transferred to a 384-well Opti-plate containing a new compound, and incubated at 25° C. for 90 minutes; to a UNIFILTER-96 GF/B filter plate, 100 μL of 0.5% polyethyleneimine solution (PEI) was added to soak at 4° C. for 90 min; then Cell Harvester was used to transfer 500 μL of washing buffer/well (50 mM Tris-HCl pH 7.4, 154 mM NaCl), and the UNIFILTER-96 GF/B filter plate was washed twice; the mixture 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 a 37° C. incubator for 3 min; 40 μL of ULTIMA GOLD scintillation solution (Perkin Elmer, Cat #77-16061) was added to each well, and MicroBeta liquid scintillation counter (PerkinElmer) was used to read CPM (count per minute) value. 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 based on the compound concentration and the specific binding percentage of [3H]CGS21680 by curve fitting.
The inhibition constant (Ki) value was calculated from the IC50 value according to the Cheng and Prusoff equation, Ki=IC50/(1+[S]/Km), in which [S] was the concentration of the radioligand (25 nM), and Km was the human A2AAR dissociation constant (22 nM) of [3H]CGS21680. The inhibition constant Ki values for Compounds 1 to 20 of the present invention binding to A2A adenosine receptor were shown in Table 1.
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 purchased from Gibico, Hyclone and Sigma, respectively. 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 white solid plate were purchased from Labcyte and Corning, respectively.
Experimental instruments: TECAN automated pipetting workstation, Echo ultrasonic pipetting system, and EnVison microplate reader were purchased from TECAN, Labcyte and Envision, respectively.
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., 5% CO2 environment. Before the experiment, the cells were digested with Versene solution, and the cells were collected by centrifugation at 200 g at room temperature for 5 minutes, and finally resuspended with detection buffer (Hank's buffered saline solution, containing 5 mM HEPES, 0.1% BSA stabilizer and 10 μM Rolipram, pH 7.4). TECAN automated pipetting workstation was used to prepare a compound source plate by 3-fold diluting the compound in a 384-well polypropylene microplate with DMSO to form 11 concentration points, in which the 11 concentration points of the compound were 10 mM, 3.33 mM, 1.11 mM, 0.37 mM, 0.12 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, respectively. Echo ultrasonic pipetting system (Labcyte) was used to transfer the test compound from the compound source plate to the detection plate, in which the volume of the compound transferred was 10 nl/well. The hADORA2A-HEK293 cell suspension was diluted with detection buffer to 30,000 cells/ml, and the cell suspension was transferred to the detection plate at a volume of 10 μl/well (300 cells/well). The detection plate was centrifuged at 150 g for 1 minute and pre-incubated at room temperature for 30 minutes. Eu-cAMP tracer working solution (40 μl of Eu-cAMP tracer, 1.96 ml of cAMP detection buffer) was added to the detection plate (5 μl/well), and then Ulight-anti-cAMP working solution (13 μl of Ulight-anti-cAMP reagent, and 1.95 ml of cAMP detection buffer) was added to the detection plate (5 μl/well). The detection plate was rotated at 150 g for 30 seconds, and incubated at room temperature for 30 minutes. EnVison microplate reader (EnVision multimode plate reader, PerkinElmer) was used to test 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 A2A adenosine receptor to stimulate the production of a level of cyclic adenosine monophosphate was calculated. The compound A2A receptor agonist titer was expressed as the EC50 (nM) value of the compound interacting with the A2A adenosine receptor to stimulate the production of a level of cyclic adenosine monophosphate.
The EC50 (nM) values of the test compounds interacting with A2AAR to stimulate AMP level were shown in Table 2. The results showed that Compounds 7, 15 and 16 prepared by the present invention were all hA2AAR agonists. When Compounds 7, 15 and 16 interacted with A2AAR, their inhibition constant Ki values and EC50 values of stimulating cAMP were basically in the same nanomolar range.
Fluorescein-labeled dextran FITC-Dextran (CAS: 60842-46-8) with a molecular weight of 10,000 MW was purchased from Tixiai (Shanghai) Chemical Industry Development Co., Ltd.; PBS solution and experimental animal SD rats were obtained from commercial sources.
FITC-Dextran solution was prepared with PBS to obtain six concentration gradients (0.001, 0.01, 0.1, 1, 0.5, 10 μg/ml), and a FITC-Dextran concentration standard curve was prepared by using microplate reader (λex=490 nm, λem=520 nm); 10 mg/ml FITC-Dextran solution was separately prepared, Compound 5 was added to PBS solution to make 1 mg/ml solution, 1 ml of 10 mg/ml FITC-Dextran solution and 1 ml of 1 mg/ml Compound 5 PBS solution were taken to make an administration solution; 1 ml of 10 mg/ml FITC-Dextran solution and 1 ml of PBS solution taken to make a blank control solution; 6 SD rats were injected with 2 ml of the administration solution respectively in the tail vein, while another 6 SD rats were injected with 2 ml of the blank control solution in the tail vein; after 30 minutes, the brain tissues of all SD rats were taken out, homogenized and centrifuged at 10,000 rpm for 15 minutes, and the supernatants were taken for testing; and a microplate reader (λex=490 nm, λem=520 nm) was used for the fluorescence detection of the solutions to be tested.
The fluorescence values measured by the microplate reader were converted into the corresponding FITC-Dextran average concentrations according to the obtained FITC-Dextran concentration standard curve. The results were shown in Table 3. The results showed that the macromolecule FITC-Dextran itself could pass through the blood-brain barrier, while the FITC-Dextran added with Compound 5 could enter the brain through the BBB, indicating that Compound 5 could open the blood-brain barrier.
Although the specific embodiments of the present disclosure 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 within the protection scope of the present disclosure. The full scope of the disclosure is given by the appended claims and any equivalents thereof. The publications and patent documents cited in this disclosure are incorporated herein by reference.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2019 10542462.9 | Jun 2019 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/097427 | 6/22/2020 | WO |
