CN107286146B - 4-aminopyrimidine derivatives as adenosine A2A receptor antagonists and uses thereof - Google Patents

Abstract

The invention discloses a compound serving as adenosine A_2A4-aminopyrimidine derivatives of receptor antagonists having the general structural formula (i):

wherein R is₁Selected from halogen, cyano or trifluoromethyl; r₂Selected from pyrazolyl, pyrrolidinyl, or substituted by one or more halogens or C_1‑3Alkyl-substituted pyrazolyl or pyrrolidinyl; r₃Selected from oxazolyl, oxadiazolyl, triazolyl, or substituted with one or more halogens or C_1‑3An alkyl-substituted oxazolyl group. The 4-aminopyrimidine derivative provided by the invention is human adenosine A_2AThe receptor has obvious antagonistic effect, and can be used for treating A_2AAntagonism-responsive diseases or conditions, particularly neurodegenerative diseases, extra-pyramidal syndrome, depression, hyperactivity syndrome, sleep disorders, anxiety, diabetes or tumours.

Description

As adenosine A2A4-aminopyrimidine derivatives of receptor antagonists and their use

Technical Field

The invention relates to the field of medicine and organic chemistry, in particular to adenosine A_2A4-aminopyrimidine derivatives of receptor antagonists and their use.

Background

As endogenous regulators of many physiological functions in the Central (CNS) and peripheral nervous systems, adenosine is widely distributed in multiple tissues and organs such as the nervous system, cardiovascular system, digestive system, respiratory system, etc., and plays a role in regulating various important physiological processes. It exerts its biological effects through a class of membrane-specific receptors belonging to the superfamily of G protein-coupled receptors, for which 4 subtypes of adenosine receptors have been identified, which are: a. the₁、A_2A、A_2BAnd A₃. Wherein A is₁And A_2AIs a highly expressed receptor which acts at physiologically low adenosine concentrations, and A_2BAnd A₃The expression level of (A) is low, and the adenosine can be activated only when the adenosine is greatly increased under pathological conditions, so that a series of pathological effects are generated. A. the₁And A₃Receptors down-regulate cellular levels by coupling them to G proteins that inhibit adenylate cyclase; in contrast, A_2AAnd A_2BThe receptor is coupled to Gs proteins that activate adenylate cyclase and increase intracellular levels. Through the action of these receptors, adenosine is capable of achieving a wide range of modulation of physiological functions.

A_2AReceptors are mostly found in dopamine rich areas such as basal ganglia elements, striatum and globus pallidus in various mammals including humans. The basal ganglia and striatum asThe central element, which is involved in the integration of information in the cortex, thalamus and peripheral lobes to produce motor behavior. A has been found in striatum_2AThe receptor and dopamine D2 receptor are tightly co-localized to striatal globus pallidus gabaergic neurons, forming a so-called indirect export pathway from the striatum that is associated with inhibition of motility. A. the_2AThe receptor regulates the neurotransmission of gamma-aminobutyric acid, dopamine, acetylcholine and glutamate through a variety of pathways facilitating control of motor behaviour. In general, A_2ABy interaction with the D2 receptor, especially A_2AThe effect as an antagonist is of great benefit for the treatment of parkinson's disease, which can lead to a reduction in dopamine levels. A. the_2AThe receptor interacts tightly and antagonistically with the D2 receptor, causing a decrease in the affinity of the D2 receptor for dopamine when stimulated. Thus, A_2AReceptor antagonists can potentiate the effects of endogenous dopamine as well as clinically used dopamine receptor agonists and increase the drug response time limit of dopaminergic activity.

Selectivity A_2AReceptor agonists and antagonists have been extensively described in experiments of pharmacology, behavior, and neuroprotection in rodents and non-human primates. At D2 receptor antagonist and A_2AD2 and A can be clearly exemplified in the model of receptor agonist-induced catalepsy_2AThe close interaction of the receptor in catalepsy models, which is mediated by A_2AReceptor antagonists and D2 receptor agonists. At present, a number of researchers have reported that_2APotential of receptor antagonists against defibrillation. For example, adenosine A_2AThe receptor antagonists SCH58261 and KW-6002, both enhanced bilateral rotation induced by sub-domain doses of levodopa in unilateral 6-hydroxydopamine (6-OHDA) injured mice and rats. In addition, adenosine A has been generally reported_2AThe receptor antagonist KW-6002 significantly ameliorates non-dyskinetic motor impairment induced by 1-methyl-4-phenyl-1, 2,3, 6-tetrahydropyridine (MPTP) in non-human primates by long-term treatment with the dopamine receptor agonist levodopa. Thus, since it not only reverses motor impairment but also can slow or stop disease progression by extending cell life, a_2AReceptor antagonists as parpFuture drugs for long-term use by patients with parkinson's disease show great potential.

Several preclinical studies have shown adenosine A_2AReceptor antagonists are useful for the treatment of neurodegenerative diseases such as parkinson's disease, huntington's disease or alzheimer's disease. And has been reported to be A_2AReceptor antagonists have neuroprotective effects in vivo and in vitro models of different neurodegenerative diseases. In summary, A_2AReceptor antagonists are effective in protecting different neurons from various forms of injury-induced neurodegeneration.

Has the research finding A_2AReceptor knockout mice are less sensitive to "inhibitor" challenge than their wild-type counterparts. Consistent with this study, A was tested in the mouse tail suspension experiment_2AReceptor antagonists SCH58261 and KW6002 reduced total immobility time. It was also found that the antagonists SCH8261 and ZM241385 reduced immobility when administered to previously screened mice for high immobility time, while SCH58261 reduced immobility in mice selectively raised for their "helpless" in this model. With A_2AKnockout mouse studies have shown that these animals are sluggish to psychostimulants such as amphetamine and ***e. Thus, there is evidence to suggest that adenosine A_2AReceptor antagonists may have antidepressant and/or antipsychotic properties via a dopaminergic pathway that modulates the mesostriatum or the mesocorticoid. A. the_2AReceptor activation can contribute to the amelioration of a range of neuropsychiatric diseases and disorders, such as depression, excessive daytime sleepiness, restless leg syndrome (R L S), attention deficit hyperactivity disorder and cognitive fatigue.

Extrapyramidal syndrome (EPS) is a generic term for a series of adverse neurological reactions associated with the use of antipsychotics. There are 6 different classes of EPS-associated neurological syndromes, of which 4, dystonia, akathisia, pseudoparkinsonism (Parkinson's syndrome) and delayed dyskinesia, are particularly prevalent in patients treated with antipsychotic drugs. Dystonia is a painful spasm of the muscle groups, particularly the neck, chin, back, pharynx and larynx. It is most commonly used in young men treated with antipsychotics, but may also be associated with the use of ***e, tricyclic antidepressants, lithium salt anticonvulsants (e.g., phenytoin and carbamazepine). Pseudoparkinsonism manifests itself as akinesia (rigidity, stiffness and slow voluntary movement, humpback, trails) and tremor, and these symptoms occur weeks or months after the start of therapy. Sedentary sitting fails to manifest itself as hyperkinetic movement, subjective internal sensory distress or discomfort, often misinterpreted as agitation or anxiety, a common syndrome that is often undiagnosed and minimally responsive to treatment. Prolonged dyskinesia is a later-occurring syndrome associated with chronic use of neuroleptic drugs. It occurs more often in elderly patients and is characterized by stereotypic, repetitive, involuntary, rapid chorea-like movements of the face, eyelids, mouth, tongue, limbs and body.

Akathisia is also characteristic of R L S and P L MS (periodic limb movements during sleep) and P L MD (periodic leg (or limb) movement disorders) R L S is a general disorder that causes patients to have an intolerant and unpleasant desire to move their legs, often times develops at rest and/or at night, and may disturb sleep patients who do not have the typical R L S symptoms, but who exhibit intermittent leg movements that adversely affect sleep are diagnosed as P L ms.r L S and P L MS treatments have included levodopa/carbidopa, levodopa/benserazide, dopamine agonists (such as pramipexole and ropinirole), benzodiazepines

Opioids, anticonvulsants and iron (ferrous sulfate).

In the CNS, data show A_2AReceptors are present in high density in the basal ganglia, which are important in controlling good movement. Furthermore, A_2ASelective antagonists of the receptor are pharmacologically important because they exhibit efficacy in reducing motor impairment, thereby improving function in neurodegenerative diseases, such as parkinson's disease and related movement disorders (e.g., huntington's chorea). Compared to current dopaminergic treatments that bring about an increase in the therapeutic index, A_2AAntagonists appear to exhibit a decrease inLow propensity for side effects (e.g., no dyskinesia). A. the_2AAntagonists also have antidepressant properties and stimulate cognitive function.

Thus, it is desirable to find novel, highly effective and selective adenosine A_2AReceptor antagonists are of increasing interest. Some effective adenosine A discovered by pharmaceutical companies_2AAntagonists have entered clinical trials and show positive results, indicating a_2AReceptor antagonists are expected to be useful not only in the treatment of neurodegenerative diseases such as parkinson's disease, huntington's disease or alzheimer's disease, but also in the treatment of other CNS related diseases such as depression, hyperactivity syndrome, sleep disorders and anxiety disorders.

In addition, adenosine A_2AReceptors are also closely related to immunomodulation. Immunoregulation is an important means for keeping the body homeostasis and resisting external harmful stimuli. Adenosine, an important transmitter and modulator of the body, can be greatly increased in metabolic disturbance and cell injury, activates adenosine receptors to play a biological effect, and participates in the immune regulation of the body. Recent studies have shown that adenosine A is involved in many pathological processes such as ischemic hypoxia, inflammation, trauma, transplantation, etc_2AActivation of the receptor may play an important immunomodulatory role, possibly in conjunction with A_2AThe receptor is related to high expression level on various immune cells such as T cells, B cells, mononuclear macrophages, neutrophils and the like.

A_2AThe receptor is closely related to the tumor. Normally, the body can rely on an intact immune mechanism to effectively monitor and reject cancerous cells, such as: in the aspect of cellular immunity, T lymphocytes, antibody-dependent cytotoxic cells (K cells), NK cells and macrophages all have killing effects on tumor cells. However, if the function of the cancer cells themselves or the above immune cells is changed, the cancer cells may escape the immune system and may be malignant to form tumor. Studies have shown that A_2AThe activation of the receptor can promote the organism to generate immune tolerance, and is closely involved in the formation of 'immune escape' or 'immune suppression' of tumor cells, thereby creating favorable conditions for the occurrence and development of tumors. A. the_2AReceptor activation can be achieved by inhibiting vascular endotheliumThe expression of the cellular thrombospondin promotes angiogenesis in a dose-dependent manner, so that a favorable environment is created for the growth of the blood vessel-dependent tumor; a. the_2AThe activation of the receptor can also inhibit the killing of natural killer cells to tumor cells by raising cAMP and activating PKA; can promote proliferation of tumor cells such as melanoma A375 cells, fibroblast NIH3T3 cells and pheochromocytoma PC12 cells. It has been shown that A_2AThe receptor antagonist can be used for treating various tumors, such as lung cancer, especially non-small cell lung cancer.

Is suitable for A_2ACandidate products for receptor antagonists requiring potent co-A_2AReceptor binding but not strong binding to other adenosine receptors, i.e. higher A is required_2AReceptor subtype selectivity, which helps to reduce potential side effects. A number of small molecules of adenosine A are now available_2AReceptor antagonists are entering clinical phase I studies for the treatment of tumors. Such as CPI-444 from Corvus corporation, showing binding A_2AThe affinity (Ki) of the receptor was 3.5nm, A₁The receptor affinity (Ki) was 192nm for A₁Based on these results, researchers believe that CPI-444 has sufficient safety and potential efficacy at the currently expected dose levels over a reasonable dose range, CPI-444 has been tested in 3 mouse models of different growing tumors, namely the E L-4 lymphoma model, the MC38 colon tumor model and the CT26 colon tumor model, and has shown a significant reduction in the number of lymph nodes at the cancer cells, or a significant reduction in tumor volume at the primary site, or a steady or regressive tumor volume, or even some mice have shown complete cure_2AThe receptor antagonist can be used for treating tumor.

Furthermore, A_2AThe immunomodulatory effects of receptors in chronic inflammatory diseases such as asthma and atherosclerosis are also increasingly recognized and appreciated, a_2AReceptors are also closely related to wound healing or atrial fibrillation. A number of studies have shown that A_2AReceptor antagonists may also be useful in the treatment of diabetes。

Chinese invention patent (CN102892761) provides a new adenosine A_2A4-aminopyrimidine derivatives of receptor antagonists:

wherein R is¹Represents a pyrazole, thiazole or triazole ring optionally substituted by one or two halogen atoms or by one or two methyl or trifluoromethyl groups. The compound is useful for adenosine A_2AThe receptor has better antagonism and is shown to be A_2AIs stronger, but it is for A₁Also, the selectivity of (A) is low, resulting in₁In the presence of an acceptor, which is to A_2AThe selectivity of the receptor is low. Meanwhile, the pyrazole compound has poor pharmacokinetic characteristics, high plasma clearance rate and short half-life period in a rat body.

Disclosure of Invention

It is an object of the present invention to provide adenosine A as_2A4-aminopyrimidine derivatives of receptor antagonists and their use in the treatment of A_2AAntagonizing the responsive disease or condition.

In order to achieve the above object, the present invention provides a compound which is adenosine A_2A4-aminopyrimidine derivatives of receptor antagonists having the general structural formula (i):

wherein:

R₁selected from halogen, cyano or trifluoromethyl;

R₂selected from pyrazolyl, pyrrolidinyl, or substituted by one or more halogens or C_1-3Alkyl-substituted pyrazolyl or pyrrolidinyl;

R₃selected from oxazolyl, oxadiazolyl, triazolyl, or substituted with one or more halogens or C_1-3An alkyl-substituted oxazolyl group.

Preferably, the aboveAs adenosine A_2A4-aminopyrimidine derivatives of receptor antagonists, wherein R₂Selected from pyrazolyl or pyrrolidinyl; r₃Selected from oxazolyl, oxadiazolyl, triazolyl, or via a C_1-3An alkyl-substituted oxazolyl group.

Preferably, the above is adenosine A_2AA 4-aminopyrimidine derivative of a receptor antagonist, wherein the 4-aminopyrimidine derivative is selected from the following compounds:

preferably, the 4-aminopyrimidine derivative is selected from the following compounds: (1) (5), (8), (10), (13), (14), (17) or (18).

The invention also provides the application of the 4-aminopyrimidine derivative in preparing the medicine for treating A_2AAntagonizing a disease or condition responsive thereto.

Further, the disease or condition comprises any one or more of a neurodegenerative disease, extra pyramidal syndrome, depression, hyperactivity syndrome, sleep disorders, anxiety, diabetes, or a tumor.

Further, the tumor is lung cancer, ovarian cancer, pancreatic cancer, gastric cancer, breast cancer, glioblastoma, melanoma, renal cell carcinoma, triple negative breast cancer, colorectal cancer, head and neck cancer, bladder cancer, prostate cancer, hepatocellular carcinoma, or cholangiocarcinoma.

Further, the lung cancer is non-small cell lung cancer.

The invention also provides a pharmaceutical composition, wherein the pharmaceutical composition contains the adenosine A serving as the adenosine A_2A4-aminopyrimidine derivatives of receptor antagonists, and pharmaceutically acceptable excipients therefor.

The invention also provides a compound medicine, which comprises the 4-aminopyrimidine derivative and a medicine capable of being combined with the derivative.

Further, the combination is a compound for the treatment of the following diseases or conditions: neurodegenerative diseases, extrapyramidal syndrome, depression, hyperactivity syndrome, sleep disorders, anxiety disorders, diabetes or tumors.

The invention has the following beneficial effects: the 4-aminopyrimidine derivative provided by the invention is adenosine A_2AThe receptor has obvious selective antagonism and better pharmacokinetic characteristics, and can be applied to the treatment of A_2AAntagonism-responsive diseases or conditions, particularly neurodegenerative diseases, extra-pyramidal syndrome, depression, hyperactivity syndrome, sleep disorders, anxiety, diabetes or tumours.

Detailed Description

The present invention is further described by the following specific examples, which are only for illustrating the present invention and are not intended to limit the scope of the present invention.

The invention provides a compound serving as adenosine A_2A4-aminopyrimidine derivatives of receptor antagonists having the general structural formula (i):

wherein:

R₁selected from halogen, cyano or trifluoromethyl;

R₂selected from pyrazolyl, pyrrolidinyl, or substituted by one or more halogens or C_1-3Alkyl-substituted pyrazolyl or pyrrolidinyl;

R₃selected from oxazolyl, oxadiazolyl, triazolyl, or substituted with one or more halogens or C_1-3An alkyl-substituted oxazolyl group.

Preferably, the above is adenosine A_2A4-aminopyrimidine derivatives of receptor antagonists, wherein R₂Selected from pyrazolyl or pyrrolidinyl; r₃Selected from oxazolyl, oxadiazolyl, triazolyl, or via a C_1-3An alkyl-substituted oxazolyl group.

Preferably, the above is adenosine A_2A4-aminopyrimidines as receptor antagonistsA pyridine derivative, wherein the 4-aminopyrimidine derivative is selected from the following compounds:

preferably, the 4-aminopyrimidine derivative is selected from the following compounds: (1) (5), (8), (10), (13), (14), (17) or (18).

The invention also provides the application of the 4-aminopyrimidine derivative in preparing the medicine for treating A_2AAntagonizing a disease or condition responsive thereto.

Further, the disease or condition comprises any one or more of a neurodegenerative disease, extra pyramidal syndrome, depression, hyperactivity syndrome, sleep disorders, anxiety, diabetes, or a tumor.

Further, the tumor is lung cancer, ovarian cancer, pancreatic cancer, gastric cancer, breast cancer, glioblastoma, melanoma, renal cell carcinoma, triple negative breast cancer, colorectal cancer, head and neck cancer, bladder cancer, prostate cancer, hepatocellular carcinoma, or cholangiocarcinoma.

Further, the lung cancer is non-small cell lung cancer.

The invention also provides a pharmaceutical composition, wherein the pharmaceutical composition contains the adenosine A serving as the adenosine A_2A4-aminopyrimidine derivatives of receptor antagonists, and pharmaceutically acceptable excipients therefor.

The invention also provides a compound medicine, which comprises the 4-aminopyrimidine derivative and a medicine capable of being combined with the derivative.

Further, the combination is a compound for the treatment of the following diseases or conditions: neurodegenerative diseases, extrapyramidal syndrome, depression, hyperactivity syndrome, sleep disorders, anxiety disorders, diabetes or tumors.

Provided by the invention as adenosine A_2A4-aminopyrimidine derivatives of receptor antagonists can be synthesized by one of the following synthetic routes:

scheme 1:

the reagent and the condition are (a) acetic anhydride and reflux, (b) pyrazole, cesium carbonate, Dimethylformamide (DMF) at the temperature of 80 ℃, (c) trans- β -styrene boric acid, sodium carbonate, dioxane and room temperature, and tetrakis (triphenylphosphine) palladium (Pd (PPh)₃)₄) At 90 ℃; (d) ozone, methanol (MeOH)/dichloromethane (CH)₂Cl₂) -78 deg.C; (e) p-toluenesulfonylmethyl isonitrile (TOSMIC), potassium carbonate (K)₂CO₃) MeOH, 80 ℃; (f), N-bromosuccinimide and DMF.

The preparation of the compounds (1) - (3), (6) - (8), (11) - (13), (15) - (17) is carried out according to scheme 1. See example 1 for a specific method of implementation. Compounds in which the 5-position fluorine of the pyrimidine is substituted may also be prepared starting from fluorine atom-containing starting materials.

Scheme 2:

the reagent and the conditions are (a), m-chloroperbenzoic acid, dichloromethane, room temperature, (b), N-bromosuccinimide (NBS), DMF, room temperature, (c), 1, oxazole, butyl lithium (N-Bu L i), Tetrahydrofuran (THF), 78-20 ℃,2, Pd (PPh)₃)₄At the temperature of 80 ℃; (d) 1H-pyrazole, cesium carbonate, DMF, 90 ℃; (e) pyrrolidine, cesium carbonate, DMF, 90 ℃.

The sulfoxide of formula (G) is reacted with various commercially available five-membered heterocyclic (e.g., pyrazole, oxazole, oxadiazole or triazole) derivatives in the presence of a base (e.g., cesium carbonate or butyllithium) using DMF as a solvent at room temperature or low temperature to produce a derivative substituted at the 2-position with a five-membered heterocyclic ring. For example, reaction of an intermediate of formula (G) with oxazole under these conditions affords a derivative of formula (H).

The 6-position chlorine atom of the pyrimidine derivative is further replaced by a five-membered heterocyclic derivative (e.g., pyrazole or pyrrolidine) using DMF as a solvent in the presence of a base (e.g., cesium carbonate or sodium methoxide). For example, reaction of derivative (H) with pyrazole or pyrrolidine under these conditions can produce compounds (19) and (23).

The preparation of the compounds (19), (20), (23-25) is carried out according to scheme 2. See example 15 for a specific method of implementation.

Scheme 3:

reagents and conditions: (a) cuprous cyanide, pyridine, Microwave (MW), reaction at 250 ℃ for 20 minutes. (b) Potassium fluoride (KF), bis (dibenzylideneacetone) palladium (Pd (dba)₂)2- (dicyclohexylphosphonium) -3, 6-dimethoxy-2 ',4',6 '-triisopropyl-1, 1' -biphenyl, trimethyl (trifluoromethyl) silane, dioxane, 20h, 140 ℃.

For the synthesis of the substituent R as defined above in the 5-position of the pyrimidine₁Pyrimidine derivatives, which are cyano or trifluoromethyl, can be prepared using analogous precursor compounds as shown for compound (1) using the methods described in scheme 3, respectively.

The preparation of the compounds (4), (5), (9), (10), (14), (18), (21), (22) is carried out according to scheme 3. See examples 4 and 5 for specific methods of implementation.

Example 1:

preparation of Compound (1), the structural formula of which is as follows:

preparation of compound (1) is carried out according to scheme 1 above:

first, preparation of intermediate (A)

Suspending 4-amino-2, 6-dichloropyrimidine (DCAP, 4g, 24.4mmol) in acetic anhydride (80m L, 860mmol), heating under reflux for 4 hours under stirring, cooling the reaction solution, concentrating under vacuum, adding toluene to the residual acetic anhydride, distilling off, dissolving the residue in ethyl acetate and water, adding 10% NaHCO₃Solution to pH of solution7. Washing the organic layer with saturated brine, recovering solvent, dissolving the residue in acetic anhydride (40M L), stirring at 0-5 deg.C for 2 hr, filtering, collecting precipitate, and vacuum drying at 40 deg.C to obtain intermediate (A), MS M/z (ESI) 206.0[ M +1 ]]⁺。

Second step, preparation of intermediate (B)

Dissolving intermediate (A) (1g, 5mmol) in anhydrous DMF (15M L), adding pyrazole (340mg, 5mmol) and cesium carbonate (1.6g, 5mmol), stirring at 80 deg.C for 2 hr, pouring into water, extracting with ethyl acetate, washing the organic layer with water and saturated brine, drying over anhydrous sodium sulfate, removing solvent under reduced pressure, separating and purifying the residue by silica gel column chromatography (3% methanol: dichloromethane) to obtain product (B), MS M/z (ESI) 238.0[ M +1 ]: 238.0]⁺。

Third step, preparation of intermediate (C)

Intermediate (B) (0.4g, 1.68mmol), trans- β -styreneboronic acid (0.5g, 3.36mmol) and sodium carbonate (1.08g, 10.1mmol) were added to a dioxane/water solution, nitrogen was purged for about 30min, and Pd (PPh) was added₃)₄(0.2g, 0.16mmol), the mixture was heated to 90 ℃ and stirred for 20 hours, then poured into water and extracted with ethyl acetate. The organic layer was washed with water and saturated brine in this order, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was subjected to silica gel column chromatography (3% methanol: dichloromethane) to obtain the product (C). MS M/z (ESI) 306.1[ M +1 ]]⁺。

The fourth step, preparation of intermediate (D)

Intermediate (C) (0.4g, 1.6mmol) was dissolved in a mixed solution of methanol/dichloromethane (4/1, 20ml), cooled to-78 deg.C, and ozone was introduced for 10 minutes. And after the reaction is finished, introducing nitrogen for 20 minutes, adding dimethyl ether, heating the reaction solution to room temperature, and introducing nitrogen to completely volatilize the solvent to obtain a crude product (D). MS M/z (ESI) 232.1[ M +1 ]]⁺。

Fifth step, preparation of intermediate (E)

Intermediate (D) (460mg, 2mmol), TOSMIC (p-toluenesulfonylmethylisocyan, 50mg, 4mmol), potassium carbonate (860mg, 6mmol) were mixed with methanol, heated to 80 ℃ and reacted for 16 hours, after which methanol was recovered and the residue was poured into water, ethylAnd (5) extracting with ethyl acetate. The organic layer was washed with water, saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. Chromatography of the residue on a silica gel column (5% methanol: dichloromethane) gave the product (E). MS M/z (ESI) 229.1[ M +1 ]]⁺。

Fifth step, Compound (1) is prepared

0.2g (1.25mmol) NBS (N-bromosuccinimide) was slowly added to a cold DMF solution in which 0.2g of intermediate (E) was suspended. After stirring at room temperature for 1 hour, the solvent was removed under reduced pressure. The residue was poured into water and extracted with ethyl acetate. The organic layer was washed with water, saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (5% methanol: dichloromethane) to obtain compound (1). MS M/z (ESI) 307.1[ M +1 ]]⁺,¹HNMR(400MHz,DMSO-d6) 8.55(d,1H),8.32(s,1H),7.84(d,1H),7.53(s,1H),7.58(s,2H),6.58(dd, 1H)。

Example 2:

preparation of Compound (2), having the formula:

preparation of compound (2) is carried out according to scheme 1, with specific methods as referenced in example 1. Substituting NCS (N-chlorosuccinimide) for NBS (N-bromosuccinimide) in the reaction of the step 6 to perform chlorination to obtain a compound (2). MS M/z (ESI) 263.0[ M +1 ]]⁺,¹HNMR(400MHz,DMSO-d6) 8.53(d,1H),8.26(s,1H),7.82(d,1H),7.49(s,1H),7.54(s,2H),6.55(dd, 1H)。

Example 3

Preparation of Compound (3), the structural formula of which is as follows:

compound (3) is prepared according to scheme 1. MS M/z (ESI) 247.0[ M +1 ]]⁺,¹HNMR (400MHz,DMSO-d6)8.51(d,1H),8.27(s,1H),7.83(d,1H),7.48(s,1H), 7.55(s,2H),6.54(dd,1H)。

Example 4:

preparation of Compound (4), having the formula:

a mixture of 11.3mg (20. mu. mol) Pd (dba)₂And 15.8mg (29.4. mu. mol) of 2- (dicyclohexylphosphorus) -3, 6-dimethoxy-2 ',4',6 '-triisopropyl-1, 1' -biphenyl were added to 3ml of dioxane, and then the mixed solution was added to a mixture containing 0.1g (0.33mmol) of the compound (1) (example 1), 0.04g (0.65mmol) of potassium fluoride, 0.093g (0.65mmol) of trimethyl (trifluoromethyl) silane was added, the reaction solution was stirred at 140 ℃ for 20 hours and then filtered through celite, and the filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography (dichloromethane: methanol) to give compound (4). MS M/z (ESI) 297.1[ M +1 ]]⁺,¹HNMR(400MHz,DMSO-d6)8.51(d,1H),8.31 (s,1H),7.85(d,1H),7.52(s,1H),7.55(s,2H),6.58(dd,1H)。

Example 5:

preparation of Compound (5), having the formula:

the preparation method is as shown in synthetic route 3, the compound (1) and 0.06g (0.72mmol) cuprous cyanide are added into pyridine, the mixture is placed in a microwave reactor for reaction at 250 ℃ for 20 minutes, after the reaction is monitored by thin layer chromatography (T L C), ethyl acetate is added, then the mixture is filtered by using kieselguhr, saturated sodium bicarbonate solution and saturated common salt water are used for washing in sequence, an organic layer is dried by anhydrous magnesium sulfate, and the product (5) is obtained by concentration, wherein the mass ratio of MS M/z (ESI) is 254.1[ M +1 ]]⁺,¹HNMR(400MHz,DMSO-d6)8.52(d,1H),8.29(s,1H),7.83 (d,1H),7.51(s,1H),7.57(s,2H),6.59(dd,1H)。

Example 6:

preparation of Compound (6), having the formula:

compound (6) was prepared according to scheme 1, wherein the reaction of step 5 replaced p-toluenesulfonylmethyl isocyan (TOSMIC) with methyl-p-toluenesulfonylmethyl isocyan (Me-TOSMIC) to prepare an analog of compound (E). MS M/z (ESI) 261.1[ M +1 ]]⁺,¹HNMR(400MHz, DMSO-d6)8.55(d,1H),8.36(s,1H),7.84(d,1H),7.57(s,2H),6.61(dd,1H), 2.16(s,3H)。

Example 7:

preparation of Compound (7), having the formula:

prepared according to synthetic route 1. Substitution of TOSMIC with Me-TOSMIC at the reaction of step 5 an analog of Compound (E) was prepared, followed by chlorination with NCS (N-chlorosuccinimide) in place of NBS (N-bromosuccinimide) to give Compound (7). MS M/z (ESI) 277.1[ M +1 ]]⁺,¹HNMR (400MHz,DMSO-d6)8.52(d,1H),8.33(s,1H),7.82(d,1H),7.55(s,2H), 6.60(dd,1H),2.14(s,3H)。

Example 8:

preparation of Compound (8), having the formula:

prepared according to synthetic route 1. Substitution of TOSMIC with Me-TOSMIC at the reaction of step 5 an analog of compound (E) was prepared and then brominated with NBS (N-bromosuccinimide) to give compound (8). MS M/z (ESI) 321.0[ M +1 ]]⁺,¹HNMR(400MHz,DMSO-d6)8.53(d,1H),8.34 (s,1H),7.82(d,1H),7.56(s,2H),6.59(dd,1H),2.12(s,3H)。

Example 9:

preparation of Compound (9), having the formula:

replacing compound (1) with compound (8)Compound (9) was prepared by the method of example 4. MS M/z (ESI) 311.1[ M +1 ]]⁺,¹HNMR(400MHz,DMSO-d6)8.50(d,1H),8.31 (s,1H),7.79(d,1H),7.55(s,2H),6.57(dd,1H),2.13(s,3H)。

Example 10:

preparation of Compound (10), having the following structural formula:

compound (10) was prepared by the method of example 5, substituting compound (8) for compound (1). MS M/z (ESI) 268.1[ M +1 ]]⁺,¹HNMR(400MHz,DMSO-d6)8.51(d,1H),8.30 (s,1H),7.81(d,1H),7.53(s,2H),6.58(dd,1H),2.16(s,3H)。

Example 11:

preparation of Compound (13), having the following structural formula:

prepared according to synthetic route 1. In the second step, pyrrolidine is used to replace pyrazole to obtain an analogue of the intermediate (B), and the compound (13) is prepared by coupling, cyclization, bromination and other reactions. MS M/z (ESI) 310.0 [ M +1 ]]⁺,¹HNMR(400MHz,DMSO-d6)8.51(d,1H),7.82(d,1H),6.90(s, 2H),3.51-3.74(m,4H),1.82-1.85(m,4H)。

Example 12:

preparation of Compound (14), having the following structural formula:

compound (14) was obtained in the same manner as in example 5 except that Compound (1) was replaced with Compound (13). MS M/z (ESI) 257.1[ M +1 ]]⁺,¹HNMR(400MHz,DMSO-d6)8.50(d,1H),7.81 (d,1H),6.94(s,2H),3.53-3.75(m,4H),1.83-1.87(m,4H)。

Example 13:

preparation of Compound (17), having the formula:

prepared according to synthetic route 1. The step 2 reaction replaces pyrazole with pyrrolidine to prepare an analog of intermediate (B), the step 5 reaction replaces TOSMIC with Me-TOSMIC to prepare an analog of intermediate (E), and then bromination with NBS (N-bromosuccinimide) affords compound (17). MS M/z (ESI) 324.0[ M +1 ]]⁺,¹HNMR(400MHz,DMSO-d6)8.50(s,1H),7.70(s,2H),3.52- 3.76(m,4H),2.16(s,3H),1.82-1.86(m,4H)。

Example 14:

preparation of Compound (18), having the structural formula:

compound (17) in place of compound (1), compound (18) was obtained in the same manner as in example 5. MS M/z (ESI) 271.1[ M +1 ]]⁺,¹HNMR(400MHz,DMSO-d6)8.52(d,1H),7.69 (s,2H),3.53-3.77(m,4H),1.82-1.86(m,4H)。

Example 15:

preparation of Compound (19), having the formula:

prepared according to synthetic route 2.

The first step is as follows: preparation of intermediate 6-chloro-2- (methylsulfinyl) pyrimidin-4-amine (F)

To a solution of 1.0g (5.7mmol) of 6-chloro-2- (methylthio) pyrimidin-4-amine in 50ml of dichloromethane was added a solution of 1.5g (6.9mmol) of m-chloroperbenzoic acid (77%) in 30ml of dichloromethane over 30 minutes. The reaction mixture was stirred at room temperature for 4 hours. The resulting white precipitate was filtered, washed several times with dichloromethane and dried to obtain 1.0g of intermediate (F). MS M/z (ESI) 192.0 [ M +1 ]]⁺。

The second step is that: preparation of intermediate 5-bromo-6-chloro-2-(methylsulfinyl) pyrimidin-4-amine (G): 1.12G (6.3 mmol) of N-bromosuccinimide (NBS) was slowly added to a cooled suspension of 1.0G (5.3mmol) of 6-chloro-2- (methylsulfinyl) pyrimidin-4-amine in 30ml of DMF, stirred at room temperature for 50 minutes, the precipitate was filtered, washed with cold DMF, washed several times with cold water, and dried under vacuum to give (G), MS M/z (ESI):271.9[ M +1 ]]⁺。

The third step: preparation of intermediate 5-bromo-6-chloro-2- (oxazol-2-yl) pyrimidin-4-amine (H):

oxazole (260mg, 3.7mmol) was dissolved in anhydrous THF at-78 deg.C, nBu L i (1.6M in n-hexane) was added and stirred for 15 minutes, the temperature was naturally raised to-20 deg.C, 1G (3.7mmol) of intermediate (G) and Pd (PPh)₃)₄Added to anhydrous THF. And then added to the above oxazole solution. After the mixture was reacted at 80 ℃ for 2 hours, the solution was poured into 1N hydrochloric acid, extracted with ethyl acetate, the organic layer was washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, and subjected to silica gel column chromatography (5% methanol in dichloromethane) to obtain the desired product. MS M/z (ESI) 276.9[ M +1 ]]⁺。

The fourth step: preparation of Compound (19)

To a solution of 0.15g of intermediate (H) in 3ml of DMF were added 0.11g (1.64mmol) of 1H-pyrazole and 0.18g (0.55mmol) of cesium carbonate. The mixture was stirred at 90 ℃ for 24 hours. The solvent DMF was concentrated under reduced pressure. The crude product was washed with water and dried to give compound (19). MS M/z (ESI) 307.1[ M +1 ]]⁺。

Example 16:

preparation of Compound (25), the structural formula of which is as follows:

prepared according to scheme 2 above, in a manner similar to that described in example 15. In the third step of reaction, the intermediate (G) and the 1H-triazole are stirred in a DMF solution for reaction for several hours at room temperature under the action of cesium carbonate to prepare the corresponding intermediate (H) analogue. The 6-chloro position of pyrimidine was replaced with pyrazole in a similar manner to that in example 15 to give compound (25). MS M/z (ESI) 307.1[ M +1 ]]⁺,¹HNMR(400MHz, DMSO-d6)9.14(s,1H),8.60(s,1H),8.45(d,1H),7.64(d,1H),6.55(dd,1H)。

Pharmacological activity:

(1) para adenosine A_2AReceptor binding affinity assay:

by determination of human adenosine A using standard techniques_2AReceptor selective radioligands

[³H]Conversion of CGS-21680 to determine in vitro binding of the Compounds of the invention to human adenosine A_2AThe binding affinities of the receptors and the results are summarized in table 1.

By encoding human adenosine A_2APlasmids of the receptor were stably transfected on HEK-293 cells, which were used to prepare membranes using standard techniques in Tris (hydroxymethyl) aminomethane (Tris-HCl) buffer (pH 7.4). About 15. mu.g of a membrane protein and 50nM of a radioligand³H]CGS-21680, 10. mu.M test compound were mixed and incubated for 90 min at 25 ℃. Nonspecific binding was determined by the addition of 50 μ M NECCA (adenosine-5' -N-ethylcarboxyamide). The membrane was filtered and washed 3 times to remove unbound radioligand. The filter measures bound ligand with a scintillation counter. The concentration-response binding competition curve was determined by analyzing a plurality of different concentrations. Calculating IC using a non-linear fitting program₅₀The value is obtained. The inhibition constant (Ki) of the compounds was calculated by Cheng-Prusoff equation (II).

Ki＝IC₅₀/(1+[L]/KD) (Ⅱ)

Wherein the IC₅₀Is the concentration of compound that converts 50% of radioligand binding, [ L]Is the free concentration of the radioligand and KD is the dissociation constant of the radioligand. IC (integrated circuit)₅₀Values were obtained by Prism software fitting the data with non-linear regression. The smaller the Ki value indicates that the compound is at human adenosine A_2AThe more pronounced the antagonistic effect of the receptor.

(2) Para adenosine A₁Receptor binding affinity assay:

the compound DPCPX (1, 3-dipropyl-cyclopentaxanthine) is known as highly active adenosine A₁Receptor antagonists (reported in the literature as Ki 0.45 nM). By determination of human adenosine A using standard techniques₁Receptor-selective radioligand [ alpha ]³H]Determination of binding of Compounds of the invention to human adenosine A in vitro₁Binding affinity of receptor to determine the Compound of the present invention to A_2AThe selective strength of the receptor. The results are summarized in table 1.

By encoding human adenosine A₁The plasmid of the receptor was stably transfected on CHO cells, and the cells were used to prepare membranes using standard techniques in modified HEPES buffer (pH 7.4). About 10. mu.g of a membrane protein and 1nM of a radioligand³H]DPCPX, 10. mu.M test compound were mixed and incubated for 90 minutes at 25 ℃. Nonspecific binding was determined by adding 100. mu. M R (-) -PIA (R-phenylisopropyladenosine). The membrane was filtered and washed 3 times to remove unbound radioligand. The filter measures bound ligand with a scintillation counter. The concentration-response binding competition curve was determined by analyzing a plurality of different concentrations. The inhibition constants (Ki) of the compounds were calculated as described previously.

(3) Human adenosine A_2AAnalysis of anti-platelet aggregation activity of receptor:

CGS-21680 is adenosine A_2AHigh activity agonists of the receptor acting on adenosine A_2AThe receptor promotes platelet aggregation. Adenosine A at the cellular level was obtained by testing compounds to inhibit CGS-21680-induced platelet aggregation_2AAntagonistic activity of the receptor. The results are summarized in table 1.

At 37 deg.C, plasma rich in human blood platelets is in thromboxane A₂Platelet-rich plasma (6 × 10) in the upper layer under the action of receptor agonist U-46619(10 μ M)⁸Platelets/ml) to produce aggregation, detected by optical agglutination. Using 1 μ M CGS-21680 as a control, it was shown that the test substance may have adenosine A if platelet aggregation reached 50% or more (. gtoreq.50%) in the test compound (30 μ M) system within 5 minutes_2AReceptor agonist activity.

When no significant agonistic activity was observed at a certain test substance concentration, it reduced CGS-21680 (1. mu.M) induced inhibitory response by 50% or more (. gtoreq.50%), indicating that the test compound has adenosine A_2AReceptor antagonistic activity. According to the above formulaThe inhibition constant (Ki) of the compound is calculated by the method, and the smaller the Ki value is, the lower the inhibition constant (Ki) of the compound is, the human adenosine A of the compound is shown_2AThe more pronounced the antagonistic effect of the receptor.

TABLE 1 human adenosine A obtained in affinity assay and platelet aggregation assay for Compounds (1) - (25)_2AAnd A₁Inhibition constant of receptor ("/" indicates not tested)

As can be seen from Table 1, the compounds prepared according to the present invention were found to be able to express human adenosine A at nM concentration level_2AThe receptor has obvious antagonism. And when R is₁The group has enhanced charge absorption, such as when the halogen atom is changed from bromine to chlorine or when the halogen atom is changed from halogen atom to cyano, the compound of the invention is used for preparing the human adenosine A_2AThe antagonistic activity of the receptor is obviously improved.

Furthermore, the compound VI is the compound of example 1 in CN102892761, which is a pyrazole compound having the following structural formula:

it is now used as a positive control in the present invention. In patent CN102892761, the compound VI is reported to be of human origin A_2AThe receptor has an affinity Ki of 12nM at CHO-A_2AKi values for cAMP on cells were 25 nM. The present invention adopts the above test method, and the antagonistic activity of the tested compound VI is poorer than that reported in the patent CN102892761 (see Table 1).

Among the 4 receptor subtypes of adenosine, due to A₁And A_2AIs a highly expressed receptor which acts at physiologically low adenosine concentrations, and A_2BAnd A₃Is low, and can be activated to cause diseases only when adenosine is greatly increased under pathological conditionsPhysical effect. The oxazole compound of the invention is relative to A₁Receptor, pair A_2AThe receptor has obvious high selectivity (the selectivity coefficient is that the compound in the table 1 is in the human adenosine A)₁Inhibition constant of receptor affinity and inhibition of human adenosine A_2AThe ratio of the inhibition constants of the receptor affinities), and the selectivity coefficient of the partial compound is 3-4 times that of compound VI reported in CN102892761, which is considered to be a significant advance in the art.

Human adenosine A by the compounds of the invention_2AAnalysis of the anti-platelet aggregation activity of the receptors proves that the receptors have antagonism A on the cellular level_2AFunctional role of the receptor. Most of the tested compounds of the present invention showed stronger cellular activity than the pyrazole compound VI described in CN102892761, e.g. compound (5) showed about 4.5 times stronger cellular activity than VI.

Therefore, compared with the pyrazole compound VI described in CN102892761, the oxazole compound provided by the invention is used for human adenosine A_2AThe antagonistic activity and the functional activity of the receptor are equal to or better than those of the receptor, and the receptor is human adenosine A₁The affinity of the receptor is obviously reduced and shows that the receptor has the affinity for A_2AHigh selectivity of the receptor.

Evaluation of pharmacokinetics:

the compounds (1), (5), (8), (10), (13) of the present invention were tested for pharmacokinetics.

According to the conventional method, SD rats are used as test animals, the L C/MS/MS method is used for measuring the drug concentration in the blood plasma of the rats at different moments after the rats are administrated with the compounds (1), (5), (8), (10) and (13) by intravenous injection, the pharmacokinetic behavior of the compounds in the rats is researched, and the pharmacokinetic characteristics of the compounds are evaluated.

TABLE 2 pharmacokinetic parameters of the Compounds of the invention (1), (5), (8), (10), (13) (SD rat intravenous 1.0mg/kg)

The results in table 2 show that the oxazole compound of the present invention has better pharmacokinetic characteristics, and has a significantly lower plasma clearance than the pyrazole compound VI in CN102892761, such that a higher blood concentration and a longer effective time can be maintained; the half-life period of the oxazole compound in a rat body is more than 3 times of that of a compound VI, the half-life period is prolonged by 20-40% in the technical field to be considered as better, and the half-life period is prolonged by more than 1 time to be considered as obvious progress; therefore, compared with the pyrazole compound VI described in CN102892761, the compound of the invention has remarkable improvement in maintaining higher blood concentration and longer effective treatment time.

In conclusion, the 4-aminopyrimidine derivative provided by the invention is human adenosine A_2AThe receptor has obvious antagonism and shows high A_2AReceptor selectivity and good metabolic absorption in rat body, and can be used for treating A_2AAntagonism-responsive diseases or conditions, particularly neurodegenerative diseases, extra-pyramidal syndrome, depression, hyperactivity syndrome, sleep disorders, anxiety, diabetes or tumours.

While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.

Claims (8)

1. A compound used as adenosine A_2A4-aminopyrimidine derivatives of receptor antagonists, characterized by the general structural formula (i) below:

wherein:

R₁selected from halogen, cyano;

R₂selected from pyrazolyl, pyrrolidinyl;

R₃selected from oxazolyl or C_1-3An alkyl-substituted oxazolyl group;

the 4-aminopyrimidine derivative is selected from the following compounds:

2. use of a 4-aminopyrimidine derivative according to claim 1 for the preparation of a medicament for the treatment of para-a_2AAntagonizing a disease or condition responsive thereto.

3. The use of claim 2, wherein the disease or condition comprises any one or more of a neurodegenerative disease, extra-pyramidal syndrome, depression, hyperactivity syndrome, sleep disorders, anxiety, diabetes or tumors.

4. The use of claim 3, wherein the tumor is lung cancer, ovarian cancer, pancreatic cancer, gastric cancer, breast cancer, glioblastoma, melanoma, renal cell carcinoma, colorectal cancer, head and neck cancer, bladder cancer, prostate cancer, hepatocellular carcinoma, or cholangiocarcinoma.

5. The use of claim 4, wherein the lung cancer is non-small cell lung cancer.

6. A pharmaceutical composition comprising as adenosine a according to claim 1_2A4-aminopyrimidine derivatives of receptor antagonists, and pharmaceutically acceptable excipients therefor.

7. A combination drug comprising the 4-aminopyrimidine derivative according to claim 1 in combination with a drug.

8. The combination according to claim 7, wherein the combination is a compound for the treatment of: neurodegenerative diseases, extrapyramidal syndrome, depression, hyperactivity syndrome, sleep disorders, anxiety disorders, diabetes or tumors.