PYRROLO[2,3d]PYRIMIDINE COMPOSITIONS AND THEIR USE

专利摘要:
A new adesar purine useful for the treatment of adenosine receptor-stimulating diseases has been disclosed.
公开号:KR20010083042A
申请号:KR1020007013675
申请日:1999-06-01
公开日:2001-08-31
发明作者:알린도 엘. 카스텔하노；브라이언 맥키벤；데이비드 제이. 휘털
申请人:추후제출；오에스아이 파마슈티컬스, 인코포레이티드；
IPC主号:

专利说明:

Pyrrolo [2,3-d] pyrimidine compositions and uses thereof.
[1] Adenosine is an ubiquitous modulator, especially for many physiological activities within the cardiovascular and nervous system. The effect of adenosine appears to be mediated by specific cell surface receptor proteins. Adenosine modulates a variety of physiological activities including sedation, vasodilation, inhibition of heart rate and contraction, inhibition of platelet aggregation, stimulation of glucose neogenesis and inhibition of lipolysis. In addition to the effects on adenylate cyclase, adenosine appears to open calcium channels, reduce flow through the calcium channel, and inhibit or stimulate phosphoenotid turnover through receptor-mediated mechanisms Adenosine Receptor Antagonists, Structures and Potential Therapeutic Applications, Current Pharmaceutical Design, 2: 501 (1996) and CE Muller "A ₁ -Adenosine Receptor Antagonists," Exp. Opin. Ther. Patents 7 (5): 419 (1997)).
[2] Adenosine receptors belong to the purine receptor family, which is now further subdivided into P ₁ (adenosine) and P ₂ (ATP, ADP and other nucleotide receptors) receptors. To date, four subtype receptors for the nucleoside adenosine have been cloned from various species, including humans. The two receptor subtypes (A ₁ and A ₂ ) show affinity for adenosine in the nanomolar range, while the other two subtypes A _2b and A ₃ are low affinity receptors with affinity for adenosine in the low micromolar range . The A < ₁ > and A < ₃ > adenosine receptor activities cause inhibition of adenylate cyclase activity whereas A _2a and A _2b activity cause stimulation of adenylate cyclase.
[3] Several A ₁ pathway antibodies have been developed to treat cognitive disorders, renal disease, and cardiac arrhythmias. A _2a antagonists have been suggested to be useful for patients with morbus Parkinson (Parkinson's disease). In particular, in view of the utility of local delivery, adenosine receptor antagonists may be useful in the treatment of allergic inflammation and asthma. In this pathophysiological context, the A ₁ antagonists are found under the respiratory epithelial cells (see, for example, Myce & Metzger "DNA antisense Therapy for Asthma in an Animal Model" While A _2b or A ₃ receptor antagonists mitigate the release of histamine and other inflammatory mediators while blocking mast cell degranulation. A _2b receptors were found in the gastrointestinal tract, especially in the large intestine and small intestine epithelium. A _2b receptors are known to mediate the cAMP response (Strohmeier et al., J. Bio. Chem. (1995) 270: 2387-94).
[4] Adenosine receptors are known to be present in the retina of various mammalian species such as cattle, pigs, monkeys, rats, guinea pigs, mice, rabbits and humans (cf. Blazynski et al., Discrete Distributions of Adenosine Receptors in Mammalian Retina, Journal of Neurology, Vol. 54, pp. 648-655 (1990); Woods et al., Characterization of Adenosine A ₁ -Receptor Binding Sites in Bovine Retinal Membranes, Experimental Eye Research, Vol. 53, pp. 325-331 (1991) , Endogenous adenosine and adenosine receptors localized to ganglion cells of the retina, Proceedings of the National Academy of Science, Vol. 84, pp. 3906-3910 (1987)). Recently, Williams reported that adenosine delivery sites were found in cultured human retinal cell lines (Williams, et al., Nucleoside Transport Sites in a Cultured Human Retinal Cell Line Established By SV-40T Antigen Gene, Current Eye Research, 109-118 (1994)).
[5] Previously, compounds that modulate adenosine uptake have been proposed as useful therapeutic agents for treating retinal and optic nerve head injuries. In U.S. Patent No. 5,780,450 to Sade, the inventors are discussing the use of adenosine sorbent degradation to treat ocular disease. Shade does not disclose the use of specific A ₃ receptor degradation. The entire contents of U.S. Patent No. 5,780,450 are hereby incorporated by reference.
[6] Additional adenosine receptor antagonists are needed as pharmaceutical tools and considerable interest has been expressed as drugs for the above mentioned disease states and / or conditions.
[7] SUMMARY OF THE INVENTION
[8] The present invention is based, at least in part, on the ability to use any N-6 substituted 7-deazapurine, described below, to treat an N-6 substituted 7-deazapurine responsive condition. Examples of such conditions include those in which the activity of adenosine derivatives is increased, i.e., bronchitis, gastrointestinal disease or asthma. This condition is characteristic in that adenosine receptor activity leads to inhibition or stimulation of the idenylate cyclase activity. The compositions and methods of the present invention comprise enantiomerically or diastereomerically pure N-6 substituted 7-deaza-purines. Preferred N-6 substituted 7-deazapurines include those having urea moieties attached to N-6 nitrogen via acetamide, carboxamide, substituted cyclohexyl, e.g., cyclohexanol, ≪ / RTI >
[9] The present invention relates to a method of modulating an adenosine receptor in a mammal, wherein a therapeutically useful amount of N-6 substituted 7-deazapurine is administered to the mammal to control the activity of the adenosine receptor. Suitable adenosine receptors include the group A ₁ , A ₂ , or A ₃ . In a preferred embodiment, the N-6 substituted 7-deazapurine is an adenosine receptor antagonist.
[10] The present invention also relates to a method of treating N-6 substituted 7-deazapurine diseases in mammals, such as, for example, the administration of a therapeutically useful amount of N-6 substituted 7-deazapurine to a mammal, For example, asthma, bronchitis, allergic rhinitis, chronic obstructive pulmonary disease, retinal disease, gastrointestinal disease and ocular disease. Suitable N-6 substituted 7-deazapurines include the compounds represented by the following general formula (I) and their pharmaceutically acceptable salts.
[11] (I)
[12] R ₁ and R ₂ are each a hydrogen atom or a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety, or form a substituted or unsubstituted heterocyclic ring. R ₃ is a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety. R ₄ is a hydrogen atom or a substituted or unsubstituted alkyl, aryl or alkylaryl moiety. R ₅ and R ₆ are independently a halogen atom, for example chlorine, fluorine or hydrogen, or a hydrogen atom or a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety, or R ₄ and R ₅ or R ₅ , R < ₆ > together form a substituted or unsubstituted heterocyclic or carbocyclic ring.
[13] In certain embodiments, R ₁ and R ₂ can each independently be a substituted or unsubstituted cycloalkyl or heteroarylalkyl moiety. In another embodiment, R ₃ is a hydrogen atom or a substituted or unsubstituted heteroaryl moiety. In yet another embodiment, R ₄ , R _5, and R ₆ can each independently be a heteroaryl moiety. In a preferred embodiment, R ₁ is a hydrogen atom, R ₂ is cyclohexanol, such as trans-cyclohexanol, R ₃ is phenyl, R ₄ is a hydrogen atom, R ₅ is a methyl group, R ₆ is a methyl group. In another embodiment, R ₁ is a hydrogen atom, R ₂ is R ₃ is phenyl, R ₄ is a hydrogen atom, and R ₅ and R ₆ are methyl groups.
[14] The present invention also relates to pharmaceutical compositions for treating N-6 substituted 7-deazapurine responsive conditions in mammals, for example asthma, bronchitis, allergic rhinitis, chronic obstructive pulmonary disease, retinal disease, gastrointestinal disease and eye disease . The pharmaceutical composition comprises a therapeutically useful amount of N-6 substituted 7-deazapurine and a pharmaceutically acceptable carrier.
[15] The present invention also relates to packaged pharmaceutical compositions for treating N-6 substituted 7-deazapurine responsive conditions in mammals. The packaged pharmaceutical composition comprises a container having a pharmaceutically effective amount of at least one N-6 substituted 7-deazapurine and an N-6 substituted 7-tertiary aminopurine compound to treat an N-6 substituted 7-deazapurine responsive condition in a mammal Includes instructions for using azapurine.
[16] The invention also relates to compounds of formula 1 wherein R < ₁ > is hydrogen; R ₂ is substituted or unsubstituted cycloalkyl, substituted or unsubstituted alkyl, or R ₁ and R ₂ together form a substituted or unsubstituted heterocyclic ring; R ₃ is unsubstituted or substituted aryl, and R ₄ is hydrogen; And R ₅ and R ₆ are each independently hydrogen or alkyl, and pharmaceutically acceptable salts thereof. The deazapurines of this embodiment may advantageously be selective A ₃ receptor antagonists. Such compounds may be useful in many therapeutic applications, for example, in the treatment of asthma, kidney disease associated with heart disease, and glaucoma. In a particularly preferred embodiment, the deazapurine is a water-soluble prodrug (prodrug) which can be metabolized to the active drug by, for example, esterase catalytic hydrolysis in vivo.
[17] In another embodiment, the present invention provides methods for inhibiting the activity of an adenosine receptor (e.g., A ₃ ) in a cell by contacting the cell with an N-6 substituted 7-deazapurine (e.g., preferably an adenosine receptor antagonist) The method comprising the steps of:
[18] In another aspect, the invention features a method of treating ocular damage in an animal (e.g., a human) by administering to the animal an effective amount of N-6 substituted 7-deazapurine of formula (I). Preferably, said N-6 substituted 7-deazapurine is an antagonist of the A ₃ adenosine receptor in the cell of said animal. The damage may be in the retina or optic nerve head and may be acute or chronic. Such damage may be, for example, the result of glaucoma, edema, ischemia, hypoxia or injury.
[19] The present invention also features a pharmaceutical composition comprising an N-6 substituted 7-deazapurine of formula (I). Preferably, the pharmaceutical formulation is an ophthalmic drug (e. G., Ocular, retrobulbar, introocular, systemic, or surgical cleaning solution).
[20] In another embodiment, the invention features deazapurine having formula (II).
[21] (II)
[22] In this formula,
[23] X is N or CR < ₆ >;
[24] R ₁ and R ₂ are each independently hydrogen or a substituted or unsubstituted alkoxy, aminoalkyl, alkyl, aryl, or alkylaryl, or, provided that R ₁ and R ₂ are not both hydrogen, Form a click ring;
[25] R < ₃ > is substituted or unsubstituted alkyl, arylalkyl or aryl;
[26] R ₄ is hydrogen or substituted or unsubstituted C ₁ -C ₆ alkyl;
[27] L is hydrogen, substituted or unsubstituted alkyl or R ₄ and L form a substituted or unsubstituted heterocyclic or carbocyclic ring with each other;
[28] R < ₆ > is hydrogen, substituted or unsubstituted alkyl or halogen;
[29] Q is CH ₂ , O, S or NR ₇ wherein R ₇ is hydrogen or substituted or unsubstituted C ₁ -C ₆ alkyl; And
[30] W is an unsubstituted or substituted alkyl, cycloalkyl, aryl, arylalkyl, biaryl, heteroaryl, substituted carbonyl, substituted thiocarbonyl or substituted sulfonyl wherein R ₃ is pyrrolidino, then R ₄ is Methyl is not satisfied. The present invention also relates to pharmaceutically acceptable salts and prodrugs of the compounds of the present invention.
[31] In a useful embodiment, in formula (II), X is CR _6, and Q is Ch _2, O, S or NH, wherein R ₆ is as defined above.
[32] In another example of formula (II), X is N.
[33] The invention also extends to a method of inhibiting the activity of an adenosine receptor (e. G., An A _2b adenosine receptor) in a cell by contacting the cell with a compound of the invention. Preferably, the compound is an antagonist of the receptor.
[34] The present invention also provides compounds of the animals formula (II) gastrointestinal disease in an animal by administering an effective amount of (antagonists of A _2b) (e. G., Diarrhea) or a respiratory disorder (e.g., allergic rhinitis, Chronic Obstructive Pulmonary Disease), therapy for . Preferably the animal is a human.
[35] The features and other details of the invention will be more particularly described and pointed out in the claims. It will be appreciated that the particular embodiments of the invention are shown by way of example and not as limitations of the invention. The central features of the invention are set forth in various embodiments without departing from the scope of the invention.
[36] The present invention relates to a method of treating an N-6 substituted 7-deazapurine responsive condition in a mammal. The method comprises administering to the mammal a therapeutically effective amount of N-6 substituted 7-deazapurine as described below to cause the treatment of the N-6 substituted 7-desazapurine responsive state in the animal.
[37] The term " N-6 substituted 7-deazapurin responsive state " encompasses a disease state or condition characterized by a susceptibility to treatment with an N-6 substituted 7-deazapurine of the present invention as described below , The treatment comprises a significant reduction in at least one symptom or effect of the condition achieved with the N-6 substituted 7-deazapurine of the present invention. Typically, such a condition is associated with an increase in adenosine in the host, such host being occasionally non-limiting examples of which include toxin release, inflammation, coma, water retention, weight gain or loss, pancreatitis, , Rheumatoid arthritis, osteoarthritis, multiple organ disease, pediatric and adult respiratory distress syndrome, allergic rhinitis, chronic obstructive pulmonary disease, eye disease, gastrointestinal disease, skin tumor promotion, immunodeficiency and asthma , For example, CE Muller and B. Stein "Adenosine Receptor Antagonists: Structures and Potential Therapeutic Applications, Current Pharmaceutical Design, 2: 501 (1996) and CE Muller" A ₁ -Adenosine Receptor Antagonists, Patients 7 (5); 419 (1997) and I. Feoktistove, R. Polosa, ST Holgate, and I. Biaggioni "Adenosine A _2b receptors: a novel therapeutic target in asthma" TiPS 19; The effects associated with such symptoms include, but are not limited to, fever, shortness of breath, nausea, diarrhea, weakness, headache and even death. In one embodiment, the N-6 substituted 7-deazapurin susceptible state is a stimulus of adenosine receptors, e.g., A ₁ , A _2a , A _2b , A ₃ , Lt; RTI ID = 0.0 > C), < / RTI > In a preferred embodiment, the N-6 substituted 7-deazapurine responsive state is associated with the adenosine receptor (s), e.g., N-6 substituted 7-deazapurine acts as an antagonist. Examples of suitable susceptibility conditions that can be treated by the compounds of the present invention, e. G., Adenosine receptor agonists mediating biological effects include central nervous system (CNS), cardiovascular, retinal, respiratory, Symptoms, gastrointestinal symptoms, and metabolic symptoms. The relative amount of adenosine in a subject is related to the symptoms described below: an increased amount of adenosine may trigger a symptom, for example, an undesirable physiological response, such as an asthma attack.
[38] CNS symptoms include reduced delivery of substance (A ₁ ), sedation (A ₁ ), reduced locomotor activity (A _2a ), anticonvulsive action, chemoreceptor stimulation (A ₂ ) and hyperalgesia. Therapeutic applications of the compounds of the present invention include dementia, Alzheimer's disease, and memory enhancing treatment.
[39] Cardiovascular symptoms vasodilation _{(A 2a), (A 2b} ) and (A _3), vasoconstriction (A _1), simdong disease (A _1), platelet inhibition (A _2a), negative cardiac contraction and transformation (A ₁₎ , Arrhythmia, cardiac hypertrophy, and angiogenesis. Therapeutic applications of the compounds of the present invention include, for example, prevention of ischemic heart disease of the heart and recovery of cardiovascular drugs, myocardial tissue protection and cardiac function.
[40] Retinal symptoms include reduced GFR (A ₁ ), vascular hepatocyte contraction (A ₁ ), antidiuretic hypertrophy (A ₁ ) and renin release inhibition (A ₁ ). Appropriate therapeutic applications of the combination fire of the present invention include use as a diuretic agent, a sodium urinary excretion enhancer, a calcium deficiency of an acute renal disease, an anti-renal / prophylactic agent, an antihypertensive agent, an anti-edema agent and an anti-nephritic agent.
[41] Respiratory symptoms include bronchodilation (A ₂ ), bronchoconstriction (A ₁ ), chronic obstructive pulmonary disease, allergic rhinitis, mucus secretion and respiratory depression. Suitable applications of the compounds of the present invention include anti-asthmatic applications, treatment of pulmonary disease after transplantation, and respiratory diseases.
[42] Immune symptoms include immunosuppression (A ₂ ), neutrophil chemotaxis (A ₁ ), neutrophil superoxide generation (A _2a ), and subcellular degranulation (A _2b and A ₃ ). Therapeutic applications of antagonists include allergic and non-allergic inflammation, for example, the release of histamine and other inflammatory mediators.
[43] Gastrointestinal symptoms include inhibition of acid secretion (A ₁ ). Therapeutic applications include reflux and ulcerative conditions. Gastrointestinal symptoms also include diarrheal diseases (A _2b ) associated with colonic, intestinal and diarrheal diseases, for example, small intestinal inflammation.
[44] Ophthalmic diseases include retinitis and optic nerve head injuries and wound related diseases (A ₃ ). In a preferred embodiment, the ocular disease is glaucoma.
[45] Other therapeutic applications of the compounds of the present invention include use as an obesity (lipolytic characterization) treatment, hypertension, depressive sedative, anxiolytic, inducing and locomotor agents, such as agents that perform mobility without developing diarrhea .
[46] The term " disease state " is intended to include conditions caused by or associated with undesirable concentrations of asynes, adenylate cyclase activity, increased physiological activity associated with abnormal stimulation of adenosine receptors, and / or increased cAMP . In one embodiment, the disease state is, for example, asthma, chronic obstructive pulmonary disease, allergic rhinitis, bronchitis, retinal disease, gastrointestinal disease or ophthalmic disease. Other examples include chronic bronchitis and cystic fibrosis. Suitable examples of inflammatory diseases are non-lymphoid leukemia, myocardial ischemia, angina, infarction, cerebral vascular ischemia, intermittent claudication, critical limb ischemia, venous hypertension, varicose veins, venous ulceration, and arteriosclerosis . Damaged reperfusion conditions include any post-surgical wound, such as, for example, reconstructive surgery, thrombotic collapse, or angioplasty.
[47] The term " treatment of the N-6 substituted 7-deazapurine susceptible condition " or " treating the N-6 substituted 7-desazapurine susceptible condition " includes a change in the disease state or condition, The term also encompasses the modulation, prevention or inhibition of physiological symptoms or symptoms associated with abnormal amounts of adenosine. In a preferred embodiment, the modulation of the disease state or condition In another preferred embodiment, the modulation is selected such that an abnormal amount of adenosine receptor activity is modulated, but other physiological systems and factors are not affected.
[48] The term " N-6 substituted 7-deazapurine " is art-recognized and refers to compounds having formula (I)
[49] (I)
[50] &Quot; N-substituted 7-deazapurine " and pharmaceutically acceptable salts thereof, and in one embodiment also includes certain N-6 substituted purines described herein.
[51] In an embodiment, the N-6 substituted 7-deazapurine is not an N-6 benzyl or N-6 phenylethyl substituent. In another embodiment, R < ₄ > is not a benzyl or phenylethyl substituent. In a preferred embodiment, R < ₁ > and R < ₂ > are not both hydrogen atoms. In another preferred embodiment, R < ₃ > is not a hydrogen atom.
[52] The term " therapeutically effective amount " of the substituted N-6 substituted 7-deazapurine refers to the amount of N-6 substituted 7-deazapurine sensitive in mammals, Quot; means the amount of therapeutic compound necessary or sufficient to treat a condition or disease state. The effective amount of the therapeutic compound will depend on the amount of the causative agent already present in the mammal, the age, sex, weight, and ability of the therapeutic compound of the present invention to affect the N-6 substituted 7-deazapurin responsive state in the animal It can vary depending on the same factors. One of ordinary skill in the art will be able to study the above factors and make decisions regarding the effective amount of therapeutic compound without undue experimentation. An " effective amount " of the therapeutic compound can be determined using in vitro or in vivo evaluation experiments, as described below. One of ordinary skill in the art will be able to select the appropriate amount of the therapeutic compound for use in the evaluation experiments described above or as a therapeutic treatment.
[53] The therapeutically effective amount is preferably at least about 20% (more preferably at least about 40%, more preferably at least about 40%, more preferably at least about 40%, more preferably at least about 40% Even more preferably at least about 60%, more preferably at least about 80%). Assessment experiments may be devised by those skilled in the art to measure such symptoms and / or diminishment of symptoms. Any technically recognized method capable of specifying such a variable is intended to be included as part of the present invention. For example, if asthma is a disease state to be treated, the amount of air used from the subject's lung is measured before and after treatment using techniques known in the art to measure the increase in air volume. Similarly, if the inflammation is a disease state to be treated, the site of inflammation can be measured before and after treatment using techniques known in the art to determine the reduced state at the site of inflammation.
[54] The term " cell " includes both prokaryotic and eukaryotic cells.
[55] The term " animal " includes any organism with an adenosine receptor or any organism sensitive to the N-6 substituted 7-deazapurine responsive state. Examples of such animals include yeast, mammals, reptiles, and algae. It also includes transgenic animals.
[56] The term "mammal" is intended to include animals, more preferably warm-blooded animals, most preferably cows, sheep, pigs, horses, dogs, cats, rats, mice, and humans, . N-6 substituted 7-deazapurine Mammals susceptible to a responsive state, inflammation, model, asthma, central nervous system conditions, or acute respiratory distress are included as part of the present invention.
[57] In yet another aspect, the present invention relates to a method of modulating adenosine receptors that causes a mammal to be administered a therapeutically effective amount of N-6 substituted 7-deazapurin to cause modulation of the adenosine receptor in the mammal. Suitable adenosine receptors include the group A ₁ , A _2, or A ₃ . In a preferred embodiment, the N-6 substituted 7-deazapurine is an acylated receptor antagonist.
[58] The term " adenosine receptor modulating " includes such cases in which the compound acts with adenosine receptors, resulting in increased, decreased, or abnormal physiological activity associated with adenosine receptors or subsequent cascade symptoms resulting from the modulation of adenosine receptors do. Physiological activities associated with adenosine receptors include induction of sedation, vasodilation, suppression of heart rate, and contraction, inhibition of platelet aggregation, stimulation of glucose production, inhibition of lipolysis, opening of calcium channels, reduction of calcium channels, and the like.
[59] The terms " regulate ", " modulate " and " modulate " are intended to include, for example, prevent, eliminate, or prevent, in the context of the therapeutic crime of the present invention, the consequence of increased undesired physiological activity associated with abnormal stimulation of adenosine receptors Including inhibition. In other embodiments, the term modulation includes antagonist action, e. G., Reduction in the activity or production of allergic and allergic inflammatory mastheaders resulting from transient stimulation of the adenosine receptors. For example, therapeutic deazapurines of the present invention may interact with adenosine receptors to inhibit, for example, adenylate cyclase activity.
[60] The term " conditions characterized by abnormal adenosine receptor activity " refers to a condition that is associated with an abnormal stimulation of the adenosine receptor, such that the stimulation of the receptor causes a series of biochemical and / or physiological events linked directly or indirectly to the disease, And includes such disease, disorder or condition. This stimulation of the adenosine receptors is not due to the sole causative agent of the disease, disorder or condition, but rather to simply causing a part of the symptoms typically associated with the disease, disorder or condition to be treated. Abnormal stimulation of the receptor may be a single agent or may involve a condition in which at least one other agent is to be treated. Examples of conditions include the abovementioned disease states, such as those manifested by the presence of inflammation, gastrointestinal disease and increased adenosine receptor activity. Preferred examples include those associated with asthma, allergic rhinitis, refractory pulmonary disease, edema, bronchitis, gastrointestinal disease and glaucoma.
[61] The term " treatment of conditions characterized by abnormal adenosine receptor activity " is intended to include alleviating or reducing at least one symptom typically associated with the condition. The treatment also includes alleviation or reduction of more than one symptom. Preferably, the treatment is to ameliorate, e.g., significantly, the symptoms associated with the condition.
[62] The present invention relates to a compound N-6 substituted 7-deazapurine having formula (I)
[63] (I)
[64] In this formula,
[65] R ₁ and R ₂ are each a hydrogen atom or a substituted or unsubstituted alkyl, aryl or alkylaryl moiety, or together form a substituted or unsubstituted heterocyclic ring;
[66] R ₃ is a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety;
[67] R ₄ is a hydrogen atom or a substituted or unsubstituted alkyl, aryl or alkylaryl moiety. R ₅ and R ₆ are independently a halogen atom, for example chlorine, fluorine or hydrogen, or a hydrogen atom or a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety, or R ₄ and R ₅ or R ₅ , R < ₆ > together form a substituted or unsubstituted heterocyclic or carbocyclic ring. Also included are pharmaceutically acceptable salts of said N-6 substituted 7-deazapurine.
[68] In certain embodiments, R ₁ and R ₂ can each independently be a substituted or unsubstituted cycloalkyl or heteroarylalkyl moiety. In another embodiment, R ₃ is a hydrogen atom or a substituted or unsubstituted heteroaryl moiety. In yet another embodiment, R ₄ , R _5, and R ₆ can each independently be a heteroaryl moiety.
[69] In one embodiment, R ₁ is a hydrogen atom, R ₂ is a substituted or unsubstituted cyclohexane, cyclopentyl, cyclobutyl or cyclopropane moiety, R ₃ is a substituted or unsubstituted phenyl moiety, R ₄ is hydrogen And R < ₅ > and R < ₆ > are both methyl groups.
[70] In another embodiment, R ₂ is cyclohexanol, cyclohexanediol, cyclohexylsulfonamide, cyclohexanamide, cyclohexyl ester, cyclohexene, cyclopentanol or cyclopentane diol and R ₃ is a phenyl moiety.
[71] In another embodiment, R ₁ is a hydrogen atom, R ₂ is cyclohexanol, R ₃ is substituted or unsubstituted phenyl, pyridine, furan, cyclopentane, or thiophene moiety, R ₄ is a hydrogen atom, a substituted Alkyl, aryl or arylalkyl moiety, and R ₅ and R ₆ are each independently a hydrogen atom, or a substituted or unsubstituted alkyl, aryl or alkylaryl moiety.
[72] In another embodiment, R ₁ is a hydrogen atom and R ₂ is selected from the group consisting of substituted or unsubstituted alkylamines, arylamides or alkylarylamides, substituted or unsubstituted alkylsulfonamides, arylsulfonamides or alkylarylsulfonamides, Substituted or unsubstituted alkylcarbamates, arylcarbamates or alkylaryl carbamates, substituted or unsubstituted alkylcarboxylic acids, arylcarboxylic acids or alkylarylcarboxylic acids, and R ₃ is a substituted or unsubstituted phenyl moiety, R ₄ is a hydrogen atom, and R ₅ and R ₆ are both methyl groups.
[73] In another embodiment, R < _{2 >} is guanidine, modified guanidine, cyanoguanidine, thiourea, thioamide or amidine.
[74] In one embodiment, R < _{2 >} is ego,
[75] Wherein R _2a -R _2c are each independently a hydrogen atom or a saturated or unsaturated alkyl, aryl or alkylaryl moiety and R _2d is a hydrogen atom or a saturated or unsaturated alkyl, aryl or alkylaryl moiety, NR _2e R _2f or OR _2g , Wherein R _2e -R _2f are each independently a hydrogen atom or a saturated or unsaturated alkyl, aryl or alkylaryl moiety. Alternatively, R _2a and R _2b together form a carbocyclic or heterocyclic ring having a ring size of about 3 to 8 members, for example, a cyclopropyl, cyclopentyl, cyclohexyl group.
[76] In one aspect of the present invention, both R ₅ and R ₆ are not methyl groups, and preferably one of R ₅ and R ₆ is an alkyl group, for example, a methyl group and the other is a hydrogen atom.
[77] In another aspect of the invention, when R ₄ is 1-phenylethyl and R ₁ is a hydrogen atom, R ₃ is phenyl, 2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl, 3-methoxyphenyl or 4-methoxyphenyl, when R ₄ and R ₁ are 1-phenylethyl, R ₃ is not a hydrogen atom, or when R ₄ is a hydrogen atom and R ₃ is phenyl, R ₁ Is not phenylethyl.
[78] In another aspect of the invention, R ₅ and R ₆ together are a carbocyclic ring, for example, , When R ₃ is not phenyl, R ₄ is 1- (4-methylphenyl) ethyl, phenylisopropyl, phenyl or 1-phenylethyl, or R ₃ is When not a hydrogen atom, R < ₄ > is 1-phenylethyl. The carbocyclic ring formed by R < ₅ > and R < ₆ > may be aromatic or aliphatic and may have, for example, naphthyl, phenylcyclohexyl, etc. having an atom between 4 and 12 carbon atoms, Lt; / RTI > carbon atoms, for example, cyclopentyl or cyclohexyl. Alternatively, R ₅ and R ₆ together can form a heterocyclic ring as described below. Representative heterocyclic rings contain from 4 to 12 carbon atoms, preferably from 5 to 7 carbon atoms, and may be aromatic or aliphatic. The heterocyclic ring may be further substituted, including substituting at least one carbon atom of the ring structure with one or more heteroatoms.
[79] In another aspect of the invention, R ₁ and R ₂ form a heterocyclic ring. Representative examples include the heterocyclic rings described below, such as, for example, 4-hydroxypiperidine, 4-aminopiperidine, such as morpholino, piperazine and the like. R ₁ and R ₂ together form a pyrazinino group, , R ₇ is a hydrogen atom or a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety.
[80] In another aspect of the invention, R ₄ and R ₆ together are a heterocyclic ring, for example, Can be formed. The heterocyclic ring may be aromatic or aliphatic and may form a ring having 4 to 12 carbon atoms, such as naphthyl, pentylcyclohexyl, and the like, and may be aromatic or aliphatic, such as cyclohexyl, Cyclopentyl. The heterocyclic ring may be further substituted, including substituting the carbon atom of the ring structure with one or more heteroatoms. Alternatively, R ₄ and R ₅ may together form a heterocyclic ring as described below.
[81] In certain embodiments, the N-6 substituted 7-deazapurine is not N-6 benzyl or N-6 phenylethyl substituted. In other embodiments, R < ₄ > is not benzyl or phenylethyl substituted. In a preferred embodiment, R < ₁ > and R < ₂ > are not both hydrogen atoms. In another preferred embodiment, R < ₃ > is not a hydrogen atom.
[82] The compounds of the present invention include, for example, water-soluble prodrugs that are metabolized in vivo to the active drug by esterase catalytic hydrolysis. Examples of possible prodrugs include, for example, deazapurines with R ₂ as cycloalkyl substituted with -OC (O) (Z) NH ₂ , wherein Z is a natural or an unnatural occurring amino acid Or an analogue thereof, an , , Or Amino acid, or a side chain of a dipeptide. Preferred amino acid side chains are glycine, alanine, valine, leucine, isoleucine, lysine, alpha -methylalanine, aminocyclopropanecarboxylic acid, azetidine-2- carboxylic acid, beta-alanine, gamma -aminobutyric acid , Alanine-alanine, or glycine-alanine.
[83] In another embodiment, the invention features diazapurine of formula (I) and a pharmaceutically acceptable salt thereof,
[84] In this formula,
[85] R ₁ is hydrogen;
[86] R ₂ is substituted or unsubstituted cycloalkyl, substituted or unsubstituted alkyl, or R ₁ and R ₂ together form a substituted or unsubstituted heterocyclic ring;
[87] R ₃ is unsubstituted or substituted aryl;
[88] R < ₄ > is hydrogen; And
[89] R ₅ and R ₆ are each independently hydrogen or alkyl. The deazapurines of this embodiment may be potentially selective A ₃ receptor antagonists.
[90] In one embodiment, R ₂ is substituted (e. G., Hydroxy substituted) or unsubstituted cycloalkyl. In particular embodiments thereof, R ₁ and R ₄ are hydrogen, R ₃ is unsubstituted or substituted phenyl, and R ₅ and R ₆ are each alkyl. Preferably R < _{2 >} is mono-hydroxycyclopentyl or mono-hydroxycyclohexyl. R ₂ can also be substituted with -NH-C (= O) E, where E is a substituted or unsubstituted C ₁ -C ₄ alkyl (e.g., alkylamine, e.g., ethylamine).
[91] R ₁ and R ₂ may also form a substituted or unsubstituted heterocyclic ring, and the ring may be substituted with an amine or an acetamino group.
[92] In another aspect, R ₂ can be -A-NHC (= O) B where A is unsubstituted C ₁ -C ₄ alkyl (eg, ethyl, propyl, butyl) and B is substituted or unsubstituted substituted C ₁ -C ₄ alkyl (e.g., methyl, aminoalkyl, e. g., amino example a methyl or aminoethyl, alkylamino, e.g., methylamino, ethylamino), preferably R ₁ and R ₄ Is hydrogen, R < ₃ > is an unsubstituted or substituted phenylmide and R < ₅ > and R < ₆ > are each alkyl. B may be a substituted or unsubstituted cycloalkyl, for example, cyclopropyl or 1-amino-cyclopropyl. Preferably, R ₃ is substituted with one or more substituents (e.g., o-, m- or p- Phenyl, o-, m- or p-fluorophenyl).
[93] Advantageously, R ₃ is substituted or unsubstituted heteroaryl, preferably when R ₅ and R ₆ are each alkyl. Examples of heteroaryl groups include pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, pyrrolyl, thiazolyl, thiazolyl, oxazolyl, oxadiazolyl, furanyl, methylenedioxyphenyl and thiophenyl . Preferably, R ₃ is 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl or 3-pyrimidyl.
[94] Preferably, in one embodiment, each of R ₅ and R ₆ is hydrogen. In another embodiment, R < ₅ > and R < ₆ > are each methyl.
[95] In a particularly preferred embodiment, the deazapurines of the present invention are water-soluble prodrugs that can be metabolized to the active drug in vivo, for example, by esterase catalytic hydrolysis. Preferably, the prodrug comprises a R ₂ group that is cycloalkyl substituted with -OC (O) (Z) NH ₂ , wherein Z is a naturally occurring or naturally occurring amino acid or analog thereof, or amino acid, or the side chain of the dipeptide. Preferred amino acid side chains are glycine, alanine, valine, leucine, isoleucine, lysine, alpha -methylalanine, aminocyclopropanecarboxylic acid, azetidine-2- carboxylic acid, beta-alanine, gamma -aminobutyric acid , Alanine-alanine, or glycine-alanine.
[96] In a particularly preferred embodiment, Z is a side chain of glycine, R ₂ is cyclohexyl, R ₃ is phenyl, and R ₅ and R ₆ are methyl.
[97] In another embodiment, the deazapurine is 4- (cis-3-hydroxycyclopentyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.
[98] In another embodiment, the deazapurine is 4- (cis-3- (2-aminoacetoxy) cyclopentyl) amino-5,6-dimethyl- Trifluoroacetic acid salt.
[99] In another embodiment, the deazapurine is 4- (3-acetamino) piperidinyl-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.
[100] In another embodiment, the deazapurine is 4- (2-N'-methyl urea propyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.
[101] In another embodiment, the deazapurine is 4- (2-acetaminobutyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.
[102] In another embodiment, the deazapurine is 4- (2-N'-methyl urea butyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.
[103] In another embodiment, the deazapurine is 4- (2-aminocyclopropylacetaminoethyl) amino-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.
[104] In another embodiment, the deazapurine is 4- (trans-4-hydroxycyclohexyl) amino-2- (3-chlorophenyl) -7H-pyrrolo [2,3d] pyrimidine.
[105] In another embodiment, the deazapurine is 4- (trans-4-hydroxycyclohexyl) amino-2- (3-fluorophenyl) -7H-pyrrolo [2,3d] pyrimidine.
[106] In another embodiment, the deazapurine is 4- (trans-4-hydroxycyclohexyl) amino-2- (4-pyridyl) -7H-pyrrolo [2,3d] pyrimidine.
[107] In another embodiment, the invention provides a method of treating an adenosine receptor (e. G., A ₁ , A, A) in a cell by contacting the cell with an N-6 substituted 7-deazapurine (e. G., Preferably an adenosine receptor antagonist) _2A , A _2B, or preferably A ₃ ).
[108] In another aspect, the invention features a method of treating an eye injury to an animal (e.g., a human) by administering to the animal an effective amount of N-6 substituted 7-deazapurine. Preferably, the N-6 substituted 7-deazapurine is an antagonist of the A ₃ adenosine receptor in animal cells. The injury is in the retina or optic nerve head and can be acute or chronic. The injury may be a result of glaucoma, edema, ischemia, hypoxia or scarring.
[109] In a preferred embodiment, the present invention is characterized by deazapurine having the formula (II)
[110] X is N or CR < ₆ >;
[111] R ₁ and R ₂ are each independently hydrogen or a substituted or unsubstituted alkoxy, aminoalkyl, alkyl, aryl, or alkylaryl, or both, together with the proviso that they are not hydrogen, together form a substituted or unsubstituted heterocyclic ring;
[112] R < ₃ > is substituted or unsubstituted alkyl, arylalkyl or aryl;
[113] R ₄ is hydrogen or substituted or unsubstituted C ₁ -C ₆ alkyl;
[114] L is hydrogen, substituted or unsubstituted alkyl or R ₄ and L form a substituted or unsubstituted heterocyclic or carbocyclic ring with each other;
[115] R < ₆ > is hydrogen, substituted or unsubstituted alkyl or halogen;
[116] Q is CH ₂ , O, S or NR ₇ wherein R ₇ is hydrogen or substituted or unsubstituted C ₁ -C ₆ alkyl; And
[117] W is an unsubstituted or substituted alkyl, cycloalkyl, aryl, arylalkyl, biaryl, heteroaryl, substituted carbonyl, substituted thiocarbonyl or substituted sulfonyl wherein R ₃ is pyrrolidino, then R ₄ is Methyl is not satisfied.
[118] In one embodiment, in the compound of formula (II), X is CR _6, and and Q is CH _2, O, S or NH. In another embodiment, X is N.
[119] In another embodiment of the compounds of formula (II), W is substituted or unsubstituted aryl, 5- or 6-membered heteroaryl or biaryl. W may be substituted with one or more substituents. Examples of substituents are: halogen, hydroxy, alkoxy, amino, amino-alkyl, amino-carboxylic neck when _{amide, CN, CF 3, CO 2} R 8, CONHR 8, CONR 8 R 9, SOR 8, SO 2 R 8, and SO ₂ NR ₈ R ₉ , wherein R ₈ and R ₉ are each independently hydrogen or substituted or unsubstituted alkyl, cycloalkyl, aryl, or arylalkyl. Preferably, W is a substituted or unsubstituted phenyl, such as methylenedioxyphenyl. W can also be a substituted or unsubstituted 5-membered heteroaryl ring such as pyrrole, pyrazole, oxazole, imidazole, triazole, tetrazole, furan, thiophene, thiazole, and oxadiazole. Preferably, W is a 6-membered heteroaryl ring such as pyridyl, pyrimidyl, pyridazinyl, pyrazinyl and thiophenyl. In a preferred embodiment, W is 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, or 5-pyrimidyl.
[120] In one useful embodiment of the compound of formula (II), Q is HN and W is a 3-pyrazol ring, which is N-substituted by unsubstituted or substituted or unsubstituted alkyl, cycloalkyl, aryl or arylalkyl.
[121] In another embodiment of the compounds of formula (II), Q is oxygen and W is a 2-thiazole ring, which is unsubstituted or substituted by substituted or unsubstituted alkyl, cycloalkyl, aryl or arylalkyl.
[122] In another embodiment of the compounds of formula (II), W is substituted or unsubstituted alkyl, cycloalkyl, e.g. cyclopentyl or arylalkyl. Examples of substituents are halogen, hydroxy, substituted or unsubstituted alkyl, cycloalkyl, aryl, arylalkyl or NHR _{10 wherein} R ₁₀ is hydrogen or substituted or unsubstituted alkyl, cycloalkyl, aryl or arylalkyl.
[123] In another embodiment, the invention features deazapurines of formula (II) wherein W is - (CH ₂ ) _a -C (= O) Y or - (CH ₂ ) _a -C ) Y, and, a is an integer from 0 to 3, Y is as in aryl, alkyl, arylalkyl, cycloalkyl, heteroalkyl, alkynyl, NHR or ₁₁ R _12, Q is NH condition, oR _13, where R ₁₁ , R ₁₂ and R ₁₃ are each independently hydrogen or substituted or unsubstituted alkyl, aryl, arylalkyl, or cycloalkyl. Preferably, Y is a 5- or 6-membered heteroaryl ring.
[124] Furthermore, W can be - (CH ₂ ) _b -S (═O) _j Y where j is 1 or 2, b is 0, 1, 2, or 3 and Y is aryl, R ₁₄ , R _15, and R ₁₆ are each independently hydrogen or a substituted or unsubstituted alkyl, cycloalkyl, alkynyl, heteroaryl, or NHR ₁₄ R _{15 wherein} b is 1 when Q is CH ₂ , Unsubstituted alkyl, aryl, arylalkyl or cycloalkyl.
[125] In another embodiment, R ₃ is selected from the group consisting of substituted or unsubstituted phenyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, pyrrolyl, triazolyl, thiazolyl, oxazolyl, oxadiazolyl, pyrazolyl, furanyl, Methylenedioxyphenyl, and thiophenyl. ≪ / RTI > When R < ₃ > is phenyl, it may be, for example, hydroxyl, alkoxy (e.g. methoxy), alkyl Phenyl, or o-, m-, or p-chlorophenyl). Advantageously, R ₃ can be 2-, 3- or 4-pyridyl or 2- or 3-pyrimidyl.
[126] The invention also relates to R ₆ is hydrogen or C ₁ -C ₃ alkyl having aza purines. Preferably R < ₆ > is hydrogen.
[127] The invention also relates to compounds of formula I wherein R ₁ is hydrogen and R ₂ is substituted or unsubstituted alkyl or alkoxy, substituted or unsubstituted alkylamine, arylamine, or alkylarylamine, substituted or unsubstituted aminoalkyl, aminoaryl, Substituted or unsubstituted alkylamides, substituted or unsubstituted alkylamides, arylamides or alkylarylamides, substituted or unsubstituted alkylsulfonamides, arylsulfonamides or alkylarylsulfonamides, substituted or unsubstituted alkylureas, arylureas or alkylarylureas, substituted or unsubstituted alkyl Carbamates, aryl carbamates or alkylaryl carbamates, or substituted or unsubstituted alkylcarboxylic acids, allylic carboxylic acids or alkylcarboxylic acids. Preferably, R ₂ is selected from substituted or unsubstituted cycloalkyl, such as mono- or dihydroxy-substituted cyclohexyl or cyclopentyl (preferably monohydroxy-substituted cyclohexyl or monohydroxy-substituted cyclo Pentyl).
[128] Advantageously, R < ₂ > may be of the formula:
[129]
[130] Wherein A is selected from the group consisting of C ₁ -C ₆ alkyl, C ₃ -C ₇ cycloalkyl, a chain of one to seven atoms, or a ring of three to seven atoms, optionally substituted with C ₁ -C ₆ alkyl, A hydroxyl, a carboxyl, a thio or an amino group;
[131] B is selected from the group consisting of methyl, N (Me) 2, N (Et) 2, NHMe, NHMe, NHEt, (CH2) rNH3 +, NH (CH2) rCH3, (CH2) rNH2, (CH2) rCHCH3NH2, wherein r is an integer from 0 to 2, m is 1 or 2, R ₁₈ is alkyl, and R ₁₉ is NH ₃ ⁺ or CO ₂ (CH 2) H or R < ₁₈ > and R < ₁₉ & , Wherein p is 2 or 3; And
[132] R ₁₇ is selected from the group consisting of C ₁ -C ₆ alkyl, C ₃ -C ₇ cycloalkyl, a chain of one to seven atoms, or a _three- to seven-membered ring, optionally substituted with C ₁ -C ₆ alkyl, halogens, , Carboxyl, thio or amino group.
[133] Advantageously, A is substituted or unsubstituted C ₁ -C ₆ alkyl. B may be substituted or unsubstituted C ₁ -C ₆ alkyl.
[134] In a preferred embodiment, R ₂ is formula-A-NHC (= O) B. In a particularly useful embodiment, A is -CH ₂ CH ₂ - and B is methyl.
[135] The compounds of the invention may, for example, comprise a water-soluble prodrug metabolised in vivo by the esterase catalytic hydrolysis into the active drug. Examples of possible prodrugs include, for example, deazapurines with R ₂ as cycloalkyl substituted with -OC (O) (Z) NH ₂ , wherein Z is a natural or an unnatural occurring amino acid Or an analogue thereof, an , , Or Amino acid, or a side chain of a dipeptide. Preferred amino acid side chains are glycine, alanine, valine, leucine, isoleucine, lysine, alpha -methylalanine, aminocyclopropanecarboxylic acid, azetidine-2- carboxylic acid, beta-alanine, gamma -aminobutyric acid , Alanine-alanine, or glycine-alanine.
[136] In other embodiments, R ₁ and R ₂ are together As a result,
[137] Wherein n is 1 or 2 and wherein the ring is optionally substituted with one or more hydroxyl, amino, thiol, carboxyl, halogen, CH ₂ OH, CH ₂ NHC (═O) alkyl or CH ₂ NHC Lt; / RTI > group. Preferably n is 1 or 2 and the ring is substituted with -NHC (+ O) alkyl.
[138] In one useful embodiment, R ₁ is hydrogen, R ₂ is substituted or unsubstituted C ₁ -C ₆ alkyl, Q is O, S or NR ₇ , R ₇ is hydrogen or a substituted or unsubstituted C ₁ - a C ₆ alkyl, and W is a substituted or unsubstituted aryl group. Preferably, R ₂ is -A-NHC (= O) B, wherein A and B are each independently substituted or unsubstituted C ₁ -C ₄ alkyl. For example, A may be CH ₂ CH ₂ . B may be, for example, alkyl (e.g., methyl), or aminoalkyl (e.g., aminomethyl). Preferably, R < ₃ > is unsubstituted phenyl and L is hydrogen. R ₆ can be methyl and is preferably hydrogen. Preferably, Q is O, S or NR ₇ , wherein R ₇ is hydrogen or substituted or unsubstituted C ₁ -C ₆ alkyl, such as methyl. W is substituted or unsubstituted phenyl (e.g., alkoxy , Halogen substituted). Preferably W is p-fluorophenyl, p-chlorophenyl, or p-methoxyphenyl. W can also be heteroaryl, for example, 2-pyridyl.
[139] In a particularly preferred embodiment, the deazapurine is 4- (2-acetylaminoethylamino-6-phenoxymethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.
[140] In another particularly preferred embodiment, the deazapurine is selected from the group consisting of 4- (2-acetylaminoethyl) amino-6- (4-fluorophenoxy) methyl-2- phenyl-7H- pyrrolo [2,3d] pyrimidine to be.
[141] In another particularly preferred embodiment the deazapurine is 4- (2-acetylaminoethyl) amino-6- (4-chlorophenoxy) methyl-2-phenyl-7H- pyrrolo [2,3d] pyrimidine .
[142] In another particularly preferred embodiment, the deazapurine is selected from the group consisting of 4- (2-acetylaminoethyl) amino-6- (4-methoxyphenoxy) methyl-2-phenyl-7H- pyrrolo [2,3d] pyrimidine to be.
[143] In another particularly preferred embodiment, the deazapurine is 4- (2-acetylaminoethyl) amino-6- (2-pyridyloxy) methyl-2- phenyl-7H-pyrrolo [2,3d] pyrimidine .
[144] In another particularly preferred embodiment, the deazapurine is 4- (2-acetylaminoethyl) amino 6- (N-phenylamino) methyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.
[145] In another particularly preferred embodiment the deazapurine is 4- (2-acetylaminoethyl) amino-6- (N-methyl-N-phenylamino) methyl- Pyrimidine.
[146] In another particularly preferred embodiment, the deazapurine is 4- (2-N'-methyl ureaethyl) amino-6-phenoxymethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.
[147] The present invention also relates to a method of inhibiting the activity of an adenosine receptor (e.g., an A _2b -inceptor) in a cell by contacting the cell with a compound of the invention. Preferably, the compound is an antagonist of the receptor.
[148] The invention also relates to a method of treating gastrointestinal disorders (e.g. diarrhea) in an animal by administering to the animal an effective amount of a compound of the invention (e.g., an antagonist of A _2b ). Preferably the animal is a human.
[149] In another embodiment, the present invention relates to a pharmaceutical composition comprising the N-6 substituted 7-deazapurine of the present invention and a pharmaceutically acceptable carrier.
[150] The present invention also relates to a method for the treatment of N-6 substituted 7-deazapurin responsive states in a mammal, comprising administering to the animal a therapeutically effective amount of the inventive deazapurine to induce N-6 substituted 7-deazapurine responsive Lt; / RTI > Advantageously, such disease states can be mediated by adenosine. Examples of preferred disease states include central nervous system diseases, cardiovascular diseases, retinal diseases, inflammatory diseases, allergic diseases, gastrointestinal diseases, ophthalmic diseases and respiratory diseases.
[151] The term " alkyl " refers to a portion of a saturated aliphatic group, such as a straight chain alkyl group, a branched alkyl group, a cycloalkyl (alicyclic) group, an alkyl substituted cycloalkyl group, and a cycloalkyl substituted alkyl group. The term alkyl is an alkyl group, which may further contain an oxygen, nitrogen, sulfur or phosphorus atom replacing one or more carbons on the hydrocarbon backbone. In a preferred embodiment, the straight chain or branched alkyl has up to 30 carbon atoms (e.g., C ₁ -C ₃₀ straight chain, C ₃ -C ₃₀ branched chain) on the backbone, and more preferably up to 20 carbons Have an atom. Similarly, preferred cycloalkyls have 4-10 carbon atoms in the ring structure, more preferably 5, 6 or 7 carbons.
[152] The term alkyl, as used throughout the specification and claims, is intended to include " unsubstituted alkyl " and " substituted alkyl ", the latter being alkyl having a substituent substituting hydrogen on one or more carbons of the hydrocarbon backbone Powder means. Such substituents include, for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyl, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, Alkylamino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbamoyl, alkylcarbamoyl, Alkylthio, arylthio, thiocarboxylate, sulfate, sulfonato, sulfamoyl, sulfonamido, nitro, aminocarbonylamino, Trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or aromatic or heteroaromatic powders. It is obvious to those skilled in the art that the powder itself substituted on the hydrocarbon chain can be suitably substituted. Cycloalkyl can be further substituted, for example, with the substituents described above. An " alkylaryl " powder is alkyl substituted with aryl (e.g., phenylmethyl (benzyl)). The term " alkyl " is also an unsaturated aliphatic group which is similar in length and which is capable of substitution with the alkyl groups described above, but which has at least one double or triple bond, respectively.
[153] As used herein, the term " aryl " refers to 5- and 6-membered monocyclic aromatic groups that may include from zero to four heteroatoms such as benzene, pyrrole, furan, thiophene, imidazole, Means an aryl group, such as benzoxazole, benzothiazole, triazole, tetrazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine. The aryl group also includes polycyclic fused aromatic groups such as naphthyl, quinolyl, indolyl, and the like. Such aryl groups having heteroatoms in the ring structure are also referred to as "aryl heterocycle", "heteroaryl" or "heteroaromatic". The aromatic ring may be substituted at one or more ring positions with such substituents as described above, for example, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, (Alkylamino, dialkylamino, arylamino, diarylamino, and alkyl (alkenyl) groups which are optionally substituted with one or more substituents selected from the group consisting of alkyl, alkoxy, (Including alkylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, sulfo Alkaryl, or aromatic or heteroaromatic powders, such as, for example, sodium, potassium, sodium, potassium, sodium, potassium, The aryl group may be substituted with a substituent also it is alicyclic or heterocyclic non-aromatic rings fused or bridge to form a poly cycle (e.g., tetralin).
[154] The terms " alkenyl " and " alkynyl " refer to unsaturated aliphatic groups similar in length and capable of substitution with the abovementioned alkyl, but which, respectively, have at least one double or triple bond. For example, the present invention uses cyano and propargyl groups.
[155] Unless otherwise specified, the term "lower alkyl" as used herein refers to an alkyl group having from one to ten carbons, more preferably from one to six carbon atoms in the backbone structure, even more preferably from one to three carbons in the backbone structure Quot; means an alkyl group having an atom. Similarly, "lower alkenyl" and "lower alkynyl" have pseudo-chain lengths.
[156] The terms " alkoxyalkyl ", " polyaminoalkyl ", and " thioalkoxyalkyl " refer to alkyl groups as described above, which substitute one or more carbons of the hydrocarbon backbone and further include oxygen, nitrogen or sulfur atoms.
[157] The term " polycyclyl " or " polycyclic radical " refers to a radical of two or more cyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and / or heterocycles) Quot; refers to the fact that more than two carbons are common to two adjacent rings, that is, the rings are " fused rings ". Rings connected through non-adjacent atoms are referred to as " bridged " Each of the polycycle rings is optionally substituted with one or more substituents selected from the group consisting of halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyl, aryloxycarbonyloxy, carbonylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl (Including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkyl, alkoxy, Alkylthio, arylthio, thiocarboxylate, sulfate, sulfonato, sulfamoyl, sulfonamido, nitro (including but not limited to carbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, , Trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or aromatic or heteroaromatic powder.
[158] The term " heteroatom " as used herein means an atom of any other element besides carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur and phosphorus.
[159] The term " amino acid " refers to an amino acid that occurs naturally or naturally in a protein, such as glycine, alanine, valine, cysteine, leucine, isoleucine, serine, threonine, methionine, glutamic acid, Glutamine, asparagine, lysine, arginine, proline, histidine, phenylalanine, tyrosine, and tryptophan. Amino acid analogs include extended or shortened side chains with appropriate functionalities or amino acids with various side chains. Amino acids also include D and L stereoisomers of amino acids when the sphere of the amino acid is in the stereostructural form. The term " dipeptide " includes two or more amino acids linked together. Preferably, the dipeptide is two amino acids linked through a peptide linkage. Particularly preferred dipeptides include, for example, alanine-alanine or glycine-alanine.
[160] The structure of some of the compounds of the present invention is meant to include asymmetric carbon atoms. Thus, unless stated otherwise, it is to be understood that isomers arising from such asymmetry (e. G., All enantiomers and diastereomers) are within the scope of the present invention. Such isomers can be obtained in fairly pure form by conventional separation techniques and stereochemically controlled synthesis.
[161] The present invention also relates to the use of a compound of formula I in the manufacture of a medicament for the treatment or prophylaxis of N-6 substituted 7-deazapurin responsive conditions in mammals, for example respiratory diseases (e.g. asthma, bronchitis, chronic obstructive pulmonary disease and allergic rhinitis), retinal diseases, And pharmaceutical compositions for treating ophthalmic diseases. The pharmaceutical composition, as described above, comprises a therapeutically effective amount of N-6 substituted 7-deazapurine and a pharmaceutically acceptable carrier. It should be understood that all of the above-mentioned deazapurines are included for therapeutic treatment. It is also to be understood that the deazapurines of the present invention may be used alone or in combination with other deazapurines of the present invention or in combination with additional therapeutic compounds such as antibiotics, anti-inflammatory agents or anti-cancer agents.
[162] The term " antibiotics " is art-recognized and refers to those substances that are produced by growing microorganisms that remove or inhibit the growth of pathogens and which are selectively toxic to pathogens, but which exhibit minimal toxicity or non-toxic effects on host subjects infected, And synthetic derivatives thereof. Suitable examples of antibiotics include, but are not limited to, aminoglycosides, cephalosporins, chloramphenicol, fusidic acid, macrolide, penicillin, polyamicin, tetracycline and streptomycin.
[163] The term " anti-inflammatory agent " is art-recognized and includes those agents that act on the body mechanism without directly antagonizing the causative agent of inflammation, such as glucocorticoid, aspirin, ibuprofen, NSAIDS, It is intended.
[164] The term " anti-cancer agent " is intended to include such agents that are known in the art, and preferably do not adversely affect other physiological functions, and that reduce, eliminate or prevent the growth of cancer cells. Representative examples include cisplatin and cyclophosphamide.
[165] When the compounds of the present invention are administered to humans and mammals as pharmaceuticals, they may be given as such or may contain, for example, 0.1 to 99.5% (more preferably 0.5 to 90%) of the active ingredient, In combination with a carrier.
[166] As used herein, the phrase " pharmaceutically acceptable carrier " refers to a carrier or delivery of a compound of the present invention to or into a subject, such as a liquid, solid filler, diluent, excipient, solvent or encapsulating material, Quot; means a pharmaceutically acceptable material, composition, or medicament that causes the subject to perform the treatment. Typically, such compounds are delivered or delivered from one organ or portion of the body to another organ or portion of the body. Such carriers must be " acceptable " in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Some examples of materials that can function as pharmaceutically acceptable carriers include: Sugars such as lactose, glucose and sucrose; Corn starch and potato starch; Cellulose and derivatives thereof such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; Powdered tragacanth; malt; gelatin; Talc; Excipients such as cocoa butter and suppository wax; Peanut oil, cottonseed oil; Oils such as safflower oil, sesame oil, olive oil, corn oil and soybean oil; Glycols such as proglycols; Polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; Esters such as ethyl oleate and ethyl laurate; Buffers such as magnesium hydroxide and aluminum hydroxide; Alginic acid; Sterile water; Physiological isotope; Ringer's solution; Ethyl alcohol; Phosphate buffer; And other non-toxic, affinity materials used in pharmaceutical preparations.
[167] As noted above, certain embodiments of the present invention may include basic functional groups such as amino or alkylamino, thus forming a pharmaceutically acceptable salt with a pharmaceutically acceptable acid. In this context, the term " pharmaceutically acceptable salts " refers to the relatively non-toxic inorganic and organic acid addition salts of the compounds of the present invention. Such salts can be formed in situ during the final separation and filtration of the compounds of the present invention or by reacting the compounds of the present invention in purified, anhydrous form with the appropriate organic or inorganic acids and separating the resulting salts have. Representative salts include, but are not limited to, hydrogen fluoride, hydrogen chloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, (See Berge et al. (1997) " Pharmaceutical Salts, " Berge et al., (1997) J. Pharm Sci. 66: 1-19).
[168] In other instances, the compounds of the present invention may contain one or more functional groups, thus forming a pharmaceutically acceptable salt with a pharmaceutically acceptable base. In this case, the term " pharmaceutically acceptable salts " refers to the relatively non-toxic, inorganic and organic base addition salts of the compounds of the present invention. Such salts may be prepared by reacting a purified compound that is in the free acid form or during the final isolation and purification of the compound, with a suitable base such as a hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, or with a pharmaceutically acceptable organic, primary, Amine. ≪ / RTI > Representative alkali or alkaline earth metal salts include lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for base addition salt formation include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like.
[169] The term " pharmaceutically acceptable esters " refers to relatively non-toxic, esterified products of the compounds of the present invention. Such esters can be prepared as such during the final isolation and purification of the compound or by reacting the purified compound or hydroxy in free acid form with a suitable esterifying agent. The carboxylic acid can be converted to the ester by treatment with an alcohol in the presence of a catalyst. Hydroxyl-containing derivatives can be converted to esters by treatment with an esterifying agent such as an alkanoyl halide. The term is intended to include lower hydrocarbon groups which may be solvated under physiological conditions, such as alkyl esters, methyl, ethyl and propyl esters (see, for example, Berge et al., Supra).
[170] The present invention is directed to the use of prodrugs which are converted in vivo to the therapeutic compounds of the invention (see, for example, RB Silverman, 1992, " The Organic Chemistry of Drug Design and Drug Action ", Academic Press, Chp. 8). Such prodrugs can be used to alter the biodistribution of the therapeutic compound (e. G., To prevent entry into the reactive site of the protease) or pharmacokinetic properties. For example, the carboxylic acid group can be esterified by esterification using, for example, a methyl group or an ethyl group. When the ester is administered to a subject, the ester is enzymatically or non-enzymatically, either reductively or hydrolytically cleaved to give an anionic group. The anionic group is esterified with a powder (for example, acyloxymethyl esters) and is cleaved to form an intermediate compound, which is decomposed to produce the active compound. In another embodiment, the prodrug is a reduced form of a sulfate or sulfonate, e. G., A thiol, which is oxidized in vivo to the therapeutic compound. Moreover, the anionic powder can be esterified with certain groups and is actively delivered in vivo or selectively absorbed by the target organ. The ester can be selected to cause the therapeutic powder to specifically target a specific reaction site to the carrier powder as described later.
[171] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as colorants, emollients, coatings, sweeteners, flavoring agents, and fragrances, preservatives and antioxidants can be used in the composition.
[172] Examples of pharmaceutically acceptable antioxidants include: Water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; Oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha -tocopherol and the like; And metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
[173] The formulations of the present invention include those suitable for oral, nasal, topical, transdermal, oral, sublingual, rectal, vaginal and / or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be made by methods known in the art of formulation. The amount of active ingredient that can be mixed with the carrier material to produce a single dosage form will generally be that amount of the compound that exhibits a therapeutic effect. Generally, this amount ranges from 1 percent to 99 percent, preferably from about 5 percent to 70 percent, and more preferably from about 10 percent to 30 percent, of the active ingredient, among 100 percent.
[174] Such formulations and methods of preparing the compositions include the step of admixing the compound of the present invention with the carrier, optionally with one or more accessory ingredients. In general, the formulations are prepared by uniformly and delicately blending the compound of the present invention with a liquid carrier or a finely divided solid carrier, or both, and then, if necessary, formulating the product.
[175] Formulations of the invention suitable for oral administration may be presented in the form of capsules, cachets, pills, tablets, lozenges (flavored base materials, usually sucrose and acacia or tragacanth), powders, granules, or Aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil emulsion or as pastille (including gelatin and glycerin, or sucrose and acacia, Using the same inert base material), and / or as a mouthwash, and each formulation contains a predetermined amount of a compound of the present invention as an active ingredient. The compounds of the present invention may also be administered as a bolus, an electuary, or a paste.
[176] The active ingredient is mixed with one or more pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate and / or any of the following in a solid dosage form for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.) Fillers or additives such as, for example, starch, lactose, sucrose, glucose, mannitol and / or silic acid; Filler binders such as, for example, carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and / or acacia; A hygroscopic agent such as glycerol; Agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, any silicate and sodium carbonate, etc .; Solution retarders such as paraffin; Absorption promoters such as gold aluminum compounds; Wetting agents such as, for example, cetyl alcohol and glycerol monostearate; Absorbents such as kaolin and bentonite clay; Lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof; And a colorant. In the case of capsules, tablets and pills, the pharmaceutical compositions also include buffering agents. Solution compositions of a similar type may be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or cellulose, as well as high molecular weight polyethylene glycols and the like.
[177] Tablets may optionally be made by compression or molding, such as with one or more accessory ingredients. Compressed tablets can be prepared by conventional means such as binding agents (e.g., gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (e. G., Sodium starch glycolate or crosslinked sodium carboxymethylcellulose) It can be made using dispersant. Molded tablets may be made by molding a mixture of a wet compound powder and a stable liquid diluent in a suitable machine.
[178] Tablets and other solid forms of the pharmaceutical compositions of the invention, such as dragees, capsules, pills and granules, can be made into shells and shells, such as enteric coatings and other coatings known in the pharmaceutical arts have. They may also be formulated to release the active substance slowly or in moderate proportions using, for example, hydroxypropylmethylcellulose, other polymeric matrices, liposomes and / or small spheres to give the desired release profile. They can be disinfected, for example, by a disinfecting solid composition that can be dissolved in disinfecting water or by filtration through a bacteria-scavenging filter, or by the introduction of any other disinfection medium immediately prior to use. Such compositions may optionally comprise a opacifying agent and may be a composition which releases only the active substance, or preferably in a particular portion of the stomach, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric materials and waxes. The active ingredient may also be in micro-capsule form, if appropriate, with one or more of the above excipients.
[179] Liquid dosage forms for oral administration of the compounds of the present invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage form may contain inert diluents commonly used in the art such as water or other solvents, solubilizers, and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate (Especially cottonseed, groundnut, corn, germ, olive, castor and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol, Glycols and fatty acid esters of sorbitan and mixtures thereof.
[180] In addition to the inert diluents, the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, coloring agents, flavoring agents and preservatives.
[181] In addition to the active compounds, the suspensions may contain, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxides, bentonites, agar- agar and tragacanth , And mixtures thereof.
[182] Formulations for rectal and vaginal administration of the pharmaceutical compositions of the present invention may be presented in the form of suppositories, which may contain one or more compounds of the present invention, for example, one containing cocoa butter, polyethylene glycol, a suppository wax or salicylate Such as a non-polar excipient or carrier, which is solid at room temperature but liquid at body temperature and therefore dissolves in the anal or vaginal cavity and releases the active compound.
[183] Formulations of the present invention suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
[184] Dosage forms for topical or transdermal administration of the compounds of the present invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed with the pharmaceutically acceptable carrier under sterile conditions and, if necessary, with any preservatives, buffers or accelerators.
[185] The ointments, pastes, creams and gels may contain excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycols, Bentonite, silic acid, talc, zinc oxide, or mixtures thereof.
[186] Powders and sprays may contain, in addition to the compounds of the present invention, excipients such as lactose, talc, silic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these materials. The spray may further comprise conventional accelerators such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, Butane and propane.
[187] Transdermal patches have the additional advantage of providing for controlled delivery of the compounds of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the compound in a suitable vehicle. Absorption enhancers may also be used to enhance the permeation of the compound through the skin. The rate of such permeation can be controlled by providing a rate controlling membrane or by dispersing the active compound in a polymer matrix or gel.
[188] Ophthalmic formulations, ointments, powders, solutions and the like are also considered to be within the scope of the present invention. Preferably, the pharmaceutical preparation is an ophthalmic formulation (e. G., An ocular, buccal or intraocular injection formulation, a systemic formulation, or a surgical wash). A variety of carriers can be used. The carrier is basically water-soluble. In the case of a formulation, an aqueous solution is generally preferred, given the ability of the patient to easily administer the composition by dropping one or two drops of the solution into the eye of the problem. However, the deazapurines of the present invention can be readily incorporated into other types of compositions, such as suspensions, viscous or viscous gels or other types of solid or semi-solid compositions. The ophthalmic compositions of the present invention may also include various other ingredients such as buffers, preservatives, co-solvents, and thickeners.
[189] Appropriate buffer systems (eg, sodium phosphate, sodium acetate or sodium borate) can be added to prevent pH changes under storage conditions.
[190] Ophthalmic products are typically packaged in multi-dose form. Preservatives are needed to prevent bacterial contamination during use. Suitable preservatives include benzalkonium chloride, thimerosal, chlorobutanol, methylparaben, propylparaben, phenylethyl alcohol, disodium edetate, sorbic acid, polyquaternium-1, or other reagents known to those skilled in the art . Such preservatives are typically used at a level of 0.001 to 1.0% weight / volume ("% w / v ").
[191] It is most preferred to use a balanced salt irritant solution as an excipient when the inventive deazapurine is administered during intraocular surgery such as retrobulbar or intraocular injections and intraocular washes or injections. A BSS ^R sterilization irritant solution and a BSS Plus ^R germicidal irritant solution (Alcon Laboratories, Fort Worth, Tex.) Are examples of physiologically balanced eye irritant solutions. The latter type of solution is described in U.S. Patent No. 4,550,022 (Garabedian et al.), Which is incorporated herein by reference in its entirety. The posterior and periocular injections are well known to those skilled in the art and are described in Ophthalmic Surgery: Principles of Practice, Ed., GL Spaeth. WB Sanders Co., Philadelphia, Pa., USA, pp 85-87 (1990).
[192] As mentioned above, the use of deazapurine to prevent or reduce damage to the retina and optic nerve head tissue at the cellular level is an important aspect of one embodiment of the invention in particular. Conditions of the eye that can be treated include injury to the retina or optic nerve due to exposure to light or surgical instruments, particularly retinopathy, retinal degeneration, ischemia, glaucoma, ischemia reperfusion injury, photochemical injury and ophthalmic surgery But not limited to, injuries to or damage to the eye tissue. The compounds may also be used as adjuvants in ophthalmic surgeries such as ophthalmic surgery to perform vitreal or subconjunctival injections. The compounds may be used in the acute treatment of adverse conditions or in the case of chronic, especially degenerative diseases. The compounds may also be used prophylactically, particularly before ophthalmic surgery or noninvasive ophthalmic surgery or other types of surgery.
[193] A pharmaceutical composition of the invention suitable for parenteral administration may be reconstituted into one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or injectable solutions or dispersions sterilized just prior to use Antioxidants, buffer solutions, bacterial growth inhibitors which are made up of one or more of the compounds of the invention in combination with a sterile powder which can be used in the form of tablets, suspensions or thickeners, ≪ / RTI >
[194] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, There are the same injectable organic esters. Proper fluidity can be maintained by the use of coating materials such as lecithin, and by the use of surfactants, by the maintenance of the required particle size in the case of dispersions.
[195] These compositions may contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Microbial action is prevented by including various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid and the like. It is also preferred to include isotonic agents such as sucrose, sodium chloride and the like in the composition. In addition, prolonged absorption of the injectable pharmaceutical form occurs by including an absorption-retarding reagent such as aluminum monostearate and gelatin.
[196] In some cases, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection to prolong the effect of the drug. This is done using a suspension of weakly soluble crystalline or amorphous material. The absorption rate of the drug depends on the dissolution rate and on the crystal size and crystal form. Alternatively, delayed absorption of the parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
[197] An injectable storage form is prepared by forming a microcapsule matrix of a compound in a polymer degradable by a microorganism, such as a polylactide-polyglycolide. Depending on the drug to polymer ratio, and the nature of the particular polymer used, the drug release rate can be controlled. Examples of degradable polymers by other microorganisms include poly (orthoesters) and poly (anhydrides). Preparations that are also storable injectable are prepared by incorporating the drug into liposomes or microemulsions that are compatible with body tissues.
[198] The preparations of the present invention may be administered orally, parenterally, topically or rectally. The preparation is prepared in a form suitable for each route of administration. For example, the preparation is administered in the form of tablets or capsules, by inhalation by injection, anion, ointment, suppository, etc., and is administered topically to the lotion or ointment by injection, infusion or inhalation into the rectum. Oral administration is preferred.
[199] The phrases " parenteral administration " and " parenterally administered " used herein refer to modes of administration other than intestinal and topical administration by injection, and include intravenous, intramuscular, intraarterial, intrathecal, But are not limited to, intravenous, intracardiac, intradermal, intraperitoneal, intracisternal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intracisternal and intrasternal injection and infusion.
[200] The phrases " systemic administration ", " systemically administered ", " peripheral administration ", and " administered spontaneously " used in the context of the present invention may be introduced into a patient's system to produce a compound, Is indirectly administered to the central nervous system, for example, subcutaneous administration.
[201] These compounds may be administered to humans and other animals for treatment by any suitable route of administration such as oral, spray, nasal, rectal, parenteral, intratracheal, intratracheal, and oral and sublingual by means of powders, ≪ / RTI >
[202] The compounds of the present invention and / or compositions of the present invention that can be used in the appropriate hydrate form, regardless of the route of administration selected, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.
[203] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention may be varied to obtain the amount of active ingredient effective to achieve the desired therapeutic result for the particular patient, the composition being non-toxic to the patient, and the mode of administration.
[204] The selected dosage level will depend upon a variety of factors including the activity of the particular compound of the invention or its ester, salt or amide used, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the time of treatment, The age, sex, weight, condition, general health of the subject being treated and the prior medical history, as well as similar factors known in the medical arts.
[205] Physicians or veterinarians involved in the art can readily determine and prescribe the effective amount of the required pharmaceutical composition. For example, a physician or veterinarian can dose a compound of the invention used in a pharmaceutical composition at a level less than the required dosage to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved have.
[206] In general, a suitable daily dose of a compound of the invention is the amount of a compound that is the minimum dose effective to achieve a therapeutic effect. Such effective dosages generally depend on the factors mentioned above. The intravenous and subcutaneous dose ranges of a compound of the present invention for a patient when used in the analgesic effect indicated are from about 0.0001 to about 200 mg / kg body weight daily, more preferably from about 0.01 to about 150 mg / kg daily, Is about 0.2 to about 140 mg / kg daily.
[207] If desired, an effective daily dose of the active compound may be administered in divided doses of 2, 3, 4, 5, 6 or more times, optionally in unit dosage form, at appropriate intervals of the day.
[208] When only the compound of the present invention to be administered is possible, it is preferable to administer the compound as a pharmaceutical composition.
[209] The present invention also relates to a packaged pharmaceutical composition for treating an N-6 substituted 7-deaza-purine response state, for example, undesired increased adenosine receptor activity in mammals. The packaged pharmaceutical compositions comprise a container containing a therapeutically effective amount of at least one of the above deazapurines and instructions for using deazapurine to treat the deazapurin responsive state in the mammal.
[210] The deazapurines of the present invention can be prepared using standard methods for organic synthesis. Deazapurine is purified by reverse phase HPLC, chromatography, recrystallization and the like, and its structure is confirmed by mass spectrum analysis, elemental analysis IR and / or NMR spectroscopy.
[211] Typically, the synthesis of the intermediates as well as the deazapurines of the invention is carried out in solution. One or more guardrails are added and removed as a typical procedure and are known to those skilled in the art. A typical synthetic reaction scheme for the preparation of the inventive deazapurine intermediates is set forth in Scheme I below.
[212] The invention is further illustrated by the following examples which are not to be construed as limiting. The contents of all references, pending patent applications, and published patent applications cited herein, including those cited in the Background section, are incorporated herein by reference. It is understood that the models in the examples are allowed models and that the efficacy of these models is also expected to be efficacious in humans.
[213] The deazapurines of the present invention can be prepared using standard methods for organic synthesis. Deazapurine is purified by reverse phase HPLC, chromatography, recrystallization and the like, and its structure is confirmed by mass spectrum analysis, elemental analysis IR and / or NMR spectroscopy.
[214] Typically, the synthesis of the intermediates as well as the deazapurines of the invention is carried out in solution. One or more guardrails are added and removed as a typical procedure and are known to those skilled in the art. An exemplary synthetic reaction scheme for the preparation of the inventive deazapurine intermediates is set forth in Scheme I below.
[215]
[216] In the above formula, R ₃ , R ₅ and R ₆ are as described above.
[217] Generally, the protected 2-amino-3-cyano-pyrrole is treated with an acyl halide to produce carboxyamido-3-cyano-pyrrole and treated with acidic methanol to afford the pyrrolo [2,3d] pyrimidine -4 (3H) -one (Muller, CE et al. J. Med. Chem. 40: 4396 (1997)). The pyrrolo protecting group is removed and then treated with a chlorinating reagent such as phosphorus oxychloride to give substituted or unsubstituted 4-chloro-7H-pyrrolo [2,3d] pyrimidines. The chloropyrimidine was treated with an amine to give 7-deazapurine.
[218] For example, as shown in Scheme I, N- (1- dl -phenylethyl) -2-amino-3-cyano-pyrrole is treated with an acyl halide in pyridine and dichloromethane. The obtained N- (1- dl-phenylethyl) -2-amino-3-carboxy-phenyl-cyano-pyrrole 10 in methanol / sulfuric acid to ring closure by treatment with a mixture of 1 dl -7H-7- (1- phenylethyl) Pyrrole [2,3d] pyrimidin-4 (3H) -one. The pyrimidine was treated with polyphosphoric acid (PPA) followed by POCl ₃ to remove the phenylethyl group to yield the key intermediate 4-chloro-7H-pyrrolo [2,3d] pyrimidine. 4-chloro-7H-pyrrolo [2,3d] pyrimidine is further treated with various amines listed in Table 1 to give compounds of formulas (I) and (II).
[219] Table 1
[220]
[221]
[222]
[223] A general scheme for preparing 6-substituted piperols is shown in the following structure (Structure II).
[224] Composition II
[225]
[226] Wherein R ₁ to R ₅ are as defined above.
[227] Transesterification and alkylation of ethyl cyanoacetate with alpha -haloketone results in the keto methyl ester being planar. The ketone was protected and then treated with an amidine (e.g., alkyl, aryl, or alkylaryl) hydrochloride to yield the ketal protected pyrimidine. After removal of the protecting group, cyclization and treatment with phosphorus oxychloride gave the chloride intermediate, which was further treated with an amine to give the amine 6-substituted pyrrole. The pyrrole nitrogen was also alkylated under conditions known in the art.
[228] A general scheme for preparing 5-substituted pyrroles is shown in the following scheme (Scheme III).
[229] Configuration III
[230]
[231] Wherein R ₁ to R ₆ are as defined above and R is a removable protecting group.
[232] After condensation of the malononitrile with an excess of ketone, the product is brominated to obtain a mixture of starting material, monobrominated and diborated product and treated with an alkylamine, arylamine or alkylarylamine. The resulting amine product was acylated with an acid chloride and the single acylated pyrrole was cyclized in the presence of acid to give the corresponding pyrimidine. The pyrrole protecting group was removed using polyphosphoric acid and treated with phosphorus oxychloride to give the chlorinated product. The next chlorinated pyrrole was treated with an amine to give the amine 5-substituted pyrrole. Under conditions known in the art, such pyrrole nitrogen could be alkylated.
[233] The methods of making the deazapurines 1 and 2 of the present invention in Forms IV and V are described.
[234]
[235] In the above formula, R ₅ and R ₆ are the same as described above, for example, CH ₃ .
[236] Special manufacture of 6-methylpyrrolopyrimidines
[237] The main reaction to 6-methylpyrrolopyrimidines (1) [R ₅ = CH ₃ ] is to cyclize cyanoacetyl with pyrimidine using benzamidine. It is believed that methyl cyanoacetate can be cyclized to the pyrimidine more efficiently with benzamidine than the corresponding ethyl ester. Thus, in the presence of NaOMe, transesterification and alkylation with ethyl cyanoacetate and a large amount of an -haloacetyl moiety, such as chloroacetone, produces the desired methyl ester (3) in 79% yield (Scheme IV) . The keto ester (3) was protected as the acetal (4) in 81% yield. A new method of cyclization to the pyrimidine (5) yielded (5) in 54% yield using amidine hydrochloride, for example, benzamidine, for example, benzamidine hydrochloride with 2 equivalents of DBU . This method uses NaOMe during cyclization with guanidines to increase the yield at 20% using known conditions. The cyclization to pyrrole-pyrimidine (6) was achieved in 78% yield by deprotection of the acetal in aqueous HCL. (6) was reacted with phosphorous oxychloride under reflux to give the corresponding 4-chloro derivative (7). Coupling was performed at 135 占 폚 in dimethylsulfoxide with trans-4-aminocyclohexanol to obtain 57% of (1) from (7). Those skilled in the art are aware that, depending on reagent selection, there is great flexibility in choosing the preferred substituent R < _{5 &} gt ;.
[238] Composition IV
[239]
[240] Special manufacture of 5-methylpyrrolopyrimidines
[241] Malononitrile and excess ketone, for example acetone, in refluxing benzene were condensed with Knoevengel to give (8) in a yield of 50% after distillation. (8) was brominated with N-bromosuccinimide in chloroform in the presence of benzoyl peroxide to give a mixture of starting material, Momo (9) and di-brominated product (5/90/5) after distillation (7 )%). This mixture was reacted with -Methylalkylamine or -Methylarylamine, for example, -Methylbenzylamine to obtain aminopyrrole (10). After passing through a short silica gel column, partially purified amines (31% yield) are diazotized with an acid chloride, e.g., benzoyl chloride, to produce mono- (11) and diaminized (12) And separated by flash chromatography. Acid hydrolysis of the dispersed pyrrole (12) produced acylpyrrole (11) with a mixed yield of 29%. (13) (23%) in concentrated sulfuric acid and DMF, and deprotected with (14) using polyphosphoric acid. Reaction of (14) with phosphorous oxychloride under reflux gave the corresponding 4-chloro derivative (15). (2) [R ₆ = CH ₃ ] was obtained as 30% from (14) by coupling with trans-4-aminocyclohexanol at 135 ° C in dimethylsulfoxide. Those skilled in the art are aware that, depending on reagent selection, there is great flexibility in choosing the preferred substituent R < _{5 &} gt ;.
[242] Configuration V
[243]
[244] R ₆- substituted pyrroles, for example, 5-methylpyrrolopyrimidines,
[245] An alternative route to R ₆ -substituted pyrroles, for example, 5-methylpyrrolopyrimidines, involves transesterification and alkylation of ethyl cyanoacetate with (16) (Scheme VI). (16) is condensed with benzamidine hydrochloride with DBU 2 equivalents to yield the pyrimidine (17). Pyridine-pyrimidine 14 is cyclized by deprotecting the acetal in aqueous HCl. (14) was reacted with phosphorous oxychloride under reflux to obtain the corresponding 4-chloro derivative (15). (2) by coupling with trans-4-aminocyclohexanol at 135 DEG C. This procedure reduces the number of reactions synthesized with the target compound (2) from 9 to 4. Further, And the yield is dramatically improved. It is also understood by those skilled in the art that, depending on the choice of reagents, there is great flexibility in choosing the preferred substituent R < _{5 &} gt ;.
[246] Composition VI
[247]
[248] des -methylpyrrole is shown in the following configuration (Scheme VII).
[249] Configuration VII
[250]
[251] Wherein R ₁ to R ₃ are as defined above.
[252] Alkyl cyanoacetate was alkylated with diethyl acetal in the presence of a base to give cyanodiethyl acetal which was treated with an amidine salt to give a methyl pyrrolopyrimidine precursor. The precursor was chlorinated and treated with an amine to yield the des -methyl pyrrolopyrimidine target described above.
[253] For example, structure VIII represents the synthesis of compound (18).
[254] Composition VIII
[255]
[256] Commercially available methyl cyanoacetate was alkylated with bromoacetaldehyde diethyl acetal in the presence of potassium carbonate and NaI to give (19). The cyclization to the pyrimidine (20) takes place in two steps. First, pyrimidine-acetal was obtained by reacting (19) with benzamidine chloride and 2 equivalents of DBU. The obtained pyrimidine-acetal was deprotected without purification by aqueous 1N HCl to obtain the aldehyde obtained by cyclization to obtain pyrrolo-pyrimidine (20), which was separated by filtration. (20) was reacted with phosphorus oxychloride under reflux to obtain the corresponding 4-chloro derivative (21). Compound (18) was obtained from compound (21) by coupling of chloro derivative with trans-4-aminocyclohexanol in DMSO at 135 占 폚.
[257] Structure II-VIII shows that it is possible to functionalize the 5- and 6-positions in the pyrrolopyrimidine ring. Various functional groups can be introduced at the 5- and 6-positions in formula (I) and formula (II), using different starting materials and modifying the reaction configuration slightly. Table 2 shows some embodiments.
[258] Table 2
[259]
[260] The invention is further illustrated by the following examples which are not to be construed as limiting. The contents of all references, pending patent applications, and published patent applications cited herein, including those cited in the Background section, are incorporated herein by reference. It is understood that the models in the examples are allowed models and that the efficacy of these models is also expected to be efficacious in humans.
[261] Example
[262] Manufacturing 1:
[263] The alkylation method of Seela and Lupke was modified and used ¹ . A solution of NaOMe (25% w / v, 58.1 mmol) was added slowly to an ice cold (0 C) solution of MeOH (20 mL) containing ethyl cyanoacetate (6.58 g, 58.1 mmol). After 10 minutes, chloro acetone (5 mL; 62.8 mmol) was slowly added. After 4 hours, the solution was removed. The brown oil was diluted with EtOAc (100 mL) and washed with H ₂ O (100 mL). The organic fraction was dried, filtered and concentrated to a brown oil (7.79 g; 79%). Oil (3) (Composition IV) is a mixture of methyl / ethyl ester product (9/1) and was used without further purification. ^{1 H NMR (200 MHz, CDCl} 3) δ4.24 (q, J = 7.2 Hz, OCH 2), 3.91 (dd, 1H, J = 7.2,7.0Hz, CH), 3.62 (s, 3H, OCH3), 3.42 (dd, 1H, J = 15.0,7.1Hz, 1xCH 2); 3.02 (dd, 1H, J = 15.0,7.0Hz, 1 x CH 2); 2.44 (s, 3H, CH ₃ ), 1.26 (t, J = 7.1 Hz, ester -CH ₃ )
[264] ¹ Seela, F .; Lupke, U.Chem. 1977, 110, 1462-1469.
[265] Manufacturing 2
[266] It was used as a method of Seela and Lupke ^1. Thus, under TsOH (100 mg) present a ketone (3) (Structure IV; 5.0 g, 32.2 mmol) ethylene glycol protected with (4 mL, 64.4 mmol) was purified by flash chromatography _{(SiO 2; 3/7 EtOAc / Hex} , R _f 0.35) to give (4) as an oil (Composition IV; 5.2 g, 81.0). This still contains ~ 5% ethyl ester. ^{1 H NMR (200 MHz, CDCl} 3) δ4.24 (q, J = 7.2 Hz, OCH 2), 3.98 (s, 4H, 2 x acetal _{-CH 2), 3.79 (s,} 3H, OCH3), 3.62 ( dd, 1H, J = 7.2,7.0Hz, CH), 2.48 (dd, 1H, J = 15.0,7.1Hz, 1 x CH 2), 2.32 (dd, 1H, J = 15.0,7.0Hz, 1 x CH 2 ); 1.35 (s, 3H, CH ₃ ), 1.26 (t, J = 7.1 Hz, ester-CH ₃ ); MS (ES): 2000.1 (M ⁺ +1).
[267] ¹ Seela, F .; Lupke, U.Chem. 1977, 110, 1462-1469.
[268] Manufacturing 3
[269] A solution of anhydrous DMF (15 mL) containing acetal (4) (compound IV, 1 g, 5.02 mmol), benzamidine (786 mg, 5.02 mmol) and DBU (1.5 mL, 10.04 mmol) And heated. The mixture was diluted with CHCl ₃ (30 mL) and washed with 0.5 N NaOH (10 mL) and H ₂ O (20 mL). The organic fraction was dried, filtered and concentrated to a brown oil. Flash chromatography (SiO ₂ ; 1/9 EtOAc / CH ₂ Cl ₂ , R _f 0.35) was carried out but the material crystallized on the column. The silica gel was washed with MeOH. The fractions containing product 5 (Composition IV) were concentrated and used without further purification (783 mg, 54.3%): ¹ H NMR (200 MHz, CDCl ₃ ) 8.24 ), 7.45 (m, 3H, Ar-H), 5.24 (brs, 2H, NH 2), 3.98 (s, 4H, 2 x acetal _{-CH 2), 3.60-3.15 (m,} 2H, CH 2), 1.38 _{(s, 3H, CH 3)} ; MS (ES): 288.1 (M + +1)
[270] Preparation 20 (compound VIII): To anhydrous DMF (10 mL) containing acetal 19 (4.43 g, 20.6 mmol) ¹ , benzamine hydrochloride (3.22 g, 20.6 mmol) and DBU (6.15 mL, 42.1 mmol) 20 mL) was heated to 85 < 0 > C for 15 h. The mixture was diluted with 100 mL of CHCl ₃ and washed with H ₂ O ( ₂ x 50 mL). The organic fraction was dried, filtered and concentrated to a dark brown oil. The dark brown oil was stirred in 1 N HCl (100 mL) at room temperature for 2 hours. The resulting slurry was filtered to yield the HCl salt of (20) as a tan solid (3.60 g, 70.6%); ^{1 H NMR (200MHz, DMSO-} d 6) 11.92 (s, 1H), 8.05 (m, 2H, Ar-H), 7.45 (m, 3H, Ar-H), 7.05 (s, 1H, pyrrole -H) ; MS (ES): 212.1 (M ^< + & gt ^; +1).
[271] Preparation 4:
[272] 1N HCl (40 mL) solution containing acetal (5) (700 mg, 2.44 mmol) was stirred at room temperature for 2 hours. By filtering the resulting slurry of 20-phenyl-6-methyl as a tan solid -7H- pyrrolo [2,3d] pyrimidin -4 (3H) - one was obtained with (498 mg, 78.0%): 1 H NMR (200MHz _{, DMSO-d 6) 11.78 (} s, 1H), 8.05 (m, 2H, Ar-H), 7.45 (m, 3H, Ar-H), 7.45 (s, 3H, Ar-H), 6.17 (s, 1H, pyrrole -H), 2.25 (s, 3H , CH 3); MS (ES): 226.1 (M + +1).
[273] Preparation 5:
[274] It was used to transform the ring closure methods, such as Chen ^1. Methylbenzylamine (12.5 mL, 97.3 mmol) was added to an ice-cooled (0 C) solution of isopropyl alcohol (60 mL) containing the compound (9) (composition V; 20.0 g, 108 mmol; 90% Was slowly added. The black solution was warmed to room temperature and stirred for 15 hours. The mixture was diluted with EtOAc (200 mL) and washed with 0.5 N NaOH (50 mL) The organic fraction was dried, filtered and concentrated to black tar (19.2 g; 94%). (SiO ₂ ; 4/96 MeOH / CH ₂ Cl ₂ , R _f 0.35) to give the compound dl-l- (l-phenylethyl) -2-amino-3-cyano- : MS (ES): 226.1 (M ⁺ +1) (19) (6.38 g, 31%).
[275] ¹ Chen YL; Mansbach, RS; Winter, SM; Brooks, E.; Collins, J.; Corman, ML; Dunaiskis, AR; Faraci, WS; Gallaschun, RJ; Schmidt, A.; Schulz, DW J. Med. Chem. 1977, 40, 1749-1754
[276] Manufacturing 6
[277] of dichloromethane (50.0 mL) containing dl-1- (1-phenylethyl) -2-amino-3-cyano-4,5-dimethylpyrrole ¹ (14.9 g, 62.5 mmol) and pyridine Was added benzoyl chloride (9.37 g, 66.7 mmol) at 0 < 0 > C. After stirring for 1 h at 0 C, hexane (10.0 mL) was added to precipitate the product. The solvent was removed in vacuo and the solid was recrystallized from EtOH / H ₂ O was dl-1- (1- phenylethyl) -2-phenyl 13.9 g (65% carbonyl-amino-3-cyano-4,5-dimethyl-pyrrole ). mp 218-221 [deg.] C; _{^{1 H NMR (200MHz, CDCl 3}} ) δ1.72 (s, 3H), 1.76 (d, J = 7.3Hz, 3H), 1.98 (s, 3H), 5.52 (q, J = 7.3 Hz, 1H), 7.14 -7.54 (m, 9H), 7.68-7.72 (dd, J = 1.4 Hz, 6.9 Hz, 2H), 10.73 (s, 1H); MS (ES): 344.4 (M ⁺
[278] ¹ Liebigs Ann. Chem. 1986, 1485-1505
[279] The following compounds were obtained by a method analogous to Preparation 6 :
[280] dl-1- (1-phenylethyl) -2- (3-pyridyl) carbonylamino-3-cyano-4,5-dimethylpyrrole. ^{1 H NMR (200 MHz, CDCl} 3) δ 1.83 (d, J = 6.8 (d, J = 6.8 z, 3H), 2.02 (s, 3H), 2.12 (s, 3H), 5.50 (q, J = 6.8 1H), 7.14-7.42 (m, 5H), 8.08 (m, 3H); MS (ES): 345.2 (M ⁺ +1).
[281] _{^{1 H NMR (200MHz, CDCl 3}} ) δ1.84 (d, J = 7.4 Hz, 3H), 1.92 (s, 3H), 2.09 (s, 3H), 5.49 (q, J = 7.4 Hz, 1H), 6.54 (dd, J = 1.8 Hz, 3.6 Hz, 1H), 7.12-7.47 (m, 7H); MS (ES): 334.2 (M ⁺
[282] ¹ H NMR (200 MHz, CDCl ₃ ) 1.80 (d, J = 7 Hz 3H), 1.89 (s, 3H), 2.05 , 1H), 7.12-7.40 (m, 6H), 7.93 (s, 1H), MS (ES): 334.1 (M + +1), 230.0
[283] dl-l (1-phenylethyl) -2-cyclopentylphenylcarbonylamino-3-cyano-4,5-diethylpyrrole. ^{1 H NMR (200 MHz, CDCl} 3) δ1.82 (d, J = 7.4Hz, 3H), 1.88 (s, 3H), 2.05 (s, 3H), 1.63-1.85 (m, 8H), 2.63 (m 1H), 5.43 (q, J = 7.4 Hz, 1 H), 6.52 (s, 1H), 7.05-7.20 (m, 5H); MS (ES): 336.3 (M ⁺
[284] dl-1 (1-phenylethyl) -2- (2-thienyl) carbonylamino-3-cyano-4,5-dimethylpyrrole. ^{1 H NMR (200 MHz, CDCl} 3) δ1.83 (d, J = 7.0 Hz, 3H), 1.99 (s, 3H), 2.12 (s, 3H), 5.49 (q, J = 7.0 Hz, 1H), 6.90 (m, 1 H), 7.18-7.36 (m, 6 H), 7.79 (m, 1 H); MS (ES): 350.2 (M ^< + & gt ^; +1), 246.1.
[285] dl-1 (1-phenylethyl) -2- (4-fluorophenyl) carbonylamino-3-cyano-4,5-dimethylphenyl. ^{1 H NMR (200 MHz, CDCl} 3) δ1.83 (d, J = 7.4 Hz, 3H), 1.96 (s, 3H), 2.08 (s, 3H), 5.51 (q, J = 7.4 Hz, 1H), 7.16-7.55 (m, 9H); MS (ES): 362.2 (M ^< + & gt ^; +1), 258.1.
[286] dl-1 (1-phenylethyl) -2- (3-fluorophenyl) carbonylamino-3-cyano-4,5-dimethylpyrrole. ^{1 H NMR (200 MHz, CDCl} 3) δ1.83 (d, J = 7.4 Hz, 3H), 1.97 (s, 3H), 2.10 (s, 3H), 5.50 (q, J = 7.4 Hz, 1H), 7.05-7.38 (m, 7H), 7.67-7.74 (m, 2H); MS (ES): 362.2 (M ^< + & gt ^; +1), 258.1.
[287] dl-1 (1-phenylethyl) -2- (2-fluorophenyl) carbonylamino-3-cyano-4,5-dimethylpyrrole. ^{1 H NMR (200 MHz, CDCl} 3) δ1.85 (d, J = 7.2 Hz, 3H), 1.94 (s, 3H), 2.11 (s, 3H), 5.50 (q, J = 7.2 Hz, 1H), 1H); MS (ES): 362.2 (M ⁺ +1), 258.0.
[288] dl-1 (1-phenylethyl) -2-isopropylcarbonylamino-3-cyano-4,5-dimethylpyrrole. ^{1 H NMR (200 MHz, CDCl} 3) δ1.19 (d, J = 7.0 Hz, 6H), 1.82 (d, J = 7.2Hz, 3h), 1.88 (s, 3H), 2.06 (s, 3H), 2.46- (m, 1H), 5.39 (m, J = 7.2 Hz, 1H), 6.64 (s, 1H), 7.11-7.36 (m, 5H); MS (ES): 310.2 (M ^< + & gt ^; +1), 206.1
[289] When acylating dl-1- (1-phenylethyl) -2-amino-3-cyano-4-methylpyrrole, the monoacylated dl- 3-cyano-4-dimethylpyrrole and diacylated pyrrole dl 1- (1-Phenylethyl) -2-amino-3-cyano-4-methylpyrrole. Monoacylated pyrrole: ¹ H NMR (200 MHz, CDCl ₃ ) 7.69 (d, 2H, J = 7.8 Hz, Ar-H), 7.58-7.12 (m, 8H, Ar- , 1H, pyrrole -H), 5.52 (q, 1H , J = 7.2 Hz, C H -CH 3), 2.05 (s, 3H, pyrrole _{-CH 3), 1.85 (d,} 3H, J = 7.2Hz, CH _{-C H 3); MS (ES} ): 330.2 (M + +1); dia misfire a ^{pyrrole: 1 H NMR (200 MHz,} CDCl 3) δ7.85 (d, 2H, J = 7.7 Hz, Ar-H 1H), 5.52 (q, IH, Ar-H), 7.74 (d, 2H, J = 7.8 Hz, Ar-H), 7,52-7.20 _{J = 7.2Hz, C H -CH 3} ), 1.77 (d, 3H, J = 7.2Hz, CH-C H 3), 1.74 (s, 3H, pyrrole _{-CH 3); MS (ES)} : 434.1 (M ⁺ +1).
[290] Preparation 7:
[291] To a solution of dl-1- (1-phenylethyl) -2-phenylcarboxamido-3-cyano-4,5-dimethylpyrrole (1.0 g, 2.92 mmol) in methanol (910.0 mL) was added concentrated sulfuric acid Lt; / RTI > The resulting mixture was refluxed for 15 hours and cooled to room temperature. The precipitate was filtered to yield 0.48 g (48%) of dl-5,6-dimethyl-2-phenyl-7H-7- (1-phenylethyl) pyrrolo [2,3d] pyrimidin- Respectively. ^{1 H NMR (200 MHz, CDCl} 3) δ2.02 (d, J = 7.4Hz, 3H), 2.04 (s, 3H), 2.41 (s, 3H), 6.25 (q, J = 7.4Hz, 1H), MS (ES): 344.2 (M ^< + & gt ^; +1).
[292] The following compounds were obtained analogously to Preparation 7 .
[293] dl-5,6-dimethyl-2- (3-pyridyl) -7H-7- (1-phenylethyl) pyrrolo [2,3d] pyrimidin-4- (3H) -one. _{^{1 H NMR (2OOMHz, CDCl 3}} ) δ2.03 (d, J = 7.2Hz, 3H), 2.08 (s, 3H), 2.42 (s, 3H), 6.24 (q, J = 7.2Hz, 1H), 7.09 MS (ES): 345.1 (M ^& lt ^; + & gt ^; +1)
[294] dl-5,6-dimethyl-2- (2-furyl) -7H-7- (1-phenylethyl) pyrrolo [2,3d] pyrimidin-4- (3H) -one. _{^{1 H NMR (2OOMHz, CDCl 3}} ) δ1.98 (d, J = 7.8Hz, 3H), 1.99 (s, 3H), 2.37 (s, 3H), 6.12 (q, J = 7.8Hz, 1H), 6.48 (dd, J = 1.8 Hz, 3.6Hz, 1H) 7.17-7.55 (m, 7H), 9.6 (s, 1H); MS (ES): 334.2 (m + +1)
[295] dl-5,6-dimethyl-2- (3-furyl) -7H-7- (1-phenylethyl) pyrrolo [2,3d] pyrimidin-4- (3H) -one. _{^{1 H NMR (2OOMHz, CDCl 3}} ) δ1.99 (d, J = 7Hz, 3H), 2.02 (s, 3H), 2.42 (s, 3H), 6.24 (q, J = 7Hz, 1H), 7.09 (s , 7.18-7.32 (m, 5H), 7.48 (s, 1H), 8.51 (s, 1H); MS (ES): 334.2 (M ⁺ +1).
[296] dl-5,6-dimethyl-2-cyclopentyl-7H-7 (1-phenylethyl) pyrrolo [2,3d] pyrimidin-4 (3H) -one. _{^{1 H NMR (2OOMHz, CDCl 3}} ) δ1.95 (d, J = 7.4Hz, 3H), 2.00 (s, 3H), 2.33 (s, 3H), 1.68-1.88 (m, 8H), 2.97 (m, 1H), 6.10 (q, J = 7.4 Hz, 1H), 7.16-7.30 (m, 5H), 9.29 (s, 1H); MS (ES): 336.3 (M ⁺
[297] dl-5,6-dimethyl-2- (2-thienyl) -7H-7- (1-phenylethyl) pyrrolo [2,3d] pyrimidin-4- (3H) -one. _{^{1 H NMR (2OOMHz, CDCl 3}} ) δ2.02 (d, J = 7.2Hz, 3H), 2.06 (s, 3H), 2.41 (s, 3H), 6.13 (q, J = 7.2 Hz, 1H), 7.12 (d, J = 4.8,2.8 Hz, 1H), 7.26-7.32 (m, 5H), 7.44 (d, J = 4.8 Hz, 1H), 8.01 1H); MS (ES): 350.2 (M ^< ⁺ > +1).
[298] dl-5,6-dimethyl-2- (3-thienyl) -7H-7- (1-phenylethyl) pyrrolo [2,3d] pyrimidin-4- (3H) -one. _{^{1 H NMR (2OOMHz, CDCl 3}} ) δ2.00 (d, J = 7.4Hz, 3H), 2.05 (s, 3H), 2.43 (s, 3H), 6.24 (q, J = 7.4 Hz, 1H), 7.24 (M, 5H), 7.33-7.39 (m, IH) 7.85 (m, IH), 8.47 (m, IH), 12.01 (s, IH); MS (ES): 350.2 (M ⁺ +1).
[299] dl-5,6-dimethyl-2- (4-fluorophenyl) -7H-7- (1-phenylethyl) pyrrolo [2,3d] pyrimidin-4- (3H) -one. _{^{1 H NMR (2OOMHz, CDCl 3}} ) δ2.01 (d, J = 6.8Hz, 3H), 2.05 (s, 3H), 2.42 (s, 3H), 6.26 (q, J = 6.8 Hz, 1H), 7.12 (S, 1H); MS (ES): 362.3 (M ⁺ +).
[300] dl-5,6-dimethyl-2- (3-fluorophenyl) -7H-7- (1-phenylethyl) pyrrolo [2,3d] pyrimidin-4- (3H) -one. _{^{1 H NMR (2OOMHz, CDCl 3}} ) δ2.02 (d, J = 7.4Hz, 3H), 2.06 (s, 3H), 2.44 (s, 3H), 6.29 (q, J = 7.4 Hz, 1H), 7.13 (S, 1H); MS (ES): 362.2 (M ^< + & gt ^; +1).
[301] dl-5,6-dimethyl-2- (2-fluorophenyl) -7H-7- (1-phenylethyl) pyrrolo [2,3d] pyrimidin-4- (3H) -one. _{^{1 H NMR (2OOMHz, CDCl 3}} ) δ2.00 (d, J = 7.2Hz, 3H), 2.05 (s, 3H), 2.38 (s, 3H), 6.24 (q, J = 7.2 Hz, 1H), 7.18 (M, 8H), 8.21 (m, 1H), 9.54 (s, 1H); MS (ES): 362.2 (M ⁺ +1).
[302] dl-5,6-Dimethyl-2-isopropyl-7H-7- (1-phenylethyl) pyrrolo [2,3d] pyrimidin-4 (3H) -one. ^{1 H NMR (2OO MHz, CDCl} 3) δ1.30 (d, J = 6.8Hz, 3H), 1.32 (d, J = 7.0Hz, 3H), 2.01 (s, 3H), 2.34 (s, 3H), 2.90 (m, 1H), 6.13 (m, 1H), 7.17-7.34 (m, 5H), 10.16 (s, 1H); MS (ES): 310.2 (M ^< + & gt ^; +1).
[303] Manufacturing 8
[304] (13 mL) containing dl-1- (1-phenylethyl) -2-benzoylamino-3-cyano-4- dimethylpyrrole (785 mg, 2.38 mmol) and concentrated H ₂ SO ₄ (1 mL) Was stirred at 130 < 0 > C for 48 hours. The black solution was diluted with CHCl ₃ (100 mL) and washed with 1 N NaOH (30 mL) and brine (30 mL). Dry the organic fractions were filtered and concentrated hanhu, flash chromatography _{(SiO 2; 8/2 EtOAc / Hex} , R f 0.35) to yield a black solid compound dl-5- methyl-2-phenyl -7H-7- ( Pyrrolo [2,3d] pyrimidin-4 (3H) -one (184 mg, 24%). ¹ H NMR (200 MHz, CDCl ₃ ) 8.18 (m, 2H, Ar-H), 7.62-7.44 (m, 3H, Ar-H), 7.40-7.18 (s, 1H, pyrrole -H), 6.28 (q, 1H , J = 7.2Hz, C H -CH 3), 2.18 (s, 3H, pyrrole _{-CH 3), 2.07 (d,} 3H, J = 7.2Hz _{, CH-C H 3);} MS (ES): 330.2 (M + +1).
[305] Manufacturing 9
[306] A mixture of dl-1- (1-phenylethyl) -2-amino-3-cyano-4,5-dimethylpyrrole (9.60 g, 40.0 mmol) and formic acid (50.0 mL, 98%) was refluxed for 5 hours. After cooling to room temperature and scratching the sides of the flask, many precipitates were obtained and filtered. This material was washed with water until a neutral pH was seen by washing to yield dl-5,6-dimethyl-7H-7- (1-phenylethyl) pyrrolo [2,3d] pyrimidin-4 (3H) Respectively. ^{1 H NMR (200 MHz, CDCl} 3) δ1.96 (d, J = 7.4 Hz, 3H), 2.00 (s, 3H), 2.38 (s, 3H), 621 (q, J = 7.4Hz, 1H), 1H); MS (ES): 268.2 (M ⁺ 1).
[307] Manufacturing 10
[308] (3H) -one (1.0 g, 2.91 mmol) was added to a solution of polyphosphoric acid (30.0 g, mL_ The mixture was heated for 4 hours at 100 DEG C. The hot suspension was poured onto ice water and the suspension was dispersed by vigorous stirring and alkalized with KOH to pH 6. The resulting solid was filtered And collected to obtain 0.49 g (69%) of 5,6-dimethyl-2-phenyn-7H-pyrrolo [2,3d] pyrimidin-4 (3H) -one.
[309] ^{1 H NMR (200 MHz, DMSO} -d 6) δ2.17 (s, 3H), 2.22 (s, 3H), 7.45 (br, 3H), 8.07 (br, 2H), 11.49 (s, 1H), 11.82 (s, 1H); MS (ES): 344.2 (M ^< + & gt ^; +1).
[310] The following compounds were obtained by a method similar to that of Preparation 10
[311] 5-Methyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidin-4 (3H) -one. MS (ES): 226.0 (M ^< + & gt ^; +1).
[312] 5,6-Dimethyl-2- (3-dyridyl) -7H-pyrrolo [2,3d] pyrimidin-4 (3H) -one. MS (ES): 421.1 (M ^< + & gt ^; +1).
[313] 5,6-Dimethyl-2 (2-furyl) -7H-pyrrolo [2,3d] pyrimidin-4 (3H) -one. ^{1 H NMR (200MHz, DMSO-} d 6) δ2.13 (s, 3H), 2.18 (s, 3H), 6.39 (dd, J = 1.8,3.6Hz, 1H), 6.65 (dd, J = 1.8Hz, 1H), 7.85 (dd, J = 1.8, 3.6 Hz, 1H), 11.45 (s, 1H), 11.60 (s, 1H); MS (ES): 230.1 (M ⁺
[314] 5,6-Dimethyl-2-cyclopenty-7H-pyrrolo [2,3d] pyrimidin-4 (3H) -one. ^{1 H NMR (200 MHz, DMSO} -d 6) δ1.57-1.91 (m, 8H), 2.12 (s, 3H), 2.16 (s, 3H), 2.99 (m, 1H), 11.24 (s, 1H) , 11.38 (s, 1H); MS (ES): 232.2 (M ^< + & gt ^; +1).
[315] 5,6-Dimethyl-2- (2-thienyl) -7H-pyrrolo [2,3d] pyrimidin-4 (3H) -one. ^{1 H NMR (200 MHz, DMSO} -d 6) δ2.14 (s, 3H), 2.19 (s, 3H), 7.14 (dd, J = 3.0,5.2 Hz, 1H), 7.70 (d, J = 5.2Hz 1H), 8.10 (d, J = 3.0 Hz, 1H), 11.50 (s, 1H); MS (ES): 246.1 (M ⁺
[316] 5,6-Dimethyl-2- (3-thienyl) -7H-pyrrolo [2,3d] pyrimidin-4 (3H) -one. ¹ H NMR (200 MHz, DMSO- d ₆ ) 2.17 (s, 3H), 2.21 (s, 3H), 7.66 (m, (s, 1 H); MS (ES): 258.2 (M ^< + & gt ^; +1).
[317] 5,6-Dimethyl-2- (3-fluorophenyl) -7H-pyrrolo [2,3d] pyrimidin-4 (3H) -one. ^{1 H NMR (200 MHz, DMSO} -d 6) δ2.18 (s, 3H), 2.21 (s, 3H), 7.33 (m, 1H), 7.52 (m, 1H), 7.85-7.95 (m, 2H) , 11.56 (s, 1H), 11.80 (s, 1H); MS (ES): 258.1 (M ⁺ +1).
[318] 5,6-Dimethyl-2- (2-fluorophenyl) -7H-pyrrolo [2,3d] pyrimidin-4 (3H) -one. ^{1 H NMR (200 MHz, DMSO} -d 6) δ2.18 (s, 3H), 2.22 (s, 3H), 7.27-7.37 (m, 2H), 7.53 (m, 1H), 7.68 (m, 1H) , 11.54 (s, 1H), 11.78 (s, 1H); MS (ES): 258.1 (M ⁺ +1).
[319] 5,6-Dimethyl-2-isopropyl-7H-pyrrolo [2,3d] pyrimidin-4 (3H) -one. ^{1 H NMR (2000 MHz, DMSO} -d 6) δ1.17 (d, J = 6.6Hz, 6H), 2.11 (s, 3H), 2.15 (s, 3H), 2.81 (m, 1H), 11.20 (s , 1 H), 11.39 (s, 1 H); MS (ES): 206.1 (M ⁺ +1).
[320] 5,6-Dimethyl-7H-pyrrolo [2,3d] pyrimidin-4 (3H) -one. ¹ H NMR (200 MHz, DMSO-d ₆ ) 2.13 (s, 3H), 2.17 (s, 3H), 7.65 (s, 1H); MS (ES): 164.0 (M ⁺
[321] Manufacturing 11
[322] A solution of phosphorus oxychloride (25.0 mL) containing 5,6-dimethyl-2-phenyl-7H-pyrro [2,3d] pyrimidin-4 (3H) -one (1.0 g, 4.2 mmol) After refluxing, it was concentrated to dryness in vacuo. Water was added to the residue to induce staining and the resulting solid was filtered and collected to give 0.90 g (83%) of 4-chloro-5,6-dimethyl-2-phenyl-7H- pyrrolo [2,3d] pyrimidine %). ^{1 H NMR (200 MHz, DMSO} -d 6) δ2.33 (s, 3H), 2.33 (s, 3H), 7.46-7.49 (m, 3H), 8.30-8.35 (m, 2H), 12.20 (s, 1H); MS (ES): 258.1 (M ^< + & gt ^; +1)
[323] The following compounds were obtained by a method analogous to Preparation 11 :
[324] 4-Chloro-5-methyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. MS (ES): 244.0 (M⁺+ One).
[325] 4-Chloro-6-methyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. MS (ES): 244.0 (M⁺+ One).
[326] 4-Chloro-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. ^{1 H NMR (200 MHz, DMSO} -d 6) 8.35 (2,2H), 7.63 (br s, 1H), 7.45 (m, 3H), 6.47 (br s, 1H); MS (ES): 230.0 (M ⁺ +1).
[327] 4-Chloro-5,6-dimethyl-2- (3-pyridyl) -7H-pyrrolo [2,3d] pyrimidine. MS (ES): 259.0 (M ^< + & gt ^; +1).
[328] 4-Chloro-5,6-dimethyl-2- (2-furyl) -7H-pyrrolo [2,3d] pyrimidine. ^{1 H NMR (200MHz, DMSO-} d 6) δ2.35 (s, 3H), 2.35 (s, 3H), 6.68 (dd, J = 1.8,3.6Hz, 1H), 7.34 (dd, J = 1.8 Hz, 1H), 7.89 (dd, J = 1.8, 3.6 Hz, 1H); MS (ES): 248.0 (M ⁺ +1).
[329] 4-Chloro-5,6-dimethyl-2- (3-furyl) -7H-pyrrolo [2,3d] pyrimidine. ¹ H NMR (200 MHz, DMSO- d ₆ ) 2.31 (s, 3H), 2.31 (s, 3H), 6.62 (s, ES): 248.1 (M ^< + & gt ^; +1).
[330] 4-Chloro-5,6-dimethyl-2-cyclopentyl-7H-pyrrolo [2,3d] pyrimidine. ^{1 H NMR (200MHz, DMSO-} d 6) δ1.61-1.96 (m, 8H), 2.27 (s, 3H), 2.27 (s, 3H), 3.22 (m, 1H), 11.97 (s, 1H); MS (ES): 250.1 (M ^< + & gt ^; +1).
[331] 4-Chloro-5,6-dimethyl-2- (2-thienyl) -7H-pyrrolo [2,3d] pyrimidine. ^{1 H NMR (200MHz, DMSO-} d 6) δ2.29 (s, 3H), 2.31 (s, 3H), 7.14 (dd, J = 3.1Hz, 4.0Hz, 1H), 7.33 (d, J = 4.9 Hz , 7.82 (d, J = 3.1 Hz, 1H), 12.19 (s, 1H); MS (ES): 264.0 (M ⁺ +1).
[332] 4-Chloro-5,6-dimethyl-2- (4-fluorophenyl) -7H-pyrrolo [2,3d] pyrimidine. ¹ H NMR (200 MHz, DMSO- d ₆ ) 2.23 (s, 3H), 2.33 (s, 3H), 7.30 (m, 2H), 8.34 (m, 2H), 12.11 (s, 1H); MS (ES): 276.1 (M ^< + & gt ^; +1).
[333] 4-Chloro-5,6-dimethyl-2- (3-fluorophenyl) -7H-pyrrolo [2,3d] pyrimidine. ^{1 H NMR (200MHz, DMSO-} d 6) δ2.31 (s, 3H), 2.33 (s, 3H), 7.29 (m, 1H), 7.52 (m, 1H), 7.96 (m, 1H), 8.14 ( m, 1 H), 11.57 (s, 1 H); MS (ES): 276.1 (M ⁺ +1).
[334] 4-Chloro-5,6-dimethyl-2- (2-fluorophenyl) -7H-pyrrolo [2,3d] pyrimidine. ^{1 H NMR (200MHz, DMSO-} d 6) δ2.34 (s, 3H), 2.34 (s, 3H), 7.33 (m, 2H), 7.44 (m, 1H), 7.99 (m, 1H), 12.23 ( s, 1H); MS (ES): 276.1 (M ^< + & gt ^; +1).
[335] 4-Chloro-5,6-dimethyl-2-isopropyl-7H-pyrrolo [2,3d] pyrimidine. ^{1 H NMR (200MHz, DMSO-} d 6) δ1.24 (d, J = 6.6Hz, 6H), 2.28 (s, 3H), 2.28 (s, 3H), 3.08 (q, J = 6.6Hz, 1H) , 11.95 (s, 1H); MS (ES): 276.1 (M ^< + & gt ^; +1).
[336] 4-Chloro-5,6-dimethyl-7H-pyrrolo [2,3d] pyrimidine. ¹ H NMR (200 MHz, DMSO-d ₆ ) 2.31 (s, 3H), 2.32 (s, 3H), 8.40 (s, 1H); MS (ES): 182.0 (M ⁺
[337] dl-4-Chloro-5,6-dimethyl-2-phenyl-7H-7- (1-phenylethyl) pyrrolo [2,3d] pyrimidine.
[338] Preparation 12:
[339] To a solution of dioxane (1000.0 mL) and water (100.0 mL) containing dl-, 2-diaminopropane (1.48 g, 20.0 mmol) and sodium carbonate (2.73 g, 22.0 mmol) (4.80 g, 22.0 mmol) at room temperature. The resulting mixture was aged for 14 hours. The dioxane was removed in vacuo. The precipitate was filtered off and the filtrate was concentrated to dryness in vacuo. The residue was triturated with EtOAc and filtered. The filtrate was concentrated to dryness in vacuo to give dl-1-amino-2- (1,1-dimethylethoxy) carbonylamino-propane and dl- 2- amino-1- (1,1-dimethylethoxy) Amino-propane, which could not be separated by normal chromatographic methods. This mixture was used in the reaction in Example 8 .
[340] Manufacturing 13:
[341] Dimethylformamide was added to a solution of Fmoc- beta -la-OH (1.0 g, 3.212 mmol) and oxalyl chloride (0.428 g, 0.29 mL, 3, 373 mmol) in dichloromethane Lt; / RTI > The mixture was stirred at room temperature for 1 hour and then cyclopropylmethylamine (0.229 g, 0.28 mL, 3.212 mmol) and triethylamine (0.65 g, 0.90 mL, 6.424 mmol) were added. After 10 minutes, the mixture was treated with 1 M hydrochloride (10.0 mL) and the resulting aqueous mixture was extracted with dichloromethane (3 x 30.0 mL). The organic solution was concentrated to dryness in vacuo. The residue was treated with a solution of N, N-dimethylformamide (20.0 mL) containing 20% piperidine for 0.5 hour. After removal of the solvent in vacuo, the residue was treated with 1 M hydrochloride 920.0 mL) and ethyl acetate (20.0 mL). The mixture was triturated and the aqueous layer was alkalized with solid sodium hydroxide at pH = 8. The precipitate was filtered off and the aqueous solution was subjected to ion exchange column elution with 20% pyridine to give 0.262 g (57%) of N-cyclopropylmethyl β-alanine amide. ^{1 H NMR (200 MHz, CD} 3 OD) δ0.22 (m, 2H), 0.49 (m, 2H), 2.40 (t, 2H), 2.92 (t, 2H), 3.05 (d, 2H); MS ( ES): 143.1 (M ^< + & gt ^; +1).
[342] Preparation 14:
[343] N-tert-butoxycarbonyl-trans-1,4-cyclohexyldiamine.
[344] Trans-1,4-cyclohexenediamine (6.08 g, 53.2 mmol) was dissolved in dichloromethane (100 mL). A solution of dichloromethane (40 mL) containing di-t-butyl dicarbonate (2.32 g, 10.65 mmol) was added via a cannula. After 20 period, the reaction was partitioned between CHCl ₃ and water. The layers were separated and the aqueous layer was extracted with CHCl ₃ (x3). Dry the combined organic layers over MgSO _4, filtered and concentrated to yield a white solid 1.20 g (53%). _{^{1 H NMR (200MHz, CDCl 3}} ): δ1.0-1.3 (m, 4H), 1.44 (s, 9H), 1.8-2.1 (m, 4H), 2.62 (brm, 1H), 3.40 (brs, 1H) , 4.37 (br s, 1H); MS (ES): 215.2 (M ⁺ +1).
[345] 4 (N-acetyl) -N-tert-butoxycarbonyl-trans-1,4-cyclohexyldiamine.
[346] N-tert-Butoxycarbonyl-trans-1,4-cyclodiamine (530 mg, 2.47 mmol) was dissolved in dichloromethane (20 mL). Acetic anhydride (250 mg, 2.60 mmol) was added dropwise. After 16 hours, the reaction solution was diluted with water and CHCl _3. The layers were separated and the aqueous layer was extracted with CHCl ₃ (x3). Dry the combined organic layers over MgSO _4, filtered and concentrated. Recrystallization (EtOH / H ₂ O) gave 190 mg (30%) of white crystals. _{^{1 H NMR (200MHz, CDCl 3}} ): δ0.9-1.30 (m, 4H), 1.43 (s, 9H), 1.96-2.10 (m, 7H), 3.40 (brm, 1H), 3.70 (brs, 1H) , 4.40 (br s, 1H), 4.40 (brs, 1H); MS (ES): 257.2 (M ⁺ +1), 242.1 (M ⁺ -15), 201.1 (M ⁺
[347] 4- (4-trans-acetamidocyclohexyl) amino-5,6-dimethyl-2-phenyl-7H- (1-phenylethyl) pyrrolo [2,3d] pyrimidine.
[348] 4-cyclohexyldiamine (190 mg, 0.74 mmol) was dissolved in dichloromethane (5 mL) and diluted with TFA (6 ml) . After 16 hours, the reaction solution was concentrated. Crude solid, DMSO (2 mL), NaHCO 3 (200 mg, 2.2 mmol) and 4-chloro-5,6-dimethyl--2-7H- blood rolrol [2,3d] pyrimidine (35 mg, 0.14 mmol) Were mixed in a flask and heated to 130 < 0 > C. After 4.5 hours, the reaction was cooled to room temperature and diluted with EtOAc and water. The layers were separated and the aqueous layer was extracted with CHCl ₃ (x3). Dry the combined organic layers over MgSO _4, filtered and concentrated. Chromatography; was calculated (silica purification plate 20: EtOH: 1 CHCl ₃₎ to a tan solid 0.3 mg (1% yield). MS (ES): 378.2 (M ^< + & gt ^; +1).
[349] 4 (N-methanesulfonyl) -N-tert-butoxycarbonyl-trans-1,4-cyclohexyldiamine.
[350] Trans-1,4-cyclohexyldiamine (530 mg, 2.47 mmol) was dissolved in dichloromethane (20 ml) and diluted with pyridine (233 mg, 3.0 mmol). Methanesulfonyl chloride (300 mg, 2.60 mmol) was added dropwise. After 16 hours, the reaction was diluted with water and CHCl _3. The layers were separated and the aqueous layer was extracted with CHCl ₃ (x3). Dry the combined organic layers over MgSO _4, filtered and concentrated. Recrystallization (EtOH / H ₂ O) gave 206 mg (29%) of white crystals. _{^{1 H NMR (200MHz, CDCl 3}} ): δ1.0-1.40 (m, 4H), 1.45 (s, 9H), 2.00-2.20 (m, 4H), 2.98 (s, 3H), 3.20-3.50 (brs, 2H), 4.37 (br s, 1H); MS (ES): 239.1 (M ⁺ +1), 278.1 (M ⁺ -15), 237.1 (M ⁺
[351] Amino-5,6-dimethyl-2-phenyl-7H- (1-phenylethylpyrrolo [2,3d] pyrimidine
[352] To a solution of 206 mg (0.71 mmol) of 4- (N-sulfonyl) -N-tert-butoxycarbonyl-trans-1,4-cyclohexyldiamine in 5 ml of dichloromethane was diluted with TFA . After 16 hours, the reaction solution was concentrated. Mixed in the crude reaction mixture, DMSO (2 mL), NaHCO 3 (100 mg, 1.1 mmol) and 1-chloro-5,6-dimethyl--2-7H--pyrrolo [2,3d] pyrimidine flask and 130 Lt; 0 > C. After 1.5 h, the reaction was cooled to room temperature and extracted with EtOAc (x3). Dry the combined organic layers over MgSO _4, filtered and concentrated. Chromatography; was calculated (for example, silica criticism 20: EtOH: 1 CHCl ₃₎ to a tan solid 2.6 mg (5% yield). MS (ES): 414.22 (M ^< + & gt ^; +1).
[353] Example 1
[354] Pyrrolo [2,3d] pyrimidine (0.50 g, 1.94 mmol) and 4-trans-hydroxycyclohexylamine (2.33 g, 19.4 mmol) were added to a solution of 4-chloro-5,6- (10.0 mL) was heated at 130 < 0 > C for 5 hours. After cooling to room temperature, water (10.0 ml) was added and the resulting aqueous solution was extracted with EtOAc (3 x 10.0 mL). The combined EtOAc solution was dried (MgSO ₄ ) and filtered, and the filtrate was concentrated to dryness in vacuo and the residue was chromatographed on silica gel to give 4- (4-trans-hydroxycyclohexyl) amino- ¹ H NMR (200 MHz, CDCl ₃ ) 1.25-1.59 (m, 8H (CDCl3) ), 2.08 (s, 3H), 2.29 (s.3H), 3.68-3.79 (m, IH), 4.32-4.38 (m, IH), 4.88 (d, J = 8 Hz, 1H), 7.26-7.49 , 3H), 8.40-8.44 (dd, J = 2.2, 8Hz, 2H), 10.60 (s, 1H); MS (ES): 337.2 (M ⁺
[355] The following compounds were obtained by a method analogous to that of Example 1 :
[356] 4- (4-trans-hydroxycyclohexyl) amino-6-methyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. ¹ H NMR (200 MHz, CDCl ₃ ) 11.37 (s, IH, pyrrole -NH), 8.45 (m, 2H, Ar- (M, 2H), 2.19-1.98 (m, 2H), 4.90 (br d, m.2H), 2.25 (s, 3H , CH 3) 1.68-1.20 (m, 4H); MS (ES): 323.2 (m + +1).
[357] 4- (4-trans-hydroxycyclohexyl) amino-5-methyl-2-phenyl-7H-pyrrole [2,3d] pyrimidine. ¹ H NMR (200 MHz, CDCl ₃ ) 11.37 (s, IH, pyrrole -NH), 8.40 (m, 2H, Ar- (M, 2H), 2.18-1.98 (m, 2H), 4.90 (br d, m.2H), 2.00 (s, 3H , CH 3) 1.68-1.20 (m, 4H); MS (ES): 323.2 (m + +1).
[358] 4- (4-trans-hydroxycyclohexyl) amino-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. mp 245.5-246.5 DEG C .; _{^{1 H NMR (200MHz, CD 3}} OD) δ8.33 (m, 2H, Ar-H), 7.42 (m, 3H, Ar-H), 7.02 (d, 1H, J = 3.6Hz, pyrrole -H), 1H, CH-N), 2.30-2.12 (m, 2H) 2.12-6 (m, m, 2H), 1.64-1.34 (m, 4H); MS, M + 1 = 309.3; Anal (C ₁₈ H ₂₀ N ₄ O) C, H, N.
[359] 4- (4-trans-hydroxycyclohexyl) amino-5,6-dimethyl-2- (3-pyridyl) -7H-pyrrolo [2,3d] pyrimidine. ¹ H NMR (200 MHz, CDCl ₃ ) 1.21-1.54 (m, 8H); 2.28 (s, 3H); 2.33 (s, 3H); (M, 2H), 9.64 (m, 1H); MS (ES): 338.2 (M, ⁺ +1).
[360] 4- (4-trans-hydroxycyclohexyl) amino-5,6-dimethyl-2- (2-furyl) -7H-pyrrolo [2,3d] pyrimidine. ¹ H NMR (200 MHz, CDCl ₃ ) 1.26-1.64 (m, 8H); 2.22 (s, 3H); 2.30 (s, 3H); 1H), 7.52 (m, 1H), 7.52 (m, 1H), 4.23 (m, (ES): 327.2 (M ^< + & gt ^; +1).
[361] 4- (4-trans-hydroxycyclohexyl) amino-5,6-dimethyl-2- (3-furyl) -7H-pyrrolo [2,3d] pyrimidine. ^{1 H NMR (200 MHz, CDCl} 3) δ1.25-1.63 (m, 8H), 2.11 (s, 3H), 2.27 (s, 3H), 3.71 (m, 1H), 4.20 (m, 1H), 4.84 (d, 1H), 7.03 ( m, 1H), 7.45 (m, 1H), 8.13 (m, 1H), 10.38 (m, 1H); MS (ES); 327.2 (m + +1).
[362] 4- (4-trans-hydroxycyclohexyl) amino-5,6-dimethyl-2-cyclopentyl-7H-pyrrolo [2,3d] pyrimidine. ^{1 H NMR (200 MHz, CDCl} 3) δ1.26-2.04 (m, 16H), 2.26 (s, 3H), 2.27 (s, 3H), 3.15 (m, 1H), 3.70 (m, 1H), 4.12 (d, 1H), 4.75 (d, 1H), MS (ES): 329.2 (M ⁺ +1).
[363] 4- (4-trans-hydroxycyclohexyl) amino-5,6-dimethyl-2- (2-thienyl) -7H-pyrrolo [2,3d] pyrimidin- ^{1 H NMR (200 MHz, CDCl} 3) δ1.28-1.59 (m, 8H), 2.19 (s, 3H), 2.29 (s, 3H), 3.74 (m, 1H), 4.19 (m, 1H), 4.84 (d, 1H), 7.09 ( m, 1H), 7.34 (m, 1H), 7.85 (m, 1H), 9.02 (s, 1H); MS (ES); 342.2 (m + +1).
[364] 4- (4-trans-hydroxycyclohexyl) amino-5,6-dimethyl-2- (3-thienyl) -7H-pyrrolo [2,3d] pyrimidine. ^{1 H NMR (200 MHz, CDCl} 3) δ1.21-1.60 (m, 8H), 1.98 (s, 3H), 2.23 (s, 3H), 3.66 (m, 1H), 4.22 (m, 1H), 7.27 (m, 1H), 7.86 (m, 1H), 8.09 (m, 1H), 11.23 (s, 1H; MS (ES): 342.2 (M ⁺
[365] 4- (4-trans-hydroxycyclohexyl) amino-5,6-dimethyl-2- (4-fluorophenyl) -7H-pyrrolo [2,3d] pyrimidine. ¹ H NMR (200 MHz, CDCl ₃ ) 1.26-1.66 (m, 8H), 1.94 (s, 3H), 2.28 (d, 1H), 7.13 (m, 2H), 8.41 (m, 2H), 11.14 (s, 1H); MS (ES): 355.2 (M ⁺
[366] 4- (4-trans-hydroxycyclohexyl) amino-5,6-dimethyl-2- (3-fluorophenyl) -7H-pyrrolo [2,3d] pyrimidine. ^{1 H NMR (200 MHz, CDCl} 3) δ1.26-1.71 (m, 8H), 2.06 (s, 3H), 2.30 (s, 3H), 3.72 (m, 1H), 4.30 (m, 1H), 4.90 (d, 1H), 7.09 ( m, 1H), 7.39 (m, 1H), 8.05 (m, 1H), 8.20 (m, 1H), 10.04 (s, 1H); MS (ES); 355.2 (m + +1).
[367] 4- (4-trans-hydroxycyclohexyl) amino-5,6-dimethyl-2- (2-fluorophenyl) -7H-pyrrolo [2,3d] pyrimidine. ^{1 H NMR (200 MHz, CDCl} 3) δ1.30-1.64 (m, 8H), 2.17 (s, 3H), 2.31 (s, 3H), 3.73 (m, 1H), 4.24 (m, 1H), 4.82 (d, IH), 7.28 (m, 2H), 8.18 (m, IH), 9.02 (m, IH), 12.20 (s, IH); MS (ES): 355.3 (M ⁺
[368] 4- (4-trans-hydroxycyclohexyl) amino-5,6-dimethyl-2-isopropy-7H-pyrrolo [2,3d] pyrimidine. ^{1 H NMR (200 MHz, CDCl} 3) δ1.31 (d, J = 7.0Hz, 6H), 1.30-1.65 (m, 8H), 2.27 (s, 3H), 2.28 (s, 3H), 3.01 (m , J = 7.0 Hz, 1H), 4.14 (m, 1H), 4.78 (d, 1H); MS (ES): 303.2.
[369] dl-4- (2- trans-hydroxycyclohexyl) amino-5,6-dimethyl-2-isopropyl -7H- pyrrolo [2,3d] pyrimidine _{^{1 H NMR (200MHz, CDCl 3}} ) d, 1.31 1H), 5.08 (d, IH), 1.42 (m, IH) ), 7.41-7.48 (m, 3H), 8.30 (m, 2H), 10.08 (s, 1H); MS (ES): 337.2 (M ⁺ +1).
[370] 4- (3,4-trans-dihydroxycyclohexyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. MS (ES): 353.2 (M ^< + & gt ^; +1).
[371] 4- (3,4-cis-dihydroxycyclohexyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. MS (ES): 353.2 (M ^< + & gt ^; +1).
[372] 4- (2-Acetylaminoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. mp 196-199 [deg.] C .; ^{1 H NMR (200 MHz, CDCl} 3) δ1.72 (s, 3H), 1.97 (s, 3H), 2.31 (s, 3H), 3.59 (m, 2H), 3.96 (m, 2H), 5.63 (br 1H), 7.44-7.47 (m, 3H), 8.36-8.43 (dd, J = 1 Hz, 7 Hz, 2H), 10.76 (s, 1H); MS (ES): 324.5 (M ⁺
[373] dl-4- (2-trans-hydroxycyclopentyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.
[374] ^{1 H NMR (200 MHz, CDCl} 3) δ1.62 (m, 2H), 1.79 (br, 4H), 1.92 (s, 3H), 2.29 (s, 3H), 4.11 (m, 1H), 4.23 (m 1H), 5.28 (d, 1H), 7.41-7.49 (m, 3H), 8.22 (m, 2H), 10.51 (s, 1H); MS (ES): 323.2 (M ^< + & gt ^; +1).
[375] Preparation of ¹ -trans-hydroxycyclopentylamine is described in PCT 9417090.
[376] dl-4- (3-trans-hydroxycyclopentyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.
[377] ^{1 H NMR (200 MHz, CDCl} 3) δ1.58-1.90 (br, 6H), 2.05 (s, 3H), 2.29 (s, 3H), 4.48-4.57 (m, 1H), 4.91-5.01 (m, 2H), 7.35-7.46 (m, 3H), 8.42-8.47 (m, 2H), 10.11 (s, 1H); MS (ES): 323.2 (M ^< + & gt ^; +1).
[378] ¹ Preparation of 3-trans-hydroxycyclopentylamine is described in EP-A-322242.
[379] dl-4- (3-cis-hydroxycyclopentyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. ^One
[380] ^{1 H NMR (200 MHz, CDCl} 3) δ1.82-2.28 (br, 6H), 2.02 (s, 3H), 2.30 (s, 3H), 4.53-4.60 (m, 1H), 4.95-5.08 (m, 1H), 5.85-5.93 (d, 1H), 7.35-7.47 (m, 3H), 8.42-8.46 (m, 2H), 10.05 (s, 1H); MS (ES): 323.2 (M ⁺
[381] ¹ Preparation of 3-cis-hydroxycyclopentylamine is described in EP-A-322242.
[382] 4- (3,4-trans-dihydroxycyclopentyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. ^{1 1 H NMR (200 MHz,} CDCl 3) δ1.92-1.99 (br, 2H), 2.14 (s, 3H), 2.20 (br, 2H), 2.30 (s, 3H), 2.41-2.52 (br, 2H 2H), 4.98 (m, 2H), 7.38-7.47 (m, 2H), 2.14 (s, 3H) , 3H), 8.38-8.42 (m, 2 H) 9.53 (s, 1 H); MS (ES): 339.2 (M ^< + & gt ^; +1).
[383] Preparation of ^1, 3,4-trans-dihydroxycyclopentylamine is described in PCT 9417090.
[384] 4- (3-Amino-3-oxopropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.
[385] ^{1 H NMR (200 MHz, CDCl} 3) δ2.02 (s, 3H), 2.29 (s, 3H), 2.71 (t, 2H), 4.18 (m, 2H), 5.75-5.95 (m, 3H), 7.38 : MS (ES): 310.1 (M ⁺ 1) &^lt; RTI ID ⁼ 0.0 &^gt;
[386] 4- (3-N-cyclopropylmethylamino-3-oxopropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.
[387] ^{1 H NMR (200 MHz, CD} 3 OD) δ0.51 (q, 2H), 0.40 (q, 2H), 1.79-1.95 (br, 1H), 2.36 (s, 3H), 2.40 (s, 3H), MS (ES): 364.2 (M ^< + ^> +2) ).
[388] 4- (2-amino -2-oxo-ethyl) amino-5,6-dimethyl-2-phenyl -7H- pyrrolo [2,3d] pyrimidine ^{1 H NMR (200 MHz, CD} 3 OD) δ2.31 ( (s, 3H), 2.38 (s, 3H), 4.26 (s, 2H), 7.36 (m, 3H), 8.33 (m, 2H); MS (ES): 396.1 (M ⁺
[389] Pyrrolo [2,3d] pyrimidine 1H NMR (200 MHz, CDCl3) ^{1. 1} H-NMR (CDCl ₃ ) 1H), 7.41-7.48 (m, 3H), 6.99 (s, 3H), 2.17 (s, 3H) , 8.40 (m, 2H), 10.66 (s, 1H); MS (ES): 310.1 (M ⁺ +1).
[390] 4- (3-tert-Butyloxyl-3-oxopropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. ^{1 H NMR (200 MHz, CDCl} 3) δ1.45 (s, 9H), 1.96 (s, 3H), 2.29 (s, 3H), 2.71 (t, 2H), 4.01 (q, 2H), 5.78 (t , 7.41-7.48 (m, 3H), 8.22-8.29 (m, 2H); MS (ES): 367.2 (M ⁺ +1).
[391] 4- (2-hydroxyethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. ^{1 H NMR (200 MHz, CDCl} 3) δ1.92 (s, 3H), 2.29 (s, 3H), 3.81-3.98 (br, 4H), 5.59 (t, 1H), 7.39-7.48 (m, 3H) , 8.37 (m, 2 H), 10.72 (s, 1 H); MS (ES): 283.1 (M ^< + & gt ^; +1).
[392] 4- (3-Hydroxypropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. ^{1 H NMR (200 MHz, CDCl} 3) δ1.84 (m, 2H), 1.99 (s, 3H), 2.32 (s, 3H), 3.62 (t, 2H), 3.96 (m, 2H), 3.35 (t MS (ES): 297.2 (M ^< + & gt ^; +1), 7.39-7.48 (m, 3H), 8.36
[393] 4- (4-Hydroxybutyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. ^{1 H NMR (200 MHz, CDCl} 3) δ1.71-1.82 (m, 4H), 1.99 (s, 3H), 2.31 (s, 3H), 3.68-3.80 (m, 4H), 5.20 (t.1H) , 7.41-7.49 (m, 3H), 8.41 (m, 2H), 10.37 (s, 1H); MS (ES): 311.2 (M ⁺
[394] 4- (4-trans-acetylaminocyclohexyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.
[395] 4- (4-trans-methylsulfonylaminocyclohexyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.
[396] 4- (2-Acetylaminoethyl) amino-5,6-dimethyl-2-phenyl-7H-7 (1-phenylethyl) pyrrolo [2,3d] pyrimidine.
[397] 4- (4-trans-hydroxycyclohexyl) amino-5,6-dimethyl-2-phenyl-7H- (1-phenylethyl) pyrrolo [2,3d] pyrimidine.
[398] 6-dimethyl-2-phenyl-7H-7 (1-phenylethyl) pyrrolo [2,3d] pyrimidine.
[399] 4- (2-methylpropyl) amino-5,6-dimethyl-2-phenyl-7H-7- (1-phenylethyl) pyrrolo [2,3d] pyrimidine.
[400] Example 2
[401] To a stirred suspension of THF (1.0 mL) cooled to 0 C containing triphenylphosphine (0.047 g, 0.179 mmol) and bezoic acid (0.022 g, 0.179 mmol) was added 4- (4-trans- hydroxycyclohexyl) Amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine (0.05 g, 0.149 mmol) at 0 <0> C. The following diethyl azodicarboxylate (0.028 ml, 0.179 mmol) was added dropwise over 10 minutes. Then, the reaction solution was warmed to room temperature. After completion of the reaction by TLC, the reaction mixture was cooled with aqueous sodium bicarbonate (3.0 mL). The aqueous phase was separated and extracted with ether (2x5.0 mL). The organic extracts were combined, dried and concentrated to dryness in vacuo. Ether (2.0 mL) and hexane (5.0 mL) were added to the residue, and then triphenylphosphine oxide was filtered off. The filtrate was concentrated to give viscous cucumber which was purified by column chromatography (hexane: ethyl acetate = 4: 1) to give 4- (4-cis-benzoyloxycyclohexyl) amino-5,6- MS (ES): 441.3 (M ^& lt ^; + & gt ^; +1). This reaction solution also produced 50.0 mg (84%) of 4- (3-cyclohexenyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. MS (ES): 319.2 (M ^&lt; + & gt ^; +1).
[402] Example 3:
[403] Ethanol (1.0 mL) containing 4- (4-cis-benzoyloxycyclohexyl) amino-5,6-dimethyl-2- phenyl-7H- pyrrolo [2,3d] pyrimidine (5.0 mg, 0.0114 mmol) To the solution was added 10 drops of 2 M sodium hydroxide. After 1 h, the reaction mixture was extracted with ethyl acetate (3 x 5.0 mL) and the organic layer was dried, filtered and concentrated to dryness in vacuo. The residue was purified by column chromatography (hexane: ethyl acetate = 4: 1) to give 4- (4-cis-hydroxycyclohexyl) amino-5,6-dimethyl- Pyrimidine 3.6 mg (94%). MS (ES): 337.2 (M ^&lt; + & gt ^; +1).
[404] The following compound was obtained in analogy to example 3 :
[405] 4- (3-N, N-dimethyl-3-oxopropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.
[406] _{^{1 H NMR (200MHz, CDCl 3}} ) δ2.01 (s, 3H), 2.31 (s, 3H), 2.73 (t, 2H), 2.97 (s, 6H), 4.08 (m, 2H), 6.09 (t, 1H), 7.41-7.48 (m, 3H), 8.43 (m, 2H), 10.46 (s, 1H); MS (ES): 338.2 (M ⁺
[407] 4- (2-formylaminoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.
[408] _{^{1 H NMR (200MHz, CDCl 3}} ) δ2.26 (s, 3H), 2.37 (s, 3H), 3.59-3.78 (m, 2H), 3.38-4.01 (m, 2H), 5.48-5.60 (m, 1H ), 7.38-7.57 (m, 3H), 8.09 (s, 3H), 8.30-8.45 (m, 2H) 8.82 (s, 1H); MS (ES): 310.1 (M ⁺
[409] 4- (3-Acetylaminopropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. MS (ES): 338.2 (M ^&lt; + & gt ^; +1).
[410] Example 4
[411] Pyrrolo [2,3d] pyrimidine (70.0 mg, 0.191 mmol) was dissolved in trifluoroacetic acid (5 ml) and the mixture was stirred at room temperature for 3 hours. Acid: Dichloromethane (1: 1, 5.0 mL). The resulting solution was stirred at room temperature for 1 hour and refluxed for 2 hours. After cooling to room temperature, the mixture was concentrated to dryness in vacuo. The residue was purified by thin layer chromatography (EtOAc: hexane: AcOH = 7: 2.5: 0.5) to give 4- (3-hydroxy-3-oxopropyl) amino-5,6- [2,3d] pyrimidine (40.0 mg, 68%). _{^{1 H NMR (200MHz, CD 3}} OD) δ2.32 (s, 3H), 2.38 (s, 3H), 2.81 (t, 2H), 4.01 (t, 2H), 7.55 (m, 3H), 8.24 (m , 2H); MS (ES): 311.1 (M ^&lt; + & gt ^; +1).
[412] MS (ES): 296.1 (M ⁺ +1), 279.1 (M ^{& lt ;} + &gt;). NH ₃ ).
[413] Example 5
[414] Pyrrolo [2,3d] pyrimidine (50.0 mg, 0.161 mmol) was dissolved in N, N-dimepyl (4-hydroxyphenyl) Was dissolved in a mixture of formamide (0.50 mL), dioxane (0.50 mL) and water (0.25 mL). To this solution was added methylamine (0.02 mL, 40% aqueous solution, 0.242 mmol), triethylamine (0.085 mL) and N, N, N ', N'- tetramethyluronium tetrafluoroborate (61.2 mg, 0.203 mmol ). After stirring at room temperature for 10 minutes, the solution was concentrated and the residue was purified by thin layer chromatography (EtOAc) to give 4 (3-N-methyl-3-oxopropyl) amino-5,6-dimethyl- -Pyrrolo [2,3d] pyrimidine (35.0 mg, 67%).
[415] 1H NMR (200MHz, CDCl ₃ ) 1.92 (s, 3H), 2.30 (s, 3H), 2.65 (t, 2H), 4.08 , 7.45 (m, 3H), 8.41 (m, 2H), 10.68 (s, 1H); MS (ES): 311.1 (M ⁺ +1).
[416] The following compounds were obtained in analogy to example 5 :
[417] MS (ES): 350.2 (M ^&lt; + & gt ^; +1).
[418] MS (ES): 352.2 (M ^&lt; + & gt ^; +1).
[419] 4- (3-propionylaminopropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. _{^{1 H NMR (200MHz, CDCl 3}} ) δ1.00-1.08 (t, 3H), 1.71-2.03 (m, 4H), 2.08 (s, 3H), 2.37 (s, 3H) 3.26-3.40 (m, 2H) 2H), 9.69 (s, 1H), 7.39-7.57 (m, 3H), 8.31-8.39 1H); MS (ES): 352.2 (M ^&lt; ⁺ &gt; +1).
[420] 4- (2-methylsulfonylaminoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. _{^{1 H NMR (200MHz, CDCl 3}} ) δ2.18 (s, 3H), 2.27 (s, 3H), 2.92 (s, 3H), 3.39-3.53 (m, 2H) 3.71-3.88 (m, 2H), 5.31 (M, 2H), 9.52 (s, 1H); MS (ES): 360.2 (m, 2H), 7.32-7.43 ⁺ +1).
[421] Example 6
[422] A mixture of 4-chloro-5,6-dimethyl-2-phenyl-7H- pyrrolo [2,3d] pyrimidine (0.70 g, 2.72 mmol) and 1,2- diaminoethane (10.0 mL, And refluxed in an inert atmosphere for 6 hours. Excess amine was removed in vacuo and the residue was washed sequentially with ether and hexane to give 4- (2-aminoethyl) amino-5,6-dimethyl-2-phenyl-7H- pyrrolo [2,3d ] Pyrimidine (0.75 g, 98%). MS (ES); ^{282.2 (M + +1), 2651.1} (M + -NH 3).
[423] Example 7
[424] Pyrrolo [2,3d] pyrimidine (70.0 mg, 0.249 mmol) and triethylamine (50.4 mg, 0.498 mmol) were added to a solution of 4- (2-aminoethyl) (2.0 mL) was added propionyl chloride (25.6 mg, 0.024 mL, 0.274 mmol) at 0 &lt; 0 &gt; C. After 1 h, the mixture was concentrated in vacuo and the residue was purified by thin layer chromatography (EtOAc) to give 4- (2-propionylaminoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [ , 3d] pyrimidine (22.0 mg, 26%). MS (ES): 338.2 (M ^&lt; + & gt ^; +1).
[425] The following compounds were obtained in analogy to example 7 :
[426] 4- (2-N'-methylureaethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.
[427] _{^{1 H NMR (200MHz, CDCl 3}} ) δ2.13 (s, 3H), 2.32 (s, 3H), 3.53 (d, 3H), 3.55 (m, 2H), 3.88 (m, 2H), 4.29 (m, 1H), 5.68 (t, IH), 5.84 (m, IH), 7.84 (m, IH), 7.42 (m, 3H), 8.36 (dd, 2H), 9.52 MS (ES): 339.3 (M ^&lt; + & gt ^; +1).
[428] 4- (2-N'-ethylureaethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. MS (ES): 353.2 (M ^&lt; + & gt ^; +1).
[429] Example 8:
[430] (41.1 mg, 0.215 mmol), dimethylaminopyridine (2.4 mg, 0.020 mmol) and pyruvic acid (18.9 mg, 0.015 mL, 0.215 mmol) were added to a solution of 1- (3- dimethylaminopropyl) -3- ethylcarbodiimide hydrochloride (55.0 mg, 0.196 mmol) was added to a solution of 4- (2-aminoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine Respectively. This mixture was stirred at room temperature for 4 hours. The usual work-up and column chromatography (EtOAc) were performed to give 4- (2'-Pyrroylamidoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. MS (ES): 352.2 (M ^&lt; + & gt ^; +1).
[431] Example 9:
[432] To a solution of dichloromethane (2.0 mL) containing 4- (2-aminoethyl) amino-5,6-dimethyl-2-phenyl-7H- pyrrolo [2,3d] pyrimidine (60.0 mg, 0.213 mmol) -Trimethylsilylisocyanate (43.3 mg, 0.051 mL, 0.320 mmol). The mixture was stirred at room temperature for 3 hours, and sodium bicarbonate solution was added. After filtration through a small amount of silica gel, the filtrate was concentrated to dryness in vacuo to give 4- (2-ureaethyl) amino-5,6-dimethyl-2-phenyl-7H- pyrrolo [2,3d] pyrimidine 9.8 mg (14%). MS (ES): 325.2 (M ^&lt; + & gt ^; +1).
[433] The following compounds were obtained in analogy to example 9 :
[434] dl-4- (2-Acetylaminopropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. _{^{1 H NMR (200MHz, CDCl 3}} ) δ1.28-1.32 (d, J = 8Hz, 3H), 1.66 (s, 3H), 1.96 (s, 3H), 2.30 (s, 3H), 3.76-3.83 (m (M, 2H), 10.83 (s, 1H), 5.60-5.66 (t, J = 6 Hz, 1H), 7.40-7.51 (m, 3H), 8.36-8.43 ; MS (ES): 338.2 (M ^&lt; + & gt ^; +1).
[435] (R) -4- (2-Acetylaminopropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. _{^{1 H NMR (200MHz, CDCl 3}} ) δ1.31 (d, 3H), 1.66 (s, 3H), 1.99 (s, 3H), 2.31 (s, 3H), 3.78-3.83 (m, 2H), 4.17- MS (ES): 338.2 (M ^&lt; + & gt ^; +1).
[436] (R) -4- (l-methyl-2-acetylaminoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. _{^{1 H NMR (200MHz, CDCl 3}} ) δ1.41 (d, 3H), 1.68 (s, 3H), 2.21 (s, 3H), 2.34 (s, 3H), 3.46-3.52 (br, m, 2H), (M, 2H), 8.93 (s, 1H); MS (ES): 338.2 (M ⁺ ).
[437] (S) -4- (2-Acetylaminopropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. _{^{1 H NMR (200MHz, CDCl 3}} ) δ1.31 (d, 3H), 1.66 (s, 3H), 2.66 (s, 3H), 2.35 (s, 3H), 3.78-3.83 (m, 2H), 4.17- MS (ES): 338.2 (M ⁺ l +). ^& Lt; / RTI &gt;
[438] (S) -4- (l-methyl-2-acetylaminoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. _{^{1 H NMR (200MHz, CDCl 3}} ) δ1.41 (d, 3H), 1.68 (s, 3H), 2.05 (s, 3H), 2.32 (s, 3H), 3.46-3.52 (m, 2H), 4.73 ( 1H), 5.22 (d, 1H), 7.41-7.46 (m, 3H), 8.36-8.40 (m, 2H), 10.13 (s, 1H); MS (ES): 338.2 (M ⁺
[439] Example 10:
[440] Dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine and dl-1-amino-2- (1,1- dimethylethoxy) carbonylamino- -2-amino-1- (1,1-dimethylethoxy) carbonylamino-propane was carried out in analogy to Example 1 . This reaction was carried out in the same manner as in Example 1 except that dl-4- (1-methyl-2- (1,1-dimethylethoxy) carbonylamino) ethylamino) -5,6-dimethyl- Methyl-2- (1,1-dimethylethoxy) carbonylamino) ethylamino-5,6-dimethyl-7H-pyrrolo [2,3-d] Methylene chloride (1: 3), which was isolated by column chromatography (EtOAc; hexane = 1: 3). Dimethyl-2-phenyl-7H-pyrrolo [2,3-d] pyrimidin- ^{pyrimidine: 1 H NMR (200MHz, CDCl} 3) δ1.29-1.38 (m, 12H), 1.95 (s, 3H), 2.31 (s, 3H), 3.34-3.43 (m, 2H), 4.62-4.70 ( (m, 3H), 8.37-8.44 (m, 2H), 10.75 (s, 1H); MS 396.3 (M ^&lt; + & gt ^; +1). The second fraction is a mixture of dl-4- (2- (1,1-dimethylethoxy) carbonylaminopropyl 0 amino-5,6-dimethyl-2-phenyl-7H- pyrrolo [2,3d] pyrimidine:
[441] _{^{1 H NMR (200MHz, CDCl 3}} ) δ1.26-1.40 (m, 12H), 2.00 (s, 3H), 2.31 (s, 3H), 3.60-3.90 (m, 2H), 3.95-4.10 (m, 1H ), 5.41-5.44 (d, J = 6.0 Hz, 1H), 5.65 (br, 1H), 7.40-7.46 (m, 3H), 8.37-8.44 (m, 2H), 10.89 ES): 396.2 (M ^&lt; + & gt ^; +1).
[442] The following compounds were obtained in analogy to example 9 :
[443] (S, S) -4- (2-Acetylaminocyclohexyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. _{^{1 H NMR (200MHz, CDCl 3}} ) δ1.43 (m, 4H), 1.60 (s, 3H), 1.83 (m, 2H), 2.18 (s, 3H), 2.30 (m, 2H), 2.32 (s, (M, 3H), 3.73 (br, IH), 4.25 (br, IH), 5.29 (d, IH), 7.43-7.48 (m, 3H), 8.35-8.40 (m, 2H), 9.05
[444] 4- (2-Methyl-2-acetylaminopropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. _{^{1 HNMR (200MHz, CDCl 3)}} δ1.51 (m, 6H), 1.56 (s, 3H), 2.07 (s, 3H), 2.36 (s, 3H), 3.76 (d, 2H), 5.78 (t, 1H ), 7.41-7.48 (m, 3H), 7.93 (s, 1H), 8.39 (m, 2H), 10.07 (s, 1H); MS (ES): 352.3 (M ⁺
[445] Example 11
[446] dl-4 (1-methyl-2- (1,1-dimethylethoxy) carbonylaminoethyl) amino-5,6- dimethyl-2-phenyl-7H- pyrrolo [2,3d] pyrimidine , 0.153 mmol) was treated with trifluoroacetic acid in dichloromethane (2.0 mL) for 14 hours. The organic solvent was removed in vacuo to dryness. The residue was dissolved in N, N-dimethylformamide (2.0 mL) and dissolved in triethylamine (2.0 mL). To this solution was added acetic anhydride (17.2 mg, 0.016, 0.16 mmol) at 0 &lt; 0 &gt; C. The resulting mixture was stirred at room temperature for 48 hours and then concentrated to dryness in vacuo. The residue was purified by thin layer chromatography (EtOAc) to give dl-4- (l-methyl-2-acetylaminoethyl) amino-5,6-dimethyl-2-phenyl-7H- pyrrolo [2,3d] (52%) of the title compound. _{^{1 H NMR (200MHz, CDCl 3}} ) δ1.38-1.42 (d, J = 8HZ, 3H), 1.69 (s, 3H), 2.01 (s, 3H), 2.32 (s, 3H), 3.38-3.60 (m (M, 2H), 10.44 (s, 1H), 7.60-7.51 (m, 3H), 8.37-8.43 MS (ES): 338.2 (M ^&lt; + & gt ^; +1).
[447] Example 12
[448] Pyrrolo [2,3d] pyrimidine (0.15 g, 0.583 mmol) and (lR, 2R) - (-) - 1,2-diaminocyclo- (2-aminocyclohexyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-d] pyrimidine, prepared in analogy to example 1, 2,3d] pyrimidine was treated with triethylamine (0.726 g, 7.175 mmol) and acetic anhydride (0.325 g, 3.18 mmol) in N, N dimethylformamide (10.0 mL) at room temperature for 2 hours. After removing the solvent in vacuo, ethyl acetate (10.0 mL) and water (10.0 mL) were added to the residue. The mixture was separated and the aqueous layer was extracted with ethyl acetate (2x10.0 mL). The combined ethyl acetate solution was dried (MgSO ₄ ) and evaporated. The filtrate was concentrated to dryness in vacuo and the residue was treated with column chromatography (EtOAc: hexane = 1: 1) to give (R, R) -4- (2-acetylaminocyclohexyl) amino- Methyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine (57.0 mg, 26%). _{^{1 H NMR (200MHz, CDCl 3}} ) δ1.43 (m, 4H), 1.60 (s, 3H), 1.84 (s, 2H), 2.22 (s, 3H), 2.30 (m, 2H), 2.33 (s, (M, 3H), 8.35-8.39 (m, 2H), 8.83 (s, 1H): MS (ES): 378.3 (M ^&lt; + & gt ^; +1).
[449] Example 13
[450] To a solution of pyridine (1.0 mL) containing 4- (2-hydroxyethyl) amino-5,6-dimethyl-2- phenyl-7H- pyrrolo [2,3d] pyrimidine (40.0 mg, 0.141 mmol) Acetic anhydride was added. The mixture was stirred at room temperature for 4 hours and the solvent was removed in vacuo. The residue was purified by preparative thin layer chromatography (EtOAc: hexane = 1: 1) to give 4- (2-acetyloxyethyl) Pyrimidine &lt; / RTI &gt; 32.3 mg (71%). _{^{1 H NMR (200MHz, CDCl 3}} ) δ1.90 (s, 3H), 2.08 (s, 3H), 2.31 (s, 3H), 4.05 (m, 2H), 4.45 (t, 2H), 5.42 (m, 1H), 7.41-7.49 (m, 3H), 8.42 (m, 2H), 8.42 (m, 2H), 11.23 (s, 1H).
[451] Example 14:
[452] A solution of dichloromethane (4.0 mL) containing Fmoc-β-Ala-OH (97.4 mg, 0.313 mmol) and oxalyl chloride (39.7 mg, 27.3 uL, 0.313 mmol) was dropwise added dropwise N, N-dimethylformamide Pyrrolo [2,3-d] pyrimidine (80.0 mg, 0.285 mmol) and triethyl (2-aminoethyl) Amine (57.6 mg, 79.4 uL, 0.570 mmol) at 0 &lt; 0 &gt; C. After 3 h, the mixture was concentrated in vacuo and the residue was treated with a solution of N, N-dimethylformamide (2.0 mL) containing 20% piperidine for 0.5 h. The solvent was removed in vacuo and the residue was washed with diethyl ether: hexane (1: 5) to give 4- (6-amino-3-aza-4-oxohexyl) amino- 7H-pyrrolo [2,3d] pyrimidine (3.0 mg, 3%). MS (ES): 353.2 (M ^&lt; + & gt ^; +1)
[453] Example 15:
[454] Pyrrolo [2,3d] pyrimidine (70.0 mg, 0.249 mmol) and anhydrous succinic acid (27.0 mg, 0.274 mmol) were added to a solution of 4- (2- aminoethyl) amino-5,6- (4.0 mL) was added dropwise N, N-dimethylformamide, and the mixture was stirred at room temperature for 4 hours. The reaction mixture was extracted with 20% sodium hydroxide (3 x 5.0 mL). This aqueous solution was acidified to pH = 7.0 with 3 M hydrogen chloride. The entire mixture was extracted with ethyl acetate (3 x 10 mL). The combined organic solutions were dried (MgSO ₄ ) and filtered. The filtrate was concentrated to dryness in vacuo to give 4- (7-hydroxy-3-aza-4,7-dioxoheptyl) amino-5,6-dimethyl-2-phenyl-7H- pyrrolo [2,3- (16%) of the title compound. MS (ES): 382.2 (M ^&lt; + & gt ^; +1)
[455] Example 16:
[456] 700 mg of 4-cis-3-hydroxycyclopentyl) amino-2-phenyl-5,6-dimethyl-7H-pyrrolo [2,3d] pyrimidine was added at room temperature to 10 mL of methylformamide (DMF) Thereafter, 455 mg of N-Noc glycine, 20 mg of N, N-dimethylaminopyridine (DMAP), 293 mg of hydroxybenzotriazole (HOBT), and 1- (3-dimethylaminopropyl) 622 mg of midhydrochloride (EDCl) was added. The reaction mixture was allowed to stand overnight with stirring. The DMF was then removed under reduced pressure and the reaction mixture was partitioned between 20 mL of ethyl acetate and 50 mL of water. The aqueous was further extracted with ethyl acetate (2 x 20 mL) and the combined organics washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated. Purification on silica gel eluting with ethyl acetate / hexanes gave the desired product: 4- (cis-3- (Nt-butoxycarbonyl-2-aminoacetoxy) cyclopentyl) amino- -7H-pyrrole [2,3d] pyrimidine. MS (ES) (M ^&lt; + & gt ^; +1) = 480.2. The ester was then treated with 5 mL of dichloromethane containing 20% trifluoroacetic acid at room temperature, allowed to stand overnight and then concentrated. Trituration with ethyl acetate afforded 300 mg of an off-white solid; Pyrrolo [2,3d] pyrimidine trifluoroacetic acid salt as a colorless solid. MS (m / z) ES) (M ^&lt; + & gt ^; +1) = 380.1
[457] Those skilled in the art will recognize that the following compounds can be synthesized by the methods described above.
[458] (Cis-3-Hydroxycyclopentyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine MS (ES) (M ⁺ +1) = 323.1.
[459] Pyrido [2,3-d] pyrimidine trifluoroacetic acid salt MS (ES) m / e calcd for &lt; RTI ID = (M ^&lt; + & gt ^; +1) = 380.1.
[460] MS (ES) (M ⁺ +1) = 364.2 &lt; RTI ID = 0.0 &^gt; (M ⁺ 1) &^lt; / RTI &gt;
[461] MS (ES) (M ^&lt; + & gt ^; +1) = 353.4 (M ⁺ H +).
[462] MS (ES) (M ^&lt; + & gt ^; +1) = 352.4.
[463] 2,3-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine MS (ES) (M ⁺ +1) = 367.5.
[464] MS (ES) (M ⁺ +1) = 309.1 (M ⁺ H +), was prepared in accordance with the general method of example 1, step 3 using 4- (2-aminocyclopropylacetamidoethyl) amino-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.
[465] MS (ES) (M ⁺ +1) = 342.8. Synthesis of 4- (trans-4-hydroxycyclohexyl) amino-2- (3-chlorophenyl) -7H-pyrrolo [2,3d] pyrimidine.
[466] MS (ES) (M ⁺ +1) = 327.2 (M ⁺ H +).
[467] (Trans-4-hydroxycyclohexyl) amino-2- (4-pyridyl) -7H-pyrrolo [2,3d] pyrimidine MS (ES) (M ⁺ +1) = 310.2.
[468] Example 17
[469] Configuration IX
[470]
[471] (7) (Structure IX) was protected with di-t-butyl dicarbonate under basic conditions to give the corresponding carbamate (22). (22) was selectively radically brominated to yield bromide (23). In general, compound (23) serves as a major nucleophilic intermediate for various nucleophilic coupling partners. This alkyl bromide was substituted with sodium phenolate trihydrate to give compound (24). Subsequent displacement of the aryl chloride and removal of the t-butyl carbamate protecting group were carried out in one step to give the desired compound (25).
[472] Synthesis of compounds (22) - (25) according to composition IX
[473]
[474] Di-t-butyl dicarbonate (5.37 g, 24.6 mmol) and dimethylaminopyridine (1.13 g, 9.2 mmol) were added to a solution containing 7 (1.50 g, 6.15 mmol) and pyridine (30 mL). After 20 h, the reaction was concentrated and the residue was partitioned between CH ₂ Cl ₂ and water. The CH ₂ Cl ₂ layer was separated, dried over MgSO ₄ , filtered and concentrated to give a black solid. By flash chromatography by (SiO ₂ 1/9 EtOAc / hexanes, R _f 0.40) as a white solid (22) was calculated 1.70 g (80%). _{^{1 H NMR (200MHz, CDCl 3}} ) δ8.50 (m, 2H, Ar-H), 7.45 (m, 3H, Ar-H), 6.39 (s, 1H, pyrrole -H), 2.66 (s, 3H , pyrrole _{-CH 3), 1.76 (s,} 9H, carbamate-CH 3); MS, M + 1 = 344.1; Mpt = 175-177 ° C.
[475] N-Bromosuccinimide (508 mg, 2.86 mmol) and AIBN (112 mg, 0.68 mmol) were added to a solution containing (22) (935 mg, 2.71 mmol) and CCl ₄ (50 mL). The solution was heated and condensed. After 2 h, the reaction was cooled to room temperature and concentrated in vacuo to yield a white solid. Flash chromatography (SiO ₂ ; 1/1 CH ₂ Cl ₂ / hexane, R _f 0.30) yielded 960 mg (84%) of a white solid (23). _{^{1 H NMR (200MHz, CDCl 3}} ) δ8.52 (m, 2H, Ar-H), 7.48 (m, 3H, Ar-H), 6.76 (s, 1H, pyrrole -H), 4.93 (s, 2H , pyrrole _{-CH 2 Br), 1.79 (s} , 9H, carbamate-CH 3); MS, M + 1 = 423.9; Mpt = 155-157 ° C.
[476]
[477] Sodium phenoxide trihydrate (173 mg, 1.02 mmol) was added in portions to a solution of the bromide 23 (410 mg, 0.97 mmol) in CH ₂ Cl ₂ (5 mL) and DMF (10 mL). After 2 h, the reaction solution was partitioned between CH ₂ Cl ₂ and water. The water layer was extracted with CH ₂ Cl ₂ . The combined CH ₂ Cl ₂ layers were washed with water, dried over MgSO ₄ , filtered and concentrated to give a yellow solid. Purification by flash chromatography (SiO _2; 1/6 EtOAc / hexanes, R _f 0.30) to a white solid (24) 210 mg (50% ) were calculated. _{^{1 H NMR (200MHz, CDCl 3}} ) δ8.53 (m, 2H, Ar-H), 7.48 (m, 3H, Ar-H), 7.34 (s, 2H, Ar-H), 7.03 (s, 3H, Ar-H), 6.83 (s , 1H, pyrrole -H), 5.45 (s, 2H , ArCH 2 O), 1.76 (s, 9H, carbamate-CH 3); MS, M + = 436.2.
[478]
[479] 25
[480] (24) (85 mg, 0.20 mmol), N-acetylethylenediamine 9201 mg, 1.95 mmol) and DMSO (3 mL) was heated to 100 &lt; 0 &gt; C. After 1 hour, the temperature was raised to 130 占 폚. After 3 hours, the reaction was cooled to room temperature and partitioned between EtOAc and water. Water was extracted with a layered EtOAc (x2). Wash the combined EtOAc layer with water, dried with MgSO ₄ during gyeotda sulfate, filtered and concentrated. Purification by flash chromatography _{(SiO 2; 1/9 EtOH / CHCl} 3, R f 0.25) to foaming white 73 mg (93%) a solid (25) was calculated. ^{1 H NMR (200MHz, DMSO-} d 6) δ11.81 (brs, 1H, NH), 8.39 (m, 2H, Ar-H), 8.03 (br t, 1H, NH), 7.57 (brt, 1H, NH ), 7.20-7.50 (m, 5H, Ar-H), 6.89-7.09 (m, 3H, Ar-H), 6.59 (s, 1H, pyrrole -H), 5.12 (s, 2H , ArCH 2 O), _{3.61 (m, 2H, NCH 2} ), 3.36 (m, 2H, NCH 2), 1.79 (s, 3H, COCH 3); MS, M + 1 = 402.6
[481] The following compound was obtained analogously to Example 17:
[482] 4- (2-Acetylaminoethyl) amino-6-phenoxymethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. mp 196-197 [deg.] C; MS (ES): 401.6 (M ^&lt; + & gt ^; +1).
[483] 4- (2-Acetylaminoethyl) amino-6- (4-fluorophenoxy) methyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. MS (ES): 420.1 (M ^&lt; + & gt ^; +1).
[484] 4- (2-Acetylaminoethyl) amino-6- (4-chlorophenoxy) methyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. MS (ES): 436.1 (M ^&lt; + & gt ^; +1).
[485] 4- (2-Acetylaminoethyl) amino-6- (4-methoxyphenoxy) methyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. MS (ES): 432.1 (M ^&lt; + & gt ^; +1).
[486] 4- (2-Acetylaminoethyl) amino-6- (N-pyridin-2-one) methyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. MS (ES): 403.1 (M ^&lt; + & gt ^; +1).
[487] 4- (2-Acetylaminoethyl) amino-6- (N-phenylamino) methyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. MS (ES): 400.9 (M ^&lt; + & gt ^; +1).
[488] 4- (2-Acetylaminoethyl) amino-6- (N-methyl-N-phenylamino) methyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. MS (ES): 414.8 (M ^&lt; + & gt ^; +1).
[489] 4- (2-N'-methylureaethyl) amino-6-phenoxymethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. MS (ES): 416.9 (M ^&lt; + & gt ^; +1).
[490] Analysis of yeast β-galactosidase reporter gene for human adenosine A1 and A2a receptors
[491] The yeast strain S. cerevisiae was transformed with human adenosine A1 (A1R; CADUS strain CY12660) or human A2a (A2a; CADUS strain CY8362) and lacZ ( -Galactosidase) Respectively. A full description of the transformation is given below (see yeast strains). NECA (5'-N-ethylcarboxamidoadenosine), an efficacious adenosine receptor agonist with similar affinity for the A1 and A2a receptors, was used as all analytical ligands. The ability of the test compounds to inhibit NECA-induced beta -galactosidase activity by CY12660 or CY8362 at eight concentrations (0.1-10,000 nM) was investigated.
[492] Preparation of yeast stock culture Each yeast strain, CY12660 and CY8362, was streaked on an LT agar plate and cultured at 30 DEG C to observe the colonies. Yeasts from these colonies were added to a LT liquid (pH 6.8) and grown overnight at 30 占 폚. Each yeast strain was measured by spectrophotometry (molecular device VMAX) and diluted to OD ₆₀₀ = 1.0-2.0 (approximately 1-2 x 10 ⁷ cells / ml). For each 6 ml yeast broth, 4 ml of 40% glycerol (1: 1.5 volume: volume) was added ("yeast / glycerol stock"). From this yeast / glycerol stock, 10 1 ml aliquots were stored at-80 C until needed to make and analyze.
[493] Yeast A1R and A2aR Assay One vial of each CY8362 and CY12660 yeast / glycerol stock was thawed and resuspended in supplemented LT liquid medium (92 ml LT medium, 5 ml of 40% glucose, 0.45 ml of 1 M KOH and 2.5 ml of pipette ) Were used for inoculation. The liquid medium was grown at 30 占 폚 for 16-18 hours (overnight). An aliquot from the culture overnight incubated was then diluted in LT medium containing 4 U / ml adenosine deaminase (type VI or VII from the calf intestinal mucosa, Sigma) to give OD ₆₀₀ = 0.15 for CY8362 (A2aR) (1.5 × 10 ⁶ cells / ml) and OD ₆₀₀ = 0.50 (5 × 10 ⁶ cells / ml) for CY12660 (A1R).
[494] Analysis was performed on a 96-well microtiter plate to a final volume of 100 ul, resulting in a final concentration of 2% DMSO in all wells. For primary screening, 1-2 concentrations (10 uM, 1 μM) of the test compound were used. For the compound profile, it was tested at eight concentrations (10000, 1000, 500, 100, 50, 10, 1 and 0.1 nM). For each microtiter plate, 10 ul of 20% DMSO was added to the "control" and "whole" wells and 10 ul of the test compound (in 20% DMSO) was added to the "unknown" well. Then, 10 ul of NECA (5 uM for A1R and 1 uM for A2aR) was added to the "total" and "unknown" wells; 10ul PBS was added to " control " wells. In the final addition, 80 ul of yeast strain CY8362 or CY12660 was added to all wells. All plates were briefly agitated (laparoscope orbital shaker for 2-3 minutes) and incubated in a drying oven at 30 ° C for 4 hours.
[495] The -galactosidase activity can be quantitated using either a colorimetric substrate (eg, ONPG, CPRG), a luminescent substrate (eg, galactin-star), or a fluorogenic substrate (eg, FDG, resorufin). Fluorescence detection is desirable due to its excellent signal to noise ratio due to its low interference and low cost. Fluorescein digalactopyranoside (FDG, molecular probe or marker gene technology), fluorescent β-galactosidase substrate was added to all wells (final concentration = 80 uM) at 20 ul / well. Plates were shaken for 5-6 seconds (laparoscopic oviduct) and incubated for 90 min at 37 ° C (95% O ₂ /5% CO ₂ incubator). After 90 minutes of incubation time, the -Galactosidase activity was stopped using 20 μl / well of 1 M Na ₂ CO ₃ and all plates were shaken for 5-6 seconds. The plate was then stirred for 6 seconds and the relative fluorescence intensity was measured using a Tecan Spectrafluor (excitation = 485 nm, emission = 535 nm).
[496] The relative fluorescence values for the calculated " control " wells were based on and subtracted from the " total " and " unknown " The compound profile was analyzed via log-transform (x-axis: compound concentration) followed by a fractional competition curve appropriate for IC ₅₀ values (GraphPad Prism).
[497] Yeast strain Saccharomyces cerevisiae strain CY12660 [farl * 1442 tbt1-1 fusl-HIS3can1 ste14 :: trp1 :: LYS2 ste3 * 1156 gpa1 (41) -Gαi3 lys2 ura3 leu2 trp1: his3; LEU2 PGKp-Mfα1 Leader- HLA1R-PHO5term 2mu-orig REP3 Ampr] and CY8362 [gpa1prGαsE10K far1 * 1442 tbt1-1 fus1-HIS3 can1 ste14 :: trp1: LYS2 ste3 * 1156lys2 ura3 leu2 trpl his3; LEU2 PGKp-hA2aR 2mu-ori REP3 Ampr]
[498] LT medium The LT medium (Leu-Trp supplement) medium contained 1.0 g of valine, 1.0 g of aspartic acid, 0.75 g of phenylalanine, 0.9 g of lysine, 0.45 g of tyrosine, 0.45 g of isoleucine, 0.3 g of methionine, 0.6 g of adenine, 0.3 g of proline, 0.3 g of cysteine, 0.3 g of arginine, 0.9 g of histidine and 1.0 g of threonine.
[499] Structure of yeast strains expressing human A1 adenosine receptor
[500] In this example, the structure of yeast strains expressing human A1 adenosine receptors functionally integrated into the yeast pheromone system pathway is described.
[501] 1. Expression vector structure
[502] To construct a yeast expression vector for the human A1 adenosine receptor, the A1 adenosine receptor cDNA was amplified by reverse transcriptase PCR of human hippocampal mRNA using primers designed based on standard techniques and published sequences of human A1 adenosine receptors . The PCR product was subcloned into the Nco I and Xba I sites of the yeast expression plasmid pMP15.
[503] The pMP15 plasmid originated from the following pLPXt: Xba I site of YEP51 (Broach, JR et al. (1983) "Vectors for high-level, inducible expression of cloned genes in yeast" (Ed.), Experimental Manipulation of Gene Expression. Academic Press, New York) was removed by restriction enzyme cleavage, terminal peeling and re-ligation to obtain Yep51 Nco D Xba . Different Xba I sites are restriction enzyme digestion, end-filling by BamH I, linker (New England Biolabs, # 1081) ligation, Xba I to disassemble and re-ligation occurs in the BamH I to obtain a YEP51NcoXt. Decomposition of this plasmid with Esp 31 and Nco I and was ligated to Leu2 and PGKp fragments obtained by PCR. A 2 kb Leu2 PCR product was obtained by amplification from YEP51Nco using a primer containing the Esp31 and BglII sites. A 660 base pair PGKp PCR product was amplified from pPGKa s (Kang, Y.-S. et al . (1990) Mol. Cell. Biol. 10 : 2582-2590) as a PCR primer containing Bgl II and Nco I sites . The plasmid obtained is referred to as pLPXt. pLPXt is modified by inserting the coding region of the a-factor prepro-leader into the Nco I site. By inserting the prepro- leader, the Nco I cloning site was retained at the 3 'end of the leader but not at the 5' end. In this way, the receptor can be cloned by digestion of the plasmid by Nco I and Xba I. The resulting plasmid is referred to as pMP15.
[504] The human A1 adenosine receptor cDNA inserted into pMP15 is referred to as p5095. In this vector, the receptor cDNA is fused to the 3 ' end of the yeast a-factor prepro- leader. During protein aging, the prepropeptide sequence is cleaved to yield a mature, full-length receptor. This occurs during processing of the receptor via the yeast secretory pathway. This plasmid is maintained with Leu selection (i.e., grown on leucine-free medium). The sequence of the cloned coding region was determined and found to be the same as in the published literature (GenBank accession numbers S45235 and S56143).
[505] 2. Yeast strain structure
[506] Yeast strain CY7967 was used as a starting parent strain in order to construct a yeast strain expressing the human A1 adenosine receptor. The genotype of CY7967 is as follows.
[507] MATα gpaD1163 gpal (41) gαi3 far lD1442 tbt-1 FUSI-HIS3 canl ste14 :: trp1 :: LYS2 ste3D1156 lys2 ura3 leu2 trp1 his3.
[508] Genetic markers are as follows:
[509] MATa : Mating a.
[510] gpalD1163 : Endogenous yeast G-protein GPA1 gene was deleted.
[511] gpal (41) Gai3 : gpal (41) -Gai3 was integrated into the yeast genome. The chimera Ga
[512] The protein is an endogenous yeast fused to the mammalian G-protein Gai3
[513] It consists of the first 41 amino acids of the subunit GPA1,
[514] The N-terminal amino acid was deleted.
[515] farlD1442 : the FAR1 gene (cell cycle arrest) was deleted (thereby causing pheromone
[516] Preventing cell cycle arrest during activation of the pathway)
[517] tbt-1 : a strain having high transformation efficiency by electroporation
[518] FUS1-HIS3 : fusion between FUS1 promoter and HIS3 coding region
[519] Pheromone-derived HIS3 gene).
[520] can 1 : arginine / cannabinin permease enzyme.
[521] ste14 :: trp1 :: LYS2 : STE14, gene disruption of C-farnesyl methyltransferase
[522] Basic signal degradation through the pheromone pathway).
[523] ste3D1156 : endogenous yeast STR, a factor pheromone receptor (STE3) disrupted.
[524] lys2 : defect in 2- aminocapidate reductase, yeast grows
[525] Lysine is needed.
[526] ura3 : Defects in orotidine -5'-phosphate decarboxylase, yeast
[527] We need uracil to grow.
[528] Defects in leu2 : b-isopropyl maleate dehydrogenase, yeast
[529] I need leucine to grow.
[530] trp1 : Defect in phospholybosilanthranilate, yeast grows
[531] Requires tryptophan.
[532] his3 : defect in imidazole glycerophosphate dehydrogenase,
[533] Yeast needs histidine to grow.
[534] Two plasmids were transformed by electroporation into strain CY7967: plasmid p5095 (coding for the human A1 adenosine receptor; see above) and the plasmid p1584 which is a FUS1-beta-galactosidase reporter gene plasmid. Plasmid p1584 was derived from the plasmid pRS426 (Christianson, T. W. et al. (1992) Gene 110: 119-1122). Plasmid pRS426 contains a polylinker at nucleotide 2004-2016. The fusion between the FUS1 promoter and the -Galactosidase gene was inserted at restriction sites EagI and XhoI to obtain plasmid p1584. The p1584 plasmid is maintained by Trp selection (i. e., grown on leucine-free medium).
[535] The resulting strain carrying p5095 and p1584, referred to as CY12660, expresses human A1 adenosine receptors. To grow this strain on a liquid or agar plate, a minimal medium without leucine and tryptophan was used. To perform the growth assay (FUS1-HIS3) on the plate, the plate pH was 6.8, containing 0.5-2.5 mM 3-amino-1,2,4-triazole and no leucine, tryptophan and histidine. In contrast to the specificity, comparisons with one or more different yeast groups and seven transmembrane receptor screens were included in all experiments.
[536] Structure of yeast strains expressing human A2a adenosine receptors
[537] In this example, the structure of a yeast strain expressing human A2a adenosine receptors functionally integrated into the yeast pheromone system pathway is described.
[538] 1. Expression vector structure
[539] To construct a yeast expression vector for the human A2a adenosine receptor, human A2a receptor cDNA was obtained from Dr. Phil Murphy (NIH). Upon acceptance of this clone, the A2a receptor insert was sequenced and found to be identical to the published sequence (GenBank Accession No. S46950). The receptor cDNA is deleted from the plasmid by the VENT polymerase and cloned into the plasmid pLPBX and allowed to express the receptor by the phosphoglycerate kinase (PGK) promoter, a component in yeast. The sequence of the entire insert was rearranged and found to be identical to the published sequence. However, there were three amino acids, GlySerVal, attached at the carboxy terminus of the receptor by the cloning method used.
[540] 2. Yeast strain structure
[541] In order to obtain a yeast strain expressing human A2a adenosine receptor, yeast strain CY8342 was used as the starting parent strain. The genotype of CY8342 is as follows:
[542] (Or gpalp-rGαsD229S or pgalp-rGαsE10K + D229S) in the same manner as MATαfar1D1442 tbt1-1 lys2 ura3 leu2 trpl his3 fus1-HIS3 can1 ste3D1156 pgaD1163 ste14 :: trp1 :: LYS2 gpa1p-
[543] The genetic markers are as described in Example 1 except for the G-protein mutation. Yeast strains were used for human A2a receptor expression and the endogenous yeast G protein GPA1 was deleted and replaced by G _{alpha s} of the mammal. Three rat G _S mutants were used. These variants include mutations of one or two parts that convert to a protein that efficiently binds to yeast beta gamma. These are identified as G _S E10K (replaced by glutamic acid lysine at position 10), G _S D229S (replaced by aspartic acid is serine at position 229), and G _S E10K + D229S (including mutations in the two parts).
[544] Strain CY8342 (3 of mutant rat G _αs proteins carrying one of a) the parent vector pLPBX (Receptor ^-) or were transformed with any one of pLPBX-A2a (Receptor ^+). A plasmid containing the FUS1 promoter fused to the -galactosidase coding sequence (described above) was added to evaluate the activation degree of the pheromone reaction pathway.
[545] Functional analysis using yeast strains expressing human A1 adenosine receptors
[546] In this example, the phenomenon of functional screening analysis in yeast for the regulator of the human A1 adenosine receptor is described.
[547] 1. The ligand used in the assay
[548] Was used to analyze two other synthetic agonists as well as adenosine, a natural agonist for this receptor. (-) - N6- (2-phenylisopropyl) -adenosine (PIA) with an EC ₅₀ of about 75 nM and adenosine with an affinity of about 50 nM were used in the experiments. 5'-N-ethylcarboxamido-adenosine (NECA) was used for all growth assays. Adenosine deaminase (4 U / ml) was added to all assays to prevent signaling due to the presence of adenosine in the growth medium.
[549] 2. Biological response in yeast
[550] The ability of the A1 adenosine receptor to functionally couple to the heterologous yeast system was evaluated by introducing an A1 receptor expression vector (p5095, supra) into a series of yeast strains expressing different G protein subunits. The majority of these transformants were expressing the G _αi or G _αo subtype of G _α subunit. Additional G _alpha proteins were tested for possible identification of mixed receptor-G _alpha protein coupling. In various strains, STE18 or chimeric STE18- G ₂ constructs were integrated into the yeast genome. Yeast strains protect the incomplete HIS3 gene and integrate a replica of FUS1-HIS3 into 3-amino-1,2,4-triazole (tested at 0.2, 0.5 and 1.0 mM) and screened in histidine-free selection medium Respectively. The transformants were separated and a monolayer was prepared on a medium containing 3-amino-1,2,4-triazole, 4 U / ml adenosine deaminase and no histidine. Five microliters of various concentrations of the ligand (e.g., NECA at 0, 0.1, 1.0, and 10 mM) were applied. Growth was monitored for 2 days. The ligand-dependent growth response was tested in this manner in various yeast strains. The results are summarized in Table 1 below. The symbol (-) indicates that no ligand-dependent receptor activity is detected and (+) indicates a ligand-dependent response. The term " LIRMA " refers to ligand-independent receptor mediated activity.
[551] Table 3
[552] Yeast strainG SubunitG SubunitStrain variantresult CY1316GPA1STE18 -GPA41-G _αi1 +GPA41-G _αi2 +GPA41-G _αi3 +GPA41-G _ai2- G _alphaOB LIRMAGPA41-G _αSE10K -GPA41-G _αSD229S - CY7967GPA41-G _αI3 Intergrated +++ CY2120GPA1STE18sst2Δ+GPA41-G _αil +GPA41-G _αi2 +GPA41-G _αi3 +GPA41-G _αi2 -Gα _OB LIRMAGPA41-G _αSE10K _GPA41-G _αSE229S _ CY9438GPA1STE18-G 2 _GPA41-G _αil +GPA41-G _αi3 +GPA41-G _αi3 +GPA41-G _αi2 -Gα _OB LIRMAGPA41-G _αSE10K _GPA41-G _αSE229S _ CY10560GPA1-integratedSTE18-G 2sst2Δ++
[553] As indicated in Table 3, the most robust signal occurs in yeast strains expressing GPA1 (41) -G [ _{alpha] i3} chimeras.
[554] III. fus1-LacZ assay
[555] To characterize the activity of the pheromone response pathway in more detail, the synthesis of -Galactosidase through fus1LacZ corresponding to agonist stimulation was measured. In order to carry out the -galactosidase analysis, the ligand was _added to the intermediate logarithmic growth medium solution of human A1-asynergic receptor expressed in a yeast strain expressing both Ste18-G 2 chimeric and GPA ₄₁ -G _3i . The transformants were isolated and incubated overnight with 4 U / ml of histidine and adenosine deaminase. After culturing for 5 hours with 4 U / ml of asindiaminase and a ligand, the induction of -Galactosidase was measured using CPRG as a -Galactosidase group. 5x10 &lt; ⁵ &gt; cells were used for each analysis.
[556] The results obtained by NECA stimulation indicate that the β-galactosidase activity is stimulated about twice as much at a concentration of 10 ^-8 M NECA. In addition, about 10-fold stimulation index was observed at 10 ^-5 M NECA concentration.
[557] The usefulness of this assay can be further explored by confirming the activity of the antagonist on these strains. Two known asynergic antibodies, XAC and DPCPX, were tested for their ability to be activated against NECA (at 5 mM) in the beta -galactosidase assay. In this assay, β-galactosidase induction was measured using 1.6 × 10 ⁵ cells per assay using FDG as a substrate. The results show that both XAC and DPCPX have IC ₅₀ values of 44 nM and 49 nM, respectively, and acted as potent antagonists of the yeast-expressing A1 asynergic receptor.
[558] To determine if this Henan inhibitory effect is specific to the A subtype, a series of adjuvant experiments were performed using yeast-based A2a receptor analysis (described in Example 4). Results from A2a yeast-based assays indicate that XAC is a relatively effective A2a receptor antagonist, consistent with published reports. At night, DPCPX was relatively inactive at these receptors, as expected from published reports.
[559] IV. Radioligand binding
[560] The radioligand binding parameter of the receptor was measured to further elucidate the A1 asynergic receptor assay. Using membranes prepared from yeast expressing the human A1 asynergic receptor, it was analyzed that [ ³ H] CPX was substituted by several acceptor reference compounds XAC, DPCPX and CGS. The binding specificity was investigated by comparing the results with yeast membranes assaying human A1 asynergic receptors to those from yeast membranes expressing human A2a or human A3 receptors. To perform this assay, 50 mg of membrane was incubated with 0.4 nM [ ³ H] CPX and increasing the ash receptor ligand concentration. The incubation was carried out at room temperature for 60 minutes using 50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 10 mM MgCl ₂ , 0.25% BSA and 2 U / ml ash diaminase in the presence of protease degradation. Binding was terminated by the addition of 10 mM MgCl ₂ to ice-cold 50 mM Tris-HCl, pH 7.4, and the protein was then separated on a GF / B filter prewetted with 0.5% polyethyleneimine using a Packard 96- And quickly filtered. Data was analyzed using a nonlinear least-squares fitting method using Prism 2.01 software. The IC ₅₀ values obtained in this experiment are summarized in Table 4 below.
[561] Table 4
[562] IC 50 [nM] compound hA1R hA2aR hA3R XAC6.611.753.1 DPCPX8.5326.41307.0 CGS-1594313.115.855.5 NECA215.5294.934.9 R-PIA67.6678.123.6 IB-MECA272.7859.43.1 Allozacin1072.01934.08216.0
[563] These data show that the reference compound has an affinity that matches that reported in the literature. This data also indicates that the yeast-based assay is sensitive enough to distinguish receptor subtype specificity.
[564] Functional analysis using yeast strains expressing human A2a asynergic receptor
[565] In this example, the development of a functional screening assay in yeast for the regulator of the human &lt; RTI ID = 0.0 &gt; A1 &lt; / RTI &gt;
[566] I. Ligands used in the assay
[567] The natural ligand as well as other fully characterized and commercially available ligands have been used for the study of human A2a receptors that are functionally expressed in yeast. Three ligands were used to run this assay.
[568] Ligand Reported Ki function
[569] Asin 500 n agonist
[570] 5'-N-ethylcarboxamidoacin (NECA) 10-15 nM agonist
[571] (-) - N6 (2-phenylisopropyl) -acin (PIA) 100-125 nM agonist
[572] Asymidine (4U / ml) was used in all assays to prevent signaling by asymmetry in the growth medium.
[573] II. Biological response in yeast
[574] A2a receptor agonists were transformed with A2a receptor expressing plasmids and analyzed for their ability to exclude the pheromone response pathway in yeast expressing G _S E10K, G _S D229S, or G _S E10K + D229S. A change in the yeast phenotype can lead to a ligand's ability to stimulate the pheromone response pathway in a receptor-dependent manner. Receptor activation alters the phenotype from the histidine auxotrophy to the histidine basal olfactory bulb (activation of fus1-HIS3). Three independent transcripts were isolated and incubated overnight in the presence of histidine. Cells were streaked to remove histidine and diluted to 2x10 ⁶ cells / ml. 5 [mu] l of each transformant was placed on non-selective medium (with histidine) or selective medium (1 mM AT) with or without 4 U / ml adenosine deaminase. Plates were incubated at 30 ° C for 24 hours. Under the presence of histidine, Receptor ⁺ (R ⁺⁾ and Receptor ^- (R ^-) strains were able to grow both. However, in the absence of histidine, only R ⁺ cells were grown. Two explanations for this result are possible as long as no ligand is added to this plate. One possible interpretation is that the receptor-bearing yeast was beneficial to growth by ligand independent receptor mediated activation (LIRMA). Alternatively, the yeast may have synthesized the ligand ash. To distinguish between these two possibilities, the enzyme digesting the ligand, asindiaminase (ADA), was added to the growth enzyme and plate. Under the presence of asymindiaminase, R ⁺ cells no longer grow under histidine deficiency, indicating that the enzyme was indeed synthesizing a ligand.
[575] This interpretation is confirmed by liquid phase A2a growth analysis. R ⁺ yeast in these experiments (G _αS E10K strain expressing Aea receptor) the Aisin deaminase exist three concentrations under (4 U / ml) or absence (1x10 ⁶ cells / ml; 3x10 ⁵ cells / ml; or 1x10 ⁵ cells / ml). (0, 0.1, 0.2 or 0.4 mM) of the protein product of 3-amino-1,24-triazole (AT), imidazole glycerol-P dihydratase, HIS3 gene . In the presence of the asymindiaminase and 3-amino-1,2,4-triazole, the yeast grew less vigorously. However, in the absence of 3-amino-1, 24-triazole, the asymindiaminase did not work. Thus, the asindiaminase itself does not directly affect the pheromone response pathway.
[576] An alternative method of measuring growth and which can be scaled down for high-level screening is the A2a receptor ligand spot assay. A2a receptor (A2aR ⁺⁾ or receptor expression (R ^-) the lack G _αS E10K strain under histidine and 4U / ml Aisin deaminase there were incubated overnight. Cells were washed to remove histidine and diluted to 5 x ¹⁰ cells / ml. 1x10 &lt; ⁶ &gt; cells were dispersed on a selective place containing 4 U / ml ash diaminase and 0.5 or 1.0 mM 3-amino-1,2,4-triazole (AT) and dried for 1 hour. 5 μl of the following reagents were applied to the monolayer: 10 mM asine, 38.7 mM histidine, dimethylsulfoxide (DMSO), 10 mM PIA or 10 mM NECA. The cells were grown at 30 ° C for 24 hours. The result is that cells lacking the receptor can only grow when histidine is added to the medium. On the other hand, R ⁺ cells only grew in the area where the A2a receptor ligand PIA and NECA were spotted. Since the plate contains an asymindiaminase, the growth factor at which the asymmetry is spotted confirms that the asymindiaminase is active.
[577] III. fus1LacZ analysis
[578] To quantify the activation of the yeast mating pathway, the synthesis of beta -galactosidase through fus1LacZ was measured. G _αS E10K, G _αS D229S or G _αS E10K + D229S a yeast strain expressing the human A2a receptor or plasmid encoding a receptor (R ^{+) (_} R) was transformed with a plasmid lacking. The transformants were isolated and grown overnight in the presence of histidine and 4 U / ml ash diaminase. 1x10 &lt; ⁷ &gt; cells were diluted to 1x10 &lt; ⁶ &gt; cells / ml and incubated for 4 hours with increasing concentration of NECA, and then the [beta] -galactosidase activity in the cells was measured. The results show that β-galactosidase activity is essentially absent in the R-strain, but as the concentration of NECA is increased, the β-galactosidase activity in the R ⁺ strain expressing G _αS E10K, G _αS D229S or G _αS E10K + D229S An increase in lactocidase activity is measured, which shows a dose-dependent increase in the measured units of -Galactosidase relative to exposure to increased ligand concentrations. This dose dependence is observed only in A2a receptor expressing cells. Moreover, the most powerful G _αS preparations for the A2a receptor was G _αS E10K. G _αS D229S preparation and the powerful G _αS preparations for the second A2a receptor, G _αS E10K + D229S, although G _αS E10K + D229S preparation is stimulated by the size of the extent to instantly detect β- galactosidase activity , The lowest among the three G _αS products tested.
[579] A more detailed description of the identified assay method can be found in U.S. Pat. No. 6,301,302, filed June 2, 1998 (Attorney Docket No. CPI-093), entitled " Functional Expression of Ascine Receptor in Yeast " See Application Serial No. 09/088985. The entire contents of which are incorporated herein by reference.
[580] Pharmacokinetic Characterization of Human Ascine Receptor Subtypes
[581] Materials and methods
[582] material. ^[3 H] -DPCPX [Cyclopentyl-1,3-deploy to pilkeu Santin, 8- [dipropyl ^{-2,3- 3 H (N)] (} 120.o Ci / mmol); [3 H] -CGS (Carboxyethyl- ³ H (N)] (30 Ci / mmol) and [ ¹²⁵ I] -AB-MECA ([ ¹²⁵ I] -4-aminobenzyl-5'N- 2,200 Ci / mmol) were purchased from New England Nuclear (Boston, Mass.). XAC (xanthine amine analog); NECA (5'-N-ethylcarboxydeoacin); And IB-MECA were purchased from Research Biochemicals International (RBI, Natick, Mass.). The asymindiaminase and the exchange protease inhibitor cocktail tablets were purchased from Boehringer Mannheim Corp. (Indianapolis, Ind.). HEK-293 cell-derived cell membrane stably expressing the heterosexamine 2a [RB-HA2a]; Each of the asine 2b [RB-HA2b] or asyn 3 [RB-HA3] receptor subtypes was purchased from Receptor Biology (Beltsville, Md.). Cell culture reagents were purchased from Life Technologies (Gand Island, NY) and single serum was purchased from Hyclone (Logan, UT).
[583] Yeast strain. Saccharide in my process three Levy Jia strains CY12660 [far1 * 1442 tbt1-1 fus1- HIS3 can1 ste14 :: trp1 :: LYS2 ste3 * 1156 gpa1 (41) -Gαi3 lys2 ura3 leu2 trp1: his3; LEU2 PGKp-Mfα1leader-hA1R- LYS2 ste3 * 1156 lys2 ura3 leu2trp1 his3; LEU2 PGKp-hA2aR2mu-ori REP3 Ampl] was prepared as described above Respectively.
[584] Yeast culture Transformed yeast was grown in Leu-Trp [LT] medium (pH 5.4) supplemented with 2% glucose. To prepare the cell membranes, 1-2x10 ⁶ cells / ml were inoculated from 30 ml culture medium overnight in 250 ml of LT medium and incubated at 30 ° C. under an inverted oxygen supply by spinning. After 16 hours of growth, cells were harvested by centrifugation and cell membranes were obtained as described below.
[585] Mammalian Tissue Culture HEK-293 cells stably expressing human asine 2a receptor subtype (Cadus clone No. 5) were cultured on a minimal medium (DMEM) supplemented with 10% calf serum and 1X penicillin / streptomycin The culture conditions were 5% CO ₂ atmosphere at 37 ° C under 500 mg / ml G418 antibiotic at a selected pressure.
[586] Yeast cell membrane preparation After overnight incubation, the 250 ml culture was centrifuged at 2,000xg speed on a Sorval RT6000 centrifuge to collect cells. Cells were washed in ice-cold water, centrifuged at 4 ° C, and the pellet was resuspended in ice-cold lysis buffer [5 mM Tris-HCl, pH 7.5; 1 ml) supplemented with protease degradation cocktail tablets (1 tablet per 25 ml buffer) 5 mM EDTA; And 5 mM EGTA]. Glass beads 917 g; Mesh 400-500; Sigma) was added to the suspension and the cells were vigorously vortexed at 4 ° C for 5 minutes to destroy the cells. The microbial cells were diluted with an additional 30 ml of lysis buffer and protease inhibitor and centrifuged at 3,000 xg for 5 minutes. Subsequently, the cell membrane was pelleted at 36,000 x g (Sorvall RC5B, extruded SS34 rotor) for 45 minutes. The resulting cell membrane pellet was resuspended in 5 ml cell membrane buffer [50 mM Tris-HCl, pH 7.5; 0.6 mM EDTA; And 5 mM MgCl ₂ ] and stored at -80 ° C for further experiments.
[587] Preparation of mammalian cell membrane HEK-293 cell membranes were prepared as previously described (Duzic E et al., J. Biol. Chem., 267, 9844-9851, 1992). In summary, cells were washed with PBS and collected with a rubber policeman. Cells were pelleted at 4O &lt; 0 &gt; C at a rate of 200 xg on a Sorvall RT6000 centrifuge. The pellet was resuspended in lysis buffer (5 mM Tris-HCL, pH 7.5; 5 mM EDTA; 5 mM EGTA; 0.1 mM phenylmethylsulfonyl fluoride, 10 mg / ml pepstatin A; and 10 mg / ml aprotinin) 5 ml / dish and homogenized in a Dounce homogenizer. Next, the cell lysate was centrifuged for 45 minutes in a 36,000 xg (Sorvall RC5B, type SS34 rotor) and resuspended in cell membrane buffer [50 mM Tris-HCl, pH 7.5; 0.6 mM EDTA; 5 mM MgCl _2; 0.1 mM phenylmethylsulfonyl fluoride, 10 mg / ml pepstatin A; And 10 mg / ml aprotinin) and stored at -80 ° C for further experiments.
[588] The total protein concentration in yeast and mammalian cell membranes was determined using a Bio-Rad protein assay kit based on the Bradford dye-binding method (Bradford, M .: Anal. Biochem. 72: 248 (1976)).
[589] Acin 1 receptor subtype saturation and competing radioligand binding
[590] Saturated and competitive binding was carried out on the cell membrane of yeast cells transformed with the human A1 receptor subtype using the antagonist [ ³ H] DPCPX as the radiolabeled ligand. Cell membranes were washed with binding buffer [50 mM Tris-HCl, pH 7.4; 10 mM MgCl _2; 1.0 mM EDTA; 0.25% BSA; 2 U / ml &lt; / RTI &gt; acindiaminase and 1 protease inhibitor cocktail tablet / containing 50 ml] at a concentration of 1.0 mg / ml. For saturating binding, cell membranes (50 μg / well) were incubated for 1 h at 25 ° C. in binding buffer with a final volume of 100 ml, such as [ ³ H] -DPCPX (1.0 nM), on a 96-well microtiter plate Cultured in the presence and absence of 10 μM unlabeled XAC or increasing concentrations of competing compounds.
[591] Competitive radioactive ligand binding
[592] Competition binding was performed on the cell membrane of HEK293 cells stably expressing the human A2a receptor subtype using the agonist [ ³ H] CGS-21680 as the radiolabeled ligand. Cell membranes were washed with binding buffer [50 mM Tris-HCl, pH 7.4; 10 mM MgCl _2; 1.0 mM EDTA; 0.25% BSA; 2 U / ml ash diaminase and 1 protease inhibitor cocktail tablet / containing 50 ml] at a concentration of 0.2 mg / ml. On a 96-well microtiter plate, cell membranes (10 μg / well) were incubated for 1 hour at 25 ° C. in binding buffer with a final volume of 100 ml, such as [ ³ H] -CGS-21680 Incubate in the presence and absence of 50 μM unlabeled NECA members or increasing the concentration of competing compounds.
[593] Asynine 3 receptor competitive radioligand binding
[594] Using agonist [ ¹²⁵ I] AB-MECA as radiolabeled ligand, competing binding on the cell membrane of HEK293 cells stably expressing human A3 receptor subtype was performed. Cell membranes were washed with binding buffer [50 mM Tris-HCl, pH 7.4; 10 mM MgCl _2; 1.0 mM EDTA; 0.25% BSA; 2 U / ml ash diaminase and 1 protease inhibitor cocktail tablet / containing 50 ml] at a concentration of 0.2 mg / ml. On a 96-well microtiter plate, cell membranes (10 μg / well) were incubated at 25 ° C. for 1 hour in binding buffer with a final volume of 100 μl, such as [ ¹²⁵ I] AB- MECA (0.75 nM) mu] m in the presence and absence of unlabeled IB-MECA members or with increasing concentrations of competing compounds.
[595] The end of the culture, 10 mM MgCl ₂ is added to an ice-cooled 50 mM Tris -HCl (pH 7.4) supplemented by, terminate the A1, A2a and A3 receptor subtypes radioligand binding assays, and Filtermate 196 cell harvester (Packard) (96-well GF / B UniFilters, Packard) previously wetted with 0.5% polyethyleneimine. The filter plates were dried, covered with 50 [mu] l / well scintillation fluid (MicroScint-20, Packard) and counted on a TopCount (Packard). The analysis was carried out three times. Nonspecific binding was 5.6 ± 0.5%, 10.8 ± 1.4% and 15.1 ± 2.6% of total binding in AiR, A2aR and A3R.
[596] Asynine 2b receptor subtype Competitive radioligand binding
[597] Using agonist [ ¹²⁵ I] DPCPX as a radiolabeled ligand, competition binding on the cell membrane of HEK293 cells stably expressing the human A2b receptor subtype was performed. Cell membranes were resuspended in binding buffer [10 mM Hepes-KOH, pH 7.4; 1.0 mM EDTA; 0.1 mM benzamidine and 2 U / ml acindiaminase] at a concentration of 0.3 mg / ml. On a 96-well microtiter plate, cell membranes (15 μg / well) were incubated for 1 hour at 25 ° C. in binding buffer with a final volume of 100 μl, such as [ ³ H] DPCPX (15 nM) Cultured in the presence and in the presence of unlabeled XAC or with increasing concentrations of competitive compounds. At the end of the incubation, the binding assay was terminated by adding ice-cold 10 mM Hepes-KOH (pH 7.4) and the cells were washed three times with a filter glass 196 cell harvester (Packard) Well GF / B UniFilters, Packard). The filter plates were dried, covered with 50 [mu] l / well scintillation fluid (MicroScint-20, Packard) and counted on a TopCount (Packard). The analysis was carried out three times. Nonspecific binding was 14.3 ± 2.3% of total binding.
[598] [&Lt; ³ &gt; H] DPCPX; The specific binding of [ ³ H] CGS-21680 and [ ¹²⁵ I] AB-MECA is defined as the difference between total binding and non-specific binding. The percent inhibition of compound was calculated for total binding. Competitor data was analyzed by applying a repetition curve to one site model, and K ₁ values were calculated from IC ₅₀ values using GraphPad Prizm 2.01 software. (Cheng and Prusof, Biochem. Pharmacol. 22, 3099-3109, 1973)
[599] result
[600] The primary function of any cell surface receptor is to recognize the appropriate ligand. Thus, we determined the ligand binding affinity and established the functional entity of the asis 1 receptor subtype expressed in yeast. The crude cell membrane prepared from Saccharomyces cerevisiae transformed with the human asexin 1 receptor subtype product had a K _D of 4.0 ± 0.19 nM and showed specific binding of [ ³ H] DPCP. The K _D and B _max values were calculated from saturated isotherms, and if the data were scatched, the binding sites were found to be a single group. The density of the asymptotic portion in the yeast cell membrane preparation was estimated as 716.8 ± 43.4 fmol / mg membrane protein.
[601] The pharmacokinetic properties of recombinant yeast cells transformed with the human A1 receptor subtype were compared with those of the human recombinant yeast cell line with the subtype selective ash ligand (XAC, DPCPX; CGS-15943; CDS-0646142; CDS-046123; NECA, -MECA and alloxazine). It competed with [ ₃ H] DPCPX in the expected ranking order. The displacement curves recorded using these compounds show a general slope for all ligands and data for each ligand could be modeled by single site pits. The apparent dissociation constants (Table 5) estimated for the individual compounds from the curve are consistent with the published values for the receptors from other sources.
[602] Table 5
[603] Ki values for cell membranes of yeast cells transformed with the human A1 receptor subtype
[604] LigandK _I (nM) XAC5.5 DPCPX7.1 CGS-159410.8 NECA179.6 (R) -PIA56.3 IB-MECA606.5 Allok Photo894.1 CDS-04614213.9 CDS-0461239.8
[605] Tables 6 to 12 show the efficacy and structural activity profile of deazapurine of the present invention. Tables 13 and 14 show that selectivity can be achieved by controlling the functionality on the deazapurine structure by eliminating the anhyangase receptor site. Table 14 also shows the surprising finding that the compounds set have sub-nanomolar activity and have a higher selectivity for the A _2b receptor when compared to the compounds in Table 13.
[606] Table 6
[607]
[608] Activity of CDS-046142 series: Effect of N ₆ -substituents
[609]
[610]
[611]
[612] Table 7
[613]
[614] Activity of CDS-046142 series: Effect of N ₂ - substituent
[615]
[616]
[617] Table 8
[618]
[619] Activity of CDS-046142 series: effect of pyrrole ring substituent
[620]
[621]
[622] Table 9
[623]
[624]
[625] Table 10
[626]
[627] Activity of CDS-046143 series: Effect of N ₆ -substituents
[628]
[629]
[630] Table 11
[631]
[632] Activity of CDS-046143 series: Effect of N ₆ -substituents
[633]
[634]
[635] Table 12
[636]
[637] The " retro-amide " analog of CDS-046123
[638]
[639]
[640] Table 13
[641] Profile of Selective Ascidia Antibody
[642]
[643]
[644]
[645]
[646] ¹ 2-thienyl-2-yl; ^{2 is} C- ₅ ; ³ water soluble; ⁴ R ₅ and R ₆ are hydrogen; ⁵ R ₃ is 3-fluorophenyl
[647] ⁶R₃Is 3-chlorophenyl;⁷R₃ Is-4-pyridyl;⁸% Active @ 10 μM
[648] Table 14
[649] Profiles of selectivity A _2b antagonists
[650]
[651] codeXR ₁ R ₂ The combined data K _i (nK) A ₁ A _2A A _2B A ₃CDS-129851-O-phMe41.7210.314.6 CDS-143995-O-ph (p) FMe33580.0118 CDS-143994-O-pH (p) ClMe8255910.360 CDS-143988-N-pyridin-2-oneMe60414748 CDS-143996-NH-phMe493110957
[652] Reference Merger
[653] All patents, published patent applications and other references mentioned herein are incorporated herein by reference.
[654] Equivalent
[655] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be included within the scope of the following claims.

权利要求:
Claims (186)
[1" claim-type="Currently amended] 6-substituted 7-tert-butyl-7-tert-butoxycarbonyl-N-6-substituted 7-deazapurine in a mammal, wherein a therapeutically effective amount of N-6 substituted 7-deazapurine is administered to the mammal, Methods for treating azafurine susceptibility states
[2" claim-type="Currently amended] 7. The method of claim 1, wherein the N-6 substituted 7-deazapurin responsive state is a disease state and the disease state is a disease mediated by adenosine.
[3" claim-type="Currently amended] 6. The method of claim 2, wherein the N-6 substituted 7-deazapurine is not N-6 benzyl or N-6 phenylethyl substituted.
[4" claim-type="Currently amended] The method according to claim 2, wherein the disease state is a central nervous system disease, a cardiovascular disease, a retinal disease, an inflammatory disease, an allergic disease, a gastrointestinal disease, an ophthalmic disease or a respiratory disease.
[5" claim-type="Currently amended] The method of claim 1, wherein the N-6 substituted 7-deazapurine has the formula 1.
(I)
In this formula,
R ₁ and R ₂ are each a hydrogen atom or a substituted or unsubstituted alkyl, aryl or alkylaryl moiety or form a substituted or unsubstituted heterocyclic ring;
R ₃ is a hydrogen atom or a substituted or unsubstituted alkyl, aryl or alkylaryl moiety;
R ₄ is a hydrogen atom or a substituted or unsubstituted alkyl, aryl or alkylaryl moiety;
R ₅ and R ₆ are independently a halogen atom, a hydrogen atom or a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety, or R ₄ and R ₅ or R ₅ and R ₆ together form a substituted or unsubstituted heterocycle To form a carbocyclic or heterocyclic ring.
[6" claim-type="Currently amended] A method of modulating an adenosine receptor in a mammal, wherein a therapeutically useful amount of N-6 substituted 7-deazapurine is administered to the mammal to modulate the activity of the adenosine receptor.
[7" claim-type="Currently amended] 7. The method of claim 6, wherein the adenosine receptor is A ₁ , A ₂ , A _2a, A _2b or A ₃ .
[8" claim-type="Currently amended] 7. The method of claim 6, wherein the adenosine receptor is associated with a central nervous system disease, cardiovascular disease, retinal disease, inflammatory disease, allergic disease, gastrointestinal disease, ophthalmic disease or respiratory disease.
[9" claim-type="Currently amended] 7. The method of claim 6, wherein the N-6 substituted 7-deazapurine has formula (I).
(I)
In this formula,
R ₁ and R ₂ are each a hydrogen atom or a substituted or unsubstituted alkyl, aryl or alkylaryl moiety or form a substituted or unsubstituted heterocyclic ring;
R ₃ is a hydrogen atom or a substituted or unsubstituted alkyl, aryl or alkylaryl moiety;
R ₄ is a hydrogen atom or a substituted or unsubstituted alkyl, aryl or alkylaryl moiety;
R ₅ and R ₆ are independently a halogen atom, a hydrogen atom or a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety, or R ₄ and R ₅ or R ₅ and R ₆ together form a substituted or unsubstituted heterocycle To form a carbocyclic or heterocyclic ring.
[10" claim-type="Currently amended] A method of treating asthma in a mammal, wherein a therapeutically effective amount of N-6 substituted 7-deazapurine is administered to the mammal to treat asthma in the mammal.
[11" claim-type="Currently amended] N-6 substituted 7-deazapurines having the formula (I) and pharmaceutically acceptable salts thereof
(I)
In this formula,
R ₁ and R ₂ are each a hydrogen atom or a substituted or unsubstituted alkyl, aryl or alkylaryl moiety or form a substituted or unsubstituted heterocyclic ring;
R ₃ is a hydrogen atom or a substituted or unsubstituted alkyl, aryl or alkylaryl moiety;
R ₄ is a hydrogen atom or a substituted or unsubstituted alkyl, aryl or alkylaryl moiety;
R ₅ and R ₆ are independently a halogen atom, a hydrogen atom or a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety, or R ₄ and R ₅ or R ₅ and R ₆ together form a substituted or unsubstituted heterocycle click or e, R ₄ to form a carbocyclic ring is a condition not a 1-phenylethyl.
[12" claim-type="Currently amended] 12. The method of claim 11,
R &lt; ₁ &gt; is hydrogen;
R ₂ is substituted or unsubstituted cycloalkyl, substituted or unsubstituted alkyl, or R ₁ and R ₂ together form a substituted or unsubstituted heterocyclic ring;
R ₃ is substituted or unsubstituted alkyl;
R ₄ is a hydrogen atom; And
R ₅ and R ₆ are independently a halogen atom or an alkyl.
[13" claim-type="Currently amended] 13. The deazapurine of claim 12, wherein R &lt; ₂ &gt; is substituted or unsubstituted cycloalkyl.
[14" claim-type="Currently amended] 14. The deazapurine of claim 13, wherein R ₁ and R ₄ are hydrogen, R ₃ is substituted or unsubstituted phenyl, and R ₅ and R ₆ are each alkyl.
[15" claim-type="Currently amended] 15. The deazapurine of claim 14 wherein R &lt; ₂ &gt; is substituted with at least one hydroxy group.
[16" claim-type="Currently amended] 16. The process of claim 15, wherein R &lt; _{2 &gt;} is mono-hydroxycyclopentyl.
[17" claim-type="Currently amended] 16. The process of claim 15, wherein R &lt; _{2 &gt;} is mono-hydroxycyclohexyl.
[18" claim-type="Currently amended] 15. The deazapurine of claim 14, wherein R ₂ is -NH-C (= O) E, wherein E is substituted or unsubstituted C ₁ -C ₄ alkyl.
[19" claim-type="Currently amended] 19. The deazapurine of claim 18, wherein E is an alkylamine.
[20" claim-type="Currently amended] 20. The deazapurine of claim 19, wherein E is ethylamine.
[21" claim-type="Currently amended] 13. The deazapurine of claim 12, wherein R ₁ and R ₂ together form a substituted or unsubstituted heterocyclic ring.
[22" claim-type="Currently amended] 22. The deazapurine of claim 21, wherein said heterocyclic ring is substituted with an amine.
[23" claim-type="Currently amended] 22. The compound of claim 21, wherein said heterocyclic ring is substituted with acetamido.
[24" claim-type="Currently amended] 25. The deazapurine of claim 24, wherein R ₁ and R ₄ are hydrogen, R ₃ is substituted or unsubstituted phenyl, and R ₅ and R ₆ are each alkyl.
[25" claim-type="Currently amended] 27. The deazapurine of claim 24, wherein R ₁ and R ₄ are hydrogen, R ₃ is substituted or unsubstituted phenyl, and R ₅ and R ₆ are each alkyl.
[26" claim-type="Currently amended] 26. The method of claim 25, wherein A is a purine-aza to characterized in that the CH ₂ CH _2.
[27" claim-type="Currently amended] 26. The method of claim 25, wherein A is a purine-aza to characterized in that the CH ₂ CH ₂ CH _2.
[28" claim-type="Currently amended] 26. The method of claim 25, wherein A is a purine-aza to characterized in that the _{CH 2 CH 2 CH 2 CH 2} .
[29" claim-type="Currently amended] 26. The deazapurine of claim 25, wherein B is methyl.
[30" claim-type="Currently amended] 26. The deazapurine of claim 25, wherein B is aminoalkyl.
[31" claim-type="Currently amended] 31. The deazapurine of claim 30, wherein B is aminomethyl.
[32" claim-type="Currently amended] 31. The deazapurine of claim 30, wherein B is aminoethyl.
[33" claim-type="Currently amended] 26. The deazapurine of claim 25, wherein B is alkylamino.
[34" claim-type="Currently amended] 34. The deazapurine of claim 33, wherein B is methylamino.
[35" claim-type="Currently amended] 34. The deazapurine of claim 33, wherein B is ethylamino.
[36" claim-type="Currently amended] 26. The deazapurine of claim 25, wherein B is substituted or unsubstituted cycloalkyl.
[37" claim-type="Currently amended] 37. The deazapurine of claim 36, wherein B is cyclopropyl.
[38" claim-type="Currently amended] 37. The deazapurine of claim 36, wherein B is 1-amino-cyclopropyl.
[39" claim-type="Currently amended] 13. The deazapurine of claim 12, wherein R &lt; ₃ &gt; is substituted or unsubstituted phenyl.
[40" claim-type="Currently amended] 40. The deazapurine of claim 39 wherein R &lt; ₅ &gt; and R &lt; ₆ &gt; are each alkyl.
[41" claim-type="Currently amended] 41. The deazapurine of claim 40, wherein R &lt; ₃ &gt; is unsubstituted phenyl.
[42" claim-type="Currently amended] 41. The deazapurine of claim 40, wherein R &lt; ₃ &gt; is substituted phenyl.
[43" claim-type="Currently amended] 43. The deazapurine of claim 42 wherein R &lt; ₃ &gt; is phenyl having at least one substituent.
[44" claim-type="Currently amended] 44. The deazapurine of claim 43, wherein R &lt; ₃ &gt; is o-, m- or p-chlorophenyl.
[45" claim-type="Currently amended] 44. The deazapurine of claim 43, wherein R &lt; ₃ &gt; is o-, m- or p-fluorophenyl.
[46" claim-type="Currently amended] 13. The deazapurine of claim 12, wherein R &lt; ₃ &gt; is substituted or unsubstituted heteroaryl.
[47" claim-type="Currently amended] 47. The deazapurine of claim 46 wherein R &lt; ₅ &gt; and R &lt; ₆ &gt; are each alkyl.
[48" claim-type="Currently amended] The method of claim 47, wherein R ₃ is pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, pyrrolyl, triazolyl, thiophenyl Oh thiazolyl, oxazolyl, oxadiazolyl, furanyl, methylenedioxyphenyl and thiophenyl &Lt; RTI ID = 0.0 &gt; deazapurine. &Lt; / RTI &gt;
[49" claim-type="Currently amended] 49. The deazapurine of claim 48, wherein R &lt; ₃ &gt; is 2-pyridyl, 3-pyridyl or 4-pyridyl.
[50" claim-type="Currently amended] 49. The deazapurine of claim 48, wherein R &lt; ₃ &gt; is 2-pyrimidyl or 3-pyrimidyl.
[51" claim-type="Currently amended] 13. The deazapurine of claim 12 wherein R &lt; ₅ &gt; and R &lt; ₆ &gt; are each hydrogen.
[52" claim-type="Currently amended] 13. The deazapurine of claim 12 wherein R &lt; ₅ &gt; and R &lt; ₆ &gt; are each methyl.
[53" claim-type="Currently amended] 13. The compound according to claim 12, characterized in that the compound is 4- (cis-3-hydroxycyclopentyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine Azapurine.
[54" claim-type="Currently amended] 13. A compound according to claim 12, wherein said compound is selected from the group consisting of 4- (cis-3- (2-aminoacetoxy) cyclopentyl) amino-5,6-dimethyl- Lt; / RTI &gt; is a fluoroacetic acid salt.
[55" claim-type="Currently amended] 13. The compound according to claim 12, wherein the compound is 4- (3-acetamino) piperidinyl-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine .
[56" claim-type="Currently amended] The compound according to claim 12, characterized in that the compound is 4- (2-N'-methyl urea propyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine Azapurine.
[57" claim-type="Currently amended] 13. The deazapurine of claim 12, wherein the compound is 4- (2-acetaminobutyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.
[58" claim-type="Currently amended] 14. The compound according to claim 13, characterized in that the compound is 4- (2-N'-methylureabutyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine Azapurine.
[59" claim-type="Currently amended] 13. The deazapurine of claim 12, wherein the compound is 4- (2-aminocyclopropylacetaminoethyl) amino-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.
[60" claim-type="Currently amended] 13. The compound according to claim 12, wherein the compound is 4- (trans-4-hydroxycyclohexyl) amino-2- (3- chlorophenyl) -7H- pyrrolo [2,3d] pyrimidine Purine.
[61" claim-type="Currently amended] The compound according to claim 12, characterized in that the compound is 4- (trans-4-hydroxycyclohexyl) amino-2- (3-fluorophenyl) -7H-pyrrolo [2,3d] pyrimidine Azapurine.
[62" claim-type="Currently amended] 13. The compound of claim 12, wherein the compound is 4- (trans-4-hydroxycyclohexyl) amino-2- (4-pyridyl) -7H-pyrrolo [2,3d] pyrimidine Purine.
[63" claim-type="Currently amended] Deazapurine with formula (II)
(II)
In this formula,
X is N or CR &lt; ₆ &gt;;
R ₁ and R ₂ are each independently hydrogen or a substituted or unsubstituted alkoxy, aminoalkyl, alkyl, aryl, or alkylaryl, or, provided that R ₁ and R ₂ are not both hydrogen, Form a click ring;
R &lt; ₃ &gt; is substituted or unsubstituted alkyl, arylalkyl or aryl;
R ₄ is hydrogen or substituted or unsubstituted C ₁ -C ₆ alkyl;
L is hydrogen, substituted or unsubstituted alkyl or R ₄ and L form a substituted or unsubstituted heterocyclic or carbocyclic ring with each other;
R &lt; ₆ &gt; is hydrogen, substituted or unsubstituted alkyl or halogen;
Q is CH ₂ , O, S or NR ₇ wherein R ₇ is hydrogen or substituted or unsubstituted C ₁ -C ₆ alkyl; And
W is unsubstituted or substituted alkyl, cycloalkyl, aryl, arylalkyl, biaryl, heteroaryl, substituted carbonyl, substituted thiocarbonyl or substituted sulfonyl,
In the above, when R ₃ is pyrrolidino, the condition that R ₄ is not methyl is satisfied.
[64" claim-type="Currently amended] Deazapurine (58) having the formula (III)
(III)
Wherein Q is CH ₂ , O, S or NH.
[65" claim-type="Currently amended] The method of claim 64, wherein R ₄ is hydrogen, L is hydrogen or methyl, R ₃ is a purine-aza to characterized in that the substituted or unsubstituted aryl group.
[66" claim-type="Currently amended] 66. The deazapurine of claim 65, wherein W is substituted or unsubstituted aryl, 5- or 6-membered heteroaryl or biaryl.
[67" claim-type="Currently amended] 67. The method of claim 66, wherein, W is halogen, hydroxy, alkoxy, amino, aminoalkyl, amino, _{carboxamide, CN, CF 3, CO 2} R 8, CONHR 8, CONR 8 R 9, SOR 8, SO 2 R 8, And SO ₂ NR ₈ R ₉ , wherein R ₈ and R ₉ are each independently hydrogen or substituted or unsubstituted alkyl, cycloalkyl, aryl, or arylalkyl, optionally substituted with one or more substituents selected from the group consisting of Featuring deaza purine.
[68" claim-type="Currently amended] 66. The deazapurine of claim 66, wherein W is methylene dioxyphenyl.
[69" claim-type="Currently amended] 67. The deazapurine of claim 66, wherein W is substituted or unsubstituted phenyl.
[70" claim-type="Currently amended] 66. The deazapurine of claim 66, wherein W is a substituted or unsubstituted 5-membered heteroaryl ring.
[71" claim-type="Currently amended] 66. The compound of claim 66, wherein W is pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, pyrrolyl, triazolyl, thiazolyl, oxazolyl, oxadiazolyl, pyrazolyl, furanyl, Lt; RTI ID = 0.0 &gt; deazapurine. &Lt; / RTI &gt;
[72" claim-type="Currently amended] 74. The desazapurine of claim 71, wherein Q is NH and W is a 3-pyrazole ring that is unsubstituted or N-substituted by substituted or unsubstituted alkyl, cycloalkyl, aryl, or arylalkyl.
[73" claim-type="Currently amended] 74. The compound of claim 71, wherein Q is oxygen and W is a 2-thiazole ring unsubstituted or substituted by unsubstituted alkyl, cycloalkyl, aryl, or arylalkyl.
[74" claim-type="Currently amended] 66. The deazapurine of claim 66, wherein W is a 6-membered heteroaryl ring.
[75" claim-type="Currently amended] 74. The desazapurine of claim 74, wherein W is selected from the group consisting of 2-pyridyl, 3-pyridyl, and 4-pyridyl.
[76" claim-type="Currently amended] 74. The desazapurine of claim 74, wherein W is selected from the group consisting of 2-pyridyl, 3-pyridyl, and 5-pyridyl.
[77" claim-type="Currently amended] 66. The deazapurine of claim 65, wherein W is substituted or unsubstituted alkyl, cycloalkyl, alkynyl or arylalkyl.
[78" claim-type="Currently amended] 78. The desazapurine of claim 77, wherein W is alkynyl.
[79" claim-type="Currently amended] The method of claim 78, wherein, W is substituted with one or more substituents selected from halogen, hydroxy, substituted or unsubstituted alkyl, a seek roll alkyl, aryl, arylalkyl or NHR group consisting of _10, R ₁₀ is hydrogen or a substituted Or unsubstituted alkyl, cycloalkyl, aryl or arylalkyl.
[80" claim-type="Currently amended] 77. The desazapurine of claim 77, wherein W is substituted or unsubstituted cyclopentyl.
[81" claim-type="Currently amended] 66. The compound of claim 65, wherein W is - (CH ₂ ) _a -C (= O) Y or - (CH ₂ ) _a -C (= S) Y wherein a is an integer from 0 to 3, alkyl, arylalkyl, cycloalkyl, heteroalkyl, alkynyl, NHR or ₁₁ R _12, in which Q is NH conditions, as oR _13, where R _11, R ₁₂ and R ₁₃ is hydrogen or a substituted or unsubstituted, each independently Alkyl, aryl, arylalkyl, or cycloalkyl.
[82" claim-type="Currently amended] 83. The deazapurine of claim &lt; RTI ID = 0.0 &gt; 81, &lt; / RTI &gt;
[83" claim-type="Currently amended] 83. The deazapurine of claim 81, wherein Y is a 5- or 6-membered heteroaryl ring.
[84" claim-type="Currently amended] 66. The method of claim 65, wherein W is - (CH ₂ ) _b -S (= O) _j Y wherein j is 1 or 2, b is 0, 1, 2, or 3, Q is CH ₂ where R ₁₄ , R ₁₅ and R ₁₆ are each independently hydrogen or an optionally substituted group selected from the group consisting of hydrogen, alkyl, cycloalkyl, cycloalkyl, alkynyl, heteroaryl, or NHR ₁₄ R ₁₅ ; Substituted or unsubstituted alkyl, aryl, arylalkyl or cycloalkyl.
[85" claim-type="Currently amended] The method of claim 64 wherein, R ₃ is a substituted or unsubstituted phenyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, pyrrolyl, triazolyl, thiophenyl Oh thiazolyl, oxazolyl, oxadiazolyl, pyrazolyl, furanyl &Lt; / RTI &gt; methylene dioxyphenyl, and thiophenyl.
[86" claim-type="Currently amended] 88. The desazapurine of claim 85, wherein R &lt; ₃ &gt; is unsubstituted phenyl.
[87" claim-type="Currently amended] The method of claim 85 wherein, R ₃ is wherein the phenyl has at least one substituent.
[88" claim-type="Currently amended] 87. The desazapurine of claim 87, wherein said substituent is selected from the group consisting of hydroxyl, alkoxy, alkyl and halogen.
[89" claim-type="Currently amended] 90. The deazapurine of claim 88, wherein said substituent is halogen.
[90" claim-type="Currently amended] 90. The desazapurine of claim 89, wherein R &lt; ₃ &gt; is o-, m-, or p-fluorophenyl.
[91" claim-type="Currently amended] The desazapurine of claim 89, wherein R ₃ is o-, m-, or p-chlorophenyl.
[92" claim-type="Currently amended] 88. The deazapurine of claim 88, wherein R &lt; ₃ &gt; is an alkyl substituted phenyl.
[93" claim-type="Currently amended] 93. The deazapurine of claim 92, wherein R &lt; ₃ &gt; is tolyl.
[94" claim-type="Currently amended] 88. The deazapurine of claim 88, wherein R &lt; ₃ &gt; is alkoxy substituted phenyl.
[95" claim-type="Currently amended] 95. The desazapurine of claim 94, wherein R &lt; ₃ &gt; is methoxyphenyl.
[96" claim-type="Currently amended] The method of claim 85 wherein, R ₃ is a purine-aza used, characterized in that 2-, 3- or 4- pyridyl.
[97" claim-type="Currently amended] 88. The deazapurine of claim 85, wherein R &lt; ₃ &gt; is 2- or 3-pyrimidyl.
[98" claim-type="Currently amended] The method of claim 64, wherein, R ₆ is a purine having aza, characterized in that hydrogen or C ₁ -C ₃ alkyl.
[99" claim-type="Currently amended] 98. The use of claim 98, wherein R &lt; ₆ &gt; is hydrogen.
[100" claim-type="Currently amended] The method of claim 64, wherein R ₁ is hydrogen and R ₂ is substituted or unsubstituted alkyl or alkoxy, substituted or unsubstituted alkylamine, arylamine, or alkylarylamine, substituted or unsubstituted aminoalkyl, Substituted or unsubstituted alkylene, aryl, substituted or unsubstituted alkylamide, arylamide or alkylarylamide, substituted or unsubstituted alkylsulfonamide, arylsulfonamide or alkylarylsulfonamide, substituted or unsubstituted alkylurea, arylurea or alkylarylurea, Substituted alkylcarbamates, arylcarbamates or alkylarylcarbamates, or substituted or unsubstituted alkylcarboxylic acids, allylic carboxylic acids or alkylcarboxylic acids.
[101" claim-type="Currently amended] The method of claim 100, wherein, R ₂ is a purine-aza having a substituted or unsubstituted cycloalkyl.
[102" claim-type="Currently amended] 102. The deazapurine of claim 101, wherein R &lt; _{2 &gt;} is mono- or dihydroxy-substituted cyclohexyl.
[103" claim-type="Currently amended] 103. The deazapurine of claim 102, wherein R &lt; _{2 &gt;} is monohydroxy-substituted cyclohexyl.
[104" claim-type="Currently amended] 102. The deazapurine of claim 101, wherein R &lt; _{2 &gt;} is mono- or dihydroxy-substituted cyclopentyl.
[105" claim-type="Currently amended] 104. The desazapurine of claim 104, wherein R &lt; _{2 &gt;} is monohydroxy-substituted cyclopentyl.
[106" claim-type="Currently amended] 112. The compound of claim 100, wherein R &lt; ₂ &gt; ego,
In this formula,
A is selected from the group consisting of C ₁ -C ₆ alkyl, C ₃ -C ₇ cycloalkyl, a chain of one to seven atoms, or a ring of three to seven atoms, optionally substituted with C ₁ -C ₆ alkyl, halogens, Carboxyl, thio or amino group;
B is selected from the group consisting of methyl, N (Me) 2, N (Et) 2, NHMe, NHMe, NHEt, (CH2) rNH3 +, NH (CH2) rCH3, (CH2) rNH2, (CH2) rCHCH3NH2, wherein r is an integer from 0 to 2, m is 1 or 2, R ₁₈ is alkyl, and R ₁₉ is NH ₃ ⁺ or CO ₂ (CH 2) H or R &lt; ₁₈ &gt; and R &lt; ₁₉ & , Wherein p is 2 or 3; And
R ₁₇ is selected from the group consisting of C ₁ -C ₆ alkyl, C ₃ -C ₇ cycloalkyl, a chain of one to seven atoms, or a _three- to seven-membered ring, optionally substituted with C ₁ -C ₆ alkyl, halogens, , Carboxyl, thio, or amino group.
[107" claim-type="Currently amended] The method of claim 106, wherein, A is a purine having aza, characterized in that the substituted or unsubstituted C ₁ -C ₆ alkyl.
[108" claim-type="Currently amended] The method of claim 106, wherein, B is a purine-aza used, characterized in that the substituted or unsubstituted C ₁ -C ₆ alkyl.
[109" claim-type="Currently amended] The method of claim 106, wherein, R ₂ is-aza purine de, characterized in that -A-NHC (= O) in B.
[110" claim-type="Currently amended] The method of claim 109, wherein, A is -CH ₂ CH ₂ - aza purine to as B and is characterized in that methyl.
[111" claim-type="Currently amended] 11. A deazapurine according to claim 11, 12, 65 or 66, characterized in that it comprises a water-soluble prodrug which is metabolized in vivo to an active drug.
[112" claim-type="Currently amended] 111. The dezapurine of claim 111, wherein the prodrug is metabolized in vivo by esterase catalytic hydrolysis.
[113" claim-type="Currently amended] The method of claim 111, wherein, R ₂ is -OC (O) (Z) as a cycloalkyl substituted with NH _2, where, Z is an amino acid or an analog of the naturally occurring or unnatural, α, β, γ, or ω amino acids , Or a side chain of a dipeptide.
[114" claim-type="Currently amended] Wherein Z is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, lysine, alpha -methylalanine, aminocyclopropanecarboxylic acid, azetidine- Aminobutyric acid, alanine-alanine, or glycine-alanine.
[115" claim-type="Currently amended] The method of claim 64 wherein, R ₁ and R ₂ are together Wherein n is 1 or 2 and wherein the ring is optionally substituted with one or more hydroxyl, amino, thiol, carboxyl, halogen, CH ₂ OH, CH ₂ NHC (═O) alkyl or CH ₂ NHC Lt; RTI ID = 0.0 &gt; = NH) &lt; / RTI &gt; alkyl group.
[116" claim-type="Currently amended] 116. The desazapurine of claim 115, wherein n is 1 or 2 and said ring is substituted with -NHC (+ O) alkyl.
[117" claim-type="Currently amended] The method of claim 64 wherein, R ₁ is hydrogen, R ₂ is a substituted or an unsubstituted C ₁ -C ₆ alkyl, R ₃ is a substituted or unsubstituted phenyl, R ₄ is hydrogen, L is hydrogen, substituted or unsubstituted and substituted C ₁ -C ₆ alkyl, Q is O, an S, or NR ₇ where R ₇ is hydrogen or a substituted or unsubstituted C ₁ -C ₆ alkyl; And W is an unsubstituted or substituted aryl.
[118" claim-type="Currently amended] 117. The desazapurine of claim 117, wherein R ₂ is -A-NHC (= O) B, wherein A and B are independently unsubstituted C ₁ -C ₄ alkyl.
[119" claim-type="Currently amended] The method of claim 118, wherein, A is-aza purine to characterized in that the CH ₂ CH _2.
[120" claim-type="Currently amended] 119. The deazapurine of claim 118, wherein B is methyl.
[121" claim-type="Currently amended] 119. The deazapurine of claim 118, wherein B is aminoalkyl.
[122" claim-type="Currently amended] 121. The deazapurine of claim 121, wherein B is aminomethyl.
[123" claim-type="Currently amended] 118. The deazapurine of claim 117, wherein R &lt; ₃ &gt; is unsubstituted phenyl.
[124" claim-type="Currently amended] 117. The desazapurine of claim 117, wherein L is hydrogen.
[125" claim-type="Currently amended] 118. The deazapurine of claim 117, wherein R &lt; ₆ &gt; is hydrogen or methyl.
[126" claim-type="Currently amended] 128. The desazapurine of claim 125, wherein R &lt; ₆ &gt; is hydrogen.
[127" claim-type="Currently amended] 117. The desazapurine of claim 117, wherein Q is O.
[128" claim-type="Currently amended] 116. The desazurin according to claim 117, wherein Q is S.
[129" claim-type="Currently amended] The method of claim 117, wherein Q a is NR ₇ wherein R ₇ is-aza purine de, characterized in that hydrogen or a substituted or unsubstituted C ₁ -C ₆ alkyl.
[130" claim-type="Currently amended] 127. The desazurin according to claim 129, wherein R &lt; ₇ &gt; is hydrogen.
[131" claim-type="Currently amended] 129. The desazapurine of claim 129, wherein R &lt; ₇ &gt; is methyl.
[132" claim-type="Currently amended] 118. The deazapurine of claim 117, wherein W is unsubstituted phenyl.
[133" claim-type="Currently amended] 118. The deazapurine of claim 117, wherein W is phenyl having at least one substituent.
[134" claim-type="Currently amended] 133. The desazapurine of claim 133, wherein said substituent is halogen.
[135" claim-type="Currently amended] 134. The desazapurine of claim 134, wherein W is p-fluorophenyl.
[136" claim-type="Currently amended] 138. The desazapurine of claim 134, wherein W is p-chlorophenyl.
[137" claim-type="Currently amended] 133. The deazapurine of claim 133, wherein said substituent is alkoxy.
[138" claim-type="Currently amended] 138. The desazapurine of claim 137, wherein W is p-methoxy.
[139" claim-type="Currently amended] 117. The desazapurine of claim 117, wherein W is heteroaryl.
[140" claim-type="Currently amended] 140. The desazapurine of claim 139, wherein W is 2-pyridyl.
[141" claim-type="Currently amended] 118. The method of claim 117, wherein the deazapurine is 4- (2-acetylaminoethylamino-6-phenoxymethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine .
[142" claim-type="Currently amended] 118. The compound of claim 117, wherein the deazapurine is 4- (2-acetylaminoethyl) amino-6- (4-fluorophenoxy) methyl-2- phenyl-7H-pyrrolo [2,3d] pyrimidine Characterized in that the deazapurine.
[143" claim-type="Currently amended] The method of claim 117, wherein the deazapurine is 4- (2-acetylaminoethyl) amino-6- (4-chlorophenoxy) methyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine Featuring deaza purine.
[144" claim-type="Currently amended] 118. The method of claim 117, wherein the deazapurine is 4- (2-acetylaminoethyl) amino-6- (4-methoxyphenoxy) methyl-2-phenyl-7H- pyrrolo [2,3d] pyrimidine Deazapurine.
[145" claim-type="Currently amended] Wherein the deazapurine is 4- (2-acetylaminoethyl) amino-6- (2-pyridyloxy) methyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine Azapurine.
[146" claim-type="Currently amended] Wherein the deazapurine is selected from the group consisting of 4- (2-acetylaminoethyl) amino 6- (N-phenylamino) methyl-2-phenyl-7H- pyrrolo [2,3d] pyrimidine, .
[147" claim-type="Currently amended] 117. The method of claim 117, wherein the deazapurine is selected from the group consisting of 4- (2-acetylaminoethyl) amino-6- (N-methyl-N-phenylamino) methyl- Middin Indaazapurine.
[148" claim-type="Currently amended] 117. The method of claim 117, wherein said deazapurine is selected from the group consisting of 4- (2-N'-methylureaethyl) amino-6-phenoxymethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine, .
[149" claim-type="Currently amended] A method of inhibiting the activity of an adenosine receptor in a cell, said method comprising contacting said cell with deazapurine of claim 11, 12, 14, 25, 63 or 65.
[150" claim-type="Currently amended] 155. The method of claim 149, wherein the deazapurine is selected from the group consisting of 4- (2-acetylaminoethylamino-6-phenoxymethyl-2-phenyl-7H- pyrrolo [2,3d] pyrimidine, 4- Ethyl) amino-6- (4-fluorophenoxy) methyl-2-phenyl-7H-pyrrolo [2,3- d] pyrimidine, 4- (4-methoxyphenoxy) methyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine, Pyrrolo [2,3d] pyrimidine, 4- (2-pyridyloxy) methyl-2-phenyl- 2-phenyl-7H-pyrrolo [2,3-d] pyrimidine, 4- (2-acetylaminoethyl) amino-6- (N-methyl Phenyl-7H-pyrrolo [2,3d] pyrimidine, 4- (2-N'-methylureaethyl) amino-6-phenoxymethyl- Pyrrolo [2,3d] pyrimidine, 4- (cis-3-hydroxycyclopentyl) amino-5,6-dimethyl- 2-phenyl-7H-pyrrolo [2,3-d] pyrimidin- Pyrimidine trifluoroacetic acid salt, 4- (3-acetamino) piperidinyl-5,6-dimethyl-2-phenyl-7H- pyrrolo [2,3- Methyl-2-phenyl-7H-pyrrolo [2,3-d] pyrimidine, 4- (2-acetaminobutyl) amino-5,6- Dimethyl-2-phenyl-7H-pyrrolo [2,3-d] pyrimidine, 4- (2-pyridinylmethyl) - (2-aminocyclopropylacetaminoethyl) amino-2-phenyl-7H-pyrrolo [2,3d] pyrimidine, 4- (trans-4- hydroxycyclohexyl) ) -7H-pyrrolo [2,3d] pyrimidine, and deazapurine was obtained in the same manner as in 4- (trans-4-hydroxycyclohexyl) amino- ] Pyrimidine, and 4- (trans-4-hydroxycyclohexyl) amino-2- (4-pyridyl) -7 H-pyrrolo [2,3d] pyrimidine. &Lt; / RTI &gt;
[151" claim-type="Currently amended] 153. The method of claim 149, wherein the adenosine receptor is an A &_lt; 2 &gt; adenosine receptor.
[152" claim-type="Currently amended] According to claim 151, wherein the de-aza purine is characterized in that the A _2b adenosine receptor antagonist of the chain.
[153" claim-type="Currently amended] 155. The method of claim 149, wherein the adenosine receptor is an A &lt; ₃ &gt; adenosine receptor.
[154" claim-type="Currently amended] The method of claim 153, wherein wherein the antagonist chain of the N-6 substituted 7-aza-purine is to the A ₃ adenosine receptor.
[155" claim-type="Currently amended] A method of treating gastrointestinal disease in a mammal, comprising administering to the mammal an effective amount of dezapurine of claim 63 or 65.
[156" claim-type="Currently amended] 155. The method of claim 155, wherein the deazapurine is selected from the group consisting of 4- (2-acetylaminoethylamino-6-phenoxymethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine, 4- Ethyl) amino-6- (4-fluorophenoxy) methyl-2-phenyl-7H-pyrrolo [2,3- d] pyrimidine, 4- (4-methoxyphenoxy) methyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine, Pyrrolo [2,3d] pyrimidine, 4- (2-pyridyloxy) methyl-2-phenyl- 2-phenyl-7H-pyrrolo [2,3-d] pyrimidine, 4- (2-acetylaminoethyl) amino-6- (N-methyl Phenyl-7H-pyrrolo [2,3d] pyrimidine and 4- (2-N'-methylureaethyl) amino-6-phenoxymethyl- Pyrrolo [2,3d] pyrimidine. &Lt; / RTI &gt;
[157" claim-type="Currently amended] 155. The method of claim 155, wherein the disease is diarrhea.
[158" claim-type="Currently amended] The method of claim 155, wherein the animal is a human.
[159" claim-type="Currently amended] The method of claim 155, wherein the de-aza purine is characterized in that antagonistic chains of A _2b adenosine receptor in the cells of the animal.
[160" claim-type="Currently amended] A method of treating respiratory disease in an animal, comprising administering to the animal an effective amount of dezapurine of claim 63 or 64.
[161" claim-type="Currently amended] 155. The method of claim 160, wherein the deazapurine is selected from the group consisting of 4- (2-acetylaminoethylamino-6-phenoxymethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine, 4- Ethyl) amino-6- (4-fluorophenoxy) methyl-2-phenyl-7H-pyrrolo [2,3- d] pyrimidine, 4- (4-methoxyphenoxy) methyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine, Pyrrolo [2,3d] pyrimidine, 4- (2-pyridyloxy) methyl-2-phenyl- 2-phenyl-7H-pyrrolo [2,3-d] pyrimidine, 4- (2-acetylaminoethyl) amino-6- (N-methyl Phenyl-7H-pyrrolo [2,3d] pyrimidine and 4- (2-N'-methylureaethyl) amino-6-phenoxymethyl- Pyrrolo [2,3d] pyrimidine. &Lt; / RTI &gt;
[162" claim-type="Currently amended] 160. The method of claim 160, wherein the disease is asthma, chronic obstructive pulmonary disease, allergic rhinitis, or upper respiratory disease.
[163" claim-type="Currently amended] 160. The method of claim 160, wherein the animal is a human.
[164" claim-type="Currently amended] The method of claim 160, wherein the de-aza purine is characterized in that antagonistic chains of A _2b adenosine receptor in the animal cell.
[165" claim-type="Currently amended] A method of treating an N-6 substituted 7-deazapurin responsive state in an animal comprising administering to the animal a therapeutically effective amount of deazapurine of claim 11, 12, 14, 25, 63 or 64, &Lt; / RTI &gt; wherein the N-6 substituted 7-deazapurine responsive condition treatment occurs.
[166" claim-type="Currently amended] 165. The method of claim 165, wherein the N-6 substituted 7-deazapurine responsive state is a disease state, which is an adenosine mediated disease.
[167" claim-type="Currently amended] 166. The method according to claim 166, wherein the disease state is a central nervous system disease, a cardiovascular disease, a retinal disease, an inflammatory disease, an allergic disease, a gastrointestinal disease or a respiratory disease.
[168" claim-type="Currently amended] A method for treating an animal-borne injury, comprising administering to the animal an effective amount of N-6 substituted 7-deazapurine of claim 11, 12, 14 or 25.
[169" claim-type="Currently amended] 178. The compound of claim 168, wherein the N-6 substituted 7-deazapurine is 4- (cis-3-hydroxycyclopentyl) amino-5,6-dimethyl- Pyrrolo [2,3d] pyrimidine trifluoroacetic acid salt, 4 (4-cyanophenoxy) cyclopentyl) amino-5,6-dimethyl- (2-N'-methyl urea propyl) amino-5,6-dihydro-7H-pyrrolo [2,3-c] pyrimidine Dimethyl-2-phenyl-7H-pyrrolo [2,3-d] pyrimidine, Methyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine, 4- (2-aminocyclopropylaminoethyl) (3-chlorophenyl) -7H-pyrrolo [2,3-d] pyrimidine, Pyrimidine, deazapurine is 4- (trans-4-hydroxycyclohexyl) amino (4-pyridyl) -7H-pyrrolo [2,3d] pyrimidine, and 4- (trans-4-hydroxycyclohexyl) amino- Pyrrolo [2,3d] pyrimidine. &Lt; / RTI &gt;
[170" claim-type="Currently amended] 173. The method of claim 168, wherein the injury is a retinal or optic nerve injury.
[171" claim-type="Currently amended] 168. The method of claim 168, wherein the injury is acute or chronic.
[172" claim-type="Currently amended] 174. The method of claim 168, wherein the injury is a result of glaucoma, edema, ischemia, hypoxia or scarring.
[173" claim-type="Currently amended] 173. The method of claim 168, wherein the animal is a human.
[174" claim-type="Currently amended] The method of claim 168, wherein said N-6 substituted 7-to-aza purine is characterized in that antagonistic chains of A ₃ adenosine receptors in cells of the animal.
[175" claim-type="Currently amended] 14. A pharmaceutical composition comprising a therapeutically effective amount of deazapurine of claim 11, 12, 14, 25, 63 or 64 and a pharmaceutically acceptable carrier.
[176" claim-type="Currently amended] 178. The method of claim 175, wherein the deazapurine is selected from the group consisting of 4- (2-acetylaminoethylamino-6-phenoxymethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine, 4- Ethyl) amino-6- (4-fluorophenoxy) methyl-2-phenyl-7H-pyrrolo [2,3- d] pyrimidine, 4- (4-methoxyphenoxy) methyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine, Pyrrolo [2,3d] pyrimidine, 4- (2-pyridyloxy) methyl-2-phenyl- 2-phenyl-7H-pyrrolo [2,3-d] pyrimidine, 4- (2-acetylaminoethyl) amino-6- (N-methyl Phenyl-7H-pyrrolo [2,3d] pyrimidine, 4- (2-N'-methylureaethyl) amino-6-phenoxymethyl- Pyrrolo [2,3d] pyrimidine, 4- (cis-3-hydroxycyclopentyl) amino-5,6-dimethyl- 2-phenyl-7H-pyrrolo [2,3-d] pyrimidin- Pyrimidine trifluoroacetic acid salt, 4- (3-acetamino) piperidinyl-5,6-dimethyl-2-phenyl-7H- pyrrolo [2,3- Methyl-2-phenyl-7H-pyrrolo [2,3-d] pyrimidine, 4- (2-acetaminobutyl) amino-5,6- Dimethyl-2-phenyl-7H-pyrrolo [2,3-d] pyrimidine, 4- (2-pyridinylmethyl) - (2-aminocyclopropylacetaminoethyl) amino-2-phenyl-7H-pyrrolo [2,3d] pyrimidine, 4- (trans-4- hydroxycyclohexyl) ) -7H-pyrrolo [2,3d] pyrimidine, and deazapurine was obtained in the same manner as in 4- (trans-4-hydroxycyclohexyl) amino- ] Pyrimidine, and 4- (trans-4-hydroxycyclohexyl) amino-2- (4-pyridyl) -7 H-pyrrolo [2,3d] pyrimidine. &Lt; / RTI &gt;
[177" claim-type="Currently amended] 198. The pharmaceutical composition of claim 175, wherein said therapeutically effective amount is effective in treating respiratory or gastrointestinal disorders.
[178" claim-type="Currently amended] 177. The pharmaceutical composition according to claim 177, wherein the gastrointestinal disorder is diarrhea.
[179" claim-type="Currently amended] 177. The pharmaceutical composition according to claim 177, wherein the respiratory disease is asthma, allergic rhinitis or chronic obstructive pulmonary disease.
[180" claim-type="Currently amended] 198. The pharmaceutical composition according to claim 175, wherein the pharmaceutical preparation is an ophthalmic formulation.
[181" claim-type="Currently amended] 198. The pharmaceutical composition according to claim 180, wherein the pharmaceutical preparation is an ophthalmic, oral or intraocular formulation.
[182" claim-type="Currently amended] 198. The pharmaceutical composition of claim 180, wherein the pharmaceutical formulation is a systemic formulation.
[183" claim-type="Currently amended] 198. The pharmaceutical composition of claim 180, wherein the pharmaceutical preparation is a surgical cleaning solution.
[184" claim-type="Currently amended] A packaged pharmaceutical composition for treating an N-6 substituted 7-deazapurine responsive condition in a mammal,
A container holding a therapeutically effective amount of at least one deazapurine of claim 11, 12, 14, 25, 63 or 64; And
Lt; RTI ID = 0.0 &gt; N-6 &lt; / RTI &gt; substituted 7-deazapurine responsive state in an animal.
[185" claim-type="Currently amended] In the production method of the present invention,
a) And Gt; &Lt; / RTI &gt; wherein P is lower alkyl or a protecting group;
b) cyclizing the product of step a) ;
c) chlorinating the product of step b) ; And
d) treating the product of step c) The method comprising the steps of:
In this formula,
R ₁ and R ₂ are each a hydrogen atom or a substituted or unsubstituted alkyl, aryl or alkylaryl moiety or form a substituted or unsubstituted heterocyclic ring,
R ₃ is a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety; And
R &lt; ₅ &gt; is a halogen atom, a hydrogen atom or a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety.
[186" claim-type="Currently amended] A method for producing
a) And Gt; Lt; / RTI &gt; wherein P is a removable protecting group;
b) treating the product of step a) in cyclization conditions to give ;
c) treating the product of step b) under suitable conditions ; And
d) The chlorinated product of step c) is treated with an amine The method comprising the steps of:
In this formula,
R ₁ and R ₂ are each a hydrogen atom or a substituted or unsubstituted alkyl, aryl or alkylaryl moiety or form a substituted or unsubstituted heterocyclic ring,
R ₃ is a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety; And
R &lt; ₆ &gt; is a halogen atom, a hydrogen atom or a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety.

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同族专利:

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公开号 | 公开日

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TWI242435B|2005-11-01|

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US20050043332A1|2005-02-24|

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HU0103836A3|2003-05-28|

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AU4226599A|1999-12-20|

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HU0103836A2|2002-02-28|

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KR100722194B1|2007-05-29|

---

PL347020A1|2002-03-11|

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BR9911612A|2001-02-06|

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ID27600A|2001-04-12|

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EP1082120A1|2001-03-14|

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EP2011499A3|2010-10-20|

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NZ508314A|2004-03-26|

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AP1411A|2005-05-10|

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NO20006090D0|2000-11-30|

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PL204628B1|2010-01-29|

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AP200002015A0|2000-12-31|

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NO325233B1|2008-03-03|

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US7429574B2|2008-09-30|

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OA12147A|2006-05-05|

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CA2334200A1|1999-12-09|

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EA200001245A1|2001-08-27|

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EP2011499A2|2009-01-07|

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US6800633B2|2004-10-05|

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US20020028782A1|2002-03-07|

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MXPA00011889A|2003-04-25|

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TR200003513T2|2001-06-21|

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CZ302486B6|2011-06-15|

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CA2334200C|2011-01-25|

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US20090082369A1|2009-03-26|

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DZ2805A1|2005-01-30|

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EA003604B1|2003-06-26|

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YU76100A|2003-12-31|

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CN100528874C|2009-08-19|

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WO1999062518A1|1999-12-09|

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AR020590A1|2002-05-22|

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JP4611524B2|2011-01-12|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
1998-06-02|Priority to US8770298P

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1999-03-08|Priority to US12321699P

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1999-03-26|Priority to US60/126,527

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1999-03-26|Priority to US60/123,216

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1999-03-26|Priority to US12652799P

---

1999-03-26|Priority to US60/087,702

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1999-06-01|Application filed by 추후제출, 오에스아이 파마슈티컬스, 인코포레이티드

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2001-08-31|Publication of KR20010083042A

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2007-05-29|Application granted

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2007-05-29|Publication of KR100722194B1

优先权:

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申请号 | 申请日 | 专利标题

---

US8770298P| true| 1998-06-02|1998-06-02|

---

US12321699P| true| 1999-03-08|1999-03-08|

---

US12652799P| true| 1999-03-26|1999-03-26|

---

US60/087,702|1999-03-26|

---

US60/126,527|1999-03-26|

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US60/123,216|1999-03-26|