CA2296485A1 - N6-substituted-adenosine-5'-uronamides as adenosine receptor modulators - Google Patents
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Abstract
A series of adenosine-5'-uronamide derivatives bearing N6-arylurea, alkarylurea, heteroarylurea, arylcarbonyl, alkarylcarbonyl or heteroarylcarbonyl groups which have affinity and, in some cases, selectivity for the adenosine A1 or A3 receptors are disclosed. These compounds can be used in a pharmaceutical composition to treat disorders caused by excessive activation of the A1 or A3 receptors, or can be used in a diagnostic application to determine the relative binding of other compounds to the A1 or A3 receptors.
Description
N6-Substituted-Adenosine-5'-Uronamides as Adenosine Receptor Modulators FIELD OF THE INVENTION
The present invention relates to certain N6-substituted-adenosine-S'-uronamide derivatives and their use in the practice of medicine as compounds with activity as agonists of adenosine receptors, in particular, the adenosine A 1 and A3 receptors.
BACKGROUND OF THE INVENTION
Three major classes of adenosine receptors, classified as A~, A2, and A3, have been characterized pharmacologically and have been defined on the basis of cloned sequenced-4. A1 receptors are coupled to the inhibition of adenylate cyclase through G~ proteins and have also been shown to couple to other secondary messenger systems, including inhibition or stimulation of phosphoinositol turnover and activation of ion channels. A2 receptors are further divided into two subtypes, A2A and A2B, at which adenosine agonists activate adenylate cyclase with high and low affinity, respectively. The A.~
receptor sequence was first identified in a rat testes cDNA library, and this sequence, later cloned by homology to other G-protein coupled receptors from a rat brain cDNA library, was shown to correspond to a novel, functional adenosine receptor.
Many selective agonists and antagonists have been developed for the A1 to-15 and A2a16-19 receptor subtypes. Some of these have shown promise as potential therapeutic agents in the treatment of hypertensionl~, Parkinson's disease2~, cognitive deficits''1, schizophrenia''', epilepsy and renal failure23. Selective and/or high affinity agonists and antagonists for the A2b receptor are not well known.
The discovery of the A3 receptor has opened new therapeutic vistas in the purine field. In particular, the A3 receptor mediates processes of inflammation, hypotension, and mast cell degranulation. This receptor apparently also has a role in the central nervous system. The A3 selective agonist IB-MECA induces behavioral depression and upon chronic administration protects against cerebral ischemia. A3 selective agonists at high concentrations were also found to induce apoptosis in HL-60 human leukemia cells. These and other findings have made the A3 receptor a promising therapeutic target. Selective antagonists for the A3 receptor are sought as potential antiinflammatory or possibly antiischemic agents in the brain. Recently, A3 antagonists have been under development as antiasthmatic, antidepressant, antiarrhythmic, renal protective, antiparkinson and cognitive enhancing drugs.
Selective agonist24 and antagonist25''-8 ligands have been developed for the A3 receptor. In the agonist field, 1 B-MECA (N6-(3-iodobenzyl)adenosine-5'-methyluronamide) shows a Ki value of 1.1 nM at rat A3 receptors and a 50-fold selectivity versus either A1 or A2a receptorsZ9.
The related agonist, [I25I]AB-MECA (N6-(4-amino-3-iodobenzyl)adenosine-5'-methyluronamide)3~ has become a useful radioligand for the screening of new derivatives at cloned A3 receptors.
More recently, N6-(substituted phenylcarbamoyl)adenosine-5'-uronamides, where the substituent is 2-chloro, 3-chloro or 4-methoxy, have been prepared (Baraldi et al., A~Lyance ACS Abstracts, December 15, 1995) and demonstrated affinity at A3 receptors in the low nanomolar range (Ki values less than 10 nm). However, other closely related substituents, such as 3-bromo, showed a ten-fold loss in activity and affinity for the A3 receptor (Baraldi et al.,1. Med Chem., 39:802-806 (1996). Further, while the 2-chloro, 3-chloro and 4-methoxy substituents showed high affinity for the A3 receptor and high selectivity relative to the A., receptor, the selectivity for the A1 receptor was not as high (the ratio of A3 to A1 activity was between 4 and 1 S).
A number of A3 adenosine receptor agonists which have been S previously synthesized are structurally related to adenosine itself, in which the ribose moiety is mainly intact. On the ribose, S'-alkyluronamide groups are generally tolerated. Positions on the structure of adenosine providing flexibility of substitution, in general for adenosine agonists, have been the and C2 position. At the N6 position, most alkyl or aryl derivatives are A1 selective. At the C2 position, many C-, N-, or O-derivatives are A2a selective. Benzyl derivatives at the N6 position have been shown to be A3 selective.
It would be advantageous to provide other modulators of the A1 and 1S A3 receptors, with high affinity and selectivity for these receptors with respect to the other adenosine receptors.
It is therefore an object of the present invention to provide compounds and methods of preparation and use thereof, which are agonists or partial agonists of the adenosine receptors, in particular, the adenosine and A3 receptors.
SUMMARY OF THE INVENTION
2S Compounds useful as potent, and in some cases, selective agonists of the adenosine A~ and A3 receptors, and methods of preparation and use thereof, are disclosed.
The present invention relates to certain N6-substituted-adenosine-S'-uronamide derivatives and their use in the practice of medicine as compounds with activity as agonists of adenosine receptors, in particular, the adenosine A 1 and A3 receptors.
BACKGROUND OF THE INVENTION
Three major classes of adenosine receptors, classified as A~, A2, and A3, have been characterized pharmacologically and have been defined on the basis of cloned sequenced-4. A1 receptors are coupled to the inhibition of adenylate cyclase through G~ proteins and have also been shown to couple to other secondary messenger systems, including inhibition or stimulation of phosphoinositol turnover and activation of ion channels. A2 receptors are further divided into two subtypes, A2A and A2B, at which adenosine agonists activate adenylate cyclase with high and low affinity, respectively. The A.~
receptor sequence was first identified in a rat testes cDNA library, and this sequence, later cloned by homology to other G-protein coupled receptors from a rat brain cDNA library, was shown to correspond to a novel, functional adenosine receptor.
Many selective agonists and antagonists have been developed for the A1 to-15 and A2a16-19 receptor subtypes. Some of these have shown promise as potential therapeutic agents in the treatment of hypertensionl~, Parkinson's disease2~, cognitive deficits''1, schizophrenia''', epilepsy and renal failure23. Selective and/or high affinity agonists and antagonists for the A2b receptor are not well known.
The discovery of the A3 receptor has opened new therapeutic vistas in the purine field. In particular, the A3 receptor mediates processes of inflammation, hypotension, and mast cell degranulation. This receptor apparently also has a role in the central nervous system. The A3 selective agonist IB-MECA induces behavioral depression and upon chronic administration protects against cerebral ischemia. A3 selective agonists at high concentrations were also found to induce apoptosis in HL-60 human leukemia cells. These and other findings have made the A3 receptor a promising therapeutic target. Selective antagonists for the A3 receptor are sought as potential antiinflammatory or possibly antiischemic agents in the brain. Recently, A3 antagonists have been under development as antiasthmatic, antidepressant, antiarrhythmic, renal protective, antiparkinson and cognitive enhancing drugs.
Selective agonist24 and antagonist25''-8 ligands have been developed for the A3 receptor. In the agonist field, 1 B-MECA (N6-(3-iodobenzyl)adenosine-5'-methyluronamide) shows a Ki value of 1.1 nM at rat A3 receptors and a 50-fold selectivity versus either A1 or A2a receptorsZ9.
The related agonist, [I25I]AB-MECA (N6-(4-amino-3-iodobenzyl)adenosine-5'-methyluronamide)3~ has become a useful radioligand for the screening of new derivatives at cloned A3 receptors.
More recently, N6-(substituted phenylcarbamoyl)adenosine-5'-uronamides, where the substituent is 2-chloro, 3-chloro or 4-methoxy, have been prepared (Baraldi et al., A~Lyance ACS Abstracts, December 15, 1995) and demonstrated affinity at A3 receptors in the low nanomolar range (Ki values less than 10 nm). However, other closely related substituents, such as 3-bromo, showed a ten-fold loss in activity and affinity for the A3 receptor (Baraldi et al.,1. Med Chem., 39:802-806 (1996). Further, while the 2-chloro, 3-chloro and 4-methoxy substituents showed high affinity for the A3 receptor and high selectivity relative to the A., receptor, the selectivity for the A1 receptor was not as high (the ratio of A3 to A1 activity was between 4 and 1 S).
A number of A3 adenosine receptor agonists which have been S previously synthesized are structurally related to adenosine itself, in which the ribose moiety is mainly intact. On the ribose, S'-alkyluronamide groups are generally tolerated. Positions on the structure of adenosine providing flexibility of substitution, in general for adenosine agonists, have been the and C2 position. At the N6 position, most alkyl or aryl derivatives are A1 selective. At the C2 position, many C-, N-, or O-derivatives are A2a selective. Benzyl derivatives at the N6 position have been shown to be A3 selective.
It would be advantageous to provide other modulators of the A1 and 1S A3 receptors, with high affinity and selectivity for these receptors with respect to the other adenosine receptors.
It is therefore an object of the present invention to provide compounds and methods of preparation and use thereof, which are agonists or partial agonists of the adenosine receptors, in particular, the adenosine and A3 receptors.
SUMMARY OF THE INVENTION
2S Compounds useful as potent, and in some cases, selective agonists of the adenosine A~ and A3 receptors, and methods of preparation and use thereof, are disclosed.
The compounds have the following general formula:
O
R2 ~ N
N
N
N
R~'N
H
wherein:
R is hydrogen, alkyl, substituted alkyl, or aryl;
Ri is heteroaryl-NR-C(X), heteroaryl-C(X)-, alkaryl-NR.-C(X)-, substituted alkaryl-NR-C(X)-, aryl-NR-C(X)-, aryl-C(X)-, alkaryl-C(X)-, or substituted alkaryl-C(X)-, RZ is hydrogen, alkyl, substituted alkyl, or aryl-NH-C(X)-, and X is O, S, or NR., with the proviso that, when R' is H, R~ is aryl-NH-C(X)- and X is O, the substituents on the aryl ring are not halo, methoxy or trifluoromethyl.
R3 'and R4 are, independently selected from the group consisting of halo, ether, ester, azide, alkyl, alkoxy, carboxy, nitrite, nitro, trifluoro, aryl, alkaryl, thio, thioester, thioether, amine, amide and other substituents routinely used in the field of nucleoside chemistry to modify these positions.
Such modifications are expected to provide the compounds with activity as partial agonists.
O
R2 ~ N
N
N
N
R~'N
H
wherein:
R is hydrogen, alkyl, substituted alkyl, or aryl;
Ri is heteroaryl-NR-C(X), heteroaryl-C(X)-, alkaryl-NR.-C(X)-, substituted alkaryl-NR-C(X)-, aryl-NR-C(X)-, aryl-C(X)-, alkaryl-C(X)-, or substituted alkaryl-C(X)-, RZ is hydrogen, alkyl, substituted alkyl, or aryl-NH-C(X)-, and X is O, S, or NR., with the proviso that, when R' is H, R~ is aryl-NH-C(X)- and X is O, the substituents on the aryl ring are not halo, methoxy or trifluoromethyl.
R3 'and R4 are, independently selected from the group consisting of halo, ether, ester, azide, alkyl, alkoxy, carboxy, nitrite, nitro, trifluoro, aryl, alkaryl, thio, thioester, thioether, amine, amide and other substituents routinely used in the field of nucleoside chemistry to modify these positions.
Such modifications are expected to provide the compounds with activity as partial agonists.
When Rl is heteroaryl-NR-C(X), heteroaryl-C(X)-, aryl-C(X)-, alkaryl-C(X)-, or substituted alkaryl-C(X)-, the compounds tend to show affinity, and, in some cases, selectivity for the adenosine A1 receptor. When R~ is alkaryl-NR-C(X)-, substituted alkaryl-NR-C(X)-, or aryl-NR-C(X)-, the compounds tend to show affinity and selectivity for the A3 receptor.
Preferred substituents include p-sulfonamide, p-nitro, p-phenyl, 2,4-dichloro, p-methoxy, m-chloro, o-chloro and p-nitro.
The compounds can be used in a method for fully or partially inhibiting adenylate cyclase (A1 and A3) in a mammal, including a human.
The methods involve administering an effective amount of a compound of formula I sufficient to fully or partially inhibiting adenylate cyciase in the mammal.
Additionally, the compounds can be used in competitive binding assays to determine the activity of other compounds in their ability to bind the Al or A3 receptor.
The compounds can be used in a pharmaceutical formulation that includes a compound of formula I and one or more excipients. Various chemical intermediates, such as 2',3-isopropylidene-N-alkylcarboxamido adenosines, can be used to prepare the compounds.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is a graph showing the effect of various adenosine agonists, 5'-(N-ethylcarboxamido)adenosine {NECA), N6-sulfonamido-phenylcarbamoyl)adenosine-5'-N-ethyluronamide. ?-chloro-N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (Cl-IB-MECA), and N6-(4-amino-3-iodobenzyl)adenosine-5'N-methyluronamide (I-AB-MECA) on the binding of [35S]GTP-'y-S to membranes of RBL-2H3 rat mast cells.
Figures 2a and 2b are reaction schemes for preparing compounds of formula I.
DETAILED DESCRIPTION OF THE INVENTION
The present application discloses compounds useful as agonists or partial agonists of adenosine receptors, in particular, with activity as A1 or A3 agonists or partial agonists, and methods of preparation and use thereof.
The compounds can be used in a method for treating a mammal, including a human, with excessive activity at adenosine receptors, in particular, AI or A3 receptors. The methods involve administering to the mammal an effective amount of a compound of formula I sufficient to modulate adenosine receptors in the mammal. The compounds can be used in a pharmaceutical formulation that includes a compound of formula I and one or more excipients.
As used herein, a compound is an agonist of an adenosine A1 or A3 receptor if it is able to fully inhibit adenylate cyclase (A1 and A3) and is able to displace [1251]-AB_MECA in a competitive binding assay.
As used herein, a compound is a partial agonist of an adenosine A1 or A3 receptor if it is able to partially inhibit adenylate cyclase (AI and A3) and is able to displace [t2~I]-AB-MECA in a competitive binding assay.
As used herein, a compound is an antagonist of an adenosine A1 or A3 receptor if it is able to prevent the inhibition due to an agonist and is able to displace (1251]_AB_MECA in a competitive binding assay.
Preferred substituents include p-sulfonamide, p-nitro, p-phenyl, 2,4-dichloro, p-methoxy, m-chloro, o-chloro and p-nitro.
The compounds can be used in a method for fully or partially inhibiting adenylate cyclase (A1 and A3) in a mammal, including a human.
The methods involve administering an effective amount of a compound of formula I sufficient to fully or partially inhibiting adenylate cyciase in the mammal.
Additionally, the compounds can be used in competitive binding assays to determine the activity of other compounds in their ability to bind the Al or A3 receptor.
The compounds can be used in a pharmaceutical formulation that includes a compound of formula I and one or more excipients. Various chemical intermediates, such as 2',3-isopropylidene-N-alkylcarboxamido adenosines, can be used to prepare the compounds.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is a graph showing the effect of various adenosine agonists, 5'-(N-ethylcarboxamido)adenosine {NECA), N6-sulfonamido-phenylcarbamoyl)adenosine-5'-N-ethyluronamide. ?-chloro-N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (Cl-IB-MECA), and N6-(4-amino-3-iodobenzyl)adenosine-5'N-methyluronamide (I-AB-MECA) on the binding of [35S]GTP-'y-S to membranes of RBL-2H3 rat mast cells.
Figures 2a and 2b are reaction schemes for preparing compounds of formula I.
DETAILED DESCRIPTION OF THE INVENTION
The present application discloses compounds useful as agonists or partial agonists of adenosine receptors, in particular, with activity as A1 or A3 agonists or partial agonists, and methods of preparation and use thereof.
The compounds can be used in a method for treating a mammal, including a human, with excessive activity at adenosine receptors, in particular, AI or A3 receptors. The methods involve administering to the mammal an effective amount of a compound of formula I sufficient to modulate adenosine receptors in the mammal. The compounds can be used in a pharmaceutical formulation that includes a compound of formula I and one or more excipients.
As used herein, a compound is an agonist of an adenosine A1 or A3 receptor if it is able to fully inhibit adenylate cyclase (A1 and A3) and is able to displace [1251]-AB_MECA in a competitive binding assay.
As used herein, a compound is a partial agonist of an adenosine A1 or A3 receptor if it is able to partially inhibit adenylate cyclase (AI and A3) and is able to displace [t2~I]-AB-MECA in a competitive binding assay.
As used herein, a compound is an antagonist of an adenosine A1 or A3 receptor if it is able to prevent the inhibition due to an agonist and is able to displace (1251]_AB_MECA in a competitive binding assay.
As used herein, a compound is selective for the A1 receptor if the ratio of A2/A1 and A3/A1 activity is greater than about 50, preferably between SO and 100, and more preferably, greater than about 100. A
compound is selective for the A3 receptor if the ratio of A1/A3 and A2/A3 activity is greater than about 50, preferably between 50 and 100, and more preferably, greater than about 100.
As used herein, the term "alkyl" refers to monovalent straight, branched or cyclic alkyl groups preferably having from 1 to 20 carbon atoms, more preferably 1 to 10 carbon atoms ("lower alkyl") and most preferably 1 to 6 carbon atoms. This term is exemplified by groups such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, n-hexyl, and the like. The terms "alkylene" and "lower alkylene" refer to divalent radicals of the corresponding alkane. Further, as used herein, other moieties having names derived from alkanes, such as alkoxyl, alkanoyl, alkenyl, cycloalkenyl, etc when modified by "lower," have carbon chains of ten or less carbon atoms.
In those cases where the minimum number of carbons are greater than one, e.g., alkenyl (minimum of two carbons) and cycloalkyl, (minimum of three carbons), it is to be understood that "lower" means at least the minimum number of carbons.
As used herein, the term "substituted alkyl" refers to an alkyl group, having from 1 to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyi, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino aminoacyl, aminoacyloxy, oxyacylamino, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, hydroxylamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SOZ-alkyl, -S02-substituted alkyl, -S02-aryl, -SO.,-heteroaryl, and mono- and di-alkylamino, mono- and di-(substituted alkyl)amino, mono- and di-arylamino, mono- and di-heteroarylamino, mono- and di-heterocyclic amino, and unsymmetric di-substituted amines having different substituents selected from alkyl, aryl, heteroaryl and heterocyclic. As used herein, other moieties having the prefix "substituted" are intended to include one or more of the substituents listed above.
As used herein, the term "alkoxy" refers to the group "alkyl-O-", where alkyl is as defined above. Preferred alkoxy groups include, by way of example, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sa~-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.
As used herein, the term "alkenyl" refers to alkenyl groups preferably having from 2 to 10 carbon atoms and more preferably 2 to 6 carbon atoms and having at least 1 and preferably from 1-2 sites of alkenyl unsaturation.
Preferred alkenyl groups include ethenyl (-CH=CH2), n-propenyl (-CH2CH=CH2), iso-propenyl (-C(CH3)=CH.,), and the like.
As used herein, the term "alkynyl" refers to alkynyl groups preferably having from 2 to 10 carbon atoms and more preferably 2 to 6 carbon atoms and having at least 1 and preferably from 1-2 sites of alkynyl unsaturation.
As used herein, the term "acyl" refers to the groups alkyl-C(O)-, substituted alkyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, aryl-C(O)-, heteroaryl-C(O)- and heterocyclic-C(O)- where alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl and heterocyclic are as defined herein.
As used herein, the term "acylamino" refers to the group -C(O)NRR
where each R is independently hydrogen, alkyl, substituted alkyl, aryl, _g_ heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.
As used herein, the term "aryl" refers to an unsaturated aromatic carbocyclic group of from 6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple condensed (fused) rings (e.g., naphthyl or anthryi).
Preferred aryls include phenyl, naphthyl and the like. Unless otherwise constrained by the definition for the aryl substituent, such aryl groups can optionally be substituted with from 1 to 5 substituents and preferably 1 to 3 substituents selected from the group consisting of hydroxy, acyl, alkyl, alkoxy, alkenyl, alkynyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, amino, substituted amino, aminoacyl, acyloxy, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxyialkyl, cyano, halo, vitro, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO~-alkyl, -S02-substituted alkyl, -S02-aryl, -S02-heteroaryl, trihalomethyl. Preferred substituents include alkyl, alkoxy, halo, cyano, vitro, trihalomethyl, and thioalkoxy.
As used herein, the term "cycloalkyl" refers to cyclic alkyl groups of from 3 to 12 carbon atoms having a single cyclic ring or multiple condensed rings. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.
As used herein, the terms "halo" or "halogen" refer to fluoro, chloro, bromo and iodo and preferably is either fluoro or chloro.
As used herein, the term "heteroaryl" refers to an aromatic carbocyclic group of from 1 to 15 carbon atoms and 1 to 4 heteroatoms WO 99/Ob053 PCT/US98/16053 selected from the group consisting of oxygen, nitrogen and sulfur within at least one ring (if there is more than one ring).
Unless otherwise constrained~by the definition for the heteroaryl substituent, such heteroaryl groups can be optionally substituted with from 1 to 5 substituents and preferably 1 to 3 substituents selected from the group consisting of hydroxy, acyl, alkyl, alkoxy, alkenyl, alkynyl, substituted alkyl, substituted aikoxy, substituted alkenyl, substituted alkynyl, amino, substituted amino, aminoacyl, acyloxy, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halo, nitro, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO.,-alkyl, -S02-substituted alkyl, -SO~-aryl, -S02-heteroaryl, trihalomethyl. Preferred substituents include alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy. Such heteroaryl groups can have a single ring (e.g., pyridyl or furyl) or multiple condensed rings {e.g., indolizinyl or benzothienyl).
"Heterocycle" or "heterocyclic" refers to a monovalent saturated or unsaturated group having a single ring or multiple condensed rings, from 1 to 15 carbon atoms and from 1 to 4 hetero atoms selected from the group consisting of nitrogen, sulfur and oxygen within the ring.
Unless otherwise constrained by the definition for the heterocyclic substituent; such heterocyclic groups can be optionally substituted with 1 to 5 substituents selected from the group consisting of alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, aryloxy, halo, nitro, heteroaryl, thiol, thioalkoxy, substituted thioalkoxy, thioaryloxy, trihalomethyl, and the like.
Such heterocyclic groups can have a single ring or multiple condensed rings.
WO 99/Ob053 PCT/US98/16053 As to any of the above groups that contain 1 or more substituents, it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non-feasible.
"Pharmaceutically acceptable salts" refers to pharmaceutically acceptable salts of a compound of Formula I, which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like can be used as the pharmaceutically acceptable salt.
The term "protecting group" or "blocking group" refers to any group which when bound to one or more hydroxyl, amino or carboxyl groups of the compounds (including intermediates thereof such as the aminolactams, aminolactones, etc.) prevents reactions from occurnng at these groups and which protecting group can be removed by conventional chemical or enzymatic steps to reestablish the hydroxyl, amino or carboxyl group.
Preferred removable amino blocking groups include conventional substituents such as t-butyoxycarbonyl (t-BOC), benzyloxycarbonyl (CBZ), and the like which can be removed by conventional conditions compatible with the nature of the product.
The following abbreviations are used herein: Abbreviations:
[1251]~_MECA, [tZSI]N6-(4-amino-3-iodobenzyl)adenosine-5'N-methyluronamide; CHO cells, Chinese hamster ovary cells; CGS 21680, 2-[4-[(2-carboxyethyl)phenyl]ethyl-amino]-5'-N-ethylcarboxamido adenosine;
Cl-IB-MECA, 2-chloro-N6-(3-iodobenzyl)adenosine-S'-N-methyluronamide;
(R)-PIA, (R)-N6-(phenylisopropyl)adenosine; DMSO, dimethysulfoxide;
EDTA, ethylenediamine tetraacetic acid, I-AB-MECA, N6-(4-amino-3-iodobenzyl)adenosine-5'-N-methyluronamide; IB-MECA, N6-{3-iodobenzyl)adenosine-5'-N-methyluronamide; Ki, equilibrium inhibition constant; NECA, 5'-N-ethylcarboxamido adenosine; THF, tetrahydrofuran;
Tris, tris(hydroxymethyl)aminomethane.
Those skilled in the art of organic chemistry will appreciate that reactive and fragile functional groups often must be protected prior to a particular reaction, or sequence of reactions, and then restored to their original forms after the last reaction is completed. Usually groups are protected by converting them to a relatively stable derivative. For example, a hydroxyl group may be converted to an ether group and an amine group converted to an amide or carbamate. Methods of protecting and de-protecting, also known as "blocking" and "de-blocking," are well known and widely practiced in the art, e.g., see T. Green, Protective Groups in Organic Synthesis, John Wiley, New York ( 1981 ) or Protective Groacps in Organic Chemistry, Ed. J.F.W. McOmie, Plenum Press, London (1973).
The compounds can be prepared as described below. Generally, the 2'- and 3'-hydroxy groups on an N-alkyl carboxamido adenosine, such as 5'-(N-ethyicarboxamido)adenosine (NECA), are protected. A suitable protecting group is an isopropylidene ring, although other protecting groups can be used and are intended to be within the scope of the invention.
2',3'-O-isopropylidene-N6-(substituted-carbonylamino)-adenosine-S'-alkyluronamides can be prepared from the isopropylidene protected N
alkylcarboxamido adenosines by reaction of the N6-amine group with an appropriate acid chloride or other suitable activated carboxylic acid derivative, such as an anhydride, using routine amidation conditions (as shown in Figure 2a). 2',3'-O-isopropylidene-N6-(substituted-carbamoylamino)-adenosine-5'-alkyluronamides can be prepared from the isopropylidene protected N-alkylcarboxamido adenosines by reaction of the N6-amine group with an appropriate isocyanate using known chemistry {as shown in Figure 2b). The protecting groups are then removed to provide the desired compounds.
In those embodiments in which the 2' and/or 3'-hydroxy groups have been replaced with halo, ether, ester, azide, alkyl, alkoxy, carboxy, nitriIe, vitro, trifluoro, aryl, alkaryl, thio, thioester, thioether, amine, or amide groups, the chemistry proceeds in substantially the same manner, except that thio and amine groups, which would interfere with the coupling chemistry, must first be protected with suitable protecting groups prior to coupling the N6 amine group with an acid halide or isocyanate.
Methods of Icing th .om on ands The compounds can be used for all indications for which agonists and antagonists of the A1 or A3 receptor are effective.
Compounds which effectively modulate the A1 receptor can be used for:
~ Protection against hypoxia and/or ischemia induced injuries (e.g., stroke, infarction);
~ Treatment of adenosine-sensitive cardiac arrhythmias;
~ antinociception (i.e., analgesics);
~ anticonvulsants;
~ cardioprotection, short term (e.g., prior to percutaneous angioplasty (PTDA), angioplasty, and cardiac surgeries) and long term (prevention of myocardial infarction, especially in high risk patients, reduction of infarct damage, especially in high risk patients);
~ neuroprotection: stroke prevention, stroke treatment, and the treatment of epilepsy;
~ pain management generally, including different forms of neuropathic pain, e.g., diabetic neuropathy, post herpetic neuralgia;
~ antilipid uses: reduction of free fatty acids, triglycerides, glucose;
~ adjunct therapy in diabetes, including insulin and non-insulin dependent diabetes mellitus: stimulation of insulin secretion from the pancreas, increase in tissue sensitivity to insulin;
~ treatment of GI disorders such as diarrhea, irritable bowel disease, irntable bowel syndrome, incontinence;
~ treatment.of glaucoma;
~ treatment of sleep apnea;
~ treatment of cardiac disarrythmias (peroxysmal supraventricular tachycardia;
~ use in combination with anesthesia for post surgical pain;
~ treatment of inflammation;
~ diagnostic uses, for example, to determine the presence of one or more of the above described medical conditions, or in a screening assay to determine the effectiveness of other compounds for binding to the A~ Ado receptor (i.e., through competitive inhibition as determined by various binding assays), as described in Jacobson and Van Rhee, Purinergic approaches to experimental therapy, Jacobson and Jarvis, ed., Wiley, New York, 1997, pp. 101-128; Mathot et al., B
Pha~na~ol., 116:1957-1964 (1995); van der Wenden et al., J.
l~d.~hem., 38:4000-4006 (1990; and van Calenbergh, ,L
Med. C.'hem., 40:3765-3772 ( 1997), the contents of which are hereby incorporated by reference.
Compounds which effectively modulate the A3 receptor can be used for:
~ treating hypertension;
~ mast cell degranulation;
~ antitumor agents;
~ ~ treating cardiac hypoxia; and ~ protection against cerebral ischemia;
as described, for example, in Jacobson, TIPS May 1998, pp. 185-191, the contents of which are hereby incorporated by reference.
The compounds can be administered via any medically acceptable means. Suitable means of administration include oral, rectal, topical or parenteral (including subcutaneous, intramuscular and intravenous) administration, although oral or parenteral administration are preferred.
The amount of the compound required to be effective as agonist or partial agonist of an adenosine receptor will, of course, vary with the individual mammal being treated and is ultimately at the discretion of the medical or veterinary practitioner. The factors to be considered include the condition being treated, the route of administration, the nature of the formulation, the mammal's body weight, surface area, age and general condition, and the particular compound to be administered. However, a suitable effective dose is in the range of about 0.1 pg/kg to about 10 mg/kg body weight per day, preferably in the range of about 1 mg/kg to about 3 mg/kg per day.
The total daily dose may be given as a single dose, multiple doses, e.g., two to six times per day, or by intravenous infusion for a selected duration. Dosages above or below the range cited above are within the scope of the present invention and may be administered to the individuai patient if WO 99/06053 PCT/US9$/16053 desired and necessary. For example, for a 75 kg mammal, a dosage range would be about 75 mg to about 220 mg per day, and a typical dose would be about 150 mg per day. If discrete multiple doses are indicated, treatment might typically be 50 mg of a compound given 3 times per day.
The compounds described above are preferably administered in a formulation including an active compound, i.e., a compound of formula I, together with an acceptable carrier for the mode of administration. Suitable pharmaceutically acceptable carnets are known to those of skill in the art.
The compositions can optionally include other therapeutically active ingredients such as antivirals, antitumor agents, antibacterials, anti-inflammatories, analgesics, and immunosuppresants. The carnet must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
The formulations can include carnets suitable for oral, rectal, topical or parenteral (including subcutaneous, intramuscular and intravenous) administration. Preferred carriers are those suitable for oral or parenteral administration.
Formulations suitable for parenteral administration include sterile aqueous preparations of the active compound, which are preferably isotonic with the blood of the recipient. Such formulations may contain distilled water, 5% dextrose in distilled water or saline. Suitable formulations also include concentrated solutions or solids containing the compound of formula (I) which upon dilution with an appropriate solvent give a solution suitable for parental administration.
For enteral administration, the compound can be incorporated into an inert carnet in discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the active compound; as a powder or granules; or a suspension or solution in an aqueous liquid or non-aqueous liquid, e.g., a syrup, an elixir, an emulsion or a draught. Suitable carriers may be starches or sugars and include lubricants, flavorings, binders, and other materials of the same nature.
A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active compound in a free-flowing form, e.g., a powder or granules, optionally mixed with accessory ingredients, e.g., binders, lubricants, inert diluents, surface active or dispersing agents. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered active compound with any suitable Garner.
A syrup or suspension may be made by adding the active compound to a concentrated, aqueous solution of a sugar, e.g., sucrose, to which may also be added any accessory ingredients. Such accessory ingredients may include flavoring, an agent to retard crystallization of the sugar or an agent to increase the solubility of any other ingredient, e.g., as a polyhydric alcohol, for example, glycerol or sorbitol.
The compounds can also be administered locally by topical application of a solution, ointment, cream, gel, lotion or polymeric material (for example, a PluronicTM, BASF), which may be prepared by conventional methods known in the art of pharmacy. In addition to the solution, ointment, cream, gel, lotion or polymeric base and the active ingredient, such topical formulations may also contain preservatives, perfumes, and additional active pharmaceutical agents.
Formulations for rectal administration may be presented as a suppository with a conventional carrier, e.g., cocoa butter or Witepsol S55 (trademark of Dynamite Nobel Chemical, Germany), for a suppository base.
Alternatively, the compound may be administered in liposomes or microspheres {or microparticles). Methods for preparing liposomes and microspheres for administration to a patient are well known to those of skill in the art. U.S. Patent No. 4,789,734, the contents of which are hereby incorporated by reference, describes methods for encapsulating biological materials in liposomes. Essentially, the material is dissolved in an aqueous solution, the appropriate phospholipids and lipids added, along with surfactants if required, and the material dialyzed or sonicated, as necessary.
A review of known methods is provided by G. Gregoriadis, Chapter 14, "Liposomes," ~ru~ Carriers in Biolog;l and Medicine, pp. 287-341 (Academic Press, 1979). Microspheres formed of polymers or proteins are well known to those skilled in the art, and can be tailored for passage through the gastrointestinal tract directly into the blood stream. Alternatively, the compound can be incorporated and the microspheres, or composite of microspheres, implanted for slow release over a period of time ranging from days to months. See, for example, U.S. Patent Nos. 4,906,474, 4,925,673 and 3,625,214, the contents of which are hereby incorporated by reference.
Preferred microparticles are those prepared from biodegradable polymers, such as polyglycolide, polylactide and copolymers thereof. Those of skill in the art can readily determine an appropriate carrier system depending on various factors, including the desired rate of drug release and the desired dosage.
The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active compound into association with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active compound into association with a liquid earner or a finely divided solid carrier and then, if necessary, shaping the product into desired unit dosage form.
In addition to the aforementioned ingredients, the formulations may further include one or more optional accessory ingredient{s) utilized in the art of pharmaceutical formulations, e.g., diluents, buffers, flavoring agents, binders, surface active agents, thickeners, lubricants, suspending agents, preservatives (including antioxidants) and the like.
The activity of the compounds can be readily determined using no more than routine experimentation using, for example, any of the following assays.
~1-~-A2A '°'denosine Receptor Binding A
Membrane preparations:
Male Wistar rats (200-250 g) can be decapitated and the whole brain (minus brainstem, striatum and cerebellum) was dissected on ice. The brain tissues can be disrupted in a Polytron (setting 5) in 20 vols of 50 mM Tris HCI, pH 7.4. The homogenate can then be centrifuged at 48,000 g for 10 min and the pellet resuspended in Tris-HCI containing 2 ILJ/ml adenosine deaminase; type VI (Sigma Chemical Company, St. Louis., Mo., USA).
After a 30 min incubation at 37°C, the membranes can be centrifuged and the pellets stored at -70°C. Striatal tissues can be homogenized with a Polytron in 25 vol of 50 mM Tris HCI buffer containing 10 mM MgCh pH
compound is selective for the A3 receptor if the ratio of A1/A3 and A2/A3 activity is greater than about 50, preferably between 50 and 100, and more preferably, greater than about 100.
As used herein, the term "alkyl" refers to monovalent straight, branched or cyclic alkyl groups preferably having from 1 to 20 carbon atoms, more preferably 1 to 10 carbon atoms ("lower alkyl") and most preferably 1 to 6 carbon atoms. This term is exemplified by groups such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, n-hexyl, and the like. The terms "alkylene" and "lower alkylene" refer to divalent radicals of the corresponding alkane. Further, as used herein, other moieties having names derived from alkanes, such as alkoxyl, alkanoyl, alkenyl, cycloalkenyl, etc when modified by "lower," have carbon chains of ten or less carbon atoms.
In those cases where the minimum number of carbons are greater than one, e.g., alkenyl (minimum of two carbons) and cycloalkyl, (minimum of three carbons), it is to be understood that "lower" means at least the minimum number of carbons.
As used herein, the term "substituted alkyl" refers to an alkyl group, having from 1 to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyi, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino aminoacyl, aminoacyloxy, oxyacylamino, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, hydroxylamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SOZ-alkyl, -S02-substituted alkyl, -S02-aryl, -SO.,-heteroaryl, and mono- and di-alkylamino, mono- and di-(substituted alkyl)amino, mono- and di-arylamino, mono- and di-heteroarylamino, mono- and di-heterocyclic amino, and unsymmetric di-substituted amines having different substituents selected from alkyl, aryl, heteroaryl and heterocyclic. As used herein, other moieties having the prefix "substituted" are intended to include one or more of the substituents listed above.
As used herein, the term "alkoxy" refers to the group "alkyl-O-", where alkyl is as defined above. Preferred alkoxy groups include, by way of example, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sa~-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.
As used herein, the term "alkenyl" refers to alkenyl groups preferably having from 2 to 10 carbon atoms and more preferably 2 to 6 carbon atoms and having at least 1 and preferably from 1-2 sites of alkenyl unsaturation.
Preferred alkenyl groups include ethenyl (-CH=CH2), n-propenyl (-CH2CH=CH2), iso-propenyl (-C(CH3)=CH.,), and the like.
As used herein, the term "alkynyl" refers to alkynyl groups preferably having from 2 to 10 carbon atoms and more preferably 2 to 6 carbon atoms and having at least 1 and preferably from 1-2 sites of alkynyl unsaturation.
As used herein, the term "acyl" refers to the groups alkyl-C(O)-, substituted alkyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, aryl-C(O)-, heteroaryl-C(O)- and heterocyclic-C(O)- where alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl and heterocyclic are as defined herein.
As used herein, the term "acylamino" refers to the group -C(O)NRR
where each R is independently hydrogen, alkyl, substituted alkyl, aryl, _g_ heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.
As used herein, the term "aryl" refers to an unsaturated aromatic carbocyclic group of from 6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple condensed (fused) rings (e.g., naphthyl or anthryi).
Preferred aryls include phenyl, naphthyl and the like. Unless otherwise constrained by the definition for the aryl substituent, such aryl groups can optionally be substituted with from 1 to 5 substituents and preferably 1 to 3 substituents selected from the group consisting of hydroxy, acyl, alkyl, alkoxy, alkenyl, alkynyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, amino, substituted amino, aminoacyl, acyloxy, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxyialkyl, cyano, halo, vitro, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO~-alkyl, -S02-substituted alkyl, -S02-aryl, -S02-heteroaryl, trihalomethyl. Preferred substituents include alkyl, alkoxy, halo, cyano, vitro, trihalomethyl, and thioalkoxy.
As used herein, the term "cycloalkyl" refers to cyclic alkyl groups of from 3 to 12 carbon atoms having a single cyclic ring or multiple condensed rings. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.
As used herein, the terms "halo" or "halogen" refer to fluoro, chloro, bromo and iodo and preferably is either fluoro or chloro.
As used herein, the term "heteroaryl" refers to an aromatic carbocyclic group of from 1 to 15 carbon atoms and 1 to 4 heteroatoms WO 99/Ob053 PCT/US98/16053 selected from the group consisting of oxygen, nitrogen and sulfur within at least one ring (if there is more than one ring).
Unless otherwise constrained~by the definition for the heteroaryl substituent, such heteroaryl groups can be optionally substituted with from 1 to 5 substituents and preferably 1 to 3 substituents selected from the group consisting of hydroxy, acyl, alkyl, alkoxy, alkenyl, alkynyl, substituted alkyl, substituted aikoxy, substituted alkenyl, substituted alkynyl, amino, substituted amino, aminoacyl, acyloxy, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halo, nitro, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO.,-alkyl, -S02-substituted alkyl, -SO~-aryl, -S02-heteroaryl, trihalomethyl. Preferred substituents include alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy. Such heteroaryl groups can have a single ring (e.g., pyridyl or furyl) or multiple condensed rings {e.g., indolizinyl or benzothienyl).
"Heterocycle" or "heterocyclic" refers to a monovalent saturated or unsaturated group having a single ring or multiple condensed rings, from 1 to 15 carbon atoms and from 1 to 4 hetero atoms selected from the group consisting of nitrogen, sulfur and oxygen within the ring.
Unless otherwise constrained by the definition for the heterocyclic substituent; such heterocyclic groups can be optionally substituted with 1 to 5 substituents selected from the group consisting of alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, aryloxy, halo, nitro, heteroaryl, thiol, thioalkoxy, substituted thioalkoxy, thioaryloxy, trihalomethyl, and the like.
Such heterocyclic groups can have a single ring or multiple condensed rings.
WO 99/Ob053 PCT/US98/16053 As to any of the above groups that contain 1 or more substituents, it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non-feasible.
"Pharmaceutically acceptable salts" refers to pharmaceutically acceptable salts of a compound of Formula I, which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like can be used as the pharmaceutically acceptable salt.
The term "protecting group" or "blocking group" refers to any group which when bound to one or more hydroxyl, amino or carboxyl groups of the compounds (including intermediates thereof such as the aminolactams, aminolactones, etc.) prevents reactions from occurnng at these groups and which protecting group can be removed by conventional chemical or enzymatic steps to reestablish the hydroxyl, amino or carboxyl group.
Preferred removable amino blocking groups include conventional substituents such as t-butyoxycarbonyl (t-BOC), benzyloxycarbonyl (CBZ), and the like which can be removed by conventional conditions compatible with the nature of the product.
The following abbreviations are used herein: Abbreviations:
[1251]~_MECA, [tZSI]N6-(4-amino-3-iodobenzyl)adenosine-5'N-methyluronamide; CHO cells, Chinese hamster ovary cells; CGS 21680, 2-[4-[(2-carboxyethyl)phenyl]ethyl-amino]-5'-N-ethylcarboxamido adenosine;
Cl-IB-MECA, 2-chloro-N6-(3-iodobenzyl)adenosine-S'-N-methyluronamide;
(R)-PIA, (R)-N6-(phenylisopropyl)adenosine; DMSO, dimethysulfoxide;
EDTA, ethylenediamine tetraacetic acid, I-AB-MECA, N6-(4-amino-3-iodobenzyl)adenosine-5'-N-methyluronamide; IB-MECA, N6-{3-iodobenzyl)adenosine-5'-N-methyluronamide; Ki, equilibrium inhibition constant; NECA, 5'-N-ethylcarboxamido adenosine; THF, tetrahydrofuran;
Tris, tris(hydroxymethyl)aminomethane.
Those skilled in the art of organic chemistry will appreciate that reactive and fragile functional groups often must be protected prior to a particular reaction, or sequence of reactions, and then restored to their original forms after the last reaction is completed. Usually groups are protected by converting them to a relatively stable derivative. For example, a hydroxyl group may be converted to an ether group and an amine group converted to an amide or carbamate. Methods of protecting and de-protecting, also known as "blocking" and "de-blocking," are well known and widely practiced in the art, e.g., see T. Green, Protective Groups in Organic Synthesis, John Wiley, New York ( 1981 ) or Protective Groacps in Organic Chemistry, Ed. J.F.W. McOmie, Plenum Press, London (1973).
The compounds can be prepared as described below. Generally, the 2'- and 3'-hydroxy groups on an N-alkyl carboxamido adenosine, such as 5'-(N-ethyicarboxamido)adenosine (NECA), are protected. A suitable protecting group is an isopropylidene ring, although other protecting groups can be used and are intended to be within the scope of the invention.
2',3'-O-isopropylidene-N6-(substituted-carbonylamino)-adenosine-S'-alkyluronamides can be prepared from the isopropylidene protected N
alkylcarboxamido adenosines by reaction of the N6-amine group with an appropriate acid chloride or other suitable activated carboxylic acid derivative, such as an anhydride, using routine amidation conditions (as shown in Figure 2a). 2',3'-O-isopropylidene-N6-(substituted-carbamoylamino)-adenosine-5'-alkyluronamides can be prepared from the isopropylidene protected N-alkylcarboxamido adenosines by reaction of the N6-amine group with an appropriate isocyanate using known chemistry {as shown in Figure 2b). The protecting groups are then removed to provide the desired compounds.
In those embodiments in which the 2' and/or 3'-hydroxy groups have been replaced with halo, ether, ester, azide, alkyl, alkoxy, carboxy, nitriIe, vitro, trifluoro, aryl, alkaryl, thio, thioester, thioether, amine, or amide groups, the chemistry proceeds in substantially the same manner, except that thio and amine groups, which would interfere with the coupling chemistry, must first be protected with suitable protecting groups prior to coupling the N6 amine group with an acid halide or isocyanate.
Methods of Icing th .om on ands The compounds can be used for all indications for which agonists and antagonists of the A1 or A3 receptor are effective.
Compounds which effectively modulate the A1 receptor can be used for:
~ Protection against hypoxia and/or ischemia induced injuries (e.g., stroke, infarction);
~ Treatment of adenosine-sensitive cardiac arrhythmias;
~ antinociception (i.e., analgesics);
~ anticonvulsants;
~ cardioprotection, short term (e.g., prior to percutaneous angioplasty (PTDA), angioplasty, and cardiac surgeries) and long term (prevention of myocardial infarction, especially in high risk patients, reduction of infarct damage, especially in high risk patients);
~ neuroprotection: stroke prevention, stroke treatment, and the treatment of epilepsy;
~ pain management generally, including different forms of neuropathic pain, e.g., diabetic neuropathy, post herpetic neuralgia;
~ antilipid uses: reduction of free fatty acids, triglycerides, glucose;
~ adjunct therapy in diabetes, including insulin and non-insulin dependent diabetes mellitus: stimulation of insulin secretion from the pancreas, increase in tissue sensitivity to insulin;
~ treatment of GI disorders such as diarrhea, irritable bowel disease, irntable bowel syndrome, incontinence;
~ treatment.of glaucoma;
~ treatment of sleep apnea;
~ treatment of cardiac disarrythmias (peroxysmal supraventricular tachycardia;
~ use in combination with anesthesia for post surgical pain;
~ treatment of inflammation;
~ diagnostic uses, for example, to determine the presence of one or more of the above described medical conditions, or in a screening assay to determine the effectiveness of other compounds for binding to the A~ Ado receptor (i.e., through competitive inhibition as determined by various binding assays), as described in Jacobson and Van Rhee, Purinergic approaches to experimental therapy, Jacobson and Jarvis, ed., Wiley, New York, 1997, pp. 101-128; Mathot et al., B
Pha~na~ol., 116:1957-1964 (1995); van der Wenden et al., J.
l~d.~hem., 38:4000-4006 (1990; and van Calenbergh, ,L
Med. C.'hem., 40:3765-3772 ( 1997), the contents of which are hereby incorporated by reference.
Compounds which effectively modulate the A3 receptor can be used for:
~ treating hypertension;
~ mast cell degranulation;
~ antitumor agents;
~ ~ treating cardiac hypoxia; and ~ protection against cerebral ischemia;
as described, for example, in Jacobson, TIPS May 1998, pp. 185-191, the contents of which are hereby incorporated by reference.
The compounds can be administered via any medically acceptable means. Suitable means of administration include oral, rectal, topical or parenteral (including subcutaneous, intramuscular and intravenous) administration, although oral or parenteral administration are preferred.
The amount of the compound required to be effective as agonist or partial agonist of an adenosine receptor will, of course, vary with the individual mammal being treated and is ultimately at the discretion of the medical or veterinary practitioner. The factors to be considered include the condition being treated, the route of administration, the nature of the formulation, the mammal's body weight, surface area, age and general condition, and the particular compound to be administered. However, a suitable effective dose is in the range of about 0.1 pg/kg to about 10 mg/kg body weight per day, preferably in the range of about 1 mg/kg to about 3 mg/kg per day.
The total daily dose may be given as a single dose, multiple doses, e.g., two to six times per day, or by intravenous infusion for a selected duration. Dosages above or below the range cited above are within the scope of the present invention and may be administered to the individuai patient if WO 99/06053 PCT/US9$/16053 desired and necessary. For example, for a 75 kg mammal, a dosage range would be about 75 mg to about 220 mg per day, and a typical dose would be about 150 mg per day. If discrete multiple doses are indicated, treatment might typically be 50 mg of a compound given 3 times per day.
The compounds described above are preferably administered in a formulation including an active compound, i.e., a compound of formula I, together with an acceptable carrier for the mode of administration. Suitable pharmaceutically acceptable carnets are known to those of skill in the art.
The compositions can optionally include other therapeutically active ingredients such as antivirals, antitumor agents, antibacterials, anti-inflammatories, analgesics, and immunosuppresants. The carnet must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
The formulations can include carnets suitable for oral, rectal, topical or parenteral (including subcutaneous, intramuscular and intravenous) administration. Preferred carriers are those suitable for oral or parenteral administration.
Formulations suitable for parenteral administration include sterile aqueous preparations of the active compound, which are preferably isotonic with the blood of the recipient. Such formulations may contain distilled water, 5% dextrose in distilled water or saline. Suitable formulations also include concentrated solutions or solids containing the compound of formula (I) which upon dilution with an appropriate solvent give a solution suitable for parental administration.
For enteral administration, the compound can be incorporated into an inert carnet in discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the active compound; as a powder or granules; or a suspension or solution in an aqueous liquid or non-aqueous liquid, e.g., a syrup, an elixir, an emulsion or a draught. Suitable carriers may be starches or sugars and include lubricants, flavorings, binders, and other materials of the same nature.
A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active compound in a free-flowing form, e.g., a powder or granules, optionally mixed with accessory ingredients, e.g., binders, lubricants, inert diluents, surface active or dispersing agents. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered active compound with any suitable Garner.
A syrup or suspension may be made by adding the active compound to a concentrated, aqueous solution of a sugar, e.g., sucrose, to which may also be added any accessory ingredients. Such accessory ingredients may include flavoring, an agent to retard crystallization of the sugar or an agent to increase the solubility of any other ingredient, e.g., as a polyhydric alcohol, for example, glycerol or sorbitol.
The compounds can also be administered locally by topical application of a solution, ointment, cream, gel, lotion or polymeric material (for example, a PluronicTM, BASF), which may be prepared by conventional methods known in the art of pharmacy. In addition to the solution, ointment, cream, gel, lotion or polymeric base and the active ingredient, such topical formulations may also contain preservatives, perfumes, and additional active pharmaceutical agents.
Formulations for rectal administration may be presented as a suppository with a conventional carrier, e.g., cocoa butter or Witepsol S55 (trademark of Dynamite Nobel Chemical, Germany), for a suppository base.
Alternatively, the compound may be administered in liposomes or microspheres {or microparticles). Methods for preparing liposomes and microspheres for administration to a patient are well known to those of skill in the art. U.S. Patent No. 4,789,734, the contents of which are hereby incorporated by reference, describes methods for encapsulating biological materials in liposomes. Essentially, the material is dissolved in an aqueous solution, the appropriate phospholipids and lipids added, along with surfactants if required, and the material dialyzed or sonicated, as necessary.
A review of known methods is provided by G. Gregoriadis, Chapter 14, "Liposomes," ~ru~ Carriers in Biolog;l and Medicine, pp. 287-341 (Academic Press, 1979). Microspheres formed of polymers or proteins are well known to those skilled in the art, and can be tailored for passage through the gastrointestinal tract directly into the blood stream. Alternatively, the compound can be incorporated and the microspheres, or composite of microspheres, implanted for slow release over a period of time ranging from days to months. See, for example, U.S. Patent Nos. 4,906,474, 4,925,673 and 3,625,214, the contents of which are hereby incorporated by reference.
Preferred microparticles are those prepared from biodegradable polymers, such as polyglycolide, polylactide and copolymers thereof. Those of skill in the art can readily determine an appropriate carrier system depending on various factors, including the desired rate of drug release and the desired dosage.
The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active compound into association with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active compound into association with a liquid earner or a finely divided solid carrier and then, if necessary, shaping the product into desired unit dosage form.
In addition to the aforementioned ingredients, the formulations may further include one or more optional accessory ingredient{s) utilized in the art of pharmaceutical formulations, e.g., diluents, buffers, flavoring agents, binders, surface active agents, thickeners, lubricants, suspending agents, preservatives (including antioxidants) and the like.
The activity of the compounds can be readily determined using no more than routine experimentation using, for example, any of the following assays.
~1-~-A2A '°'denosine Receptor Binding A
Membrane preparations:
Male Wistar rats (200-250 g) can be decapitated and the whole brain (minus brainstem, striatum and cerebellum) was dissected on ice. The brain tissues can be disrupted in a Polytron (setting 5) in 20 vols of 50 mM Tris HCI, pH 7.4. The homogenate can then be centrifuged at 48,000 g for 10 min and the pellet resuspended in Tris-HCI containing 2 ILJ/ml adenosine deaminase; type VI (Sigma Chemical Company, St. Louis., Mo., USA).
After a 30 min incubation at 37°C, the membranes can be centrifuged and the pellets stored at -70°C. Striatal tissues can be homogenized with a Polytron in 25 vol of 50 mM Tris HCI buffer containing 10 mM MgCh pH
7.4. The homogenate can be centrifuged at 48,000 g for 10 min at 4°C
and resuspended in Tris HC1 buffer containing 2 IU/ml adenosine deaminase.
After a 30 min incubation at 37°C, the membranes can be centrifuged and the pellet stored at -70°C.
Radioligand binding assays:
Binding of [3H]-DPCPX (1,3-dipropyl-8-cyclopentylxanthine) to rat brain membranes can be performed essentially according to the method previously described by Bruns et al., Proc. Natl. Acad. Sci. L1.S.A., 77:5547-5551 (1980), the contents of which are hereby incorporated by reference. In this method, displacement experiments are performed in 0.25 mI of buffer containing 1 nM [3H]-DPCPX, 100 ~cl of diluted membranes of rat brain (100 ~g of protein/assay) and at least 6-8 different concentrations of examined compounds. Non specific binding is determined in the presence of 10 ~M of CHA (N6cyclohexyladenosine) and this is always <_ 10% of the total binding. The incubation time is typically around 120 min at 25 °C.
Bound and free radioactivity can be separated by filtering the assay mixture through Whatman GFB glass-fiber filters, using a Brandel cell harvester (Gaithersburg, MD, USA). The incubation mixture is diluted with 3 ml of ice-cold incubation buffer, rapidly vacuum filtered and the filter is washed three times with 3 ml of incubation buffer. The filter bound radioactivity can be measured by liquid scintillation spectrometry. The protein concentration can be determined using known methodology, for example, using bovine albumin as a reference standard.
Hil 3 Adenoc_ine _R_ece~tor Binding Ac~v_ Receptor binding assays: Binding assays can be carried out according to the method described by Salvatore et al., Proc. Natl_. Acad. Sci. L1.S.A., 90:10365-10369 (1993), the contents of which are hereby incorporated by reference. In saturation studies using this method, an aliquot of membranes (8 mg protein/ml) from HEK-293 cells transfected with the human recombinant A3 adenosine receptor (Research Biochemical International, *rB
Natick, MA, USA) are incubated with from 10 to 12 different concentrations of [125]AB-MECA ranging from 0.1 to 5 nM. Competition experiments are carried out in duplicate in a final volume of 100 ~cl in test tubes containing 0.3 nM [125]AB-MECA, 50 mM Tris HCI buffer, 10 mM MgCI2, pH 7.4 and 20 ~cl of diluted membranes {12.4 mg protein/ml) and at least 6 to 8 different concentrations of examined ligands. Incubation time is typically around 60 min at 37°C.
Bound and free radioactivity are separated by filtering the assay mixture through Whatman GF/B glass-fiber filters using a Brandel cell harvester. Non-specific binding is defined as binding in the presence of 50 ,uM R-PIA and can be as high as about 30%. The incubation mixture is diluted with 3 ml of ice-cold incubation buffer, rapidly vacuum filtered and the filter is washed three times with 3 ml of incubation buffer. The filter bound radioactivity is counted in a Beckman gamma SSOOB y counter. The protein concentration can be determined according to known methodology, for example, using bovine albumin as reference standard.
Another suitable method for performing binding studies is the method reported in Baraldi et al., J. Med. Chem_, 39:802-806 ( 1996).
Data Analysis Inhibitory binding constant (K~) values were calculated from those of ICS according to Cheng & Prusoff equation (Cheng and Prusoff, Phanna~l., 22:3099-3108 (1973), the contents of which are hereby incorporated by reference), Ki = ICS~/(1+[ C*]/KD*), where [C*] is the concentration of the radioligand and KD* is its dissociation constant.
A weighted non linear least-squares curve fitting program, for example, LIGAND can be used for computer analysis of saturation and inhibition experiments. Data are typically expressed as geometric mean, with 95% or 99% confidence limits in parentheses.
S All synthesized compounds have been tested for their affinity at rat A1 and A2A and human A3 receptors.
EXAMPLES
The following examples illustrate aspects of this invention but should not be construed as limitations. The symbols and conventions used in these examples are intended to be consistent with those used in the contemporary, international, chemical literature, for example, the Joa~rnal of the American Chemical Society ("J.Am.Chem.Soc. ') and Tetrahedron.
Experimental Section Chemistry: Reactions were routinely monitored by thin-layer chromatography (TLC) on silica gel (precoated F254 Merck plates) and products visualized with iodine or aqueous potassium permanganate.
Infrared spectra (IR) were measured on a Perkin Elmer 257 instrument. 1H
NMR were determined in CDC13 or DMSO-d6 solutions with a Bruker AC
200 Spectrometer, with peak positions given in parts per million (8) downfield from tetramethylsilane as internal standard, and J values are given in Hz. Light petroleum ether refers to the fractions boiling at 46-60°C.
Melting points were determined on a Buchi-Tuttoli instrument and are uncorrected. Chromatography was performed with Merck 60-200 mesh silica gel. All products reported showed IR and I H NMR spectra in agreement with the assigned structures. Organic solutions were dried over anhydrous magnesium sulfate. Elemental analyses were performed by the microanalytical laboratory of Dipartimento di Chemica, University of Ferrara, and were within 0.4% of the theoretical values for C, H and N.
Example 1: General procedure for the preparation of 2',3'-O-isopropylidene-N6-(substituted-carbonylamino)adenosine-S'-N-ethyluronamide 2',3'-isopropylidene-NECA (0.43 mmol) was dissolved in freshly distilled dioxane (4 ml) and the appropriate acid chloride (1.3 eq.) and a catalytic amount of triethylamine {2-3 drops) were added. The mixture was refluxed under argon for 15 hours. The solvent was then removed under reduced pressure and the residue was purified by flash chromatography (CH2C12-EtOAc 20%) to afford the desired compounds.
Example 2: Preparation of 2',3'-O-isopropylidene-N6-(4-biphenyl-carbonylamino)adenosine-5'-N-ethyluronamide Following the procedure outlined in Example l, the title compound was prepared in an 80% yield as a pale yellow foam. IR (neat) cm-1 3445, 1720, 1640, 1575; 1H NMR (CDC13) b: 0.84 (t, 3H, J = 7); 1.39 (s, 3H); 1.62 (s, 3H); 3.02-3.11 (m, 2H); 4.71 (d, 1H, J = 2); 5.39-x.46 (m, 2H); 6.19 (d, 1H, J = 2); 6.66 (t, 1H, J = 2); 7.40-7.74 (m, 7H); 8.09-8.16 (m, 3H); 8.74 (s, 1H); 9.44 (bs, 1H). Anal. (C28H28N605) Calc'd.: C, 63.63; H, 5.34, N, 15.90. Found: C, 63.80; H, 5.37, N, 15.82.
Example 3: Preparation of 2',3'-O-isopropylidene-N6-(2,4-dichlorobenzyl-carbonylamino)adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 1, the title compound was prepared in an 67% yield as a white foam. IR (neat) cm-1 3440, 1730, 1625, 1565, 1210; jH NMR (CDC13) 8: 0.82 (t, 3H, J = 7); 1.17 (s, 3H); 1.26 (s, 3H); 2.99-3.13 (m, 2H); 4.32 (s, 2H); 4.72 (d, 1H, J = 1.8); 5.38-5.48 (m, 2H); 6.16 (d, 1H, J = 1.8); 6.56 (t, 1 H, J = 2); 7.20-7.32 (m, 3H); 7.41 (s, 1H); 8.20 (s, 1H); 8.67 (s, 1H); Anal. (C23H24N7O5C1~) Calc'd.: C, 57.73;
H, 5.06, N, 20.49. Found: C, 57.62; H, 4.99, N, 20.42.
WO 99/06053 PCT/US9$/16053 Example 4: Preparation of 2',3'-O-isopropylidene-N6-(4-methoxyphenyl-carbonylamino)adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 1, the title compound was prepared in an 85% yield as a white solid. IR (neat) cm-1 3445, 17I S, 1640, 1555, 1210; iH NMR (CDCl3) b: 0.85 (t, 3H, J = 7); 1.40 (s, 3H); 1.63 {s, 3H); 3.03-3.12 (m, 2H); 3.90 (s, 3H); 4.72 (d, 1H, J = 2); 5.41-5.49 (m, 2H); 6.16 (d, 1H, J = 2); 6.61 (t, 1H, J = 2); 7.01 (d, 2H, J = 9); 8.02 (s, 2H, J
= 9); 8.10 (s, 1H); 8.74 (s, 1H); 9.09 (bs, 1H). Anal. (C.,3H26N606) Calc'd.:
C, 57.26; H, 5.43, N, 17.42. Found: C, 57.15; H, 5.35, N, 17.28.
Example 5: Preparation of 2',3'-O-isopropylidene-N6-(2-chlorophenyl-carbonylamino)adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 1, the title compound was prepared in an 72% yield as a white foam. IR (neat) cm-1 3435, 1720, 1620, 1550, 1230; 1H NMR (CDCl3) 8: 0.83 (t, 3H, J = 7); 1.39 (s, 3H); 1.62 (s, 3H); 3.00-3.07 {m, 2H); 4.71 (d, 1H, J = 2); 5.41-5.44 (m, 2H); 6.18 (d, 1H, J = 2); 6.67 (t, 1H, J = 2); 7.30-7.44 (m, 3H); 7.71-7.76 (m, 1 H); 8.16 (s, 1H); 8.69 (s, 1H); 10.24 (bs, 1H). Anal. {C22H23N~OSC1) Calc'd.: C, 52.75;
H, 4.63, N, 19.57. Found: C, 52.59; H, 4.58, N, 19.43.
Example 6: Preparation of 2',3'-O-isopropylidene-N6-(phenyl-carbonylamino)adenosine-5'-N-ethylu ronamide Following the procedure outlined in Example 1, the title compound was prepared in an 90% yield as a pale yellow foam. IR (neat) cm-~ 3425, 1730, 1640, 1555, 1240; 1H NMR (CDC13) b: 0.79 (t, 3H, J = 7); 1.39 (s, 3H); 1.61 (s, 3H); 2.91-3.03 (m, 2H); 4.71 (d, 1H, J = 2); 5.45-5.52 (m, 2H);
6.18 (d, 1H, J = 2); 6.40 (t, 1H, J = 2); 7.27-7.53 (m, 3H); 7.83-7.87 (m, 2H);
and resuspended in Tris HC1 buffer containing 2 IU/ml adenosine deaminase.
After a 30 min incubation at 37°C, the membranes can be centrifuged and the pellet stored at -70°C.
Radioligand binding assays:
Binding of [3H]-DPCPX (1,3-dipropyl-8-cyclopentylxanthine) to rat brain membranes can be performed essentially according to the method previously described by Bruns et al., Proc. Natl. Acad. Sci. L1.S.A., 77:5547-5551 (1980), the contents of which are hereby incorporated by reference. In this method, displacement experiments are performed in 0.25 mI of buffer containing 1 nM [3H]-DPCPX, 100 ~cl of diluted membranes of rat brain (100 ~g of protein/assay) and at least 6-8 different concentrations of examined compounds. Non specific binding is determined in the presence of 10 ~M of CHA (N6cyclohexyladenosine) and this is always <_ 10% of the total binding. The incubation time is typically around 120 min at 25 °C.
Bound and free radioactivity can be separated by filtering the assay mixture through Whatman GFB glass-fiber filters, using a Brandel cell harvester (Gaithersburg, MD, USA). The incubation mixture is diluted with 3 ml of ice-cold incubation buffer, rapidly vacuum filtered and the filter is washed three times with 3 ml of incubation buffer. The filter bound radioactivity can be measured by liquid scintillation spectrometry. The protein concentration can be determined using known methodology, for example, using bovine albumin as a reference standard.
Hil 3 Adenoc_ine _R_ece~tor Binding Ac~v_ Receptor binding assays: Binding assays can be carried out according to the method described by Salvatore et al., Proc. Natl_. Acad. Sci. L1.S.A., 90:10365-10369 (1993), the contents of which are hereby incorporated by reference. In saturation studies using this method, an aliquot of membranes (8 mg protein/ml) from HEK-293 cells transfected with the human recombinant A3 adenosine receptor (Research Biochemical International, *rB
Natick, MA, USA) are incubated with from 10 to 12 different concentrations of [125]AB-MECA ranging from 0.1 to 5 nM. Competition experiments are carried out in duplicate in a final volume of 100 ~cl in test tubes containing 0.3 nM [125]AB-MECA, 50 mM Tris HCI buffer, 10 mM MgCI2, pH 7.4 and 20 ~cl of diluted membranes {12.4 mg protein/ml) and at least 6 to 8 different concentrations of examined ligands. Incubation time is typically around 60 min at 37°C.
Bound and free radioactivity are separated by filtering the assay mixture through Whatman GF/B glass-fiber filters using a Brandel cell harvester. Non-specific binding is defined as binding in the presence of 50 ,uM R-PIA and can be as high as about 30%. The incubation mixture is diluted with 3 ml of ice-cold incubation buffer, rapidly vacuum filtered and the filter is washed three times with 3 ml of incubation buffer. The filter bound radioactivity is counted in a Beckman gamma SSOOB y counter. The protein concentration can be determined according to known methodology, for example, using bovine albumin as reference standard.
Another suitable method for performing binding studies is the method reported in Baraldi et al., J. Med. Chem_, 39:802-806 ( 1996).
Data Analysis Inhibitory binding constant (K~) values were calculated from those of ICS according to Cheng & Prusoff equation (Cheng and Prusoff, Phanna~l., 22:3099-3108 (1973), the contents of which are hereby incorporated by reference), Ki = ICS~/(1+[ C*]/KD*), where [C*] is the concentration of the radioligand and KD* is its dissociation constant.
A weighted non linear least-squares curve fitting program, for example, LIGAND can be used for computer analysis of saturation and inhibition experiments. Data are typically expressed as geometric mean, with 95% or 99% confidence limits in parentheses.
S All synthesized compounds have been tested for their affinity at rat A1 and A2A and human A3 receptors.
EXAMPLES
The following examples illustrate aspects of this invention but should not be construed as limitations. The symbols and conventions used in these examples are intended to be consistent with those used in the contemporary, international, chemical literature, for example, the Joa~rnal of the American Chemical Society ("J.Am.Chem.Soc. ') and Tetrahedron.
Experimental Section Chemistry: Reactions were routinely monitored by thin-layer chromatography (TLC) on silica gel (precoated F254 Merck plates) and products visualized with iodine or aqueous potassium permanganate.
Infrared spectra (IR) were measured on a Perkin Elmer 257 instrument. 1H
NMR were determined in CDC13 or DMSO-d6 solutions with a Bruker AC
200 Spectrometer, with peak positions given in parts per million (8) downfield from tetramethylsilane as internal standard, and J values are given in Hz. Light petroleum ether refers to the fractions boiling at 46-60°C.
Melting points were determined on a Buchi-Tuttoli instrument and are uncorrected. Chromatography was performed with Merck 60-200 mesh silica gel. All products reported showed IR and I H NMR spectra in agreement with the assigned structures. Organic solutions were dried over anhydrous magnesium sulfate. Elemental analyses were performed by the microanalytical laboratory of Dipartimento di Chemica, University of Ferrara, and were within 0.4% of the theoretical values for C, H and N.
Example 1: General procedure for the preparation of 2',3'-O-isopropylidene-N6-(substituted-carbonylamino)adenosine-S'-N-ethyluronamide 2',3'-isopropylidene-NECA (0.43 mmol) was dissolved in freshly distilled dioxane (4 ml) and the appropriate acid chloride (1.3 eq.) and a catalytic amount of triethylamine {2-3 drops) were added. The mixture was refluxed under argon for 15 hours. The solvent was then removed under reduced pressure and the residue was purified by flash chromatography (CH2C12-EtOAc 20%) to afford the desired compounds.
Example 2: Preparation of 2',3'-O-isopropylidene-N6-(4-biphenyl-carbonylamino)adenosine-5'-N-ethyluronamide Following the procedure outlined in Example l, the title compound was prepared in an 80% yield as a pale yellow foam. IR (neat) cm-1 3445, 1720, 1640, 1575; 1H NMR (CDC13) b: 0.84 (t, 3H, J = 7); 1.39 (s, 3H); 1.62 (s, 3H); 3.02-3.11 (m, 2H); 4.71 (d, 1H, J = 2); 5.39-x.46 (m, 2H); 6.19 (d, 1H, J = 2); 6.66 (t, 1H, J = 2); 7.40-7.74 (m, 7H); 8.09-8.16 (m, 3H); 8.74 (s, 1H); 9.44 (bs, 1H). Anal. (C28H28N605) Calc'd.: C, 63.63; H, 5.34, N, 15.90. Found: C, 63.80; H, 5.37, N, 15.82.
Example 3: Preparation of 2',3'-O-isopropylidene-N6-(2,4-dichlorobenzyl-carbonylamino)adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 1, the title compound was prepared in an 67% yield as a white foam. IR (neat) cm-1 3440, 1730, 1625, 1565, 1210; jH NMR (CDC13) 8: 0.82 (t, 3H, J = 7); 1.17 (s, 3H); 1.26 (s, 3H); 2.99-3.13 (m, 2H); 4.32 (s, 2H); 4.72 (d, 1H, J = 1.8); 5.38-5.48 (m, 2H); 6.16 (d, 1H, J = 1.8); 6.56 (t, 1 H, J = 2); 7.20-7.32 (m, 3H); 7.41 (s, 1H); 8.20 (s, 1H); 8.67 (s, 1H); Anal. (C23H24N7O5C1~) Calc'd.: C, 57.73;
H, 5.06, N, 20.49. Found: C, 57.62; H, 4.99, N, 20.42.
WO 99/06053 PCT/US9$/16053 Example 4: Preparation of 2',3'-O-isopropylidene-N6-(4-methoxyphenyl-carbonylamino)adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 1, the title compound was prepared in an 85% yield as a white solid. IR (neat) cm-1 3445, 17I S, 1640, 1555, 1210; iH NMR (CDCl3) b: 0.85 (t, 3H, J = 7); 1.40 (s, 3H); 1.63 {s, 3H); 3.03-3.12 (m, 2H); 3.90 (s, 3H); 4.72 (d, 1H, J = 2); 5.41-5.49 (m, 2H); 6.16 (d, 1H, J = 2); 6.61 (t, 1H, J = 2); 7.01 (d, 2H, J = 9); 8.02 (s, 2H, J
= 9); 8.10 (s, 1H); 8.74 (s, 1H); 9.09 (bs, 1H). Anal. (C.,3H26N606) Calc'd.:
C, 57.26; H, 5.43, N, 17.42. Found: C, 57.15; H, 5.35, N, 17.28.
Example 5: Preparation of 2',3'-O-isopropylidene-N6-(2-chlorophenyl-carbonylamino)adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 1, the title compound was prepared in an 72% yield as a white foam. IR (neat) cm-1 3435, 1720, 1620, 1550, 1230; 1H NMR (CDCl3) 8: 0.83 (t, 3H, J = 7); 1.39 (s, 3H); 1.62 (s, 3H); 3.00-3.07 {m, 2H); 4.71 (d, 1H, J = 2); 5.41-5.44 (m, 2H); 6.18 (d, 1H, J = 2); 6.67 (t, 1H, J = 2); 7.30-7.44 (m, 3H); 7.71-7.76 (m, 1 H); 8.16 (s, 1H); 8.69 (s, 1H); 10.24 (bs, 1H). Anal. {C22H23N~OSC1) Calc'd.: C, 52.75;
H, 4.63, N, 19.57. Found: C, 52.59; H, 4.58, N, 19.43.
Example 6: Preparation of 2',3'-O-isopropylidene-N6-(phenyl-carbonylamino)adenosine-5'-N-ethylu ronamide Following the procedure outlined in Example 1, the title compound was prepared in an 90% yield as a pale yellow foam. IR (neat) cm-~ 3425, 1730, 1640, 1555, 1240; 1H NMR (CDC13) b: 0.79 (t, 3H, J = 7); 1.39 (s, 3H); 1.61 (s, 3H); 2.91-3.03 (m, 2H); 4.71 (d, 1H, J = 2); 5.45-5.52 (m, 2H);
6.18 (d, 1H, J = 2); 6.40 (t, 1H, J = 2); 7.27-7.53 (m, 3H); 7.83-7.87 (m, 2H);
8.19 (s, 1H); 8.63 (s, 1H); 9.18 (bs, 1H). Anal. (C22H24N605) Calc'd.: C, 58.40; H, 5.35, N, 18.57. Found: C, 58.23; H, 5.28, N, 18.45.
Example 7: General procedure for the preparation of 2',3'-O-isopropylidene-N6-(substituted-carbamoylamino)adenosine-5'-N-ethyluronamide 2',3'-isopropylidene-NECA (0.43 mmol) was dissolved in freshly distilled THF (4 ml) and the appropriate isocyanate (1.3 eq.) and a catalytic amount of triethylamine (two drops) were added. The mixture was refluxed under argon for 18 hours. The solvent was then removed under reduced pressure and the residue was purified by flash chromatography (CH2C12-EtOAc 20%) to afford the desired compounds.
Example 8: Preparation of 2',3'-O-isopropylidene-N6-(benzyl carbamoylamino)adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 7, the title compound was prepared in an 82% yield as a white solid, mp 109-1 I 1 °C. IR
(KBr) cm-1 3440, 1740, 1610, 1550, 1210; 1H NMR (CDCl3) b: 0.76 (t, 3H, J = 7);
1.21 (s, 3H); 1.63 (s, 3H); 2.96-3.03 (m, 2H); 4.64 (d, 1 H, J = 6); 4.71 (d, 1H,J=
1.8); 5.44-5.48 (m, 2H); 6.16 (d, IH, J = 2); 6.52 (t, 1H, J = 2); 7.26-7.38 (m, 5H); 8.14 (s, 1H); 8.45 (s, 1H); 8.46 (s, 1H), 9.79 (bs, IH). Anal.
(C26H31N705) Calc'd.: C, 59.87; H, 5.99, N, 18.80. Found: C, 60.00; H, 6.03, N, 18.88.
Example 9: Preparation of 2',3'-O-isopropylidene-N6-{4-sulfonamido-phenylcarbamoylamino)adenosine-5'-N-ethylurouamide Following the procedure outlined in Example 7, the title compound was prepared in an 60% yield as a white solid, mp 207°C. IR (KBr) cm 1 3450-3250, 1730, 1610, 1565, 1360, 1230; IH NMR (CDC13) 8: 0.57 (t, 3H, J = 7); 1.35 (s, 3H); 1.54 (s, 3H); 2.96-3.03 (m, 2H); 4.60 (d, 1H, J = 2);
5.47-5.50 (m, 2H); 6.47 (d, 1H, J = 2); 6.60 (t, 1H, J = 2); 7.26 (d, 2H, J =
Example 7: General procedure for the preparation of 2',3'-O-isopropylidene-N6-(substituted-carbamoylamino)adenosine-5'-N-ethyluronamide 2',3'-isopropylidene-NECA (0.43 mmol) was dissolved in freshly distilled THF (4 ml) and the appropriate isocyanate (1.3 eq.) and a catalytic amount of triethylamine (two drops) were added. The mixture was refluxed under argon for 18 hours. The solvent was then removed under reduced pressure and the residue was purified by flash chromatography (CH2C12-EtOAc 20%) to afford the desired compounds.
Example 8: Preparation of 2',3'-O-isopropylidene-N6-(benzyl carbamoylamino)adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 7, the title compound was prepared in an 82% yield as a white solid, mp 109-1 I 1 °C. IR
(KBr) cm-1 3440, 1740, 1610, 1550, 1210; 1H NMR (CDCl3) b: 0.76 (t, 3H, J = 7);
1.21 (s, 3H); 1.63 (s, 3H); 2.96-3.03 (m, 2H); 4.64 (d, 1 H, J = 6); 4.71 (d, 1H,J=
1.8); 5.44-5.48 (m, 2H); 6.16 (d, IH, J = 2); 6.52 (t, 1H, J = 2); 7.26-7.38 (m, 5H); 8.14 (s, 1H); 8.45 (s, 1H); 8.46 (s, 1H), 9.79 (bs, IH). Anal.
(C26H31N705) Calc'd.: C, 59.87; H, 5.99, N, 18.80. Found: C, 60.00; H, 6.03, N, 18.88.
Example 9: Preparation of 2',3'-O-isopropylidene-N6-{4-sulfonamido-phenylcarbamoylamino)adenosine-5'-N-ethylurouamide Following the procedure outlined in Example 7, the title compound was prepared in an 60% yield as a white solid, mp 207°C. IR (KBr) cm 1 3450-3250, 1730, 1610, 1565, 1360, 1230; IH NMR (CDC13) 8: 0.57 (t, 3H, J = 7); 1.35 (s, 3H); 1.54 (s, 3H); 2.96-3.03 (m, 2H); 4.60 (d, 1H, J = 2);
5.47-5.50 (m, 2H); 6.47 (d, 1H, J = 2); 6.60 (t, 1H, J = 2); 7.26 (d, 2H, J =
9);
7.74 (d, 2H, J = 9); 8.60 (s, 1 H); 8.64 (s, I H), 9.22 (s, 1 H); 10.47 (bs, 1 H);
WO 99/06053 PCT/US98/1b053 12.05 (s, 1H). Anal. (C22H26N807S) Calc'd.: C, 48.35; H, 4.79, N, 20.50.
Found: C, 48.27; H, 4.79, N, 20.63.
Example 10: Preparation of 2',3'-O-isopropylidene-Nb-(4-acetyl-phenylcarbamoyiamino)adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 7, the title compound was prepared in an 77% yield as a white solid, mp 204°C. IR (KBr) cm 1 3430, 1740, 1720, 1630, 1545, 1240; 1 H NMR (CDC13) b: 0.83 (t, 3H, J =
7); 1.42 (s, 3H); 1.64 (s, 3H); 2.61(s, 3H); 2.99-3.08 (m, 2H); 4.74 (d, 1H, J
= 2); 5.47-5.55 (m, 2H); 6.21 (d, 1H, J = 2); 6.47 (t, 1H, J = 2); 7.74 (d, 2H, J
= 9); 7.98 (d, 2H, J = 9); 8.21 (s, 1 H); 8.63 (s, 1 H), 8.67 (bs, 1 H); 12.01 (s, 1H). Anal. (C22H26N807) Calc'd.: C, 51.36; H, 5.09, N, 21.78. Found: C, 51.35; H, 5.12, N, 21.66.
Example 11: Preparation of 2',3'-O-isopropylidene-Nb-((R)-a-phenylethyl-carbamoylamino)adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 7, the title compound was prepared in an 65% yield as a white solid, mp 110-111 °C. IR (KBr) cm-i 3430, 1730, 1620, 1555, 1230; 1H NMR (CDC13) b: 0.68 (t, 3H, J = 7);
1.41 (s, 3H); 1.63 (s, 3H); 1.64 (d, 3H, J = 7); 2.87-2.96 (m, 2H); 4.71 (d, IH, J =1.8); 5.16 (m, 1H); 5.48-5.51 (m, 2H); 6.16 (d, 1H, J = 2); 6.52 (bs, 1H); 7.25-7.408 (m, 5H); 8.15 (s, 1H); 8.51 (s, 1H); 9.84 (bs, 1H}, 10.94 (s, 1H). Anal. (C23H29N705) Calc'd.: C, 57.13; H, 6.05, N, 20.28. Found: C, 57.O1;H,5.99,N,20.33.
Example 12: Preparation of 2',3'-O-isopropylidene-Nb-((S)-a-phenylethyl-carbamoylamino)adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 7, the title compound was prepared in an 82% yield as a white solid, mp 109-111 °C. IR (ICBr) cm-1 3445, 1725, 1615, 1560, 1230; 1H NMR (CDC13) b: 0.85 (t, 3H, J = 7);
1.40 (s, 3H); I .63 (s, 3H); 1.64 (d, 3H, J = 7); 3.00-3.09 (m, 2H); 4.72 (d, 1H, J = 1.8); 5.17 (m, IH); 5.39-5.50 (m, 2H); 6.14 (d, 1H, J = 2); 6.57 (bs, 1H); 7.26-7.45 (m, 5H); 8.12 (s, 1H); 8.51 (s, 1H); 9.81 (bs, 1H); 10.87 (s, 1H). Anal. {C23H29N7~5) Calc'd.: C, 57.13; H, 6.05, N, 20.28. Found: C, 57.25; H, 6.11, N, 20.37.
Example 13: Preparation of 2',3'-O-isopropylidene-N6-(5-methyl-isoxazol-3-yl-carbamoylamino)adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 7, the title compound was prepared in an 83% yield as a white solid, mp I20°C. IR (KBr) cm t 3450, 1720, 1620, 1550, 1215; tH N~IR (CDC13) b: 0.82 (t, 3H, J = 7); 1.20 (s, 3H); 1.41 (s, 3H); 2.44 (s, 3H); 3.00-3.07 (m, 2H); 4.74 (d, 1 H, J = 2);
5.46-5.50 (m, 2H); 6.21 (d, 1H, J = 2); 6.55 (t, 1H, J = 2); 6.70 (s, 1H);
8.37 (s, 1H); 8.63 (s, 1H); 9.39 (bs, 1H); 12.34 (bs, 1H). Anal. (C2pH24N8~6) Calc'd.: C, 50.84; H, 5.12, N, 23.72. Found: C, 50.96; H, 5.18, N, 23.64.
Example 14: Preparation of 2',3'-O-isopropylidene-N6-(I,3,4-thiadiazol-2-yl-carbamoylamino)adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 7, the title compound was prepared in a 74% yield as a yellow solid, mp 148°C. IR (KBr) cm 1 3430, 1730, 1615, 1560, 1240; IH NMR (CDC13) 8: 0.86 (t, 3H, J = 7); 1.44 (s, 3H); 1.67 (s, 3H); 3.07-3.14 {m, 2H); 4.78 (d, 1H, J = 1.8); 4.78 (d, 1H, J
= 1.8); 5.47-5.51 (m, 2H); 6.24 (d, I H, J = 1.8); 6.52 (t, 1H, J = 2); 8.36 (s, 1H); 8.75 (s, 1H); 8.91 (bs, 1H), 9.40 (bs, IH); 11.72 (bs, IH). Anal.
(CtgH2tN905S) Calc'd.: C, 45.47; H, 4.45, N, 26.51. Found: C, 45.39; H, 4.44, N, 26.46.
Example 15: Preparation of 2',3'-O-isopropylidene-N6-(4-n-propyloxy-phenylcarbamoylamino)adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 7, the title compound was prepared in an 70% yield as a pale yellow foam. IR (neat) cm-1 3440, 1725, 1620, 1555, 1220; 1H NMR (CDCl3) b: 0.71 (t, 3H, J = 7); 0.90 (s, 3H, J = 7); 1.40 (s, 3H); 1.62 (s, 3H); 1.81-2.03 (m, 2H); 2.95-3.01 (m, 2H);
3.90 (t, 2H, J = 7); 4.71 (d, 1H, J = 1.8); 5.46-5.51 (m, 2H); 6.19 (d, 1H, J
=
1.8); 6.51 (t, 1H, J = 2); 6.89 (d, 2H, J = 9); 7.48 (d, 2H, J = 9); 8.25 (s, 1H);
8.56 (s, 1H); 8.80 (bs, 1H), 11.48 (s, 1H). Anal. (C22H25N705) Calc'd.: C, 56.52; H, 5.39, N, 20.97. Found: C, 56.44; H, 5.42, N, 21.03.
Example 16: Preparation of 2',3'-O-isopropylidene-N6-bis-(4-nitrophenyl-carbamoylamino)adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 7, the title compound was prepared in an 65% yield as a yellow solid, mp 261 °C. IR (KBr) crri 1 3430, 1730, 1620, 1555, 1210; 1H NMR (DMSO d6) 8: 0.56 (t, 3H, J = 7);
1.34 (s, 3H); 1.54 (s, 3H); 2.67-2.83 (m, 2H); 4.61 (d, 1H, J = 1.8); 5.47-5.50 (m, 2H); 6.47 (d, 1H, J = 1.8); 7.61 (t, 1 H, J = 2); 7.71 (d, 2H, J = 9);
7.91 (d, 2H, J = 9); 8.22 (d, 2H, J = 9); 8.24 (s, 1 H), 8.64 (d, 2H, J = 9); 9.72 (s, 1 H);
7.74 (d, 2H, J = 9); 8.60 (s, 1 H); 8.64 (s, I H), 9.22 (s, 1 H); 10.47 (bs, 1 H);
WO 99/06053 PCT/US98/1b053 12.05 (s, 1H). Anal. (C22H26N807S) Calc'd.: C, 48.35; H, 4.79, N, 20.50.
Found: C, 48.27; H, 4.79, N, 20.63.
Example 10: Preparation of 2',3'-O-isopropylidene-Nb-(4-acetyl-phenylcarbamoyiamino)adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 7, the title compound was prepared in an 77% yield as a white solid, mp 204°C. IR (KBr) cm 1 3430, 1740, 1720, 1630, 1545, 1240; 1 H NMR (CDC13) b: 0.83 (t, 3H, J =
7); 1.42 (s, 3H); 1.64 (s, 3H); 2.61(s, 3H); 2.99-3.08 (m, 2H); 4.74 (d, 1H, J
= 2); 5.47-5.55 (m, 2H); 6.21 (d, 1H, J = 2); 6.47 (t, 1H, J = 2); 7.74 (d, 2H, J
= 9); 7.98 (d, 2H, J = 9); 8.21 (s, 1 H); 8.63 (s, 1 H), 8.67 (bs, 1 H); 12.01 (s, 1H). Anal. (C22H26N807) Calc'd.: C, 51.36; H, 5.09, N, 21.78. Found: C, 51.35; H, 5.12, N, 21.66.
Example 11: Preparation of 2',3'-O-isopropylidene-Nb-((R)-a-phenylethyl-carbamoylamino)adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 7, the title compound was prepared in an 65% yield as a white solid, mp 110-111 °C. IR (KBr) cm-i 3430, 1730, 1620, 1555, 1230; 1H NMR (CDC13) b: 0.68 (t, 3H, J = 7);
1.41 (s, 3H); 1.63 (s, 3H); 1.64 (d, 3H, J = 7); 2.87-2.96 (m, 2H); 4.71 (d, IH, J =1.8); 5.16 (m, 1H); 5.48-5.51 (m, 2H); 6.16 (d, 1H, J = 2); 6.52 (bs, 1H); 7.25-7.408 (m, 5H); 8.15 (s, 1H); 8.51 (s, 1H); 9.84 (bs, 1H}, 10.94 (s, 1H). Anal. (C23H29N705) Calc'd.: C, 57.13; H, 6.05, N, 20.28. Found: C, 57.O1;H,5.99,N,20.33.
Example 12: Preparation of 2',3'-O-isopropylidene-Nb-((S)-a-phenylethyl-carbamoylamino)adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 7, the title compound was prepared in an 82% yield as a white solid, mp 109-111 °C. IR (ICBr) cm-1 3445, 1725, 1615, 1560, 1230; 1H NMR (CDC13) b: 0.85 (t, 3H, J = 7);
1.40 (s, 3H); I .63 (s, 3H); 1.64 (d, 3H, J = 7); 3.00-3.09 (m, 2H); 4.72 (d, 1H, J = 1.8); 5.17 (m, IH); 5.39-5.50 (m, 2H); 6.14 (d, 1H, J = 2); 6.57 (bs, 1H); 7.26-7.45 (m, 5H); 8.12 (s, 1H); 8.51 (s, 1H); 9.81 (bs, 1H); 10.87 (s, 1H). Anal. {C23H29N7~5) Calc'd.: C, 57.13; H, 6.05, N, 20.28. Found: C, 57.25; H, 6.11, N, 20.37.
Example 13: Preparation of 2',3'-O-isopropylidene-N6-(5-methyl-isoxazol-3-yl-carbamoylamino)adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 7, the title compound was prepared in an 83% yield as a white solid, mp I20°C. IR (KBr) cm t 3450, 1720, 1620, 1550, 1215; tH N~IR (CDC13) b: 0.82 (t, 3H, J = 7); 1.20 (s, 3H); 1.41 (s, 3H); 2.44 (s, 3H); 3.00-3.07 (m, 2H); 4.74 (d, 1 H, J = 2);
5.46-5.50 (m, 2H); 6.21 (d, 1H, J = 2); 6.55 (t, 1H, J = 2); 6.70 (s, 1H);
8.37 (s, 1H); 8.63 (s, 1H); 9.39 (bs, 1H); 12.34 (bs, 1H). Anal. (C2pH24N8~6) Calc'd.: C, 50.84; H, 5.12, N, 23.72. Found: C, 50.96; H, 5.18, N, 23.64.
Example 14: Preparation of 2',3'-O-isopropylidene-N6-(I,3,4-thiadiazol-2-yl-carbamoylamino)adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 7, the title compound was prepared in a 74% yield as a yellow solid, mp 148°C. IR (KBr) cm 1 3430, 1730, 1615, 1560, 1240; IH NMR (CDC13) 8: 0.86 (t, 3H, J = 7); 1.44 (s, 3H); 1.67 (s, 3H); 3.07-3.14 {m, 2H); 4.78 (d, 1H, J = 1.8); 4.78 (d, 1H, J
= 1.8); 5.47-5.51 (m, 2H); 6.24 (d, I H, J = 1.8); 6.52 (t, 1H, J = 2); 8.36 (s, 1H); 8.75 (s, 1H); 8.91 (bs, 1H), 9.40 (bs, IH); 11.72 (bs, IH). Anal.
(CtgH2tN905S) Calc'd.: C, 45.47; H, 4.45, N, 26.51. Found: C, 45.39; H, 4.44, N, 26.46.
Example 15: Preparation of 2',3'-O-isopropylidene-N6-(4-n-propyloxy-phenylcarbamoylamino)adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 7, the title compound was prepared in an 70% yield as a pale yellow foam. IR (neat) cm-1 3440, 1725, 1620, 1555, 1220; 1H NMR (CDCl3) b: 0.71 (t, 3H, J = 7); 0.90 (s, 3H, J = 7); 1.40 (s, 3H); 1.62 (s, 3H); 1.81-2.03 (m, 2H); 2.95-3.01 (m, 2H);
3.90 (t, 2H, J = 7); 4.71 (d, 1H, J = 1.8); 5.46-5.51 (m, 2H); 6.19 (d, 1H, J
=
1.8); 6.51 (t, 1H, J = 2); 6.89 (d, 2H, J = 9); 7.48 (d, 2H, J = 9); 8.25 (s, 1H);
8.56 (s, 1H); 8.80 (bs, 1H), 11.48 (s, 1H). Anal. (C22H25N705) Calc'd.: C, 56.52; H, 5.39, N, 20.97. Found: C, 56.44; H, 5.42, N, 21.03.
Example 16: Preparation of 2',3'-O-isopropylidene-N6-bis-(4-nitrophenyl-carbamoylamino)adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 7, the title compound was prepared in an 65% yield as a yellow solid, mp 261 °C. IR (KBr) crri 1 3430, 1730, 1620, 1555, 1210; 1H NMR (DMSO d6) 8: 0.56 (t, 3H, J = 7);
1.34 (s, 3H); 1.54 (s, 3H); 2.67-2.83 (m, 2H); 4.61 (d, 1H, J = 1.8); 5.47-5.50 (m, 2H); 6.47 (d, 1H, J = 1.8); 7.61 (t, 1 H, J = 2); 7.71 (d, 2H, J = 9);
7.91 (d, 2H, J = 9); 8.22 (d, 2H, J = 9); 8.24 (s, 1 H), 8.64 (d, 2H, J = 9); 9.72 (s, 1 H);
10.63 (bs, 1H); 12.24 (s, 1H). Anal. (C29H2gN1001o) Calc'd.: C,51.48; H, 4.17, N, 20.70. Found: C, 51.56 ; H, 4.16, N, 20.69.
Example 17: Preparation of 2',3'-O-isopropylideoe-N6-bis-(5-chloro-pyridin-2-yl-carbamoylamino)adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 7, the title compound was prepared in a 60% yield as a yellow foam. IR (neat) cm-1 3440, 1710, 1630, 1540, 1220; 1H NMR (CDCl3) b: 0.58 (t, 3H, 3 = 7); 1.35 (s, 3H); 1.54 (s, 3H); 2.71-2.85 (m, 2H); 4.60 (d, 1H, J = 2); 5.47-5.51 (m, 2H); 6.47 (d, WO 99/Ob053 PCT/US98/16053 1H, J = 2); 7.52 (t, 1H, J = 2); 7.79-7.92 (m, 4H); 8.34-8.38 (m, 2H); 8.60 (s, 1H); 8.65 (s, 1H), 10.66 (bs, 1H); 12.22 (bs, 1H). Anal. (C2~Hz6N806C12) Calc'd.: C, 51.52; H, 4.16, N, 17.80. Found: C, 51.55; H, 4.13, N, 17.84.
Example 18: General procedure for the preparation of Nb-(substituted)adenosine-5'-N-ethyluronamide A solution of the isopropylidene derivative {0.084 mmol) in aqueous 1N HCl (6 ml) and dioxane (6 ml) was stirred at 65°C for 1 hour. The solvent was then removed under reduced pressure and the residue was crystallized from ethanol to afford the desired compounds.
Example 19: Preparation of Nb-(4-biphenyl-carbonylamino)-adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 18, the title compound was prepared in a 66% yield as a white solid, mp 221 °C. IR (KBr) cm-1 3500-3100, 1720, 1615, 1550; 1H NMR (DMSO d6) s: 1.07 (t, 3H, J = 7);
3.17-3.24 (m, 2H); 4.23-4.25 (m, 1H); 4.36 {d, 1H, J = 2); 4.71-4.73 (m, 1H);
5.70 (d, 1H, J = 8); 5.79 (d, 1H, J = 4); 6.13 (d, 1H, J = 8); 7.43-7.53 (m, 3H); 7.78-7.89 (m, 4H); 8.16 (d, 2H, J = 11 ); 8.46 (t, 1 H, J = 4), 8.80 (s, 1 H);
8.82 (s, 1H); 11.34 (bs, 1H). Anal. (CZSH24N6~5) Calc'd.: C, 61.47; H, 4.95, N, 17.20. Found: C, 61.57; H, 5.00, N, 17.28.
Example 20: Preparation of Nb-(2,4-dichlorobenzyl-carbonylamino)-adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 18, the title compound was prepared in a 66% yield as a white solid, mp 132-134°C. IR (KBr) cm 3450-3050, 1730, 1635, 1545, 1230; 1H NMR (DMSO d6) b: 1.08 (t, 3H, J =
7); 3.18-3.26 (m, 2H); 3.34 (s, 2H); 4.12-4.15 (m, 1H); 4.30 (s, 1H); 4.=19-4.62 (m, 2H); 5.57 (d, IH, J = 8); 5.77 (d, 1 H, J = 4); 5.95 (d, 1 H, J = 8);
7.44-7.51 (m, 3H); 8.19 (s, 1 H); 8.39 (s, 1 H), 8.95 (bs, 1 H). Anal.
(C2~H2oN605) Calc'd.: C, 56.60; H, 4.75, N, 19.80. Found: C, 56.60; H, 4.77, N, 19.84.
Example 21: Preparation of N6-(4-methoxyphenyl-carbonylamino)-adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 18, the title compound was prepared in an 80% yield as a white solid, mp 167-169°C. IR (KBr) cm-1 3550-3150, 1710, 1640, 1530, 1270; ~H NMR (DMSO d6) 8: 1.07 (t, 3H, J
= 7); 3.15-3.23 (m, 2H); 3.86 (s, 3H); 4.20-4.24 (m, 1H); 4.35 (d, 1H, J = 2);
4.70-4.75 (m, 1H); 5.69 (d, 1H, J = 7); 5.77 (d, 1H, J = 4); 6.11 (d, 1H, J =
7); 7.08 (d, 2H, J = 9); 8.04 (d, 2H, J = 9); 8.44 (bs, 1H); 8.77 (s, 1H);
8.78 (s, 1H), 11.12 (s, 1H). Anal. (C2~H22N606) Calc'd.: C, 54.30; H, S.Oi, N, 18.99. Found: C, 54.38; H, 4.98, N, 19.02.
Example 22: Preparation of N6-(4-chlorophenyl-carbonylamino)-adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 18, the title compound was prepared in a 78% yield as a white solid, mp 179-180°C. IR. (KBr) cm-1 3510-3050, 1720, 1660, 1510, 1250; IH NMR (DMSO d6) b: 1.06 (t, 3H, J =
7); 3.15-3.22 (m, 2H); 4.22 (bs, 1H); 4.34 (s, 1H); 4.68-4.70 (m, 1H); 5.66 (d, 1 H, J = 7); 5.76 (d, 1 H, J = 4); 6.10 (d, 1 H, J = 7); 7.43-7.61 (m, 4H);
8.44 (bs, 1H); 8.67 (s, 1H); 8.82 (bs, 1H); 11.53 (bs, 1H). Anai.
(C19H19N6O5) Calc'd.: C, 55.47; H, 4.66, N, 20.43. Found: C, 55.53; H, 4.72, N, 20:50.
Example 23: Preparation of N6-(phenyl-carbonylamino)-adenosine-5'-N-ethvluronamide Following the procedure outlined in Example 18, the title compound was prepared in a 67% yield as a white solid, mp 157-159°C. IR (KBr) cm 3500-3000, 1715, 1630, 1535, 1260; 1H NMR (DMSO d6) b: 1.03 (t, 3H, J =
*rB
7); 3.16-3.19 (m, 2H); 3.74 (bs, 1H); 4.22 (bs, 1H); 4.36 (s, 1H); 4.51-4.63 (m, 1H); 6.13 (d, 1H, J = 6.8); 7.48-7.59 (m, 3H); 8.04-8.11 (m, 2H); 8.52 (bs, 1H), 8.79 (s, 1H); 8.83 (s, 1H); 11.22 (s, 1H). Anal. (C19H2oN605) Calc'd.: C, 55.34; H, 4.89, N, 20.38. Found: C, 55.47; H, 4.93, N, 20.43.
Example 24: Preparation of N6-(benzylcarbamoylamino)-adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 18, the title compound was prepared in a 70% yield as a white solid, mp 184-186°C. IR (ICBr) cm 1 3500-3100, 1675, 1620, 1565, 1520, 1310; iH NMR (DMSO d6) 8: 1.06 (t, 3H, J = 7); 3.10-3.20 (m, 2H); 4.05 (bs, 1H); 4.17-4.20 (m, 1H); 4.36 (s, 1H);
4.47-4.50 (m, 2H); 4.55-4.65 (m, 1H); 6.05-6.10 (m, 1H); 7.17-7.40 (m, 5H);
8.30-8.40 (m, 1H); 8.62 (s, 1H); 8.86 (s, 1H); 9.50-9.60 (m, 1H); 10.30 (bs, 1H). Anai. (C.,oH24N~O5) Calc'd.: C, 54.29; H, 5.47, N, 22.16. Found: C, 54.36; H, 5.52, N, 22.08.
Example 25: Preparation of N6-(4-sulfonamido-phenylcarbamoyl)-adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 18, the title compound was prepared in a 63% yield as a white solid, mp 183-185 °C. IR {KBr) cm i 3550-3050, 1715, 1630, 1545, 1370, 1250; 1H NMR (DMSO d6) 8: 1.07 (t, 3H, J = 7); 3.16-3.26 (m, 2H); 4.21 (bs, 1 H); 4.36 (s, 1 H); 4.63-4.69 (m, 1 H};
5.62 (d, 1H, J = 6.8); 5.78 (d, 1H, J = 4); 6.11 (d, 1H, J = 6.8); 7.31 (bs, 2H);
7.81 (s, 4H); 8.49 (bs, 1 H); 8.75 (s, 1 H), 8.89 (s, 1 H); 9.69 (bs, 1 H);
Example 17: Preparation of 2',3'-O-isopropylideoe-N6-bis-(5-chloro-pyridin-2-yl-carbamoylamino)adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 7, the title compound was prepared in a 60% yield as a yellow foam. IR (neat) cm-1 3440, 1710, 1630, 1540, 1220; 1H NMR (CDCl3) b: 0.58 (t, 3H, 3 = 7); 1.35 (s, 3H); 1.54 (s, 3H); 2.71-2.85 (m, 2H); 4.60 (d, 1H, J = 2); 5.47-5.51 (m, 2H); 6.47 (d, WO 99/Ob053 PCT/US98/16053 1H, J = 2); 7.52 (t, 1H, J = 2); 7.79-7.92 (m, 4H); 8.34-8.38 (m, 2H); 8.60 (s, 1H); 8.65 (s, 1H), 10.66 (bs, 1H); 12.22 (bs, 1H). Anal. (C2~Hz6N806C12) Calc'd.: C, 51.52; H, 4.16, N, 17.80. Found: C, 51.55; H, 4.13, N, 17.84.
Example 18: General procedure for the preparation of Nb-(substituted)adenosine-5'-N-ethyluronamide A solution of the isopropylidene derivative {0.084 mmol) in aqueous 1N HCl (6 ml) and dioxane (6 ml) was stirred at 65°C for 1 hour. The solvent was then removed under reduced pressure and the residue was crystallized from ethanol to afford the desired compounds.
Example 19: Preparation of Nb-(4-biphenyl-carbonylamino)-adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 18, the title compound was prepared in a 66% yield as a white solid, mp 221 °C. IR (KBr) cm-1 3500-3100, 1720, 1615, 1550; 1H NMR (DMSO d6) s: 1.07 (t, 3H, J = 7);
3.17-3.24 (m, 2H); 4.23-4.25 (m, 1H); 4.36 {d, 1H, J = 2); 4.71-4.73 (m, 1H);
5.70 (d, 1H, J = 8); 5.79 (d, 1H, J = 4); 6.13 (d, 1H, J = 8); 7.43-7.53 (m, 3H); 7.78-7.89 (m, 4H); 8.16 (d, 2H, J = 11 ); 8.46 (t, 1 H, J = 4), 8.80 (s, 1 H);
8.82 (s, 1H); 11.34 (bs, 1H). Anal. (CZSH24N6~5) Calc'd.: C, 61.47; H, 4.95, N, 17.20. Found: C, 61.57; H, 5.00, N, 17.28.
Example 20: Preparation of Nb-(2,4-dichlorobenzyl-carbonylamino)-adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 18, the title compound was prepared in a 66% yield as a white solid, mp 132-134°C. IR (KBr) cm 3450-3050, 1730, 1635, 1545, 1230; 1H NMR (DMSO d6) b: 1.08 (t, 3H, J =
7); 3.18-3.26 (m, 2H); 3.34 (s, 2H); 4.12-4.15 (m, 1H); 4.30 (s, 1H); 4.=19-4.62 (m, 2H); 5.57 (d, IH, J = 8); 5.77 (d, 1 H, J = 4); 5.95 (d, 1 H, J = 8);
7.44-7.51 (m, 3H); 8.19 (s, 1 H); 8.39 (s, 1 H), 8.95 (bs, 1 H). Anal.
(C2~H2oN605) Calc'd.: C, 56.60; H, 4.75, N, 19.80. Found: C, 56.60; H, 4.77, N, 19.84.
Example 21: Preparation of N6-(4-methoxyphenyl-carbonylamino)-adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 18, the title compound was prepared in an 80% yield as a white solid, mp 167-169°C. IR (KBr) cm-1 3550-3150, 1710, 1640, 1530, 1270; ~H NMR (DMSO d6) 8: 1.07 (t, 3H, J
= 7); 3.15-3.23 (m, 2H); 3.86 (s, 3H); 4.20-4.24 (m, 1H); 4.35 (d, 1H, J = 2);
4.70-4.75 (m, 1H); 5.69 (d, 1H, J = 7); 5.77 (d, 1H, J = 4); 6.11 (d, 1H, J =
7); 7.08 (d, 2H, J = 9); 8.04 (d, 2H, J = 9); 8.44 (bs, 1H); 8.77 (s, 1H);
8.78 (s, 1H), 11.12 (s, 1H). Anal. (C2~H22N606) Calc'd.: C, 54.30; H, S.Oi, N, 18.99. Found: C, 54.38; H, 4.98, N, 19.02.
Example 22: Preparation of N6-(4-chlorophenyl-carbonylamino)-adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 18, the title compound was prepared in a 78% yield as a white solid, mp 179-180°C. IR. (KBr) cm-1 3510-3050, 1720, 1660, 1510, 1250; IH NMR (DMSO d6) b: 1.06 (t, 3H, J =
7); 3.15-3.22 (m, 2H); 4.22 (bs, 1H); 4.34 (s, 1H); 4.68-4.70 (m, 1H); 5.66 (d, 1 H, J = 7); 5.76 (d, 1 H, J = 4); 6.10 (d, 1 H, J = 7); 7.43-7.61 (m, 4H);
8.44 (bs, 1H); 8.67 (s, 1H); 8.82 (bs, 1H); 11.53 (bs, 1H). Anai.
(C19H19N6O5) Calc'd.: C, 55.47; H, 4.66, N, 20.43. Found: C, 55.53; H, 4.72, N, 20:50.
Example 23: Preparation of N6-(phenyl-carbonylamino)-adenosine-5'-N-ethvluronamide Following the procedure outlined in Example 18, the title compound was prepared in a 67% yield as a white solid, mp 157-159°C. IR (KBr) cm 3500-3000, 1715, 1630, 1535, 1260; 1H NMR (DMSO d6) b: 1.03 (t, 3H, J =
*rB
7); 3.16-3.19 (m, 2H); 3.74 (bs, 1H); 4.22 (bs, 1H); 4.36 (s, 1H); 4.51-4.63 (m, 1H); 6.13 (d, 1H, J = 6.8); 7.48-7.59 (m, 3H); 8.04-8.11 (m, 2H); 8.52 (bs, 1H), 8.79 (s, 1H); 8.83 (s, 1H); 11.22 (s, 1H). Anal. (C19H2oN605) Calc'd.: C, 55.34; H, 4.89, N, 20.38. Found: C, 55.47; H, 4.93, N, 20.43.
Example 24: Preparation of N6-(benzylcarbamoylamino)-adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 18, the title compound was prepared in a 70% yield as a white solid, mp 184-186°C. IR (ICBr) cm 1 3500-3100, 1675, 1620, 1565, 1520, 1310; iH NMR (DMSO d6) 8: 1.06 (t, 3H, J = 7); 3.10-3.20 (m, 2H); 4.05 (bs, 1H); 4.17-4.20 (m, 1H); 4.36 (s, 1H);
4.47-4.50 (m, 2H); 4.55-4.65 (m, 1H); 6.05-6.10 (m, 1H); 7.17-7.40 (m, 5H);
8.30-8.40 (m, 1H); 8.62 (s, 1H); 8.86 (s, 1H); 9.50-9.60 (m, 1H); 10.30 (bs, 1H). Anai. (C.,oH24N~O5) Calc'd.: C, 54.29; H, 5.47, N, 22.16. Found: C, 54.36; H, 5.52, N, 22.08.
Example 25: Preparation of N6-(4-sulfonamido-phenylcarbamoyl)-adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 18, the title compound was prepared in a 63% yield as a white solid, mp 183-185 °C. IR {KBr) cm i 3550-3050, 1715, 1630, 1545, 1370, 1250; 1H NMR (DMSO d6) 8: 1.07 (t, 3H, J = 7); 3.16-3.26 (m, 2H); 4.21 (bs, 1 H); 4.36 (s, 1 H); 4.63-4.69 (m, 1 H};
5.62 (d, 1H, J = 6.8); 5.78 (d, 1H, J = 4); 6.11 (d, 1H, J = 6.8); 7.31 (bs, 2H);
7.81 (s, 4H); 8.49 (bs, 1 H); 8.75 (s, 1 H), 8.89 (s, 1 H); 9.69 (bs, 1 H);
11.92 (bs, 1H). Anal. (C19H22N807S} Calc'd.: C, 45.06; H, 4.38, N, 22.12.
Found: C, 45.15; H, 4.36, N, 22.07.
Example 26: Preparation of N6-(4-acetyl-phenylcarbamoyl)-adenosine-5'-N-ethyluronamide *rB
Following the procedure outlined in Example 18, the title compound was prepared in an 83% yield as a white solid, mp 187°C. IR (KBr) cm 1 3550-3100, 1740, 1720, 1630, 1525, 1215; 1H NMR (DMSO d6) b: 1.08 (t, 3H, J = 7); 2.55 (s, 2H); 3.16-3.23 (m, 2H); 4.18-4.20 (m, 1H}; 4.21 {d, 1H, J
S = 2); 4.45 (bs, 1 H); 4.64-4.69 (m, 1 H); 6.11 (d, 1 H, J = 7); 7.77 (d, 2H, J =
9); 7.98 (d, 2H, J = 9); 8.49 (bs, 1H), 8.76 (s, 1H); 8.88 (s, 1H); 10.62 {bs, 1H); 11.94 (bs, 1H). Anal. (C21H23N'706) Calc'd.: C, 53.73; H, 4.94, N, 20.89. Found: C, 53.80; H, 4.95, N, 20.93.
Example 27: Preparation of N6-((R)-a-phenylethylcarbamoyl)-adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 18, the title compound was prepared in a 6S% yield as a white solid, mp 153-155 °C. IR (KBr) cm 1 3550-3100, 1720, 1635, 1535, 1240; ~H NMR (DMSO d6) 8: 1.05 (t, 3H, J =
7); 1.51 (d, 3H, J = 8); 3.14-3.21 (m, 2H); 3.35-3.41 (m, 1H); 4.20 (s, 1H);
4.36 (s, 1H); 4.93-5.00 {m, 3H); 6.09 (d, 1H, J = 6.8); 7.25-7.39 (m, SH);
8.48 (bs, 1H); 8.65 (s, 1H); 8.89 (s, 1H); 9.51 (bs, 1H); 10.38 (bs, 1H).
Anal.
(C21H25N7~5) Calc'd.: C, 53.50; H, 5.34, N, 20.80. Found: C, 53.55; H, 5.38, N, 20.85.
Example 28: Preparation of N6-((S)-a-phenylethylcarbamoyl)-adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 18, the title compound _ was prepared in a 72% yield as a white solid, mp 151 °C. IR {KBr) cm-3550-3100, 1725, 1630, 1525, 1240; 1H NMR (DMSO d6) 8: 1.06 (t, 3H, J =
7); 1.49 (d, 3H, J = 8); 3.15-3.21 (m, 2H);, 3.28-3.39 {m, 1H); 4.00 (bs, 1H);
4.35 (s, 1H); 4.61-4.66 {m, 1H); 4.95-5.01 (m, 1H); 6.07 (d, 1H, J = 6.8);
7.24-7.39 (m, 5H); 8.48 (bs, 1H), 8.64 (s, 1H); 8.83 (s, 1H); 9.57 (bs, 1H);
10.12 (bs, 1H). Anal. (C21H25N705) Calc'd.: C, 53.50; H, 5.34, N, 20.80.
Found: C, 53.44; H, 5.34, N, 20.77.
Example 29: Preparation of N6-(5-methyl-isoxazol-3-yl-carbamoyl)-adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 18, the title compound was prepared in a 58% yield as a white solid, mp 197°C. IR (KBr) crri l 3550-3050, 1715, 1620, 1535, 1240; tH NMR (DMSO d6) 8: 1.06 (t, 3H, J =
7); 2.40 (s, 3H); 3.12-3.25 (m, 2H); 4.21-4.23 (m, iH); 4.36 (d, 1H, J = 2);
4.64-4.70 (m, 1 H); 5.68 (m, 2H); 6.09 (d, 1 H, J = 6.8); 6.67 (s, 1 H); 8.46 (t, 1 H, J = 6), 8.74 (s, 1 H); 8.84 (s, 1 H); 10.75 (bs, 1 H); 12.19 (bs, 1 H).
Anal.
(Cl~H2oN806) Calc'd.: C, 47.22; H, 4.66, N, 25.91. Found: C, 47.14; H, 4.69, N, 25.96.
Example 30: Preparation of N6-(1,3,4-thiadiazol-2-yl-carbamoyl)-adenosine-5'-N-ethvluronamide Following the procedure outlined in Example 18, the title compound was prepared in a 65% yield as a white solid, mp 197°C. IR (KBr) cm-I
3450-3100, 1720, 1635, 1515, 1240; 1H NMR (DMSO d6) b: 1.06 (t, 3H, J =
7); 3.15-3.22 (m, 2H); 3.72 (bs, 2H); 4.21-4.24 (m, 1H); 4.36 (d, 1H, 3 = 2);
4.65-4.70 (m, 1 H); 6.12 (d, I H, J = 6. 8); 8.45 (t, 1 H, J = 7); 8.80 (s, 1 H);
8.89 (s, 1H); 9.19 (s, 1H); 10.35 (bs, 1H); 11.38 (bs, 1H). Anal.
(C15HI~N405S) Calc'd.: C, 49.31; H, 4.69, N, 15.33. Found: C, 49.22; H, 4.72, N, 15.33.
Example 31: Preparation of N6-(4-n-propoxy-phenylcarbamoyl)-adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 18, the title compound was prepared in a 66% yield as a white solid, mp 270°C. IR (KBr) cm 1 3450-3050, 1720, 1630, 1535, 1240; 1H NMR (DMSO d6) 8: 0.95 (t, 3H, J =
7); 1.09 (t, 3H, J = 7); 1.65-1.75 (m, 2H); 3.06-3.23 (m, 2H); 3.67 (bs, 2H);
3.89 (t, 2H, J = 7); 4.18-4.20 (m, 1 H); 4.33 (s, 1 H); 4.51-4.61 (m, 1 H);
6.05 (d, 1H, J = 6.8); 6.91 (d, 2H, J = 9); 7.52 (d, 2H, J = 9); 7.92 (t, 1H, J =
7);
8.22 (s, IH); 8.52 (s, 1H); 9.65 (bs, 1H); 11.52 (bs, 1H). Anal.
(C22H2~N~O6) Calc'd.: C, 5.61; H, 5.61, N, 20.19. Found: C, 54.50; H, 5.66, N, 20.06.
Example 32: Preparation of N6-bis-(4-nitrophenylcarbamoyl~
adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 18, the title compound was prepared in a 52% yield as a white solid, mp 213 °C. IR {KBr) cm 1 3500-3050, 1720, 1630, 1545, 1340, 1220; ~ H NMR (DMSO d6) S: 1.07 (t, 3H, J = 7); 3.12-3.23 (m, 2H); 4.21-4.23 (m, IH); 4.35 (d, IH, J = 2); 4.64-4.69 (m, 1H); 5.69 (bs, 2H); 6.09 (d, 1H, J = 6.8); 7.68 (d, 2H, J = 9); 7.91 (d, 2H, J = 9); 8.18-8.29 (m, 4H); 8.83 (s, 1H); 9.69 (s, 1H); 10.68 (bs, 1H);
Found: C, 45.15; H, 4.36, N, 22.07.
Example 26: Preparation of N6-(4-acetyl-phenylcarbamoyl)-adenosine-5'-N-ethyluronamide *rB
Following the procedure outlined in Example 18, the title compound was prepared in an 83% yield as a white solid, mp 187°C. IR (KBr) cm 1 3550-3100, 1740, 1720, 1630, 1525, 1215; 1H NMR (DMSO d6) b: 1.08 (t, 3H, J = 7); 2.55 (s, 2H); 3.16-3.23 (m, 2H); 4.18-4.20 (m, 1H}; 4.21 {d, 1H, J
S = 2); 4.45 (bs, 1 H); 4.64-4.69 (m, 1 H); 6.11 (d, 1 H, J = 7); 7.77 (d, 2H, J =
9); 7.98 (d, 2H, J = 9); 8.49 (bs, 1H), 8.76 (s, 1H); 8.88 (s, 1H); 10.62 {bs, 1H); 11.94 (bs, 1H). Anal. (C21H23N'706) Calc'd.: C, 53.73; H, 4.94, N, 20.89. Found: C, 53.80; H, 4.95, N, 20.93.
Example 27: Preparation of N6-((R)-a-phenylethylcarbamoyl)-adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 18, the title compound was prepared in a 6S% yield as a white solid, mp 153-155 °C. IR (KBr) cm 1 3550-3100, 1720, 1635, 1535, 1240; ~H NMR (DMSO d6) 8: 1.05 (t, 3H, J =
7); 1.51 (d, 3H, J = 8); 3.14-3.21 (m, 2H); 3.35-3.41 (m, 1H); 4.20 (s, 1H);
4.36 (s, 1H); 4.93-5.00 {m, 3H); 6.09 (d, 1H, J = 6.8); 7.25-7.39 (m, SH);
8.48 (bs, 1H); 8.65 (s, 1H); 8.89 (s, 1H); 9.51 (bs, 1H); 10.38 (bs, 1H).
Anal.
(C21H25N7~5) Calc'd.: C, 53.50; H, 5.34, N, 20.80. Found: C, 53.55; H, 5.38, N, 20.85.
Example 28: Preparation of N6-((S)-a-phenylethylcarbamoyl)-adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 18, the title compound _ was prepared in a 72% yield as a white solid, mp 151 °C. IR {KBr) cm-3550-3100, 1725, 1630, 1525, 1240; 1H NMR (DMSO d6) 8: 1.06 (t, 3H, J =
7); 1.49 (d, 3H, J = 8); 3.15-3.21 (m, 2H);, 3.28-3.39 {m, 1H); 4.00 (bs, 1H);
4.35 (s, 1H); 4.61-4.66 {m, 1H); 4.95-5.01 (m, 1H); 6.07 (d, 1H, J = 6.8);
7.24-7.39 (m, 5H); 8.48 (bs, 1H), 8.64 (s, 1H); 8.83 (s, 1H); 9.57 (bs, 1H);
10.12 (bs, 1H). Anal. (C21H25N705) Calc'd.: C, 53.50; H, 5.34, N, 20.80.
Found: C, 53.44; H, 5.34, N, 20.77.
Example 29: Preparation of N6-(5-methyl-isoxazol-3-yl-carbamoyl)-adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 18, the title compound was prepared in a 58% yield as a white solid, mp 197°C. IR (KBr) crri l 3550-3050, 1715, 1620, 1535, 1240; tH NMR (DMSO d6) 8: 1.06 (t, 3H, J =
7); 2.40 (s, 3H); 3.12-3.25 (m, 2H); 4.21-4.23 (m, iH); 4.36 (d, 1H, J = 2);
4.64-4.70 (m, 1 H); 5.68 (m, 2H); 6.09 (d, 1 H, J = 6.8); 6.67 (s, 1 H); 8.46 (t, 1 H, J = 6), 8.74 (s, 1 H); 8.84 (s, 1 H); 10.75 (bs, 1 H); 12.19 (bs, 1 H).
Anal.
(Cl~H2oN806) Calc'd.: C, 47.22; H, 4.66, N, 25.91. Found: C, 47.14; H, 4.69, N, 25.96.
Example 30: Preparation of N6-(1,3,4-thiadiazol-2-yl-carbamoyl)-adenosine-5'-N-ethvluronamide Following the procedure outlined in Example 18, the title compound was prepared in a 65% yield as a white solid, mp 197°C. IR (KBr) cm-I
3450-3100, 1720, 1635, 1515, 1240; 1H NMR (DMSO d6) b: 1.06 (t, 3H, J =
7); 3.15-3.22 (m, 2H); 3.72 (bs, 2H); 4.21-4.24 (m, 1H); 4.36 (d, 1H, 3 = 2);
4.65-4.70 (m, 1 H); 6.12 (d, I H, J = 6. 8); 8.45 (t, 1 H, J = 7); 8.80 (s, 1 H);
8.89 (s, 1H); 9.19 (s, 1H); 10.35 (bs, 1H); 11.38 (bs, 1H). Anal.
(C15HI~N405S) Calc'd.: C, 49.31; H, 4.69, N, 15.33. Found: C, 49.22; H, 4.72, N, 15.33.
Example 31: Preparation of N6-(4-n-propoxy-phenylcarbamoyl)-adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 18, the title compound was prepared in a 66% yield as a white solid, mp 270°C. IR (KBr) cm 1 3450-3050, 1720, 1630, 1535, 1240; 1H NMR (DMSO d6) 8: 0.95 (t, 3H, J =
7); 1.09 (t, 3H, J = 7); 1.65-1.75 (m, 2H); 3.06-3.23 (m, 2H); 3.67 (bs, 2H);
3.89 (t, 2H, J = 7); 4.18-4.20 (m, 1 H); 4.33 (s, 1 H); 4.51-4.61 (m, 1 H);
6.05 (d, 1H, J = 6.8); 6.91 (d, 2H, J = 9); 7.52 (d, 2H, J = 9); 7.92 (t, 1H, J =
7);
8.22 (s, IH); 8.52 (s, 1H); 9.65 (bs, 1H); 11.52 (bs, 1H). Anal.
(C22H2~N~O6) Calc'd.: C, 5.61; H, 5.61, N, 20.19. Found: C, 54.50; H, 5.66, N, 20.06.
Example 32: Preparation of N6-bis-(4-nitrophenylcarbamoyl~
adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 18, the title compound was prepared in a 52% yield as a white solid, mp 213 °C. IR {KBr) cm 1 3500-3050, 1720, 1630, 1545, 1340, 1220; ~ H NMR (DMSO d6) S: 1.07 (t, 3H, J = 7); 3.12-3.23 (m, 2H); 4.21-4.23 (m, IH); 4.35 (d, IH, J = 2); 4.64-4.69 (m, 1H); 5.69 (bs, 2H); 6.09 (d, 1H, J = 6.8); 7.68 (d, 2H, J = 9); 7.91 (d, 2H, J = 9); 8.18-8.29 (m, 4H); 8.83 (s, 1H); 9.69 (s, 1H); 10.68 (bs, 1H);
12.26 (bs, IH). Anal. (C26H29N1o01o) Calc'd.: C, 48.67; H, 4.56, N, 21.83.
Found: C, 48.74; H, 4.60, N, 21.88.
Example 33: Preparation of N6-bis-{5-chloro-pyridin-2-yl-carbamoyl)-adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 18, the title compound was prepared in a 68% yield as a white solid, mp 220°C. IR (ICBr) cm-1 3550-3100, 1725, 1630, 1525, 1250; 1H NMR (DMSO d6) b: 1.06 (t, 3H, J =
7); 3.15-3.22 (m, 2H); 4.22-4.24 (m, 1H); 4.36 (d, 2H, J = 2); 4.64-4.69 (m, 1H); 5.95 (bs, 2H); 6.10 (d, 1H, J = 6.8); 7.80-8.06 (m, 6H); 8.39 (t, 1H, J =
7); 8.76 (s, 1H); 8.93 (s, IH); 10.39 (bs, 1H); 12.11 {bs, 1H). Anal.
{C24H22N806C12) Calc'd.: C, 48.91; H, 3.76, N, 19.01. Found: C, 49.01;
H, 3.77, N, 18.89.
Example 34. Binding Assays Procedures for preparing rat brain membranes and Chinese hamster ovary (CHO) cell membranes were as reported30, 38, 44_ For binding experients, membrane homogenates were frozen and stored at -20°C for greater than 2 months. Adenosine deaminase (ADA) was purchased from Boehringer Mannheim (Indianapolis, IN). [3H]R-PIA was purchased from Amersham (Arlington Heights, IL), and [3H]CGS 21680 was purchased from DuPont NEN
(Boston, MA). [~25I]-AB-MECA was prepared as described by Olah et al.3o The binding of [1251]_AB-MECA to CHO cells stably transfected with the rat A3 receptor clone or to HEK-293 cells stably expressing the human A3 receptor, was performed essentially as described 2g, 30, 3s, a4. Assays were performed in SO mM Tris/ 10 mM MgCl2/ 1 mM EDTA buffer (adjusted to pH
8.26 at 5 °C) in glass tubes containing 100 ul of the membrane suspension, 50 pl of [~25I]-AB-MECA (final concentration 0.3 nM), and 50 ~1 of inhibitor.
1 S Inhibitors were routinely dissolved in DMSO. Concentrations of DMSO in incubations never exceeded 1%; this concentration did not influence [~25I]-AB-MECA binding. Incubations were carried out in duplicate for 1 h at 37°C, and were terminated by rapid filtration over Whatman GF/B filters using a Brandell cell harvester (Brandell, Gaithersburg, MD). Tubes were washed three times with 3 mL of buffer. Radioactivity was determined in a Beckman gamma SSOOB y-counter.
Nonspecific binding was determined in the presence of 200 uM NECA.
Kl values were calculated according to Cheng-Prusoff~5 assuming a Kd for [1251]-AB-MECA of 1.48 nM. Binding of [3H]R-PIA to A1 receptors from rat cortical membranes and of [3H]CGS 21680 to A2A receptors for rat striatal membranes was performed as described previously.46 Adenosine deaminase (2 units/mL) was present during the preparation of the membranes. Additional deaminase was not added during incubation with the radioligand.
Bin~~ of 1~35SJC"TTP-v-The binding for [35S]GTP-y-S (Ameersham, Chicago IL, specific activity 1275 Ci/mmol) was carried out using rat RBL-2H3 mast cell membranes by the general method of Lorenzen et a1.4~ Membranes were suspended in a buffer containing 50 mM Tris, 3 units/mL adenosine deaminase, $ 100 mM NaCI, and 10 mM MgCl2 (pH 7.4) and a protein concentration of 1 to ~g per tube. The membrane suspension was preincubated with 10 GDP, R-PIA and/or riboflavin in a final volume of 125 ~cL buffer at 30°C for 60 min and then transferred to ice for 20 min. [35S]GTP-y-S was added to a final concentration of 0.1 nM in a total volume of 500 ~L and the mixture was 10 incubated for 30 min at 30°C. Non-specific binding was determined in the presence of 10 ~M GTP-y-S (Sigma, St. Louis MO). Incubation of the reaction mixture was terminated by filtration over a GFB glass filter using a Brandell cell harvester and washed with the same buffer.
RP~!!lt~ and Discussion The derivatives 3a-o (the compounds in Examples 19-33, sequentially) were tested in radioligand binding assays for affinity at rat brain A1, A2A and A3 receptors, and the results are summarized in Table 1.
a a .~
Q ~ o ,, a o a b U M c'I
f'1 a Q
+, x ,o x :.fl ._ s o , y o ~n 0 .~ ~ .. v, ~
c x ~'' - o o . ~ ~ "., M
C
O ~ Q
~ ~ .-.
a -~
_ x ' /w z 1 z ,, o O q U
/ \ O ~' C >, z z N
~
G z ~ ~ ~, o ~, , =w w z .
~
a .
Q x ~ x v a N
w CC ~
U
~n o a ~p pp ,n, -. o0 N N w0 ~J
N v'1 00 ~ ~ 'j M ~ "' d' O O (~i O d' ~~ ~ c ~ O N
a~ ~ O O O ~ ~ ~D N
O O ~ d' M fuel O ~ N O
i~ M l~ ~' M N ~ n V7 ~ M ~ 00 ~ N
O ~ - ~ O f~ M N N M d' a I~ ~f G1 ~ ~ M M ~ M N O ~ 00 M V1N 00 I~ ~O~ M V'1O
00 t~00 M ~ ~ -~M V1 ~ ~'.., ~_ \
O
..~ O ~ O O O O N ~, O
00 OO d' ~ N N M N ~ GM1N N O~pO
_ r~r~-i 'H -H 1-I~ -f-i-H-t-I~ -H
c~ ~ O GMIOOOp ~ ~ ~f _I~~ O
N N '~~D ~ ,_,~ N '~ 00 Ov N
~" N N
~N ~ ~.~ ~ M G\ N M 00 M M ~t 'cY
~' Ct ~ ~ -H -~I'~M
~ M M ~' O V7 ~ ~ N ~ ~ v7 .-.N N
x x x x x x z x x z z z ~ x ~ z z z ;,' ~~,, z c~ ' U a N z U ~ ; ~ ~ ~ M ~ .~
S ay v a~ =. o c~:.
.~.. N Z Z N
~' U O ~ ~ o x 0 U U Z,M m ' ' ~ ~ M
N a. o z H v et ~ cnU 23t3 ~ ~
d. ~
~ M
Q.
x ~ x x x x x x x x x x x x o x z z ' a U
LL ~ M M M M M M M M M '~ ~
M M C
M
M
U
~n ~n o a ~_ M w I
I
a M . ...
a M
C s w a v_ M C_ Q, O ~-I -~I N U
w.r_ Q N
O Q
r. cd p" V
v-~
"' k G
N V~ 'b C ~ G N
o " ~ U
C U ~ y<
.-.p ~.. vi U y C pp G1 ~ ~'1r, W
-H -~ ice.._c3 O
C\ ~ ~ U
N .~~.. U
y.
M
a ~
a ~
z ' w ~?
a C~ U 2 _ N
a a a N
a, a. p.
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N U
U
b a v n o ~ n _ o w o ~ M
U
~' 39 Baraldi demonstrated that certain N6-substituted-arylcarbamoyl adenosine-uronamides are full agonists in inhibiting adenylate cyclase via rat A3 receptors, but that the compounds showed relatively high activity with respect to adenosine A1 receptors39. There is a similarity between A1 and A3 receptors. Small modifications in the length of chain at the N6 position can alter the affinity versus At or A3 receptors. The present binding data demonstrate that the presence of aryl- or alkaryl-carbamoylic chains at the N6-position bring about an interaction with the A3 receptor subtype. Also, the data demonstrate that heteroaryl-carbamoyl, heteroaryl-carboxamide, aryl-carboxamide, and alkaryl-carboxamide substitutions at the N6-position bring about an interaction with the A1 receptor subtype.
In the series of urea-derivatives (Compounds 3f o) a substituted-phenyl (Compounds 3g, h and m) or substituted-benzyl (Compounds 3f, i and j) group leads to relatively high affinity and selectivity at A3 adenosine receptors as compared with the affinity for the At and A., receptors.
Derivatives 3g, 3h and 3m showed a relatively high affinity (9.7-107 nM) at A3 receptors with varying degrees of A3/At selectivity. In particular, compound 3g was Iess active than IB-MECA (9.7 nM vs. 1.1 nM) but showed selectivity for A3 vs. either A1 or AZA receptors which is comparable to IB-MECA.
Compound 3g also showed a relatively high affinity, in the nanomolar range, at human A3 adenosine receptors (56.1 ~ 9.1 nM), confirming the relatively high affinity of this compound versus this receptor subtype, independent of species. The lipophilicity of para substituents on a phenyl ring play a significant role in A3 affinity.
-~0-Substitution of the phenyl ring with a heterocycle (as in Compounds 3k-1) causes the compounds to lose affinity and selectivity for A3 receptors, and increase in affinity for the Ai receptor subtype.
Disubstitution at the N6 position (for example, Compounds 3n-o) have diminished activity at A2A and A3 receptors, but have relatively high affinity for the A t receptor.
In the benzylic series, Compounds 3 f, i, and j showed an SAR pattern comparable to the phenylic series in Baraldi39. In fact, the unsubstituted Compound 3f showed affinity and selectivity at A3 receptor very similar to the previously reported phenyl derivative.3~
The stereochemistry of substituents at the N6 position is also important in determining the affinity of these compounds for the A3 receptor.
For example, comparing the two diastereomeric Compounds 3i and 3j in which substituents at N6 are of opposite configuration, the R-isomer (3i) is more potent and selective that the S-isomer (3j) at the rat A3 adenosine receptor subtype. Those of skill in the art can separate compounds with stereocenters adjacent to the N6 position using routine enantiopurification methods and evaluate the individual stereoisomers for their affinity and selectivity using no more than routine experimentation.
A functional assay indicated that Compound 3g acted as a full agonist at rat A3 receptor. The assay involved the agonist-induced inhibition of binding of guanine nucleotide to rat RBL-2H3 mast cell membranes, which contain a high density of A3 receptors.
Figure 1 shows that compound 3g was about as effective as the potent A3 agonists I-AB-MECA and Cl-IB-MECA increased binding of [35S]GTP-y-S in a dose dependent manner and with greater potency than NECA.
Example 35. Pharmaceutical Formulations (A) Transdermal System - for 1000 patches Ingredients Amount Active compound 100g Silicone fluid 450g Colloidal silicon dioxide 2g The silicone fluid and active compound are mixed together and the colloidal silicone dioxide is added to increase viscosity. The material is then dosed into a subsequent heat sealed polymeric laminate including the following: polyester release liner, skin contact adhesive composed of silicone or acrylic polymers, a control membrane which is a polyolefin, and an impermeable backing membrane made of a polyester multilaminate. The resulting laminated sheet is then cut into 10 sq. cm patches (B) Oral Tablet - For 1000 Tablets Ingredients Amount Active compound SOg Starch ~0g Magnesium Stearate Sg The active compound and the starch are granulated with water and dried. Magnesium stearate is added to the dried granules and the mixture is thoroughly blended. The blended mixture is compressed into tablets.
(C) Injection - for 1000, 1mL Ampules *rB
Ingredients Amount Active compound log Buffering Agents q.s.
Propylene glycol 400mg S Water for injection q.s.100omL
The active compound and buffering agents are dissolved in the propylene glycol at about SO°C. The water for injection is then added with stirring and the resulting solution is filtered, filled into ampules, sealed and sterilized by autoclaving.
(D) Continuous Injection - for 1000 mL
Ingredients Amount Active compound log Buffering agents q.s.
Water for injection q.s.loooml.
25 REFERENCES:
The following references have been referred to herein. These references are incorporated herein by reference in their entirety.
1. Libert, F., Schiffmann, S.N.: Lefort, A., Parmentier, M; Gerard, C.; Dumont, J E.; Vanderhaeghen, J.J.; Vassart, G. The orphan receptor _ 43 _ cDNA RDC7 encodes an A1 adenosine receptor. E.~1991, 10, 1677-1682.
2. Maenhaut, C., Sande, J.V.; Libert, F; Abramowicz, M.;
Pannentier, M.; Vanderhaeghen, J.J.; Dumont, J.E.; Vassart, G.; Schiffmann, S. RDC8 codes for an adenosine A2 receptor with physiological constitutive activity. Biochem. Biophys. Res. .ommun. 1990, 173, 1169-1178.
3. Stehle, J.H.; Rivkees, S.A.; Lee, J.J.; Weaver, D.R.; Deeds, J.D.;
Reppert, S.M. Molecular cloning and expression of the cDNA for a novel A2-adenosine receptor subtype. Mol. Endocrinol. 1992, 6, 384-393.
4. Zhou, Q.Y.; Li, C.Y.; Olah, M.E.; Johnson, R.A.; Stiles, G.L.;
Civelli, 0. Molecular cloning and characterization of an adenosine receptor-the A3 adenosine receptor. Proc Natl Acad Sci USA 1992, 89, 7432-7436.
5. Londos, C., Cooper, D.M.; Wolff. J. Subclasses of external adenosine receptors. Prop Natl. Acad. Sci. LISA 1980, 77, 2551-2554.
6. Hamprecht, B.; Van Calker, D. Nomenclature of adenosine receptors. Trendy Pharmacol. Sci. 1985,6, 153-154.
7. Ukena, D.; Olsson, R.A.; Daly, J.W. Definition of subclasses of adenosine receptors associated with adenylate cyclase: interaction of adenosine analogs with inhibitory A1 receptors and stimulatory A., receptors.
C.'an J. Phvsiol. Pharmacol. 1987, 65. 365-376.
8. Ramkumar, V., Stiles, G.L.; Beaven, M. A.; Ali, H. The A3AR is the unique adenosine receptor which facilitates release of allergic mediators in mast cells. a~ Biol. Chem. 1993, 268, 6871-6890.
9. Abbracchio, M.P.; Brambilla, R.; Ceruti, S.: Kim, H.O.; von Lubitz, D.K.J.E.; Jacobson, K.A., Cattabeni, F. G-protein-dependent activation of phospholipase C by adenosine A3 receptors in rat brain. ~1.
pharmacol. 1995, 48, 1038-1045.
10. Bruns, R.F.; Daly, J.W.; Snyder, S.H. Adenosine receptors in brain membranes: binding of N-cyclohexyl [3H] adenosine and 1,3-diethyl-8(3H] phenylxanthine. Proc Natl Acad ~ci USA 1980, 77, 5547-5551.
11. Oisson, R.A.; Kusachi, S.; Thompson, S.D.; IJkena, D., Padgett, W.; Daly, J.W. N6-substituted N-alkyladenosine-51-uronamides: bifunctional ligands having recognition groups for A1 and A2 adenosine receptors. .L
Med. Chem. 1986, 29, 1683-1689.
12. Jacobson, K.A.; Nikodijevic, 0.; Ji, X.D.; Berkich, D.A.; Eveleth, D.; Dean, R.L.; Hiramatsu, K.; Kassei, N.F.; van Galen, P.J.M.; Lee, K.S.;
Bartus, R.T.; Daly, J.W.; Lanoue, K.F.; Maillard, M. Synthesis and biological activity of N6-(p-sulfophenyl) alkyl and N6-p-sulfoalkyl derivatives of adenosine. Water soluble and peripherally selective adenosine agonists. .1. Med. C em. 1992, 35, 4143-4149.
Found: C, 48.74; H, 4.60, N, 21.88.
Example 33: Preparation of N6-bis-{5-chloro-pyridin-2-yl-carbamoyl)-adenosine-5'-N-ethyluronamide Following the procedure outlined in Example 18, the title compound was prepared in a 68% yield as a white solid, mp 220°C. IR (ICBr) cm-1 3550-3100, 1725, 1630, 1525, 1250; 1H NMR (DMSO d6) b: 1.06 (t, 3H, J =
7); 3.15-3.22 (m, 2H); 4.22-4.24 (m, 1H); 4.36 (d, 2H, J = 2); 4.64-4.69 (m, 1H); 5.95 (bs, 2H); 6.10 (d, 1H, J = 6.8); 7.80-8.06 (m, 6H); 8.39 (t, 1H, J =
7); 8.76 (s, 1H); 8.93 (s, IH); 10.39 (bs, 1H); 12.11 {bs, 1H). Anal.
{C24H22N806C12) Calc'd.: C, 48.91; H, 3.76, N, 19.01. Found: C, 49.01;
H, 3.77, N, 18.89.
Example 34. Binding Assays Procedures for preparing rat brain membranes and Chinese hamster ovary (CHO) cell membranes were as reported30, 38, 44_ For binding experients, membrane homogenates were frozen and stored at -20°C for greater than 2 months. Adenosine deaminase (ADA) was purchased from Boehringer Mannheim (Indianapolis, IN). [3H]R-PIA was purchased from Amersham (Arlington Heights, IL), and [3H]CGS 21680 was purchased from DuPont NEN
(Boston, MA). [~25I]-AB-MECA was prepared as described by Olah et al.3o The binding of [1251]_AB-MECA to CHO cells stably transfected with the rat A3 receptor clone or to HEK-293 cells stably expressing the human A3 receptor, was performed essentially as described 2g, 30, 3s, a4. Assays were performed in SO mM Tris/ 10 mM MgCl2/ 1 mM EDTA buffer (adjusted to pH
8.26 at 5 °C) in glass tubes containing 100 ul of the membrane suspension, 50 pl of [~25I]-AB-MECA (final concentration 0.3 nM), and 50 ~1 of inhibitor.
1 S Inhibitors were routinely dissolved in DMSO. Concentrations of DMSO in incubations never exceeded 1%; this concentration did not influence [~25I]-AB-MECA binding. Incubations were carried out in duplicate for 1 h at 37°C, and were terminated by rapid filtration over Whatman GF/B filters using a Brandell cell harvester (Brandell, Gaithersburg, MD). Tubes were washed three times with 3 mL of buffer. Radioactivity was determined in a Beckman gamma SSOOB y-counter.
Nonspecific binding was determined in the presence of 200 uM NECA.
Kl values were calculated according to Cheng-Prusoff~5 assuming a Kd for [1251]-AB-MECA of 1.48 nM. Binding of [3H]R-PIA to A1 receptors from rat cortical membranes and of [3H]CGS 21680 to A2A receptors for rat striatal membranes was performed as described previously.46 Adenosine deaminase (2 units/mL) was present during the preparation of the membranes. Additional deaminase was not added during incubation with the radioligand.
Bin~~ of 1~35SJC"TTP-v-The binding for [35S]GTP-y-S (Ameersham, Chicago IL, specific activity 1275 Ci/mmol) was carried out using rat RBL-2H3 mast cell membranes by the general method of Lorenzen et a1.4~ Membranes were suspended in a buffer containing 50 mM Tris, 3 units/mL adenosine deaminase, $ 100 mM NaCI, and 10 mM MgCl2 (pH 7.4) and a protein concentration of 1 to ~g per tube. The membrane suspension was preincubated with 10 GDP, R-PIA and/or riboflavin in a final volume of 125 ~cL buffer at 30°C for 60 min and then transferred to ice for 20 min. [35S]GTP-y-S was added to a final concentration of 0.1 nM in a total volume of 500 ~L and the mixture was 10 incubated for 30 min at 30°C. Non-specific binding was determined in the presence of 10 ~M GTP-y-S (Sigma, St. Louis MO). Incubation of the reaction mixture was terminated by filtration over a GFB glass filter using a Brandell cell harvester and washed with the same buffer.
RP~!!lt~ and Discussion The derivatives 3a-o (the compounds in Examples 19-33, sequentially) were tested in radioligand binding assays for affinity at rat brain A1, A2A and A3 receptors, and the results are summarized in Table 1.
a a .~
Q ~ o ,, a o a b U M c'I
f'1 a Q
+, x ,o x :.fl ._ s o , y o ~n 0 .~ ~ .. v, ~
c x ~'' - o o . ~ ~ "., M
C
O ~ Q
~ ~ .-.
a -~
_ x ' /w z 1 z ,, o O q U
/ \ O ~' C >, z z N
~
G z ~ ~ ~, o ~, , =w w z .
~
a .
Q x ~ x v a N
w CC ~
U
~n o a ~p pp ,n, -. o0 N N w0 ~J
N v'1 00 ~ ~ 'j M ~ "' d' O O (~i O d' ~~ ~ c ~ O N
a~ ~ O O O ~ ~ ~D N
O O ~ d' M fuel O ~ N O
i~ M l~ ~' M N ~ n V7 ~ M ~ 00 ~ N
O ~ - ~ O f~ M N N M d' a I~ ~f G1 ~ ~ M M ~ M N O ~ 00 M V1N 00 I~ ~O~ M V'1O
00 t~00 M ~ ~ -~M V1 ~ ~'.., ~_ \
O
..~ O ~ O O O O N ~, O
00 OO d' ~ N N M N ~ GM1N N O~pO
_ r~r~-i 'H -H 1-I~ -f-i-H-t-I~ -H
c~ ~ O GMIOOOp ~ ~ ~f _I~~ O
N N '~~D ~ ,_,~ N '~ 00 Ov N
~" N N
~N ~ ~.~ ~ M G\ N M 00 M M ~t 'cY
~' Ct ~ ~ -H -~I'~M
~ M M ~' O V7 ~ ~ N ~ ~ v7 .-.N N
x x x x x x z x x z z z ~ x ~ z z z ;,' ~~,, z c~ ' U a N z U ~ ; ~ ~ ~ M ~ .~
S ay v a~ =. o c~:.
.~.. N Z Z N
~' U O ~ ~ o x 0 U U Z,M m ' ' ~ ~ M
N a. o z H v et ~ cnU 23t3 ~ ~
d. ~
~ M
Q.
x ~ x x x x x x x x x x x x o x z z ' a U
LL ~ M M M M M M M M M '~ ~
M M C
M
M
U
~n ~n o a ~_ M w I
I
a M . ...
a M
C s w a v_ M C_ Q, O ~-I -~I N U
w.r_ Q N
O Q
r. cd p" V
v-~
"' k G
N V~ 'b C ~ G N
o " ~ U
C U ~ y<
.-.p ~.. vi U y C pp G1 ~ ~'1r, W
-H -~ ice.._c3 O
C\ ~ ~ U
N .~~.. U
y.
M
a ~
a ~
z ' w ~?
a C~ U 2 _ N
a a a N
a, a. p.
W
N U
U
b a v n o ~ n _ o w o ~ M
U
~' 39 Baraldi demonstrated that certain N6-substituted-arylcarbamoyl adenosine-uronamides are full agonists in inhibiting adenylate cyclase via rat A3 receptors, but that the compounds showed relatively high activity with respect to adenosine A1 receptors39. There is a similarity between A1 and A3 receptors. Small modifications in the length of chain at the N6 position can alter the affinity versus At or A3 receptors. The present binding data demonstrate that the presence of aryl- or alkaryl-carbamoylic chains at the N6-position bring about an interaction with the A3 receptor subtype. Also, the data demonstrate that heteroaryl-carbamoyl, heteroaryl-carboxamide, aryl-carboxamide, and alkaryl-carboxamide substitutions at the N6-position bring about an interaction with the A1 receptor subtype.
In the series of urea-derivatives (Compounds 3f o) a substituted-phenyl (Compounds 3g, h and m) or substituted-benzyl (Compounds 3f, i and j) group leads to relatively high affinity and selectivity at A3 adenosine receptors as compared with the affinity for the At and A., receptors.
Derivatives 3g, 3h and 3m showed a relatively high affinity (9.7-107 nM) at A3 receptors with varying degrees of A3/At selectivity. In particular, compound 3g was Iess active than IB-MECA (9.7 nM vs. 1.1 nM) but showed selectivity for A3 vs. either A1 or AZA receptors which is comparable to IB-MECA.
Compound 3g also showed a relatively high affinity, in the nanomolar range, at human A3 adenosine receptors (56.1 ~ 9.1 nM), confirming the relatively high affinity of this compound versus this receptor subtype, independent of species. The lipophilicity of para substituents on a phenyl ring play a significant role in A3 affinity.
-~0-Substitution of the phenyl ring with a heterocycle (as in Compounds 3k-1) causes the compounds to lose affinity and selectivity for A3 receptors, and increase in affinity for the Ai receptor subtype.
Disubstitution at the N6 position (for example, Compounds 3n-o) have diminished activity at A2A and A3 receptors, but have relatively high affinity for the A t receptor.
In the benzylic series, Compounds 3 f, i, and j showed an SAR pattern comparable to the phenylic series in Baraldi39. In fact, the unsubstituted Compound 3f showed affinity and selectivity at A3 receptor very similar to the previously reported phenyl derivative.3~
The stereochemistry of substituents at the N6 position is also important in determining the affinity of these compounds for the A3 receptor.
For example, comparing the two diastereomeric Compounds 3i and 3j in which substituents at N6 are of opposite configuration, the R-isomer (3i) is more potent and selective that the S-isomer (3j) at the rat A3 adenosine receptor subtype. Those of skill in the art can separate compounds with stereocenters adjacent to the N6 position using routine enantiopurification methods and evaluate the individual stereoisomers for their affinity and selectivity using no more than routine experimentation.
A functional assay indicated that Compound 3g acted as a full agonist at rat A3 receptor. The assay involved the agonist-induced inhibition of binding of guanine nucleotide to rat RBL-2H3 mast cell membranes, which contain a high density of A3 receptors.
Figure 1 shows that compound 3g was about as effective as the potent A3 agonists I-AB-MECA and Cl-IB-MECA increased binding of [35S]GTP-y-S in a dose dependent manner and with greater potency than NECA.
Example 35. Pharmaceutical Formulations (A) Transdermal System - for 1000 patches Ingredients Amount Active compound 100g Silicone fluid 450g Colloidal silicon dioxide 2g The silicone fluid and active compound are mixed together and the colloidal silicone dioxide is added to increase viscosity. The material is then dosed into a subsequent heat sealed polymeric laminate including the following: polyester release liner, skin contact adhesive composed of silicone or acrylic polymers, a control membrane which is a polyolefin, and an impermeable backing membrane made of a polyester multilaminate. The resulting laminated sheet is then cut into 10 sq. cm patches (B) Oral Tablet - For 1000 Tablets Ingredients Amount Active compound SOg Starch ~0g Magnesium Stearate Sg The active compound and the starch are granulated with water and dried. Magnesium stearate is added to the dried granules and the mixture is thoroughly blended. The blended mixture is compressed into tablets.
(C) Injection - for 1000, 1mL Ampules *rB
Ingredients Amount Active compound log Buffering Agents q.s.
Propylene glycol 400mg S Water for injection q.s.100omL
The active compound and buffering agents are dissolved in the propylene glycol at about SO°C. The water for injection is then added with stirring and the resulting solution is filtered, filled into ampules, sealed and sterilized by autoclaving.
(D) Continuous Injection - for 1000 mL
Ingredients Amount Active compound log Buffering agents q.s.
Water for injection q.s.loooml.
25 REFERENCES:
The following references have been referred to herein. These references are incorporated herein by reference in their entirety.
1. Libert, F., Schiffmann, S.N.: Lefort, A., Parmentier, M; Gerard, C.; Dumont, J E.; Vanderhaeghen, J.J.; Vassart, G. The orphan receptor _ 43 _ cDNA RDC7 encodes an A1 adenosine receptor. E.~1991, 10, 1677-1682.
2. Maenhaut, C., Sande, J.V.; Libert, F; Abramowicz, M.;
Pannentier, M.; Vanderhaeghen, J.J.; Dumont, J.E.; Vassart, G.; Schiffmann, S. RDC8 codes for an adenosine A2 receptor with physiological constitutive activity. Biochem. Biophys. Res. .ommun. 1990, 173, 1169-1178.
3. Stehle, J.H.; Rivkees, S.A.; Lee, J.J.; Weaver, D.R.; Deeds, J.D.;
Reppert, S.M. Molecular cloning and expression of the cDNA for a novel A2-adenosine receptor subtype. Mol. Endocrinol. 1992, 6, 384-393.
4. Zhou, Q.Y.; Li, C.Y.; Olah, M.E.; Johnson, R.A.; Stiles, G.L.;
Civelli, 0. Molecular cloning and characterization of an adenosine receptor-the A3 adenosine receptor. Proc Natl Acad Sci USA 1992, 89, 7432-7436.
5. Londos, C., Cooper, D.M.; Wolff. J. Subclasses of external adenosine receptors. Prop Natl. Acad. Sci. LISA 1980, 77, 2551-2554.
6. Hamprecht, B.; Van Calker, D. Nomenclature of adenosine receptors. Trendy Pharmacol. Sci. 1985,6, 153-154.
7. Ukena, D.; Olsson, R.A.; Daly, J.W. Definition of subclasses of adenosine receptors associated with adenylate cyclase: interaction of adenosine analogs with inhibitory A1 receptors and stimulatory A., receptors.
C.'an J. Phvsiol. Pharmacol. 1987, 65. 365-376.
8. Ramkumar, V., Stiles, G.L.; Beaven, M. A.; Ali, H. The A3AR is the unique adenosine receptor which facilitates release of allergic mediators in mast cells. a~ Biol. Chem. 1993, 268, 6871-6890.
9. Abbracchio, M.P.; Brambilla, R.; Ceruti, S.: Kim, H.O.; von Lubitz, D.K.J.E.; Jacobson, K.A., Cattabeni, F. G-protein-dependent activation of phospholipase C by adenosine A3 receptors in rat brain. ~1.
pharmacol. 1995, 48, 1038-1045.
10. Bruns, R.F.; Daly, J.W.; Snyder, S.H. Adenosine receptors in brain membranes: binding of N-cyclohexyl [3H] adenosine and 1,3-diethyl-8(3H] phenylxanthine. Proc Natl Acad ~ci USA 1980, 77, 5547-5551.
11. Oisson, R.A.; Kusachi, S.; Thompson, S.D.; IJkena, D., Padgett, W.; Daly, J.W. N6-substituted N-alkyladenosine-51-uronamides: bifunctional ligands having recognition groups for A1 and A2 adenosine receptors. .L
Med. Chem. 1986, 29, 1683-1689.
12. Jacobson, K.A.; Nikodijevic, 0.; Ji, X.D.; Berkich, D.A.; Eveleth, D.; Dean, R.L.; Hiramatsu, K.; Kassei, N.F.; van Galen, P.J.M.; Lee, K.S.;
Bartus, R.T.; Daly, J.W.; Lanoue, K.F.; Maillard, M. Synthesis and biological activity of N6-(p-sulfophenyl) alkyl and N6-p-sulfoalkyl derivatives of adenosine. Water soluble and peripherally selective adenosine agonists. .1. Med. C em. 1992, 35, 4143-4149.
13. Shamim, M.T.; Ukena, D.; Padgett, W.L.; Hong, 0.; Daly, J.W.
8-Aryl-and $cycloalkyl-1,3-dipropylxanthines: further potent and selective antagonists for A1-adenosine receptors. ,I. Med. Chem. 1988, 31, 613-617.
8-Aryl-and $cycloalkyl-1,3-dipropylxanthines: further potent and selective antagonists for A1-adenosine receptors. ,I. Med. Chem. 1988, 31, 613-617.
14. Linden, J.; Patel, A.; Earl, C.Q.; Craig, R.H.; Daluge, S.M.
(1251]_labeled 8-phenylxanthine derivatives: antagonist radioligands for adenosine A1 receptors. J. Med. Chem. 1988, 31, 745-751.
(1251]_labeled 8-phenylxanthine derivatives: antagonist radioligands for adenosine A1 receptors. J. Med. Chem. 1988, 31, 745-751.
15. Jacobson, K.A. de la Cruz., R.; Schulick, R.; Kiriasis, K.L.;
Padgett, W.; Pfleiderer, W.; Kirk, K.L.; Neumeyer, J.L., Daly, J.W. 8-*rB
substituted xanthines as antagonists at Al and AZ adenosine receptors.
Bjochem_ P1988, 37, 3653-3661.
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Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
Claims (28)
1. A compound of the following formula:
wherein:
R is hydrogen, alkyl, substituted alkyl, alkaryl or aryl;
R1 is heteroaryl-NR-C(X), heteroaryl-C(X)-, alkaryl-NR-C(X)-, substituted alkaryl-NR-C(X)-, aryl-NR-C(X)-, aryl-C(X)-, alkaryl-C(X)-, or substituted alkaryl-C(X)-, R2 is hydrogen, alkyl, substituted alkyl, alkaryl or aryl-NH-C(X)-, R3 and R4 are independently selected from the group consisting of hydroxy, hydrogen, halo, ether, ester, azide, alkyl, alkoxy, carboxy, nitrile, nitro, trifluoro, aryl, alkaryl, thin, thioester, thioether, amine and amide, and X is O, S, or NR, with the proviso that, when R2 is H, R1 is aryl-NH-C(X)- and X is O, the substituents on the aryl ring are not halo, methoxy or trifluoromethyl.
wherein:
R is hydrogen, alkyl, substituted alkyl, alkaryl or aryl;
R1 is heteroaryl-NR-C(X), heteroaryl-C(X)-, alkaryl-NR-C(X)-, substituted alkaryl-NR-C(X)-, aryl-NR-C(X)-, aryl-C(X)-, alkaryl-C(X)-, or substituted alkaryl-C(X)-, R2 is hydrogen, alkyl, substituted alkyl, alkaryl or aryl-NH-C(X)-, R3 and R4 are independently selected from the group consisting of hydroxy, hydrogen, halo, ether, ester, azide, alkyl, alkoxy, carboxy, nitrile, nitro, trifluoro, aryl, alkaryl, thin, thioester, thioether, amine and amide, and X is O, S, or NR, with the proviso that, when R2 is H, R1 is aryl-NH-C(X)- and X is O, the substituents on the aryl ring are not halo, methoxy or trifluoromethyl.
2. The compound of claim 1 wherein R is hydrogen.
3. The compound of claim 1 wherein R1 is alkaryl-NR-C(X)-.
4. The compound of claim 1 wherein R1 is substituted alkaryl-NR-C(X)-.
5. The compound of claim 1 wherein R1 is aryl-NR-C(X)-.
6. The compound of claim 1 wherein R1 is aryl-C(X)-.
7. The compound of claim 1 wherein R1 is alkaryl-C(X)-.
8. The compound of claim 1 wherein R1 is substituted alkaryl-C(X)-.
9. The compound of claim 1 wherein R1 is heteroaryl-NR-C(X)-
10. The compound of claim 1 wherein R1 is heteroaryl-C(X)-
11. The compound of claim 1 wherein R2 is hydrogen.
12. The compound of claim 1 wherein X is O.
13. A compound selected from the group consisting of N6-(4-biphenyl-carbonylamino)-adenosine-5'-N-ethyluronamide, N6-(2,4-dichlorobenzyl-carbonylamino)-adenosine-5'-N-ethyluronamide, N6-(4-methoxyphenyl-carbonylamino)-adenosine-5'-N-ethyluronamide, N6-(4-chlorophenyl-carbonylamino)-adenosine-5'-N-ethyluronamide, N6-(phenyl-carbonylamino)-adenosine-5'-N-ethyluronamide, N6-(benzylcarbamoylamino)-adenosine-5'-N-ethyluronamide, N6-(4-sulfonamido-phenylcarbamovl)-adenosine-5'-N-ethyluronamide, N6-(4-acetyl-phenylcarbamoyl)-adenosine-5'-N-ethyluronamide, N6-((R)-.alpha.-phenylethylcarbamoyl)-adenosine-5'-N-ethyluronamide, N6-((S)-.alpha.-phenylethylcarbamoyl)-adenosine-5'-N-ethyluronamide, N6-(5-methyl-isoxazol-3-yl-carbamoyl)-adenosine-5'-N-ethyluronamide, N6-(1,3,4-thiadiazol-2-yl-carbamoyl)-adenosine-5'-N-ethyluranamide, N6-(4-n-propoxy-phenylcarbamoyl)- adenosine-5'-N-ethyluronamide, N6-bis-(4-nitrophenylcarbamoyl)-adenosine-5'-N-ethyluronamide, and N6-bis-(5-chloro-pyridin-2-yl-carbamoyl)-adenosine-5'-N-ethyluronamide.
14. A method of treating tumors, hypertension, mast cell degranulation, or cardiac hypoxia, or providing protection against cerebral ischemia, comprising administering to a patient in need of treatment thereof an effective amount of a compound of claim 1 which is an agonist or partial agonist of the A3 receptor.
15. The method of claim 14 wherein R is hydrogen.
16. The method of claim 14 wherein R1 is alkaryl-NR-C(X)-.
17. The method of claim 14 wherein R1 is substituted alkaryl-NR-C(X)-.
18. The method of claim 14 wherein R1 is aryl-NR-C(X)-.
19. The method of claim 14 wherein R2 is hydrogen.
20. The method of claim 14 wherein X is O.
21. The method of claim 14 wherein the compound is selected from the group consisting of:
N6-(benzylcarbamoylamino)-adenosine-5'-N-ethyluronamide, N6-(4-sulfonamido-phenylcarbamoyl)-adenosine-5'-N-ethyluronamide, N6-(4-acetyl-phenylcarbamoyl)-adenosine-5'-N-ethyluronamide, N6-((R)-a-phenylethylcarbamoyl)-adenosine-5'-N-ethyluronamide, N6-((S)-a-phenylethylcarbamoyl)-adenosine-5'-N-ethyluronamide, N6-(4-n-propoxy-phenylcarbamoyl)-adenosine-5'-N-ethyluronamide, and N6-bis-(4-nitrophenylcarbamoyl)-adenosine-5'-N-ethyluronamide,
N6-(benzylcarbamoylamino)-adenosine-5'-N-ethyluronamide, N6-(4-sulfonamido-phenylcarbamoyl)-adenosine-5'-N-ethyluronamide, N6-(4-acetyl-phenylcarbamoyl)-adenosine-5'-N-ethyluronamide, N6-((R)-a-phenylethylcarbamoyl)-adenosine-5'-N-ethyluronamide, N6-((S)-a-phenylethylcarbamoyl)-adenosine-5'-N-ethyluronamide, N6-(4-n-propoxy-phenylcarbamoyl)-adenosine-5'-N-ethyluronamide, and N6-bis-(4-nitrophenylcarbamoyl)-adenosine-5'-N-ethyluronamide,
22. A method for providing protection against hypoxia, ischemia induced injuries, cardioprotection, neuroprotection, pain management, reduction of free fatty acids, triglycerides, or glucose levels, adjunct therapy in diabetes, treatment of adenosine-sensitive cardiac disarrhythmias, treatment of GI disorders, treatment of convulsion, treatment of glaucoma;
treatment of sleep apnea; treatment of peroxysmal supraventricular tachycardia, or treatment of inflammation comprising administering to a patient in need of treatment thereof an effective amount to treat the disorder of a compound of Formula I which is active as an agonist or partial agonist of the A1 receptor.
treatment of sleep apnea; treatment of peroxysmal supraventricular tachycardia, or treatment of inflammation comprising administering to a patient in need of treatment thereof an effective amount to treat the disorder of a compound of Formula I which is active as an agonist or partial agonist of the A1 receptor.
23. The method of claim 23 wherein R1 is aryl-C(X)-.
24. The method of claim 23 wherein R1 is alkaryl-C(X)-.
25. The method of claim 23 wherein R1 is substituted alkaryl-C(X)-.
26. The method of claim 23 wherein R1 is heteroaryl-NR-C(X)-.
27. The method of claim 23 wherein R1 is heteraryl-C(X)-.
28. The method of claim 23 wherein the compound is selected from the group consisting of:
N6-(4-biphenyl-carbonylamino)-adenosine-5'-N-ethyluronamide, N6-(2,4-dichlorobenzyl-carbonylamino)-adenosine-5'-N-ethyluronamide, N6-(4-methoxyphenyl-carbonylamino)-adenosine-5'-N-ethyluronamide, N6-(4-chlorophenyl-carbonylamino)-adenosine-5'-N-ethyluronamide, N6-(phenyl-carbonylamino)-adenosine-5'-N-ethyluronamide, N6-(5-methyl-isoxazol-3-yl-carbamoyl)-adenosine-5'-N-ethyluronamide, N6-( 1,3,4-thiadiazol-2-yl-carbamoyl)-adenosine-5'-N-ethyluronamide, and N6-bis-(5-chloro-pyridin-2-yl-carbamoyl)-adenosine-5'-N-ethyluronamide.
N6-(4-biphenyl-carbonylamino)-adenosine-5'-N-ethyluronamide, N6-(2,4-dichlorobenzyl-carbonylamino)-adenosine-5'-N-ethyluronamide, N6-(4-methoxyphenyl-carbonylamino)-adenosine-5'-N-ethyluronamide, N6-(4-chlorophenyl-carbonylamino)-adenosine-5'-N-ethyluronamide, N6-(phenyl-carbonylamino)-adenosine-5'-N-ethyluronamide, N6-(5-methyl-isoxazol-3-yl-carbamoyl)-adenosine-5'-N-ethyluronamide, N6-( 1,3,4-thiadiazol-2-yl-carbamoyl)-adenosine-5'-N-ethyluronamide, and N6-bis-(5-chloro-pyridin-2-yl-carbamoyl)-adenosine-5'-N-ethyluronamide.
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| US60/054,064 | 1997-07-29 | ||
| PCT/US1998/016053 WO1999006053A1 (en) | 1997-07-29 | 1998-07-29 | N6-substituted-adenosine-5'-uronamides as adenosine receptor modulators |
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| CA2296485A1 true CA2296485A1 (en) | 1999-02-11 |
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| CA002296485A Abandoned CA2296485A1 (en) | 1997-07-29 | 1998-07-29 | N6-substituted-adenosine-5'-uronamides as adenosine receptor modulators |
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| JP (1) | JP2003517423A (en) |
| AU (1) | AU8764398A (en) |
| CA (1) | CA2296485A1 (en) |
| WO (1) | WO1999006053A1 (en) |
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| US7825102B2 (en) | 2004-07-28 | 2010-11-02 | Can-Fite Biopharma Ltd. | Treatment of dry eye conditions |
| ES2432113T3 (en) * | 2004-07-28 | 2013-11-29 | Can-Fite Biopharma Ltd. | Adenosine A3 receptor agonists for the treatment of dry eye disorders, including Sjogren's syndrome |
| EP1798233A1 (en) * | 2005-12-19 | 2007-06-20 | Faust Pharmaceuticals | Pyrrolo[1,2-a]quinoxaline derivatives as Adenosine A3 receptor modulators and uses thereof |
| CN101820883B (en) | 2007-10-15 | 2013-03-20 | 坎-菲特生物药物有限公司 | Uses of A3AR agonist in preparing medicine for inducing hepatocyte proliferation |
| EP2432475A1 (en) | 2009-05-17 | 2012-03-28 | Can-Fite Biopharma Ltd. | A3 adenosine receptor agonists for the reduction of intraocular pressure |
| WO2011010306A1 (en) | 2009-07-21 | 2011-01-27 | Ramot At Tel-Aviv University Ltd. | A3 adenosine receptor ligands for modulation of pigmentation |
| JP2013521274A (en) | 2010-03-03 | 2013-06-10 | ガバメント オブ ザ ユナイテッド ステイツ オブ アメリカ・アズ リプレゼンテッド バイ ザ セクレタリー・デパートメント オブ ヘルス アンド ヒューマン サービシーズ | A3AR agonists for the treatment of uveitis |
| EP2892533A1 (en) | 2012-08-09 | 2015-07-15 | Can-Fite Biopharma Ltd. | A3 adenosine receptor ligands for use in treatment of a sexual dysfunction |
| IL242723B (en) | 2015-11-23 | 2019-12-31 | Can Fite Biopharma Ltd | An a3 adenosine receptor ligand for use in treating ectopic fat accumulation |
| IL254535A0 (en) | 2017-09-17 | 2017-11-30 | Can Fite Biopharma Ltd | A3 adenosine receptor ligand for managing cytokine release syndrome |
| IL264112A (en) | 2019-01-06 | 2020-07-30 | Fishman Pnina | An a3 adenosine receptor ligand for use for reducing level of adipocytes |
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|---|---|---|---|---|
| US5310731A (en) * | 1984-06-28 | 1994-05-10 | Whitby Research, Inc. | N-6 substituted-5'-(N-substitutedcarboxamido)adenosines as cardiac vasodilators and antihypertensive agents |
| CA2289731A1 (en) * | 1997-05-09 | 1998-11-12 | The United States Of America, Represented By The Secretary, Department O F Health And Human Services | Methods and compositions for reducing ischemic injury of the heart by administering adenosine receptor agonists and antagonists |
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1998
- 1998-07-29 JP JP2000504866A patent/JP2003517423A/en active Pending
- 1998-07-29 EP EP98939156A patent/EP1019427A4/en not_active Withdrawn
- 1998-07-29 AU AU87643/98A patent/AU8764398A/en not_active Abandoned
- 1998-07-29 CA CA002296485A patent/CA2296485A1/en not_active Abandoned
- 1998-07-29 WO PCT/US1998/016053 patent/WO1999006053A1/en not_active Application Discontinuation
Also Published As
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| AU8764398A (en) | 1999-02-22 |
| EP1019427A1 (en) | 2000-07-19 |
| WO1999006053A1 (en) | 1999-02-11 |
| JP2003517423A (en) | 2003-05-27 |
| EP1019427A4 (en) | 2000-07-19 |
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