WO2009049362A1 - Amplificateurs allostériques des récepteurs de l'adénosine a1 - Google Patents

Amplificateurs allostériques des récepteurs de l'adénosine a1 Download PDF

Info

Publication number
WO2009049362A1
WO2009049362A1 PCT/AU2008/001524 AU2008001524W WO2009049362A1 WO 2009049362 A1 WO2009049362 A1 WO 2009049362A1 AU 2008001524 W AU2008001524 W AU 2008001524W WO 2009049362 A1 WO2009049362 A1 WO 2009049362A1
Authority
WO
WIPO (PCT)
Prior art keywords
optionally substituted
amino
methanone
thiophen
formula
Prior art date
Application number
PCT/AU2008/001524
Other languages
English (en)
Inventor
Peter J. Scammells
Luigi Aurelio
Arthur Christopoulos
Patrick M. Sexton
Celine Valant
Bernard L. Flynn
Joel M. Linden
Original Assignee
Monash University
University Of Virginia
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Monash University, University Of Virginia filed Critical Monash University
Publication of WO2009049362A1 publication Critical patent/WO2009049362A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D333/00—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
    • C07D333/02—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings
    • C07D333/04—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom
    • C07D333/26—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D333/30—Hetero atoms other than halogen
    • C07D333/36—Nitrogen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/06—Antiarrhythmics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D333/00—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
    • C07D333/02—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings
    • C07D333/04—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom
    • C07D333/26—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D333/38—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals

Definitions

  • the present invention relates generally to chemical compounds and methods for their use and preparation.
  • the invention relates to chemical compounds which may possess useful therapeutic activity for treating conditions where the promotion of angiogensis (blood vessel formation) is beneficial, use of these compounds in therapy and the manufacture of medicaments as well as compositions containing these compounds.
  • Adenosine is an important endogenous tissue-protective compound released during ischaemia, hypoxia or inflammation. Adenosine interacts with extracellular G protein- coupled receptors to regulate adenylate cyclase, and potassium and calcium ion channels.
  • receptor subtypes Ai, A 2A , A 2 B and A 3
  • Adenosine marketed as AdenocardTM
  • a new synthetic Ai adenosine receptor (AiAR) agonist, TecadenosinTM, is in clinical development as an anti-arrhythmic agent, while the mixed A]/A 2 AR agonist, AMP-579, reached phase II clinical trials as a cardioprotective agent.
  • adenosine receptor agonists as therapeutic agents has been limited by side-effects associated with actions at adenosine receptors located in tissues away from the desired site(s) of action, and the propensity of agonists to cause receptor desensitization upon prolonged exposure. Such problems need to be addressed in order to take advantage of the enormous therapeutic potential of the adenosine receptor system.
  • allosteric modulators may prove clinically valuable, with improved therapeutic indices owing to their site and event-selective actions.
  • Allosteric enhancers bind to an allosteric site, potentiating responses to agonist binding at the primary binding domain (or orthosteric site). Since adenosine production is highest in ischaemic or hypoxic tissue, enhancers that selectively amplify the actions of endogenous adenosine would be selective for ischaemic tissue. Since extracellular adenosine is very rapidly degraded to inactive metabolites, within the circulation and interstitial compartments, amplifying the effects of endogenous adenosine would localize its actions to those cells undergoing ischemic stress. Agents inducing such site and event specific effects clearly offer a therapeutic advantage.
  • Routine screening for adenosine antagonists by Parke-Davis identified a series of 2-amino- 3-benzoylthiophenes that enhanced agonist radioligand binding at A 1 ARs (see Bruns, et al., MoI. Pharmacol, 1990, 38, 939-949 & 950-958).
  • these compounds were found to increase binding of [ 3 H]N 6 -cyclohexyladenosine (CHA) to adenosine A 1 receptors and cause a functional enhancement of the effects of adenosine A 1 receptor activation in tissue. It appears that these "allosteric enhancers" enhance the A 1 adenosine receptor function by stabilising the high affinity state of the receptor-G-protein complex.
  • allosteric enhancers offer many therapeutic benefits, as adenosine receptor A 1 agonists in general promote angiogensis (blood vessel formation) and selective Ai allosteric enhancers may selectively stimulate angiogensis in ischemic or hypoxic tissues that produce high levels of adenoside, as opposed to tissue where the adenosine concentration is low.
  • the most effective enhancer in this series was PD81,723 (1) which proved highly selective for A]ARs, having no major effect on agonist binding at the other adenosine receptor subtypes or at the other G-protein coupled receptors that were investigated (M 2 muscarinic, ⁇ 2 adrenergic or ⁇ -opiate receptors).
  • the present invention provides novel substituted thiophenes as useful Ai adenosine receptor allosteric enhancers.
  • the present invention provides compounds of formula (I) and salts thereof;
  • n and m are independently an integer from 0 to 3; and each Ri and R 2 independently represents carboxyl, cyano, dihalomethoxy, halogen, hydroxy, nitro, pentahaloethyl, phosphono, phosphorylamino, phosphinyl, thio, - A -
  • the present invention further provides compounds of formula (II) and salts thereof;
  • n is an integer from 0 to 3;
  • Ri independently represents carboxyl, cyano, dihalomethoxy, halogen, hydroxy, nitro, pentahaloethyl, phosphono, phosphorylamino, phosphinyl, thio, sulfinyl, sulfonyl, trihaloethenyl, trihalomethanethio, trihalomethoxy, trihalomethyl, optionally substituted acyl, optionally substituted acylamino, optionally substituted acylimino, optionally substituted acyliminoxy, optionally substituted acyloxy, optionally substituted arylalkyl, optionally substituted arylalkoxy, optionally substituted alkenyl, optionally substituted alkenyloxy, optionally substituted alkoxy, optionally substituted alkyl, optionally substituted alkynyl, optionally substituted alkynyloxy, optionally substituted amino, optionally substituted aminoacyl, optionally substituted aminoacyloxy, optionally
  • R 3 represents halogen; and R 4 represents Ci -3 alkyl.
  • Figure 1 - depicts a graph of Ai adenosine receptor-mediated ERKl /2 phosphorylation in response to the agonist, R-PIA (relative to that mediated by an internal control of 3% FBS stimulation), in the absence or presence of various concentrations of Example 1.
  • Figure 2 - depicts a graph of Ai adenosine receptor-mediated [ 35 S]GTPyS binding in response to the agonist, R-PIA, in the absence or presence of various concentrations of Example 1.
  • Figure 3 - depicts a graph of Aj adenosine receptor-mediated ERKl /2 phosphorylation in response to the agonist, R-PIA (relative to that mediated by an internal control of 3% FBS stimulation), in the absence or presence of two concentrations of each of the indicated Example compounds.
  • Alkyl refers to monovalent alkyl groups which may be straight chained or branched and preferably have from 1 to 10 carbon atoms or more preferably 1 to 6 carbon atoms and most preferably 1 to 3 carbon atoms. Examples of such alkyl groups include methyl, ethyl, ⁇ -propyl, wo-propyl, «-butyl, wo-butyl, «-hexyl, and the like.
  • Aryl refers to an unsaturated aromatic carbocyclic group having a single ring (eg. phenyl) or multiple condensed rings (eg. naphthyl or anthryl), preferably having from 6 to 14 carbon atoms.
  • aryl groups include phenyl, naphthyl and the like.
  • Aryloxy refers to the group aryl-O- wherein the aryl group is as described above.
  • Arylalkyl refers to -alkylene-aryl groups preferably having from 1 to 10 carbon atoms in the alkylene moiety and from 6 to 10 carbon atoms in the aryl moiety. Such arylalkyl groups are exemplified by benzyl, phenethyl and the like.
  • Arylalkoxy refers to the group arylalkyl-O- wherein the arylalkyl group are as described above. Such arylalkoxy groups are exemplified by benzyloxy and the like.
  • Alkoxy refers to the group alkyl-O- where the alkyl group is as described above. Examples include, methoxy, ethoxy, n-propoxy, zs ⁇ -propoxy, n-bntoxy, tert-bntoxy, sec- butoxy, r ⁇ -pentoxy, n-hexoxy, 1 ,2-dimethylbutoxy, and the like.
  • Alkenyl refers to a monovalent alkenyl group which may be straight chained or branched and preferably have from 2 to 10 carbon atoms and more preferably 2 to 6 carbon atoms and have at least 1 and preferably from 1-2, carbon to carbon, double bonds.
  • Alkenyloxy refers to the group alkenyl-O- wherein the alkenyl group is as described above.
  • 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, carbon to carbon, triple bonds.
  • Alkynyloxy refers to the group alkynyl-O- wherein the alkynyl groups is as described above.
  • Acyl refers to groups H-C(O)-, alkyl-C(O)-, cycloalkyl-C(O)-, aryl-C(O)-, heteroaryl- C(O)- and heterocyclyl-C(O)-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.
  • Oxyacyl refers to groups alkyl-OC(O)-, cycloalkyl-OC(O)-, aryl-OC(O)-, heteroaryl- OC(O)-, and heterocyclyl-OC(O)-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.
  • Amino refers to the group -NR*R* where each R* is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
  • Aminoacyl refers to the group -C(0)NR*R* where each R* is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
  • Acylamino refers to the group -NR* C(O)R* where each R* is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are as described herein.
  • Acyloxy refers to the groups -OC(O)-alkyl, -OC(O)-aryl, -OC(O)-heteroaryl, and -OC(O)-heterocyclyl where alkyl, aryl, heteroaryl and heterocyclyl are as described herein.
  • Aminoacyloxy refers to the groups -OC(O)NR*-alkyl, -OC(O)NR* -aryl, -OC(O)NR* -heteroaryl, and -OC(O)NR* -heterocyclyl where R* is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
  • Oxyacylamino refers to the groups -NR*C(0)0-alkyl, -NR*C(O)O-aryl, -NR*C(O)O-heteroaryl, and NR*C(O)O-heterocyclyl where R* is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
  • Oxyacyloxy refers to the groups -OC(O)O-alkyl, -OC(O)O-aryl, -OC(O)O- heteroaryl, and -OC(O)O-heterocyclyl where alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are as described herein.
  • Acylimino refers to the groups -C(NR*)-R* where each R* is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are as described herein.
  • Acyliminoxy refers to the groups -0-C(NR*)-R* where each R* is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are as described herein.
  • Oxyacylimino refers to the groups -C(NR*)-OR* where each R* is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are as described herein.
  • Cycloalkyl refers to cyclic alkyl groups having a single cyclic ring or multiple condensed rings, preferably incorporating 3 to 8 carbon atoms.
  • Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.
  • Cycloalkenyl refers to cyclic alkenyl groups having a single cyclic ring and at least one point of internal unsaturation, preferably incorporating 4 to 8 carbon atoms.
  • suitable cycloalkenyl groups include, for instance, cyclobut-2-enyl, cyclopent-3-enyl, cyclohex-4-enyl, cyclooct-3-enyl and the like.
  • Halo or halogen refers to fluoro, chloro, bromo and iodo.
  • Heteroaryl refers to a monovalent aromatic heterocyclic group which fulfils the Huckel criteria for aromaticity (ie. contains 4n + 2 ⁇ electrons) and preferably has from
  • heteroaryl groups can have a single ring (eg. pyridyl, pyrrolyl or N-oxides thereof or furyl) or multiple condensed rings (eg. indolizinyl, benzoimidazolyl, coumarinyl, quinolinyl, isoquinolinyl or benzothienyl).
  • Heterocyclyl refers to a monovalent saturated or unsaturated group having a single ring or multiple condensed rings, preferably from 1 to 8 carbon atoms and from 1 to 4 hetero atoms selected from nitrogen, sulfur, oxygen, selenium or phosphorous within the ring.
  • heterocyclyl and heteroaryl groups include, but are not limited to, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, isothiazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7
  • Thio refers to groups H-S-, alkyl-S-, cycloalkyl-S-, aryl-S-, heteroaryl-S-, and heterocyclyl-S-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.
  • Thioacyl refers to groups H-C(S)-, alkyl-C(S)-, cycloalkyl-C(S)-, aryl-C(S)-, heteroaryl-C(S)-, and heterocyclyl-C(S)-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.
  • Oxythioacyl refers to groups HO-C(S)-, alkylO-C(S)-, cycloalkylO-C(S)-, arylO- C(S)-, heteroarylO-C(S)-, and heterocyclylO-C(S)-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.
  • Oxythioacyloxy refers to groups HO-C(S)-O-, alkylO-C(S)-O-, cycloalkylO-C(S)-O-, arylO-C(S)-O-, heteroarylO-C(S)-O-, and heterocyclylO-C(S)-O-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.
  • Phosphorylamino refers to the groups -NR* -P(O)(R* *)(OR***) where R* represents H, alkyl, cycloalkyl, alkenyl, or aryl, R** represents OR*** or is hydroxy or amino and R*** is alkyl, cycloalkyl, aryl or arylalkyl, where alkyl, amino, alkenyl, aryl, cycloalkyl, and arylalkyl are as described herein.
  • Thioacyloxy refers to groups H-C(S)-O-, alkyl-C(S)-O-, cycloalkyl-C(S)-O-, aryl- C(S)-O-, heteroaryl-C(S)-O-, and heterocyclyl-C(S)-O-, where alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are as described herein.
  • Sulfinyl refers to groups H-S(O)-, alkyl-S(O)-, cycloalkyl-S(O)-, aryl-S(O)-, heteroaryl-S(O)-, and heterocyclyl-S(O)-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.
  • Sulfonyl refers to groups H-S(O) 2 -, alkyl-S(O) 2 -, cycloalkyl-S(O) 2 -, aryl-S(O) 2 -, heteroaryl-S(O)2-, and heterocyclyl-S(O) 2 -, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.
  • Sulfmylamino refers to groups H-S(O)-NR*-, alkyl-S(O)-NR*-, cycloalkyl-S(O)- NR*-, aryl-S(O)-NR*-, heteroaryl-S(O)-NR*-, and heterocyclyl-S(O)-NR*-, where R* is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
  • “Sulfonylamino” refers to groups H-S(O) 2 -NR*-, alkyl-S(O) 2 -NR*-, cycloalkyl-S(O) 2 - NR*-, aryl-S(O) 2 -NR*-, heteroaryl-S(O) 2 -NR*-, and heterocyclyl-S(O) 2 -NR*-, where R* is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
  • Oxy sulfmylamino refers to groups HO-S(O)-NR*-, alkylO-S(O)-NR*-, cycloalkylO- S(O)-NR*-, arylO-S(O)-NR*-, heteroarylO-S(O)-NR*-, and heterocyclylO-S(O)-NR*-, where R* is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
  • Oxysulfonylamino refers to groups HO-S(O) 2 -NR*-, alkylO-S(O) 2 -NR% cycloalkylO-S(O) 2 -NR*-, arylO-S(O) 2 -NR*-, heteroarylO-S(O) 2 -NR*-, and heterocyclylO-S(O) 2 -NR*-, where R* is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
  • Aminothioacyl refers to groups R*R*N-C(S)-, where each R* is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclic and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
  • Thioacylamino refers to groups H-C(S)-NR*-, alkyl-C(S)-NR*-, cycloalkyl-C(S)- NR*-, aryl-C(S)-NR*-, heteroaryl-C(S)-NR% and heterocyclyl-C(S)-NR*-, where R* is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
  • Aminosulfinyl refers to groups R*R*N-S(0)-, where each R* is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclic and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
  • Aminosulfonyl refers to groups R*R*N-S(O) 2 -, where each R* is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclic and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.
  • a group may or may not be further substituted or fused (so as to form a condensed polycyclic group) with one or more groups selected from hydroxyl, acyl, alkyl (which may be further substituted by amino, aminoacyl, oxyacyl, hydroxy, aryl and nitro), alkoxy, alkenyl, alkenyloxy, alkynyl, alkynyloxy, amino, aminoacyl, thio, arylalkyl, arylalkoxy (which may be further substituted by halogen, hydroxy, alkyl, nitro, alkoxy, acyl and amino), aryl (which may be further substituted by halogen, hydroxy, alkyl, nitro, alkoxy, acyl and amino), aryloxy (which may be further substituted by halogen, hydroxy, alkyl, nitro, alkoxy, acyl and amino), carboxyl, acylamino,
  • R 1 includes the following groups: lower alkoxy; nitro; trihalomethyl, including trifluoromethane; and halogen, including chloro, bromo and fluoro.
  • n is 1 or 2 and in another embodiment n is 1.
  • R 2 includes the following groups: halogen, preferably chloro.
  • n is 0 or 1 and in another embodiment m is 0.
  • n is 1 or 2 and m is 0 or 1
  • n is 1 and m is 0,
  • n is 2 and m is 0,
  • n is 1 and m is 1, or
  • n is 2 and m is 1.
  • n is 1 or 2 and each Ri when present is an electron withdrawing group.
  • Most preferred electron withdrawing groups for Rj include halogen, nitro, trihaloethenyl, trihalomethanethio, trihalomethoxy, and trihalomethyl. More preferably R 1 is nitro, halo, trihalomethyl, and even more preferably R 1 is nitro, trifluoromethyl, bromo, chloro, or fluoro.
  • the Ri group is positioned ortho or meta (more preferably meta) on the phenyl ring relative to the point of attachment to the thiophene (i.e. for example 3-Cl, 3-F, 3-CF 3 or 3,5-di-CF 3 ).
  • n is 1 or 2 and R 1 is nitro, halo, trifluoromethane, wherein R 1 is positioned meta on the phenyl ring relative to the point of attachment to the thiophene.
  • n is 1
  • R 1 is halo or trifluoromethane (wherein R] is positioned meta on the phenyl ring relative to the point of attachment to the thiophene (i.e., for example, 3-F or 3-CF 3 )) and m is 0 or 1.
  • formula (I) is a compound of formula (F) or salt thereof:
  • R 1 a and R ⁇ are each independently selected from hydrogen, halo, and trifluoromethyl, provided that not both R la and Ri b are hydrogen; and R 2a is selected from hydrogen or halo.
  • Ri 3 is Cl, F 5 or CF 3 and R ]b is hydrogen and R 2a is hydrogen or chloro.
  • R 1 a and R ⁇ are both CF 3 .
  • R la is CF 3 , Cl, or F, Ru, is hydrogen and R 2a is hydrogen.
  • Ri 3 is CF 3 , Cl, or F
  • Rj b is hydrogen
  • R 2a is chloro
  • R 3 is halogen and R 4 is ethyl or methyl. Even more preferably the halogen is positioned para on the phenyl ring relative to the point of attachment to the thiophene. More preferably R 3 is chloro (wherein R 3 is positioned para on the phenyl ring relative to the point of attachment to the thiophene, (i.e. 4-chloro)) and R 4 is ethyl.
  • the compounds of formulae (I) and (II) may be prepared by the reaction sequence depicted in Scheme 1 below (wherein R 5 is -OR 4 or -Ph-(-R 2 ) m ; R 6 is -Ph-(-R 3 ); Pr is a protecting group and X is Br or I).
  • reaction sequence depicted in Scheme 1 provides not only easy access to compounds of formula I and II but also provides the artisan with access to a range of variously 5-substituted 2-amino-thiophenes for further investigating the structure activity relationship of the 5 -position.
  • the invention also provides a method of preparing compounds of formula (III) and salts thereof:
  • said method comprising the steps of:
  • Scheme 1 represents a linear synthetic sequence which may prepare both compounds of formula (I) (steps (i) and (ii)) and compounds of formula (II) (including additional steps (iii)-(vi)).
  • the thiophene core may be formed via a two step Gewald synthesis in which a substituted acetophenone (2) may be initially reacted with either a substituted benzoylacetonitrile or a Cj -3 alkyl cyanoacetate, in the presence of titanium(IV)chloride to afford the corresponding Knoevenagel product (3).
  • This olefin may be subsequently cyclised with sulphur under basic conditions to yield the desired 2- aminothiophene (for instance, compounds of formula I where R 5 is -Ph-(-R 2 ) m ).
  • the free amino product (4) may be optionally protected before proceeding, for instance using any suitable amino protecting group such as Boc protection using BoC 2 O and catalytic DMAP in dioxane or phthaloyl protection.
  • suitable protecting groups are known to those skilled in the art, for example, as described in Protective Groups in Organis Synthesis (T.W Greene and P.G.M Wutz, Wiley Interscience, New York, 3 rd edition).
  • the protected 2-aminothiophene (5) may then be converted to the corresponding 5-halo-analog (6) using a suitable halogenating agent such as N-bromosuccinimide.
  • the R 6 group may be introduced by a Pd mediated cross coupling reaction such as using Suzuki-Miyaura cross coupling conditions in DMF/H 2 0 mixtures with tribasic potassium phosphate and catalytic Pd(PPh 3 ) 4 in an inert atmosphere (N 2 ) with heating (70-80 °C) in a 24 h period or alternatively under microwave irradiation.
  • the resultant protected cross coupled product may then be deprotected, if required, to afford (7) (compounds of formula II, when R 6 is - Ph-(-R 3 ) and R 5 is -OR 4 ).
  • the crude isolated product (7) may be purified by recrystallisation or column chromatography.
  • n is an integer from 0 to 3; each Rj independently represents carboxyl, cyano, dihalomethoxy, halogen, hydroxy, nitro, pentahaloethyl, phosphono, phosphorylamino, phosphinyl, thio, sulfinyl, sulfonyl, trihaloethenyl, trihalomethanethio, trihalomethoxy, trihalomethyl, optionally substituted acyl, optionally substituted acylamino, optionally substituted acyliniino, optionally substituted acyliminoxy, optionally substituted acyloxy, optionally substituted arylalkyl, optionally substituted arylalkoxy, optionally substituted alkenyl, optionally substituted alkenyloxy, optionally substituted alkoxy, optionally substituted alkyl, optionally substituted alkynyl, optionally substituted alkynyloxy, optionally substituted amino, optionally substituted substituted
  • R 7 is optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkenyl, optionally substituted alkyl, optionally substituted alkynyl; Hal is chloro, bromo or iodo; and
  • Z is halogen, triflate, sulfonate, BrZn, Sn(alkyl) 3 , Sn(aryl) 3 or B(OR 8 ) 2 where Rs is H or alkyl.
  • step d) the coupling of formulae (UIe) and (IHf) may be carried out under a range of conditions known to those skilled in art of Pd based coupling reactions.
  • R 7 is an optionally substituted alkenyl or optionally substituted alkynyl
  • compounds where R 7 is an optionally substituted alkenyl or optionally substituted alkynyl may be prepared using standard Stille (with reactive stannanes), Hiyama or Sonogashira (with catalytic Cu) reaction conditions. Negishi conditions with organozinc reagents may afford R 7 substituents which are optionally substituted aryl.
  • Pd coupling agents examples include: Pd(PPli 3 ) 2 Cl 2 , Pd(PPh 3 ) 4 , Pd(dibenzylideneacetone) 3 , PdCl 2 (CH 3 CN) 2 , Pd(OAc) 2 , and the like.
  • the improved activity of the present invention may be as a result of the present compounds possessing enhanced allosteric effects while also minimising antagonistic activity at adenosine Aj receptors. It has now been found that the activity of thiophene based allosteric enhancers may be improved by selecting particular substitution patterns.
  • cardioprotective therapies which respond well to the promotion of angiogenesis.
  • cardioprotective therapies may include protection against conditions such as hypoxia, ischemia (including strokes, heart disease, and peripheral vascular disease), induced injury as well as adenosine-sensitive cardiac arrthythmias and seizures.
  • the invention also provides for the use of a compound of formulae (I) or (II) in the manufacture of a medicament for treating or managing pain or as a cardioprotective agent.
  • the compounds of the invention may be particularly useful in combination therapy, eg. combining the treatment with other chemotherapeutic treatments.
  • the compounds of the present invention may be used in combination with an orthosteric agonist, thus boosting the latter's efficacy.
  • the compounds of the invention can be used in the treatment of any disease state for which the enhancement of adenosine binding and functional activation of Ai adenosine receptors may be beneficial.
  • Compounds of the present invention can be formulated as a composition, particularly a pharmaceutical composition, together with a pharmaceutically acceptable additive.
  • a treatment effective amount is intended to include at least partially attaining the desired effect, or delaying the onset of, or inhibiting the progression of, or halting or reversing altogether the onset or progression of the particular disease of condition being treated.
  • the term "effective amount" relates to an amount of compound which, when administered according to a desired dosing regimen, provides the desired therapeutic activity. Dosing may occur at intervals of minutes, hours, days, weeks, months or years or continuously over any one of these periods. Suitable dosages lie within the range of about 0.1 ng per kg of body weight to 1 g per kg of body weight per dosage.
  • the dosage may be in the range of 1 ⁇ g to 1 g per kg of body weight per dosage, such as is in the range of 1 mg to 1 g per kg of body weight per dosage. In one embodiment, the dosage may be in the range of 1 mg to 500 mg per kg of body weight per dosage.
  • the dosage may be in the range of 1 mg to 250 mg per kg of body weight per dosage. In yet another preferred embodiment, the dosage may be in the range of 1 mg to 100 mg per kg of body weight per dosage, such as up to 50 mg per body weight per dosage.
  • Suitable dosage amounts and dosing regimens can be determined by the attending physician and may depend on the particular condition being treated, the severity of the condition as well as the general age, health and weight of the subject.
  • the active ingredient may be administered in a single dose or a series of doses. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a composition, preferably as a pharmaceutical composition.
  • the formulation of such compositions is well known to those skilled in the art.
  • the composition may contain any suitable carriers, diluents or excipients. These include all conventional solvents, dispersion media, fillers, solid carriers, coatings, antifungal and antibacterial agents, dermal penetration agents, surfactants, isotonic and absorption agents and the like. It will be understood that the compositions of the invention may also include other supplementary physiologically active agents.
  • compositions include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parental (including subcutaneous, intramuscular, intravenous and intradermal) administration.
  • the compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
  • compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient may also be presented as a bolus, electuary or paste.
  • a tablet may be made by compression or moulding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed i with a binder (e.g inert diluent, preservative disintegrant (e.g. sodium starch glycolate, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose) surface- active or dispersing agent.
  • a binder e.g inert diluent, preservative disintegrant (e.g. sodium starch glycolate, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose) surface- active or dispersing agent.
  • Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.
  • compositions suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured base, usually sucrose and acacia or tragacanth gum; pastilles comprising the active ingredient in an inert basis such as gelatine and glycerin, or sucrose and acacia gum; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
  • compositions suitable for topical administration to the skin may comprise the compounds dissolved or suspended in any suitable carrier or base and may be in the form of lotions, gel, creams, pastes, ointments and the like.
  • suitable carriers include mineral oil, propylene glycol, polyoxyethylene, polyoxypropylene, emulsifying wax, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • Transdermal patches may also be used to administer the compounds of the invention.
  • compositions for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter, glycerin, gelatine or polyethylene glycol.
  • compositions suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
  • Compositions suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bactericides and solutes which render the composition isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • compositions may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • sterile liquid carrier for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • Preferred unit dosage compositions are those containing a daily dose or unit, daily sub- dose, as herein above described, or an appropriate fraction thereof, of the active ingredient.
  • compositions of this invention may include other agents conventional in the art having regard to the type of composition in question, for example, those suitable for oral administration may include such further agents as binders, sweeteners, thickeners, flavouring agents disintegrating agents, coating agents, preservatives, lubricants and/or time delay agents.
  • suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine.
  • Suitable disintegrating agents include cornstarch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar.
  • Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring.
  • Suitable coating agents include polymers or copolymers of acrylic acid and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten.
  • Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite.
  • Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc.
  • Suitable time delay agents include glyceryl monostearate or glyceryl distearate.
  • the compounds of the present invention may be administered to a subject as a pharmaceutically acceptable salt.
  • Suitable pharmaceutically acceptable salts include, but are not limited to salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, maleic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benezenesulphonic, salicyclic sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic,
  • Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium.
  • the present invention includes within its scope cationic salts eg sodium or potassium salts, or alkyl esters (eg methyl, ethyl) of the phosphate group.
  • Basic nitrogen-containing groups may be quarternised with such agents as lower alkyl halide, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others.
  • lower alkyl halide such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides
  • dialkyl sulfates like dimethyl and diethyl sulfate; and others.
  • prodrug any compound that is a prodrug of a compound of formula (I) is also within the scope and spirit of the invention.
  • pro-drug is used in its broadest sense and encompasses those derivatives that are converted in vivo to the compounds of the invention. Such derivatives would readily occur to those skilled in the art, and include, for example, compounds where a free hydroxy group (for instance at the
  • Ri or R 2 position is converted into an ester, such as an acetate or phosphate ester, or where a free amino group is (for instance at the C-2 position of the thiophene or a Ri or R 2 group) converted into an amide (eg. ⁇ -aminoacid amide).
  • Procedures for esterifying, eg. acylating, the compounds of the invention are well known in the art and may include treatment of the compound with an appropriate carboxylic acid, anhydride or chloride in the presence of a suitable catalyst or base.
  • the compounds of the invention may be in crystalline form either as the free compounds or as solvates (e.g. hydrates) and it is intended that both forms are within the scope of the present invention. Methods of solvation are generally known within the art.
  • compounds of the invention may possess asymmetric centres and are therefore capable of existing in more than one stereoisomeric form.
  • the invention thus also relates to compounds in substantially pure isomeric form at one or more asymmetric centres eg., greater than about 90% ee, such as about 95% or 97% ee or greater than 99% ee, as well as mixtures, including racemic mixtures, thereof.
  • Such isomers may be prepared by asymmetric synthesis, for example using chiral intermediates, or mixtures may be resolved by conventional methods, eg., chromatography, or use of a resolving agent.
  • the compounds of the present invention may be capable of undergoing tautomerism. Accordingly, all possible tautomers of a compound of the present invention fall within the scope and spirit of the invention.
  • the synthetic methods and processes described herein to prepare the compounds of the present invention are amenable to solid phase synthetic techniques and/or combinatorial chemistry to produce individual compounds or libraries of compounds.
  • Solution phase libraries may be prepared via parallel syntheses wherein different compounds are synthesised in separate reaction vessels in parallel, often in an automated fashion.
  • attachment of the individual components employed in a synthetic sequence to an appropriate solid phase support allows for the further creation of chemical diversity by utilising not only parallel synthesis but also split synthesis wherein the solid support containing the compounds prepared in the prior step can be split into a number of batches, treated with the appropriate reagent and recombined.
  • the substrates can be attached to a solid support surface by any linkers known in the art.
  • the linkers may be any component capable of being cleaved to release the substrate or final compound from the support.
  • the solid support is a polymer support.
  • polymeric supports currently used in solid phase synthesis include: alkenyl resins: eg. REM resins; BHA resins: eg. benzhydrylamine (polymer-bound hydrochloride, 2% crosslinked), benzhydryl chloride (polymer bound); Br- functionalised resins: eg. brominated PPOA resin, brominated Wang resin; Chloromethyl resins: eg. 4-methoxybenzhydryl chloride (polymer bound); CHO-functionalised resins: eg. indole resin, formylpolystyrene; Cl-functionalised resins: eg.
  • OH- functionalised resins eg. 4-benzyloxybenzyl alcohol (polymer bound); Hydroxy methyl resins: eg. benzyl alcohol (polymer bound); HMBA resin; Oxime resins; Rink acid resin; Triazine-based resin; Trityl amine resins; Trityl resins: eg. trityl-chloride (polymer bound), 2-chlorotrityl alcohol, 1,3-diaminepropane trityl.
  • OH- functionalised resins eg. 4-benzyloxybenzyl alcohol (polymer bound); Hydroxy methyl resins: eg. benzyl alcohol (polymer bound); HMBA resin; Oxime resins; Rink acid resin; Triazine-based resin; Trityl amine resins; Trityl resins: eg. trityl-chloride (polymer bound), 2-chlorotrityl alcohol, 1,3-diaminepropane
  • individual compounds or libraries of compounds can be synthesised by initially attaching the first compound substrate to a solid support surface which can be performed by providing a plurality of solid support surfaces, suitably derivatising each of the surfaces with groups capable of reacting with either the compound substrate or a linker moiety attached thereto.
  • the various support surfaces with the attached first compound substrate can then be subjected to various reaction conditions and second compound substrates to provide a library of attached compounds, which may, if necessary, be reacted further with third and subsequent compound substrates or varying reactions conditions.
  • Attachment and detachment of substrates and products can be performed under conditions similar to those as described in Johnson, M.G., et al, Tetrahedron, 1999, 55, 11641; Han Y., et al. Tetrahedron 1999, 55, 11669; and Collini, M.D., et al, Tetrahedron Lett, 1997, 58, 7963.
  • the allosteric score (0-100%) is a measure of the maximum allosteric effect of each compound in preventing orthosteric radioligand dissociation.
  • the potency (ED 5 o) of each compound as an allosteric enhancer, and orthosteric antagonist activities for each compound are also reported.
  • the maximum score (Max in Table 1) and the ED 50 we fit a dose response curve of enhancer score vs. enhancer concentration to a 4 parameter logistic equation using Graphpad PrismTM software. From the fit to these curves we calculate the ED 50 of each AE, as a measure of potency, and the theoretical maximum score that would occur upon saturation of the AE binding site, a measure of maximum allosteric efficacy.
  • Example 1 the most active compound was more rigorously characterized in a cell-based signalling assay of adenosine Ai receptor-mediated ERK1/2 phosphorylation.
  • Figure 1 shows the results of these experiments, where it can be seen that Example 1 caused a concentration-dependent enhancement of the effects of the orthosteric agonist, R-PIA, in a recombinant cell line stably expressing the human A 1 receptor.
  • Ethyl 2-amino-4- (3-(trifluoromethyl)phenyl)thiophene-3-carboxylate had a AE score of only 8% while the 5-pyridyl analog (Reference Example 14) was inactive at the test concentration.
  • the aqueous phase is extracted with dichloromethane (2 x 30 mL) and the combined organics were washed with water then brine, dried over MgSO 4 , filtered and concentrated to a resin.
  • the resin was taken up in THF (6 mL) and elemental sulphur (3.3 mmol, 106 mg) was added followed by Et 2 NH (610 ⁇ L) and stirred at room temperature for 18 hr. The mixture is diluted with ethyl acetate and washed with water (x 2), then brine, dried over MgSO 4 , filtered and concentrated to a resin.
  • Example 1 (1.00 g, 2.88 mmol) was dissolved in dioxane (20 mL) and Boc 2 O (0.66 g, 3.02 mmol) was added to the solution followed by DMAP (35 mg, 10 mol%). The homogeneous solution was heated on an oil bath at 70 to 80 0 C for 2 h. After cooling, the solution was diluted with ether and washed with water (x 2) and then brine. The organic layer was dried (MgSO 4 ), filtered from insoluble material and evaporated to give an orange oil (1.84 g). This oil was chromatographed on silica gel column using EtOAc ⁇ et ether (5:95) as an eluent to afford a pale yellow resin (0.75 g, 58%).
  • the resin (494 mg, 1.10 mmol) was dissolved in 7 mL of CH 2 C
  • Method B Intermediate A or B (200 mg) was dissolved in 4:1 DMF / 2M K 3 PO 4 (2 mL, previously sonicated for 30 min) in a sealed vial (microwave pressure tube). The appropriate boronic acid (1.2 eq) and Pd(PPh 3 ) 2 Cl 2 (8.0 mg, 11.40 ⁇ mol, 3 mol%) were added and the mixture was heated with stirring to 150 0 C for 15 min. If the reaction was incomplete (by TLC), a further equivalent of boronic acid and 3 mol% of catalyst were added and microwave heating was continued for 10 min at 150 0 C. The solution was cooled and the toluene layer was filtered through a silica plug eluting with EtOAc and finally chloroform and concentrated to a residue.
  • Method B The residue obtained was sufficiently pure for the next step. It was dissolved in CH 2 Cl 2 (1 mL), TFA (1 mL) was added and the reaction was stirred at room temperature. After 3 h, the mixture was diluted with CHCl 3 (10 mL) and concentrated to a reddish brown residue. The residue was chromatographed on a silica gel column eluting with EtOAc:pet ether (15:85), to provide a light purple resin which slowly solidified upon standing. Complete crystallisation was induced by sonication in 50% aqueous methanol, giving a light purple powder (91 mg, 49%). Mp 112-116 °C.
  • Example 1 (0.65 mg, 1.86 mmol) and phthalic anhydride (0.33 g, 2.23 mmol) were dissolved in 17 mL of glacial acetic and refluxed overnight. The solution was concentrated and the resultant solid was recrystallised from 5 r ec-butanol/H 2 O (80:20) to give compound phthaloyl protected product as a pale yellow solid (661 mg).
  • This crude product 200 mg, 0.42 mmol
  • the binding kinetic assay of AE activity consisted of three phases: (1) binding to equilibrium of the agonist, 125 I-ABA to the AiAR-G protein ternary complex; (2) stabilization of that complex by adding vehicle or AE for 30 min, and (3) dissociation of the complex by adding a combination of an A 1 AR antagonist, 100 ⁇ M BW-1433, and 25 ⁇ M GTP ⁇ S for 10 min. Compounds were scored between 0% (no different than AE vehicle) and 100% (complete abolition of 125 I-ABA dissociation).
  • the assay employed membranes from CHO-Kl cells stably expressing the ILA 1 AR.
  • phase 1 For agonist binding to equilibrium (phase 1) the buffer consisted of 10 mM HEPES, pH 7.2, containing 0.5 mM MgCl 2 , 1 LVmL adenosine deaminase, 0.5 nM 125 I-ABA and 10 ⁇ g of membrane protein in a final volume of 100 ⁇ L applied to 96 well Millipore GF/C glass fiber filter plates. After 90 minutes at room temperature the addition of 50 ⁇ L of AE (0.1 - 50 ⁇ M, final) initiated stabilization of the ternary complex (phase 2). Thirty minutes later 50 ⁇ L solution containing BW- 1433 and GTP ⁇ S was added to initiate the dissociation of the ternary complex. Ten minutes later membranes were filtered, washed, dried and counted for residual 125 I-ABA. The percentage of specifically bound agonist remaining after 10 minutes of dissociation served as an index of AE activity:
  • % AE score 100 x (B-B 0 ) / (B eq -B 0 )
  • B residual binding (cpm) bound at the end of 10 minutes of dissociation in the presence of an AE
  • B 0 residual binding (cpm) at the end of 10 minutes of dissociation in the absence of an AE
  • B e q cpm bound at the end of 90 minutes of equilibrium binding.
  • the percentage of specific binding remaining after 10 minutes of dissociation constitutes an index of AE activity for ranking candidate compounds.
  • a score of 100 % means no dissociation and a score of zero means complete dissociation.
  • CHOKl membranes expressing human Ai adenosine receptors were resuspended at 400 ⁇ g/mL in HE buffer containing lU/mL adenosine deaminase. 50 ⁇ L of membrane solution was added to 50 ⁇ L HE buffer containing [ 3 H] CPX (2 nM). 100 ⁇ L of HE buffer with either vehicle, enhancer or NECA (to define non-specific) was added. The final drug concentrations were 10 ⁇ M for enhancer and 100 ⁇ M for NECA. Samples were incubated for 90 minutes at room temperature, filtered and counted on a liquid scintillation counter. Binding was performed in triplicate and expressed as % inhibition as compared to control binding.
  • A] receptor were approximately 90% confluent, they were harvested using trypsinization and centrifuged (300 g, 3 minutes). The pellet was then resuspended in HEPES homogenization buffer (5OmM HEPES, 2.5mM MgCl 2 , 2mM EGTA) and the centrifugation procedure repeated. The intact cell pellet was resuspended in HEPES homogenization buffer and homogenized using a Polytron homogenizer for two 10-second intervals at maximum setting, with 30 second cooling periods on ice between each burst. The homogenate was centrifuged (1000 g, 10 min, 25°), the pellet discarded and the supernatant recentrifuged (30,000 g, 30 min, 4 0 C).
  • HEPES homogenization buffer 5OmM HEPES, 2.5mM MgCl 2 , 2mM EGTA
  • the resulting pellet was resuspended in 5mL assay buffer (1OmM HEPES, 10OmM NaCl, 1OmM MgCl 2 ; pH 7.4 at 30°C) and the protein content determined using the method of Bradford (1976).
  • Membrane homogenates (15 ⁇ g) were equilibrated in a 500 ⁇ L total volume of assay buffer containing 1 O ⁇ M guanosine 5 '-diphosphate (GDP) and a range of concentrations of R-PIA (O.OlnM - lO ⁇ M) in the absence or presence of VCP171 (0.1 ⁇ M to 3 ⁇ M) at 30°C for 60 minutes. After this time, 50 ⁇ L of [ 35 S]-GTPyS (InM) was added and incubation continued for 30 minutes at 3O 0 C. Incubation was terminated by rapid filtration through Whatman GF/B filters using a Brandell cell harvester (Gaithersburg, MD).
  • GDP O ⁇ M guanosine 5 '-diphosphate
  • CHO-FIpIn cells stably expressing the human adenosine A 1 receptor were seeded into 96- well plates at a density of 50 000 cells/well. After 4 hours, cells were washed twice with PBS and maintained in DMEM containing 16mM HEPES and 50U/mL penicillin- streptomycin for at least 4 hours before assaying. Assays investigating the time course of action of ERK 1/2 phosphorylation were generated by the addition of ligand for various time periods (200 ⁇ L final volume) at 37 0 C. The agonist R-PIA peaked at 5min while Example 1 peaked at 1 Omin.
  • the time of stimulation when constructing concentration- response curves was chosen from the time to peak response as determined in the time course assays, lOmin for the allosteric stimulation and 5min for the agonist stimulation. Stimulation of cells was terminated by the removal of media and the addition of lOO ⁇ L of SureFireTM lysis buffer to each well. The plate was then agitated for 1-2 minutes. A 4:1 v/v dilution of Lysate:SureFireTM activation buffer was made in a total volume of 50 ⁇ L.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Cardiology (AREA)
  • Vascular Medicine (AREA)
  • Urology & Nephrology (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne en général des composés chimiques et des procédés pour leur utilisation et leur préparation. En particulier, l'invention concerne des composés chimiques qui peuvent posséder une activité thérapeutique utile pour traiter des conditions dans lesquelles la promotion de l'angiogenèse est bénéfique (formations de vaisseaux sanguins), l'utilisation de ces composés en thérapie et la fabrication de médicaments ainsi que de compositions contenant ces composés.
PCT/AU2008/001524 2007-10-15 2008-10-15 Amplificateurs allostériques des récepteurs de l'adénosine a1 WO2009049362A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US96080407P 2007-10-15 2007-10-15
US60/960,804 2007-10-15

Publications (1)

Publication Number Publication Date
WO2009049362A1 true WO2009049362A1 (fr) 2009-04-23

Family

ID=40566919

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU2008/001524 WO2009049362A1 (fr) 2007-10-15 2008-10-15 Amplificateurs allostériques des récepteurs de l'adénosine a1

Country Status (1)

Country Link
WO (1) WO2009049362A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012149452A1 (fr) * 2011-04-29 2012-11-01 Regents Of The University Of Michigan Composés, formulations et procédés d'inhibition de la protéine kinase c

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0350131A2 (fr) * 1988-07-07 1990-01-10 Duphar International Research B.V Dérivés de 1,4-diazénine à activité anti-ulcérogène
WO2005033102A2 (fr) * 2003-10-03 2005-04-14 Amphora Discovery Corporation Composes a base de thiophene presentant une activite d'inhibition d'enzymes utilisant l'atp, compositions contenant ces composes et utilisations

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0350131A2 (fr) * 1988-07-07 1990-01-10 Duphar International Research B.V Dérivés de 1,4-diazénine à activité anti-ulcérogène
WO2005033102A2 (fr) * 2003-10-03 2005-04-14 Amphora Discovery Corporation Composes a base de thiophene presentant une activite d'inhibition d'enzymes utilisant l'atp, compositions contenant ces composes et utilisations

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
AURELIO, L. ET AL.: "5-Substituted 2-aminothiophenes as A1 adenosine receptor allosteric enhancers.", BIOORGANIC AND MEDICINAL CHEMISTRY., vol. 16, 2008, pages 1319 - 1327, XP022453103, DOI: doi:10.1016/j.bmc.2007.10.065 *
BRUNS, R.F. ET AL.: "Structure-activity relationships for enhancement of adenosine A1 receptor binding by 2-amino-3-benzoylthiophenes.", MOLECULAR PHARMACOLOGY, vol. 38, 1990, pages 950 - 958, XP002093490 *
LUTJENS, H. ET AL.: "2-Amino-3-benzoylthiophene allosteric enhancers of A1 adenosine agonist binding: new 3, 4, and 5-modifications.", JOURNAL OF MEDICINAL CHEMISTRY, vol. 46, 2003, pages 1870 - 1877 *
ROMAGNOLI, R. ET AL.: "Synthesis and biological evaluation of 2-amino-3-(3',4',5'- trimethoxybenzoyl)-5-aryl thiophenes as a new class of potent antitubulin agents.", JOURNAL OF MEDICINAL CHEMISTRY, vol. 49, 2006, pages 3906 - 3915 *
TINNEY, F. J. ET AL.: "Synthesis and pharmacological evaluation of 2,3-dihydro-1H-thieno[2,3- e][1,4]diazepines.", JOURNAL OF MEDICINAL CHEMISTRY, vol. 17, 1974, pages 624 - 630, XP002966650, DOI: doi:10.1021/jm00252a011 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012149452A1 (fr) * 2011-04-29 2012-11-01 Regents Of The University Of Michigan Composés, formulations et procédés d'inhibition de la protéine kinase c
US8889672B2 (en) 2011-04-29 2014-11-18 The Regents Of The University Of Michigan Compounds, formulations, and methods of protein kinase C inhibition

Similar Documents

Publication Publication Date Title
TWI849107B (zh) 雙環化合物
CA2291065C (fr) Inhibiteurs de raf kinase
CN108779096B (zh) 一类氟取代的环丙胺类化合物及其制备方法、药物组合物和用途
AU725741B2 (en) Aminothiophenecarboxamides
AU2007332143B2 (en) Chemical compounds and processes
AU2001271022B2 (en) Propane-1,3-dione derivatives
EP1305294B1 (fr) DeRIVéS des IMIDAZOLYLES
DE69411317T2 (de) Indoloylguanidinderivate als Inhibitoren des Natrium-Protonen Austauschs
EP2142533B1 (fr) Dérivés d'imidazolidinone
US20070010556A1 (en) Thiophene derivatives as chk 1 inhibitors
US20040110741A1 (en) Substituted pyrazolyl compounds for the treatment of inflammation
AU2003265348A1 (en) Thiophene compounds
NZ505845A (en) Inhibition of RAF kinase using aryl and heteroaryl substituted heterocyclic ureas
EP1682538A1 (fr) Hydroxamates lies au biaryle: preparation et applications pharmaceutiques
CA2335724A1 (fr) Utilisation de diazepines pour la preparation de medicaments destines a traiter les etats pathologiques ou les maladies dans lesquels un des recepteurs de la somatostatine est implique
JP2007522142A (ja) Ｉｋｋ３に対して活性を有するベンゾイミダゾール置換チオフェン誘導体
EP1657238A1 (fr) Derive de pyrimidine fusionne et utilisation associee
BR112012007411B1 (pt) Composto, processo para sua preparação e composição farmacêutica contendo o mesmo
CA2519107A1 (fr) Triazolones fusionnees et utilisations de celles-ci
EP4324825A1 (fr) Régulateur du fxr, son procédé de préparation, composition pharmaceutique de celui-ci et son utilisation
EP1973916A1 (fr) Dérivés de pyrroloý1,2-a¨quinoxaline en tant que modulateurs du récepteur a3 de l'adénosine et applications
CZ20033018A3 (en) Sulfonamides
JP2003510320A (ja) 薬剤活性のあるスルホニルアミノ酸誘導体
EP4008716A1 (fr) Nouveaux inhibiteurs des protéines de liaison de l'arnm du facteur de croissance 2 de type insuline
EP1656360B1 (fr) Derives de thiazoline servant de modulateurs de recepteur des androgenes selectifs (sarms)

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08800157

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08800157

Country of ref document: EP

Kind code of ref document: A1