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Patent US7214665 - 2-propynyl adenosine analogs having A2A agonist activity and compositions ... - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inAdvanced Patent SearchPatentsThe invention provides compounds having the following general formula (I): wherein X, R1, R2, R7 and Z are as described here....http://www.google.com/patents/US7214665?utm_source=gb-gplus-sharePatent US7214665 - 2-propynyl adenosine analogs having A2A agonist activity and compositions thereofAdvanced Patent SearchPublication numberUS7214665 B2Publication typeGrantApplication numberUS 10/263,379Publication dateMay 8, 2007Filing dateOct 1, 2002Priority dateOct 1, 2001Fee statusPaidAlso published asCA2460911A1, CA2460911C, EP1434782A2, US7737127, US8158604, US20030186926, US20070232559, US20100152127, WO2003029264A2, WO2003029264A3Publication number10263379, 263379, US 7214665 B2, US 7214665B2, US-B2-7214665, US7214665 B2, US7214665B2InventorsJoel M. Linden, Jayson M. Rieger, Timothy L. MacDonald, Gail W. Sullivan, Lauren Jean Murphree, Robert Alan FiglerOriginal AssigneeUniversity Of Virginia Patent FoundationExport CitationBiBTeX, EndNote, RefManPatent Citations (101), Non-Patent Citations (100), Referenced by (22), Classifications (17), Legal Events (3) External Links: USPTO, USPTO Assignment, Espacenet2-propynyl adenosine analogs having A2A agonist activity and compositions thereofUS 7214665 B2Abstract The invention provides compounds having the following general formula (I):
Z is CR3R4R5 each R1 is independently hydrogen, halo, �ORa, �SRa, (C1�C8)alkyl, cyano, nitro, trifluoromethyl, trifluoromethoxy, C3-8cycloalkyl, heterocycle, hetrocycle(C1�C8)alkylene, aryl, aryl(C1�C8)alkylene-, heteroaryl, heteroaryl(C1�C8)alkylene-, �CO2Ra, RaC(═O)O�, RaC(═O)�, �OCO2Ra, RaRbNC(═O)O�, RbOC(═O)N(Ra)�, RaRbN�, RaRbNC(═O)�, RaC(═O)N(Rb)�, RaRbNC(═O)N(Rb)�, RaRbNC(═S)N(Rb)�, RaOC(═S)�, RaC(═S)�, �SSRa, RaS(═O)�, RaS(═O)2�, or �N═NRa;
each R2 is independently hydrogen, halo, (C1�C8)alkyl, (C3�C8)cycloalkyl, heterocycle, heterocycle(C1�C8)alkylene-, aryl, aryl(C1�C8)alkylene-, heteroaryl, or heteroaryl(C1�C8)alkylene-; or
R4 and R5 together with the C atom to which they are attached form a saturated or partially unsaturated, mono-, or bicyclic- ring having 3, 4, 5, 6, 7, 8, 9 or 10 ring atoms comprising 1, 2, 3, or 4 heteroatoms selected from non-peroxide oxy (�O�), thio (�S�), sulfinyl (�SO�), sulfonyl (�S(O)2�) or amine (�NRa�) in the ring;
wherein any ring comprising CR4R5 is substituted with from 1 to 14 R6 groups at any position other than at the 1-position; wherein each R6 is independently halo, �ORa, �SRa, (C1�C8)alkyl, cyano, nitro, trifluoromethyl, trifluoromethoxy, (C1�C8)cycloalkyl, (C6�C12)bicycloalkyl, heterocycle or heterocycle (C1�C8)alkylene, aryl, aryl(C1�C8)alkylene-, heteroaryl, heteroaryl(C1�C8)alkylene-, �CO2Ra, RaC(═O)O�, RaC(═O)�, �OCO2Ra, RaRbNC(═O)O�, RbOC(═O)N(Ra)�, RaRbN�, RaRbNC(═O)�, RaC(═O)N(Rb)�, RaRbNC(═O)N(Rb)�, RaRbNC(═S)N(Rb)�, RaOC(═S)�, RaC(═S)�, �SSRa, RaS(═O)�, or �NNRa, or two R6 groups and the atom to which they are attached is C═O, C═S or; two R6 groups together with the atom or atoms to which they are attached can form a carbocyclic or heterocyclic ring;
R3 is hydrogen, halo, �ORa, �SRa, (C1�C8)alkyl, cyano, nitro, trifluoromethyl, trifluoromethoxy, (C3�C8)cycloalkyl, heterocycle, hetrocycle(C1�C8)alkylene, aryl, aryl(C1�C8)alkylene-, heteroaryl, heteroaryl(C1�C8)alkylene-, �CO2Ra, RaC(═O)O�, RaC(═O)�, �OCO2Ra, RaRbNC(═O)O�, RbOC(═O)N(Ra)�, RaRbN�, RaRbNC(═O)�, RaC(═O)N(Rb)�, RaRbNC(═O)N(Rb)�, RaRbNC(═S)N(Rb)�, RaOC(═S)�, RaC(═S)�, �SSRa, RaS(═O)�, RaS(═O)2�)�, or �NNRa; or if the ring formed from CR4R5 is partially unsaturated then R3 can be absent;
each R7 is independently hydrogen, (C1�C8)alkyl, (C3�C8)cycloalkyl, aryl or aryl(C1�C8)alkylene, heteroaryl, heteroaryl(C1�C8)alkylene-;
X is �CH2ORa, �CO2Ra, �OC(O)Ra, �CH2OC(O)Ra, �C(O)NRaRb, �CH2SRa, �C(S)ORa, �OC(S)Ra, �CH2OC(S)Ra or C(S)NRaRb or �CH2N(Ra)(Rb);
wherein any of the alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl, groups of R1, R2, R3, R6 and R7 is optionally substituted on carbon with one or more substituents selected from the group consisting of halo, �ORa, �SRa, (C1�C8)alkyl, cyano, nitro, trifluoromethyl, trifluoromethoxy, (C3�C8)cycloalkyl, (C6�C12)bicycloalkyl, heterocycle or hetrocycle(C1�C8)alkylene-, aryl, aryloxy, aryl (C1�C8)alkylene-, heteroaryl, heteroaryl(C1�C8)alkylene-, �CO2Ra, RaC(═O)O�, RaC(═O)�, �OCO2Ra, RaRbNC(═O)O�, RbOC(═O)N(Ra)�, RaRbN�, RaRbNC(═O)�, RaC(═O)N(Rb)�, RaRbNC(═O)N(Rb)�, RaRbNC(═S)N(Rb)�, RaOC(═S)�, RaC(═S)�, �SSRa, RaS(═O)p��, RaRbNS(O)p�, and N═NRa;
wherein any (C1�C8)alkyl, (C3�C8)cycloalkyl, (C6�C12)bicycloalkyl, (C1�C8)alkoxy, (C1�C8)alkanoyl, (C1�C8)alkylene, or heterocycle, is optionally partially unsaturated;
Ra and Rb are each independently hydrogen, (C1�C8)alkyl, or (C1�C8)alkyl substituted with 1�3 (C1�C8)alkoxy, (C3�C8)cycloalkyl, (C1�C8)alkylthio, amino acid, aryl, aryl(C1�C8)alkylene, heteroaryl, or heteroaryl(C1�C8)alkylene; or Ra and Rb, together with the nitrogen to which they are attached, form a pyrrolidino, piperidino, morpholino, or thiomorpholino ring; and
Rc is hydrogen or (C1�C6)alkyl;
2. The compound of claim 1, wherein R1 is hydrogen, �OH, �CH2OH, �OCH3, �OC(═O)CH3, �NH2, �NHCH3, �N(CH3)2 or �NH(C═O)CH3.
3. The compound of claim 2, wherein R1 is hydrogen, �OH, �OCH3, �OC(═O)CH3, �NHCH3, �N(CH3)2 or �NHC(═O)CH3.
7. The compound of claim 1, wherein R2 is hydrogen, (C1�C8)alkyl, cyclopropyl, cyclohexyl or benzyl.
12. The compound of claim 1, wherein R3 is hydrogen, OH, OMe, �OC(═O)CH3, �NH2, NHMe, NMe2 or �NHC(═O)CH3.
17. The compound of claim 1, wherein R6 is (C1�C8)alkyl, substituted (C1�C8)alkyl, �ORa, �CO2Ra, RaC(═O)�, RaC(═O)O�, RaRbN�, RaRbNC(═O)�, or aryl.
18. The compound of claim 17, wherein R6 is (C1�C8)alkyl, �ORa, �CO2Ra, RaC(═O)�, RaC(═O)O�, RaRbN�, RaRbNC(═O)�, or aryl.
19. The compound of claim 18, wherein R6 is methyl, ethyl, butyl, OH, ORa, �CO2Ra, RaC(═O)�, OC(═O)CH2CH3, �CONRaRb, NRaRb or phenyl.
20. The compound of claim 19, wherein R6 is OH, OMe, methyl, ethyl, t-butyl, �CO2Ra, �CONRaRb, �OC(═O)CH3, NH2, NHMe, NMe2, NHEt or N(Et)2.
21. The compound of claim 20, wherein R6 is methyl, ethyl, t-butyl, phenyl, �CO2Ra �CONRaRb, or (C═O)CHR3.
22. The compound of claim 21, wherein R6 is methyl, ethyl, �CO2Ra �CONRaRb, or �OC(═O)CH3.
23. The compound of claim 22, wherein R6 is �(CH2)1-2ORa, �(CH2)1-2C(═O)ORa, �(CH2)1-2OC(═O)Ra, �(CH2)1-2C(═O)Ra, �(CH2)1-2OCO2Ra, �(CH2)1-2NHRa, �(CH2)1-2NRaRb, �(CH2)1-2OC(═O)NHRa, or �(CH2)1-2OC(═O)NRaRb.
24. The compound of claim 23, wherein R6 is �CH2OH, �CH2OC(═O)CH3, �CH2OCH3, �CH2C(═O)OCH3, �CH2OC(═O)CH3, �CH2C(═O)CH3, �CH2OCO2CH3, �CH2NH(CH3), or �(CH2)1-2N(CH3)2.
25. The compound of claim 24, wherein R6 is �CH2OH, �CH2OC(═O)CH3, �CH2C(═O)OCH3, �CH2C(═O)CH3, �CH2OCO2CH3, �CH2NH(CH3), or �(CH2)1-2N(CH3)2.
27. The compound of claim 1, wherein Ra and Rb are hydrogen, (C1�C4)alkyl, aryl or aryl(C1�C8)alkylene.
29. The compound of claim 26, wherein Ra is (C1�C8)alkyl.
33. The compound of claim 1, wherein R7 is hydrogen, alkyl, aryl or aryl(C1�C8)alkylene.
37. The compound of claim 36, wherein �N(R7)2 is amino, methylamino, dimethylamino; ethylamino; diethylamino or benzylamino.
39. The compound of claim 38, wherein X is �CH2ORa, �CO2Ra, �OC(O)Ra, �CH2OC(O)Ra, �C(O)NRaRb.
40. The compound of claim 39, wherein X is �CH2ORa or �C(O)NRaRb.
41. The compound of claim 40, wherein X is �CH2OH or �C(O)NHCH2CH3.
44. The compound of claim 1, wherein the ring comprises �C(R3)R4R5 is 4-piperidene-1-carboxylic acid, 4-piperidine-1-carboxylic acid methyl ester, 4-piperidine-1-carboxylic acid ethyl ester, 4-piperidine-1-carboxylic acid propyl ester, 4-piperidine-1-carboxylic acid tert-butyl ester, 3-piperidine-1-carboxylic acid methyl ester, 3-piperidine-1-carboxylic acid tert-butyl ester, 3-piperazine-1-carboxylic acid, 3-piperazine-1-carboxylic acid methyl ester, 3-piperazine-1-carboxylic acid ethyl ester, 3-piperazine-1-carboxylic acid propyl ester, or 3-piperidine-1-carboxylic acid tert-butylester.
CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority of U.S. provisional patent application Ser. No. 60/326,517, filed Oct. 1, 2001, and U.S. provisional patent application Ser. No. 60/383,200, filed May 24, 2002, both of which are incorporated by reference herein.
There is one report of the combination of relatively nonspecific adenosine analogs, R-phenylisopropyladenosine (R-PIA) and 2-chloroadenosine (Cl-Ado) with a phosphodiesterase (PDE) inhibitor resulting in a lowering of neutrophil oxidative activity (M. A. Iannone et al., Topics and Perspectives in Adenosine Research, E. Garlach et al., eds., Springer-Verlag, Berlin, pp. 286�298 (1987)). However, R-PIA and Cl-Ado analogs are actually more potent activators of A1 adenosine receptors than of AA2 adenosine receptors and, thus, are likely to cause side effects due to activation of A1 receptors on cardiac muscle and other tissues causing effects such as �heart block.�
SUMMARY OF THE INVENTION The present invention comprises compounds and methods of their use for the treatment of inflammatory activity in mammalian tissue. The inflammatory tissue activity can be due to pathological agents or can be due to physical, chemical or thermal trauma, or the trauma of medical procedures, such as organ, tissue or cell transplantation, angioplasty (PCTA), inflammation following ischemia/reperfusion, or grafting. The present compounds comprise a novel class of 2-alkynyladenosine derivatives, substituted at the ethyn-2-yl position by substituted cycloalkyl and heterocycle (heterocyclic) moieties. Preferably, the riboside residue is substituted at the 5′-position by an N-alkyl-(or cycloalkyl)carboxyamino (�aminocarbonyl�) moiety (�X�). Thus, the present invention provides a method for inhibiting the inflammatory response in a mammal, such as a human subject, and protecting the tissue subject to the response, by administering an effective amount of one or more compounds of the invention.
each R1 is independently hydrogen, halo, �ORa, �SRa, (C1�C8)alkyl, cyano, nitro, trifluoromethyl, trifluoromethoxy, C3-8cycloalkyl, heterocycle, hetrocycle(C1�C8)alkylene-, aryl, aryl(C1�C8)alkylene-, heteroaryl, heteroaryl(C1�C8)alkylene-, �CO2Ra, RaC(═O)O�, RaC(═O)�, �OCO2Ra, RaRbNC(═O)O�, RbOC(═O)N(Ra)�, RaRbN�, RaRbNC(═O)�, RaC(═O)N(Rb)�, RaRbNC(═O)N(Rb)�, RaRbNC(═S)N(Rb)�, �OPO3Ra, RaOC(═S)�, RaC(═S)�, �SSRa, RaS(═O)�, RaS(═O)2�, �N═NRa, or �OPO2Ra;
R4 and R5 together with the atoms to which they are attached form a saturated or partially unsaturated, mono-, bicyclic- or aromatic ring having 3, 4, 5, 6, 7, 8, 9 or 10 ring atoms optionally comprising 1, 2, 3, or 4 heteroatoms selected from non-peroxide oxy (�O�), thio (�S�), sulfinyl (�SO�), sulfonyl (�S(O)2�) or amine (�NRa�) in the ring;
wherein any ring comprising R4 and R5 is substituted with from 1 to 14 R6 groups; wherein each R6 is independently halo, �ORa, �SRa, (C1�C8)alkyl, cyano, nitro, trifluoromethyl, trifluoromethoxy, (C1�C8)cycloalkyl, (C6�C12)bicycloalkyl, heterocycle or hetrocycle (C1�C8)alkylene-, aryl, aryl (C1�C8)alkylene-, heteroaryl, heteroaryl(C1�C8)alkylene-, �CO2Ra, RaC(═O)O�, RaC(═O)�, �OCO2Ra, RaRbNC(═O)O�, RbOC(═O)N(Ra)�, RaRbN�, RaRbNC(═O)�, RaC(═O)N(Rb)�, RaRbNC(═O)N(Rb)�, RaRbNC(═S)N(Rb)�, �OPO3Ra, RaOC(═S)�, RaC(═S)�, �SSRa, RaS(═O)�, �NNRa, �OPO2Ra, or two R6 groups and the atom to which they are attached is C═O, C═S or; two R6 groups together with the atom or atoms to which they are attached can form a carbocyclic or heterocyclic ring.
R3 is hydrogen, halo, �ORa, �SRa, (C1�C8)alkyl, cyano, nitro, trifluoromethyl, trifluoromethoxy, (C3�C8)cycloalkyl, heterocycle, hetrocycle(C1�C8)alkylene-, aryl, aryl(C1�C8)alkylene-, heteroaryl, heteroaryl(C1�C8)alkylene-, �CO2Ra, RaC(═O)O�, RaC(═O)�, �OCO2Ra, RaRbNC(═O)O�, RbOC(═O)N(Ra)�, RaRbN�, RaRbNC(═O)�, RaC(═O)N(Rb)�, RaRbNC(═O)N(Rb)�, RaRbNC(═S)N(Rb)�, �OPO3Ra, RaOC(═S)�, RaC(═S)�, �SSRa, RaS(═O)�, RaS(═O)2�, �NNRa, �OPO2Ra; or if the ring formed from CR4R5 is aryl or hetreroaryl or partially unsaturated then R3 can be absent;
each R7is independently hydrogen, (C1�C8)alkyl, (C3�C8)cycloalkyl, aryl or aryl(C1�C8)alkylene, heteroaryl, heteroaryl(C1�C8)alkylene-;
wherein any of the alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl, groups of R1, R2, R3, R6 and R7is optionally substituted on carbon with one or more (e.g. 1, 2, 3, or 4) substituents selected from the group consisting of halo, �ORa, �SRa, (C1�C8)alkyl, cyano, nitro, trifluoromethyl, trifluoromethoxy, (C3�C8)cycloalkyl, (C6�C12)bicycloalkyl, heterocycle or hetrocycle(C1�C8)alkylene-, aryl, aryloxy, aryl (C1�C8)alkylene-, heteroaryl, heteroaryl(C1�C8)alkylene-, �CO2Ra, RaC(═O)O�, RaC(═O)�, �OCO2Ra, RaRbNC(═O)O�, RbOC(═O)N(Ra)�, RaRbN�, RaRbNC(═O)�, RaC(═O)N(Rb)�, RaRbNC(═O)N(Rb)�, RaRbNC(═S)N(Rb)�, �OPO3Ra, RaOC(═S)�, RaC(═S)�, �SSRa, RaS(═O)p�, RaRbNS(O)p�, N═NRa, and �OPO2Ra;
provided that when CR4R5 is a carbocyclic ring and (CR1R2) is absent or is (CH2)m, then R3 is not H, CO2Ra, RaC(═O)� or RaR6NC(═O)� when R6 is CH2OH, �CO2Ra, RaC(═O)O�, or RaRbN(C═O)�;
BRIEF DESCRIPTION OF THE FIGURES FIG. 1 illustrates the results of a comparison of the depression of blood pressure in rats using the compound ATL-146e and JR4007 at 100 ug/kg.
FIGS. 7�16 illustrate the results of the coronary blood flow for test compounds in dogs.
Specifically, (C1�C8)alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, hexyl, heptyl or octyl. As used herein, the term �cycloalkyl� encompasses bicycloalkyl (norbornyl, 2.2.2-bicyclooctyl, etc.) and tricycloalkyl (adamantyl, etc.), optionally comprising 1�2 N, O or S. Cycloalkyl also encompasses (cycloalkyl)alkyl. Thus, (C3�C6)cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like. (C1�C8)alkoxy can be methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy; (C2�C6)alkenyl can be vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, or 5-hexenyl; (C2�C6)alkynyl can be ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, or 5-hexynyl; (C1�C6)alkanoyl can be acetyl, propanoyl or butanoyl; halo(C1�C6)alkyl can be iodomethyl, bromomethyl, chloromethyl, fluoromethyl, trifluoromethyl, 2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, or pentafluoroethyl; hydroxy(C1�C6)alkyl can be hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl, 1-hydroxybutyl, 4-hydroxybutyl, 1-hydroxypentyl, 5-hydroxypentyl, 1-hydroxyhexyl, or 6-hydroxyhexyl; (C1�C6)alkoxycarbonyl (CO2R2) can be methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, or hexyloxycarbonyl; (C1�C6)alkylthio can be methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, pentylthio, or hexylthio, (C2�C6)alkanoyloxy can be acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy, or hexanoyloxy; aryl can be phenyl, indenyl, or naphthyl; and heteroaryl can be furyl, imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl, pyraxolyl, pyrrolyl, pyrazinyl, tetrazolyl, puridyl (or its N-oxide), thientyl, pyrimidinyl (or its N-oxide), indolyl, isoquinolyl (or its N-oxide) or quinolyl (or its N-oxide).
Aryl denotes a phenyl radical or an ortho-fused bicyclic carbocyclic radical having about nine to ten ring atoms in which at least one ring is aromatic. Heteroaryl denotes a radical of a monocyclic aromatic ring containing five or six ring atoms consisting of carbon and 1, 2, 3, or 4 heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(Y) wherein Y is absent or is H, O, (C1�C8)alkyl, phenyl or benzyl, as well as a radical of an ortho-fused bicyclic heterocycle of about eight to ten ring atoms derived therefrom, particularly a benz-derivative or one derived by fusing a propylene, trimethylene, or tetramethylene diradical thereto.
The term �heterocycle� generally represents a non aromatic heterocyclic group, having from 3 to about 10 ring atoms, which can be saturated or partially unsaturated, containing at least one heteroatom (e.g., 1, 2, or 3) selected from the group consisting of oxygen, nitrogen, and sulfur. Specific, �heterocycle� groups include monocyclic, bicyclic, or tricyclic groups containing one or more heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur. A �heterocycle� group also can include one or more oxo groups (═O) attached to a ring atom. Nonlimiting examples of heterocycle groups include 1,3-dioxolane, 1,4-dioxane, 1,4-dithiane, 2H-pyran, 2-pyrazoline, 4H-pyran, chromanyl, imidazolidinyl, imidazolinyl, indolinyl, isochromanyl, isoindolinyl, morpholine, piperazinyl, piperidine, piperidyl, pyrazolidine, pyrazolidinyl, pyrazolinyl, pyrrolidine, pyrroline, quinuelidine, thiomorpholine, and the like.
The term �aryl(C1�C8)alkylene� for example includes benzyl, phenethyl, naphthylmethyl and the like.
The carbon atom content of various hydrocarbon-containing moieties is indicated by a prefix designating the minimum and maximum number of carbon atoms in the moiety, i.e., the prefix Ci�Cj indicates a moiety of the integer �i� to the integer �j� carbon atoms, inclusive. Thus, for example, (C1�C8)alkyl refers to alkyl of one to eight carbon atoms, inclusive.
Specifically, (C1�C8)alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, hexyl, or heptyl; (C1�C8)alkoxy can be methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, hexyloxy, 1-methylhexyloxy, or heptyloxy; aryl can be phenyl, indenyl, or naphthyl; and heteroaryl can be furyl, imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl, pyrazolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridyl, (or its N-oxide), thienyl, pyrimidinyl (or its N-oxide), indolyl, isoquinolyl (or its N-oxide) or quinolyl (or its N-oxide).
A specific value for R2 is hydrogen or (C1�C8)alkyl, cyclopropyl, cyclohexyl or benzyl.
A specific value for R6 is (C1�C8)alkyl, or substituted (C1�C8)alkyl, �ORa, �CO2Ra, RaC(═O)�, RaC(═O)O�, RaRbN�, RaRbNC(═O)�, or aryl.
Another specific value for R6 is (C1�C8)alkyl, �ORa, �CO2Ra, RaC(═O)�, RaC(═O)O�, RaRbN�, RaRbNC(═O)�, or aryl.
Another specific value for R6 is methyl, ethyl, butyl, OH, ORa, �CO2Ra, RaC(═O)�, OC(═O)CH2CH3, �CONRaRb, �NRaRb or phenyl.
Another specific value for R6 is OH, OMe, methyl, ethyl, t-butyl, �CO2Ra, �C(═O)NRaRb, �OAc, �NH2, �NHMe, �NMe2, �NHEt or �N(Et)2.
Another specific value for R6 is �(CH2)1-2ORa, �(CH2)1-2C(═O)ORa, �(CH2)1-2OC(═O)Ra, �(CH2)1-2C(═O)Ra, �(CH2)1-2OCO2Ra, �(CH2)1-2NHRa, �(CH2)1-2NRaRb, �(CH2)1-2OC(═O)NHRa, or �(CH2)1-2OC(═O)NRaRb.
Another specific value for R6 is �CH2OH, �CH2OAc, �CH2OCH3, �CH2C(═O)OCH3, �CH2OC(═O)CH3, �CH2C(═O)CH3, �CH2OCO2CH3, �CH2NH(CH3), or �(CH2)1-2N(CH3)2.
Another specific value for R6 is methyl, ethyl, t-butyl, phenyl, �CO2Ra, �CONRaRb, or RaC(═O)�.
Another specific value for R6 is �CH2OH, �CH2OAc, �C(═O)OCH3, �C(═O)CH3, OCO2CH3�OCO2CH3, �CH2NH(CH3), or �(CH2)1-2N(CH3)2.
A more specific value for R6 is methyl, ethyl, �CO2Ra�CONRaRb, or RaC(═O)�.
A specific value for Ra and Rb is independently hydrogen, (C1�C4)alkyl, aryl or aryl(C1�C8)alkylene.
A more specific value for Ra is (C1�C8)alkyl.
A specific value for R7 is hydrogen, alkyl, aryl or aryl(C1�C8)alkylene.
A specific value for X is �CH2ORa, �CO2Ra, �OC(O)Ra, �CH2OC(O)Ra, �C(O)NRaRb.
Another specific value for X is �CH2ORa or �C(O)NRaRb.
A more specific value for X is �CH2OH or �C(O)NHCH2CH3.
A specific value for the ring comprising �C(R3)R4R5 is 2-methyl cyclohexane, 2,2-dimethylcyclohexane, 2-phenylcyclohexane, 2-ethylcyclohexane, 2,2-diethylcyclohexane, 2-tert-butyl cyclohexane, 3-methyl cyclohexane, 3,3-dimethylcyclohexane, 4-methyl cyclohexane, 4-ethylcyclohexane, 4-phenyl cyclohexane, 4-tert-butyl cyclohexane, 4-carboxymethyl cyclohexane, 4-carboxyethyl cyclohexane, 3,3,5,5-tetramethyl cyclohexane, 2,4-dimethyl cyclopentane. 4-cyclohexanecarboxylic acid, 4-cyclohexanecarboxylic acid esters, or 4-methyloxyalkanoyl-cyclohexane.
A specific value for the ring comprising �C(R3)R4R5 is 4-piperidine, 4-piperidene-1-carboxylic acid, 4-piperidine-1-carboxylic acid methyl ester, 4-piperidine-1-carboxylic acid ethyl ester, 4-piperidine-1-carboxylic acid propyl ester, 4-piperidine-1 -carboxylic acid tert-butyl ester, 1-piperidine, 1-piperidine-4-carboxylic acid methyl ester, 1-piperidine-4-carboxylic acid ethyl ester, 1-piperidine-4-carboxylic acid propyl ester, 1-piperidine-4-carboxylic acid tert-butyl ester, 1-piperidine-4-carboxylic acid methyl ester, 3-piperidine, 3-piperidene-1-carboxylic acid, 3-piperidine-1-carboxylic acid methyl ester, 3-piperidine-1-carboxylic acid tert-butyl ester, 1,4-piperazine, 4-piperazine-1-carboxylic acid, 4-piperazine-1-carboxylic acid methyl ester, 4-piperazine-1-carboxylic acid ethyl ester, 4-piperazine-1-carboxylic acid propyl ester, 4-piperazine-1-carboxylic acid tert-butylester, 1,3-piperazine, 3-piperazine-1-carboxylic acid, 3-piperazine-1-carboxylic acid methyl ester, 3-piperazine-1-carboxylic acid ethyl ester, 3-piperazine-1-carboxylic acid propyl ester, 3-piperidine-1-carboxylic acid tert-butylester, 1-piperidine-3-carboxylic acid methyl ester, 1-piperidine-3-carboxylic acid ethyl ester, 1-piperidine-3-carboxylic acid propyl ester or 1-piperidine-3-carboxylic acid tert-butyl ester.
each R1 is independently hydrogen, halo, �ORa, �SRa, (C1�C8)alkyl, cyano, nitro, trifluoromethyl, trifluoromethoxy, (C3�C8)cycloalkyl, heterocycle, heterocycle(C1�C8)alkylene-, aryl, aryl(C1�C8)alkylene-, heteroaryl, heteroaryl(C1�C8)alkylene-, �CO2Ra, RaC(═O)O�, RaC(═O)�, �OCO2Ra, RaRbNC(═O)O�, RbOC(═O)N(Ra)�, RaRbN�, RaRbNC(═O)�, RaC(═O)N(Rb)�, RaRbNC(═O)N(Rb)�, RaRbNC(═S)N(Rb)�, �OPO3Ra, RaOC(═S)�, RaC(═S)�, �SSRa, RaS(═O)�, �N═NRa, or �OPO2Ra;
each R2 independently hydrogen, (C1�C8)alkyl, (C3�C8)cycloalkyl, heterocycle, heterocycle(C1�C8)alkylene-, aryl, aryl(C1�C8)alkylene-, heteroaryl, or heteroaryl(C1�C8)alkylene-; or
R4 and R5 together with the atoms to which they are attached can form a saturated or unsaturated, mono-, bicyclic- or aromatic ring having 3, 4, 5, 6, 7 or 8 ring atoms optionally comprising 1, 2, 3, or 4 heteroatoms selected from oxy (�O�), thio (�S�), sulfinyl (�SO�), sulfonyl (�S(O)2�) or amine (�NRa�) in the ring;
wherein any ring comprising R4 and R5 is substituted with from 1 to 14 R6 groups; wherein each R6 is independently halo, �ORa, �SRa, (C1�C8)alkyl, cyano, nitro, trifluoromethyl, trifluoromethoxy, (C3�C8)cycloalkyl, (C3�C8)bicycloalkyl, heterocycle or heterocycle(C1�C8)alkylene-, aryl, aryl(C1�C8)alkylene-, heteroaryl, heteroaryl(C1�C8)alkylene-, �CO2Ra, RaC(═O)O�, RaC(═O)�, �OCO2Ra, RaRbNC(═O)O�, RbOC(═O)N(Ra)�, RaRbN�, RaRbNC(═O)�, RaC(═O)N(Rb)�, RaRbNC(═O)N(Rb)�, RaRbNC(═S)N(Rb)�, �OPO3Ra, RaOC(═S�, RaC(═S)�, �SSRa, RaS(═O)�, �NNR1 or �OPO2Ra;
R3 is hydrogen, halo, �ORa, �SRa, C1-8alkyl, cyano, nitro, trifluoromethyl, trifluoromethoxy, (C3�C8)cycloalkyl, heterocycle or heterocycle(C1�C8)alkylene-, aryl, aryl(C1�C8)alkylene-, heteroaryl, heteroaryl(C1�C8)alkylene-, �CO2Ra, RaC(═O)O�, RaC(O)�, �OCO2Ra, RaRbNC(═O)O�, RbOC(═O)N(Ra)�, RaRbN�, RaRbNC(═O)�, RaC(═O)N(Rb)�, RaRbNC(═O)N(Rb)�, RaRbNC(═S)N(Rb)�, �OPO3Ra, RaOC(═S)�, RaC(═S)�, �SSRa, RaS(═O)�, �N═NRa, �OPO2Ra; or if the ring formed from CR4R5 is aryl or heteroaryl or partially unsaturated then R3 can be absent;
each R7 is independently hydrogen, (C1�C8)alkyl, (C3�C8)cycloalkyl, aryl or aryl(C1�C8)alkylene;
wherein any of R1, R2, R3 and R6 is optionally substituted with (C1C8)alkyl, aryl, heteroaryl, heterocycle, aryloxy, (C3�C8)cycloalkyl, hydroxy, nitro, halo, cyano, (C1�C8)alkoxy, (C1�C8)alkanoyl, (C1�C8)alkoxycarbonyl, (C1�C8)alkanoyloxy, RaS(O)p�, RaRbNS(O)�, RaRbNS(O)2�, RaRbN�, or RaRbNC(═O)�;
wherein any (C1�C8)alkyl, (C3�C8)cycloalkyl, (C3�C8)bicycloalkyl, (C1�C8)alkoxy, (C1�C8)alkanoyl, (C1�C8)alkylene, or heterocycle, is optionally partially unsaturated;
Ra and Rb are each independently hydrogen, (C1�C8)alkyl, or (C1�C8)alkyl substituted with 1�3 (C1�C8)alkoxy, (C3�C8)cycloalkyl, (C1�C8)alkylthio, amino acid, aryl, aryl(C1�C8)alkylene, heteroaryl, or heteroaryl(C1�C8)alkylene;; or Ra and Rb, together with the nitrogen to which they are attached, form a pyrrolidino, piperidino, morpholino, or thiomorpholino ring; and Rc is hydrogen or C1-6 alkyl;
The preparation of alkynyl cycloalkanols is illustrated in Scheme 1A. A solution of an appropriate cycloalkanone (where j is from 0�5) is prepared in a solvent such as THF. A solution of a suitable ethynylmagnesium halide compound in a solvent is added to the cycloalkanone. After addition, the solution is allowed to stir at about 20� C. for about 20 hours. The reaction is monitored via TLC until the starting material is consumed. The reaction is quenched with water, filtered over a plug of sand and silica, washed with a solvent, such as EtOAc, and evaporated to provide the product. Typically, two products are formed, the isomers formed by the axial/equatorial addition of the alkyne (where m is as defined above, and the sum of m1 and m2 is from 0 to about 7) to the ketone. The compounds are purified via flash chromatography using EtOAc/Hexanes to provide the product.
The preparation of 2-alkynyladenosines is illustrated in Scheme 1B. A flame-dried round bottom under nitrogen is charged with 5-(6-Amino-2-iodo-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide (NECA 2-Iodoadenosine) and a solvent such as DMF. The appropriate alkyne, wherein R is a �(CR1R2)mZ group, is dissolved in acetonitrile followed by TEA, 5 mole % Pd(PPh3)4, and CuI. All solvents are thoroughly degassed.
DESCRIPTION OF PREFERRED EMBODIMENTS All melting points were determined with a Thomas Hoover capillary melting point apparatus and are uncorrected. Nuclear magnetic resonance spectra for proton (1H NMR) were recorded on a 300 MHz GE spectrophotometer. The chemical shift values are expressed in ppm (parts per million) relative to tetramethylsilane. For data reporting, s=singlet, d=doublet, t=triplet, q=quartet, and m=multiplet. Mass spectra were measured on a Finnigan LcQ Classic. High resolution mass spectrometry (HRMS) data was provided by the Nebraska Center for Mass Spectrometry. Analytical HPLC was done on a Waters 2690 Separation Module with a Waters Symmetry C8 (2.1�150 mm) column operated at room temperature. Compounds were eluted at 200 μL/min with 70:30 acetonitrile:water, containing 0.5% acetic acid, with UV detection at 214 nm using a Waters 486 Tunable Detector. Preparative HPLC was performed on a Shimadzu Discovery HPLC with a Shim-pack VP-ODS C18 (20�100 mm) column operated at room temperature. Compounds were eluted at 30 mL/min with a gradient 20�80% of water (containing 0.1% TFA) to methanol over 15 minutes with UV detection at 214 nm using a SPD10A VP Tunable detector. All final compounds presented here were determined to be greater than 98% pure by HPLC. Flash chromatography was performed on Silicyle 60A gel (230�400 mesh) or using reusable chromatography columns and system from RT Scientific, Manchester NH. Analytical thin-layer chromatography was done on Merck Kieselgel 60 F254 aluminum sheets. Preparative thin-layer chromatography was done using 1000 micron Analtech Uniplate with silica gel. All reactions were done under a nitrogen atmosphere in flame-dried glassware unless otherwise stated.
To a solution of about 10 mmol of the appropriate cyclohexanone in about 50 mL of THF is added to about 60 mL (30 mmol) of 0.5 M ethynylmagnesium bromide in THF. The solution is allowed to stir at about 20� C. for about 20 hours. After the starting material had been consumed, monitored by TLC, the reaction is quenched with about 5 mL of water, filtered over a plug of sand and silica, washed with EtOAc, and evaporated to yield a yellow oil. Usually the oil contained two spots on TLC with 20% EtOAc/Hexanes, which are visualized with Vanillin. Usually these two products are the different isomers formed by the axial/equatorial addition of the alkyne to the ketone. The compounds are purified via flash chromatography using 10% EtOAc/Hexanes to provide clear oils or white solids in a yield of about 50�80 %.
To about 100 mg of the appropriate Boc-protected piperazine/piperadine is added 2�4 mL of neat TFA. The solution is allowed to stir for 6 hours. The TFA is removed under reduced pressure to yield a yellow oil. This oil is taken up in about 10 mL of dichloromethane to which is added 10-fold excess of TEA and 3 equivalents of the appropriate acyl chloride. The yellow solution is allowed to stir at room temperature for about 12 hours, after which time the solvents are removed and the product purified using a 1.1�30 cm 14 g column from Robert Thompson Scientific with a 5%�30% gradient of ethyl acetate/hexanes.
A flame-dried 25 mL round bottom under nitrogen is charged with 5-(6-amino-2-iodo-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide (2-Iodoadenosine) (about 40 mg) (X═CH3CH2NHC(O)�) and dissolved in about 2 mL of DMF. The appropriate alkyne (approx. 0.1 mL) is then added followed by about 4 mL of acetonitrile and about 0.1 mL of TEA. All three solvents had been degassed with nitrogen for at least 24 hours. To this solution is added 5 mole percent Pd(PPh3)4 and 6 mole % copper iodide. The yellowish solution is allowed to stir for 24 hours at room temperature, or until complete by HPLC. If the reaction is not complete at this time, additional catalyst, CuI, and TEA are added. After the reaction is complete, the solvents are removed under high-vacuum and the red/black residue taken back up in a small amount of DMF. This solution is added to a preparative silica TLC plate (Analtech 1000 microns, 20 cm�20 cm) and eluted first with 120 mL of 40% Hexanes/CH2Cl2, and then again after addition of 40 mL of MeOH. The UV active band (usually yellow in color) in the middle of the plate is collected, slowly washed with 4�25 mL 20% MeOH/CH2Cl2, and concentrated. This product is then purified by RP-HPLC.
Preparation 1: [(2R,3R,4R,5R)-3,4-diacetyloxy-5-(2-amino-6-oxohyropurin-9-yl)oxolan-2-yl]methyl acetate (6.2) A suspension of 113 g (0.4 mol) of dry guanosine (6.1), acetic anhydride (240 mL, 2.5 mol), dry pyridine (120 mL) and dry DMF (320 mL) was heated for 3.75 hours at 75� C. without allowing the temperature to exceed 80� C. The clear solution was then transferred to a 3L Erlenmyer flask and filled with 2-propanol. Upon cooling the solution to room temperature crystallization was initiated and allowed to proceed at 4� C. overnight. The white solid filtrate was filtered, washed with 2-propanol and recrystallized from 2-propanol to provide 6.2 (96%). 1H NMR (300 Mhz, CDCl3) 8.20 (s, 1H, H-8), 6.17 (d, J=5.41 Hz, 1 H, H-1 ) 5.75 (t, J=5.39 Hz, 1H, H-2), 5.56 (t, J=5.0, H-3), 4.41 (m, 3H, H-4,5), 2.14 (s, 3H, Ac), 2.11 (s, 3H, Ac), 2.10 (s, 3H, Ac). 13C NMR (300 MHz, CD3OD) 171.0, 170.3, 1702, 157.7, 154.8, 152.4, 136.7, 117.7, 85.5, 80.4, 73.0, 71.3, 64.0, 31.3, 21.2, 21.0.
Preparation 2: [(2R,3R,4R,5R)-3,4-diacetyloxy-5-(2-amino-6-chloropurin-9-yl)oxolan-2-yl]methyl acetate (6.3) To a 1 L flask was added 80 g (0.195 mol) [(2R,3R,4R,5R)-3-4-diacetyloxy-5-(2-amino-6-oxohyropurin-9-yl)oxolan-2-yl]meth yl acetate (6.2), tetramethylammonium chloride (44 g, 0.4 mol), anhydrous acetonitrile (400 mL) and N,N-dimethlaniline (25 mL). The flask was placed in an ice salt bath and cooled to 2� C. To this solution was added dropwise POCl3 (107 mL 1.15 mol) at a rate that maintained the temperature below 5� C. (45 minutes). The flask was then removed from the ice bath, outfitted with a condenser, placed in an oil bath and allowed to reflux for 10 minutes. The solution changed to a red/brown color. The solvent was removed under reduced pressure to yield an oily residue which was transferred to a beaker containing 1000 g of ice and 400 mL of CHCl3 and allowed to stir for 1.5 hours to decompose any remaining POCl3. The organic phase was removed and the aqueous phase extracted with 3�50 mL of CHCl3 and pooled with the organic phase. The pooled organic layeres were back extracted with 50 mL of water followed by stirring with 200 mL of saturated NaHCO3. The organic layer was further extracted with NaHCO3 until the aqueous extract was neutral (2�). The organic layer was finally extracted with brine and dried over MgSO4 for 16 hours. To the solution was added 800 mL of 2-propanol after which the solution was concentrated under reduced pressure. To the oily solid was added 200 mL of 2-propanol and the solution was refrigerated overnight. The crystalline product was filtered, washed, and allowed to dry overnight to give 6.3 (77%). 1H NMR (300 MHz, CD3OD) 8.31 (s, 1H, H-8), 7.00 (s, 2H, NH2) 6.06 (d, J=5.8 Hz, 1H, H-1), 5.83 (t, J=6.16 Hz, 1H, H-2), 5.67 (m, 1H, H-3), 4.29 (m, 3H, H-4,5), 2.07 (s, 3H, Ac), 1.99 (s, 3H, Ac), 1.98 (s, 3H, Ac). 13C NMR (300 MHz, CD3OD) 171.0, 170.4, 170.2, 160.8, 154.6, 150.8, 142.2, 124.5, 85.8, 80.6, 72.8, 71.2, 63.9, 21.4, 21.3, 21.1.
Preparation 3: [(2R,3R,4R,5R)-3,4-diacetyloxy-5-(6-chloro-2-iodopurin-9-yl)oxolan-2-yl]methyl acetate (6.4) Isoamyl nitrite (5 mL, 37 mmol) was added to a mixture of 5.12 g (12 mmol) [(2R,3R,4R,5R)-3-,4-diacetyloxy-5-(2-amino-6-chloropurin-9-yl)oxolan-2-yl]methyl 1 acetate (6.3), I2 (3.04 g, 12 mmol), CH2I2 (10 mL, 124 mmol), and CuI (2.4 g, 12.6 mmol) in THF (60 mL). The mixture was heated under reflux for 45 minutes and then allowed to cool to room temperature. To this solution was added 100 ml of saturated Na2S2O3. This step removed the reddish color. The aqueous layer was extracted 3� with chloroform, which was pooled, dried over MgSO4, and concentrated under reduced pressure. The product was then purified over a silica gel column using CHCl3-MeOH (98:2) to collect [(2R,3R,4R,5R)-3,4-diacetyloxy-5-(6-chloro-2-iodopurin-9-yl)oxolan-2-yl]methyl acetate (6.4) (80% crystallized from EtOH). 1H NMR (300 MHz, CDCl3) 8.20 (s, 1H H-8), 6.17 (d, J=5.41 Hz, 1H, H-1), 5.75 (t, J=5.39 Hz, 1H, H-2), 5.56 (t, J=5.40Hz, 1H, H-3), 4.38 (m, 3H, H-4,5), 2.14 (s, 1H, Ac), 2.11 (s, 1H, Ac), 2.10 (s, 1H, Ac).
Preparation 4: (4S,2R,3R,5R)-2-(6-amino-2-iodopurin-9-yl)-5-(hydroxy-methyl)oxolane-3,4-diol (6.5) To a flask containing 6.0 g (11.1 mmol) [(2R,3R,4R,5R)-3,4-diacetyloxy-5-(6-chloro-2-iodopurin-9-yl)oxolan-2-yl]methyl acetate (6.4) was added 100 ml of liquid NH3 at −78� C. and the solution was allowed to stir for 6 hours. After which time it was allowed to come to room temperature overnight with concurrent evaporation of the NH3 to yield a brown oil. The product was crystallized from hot isopropanol to provide 6.5 (80%), m.p. 143�145� C., r.f.=0.6 in 20% MeOH/CHCl3. 1H NMR (300 MHz, DMSO-d6) 8.24 (s, 1H), 7.68 (s, 2H), 5.75 (d, J=6.16, 1H), 5.42 (d, J=5.40 Hz, 1H), 5.16 (d, J=4.62 Hz, 1H), 4.99 (t, J=5.39 Hz, 1H), 4.67 (d, J=4.81 Hz, 1H), 4.06 (d, J=3.37 Hz, 1H), 3.89 (m, 1H), 3.54 (m, 2H).
Preparation 5: [(1R,2R,4R,5R)-4-(6-amino-2-iodopurin-9-yl)-7-7-dimethyl-3,6,8-trioxabicyclo[3.3.0]oct-2-yl]methan-1-ol (6.6) To a solution of 2.0 g (5.08 mmol) (4S,2R,3R,5R)-2-(6-amino-2-iodopurin-9-yl)-5(hydroxymethyl)oxolane-3,4-diol (6.6) in 100 mL acetone was added 9.6 g of p-toluenesulfonic acid and 5 ml of dimethoxypropane. The reaction was stirred at room temperature for 1 hour. Solid NaHCO3, 15 g, was added to the solution. The slurry was stirred for an additional 3 hours. The residue was filtered and washed 2� with EtOAc. The filtrate was then concentrated under reduced pressure. The residue was chromatographed on a silica gel column with MeOH-CHCl3 (1:99) to give 6.6 (72%) as a solid, m.p. 185�187� C. 1H NMR (300 MHz, DMSO-d6) 8.22 (s, 1H, H-8), 7.69 (s, 2H), NH2), 6.00 (d, J=2.70 Hz, 1H, H-1), 5.21 (m, 1H, H-2), 5.07 (bs, 1H, OH), 4.88 (m, 1H, H-3), 4.13 (m, 1H, H-4), 3.47 (m, 2H, H-5), 1.49 and 1.28 (s, 3H, C(CH3)2).
Preparation 6: (2S,1R,4R,5R)-4-(6-amino-2-iodopurin-9-yl)-7,7-dimethyl-3,6,8-trioxabicyclo[3.3.0]octane-2-carboxylic acid (6.7) To a stirred solution of 1.6 g (3.7 mmol) of [(1R,2R,4R,5R)-4-(6-amino-2-iodopurin-9-yl)-7-7-dimethyl-3,6,8-trioxabicyclo[3.3.0]oct-2-yl]methan-1-ol (6.6) in 200 mL of H2O was added 0.60 g of KOH and, dropwise, a solution of 1.70 g (10.8 mml) of KMnO4 in 50 mL of H2O. The mixture was placed in the dark at room temperature for 2�4 days. The reaction mixture was then cooled to 5�10� C. and decolorized by a solution of 4 mL of 30% H2O2 in 16 mL of water, while the temperature was maintained below 10� C. using an ice-salt bath. The mixture was filtered through Celite and the filtrate was concentrated under reduced pressure to about 10 mL and then acidified to pH 4 with 2N HCl. The resulting precipitate was filtered off and washed with ether to yield 6.7 (70%) after drying as a white solid, m.p. 187�190 C. 1H NMR (300 MHz, DMSO-d6) 8.11 (s, 1H, H-8), 7.62 (s, 2H, NH2), 7.46 (s, 1H, COOH), 6.22 (s, 1H, H-1), 5.42 (d, J=5.71 Hz, 1H, H-2), 5.34 (d, J=6.16 Hz, 1H, H-3), 4.63 (s, 1H, H-4), 1.46 and 1.30 (s, 3H, C(CH3)2).
Preparation 7: (2S,3S,4R,5R)-5-(6-amino-2-iodopurin-9-yl)-3,4-dihydroxyoxolane-2-carboxylic acid (6.8) A solution of 1.72 g (3.85 mmol) of (2S,1R,4R,5R)-4-(6-amino-2-iodopurin-9-yl)-7,7-dimethyl-3,6,8-trioxabicyclo[3.3.0]octane-2-carboxylic acid (6.7) in 80 mL of 50% HCOOH was stirred at 80� C. for 1.5 hours. The reaction mixture was evaporated under reduced pressure, dissolved in H2O, and the solvent was evaporated again. This process was repeated until there was no odor of formic acid in the residue. Recrystallization from water provided 1.33 g (85%) 6.8 as a white solid, m.p. 221�223� C., dec. 1H NMR (300 MHz, DMSO-d6) 8.31 (s, 1H, H-8), 7.68 (s, 2H, NH2), 5.90 (d, J=6.55 Hz, 1H, H-1H), 4.42(m, 1H, H-2), 4.35 (d, J=2.31 Hz, 1H, H-4), 4.22 (m, 1H, H-3).
Preparation 8: [(2S,3S,4R,5R)-5-(6-amino-2-iodopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]-N-ethylcarboxamide (6.9). To a cooled (5� C.) and stirred solution of 1.29 g (3.17 mmol) of (2S,3S,4R,5R)-5-(6-amino-2-iodopurin-9-yl)-3,4-dihydroxyoxolane-2-carboxylic acid (6.8) in 150 mL of absolute ethanol was added dropwise 1.15 mL of ice-cooled SOCl2. The mixture was stirred at room temperature overnight and then brought to pH 8 with saturated aqueous NaHCO3. The mixture was filtered, and then the filtrate was concentrated under reduced pressure to yield a white solid which was dried and then redissolved in 20 mL of dry ethylamine at −20� C. for 3 hours and then at room temperature overnight. The reaction mixture was diluted with absolute ethanol, and the precipitated product was filtered off and washed with dry ether to provide 530 mg (72%) of 6.9 as a pure solid, m.p. 232�234� C. 1H NMR (300 MHz, DMSO-d6) 8.34 (s, 1H, H-8), 8.12 (t, 1H, NH), 7.73 (s, 2H, NH2), 5.85, (d, J=6.93 Hz, 1H, H-1), 4.54 (m, 1H, H-2), 4.25 (d, J=1.92 Hz, 1H, H-4), 4.13 (m, 1H, H-3), 3.28 (m, 2H, CH2CH3), 1.00 (t, J=7.2 Hz, 3H, CH2CH3).
Preparation 9: [4-(tert-Butyl-dimethyl-silanyloxymethyl)-cyclohexyl]-methanol (83) To a 100 mL-flask containing 79 (4.0 g, 27.8 mmol) in DMF (40 mL) was added TBDMSCl (3.56 g, 23.6 mmol) and imidazole (3.79 g, 55.6 mmol). The reaction was allowed to stir at 25� C. for 16 hours after which time saturated aqueous LiBr (50 mL) was added and the reaction extracted with ether (2�50 mL). The ether layers were pooled and extracted again with LiBr (2�35 mL). The ether layer became clear. The ether layer was then concentrated in vacuo and the product purified by flash chromatography, on a silica gel column, eluting with 1:2 ether/petroleum ether to yield 83 (3.80 g, 62%) as a homogenous oil. 1H NMR (CDCl3) δ 3.46 (d, J=6.2 Hz, 2H), 3.39 (d, J=6.2 Hz, 2H), 1.95�1.72 (m, 4H), 1.65 (m, 1H), 1.40 (m, 1H), 1.03�0.89 (m, 4H), 0.88 (s, 9H), 0.04 (s, 6H; 13C NMR (CDCl3) δ 69.2, 69.1, 41.2, 41.1, 29.5, 26.5, 18.9, −4.8;. APCI m/z (rel intensity) 259 (MH+, 100).
Preparation 10: Toluene-4-sulfonic acid 4-(tert-butyl-dimethyl-silanyloxymethyl)-cyclohexylmethyl ester (84) To a 100 mL-flask containing 83 (3.4 g, 13.2 mmol) in CHCl3 (30 mL) was added tosyl chloride (3.26 g, 17.1 mmol) and pyidine (3.2 mL, 39.6 mmol). The reaction was allowed to stir at 25� C. for 14 hours after which time the reaction was concentrated in vacuo to yield a wet white solid. To this solid was added ether (50 mL) and the solid was filtered and subsequently washed with additional ether (2�50 mL). The ether layers were pooled, concentrated in vacuo to yield a clear oil which was purified by flash chromatography, on a silica gel column, eluting with 1:4 ether/petroleum ether to yield 84 (4.5 g, 83%) as a white solid. 1H NMR (CDCl3) δ 7.78 (d, J=7.7, 2H), 7.33 (d, J=7.7 Hz, 2H), 3,81 (d, J=6.2 Hz, 2H), 3.37 (d, J=6.2, 2H), 2.44 (s, 3H), 1.95�1.72 (m, 4H), 1.65 (m, 1H), 1.40 (m, 1 H), 1.03�0.89 (m, 4H), 0.88 (s, 9H), 0.04 (s, 6H); 13C NMR (CDCl3) δ 145.1, 133.7, 130.3, 128.4, 75.8, 68.9, 40.7, 38.0, 29.1, 26.5, 22.1, 18.9, −4.9; APCI m/z (rel intensity) 413 (MH+, 100).
Preparation 11: (4-Prop-2-ynyl-cyclohexyl)-methanol (86) A 3-neck 250 mL-flask equipped with a gas inlet tube and dry-ice condenser was cooled to −78� C. and charged with liquid ammonia (40 mL). To the reaction mixture was added lithium wire (600 mg, 86.4 mmol) generating a deep blue solution. The mixture was allowed to stir for 1 hour. Acetylene, passed through a charcoal drying tube, was added to the ammonia until all the lithium had reacted and the solution turned colorless, at which time the flow of acetylene was stopped, the acetylene-inlet tube and condenser removed and the flask outfitted with a thermometer. DMSO (20 mL) was added and the ammonia evaporated with a warm water bath until the mixture reached a temperature of 30� C. The solution was stirred at this temperature for 2 hours until the solution stopped bubbling. The mixture was cooled to 5� C. and compound 84 (11.25 g, 27.3 mmol), in DMSO (10 mL), was added. The temperature was maintained at 5� C. The mixture was allowed to stir at 5� C. for 0.5 hours. Then the solution was gradually warmed to room temperature and stirred for an additional 18 hours. The brown/black reaction mixture was poured slowly over ice (300 g) and extracted with ether (4�100 mL), dried with anhydrous sodium sulfate, and concentrated in vacuo to yield a yellow oil. The oil was subsequently dissolved in THF (200 mL) and changed to a brownish color upon addition of TBAF hydrate (11.20 g, 35.5 mmol). The solution was allowed to stir for 24 hours under N2 atmosphere. After stirring, the reaction was quenched with water (200 mL) and extracted with ether (3�100 mL). The ether extracts were combined and concentrated in vacuo. The crude product was purified by chromatography, on a silica gel column, eluting with 1:1 ether/petroleum ether to yield 86 (3.91 g, 93%) as a yellow oil. 1H NMR (CDCl3) δ 3.45 (d, J=6.2, 2H), 2.10 (d, J=6.2, 2H), 1.9 (s, 1H), 1.94�1.69 (m, 4H), 1.52�1.34 (m, 2H), 1.16 �0.83 (m, 4H); 13C NMR (CDCl3) δ 83.8, 69.5, 69.0, 40.8, 37.7, 32.3, 29.7, 26.5.
Preparation 12: (4-prop-2-ynylcyclohexyl)methyl acetate (87) To a solution of 960 mg (6.31 mmol) of 86 in 6 mL DMF was added 0.62 mL (7.57 mmol) pyridine and 0.78 mL (8.27mmol) acetic anhydride. The reaction was allowed to stir overnight at room temperature. After 16 hours, starting material still remained. The reaction mixture was heated at 75� C. for 3 hours. The solvent was removed under reduced pressure to yield a yellow oil which was purified by flash chromatography, on silica gel, eluting with 1:3 ether/petroleum ether to yield 1.12 g (91%) of 87 as an oil. 1H NMR (CDCl3) δ 3.87 (d, J=6.2Hz, 2H), 2.06 (d, J=4.3 Hz, 2H), 2.03 (s, 3H), 1.98�1.93 (m, 1H), 1.92�1.83 (m, 2H), 1.83�1.74 (m, 2H), 1.63�1.36 (m, 2H), 1.12�0.90 (m, 4H); 13C NMR (CDCl3) δ 171.7, 83.7, 69.9, 69.6, 37.4, 37.3, 32.1, 29.7, 26.5, 21.4; APCI m/z (rel intensity) 195 (M+, 30), 153 (M+, 70), 135 (M+, 100).
Preparation 13: 4-prop-2-ynyl-cyclohexanecarboxylic acid (88) A solution of chromium trioxide (600 mg, 6.0 mmol) in 1.5 M H2SO4 (2.6 mL, 150 mmol) was cooled to 5� C. and added to a solution of 86 (280 mg, 1.84 mmol) in acetone (15 mL). The mixture was allowed to warm to room temperature and allowed to stir overnight. Isopropanol (4 mL) was added to the green/black solution, which turned light blue after 1 hr. After adding water (15 mL), the solution was extracted with CHCl3 (6�25 mL). The organic layers were pooled and concentrated in vacuo to yield a white solid. The solid was dissolved in ether (50 mL) and extracted with 1 M NaOH (2�30 mL). The basic extracts were pooled, acidified w/10% HCl, and re-extracted with ether (3�30 mL). The ether layers were combined, dried with sodium sulfate and concentrated in vacuo to yield a white solid. The product was recrystallized from acetone/water to yield 88 (222 mg, 73%) as white needles: mp 84�85� C.; 1H NMR (CDCl3) δ 2.30�2.23 (m, 1H), 2.17�2.11 (m, 2H), 2.07�2.03 (m, 2H), 1.97�1.91 (m, 3H), 1.51�1.39 (m, 3H), 1.13�1.01 (m, 2H); 13C NMR (CDCl3) δ 182.5, 83.8, 69.6, 40.7, 37.7, 32.3, 29.6, 26.5; APCI m/z (rel intensity) 165 (M−, 100).
Preparation 14: Methyl 4-prop-2-ynylcyclohexanecarboxylate (89) To a solution of 88 (240 mg, 1.45 mmol) in 7:3 CH2Cl2:MeOH (10 mL) was added TMS Diazomethane (2.0 M in hexanes) (0.9 mL, 1.8 mmol) in 0.2 ml aliquots until the color remained yellow. The reaction was allowed to stir for an additional 0.25 hours at room temperature. After stirring, glacial acetic acid was added dropwise until the solution became colorless. The reaction was concentrated in vacuo to an oil which was purified by flash chromatography on silica gel using ether:petroleum ether (1:9) to yield 89 (210 mg, 80%) as a clear oil. 1H NMR (CDCl3) δ 3.60 (s, 3H), 2.25�2.13 (m, 1H), 2.08�1.94 (m, 3H), 1.95�1.90 (m, 2H), 1.49�1.31 (m, 3H), 1.10�0.93 (m, 2H); 13C NMR (CDCl3) δ 176.7, 83.3, 69.8, 51.9, 43.4, 36.7, 31.9, 29.2, 26.3; APCI m/z (rel intensity) 181 (MH+, 100).
Preparation 15: Trans[4-(1-Propargyl)cyclohexylmethyl] methyl carbonate (90) Yield: 345 mg, 81%. 1H NMR (CDCl3) δ 0.98�1.07, 1.40�1.52, 1.57�1.70, 1.78�1.93 (4�m, 10H, cyclohexyl), 1.96 (t, 1H, acetylene), 2.10 (dd, 2H, �C6H10CH2CCH), 3.78 (s, 3H, �OCH3), 3.96 (d, �C6H10CH2O�).
Preparation 16: Trans[4-(1-Propargyl)cyclohexylmethyl] iso-butyl carbonate (91) Yield: 433 mg, 83%. 1H NMR (CDCl3) δ 0.95 (d, 4H, �OCH2CH(CH3)2), 0.98�1.09, 1.40�1.51, 1.57�1.70, 1.78�1.93 (4�m, 10H, cyclohexyl), 1.94�2.04 (m, 1H, �OCH2CH(CH3)2), 1.96 (t, 1H, acetylene), 2.10 (dd, 2H, �C6H10CH2CCH), 3.91, 3.95 (2�d, 4H, �OCH2CH(CH3)2, �C6H10CH2O�).
Preparation 17: Trans[4-(1-Propargyl)cyclohexylmethyl] benzyl carbonate (92) Yield: 340 mg, 69%. 1H NMR (CDCl3) δ 0.97�1.08, 1.40�1.49, 1.55�1.69, 1.77�1.93 (4�m, 10H, cyclohexyl), 1.96 (t, 1H, acetylene), 2.10 (dd, 2H, �C6H10CH2CCH), 3.98 (d, �C6H10CH2O�), 5.15 (s, 2H, �OCH2Ph), 7.33�7.40 (m, 5H, Ar).
Preparation 18: 4-(Toluene-4-sulfonyloxymethyl)-piperidine-1-carboxylic acid tert-butyl ester (JR3215) A solution of N-Boc-4-piperidinemethanol, 5.0 g (23.2 mmol) in chloroform, 50 mL, was prepared. Toluene sulfonyl chloride, 5.75 g (30.2 mmol), in 5.6 mL of pyridine (69.6 mmol) was added. The solution was stirred under nitrogen allowed to stir for 24 hours. Standard workup and chromatographic purification provided the title compound. Yield 6.0 g
Preparation 19: (R)-1-Ethynyl-(R)-3-methyl-cyclohexanol (JR3217A), (S)-1-Ethynyl-(R)-3-methyl-cyclohexanol (JR3217B) To a solution of 1.0 g (8.9 mmol) (R)-(+)-3-methyl-cyclohexanone in 50 mL of THF was added 54 mL (26.7 mmol) of 0.5 M ethynylmagnesium bromide in THF. The solution was allowed to stir at 20� C. for 20 hours. Analysis by TLC indicated that the starting material had been consumed. The reaction was quenched with 5 mL of water, filtered over a plug of sand and silica, washed with EtOAc, and evaporated to yield 1.15 g of a yellow oil containing two spots (r.f.'s 0.33 (minor, JR3217A) and 0.25 (major, JR3217B), 20% EtOAc/Hexanes) which were visualized with Vanillin. The compound was purified via flash chromatography using 10% EtOAc/Hexanes (225 mL silica) to provide JR3217A and JR3217B.
Preparation 20: 1-Prop-2-ynyl-piperidine-2-carboxylic acid methyl ester (JR3249) The title compound was prepared starting with 4.0 g (22.3 mmol) of methylpipecolinate hydrochloride according to general method 2.
Preparation 21: 1-Prop-2-ynyl-piperidine-4-carboxylic acid methyl ester (JR3245) To a solution of methyl isonipecotate 3.5 g (24,4 mmol, 3.30 mL) in 100 mL dichloromethane was added TEA (1.5 eq, 36.6 mmol, 5.1 mL), propargyl bromide (3.0 eq, 73.2 mmol, 6.5 ml), at room temperature for 36 hrs. The reaction was quenched with 35 mL water to yield to provide a clear solution. The solution was extracted with dichloromethane 2�25 mL, dried with Na2SO4, and the solvent evaporated to provide a yellow oil. r.f. (40% EtOAc/Hexanes) 0.26 stains faint white with Vanillin, starting material r.f. 0.05 stains yellow with Vanillin. The product appeared pure after extraction.
Preparation 22: 1-Prop-2-ynyl-piperidine-4-carboxylic acid ethyl ester (JR3271) The title compound was prepared starting with 2.0 g (12.7 mmol) of ethyl isonipecotate according to general method 2.
Preparation 23: 4-Prop-2-ynyl-piperazine-1-carboxylic acid tert-butyl ester (JR3275) To a solution of 10.0 g (54.8 mmol) of tert-butyl-1-piperazine carboxylate in 60 mL acetonitile was added 5.20 mL (60.4 mmol) propargyl bromide and 37.9 g (274 mmol) anhydrous potassium carbonate. Additional propargy bromide, 1.5 mL, was added after stirring for 36 hours at room temperature. The residue was evaporated to dryness. Dichloromethane, 50 mL, and water, 50 mL, were added. The reaction mixture was extracted with CH2Cl2, 4�40 mL, dried over magnesium sulfate, and evaporate to provide a brown oil. The oil was dissolved in dichloromethane and purify with a RT Scientific system using hexane/ethyl acetate gradient to yield 5.5 g (46%) of yellow oil, which ultimately crystallized upon standing.
Preparation 24: 4-Cyanomethyl-piperazine-1-carboxylic acid ethyl ester (JR3287) To a solution of 3 g (19.0 mmol) of ethyl N-piperazinecarboxylate in 25 mL of CH3CN was added 1.57 g (1.32 mL 20.1 mmol) of 2-chloroacetonitrile and 15.6 g (95 mmol) K2CO3.1�H2O. The suspension was stirred at room temperature for 16 hours. The reaction was analyzed using TLC (35% Ethyl acetate/Hexanes, product r.f. 0.38 vs. sm r.f. of 0.02). The analysis indicated the reaction was complete. The golden yellow solution was evaporated to dryness. The residue was extracted with CH2C12/H2O, dried with MgSO4, and concentrated.
Preparation 25: 5-Prop-2-ynyl-2,5-diaza-bicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester (JR4013) The title compound was prepared starting with 500 mg (2.52 mmol) of 2,5-Diaza-bicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester according to general method 2.
Preparation 26: 1-Cyclohexyl-4-prop-2-ynyl-piperazine (JR4019) The title compound was prepared starting with 3 g (17.9 mmol) of 1-cyclohexylpiperazine according to general method 2
Preparation 27: 1-Prop-2-ynyl-piperazine (JR4029) To a flame-dried 25 mL round bottom flask under nitrogen was added 2.1 g of 4-Prop-2-ynyl-piperazine-1-carboxylic acid tert-butyl ester. To this solid was added 5 mL of 98% TFA in 1 mL portions. The solution turned wine red, bubbled and smoked. The additional portions of TFA were added when this activity subsided. After the third portion of TFA had been added only minimal bubbling occurred. The solution was allowed to stir under nitrogen at room temperature for an additional hour and evaporated under reduced pressure to yield the product as a thick red syrup. Assumed quantitative yield of 1.16 g. The residue was suspended in 20 mL dichloromethane and used immediately without further purification for the preparation of compounds JR4031, JR4033, and JR4035.
Preparation 28: 4-Prop-2-ynyl-piperazine-1-carboxylic acid methyl ester (JR4031) The title compound was prepared starting with 385 mg (3.1 mmol) of JR4029 and using methylchloroformate according to general method 3.
Preparation 29: 4-Prop-2-ynyl-piperazine-1-carboxylic acid isobutyl ester (JR4035) The title compound was prepared starting with 385 mg (3.1 mmol) of JR4029 and using isobutylchloroformate according to general method 3.
Preparation 30: 3,3-Dimethyl-1-(4-prop-2-ynyl-piperidin-1-yl)-butan-1-one (JR4041) The title compound was prepared starting with tert-butyl ester (JR3257) and using tert-butylacetylchloride according to general method 3.
Preparation 31: 1-(4-Prop-2-ynyl-piperazin-1-yl)-ethanone (JR4043) The title compound was prepared starting with 385 mg (3.1 mmol) of JR4029 and using acetyl chloride according to general method 3.
Preparation 32: Piperidine-1,4-dicarboxylic acid mono-tert-butyl ester To a solution of piperidine-4-carboxylic acid (10 g, 77.5 mmol) and potassium carbonate (21.4 g, 155 mmol) in 150 mL of water was prepared. A solution of di-tert-butyl dicarbonate (16.9 g, 77.5 mmol) in 40 mL of THF was added dropwise via addition funnel at 0� C. The reaction was allowed to warm to room temperature gradually over 30 minutes and stirred for an additional 4 hours. The THF was removed under reduced pressure and the aqueous phase extracted with 50 mL of ether. The aqueous phase was then adjusted to pH 2 with 10% HCl and extracted with EtOAc, 4�50 mL. The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to yield 17.2 g (97%) of JR3183 as a white solid. Rf=0.2 (35% EtOAc/Hexanes stained w/vanillin). 1H NMR (CDCl3) δ 11.83 (s, 1H), 3.98 (d, J=11.8 Hz, 2H), 2.83 (t, J=11.8, 2H), 2.46 (m, 1H), 1.88 (d, J=12.9 hz, 2H), 1.2 (m, 2H), 1.42 (s, 9H). 13C NMR (CDCl3) δ 180.0, 154.8, 79.8, 42.9, 40.8, 28.3, 27.7. APCI m/z (rel intensity) M−228.2 (100).
Preparation 33 The following intermediate compounds are prepared using the general method 1 described herein and the appropriate starting materials.
(R)-1-Ethynyl-3-tert-butyl-cyclohexanol (JR3255A), (S)-1-Ethynyl-3-tert-butyl-cyclohexanol (JR3255B) Toluene-4-sulfonic acid 4-prop-2-ynyl-cyclohexylmethyl ester (JR3077) 1-Ethyl-4-prop-2-ynyl-cyclohexane (JR3083) 1-(4-Prop-2-ynyl-cyclohexyl)-ethanone (JR3115) 1,1-Dicyclohexyl-prop-2-yn-1-ol (JR3127) 1-Cyclohexyl-prop-2-yn-1-ol (JR3129) 4-Ethyl-1-ethynyl-cyclohexanol (JR3143) 1-Ethynyl-3-methyl-cyclohexanol 1-Ethynyl-3,3,5,5-tetramethyl-cyclohexanol (JR3151) 1-Ethynyl-4-phenyl-cyclohexanol (JR3153) 1-Ethynyl-2-methyl-cyclohexanol (JR3167B) 4-tert-Butyl-1-ethynyl-cyclohexanol (JR3191) 1-Ethynyl-3,3-dimethyl-cyclohexanol (JR3193) Piperidine-1,4-dicarboxylic acid 1-tert-butyl ester 4-methyl ester (JR3195) 4-Hydroxymethyl-piperidine-1-carboxylic acid tert-butyl ester (JR3199) 4-Prop-2-ynyl-piperazine-1-carboxylic acid ethyl ester (JR3211) 4-Prop-2-ynyl-piperidine-1-carboxylic acid tert-butyl ester (JR3257) 4-Prop-2-ynyl-piperidine-1-carboxylic acid ethyl ester (JR3267B) 2-(4-Prop-2-ynyl-piperazin-1-yl)-pyrimidine (JR3277) 1-(4-Prop-2-ynyl-piperidin-1-yl)-ethanone (JR4037) 2,2-Dimethyl-1-(4-prop-2-ynyl-piperidin-1-yl)-propan-1-one (JR4039) EXAMPLE 1 4-{3-[6-Amino-9-(5-ethylcarbamoyl-3,4-dihydroxytetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclohexanecarboxylic acid (109) The reaction of 110 with five equivalents of LiOH in THF/water for 6 hours gave 109 (7 mg, 72%) as a white solid which was crystallized from MeOH/H2O(0.1% TFA) after purification by reverse phase HPLC. 1H NMR (DMSO-d6) δ 8.70 (s, 1H), 8.41 (s, 1H), 7.62 (s, 2H), 5.89 (d, J=7.25 Hz, 1H), 4.53 (m, 1H), 4.27 (s, 1H), 4.08 (d, J=3.6 Hz, 1H), 2.29 (d, J=6.4 Hz, 2H), 2.15�1.99 (m, 1H), 1.92�1.76 (m, 4H), 1.52�1.38 (m, 1H), 1.38�1.19 (m, 2H), 1.02 (t, J=6.3 Hz 3H); 13C NMR (DMSO-d6) 176.7, 169.2, 155.6, 148.9, 145.2, 141.6, 119.0, 87.7, 85.0, 84.6, 81.6, 73.1, 71.9, 43.2, 35.9, 33.3, 31.2, 28.3, 25.6, 15.0. HRMS (FAB) m/z 474.2196 [(M+H)+ cacld for C22H29N6O6 474.2182].
EXAMPLE 2 4-{3-[6-Amino-9-(5-ethylcarbamoyl-3,4-dihydroxytetrahydro-furan -2-yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclohexanecarboxylic acid methyl ester (110) The reaction of 89 with 2-IodoNECA under the general conditions described above provided 110 (74 mg, 60%) as a white solid. 1H NMR (CD3OD) δ 8.23 (s, 1H), 5.92 (d, J=7.7 Hz, 1H), 4.69�4.65 (dd, J=7.7 Hz, 4.6 Hz, 1H), 4.40 (s, 1H), 4.24 (d, J=4.6 Hz, 1H), 3.59 (s, 3H), 3.49�3.31 (m, 2H), 2.31 (d, J=6.6Hz, 2H), 2.10�2.09 (m, 1H), 2.01�1.89 (m, 4H), 1.61�1.32 (m, 5H), 1.13 (t, J=7.3 Hz, 3H); 13C NMR (CD3OD) δ 177.1, 171.1, 156.3, 149.3, 146.7, 142.4, 119.7 89.6, 86.0, 85.5, 81.6, 74.0, 72.2, 51.2, 43.2, 36.8, 34.2, 31.8, 28.9, 26.2, 14.4; HRMS (FAB) m/z 487.2325 [(M+H)+ cacld for C23H31N6O6 487.2305].
EXAMPLE 3 Acetic acid 4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4- dihydroxy-tetrahydrofuran-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclohexylmethyl ester (111) The reaction of 87 with 2-IodoNECA under the general conditions described above gave 111 (78 mg, 62%) as a white solid. 1H NMR (CD3OD) δ 8.22 (s, 1H), 5.92 (d, J=8.1 Hz, 1H), 4.70�4.66 (dd, J=8.1 Hz, 4.6 Hz, 1H), 4.40 (d, J=1.2Hz, 1H), 4.25�4.23 (dd, J=4.6 Hz, 1.2 Hz, 1H), 3.83 (d, J=6.5, 2H), 3.53�3.31 (m, 2H), 2.29 (d, J=6.5 Hz, 2H), 1.97 (s, 3H), 1.93�1.89 (m, 2H), 1.79�1.75 (m, 2H), 1.64�1.42 (m, 2H), 1.12 (t, J=7.3 Hz, 3H), 1.09�0.91 (m, 4H); 13C NMR (CD3OD) δ 172.0, 171.2, 156.2, 149.3, 146.7, 142.5, 119.7, 89.6, 86.3, 85.5, 81.5, 74.0, 72.2, 69.6, 37.4, 37.2, 34.2, 32.1, 29.4, 26.4, 19.9, 14.5; HRMS (FAB) m/z 501.2469 [(M+H)+ cacld for C24H33N6O6 501.2462].
EXAMPLE 4 5-{6-Amino-2-[3-(4-hydroxymethyl-cyclohexyl)-prop-1-ynyl]-purin-9-yl}-3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide (112) The reaction of 86 (30 mg, 0.2 mmol) with 2-IodoNECA (28 mg, 0.07 mmol) under the general conditions described above gave 112 (7 mg, 24%) as a white solid. 1H NMR (CD3OD) δ 8.22 (s, 1H), 5.92 (d, J=7.7 Hz, 1H), 4.70�4.66 (dd, J=7.7 Hz, 4.8 Hz, 1H), 4.40 (d, J=1.2 Hz, 1H), 4.25�4.23 (dd, J=4.8 Hz, 1.2Hz, 1H), 3.51�3.37 (m, 2H), 3.31 (d, J=6Hz, 2H), 2.30 (d, J=6.8 Hz, 2H), 1.94�1.89 (m, 2H), 1.83�1.78 (m, 2H), 1.64�1.42 (m, 2H), 1.12 (t, J=7.3 Hz, 3H), 1.09�0.91 (m, 4H); 13C NMR (CD3OD) δ 170.3, 155.4,148.5, 146.0, 141.6, 118.8, 88.7, 85.5, 84.6, 80.6, 73.1, 71.3, 66.8, 39.6, 36.9, 33.3, 31.5, 28.6, 25.6, 13.5; HRMS (FAB) m/z 459.2373 [(M+H)+ cacld for C22H31N6O5 459.2356].
EXAMPLE 5 5-{6-Amino-2-[3-(4-ethylcarbamoyl-cyclohexyl)-prop-1-ynyl]-purin-9-yl}-3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide (JR3037) To a sealed tube containing 5 mL of freshly distilled ethylamine was added 10 mg (0.02 mmol) of ATL146e. The flask was sealed and allowed to stir at 60� C. for 80 hours. After this time the reaction was only about 50% complete by HPLC. The vessel was cooled to 0� C., opened, and the ethylamine was removed in vacuo to yield 4.5 mg (73%) of JR3037 as a white solid and the recovery of 4.0 mg of starting material after the residue was purified by RP-HPLC. 1H NMR (CD3OD-d4) δ. 13C NMR (CD3OD-d4) δ. APCI m/z (rel intensity) 500.8 (MH+, 100), 327.4(3).
EXAMPLE 6 5-{6-Amino-2-[3-(4-carbamoyl-cyclohexyl)-prop-1-ynyl]purin-9-yl}-3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide (JR3055) To a sealed tube containing 10 mL of saturated MeOH/NH3 solution was added 5 mg (0.01 mmol) of ATL146e. The flask was sealed and allowed to stir at 25� C. for 48 hours. The vessel was cooled to 0� C., opened, and the ammonia removed by bubbling N2 for 1 hour. The remaining solvent was then removed in vacuo to yield 4.0 mg (83%) of JR3055 as a white solid after the residue was purified by RP-HPLC. 1H NMR (CD3OD-d4) δ 8.41 (s, 1H), 5.98 (d, J=7.2 Hz, 1H), 4.65 (dd, J=7.3 Hz, 4.8 Hz, 1H), 4.41 (d, J=2.0 Hz, 1H), 4.28 (dd, J=4.6 Hz, 2.0 Hz, 1H), 3.35 (m, 2H), 2,37 (d, J=6,4 Hz, 2H) 2.10 (m, 1H), 1.90 (m, _H), 1.53 (m, _H_), 1.23 (m, 13H), 1,12 (t, J=7.3 Hz, 3H). 13C NMR (CD3OD-d4) δ. APCI m/z (rel intensity) 472.3 (MH+, 100), 299.4(10).
EXAMPLE 7 5-{6-Amino-2�[3-(4-methylcarbamoyl-cyclohexyl)-prop-1-ynyl]-purin-9-yl}-3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide (JR3065) To a sealed tube containing 10 mL 2.0 M methylamine in methanol was added 16.5 mg (0.03 mmol) of ATL146e. The flask was sealed and allowed to stir at 70� C. for 120 hours. The vessel was cooled to 0� C., opened, and the solvent was removed in vacuo to yield 8.0 mg (48%) of JR3065 as a white solid after the residue was purified by RP-HPLC. 1H NMR (CD3OD-d4) δ. 13C NMR (CD3OD-d4) δ. APCI m/z (rel intensity) 486.3 (MH+, 100), 313.4(35).
EXAMPLE 8 5-[6-Amino-2-(1-hydroxy-cyclopentylethynyl)-purin-9-yl]-3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide (JR3135) The title compound was prepared using the appropriate starting materials and procedures described herein. The results are as follows:
EXAMPLE 9 5-[6-Amino-2-(3,3-dicyclohexyl-3-hydroxy-prop-1-ynyl)-purin-9-yl]-3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide (JR3139) The title compound was prepared using the appropriate starting materials and procedures described herein. The results are as follows:
EXAMPLE 10 5-[6-Amino-2-(4-ethyl-1-hydroxy-cyclohexylethynyl)-purin-9-yl]-3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide (JR3149) The title compound was prepared using the appropriate starting materials and procedures described herein. The results are as follows:
EXAMPLE 11 5-[6-Amino-2-(1-hydroxy-4-phenyl-cyclohexylethynyl)-purin-9-yl]-3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide (JR3161) The title compound was prepared using the appropriate starting materials and procedures described herein. The results are as follows:
EXAMPLE 12 5-[6-Amino-2-(1-hydroxy-3,3,5,5-tetramethyl-cyclohexylethynyl)-purin-9-yl]-3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide (JR3163) The title compound was prepared using the appropriate starting materials and procedures described herein. The results are as follows:
EXAMPLE 13 5-[6-Amino-2-(1-hydroxy-2-methyl-cyclohexylethynyl)-purin-9-yl]-3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide (JR3177A, JR3177B) The reaction of 1-Ethynyl-2-methyl-cyclohexanol (JR3169B) (100 mg, 0.72 mmol) with 2-iodo-NECA (25 mg, 0.06 mmol) under the general coupling conditions gave JR3177A (8.0 mg) and JR3177B (8.2 mg) (overall yield 65%) as white solids after purification by a silica plug and RP-HPLC. JR3177A: 1H NMR (CD3OD-d4) δ 8.47 (s, 1H), 6.05 (d, J=6.9 Hz, 1H), 4.77 (dd, J=6.9 Hz, J=4.9 Hz, 1H), 4.45 (d, J=1.9 Hz, 1H), 4.34 (dd, J=4.6 Hz, J=2.1 Hz, 1H), 3.41 (m, 2H), 2.13 (d, J=12.7 Hz, 2H), 1.65 (m, 5H), 1.32 (m, 2H), 1.14 (t, J=7.0 Hz, 3H), 1.13 (d, J=6.6 Hz, 3H). 13C NMR (CD3OD-d4) δ. APCI m/z (rel intensity) 445.3 (MH+, 100), 427.4(80), 254.4(14). 1H NMR (CD3OD-d4) δ 8.49 (s, 1H), 6.05 (d, J=6.9 Hz, 1H), 4.78 (dd, J=6.4 Hz, J=4.9 Hz, 1H), 4.45 (d, J=1.9 Hz, 1H), 4.34 (dd, J=4.6 Hz, J=1.6 Hz, 1H), 3.42 (m, 2H), 2.12 (d, J=12.3 Hz, 2H), 1.65 (m, 4H), 1.35 (m, 4H), 1.14 (t, J=7.3 Hz, 3H), 1.12 (d, J=6.6 Hz, 3H). 13C NMR (CD3OD-d4) δ. APCI m/z (rel intensity) 445.7 (MH+, 100), 427.3(35), 254.4(3.5).
EXAMPLE 14 5-[6-Amino-2-(1-hydroxy-3-methyl-cyclohexylethynyl)-purin-9-yl]-3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide (JR3179) The reaction of 1-Ethynyl-3-methyl-cyclohexanol (JR3149B) (100 mg, 0.72 mmol) with 2-iodo-NECA (25 mg, 0.06 mmol) under the general coupling conditions gave JR3179 (15.0 mg, 59%) as a white solid after purification by a silica plug and RP-HPLC. 1H NMR (CD3OD-d4) δ 8.49 (s, 1H), 6.06 (d, J=6.9 Hz, 1H), 4.75 (dd, J=6.4 Hz, J=4.9 Hz, 1H), 4.46 (d, J=1.9 Hz, 1H), 4.34 (dd, J=4.9 Hz, J=2.1 Hz, 1H), 3.42 (m, 2H), 2.09 (d, J=12.3 Hz, 2H), 1.73 (m, 4H), 1.46 (m, 1H), 1.23 (m, 1H), 1.169 (t, J=7.1 Hz, 3H), 0.95 (d, J=6.2 Hz, 3H), 0.89 (m, 1H). 13C NMR (CD3OD-d4) δ. APCI m/z (rel intensity) 445.3 (MH+, 100), 427.4(40), 254.4(4).
EXAMPLE 15 4-{3-[6-Amino-9-(5-ethylcarbamoyl-3,4-dihydroxytetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-piperazine-1-carboxylic acid ethyl ester (JR3213) The title compound was prepared using the appropriate starting materials and procedures described herein. The results are as follows:
EXAMPLE 16 5-[6-Amino-2-(3-hydroxy-2-oxo-azepan-3-ylethynyl)purin-9-yl]-3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide (JR3243A, JR3243B) 35 mg (0.081 mmol) IodoNECA (62 mg alkyne, 0.41 mmol), 2 ml DMF, 4 ml Acetonitrile, 0.2 ml TEA, d(PPH3)4, CuI. Stirred overnight at room temperature (Nov. 29, 2001). R�n is tan w/ brown precipitate. TLC (20% MeOH/CH2Cl2) indicates r�n complete (r.f INECA=0.67, r.f product=0.45). Filtered mixture through celite, washed with 3�2 mL DMF, and evaporated under vacuum to brown oil. (solid precipitates out upon the addition of MeOH, thus used DMF to load on prep plate).
EXAMPLE 17 N-Ethyl 2-{3-[trans-4-(methoxycarbonyloxamethyl)-cyclohexyl]-1-propyn-1-yl}adenosine-5′-uronamide (ATL214) Yield 3.4 mg, 10%. 1H NMR (CD3OD) δ 1.18 (t, 3H, �NHCH2CH3), 1.03�1.20, 1.51�1.70, 1.79�1.85, 1.94�2.01 (4�m, 10H, cyclohexyl), 2.35 (d, 2H, �C6H10CH2CC�), 3.46 (m, 2H, �NHCH2CH3), 3.73 (s, 3H, �OCH3), 3.94 (d, 2H, �C6H10CH2O�), 4.29 (dd, 1H, 3′�H), 4.45 (d, 1H, 4′�H), 4.72 (dd, 1H, 2′�H), 5.97 (d, 1H, 1′�H), 8.27 (s, 1H, 8�H). APCI m/z 517.4 (M+H+).
EXAMPLE 18 N-Ethyl 2-{3-[trans-4-(isobutoxyoxycarbonyloxamethyl)-cyclohexyl]-1-propyn-1-yl}adenosine-5′-uronamide (ATL215) Yield 8.5 mg, 30%. 1H NMR (CD3OD) δ 0.94 (d, 4H, �OCH2CH(CH3)2), 1.18 (t, 3H, �NHCH2CH3), 1.04�1.24, 1.54�1.72, 1.79�2.03 (3�m, 11H, cyclohexyl, �OCH2CH(CH3)2), 2.38 (d, 2H, �C6H10CH2CC�), 3.43 (m, 2H, �NHCH2CH3), 3.89, 3.94 (2�d, 4H, �C6H10CH2O�, �OCH2CH(CH3)2), 4.30 (dd, 1H, 3′�H), 4.46 (d, 1H, 4′�H), 4.71 (dd, 1H, 2′�H), 6.00 (d, 1H, 1′�H), 8.37 (br s, 1H, 8�H).APCI m/z 559.5 (M+H+).
EXAMPLE 19 N-Ethyl 2-{3-[trans-4-(benzoxycarbonyloxamethyl)-cyclohexyl]-1-propyn-1-yl}adenosine-5′-uronamide (ATL216) Yield 1.0 mg, 3%. 1H NMR (CD3OD) δ 1.17 (t, 3H, �NHCH2CH3), 1.03�1.23, 1.52�1.71, 1.78�1.86, 1.93�2.02 (4�m, 10H, cyclohexyl), 2.35 (d, 2H, �C6H10CH2CC�), 3.45 (m, 2H, �NHCH2CH3), 3.97 (d, 2H, �C6H10CH2O�), 4.29 (dd, 1H, 3′�H), 4.45 (d, 1H, 4′�H), 4.72 (dd, 1H, 2′�H), 5.13 (s, 2H, �OCH2Ph), 5.97 (d, 1H, 1′�H), 7.33�7.37(m, 5H, Ar), 8.30 (br s, 1H, 8�H). APCI m/z 593.3 (M+H+).
EXAMPLE 20 4-{3-[6-Amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclohexanecarboxylic acid 2-tert-butoxycarbonylamino-ethyl ester EXAMPLE 21 5-{6-Amino-2-[3-(4-dimethylaminomethyl-cyclohexyl)-prop-1-ynyl]-purin-9-yl}-3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide (JR2023) EXAMPLE 22 4-{3-[6-Amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclohexanecarboxylic acid 2-amino-ethyl ester (JR3033) EXAMPLE 23 4-{3-[6-Amino-9-(5-ethylcarbamoyl-3,4-dihydroxytetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-1-methyl-cyclohexanecarboxylic acid methyl ester (JR3067A) EXAMPLE 24 4-{3-[6-Amino-9-(5-ethylcarbamoyl-3,4-dihydroxytetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-1-methyl-cyclohexanecarboxylic acid methyl ester (JR3067B) EXAMPLE 25 5-{6-Amino-2-[3-(4-ethyl-cyclohexyl)-prop-1-ynyl]-purin-9-yl}-3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide EXAMPLE 26 5-{2-[3-(4-Acetyl-cyclohexyl)-prop-1-ynyl]-6-aminopurin-9-yl}-3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide (JR3119) EXAMPLE 27 5-(6-Amino-2-{3-[4-(1-hydroxy-ethyl)-cyclohexyl]-prop-1-ynyl}-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide EXAMPLE 28 5-[6-Amino-2-(1-hydroxy-2-methyl-cyclohexylethynyl)-purin-9-yl]-3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide (JR3181A, JR3181B) EXAMPLE 29 5-[6-Amino-2-(1-hydroxy-3,3-dimethylcyclohexylethynyl)-purin-9-yl]-3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide (JR3201B) EXAMPLE 30 5-[6-Amino-2-(4-tert-butyl-1-hydroxycyclohexylethynyl)purin-9-yl]-3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide (JR3203) EXAMPLE 31 5-[6-Amino-2-(1-hydroxy-3-methylcyclohexylethynyl)purin-9-yl]-3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide (JR3221) EXAMPLE 32 5-[6-Amino-2-(1-hydroxy-3-methylcyclohexylethynyl)purin-9-yl]-3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide (JR3223) EXAMPLE 33 5-[6-Amino-2-(2-tert-butyl-1-hydroxycyclohexylethynyl)purin-9-yl]-3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide (JR3227) EXAMPLE 34 1-{3-[6-Amino-9-(5-ethylcarbamoyl-3,4-dihydroxytetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-piperidine-4-carboxylic acid methyl ester (JR3251) EXAMPLE 35 1-{3-[6-Amino-9-(5-ethylcarbamoyl-3,4-dihydroxytetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-piperidine-2-carboxylic acid methyl ester (JR3253) EXAMPLE 36 4-{3-[6-Amino-9-(5-ethylcarbamoyl-3,4-dihydroxytetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-piperidine-1-carboxylic acid tert-butyl ester (JR3259) EXAMPLE 37 4-{3-[6-Amino-9-(5-ethylcarbamoyl-3,4-dihydroxytetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-piperidine-1-carboxylic acid ethyl ester (JR3269) EXAMPLE 38 1-{3-[6-Amino-9-(5-ethylcarbamoyl-3,4-dihydroxytetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-piperidine-4-carboxylic acid ethyl ester (JR3279) EXAMPLE 39 4-{3-[6-Amino-9-(5-ethylcarbamoyl-3,4-dihydroxytetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-piperidine-1-carboxylic acid tert-butyl ester (JR3281) EXAMPLE 40 5-{6-Amino-2-[3-(4-pyrimidin-2-yl-piperazin-1-yl)-prop-1-ynyl]-purin-9-yl}-3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide (JR3283) EXAMPLE 41 5-[6-Amino-2-(3-piperazin-1-yl-prop-1-ynyl)purin-9-yl]-3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide (JR3289) EXAMPLE 42 1-{3-[6-Amino-9-(5-ethylcarbamoyl-3,4-dihydroxytetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-piperidine-4-carboxylic acid (JR3291) EXAMPLE 43 4-{3-[6-Amino-9-(5-ethylcarbamoyl-3,4-dihydroxytetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-piperidine-1-carboxylic acid methyl ester (JR4007) EXAMPLE 44 4-{3-[6-Amino-9-(5-ethylcarbamoyl-3,4-dihydroxytetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-piperidine-1-carboxylic acid isopropyl ester (JR4009) EXAMPLE 45 4-{3-[6-Amino-9-(5-ethylcarbamoyl-3,4-dihydroxytetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-piperidine-1-carboxylic acid isobutyl ester (JR4011) EXAMPLE 46 5-{3-[6-Amino-9-(5-ethylcarbamoyl-3,4-dihydroxytetrahydro-furan-2-yl)-9H-purin-2-yl]prop-2-ynyl}-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester (JR4015) EXAMPLE 47 5-(6-Amino-2-{3-[1-(3,3-dimethyl-butyryl)-piperidin-4-yl]-prop-1-ynyl}purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide (JR4047) EXAMPLE 48 5-(6-Amino-2-{3-[1-(2,2-dimethyl-propionyl)-piperidin-4-yl]prop-1-ynyl}-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide (JR4051) EXAMPLE 49 4-{3-[6-Amino-9-(5-ethylcarbamoyl-3,4-dihydroxytetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-piperazine-1-carboxylic acid isobutyl ester (JR4049) EXAMPLE 50 5-{2-[3-(4-Acetyl-piperazin-1-yl)-prop-1-ynyl]-6-aminopurin-9-yl}-3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide (JR4053) EXAMPLE 51 The following compounds can be prepared by following the general methods described herein and the appropriate intermediate compounds:
EXAMPLE 52 Cell Culture and Membrane Preparation Sf9 cells were cultured in Grace's medium supplemented with 10% fetal bovine serum, 2.5 μg/ml amphotericin B and 50 μg/ml gentamycin in an atmosphere of 50% N2/50% O2. Viral infection was performed at a density of 2.5�106 cells/mL with a multiplicity of infection of two for each virus used. Infected cells were harvested 3 days post-infection and washed twice in insect PBS (PBS pH 6.3). Cells were then resuspended in lysis buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 3 mM MgCl2, 1 mM β-mercaptoethanol (BME), 5 μg/mL leupeptin, 5 μg/mL pepstatin A, 1 μg/mL aprotinin, and 0.1 mM PMSF) and snap frozen for storage at −80� C. Cells were thawed on ice, brought to 30 mL total volume in lysis buffer, and burst by N2 cavitation (600 psi for 20 minutes). A low-speed centrifugation was performed to remove any unlysed cells (1000�g for 10 minutes), followed by a high-speed centrifugation (17,000�g for 30 minutes). The pellet from the final centrifugation was homogenized in buffer containing 20 mM HEPES pH 8, 100 mM NaCl, 1% glycerol, 2 μg/mL leupeptin, 2 μg/mL pepstatin A, 2 μg/mL Aprotinin, 0.1 mM PMSF, and 10 μM GDP using a small glass homogenizer followed by passage through a 26 gauge needle. Membranes were aliquoted, snap frozen in liquid N2, and stored at −80� C. Membranes from cells stably expressing the human A1 AR (CHO K1 cells) or A3 AR (HEK 293 cells) were prepared as described (Robeva et al., 1996).
EXAMPLE 53 Radioligand Binding Assays Radioligand binding to recombinant human AA2 receptors in Sf9 cell membranes was performed using either the radiolabeled agonist, 125I-APE (Luthin et al., 1995) or the radiolabeled antagonist, 125I-ZM241385 (125I-ZM). To detect the high affinity, GTPγS-sensitive state of A1 and A3 AR, we used the agonist, 125I-ABA (Linden et al., 1985; Linden et al., 1993). Binding experiments were performed in triplicate with 5 μg (A2A) or 25 μg (A1 and A3) membrane protein in a total volume of 0.1 mL HE buffer (20 mM HEPES and 1 mM EDTA) with 1 U/mL adenosine deaminase and 5 mM MgCl2 with or without 50 μM GTPγS. Membranes were incubated with radioligands at room temperature for three hours (for agonists) or two hours (for antagonists) in Millipore Multiscreen� 96-well GF/C filter plates and assays were terminated by rapid filtration on a cell harvester (Brandel, Gaithersburg, Md.) followed by 4�150 μl washes over 30 seconds with ice cold 10 mM Tris-HCl, pH 7.4, 10 mM MgCl2. Nonspecific binding was measured in the presence of 50 μM NECA. Competition binding assays were performed as described (Robeva et al., 1996) using 0.5�1 nM 125I-APE, 125I-ZM241385, or 125I-ABA. We found that it was sometimes important to change pipette tips following each serial dilution to prevent transfer on tips of potent hydrophobic compounds. The Ki values for competing compound binding to a single site were derived from IC50 values with correction for radioligand and competing compound depletion as described previously (Linden, 1982).
Linden J (1982) Calculating the Dissociation Constant of an Unlabeled Compound From the Concentration Required to Displace Radiolabel Binding by 50%. J Cycl Nucl Res 8: 163�172.
Linden J, Patel A and Sadek S (1985) [125I]Aminobenzyladenosine, a New Radioligand With Improved Specific Binding to Adenosine Receptors in Heart. Circ Res 56: 279�284.
Linden J, Taylor H E, Robeva A S, Tucker A L, Stehle J H, Rivkees S A, Fink J S and Reppert S M (1993) Molecular Cloning and Functional Expression of a Sheep A3 Adenosine Receptor With Widespread Tissue Distribution. Mol Pharmacol 44: 524�532.
Luthin D R, Olsson R A, Thompson R D, Sawmiller D R and Linden J (1995) Characterization of Two Affinity States of Adenosine AA2 Receptors With a New Radioligand, 2-[2-(4-Amino-3-[125I]Iodophenyl)Ethylamino]Adenosine. Mol Pharmacol 47: 307�313.
Robeva A S, Woodard R, Luthin D R, Taylor H E and Linden J (1996) Double Tagging Recombinant A1- and A2A-Adenosine Receptors With Hexahistidine and the FLAG Epitope. Development of an Efficient Generic Protein Purification Procedure. Biochem Pharmacol 51: 545�555.
Purified human neutrophils (2�106/ml) suspended in Hanks balanced salt solution containing 0.1% human serum albumin (HA), adenosine deaminase (1U/mL) and rolipram (100 nM) were incubated (37 C) in a water bath for 15 min with or without rhTNF(10 U/ml). Following incubation 100 L aliquots of the PMN were transferred to wells (White walled clear bottom 96 well tissue culture plates Costar #3670; 2 wells/condition) containing 501 HA and luminol (final concentration 100M) with or without adenosine agonist (final agonist concentrations 0.01�1000 nM). The plate was incubated 5 min (37 C) and then fMLP (50 1 in HA; final concentration 1M) was added to all wells.
EXAMPLE 54 Effect of AA2 Agonists on Neutrophil Oxidative Activity A. Materials
Luminol-enhanced chemiluminescence, a measure of neutrophil oxidative activity, is dependent upon both superoxide production and mobilization of the lysosomal granule enzyme myeloperoxidase. The light is emitted from unstable high-energy oxygen species generated by activated neutrophils. Purified neutrophils (5�10�105/ml) were incubated in Hanks balanced salt solution containing 0.1% human serum albumin (1 ml) with the tested AA2 agonist with or without rolipram and with or without tumor necrosis factor-alpha (1 U/ml) for 30 minutes at 37� C. in a shaking water bath. Then luminol (1�10−4 M) enhanced f-met-leu-phe (1 mcM) stimulated chemiluminescence was read with a Chronolog� Photometer (Crono-log Corp., Havertown, Pa.) at 37� C. for 2�4 minutes. Chemiluminescence is reported as relative peak light emitted (=height of the curve) compared to samples with tumor necrosis factor-alpha and without agonist or rolipram.
EXAMPLE 55 In vivo Rat Blood Pressure Experiments Sprague-Dawley rats (mean weights, 250�300 grams) were anthesthetized and jugular and carotid catheters are implanted ipsilaterally and the animals are allowed to recover 24�48 hours. Prior to each experiment a baseline blood pressure reading is established for 30 minutes with each drug injection being preceeded by a vehicle control. Drugs are injected bolus I.V. through a jugular catheter in a 200 microliter volume of saline and the catheter is flushed with an additional 300 microliters of saline. To measure blood pressure, a central line from the carotid catheter is attached to the pressure transducer of a Digi-Med Blood Pressure Analyzer. Systolic pressure, diastolic pressure, mean pressure, and heart rate are all recorded in real time at 30�60 second intervals. Data is recorded until mean blood pressure has returned to baseline and remained constant for 20 minutes. The data is presented as a fraction of the mean blood pressure averaged over the 10 minutes immediately prior to drug injection. The blood pressures are recorded and plotted over time as a means of determining potency of the compounds as well as biological half-life.
The results are illustrated in FIGS. 1�6.
EXAMPLE 56 In vivo Coronary Dog Flow Experiments Fasted, adult mongrel dogs (mean weight, 24.8�0.6 kg; range, 20.9 to 28.2 kg) were anaesthetized with sodium pentobarbital (30 mg�kg−1), tracheally intubated, and mechanically ventilated with room air on a respirator (model 613, Harvard Apparatus) with positive end-expiratory pressure of 5 cm H2O. The surgical preparation and instrumentation of the animals has been thoroughly described previously (Glover D. K. et al., Circulation 1996, 94, pages 1726�1732). Throughout each experiment, heart rate, mean arterial and left atrial pressures, ultrasonically measured LCx flow, and dP/dt were continuously monitored and recorded on a 16-channel thermal array chart recorder (K2-G, Astro-med, Inc) and digitised and stored on an IBM-compatible personal computer. All experiments were performed with the approval of the University of Virginia Animal Care and Use Committee and were in compliance with the position of the American Heart Association on the use of research animals. The compounds tested were intravenously administered by bolus injection and the parameters above were measured and recorded.
The results are illustrated in FIGS. 7�16.
EXAMPLE 57 Liver I/R Injury Protocol Mice were anesthetized by intraperitoneal injection of ketamine 100 mg/kg and xylazine 10 mg/kg. Glycopyrrolate (Robinul-V) 0.05 mg/kg was delivered subcutaneously before the operation. The ambient temperature was controlled in the range of 24� C. to 26� C. Mice were placed on a 37� C. heat pad with their core temperature monitored by a TH-8 Thermalert Monitoring Thermometer (Physitemp) and maintained at 36�37� C. by a TCAT-1A Temperature Control and Alarm Unit (Physitemp) during the entire procedure. After midline laparotomy, a microaneurysm clip was applied to the hepatic triad above the bifurcation to clamp the flow of the hepatic artery, portal vein, and bile duct. The peritoneum was closed after superfusion of 200 μl of warm saline. After 60 minutes of ischemia, the peritoneum was reopened and the microaneurysm clip was removed. Immediately after reperfusion was initiated, each mouse received a loading dose of ATL-146e (1 ug/kg) or vehicle in 200 uL warm saline, and a primed Alzet osmotic minipump was placed intraperitoneally. The surgical wound was closed with metal staples. Mice were maintained on the heat pad to monitor and maintain body temperature until the anesthetic wore off.
EXAMPLE 58 Serum Enzyme Determination Serum GPT (ALT) levels were measured using a Transaminase kit (505, Sigma). Briefly, 20 μL serum sample was mixed with 100 (L pre-heated Alanine-α-KG substrate and incubated in a 37� C. water bath for 30 minutes. Then we added 100 (L Sigma Color Reagent to the reaction and left it at room temperature for 20 minutes. We stopped the reaction with 1.0 ml 0.4N sodium hydroxide solution. Absorbance of each sample at 505 nm was measured and converted into SF unit/ml.
EXAMPLE 59 Tissue Myeloperoxidase Measurement Mouse livers were removed after 24 hours reperfusion. The tissue was immediately submerged in 10 volumes of ice-cold 50 mM KPO4 buffer, pH 7.4 and homogenized with a Tekmar tissue grinder. The homogenate was centrifuged at 15,000�g for 15 minutes at 4� C., and the supernatant was discarded. The pellet was washed twice, resuspended in 10 volumes of ice-cold 50 mM KPO4 buffer pH 7.4 with 0.5% hexadecyltrimethylammonium bromide and then sonicated. The suspension was subjected to three freeze/thaw cycles. Samples were sonicated for 10 seconds, and centrifuged at 15,000�g for 15 minutes at 4� C. The supernatant was added to an equal volume of a solution consisting of o-dianisidine (10 mg/ml), 0.3% H2O2, and 50 mM KPO4, pH 6.0. Absorbance was measured at 460 nm over a period of five minutes.
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