Method and probes for detecting nucleoside transporter and method for producing the probes

Claims

2. A compound in accordance with claim 1 wherein A is NH; R.sub.2 is benzyl or substituted benzyl; E is H; X is N; Y is C; Z is N; R.sub.3 is H; R.sub.4 is H and R.sub.5 is OH.

3. A compound in accordance with claim 2 wherein R.sub.2 is ##STR6## n is 2 and R.sub.1 is H.

4. A process for preparing a compound of the general formula: ##STR7## comprising the steps of: (a) N'1 alkylation and rearrangement of 5'-S-(2-hydroxyethyl)-5'-thioadenosine to give 5'-S-(2-hydroxyethyl-6-N-(4-nitrobenzyl)-5'-thioadenosine;

(b) treatment of the product of step (a) with diethylazodicarboxylate/triphenylphosphine followed by phthalimide to give 5'-S-(2-phthalimidoethyl)-N.sup.6 -(4-nitrobenzyl)-5'-thioadenosine;

(c) deprotection of the product of step (b) by heating with hydrazine in ethanol to give the phthaloyl hydrazide salt of 5'-S-(2-aminoethyl)-N.sup.6 -(4-nitrobenzyl)-5'-thioadenosine; and

(d) conversion of the product of step (c) to 5'-S-(2-aminoethyl)-6-N-(4-nitrobenzyl)-5'-thioadenosine HCl by ion exchange chromatography on styrene divinyl benzene (Cl.sup.-) resin.

6. A probe in accordance with claim 5 wherein R.sub.1 is a reporter moiety and a linker moiety.

7. A probe in accordance with claim 6 wherein said reporter moiety is a fluorescent moiety.

8. A probe in accordance with claim 7 wherein said reporter moiety is a fluorescent moiety selected from the group consisting of fluoresceins, rhodamines, coumarins, eosin, erythrosin, fluorescent phycobili-proteins and fluoresceinated microbeads.

9. A probe in accordance with claim 6 wherein said linker moiety is selected from the group consisting of

--CO--, --NHCO(CH.sub.2).sub.n' --CO--, 2-amino-5-(3-chloro-triazinyl) or ##STR9## wherein n' is 0 to 12; Q is --C-- and L is O or S.

10. A probe in accordance with claim 6 wherein

A is NH; R.sub.2 is benzyl or substituted benzyl; E is H; X is N; Y is C; Z is N; R.sub.3 is H; R.sub.4 is H; and R.sub.5 is OH.

11. A probe in accordance with claim 10 wherein R.sub.2 is ##STR10## and n is 2.

12. A probe in accordance with claim 11 wherein said reporter moiety is a fluorescent moiety and said linker moiety is selected from the group consisting of: --CO--, --NHCO(CH.sub.2).sub.n',--CO--, 2-amino-5-(3-chloro-triazinyl) or ##STR11## wherein n' is 0 to 12; Q is C and L is O or S.

13. A probe in accordance with claim 12 wherein said fluorescent moiety is 5-fluorescein or 6-fluorescein and said linker moiety is --CO--, --NHCS, --NHCO(CH.sub.2).sub.5 --CO--, or 2-amino-5-(3-chloro-triazinyl)--.

14. A probe in accordance with claim 13 wherein said fluorescent moiety is 5-fluorescein and said linker moiety is --NHCS--.

15. A probe in accordance with claim 5 wherein said reporter moiety is a radioactive moiety.

16. A probe in accordance with claim 15 wherein said radioactive moiety is labelled with a.gamma.-emitting isotope selected from the group consisting of.sup.123 I,.sup.125 I and.sup.99m Tc.

17. A probe in accordance with claim 5 wherein said reporter moiety is a moiety which binds specifically with a reporter-specific moiety, the reporter moiety/reporter-specific moiety binding pair being selected from the group consisting of avidin/biotin, avidin/iminobiotin, streptavidin/biotin and antigen/antibody.

18. A probe in accordance with claim 17 wherein said reporter moiety is fluorescein and said binding pair is fluorescein/antifluorescein antibody.

19. A method of determining es nucleoside transporter sites of animal cells comprising the steps of:

(a) contacting a suitable preparation of said cells with a probe in accordance with claim 5 to permit binding of said probe to said sites; and

(b) determining said reporter moiety of said probe.

20. A method of determining es nucleoside transporter sites of animal cells comprising the steps of:

(a) contacting said cells with a probe in accordance with claim 5 to permit binding of said probe to said sites;

(b) contacting said cells treated as in (a) with a reporter-specific moiety capable of binding to the reporter moiety of said probe; and

(c) determining said reporter-specific moiety.

21. A method in accordance with claim 19 wherein said animal cells are human cells.

22. A pharmaceutical composition in dosage unit form suitable for inhibiting es nucleoside transporter activity in animal cells and tissues comprising a compound in accordance with claim 1 or a pharmaceutically acceptable salt thereof in an amount effective to inhibit as nucleoside transporter activity in admixture with a suitable pharmaceutical carrier.

23. A pharmaceutical composition in accordance with claim 22 suitable for inhibiting es nucleoside transporter activity in human cells and tissues.

24. A kit for determining es nucleoside transporter sites of animal cells comprising:

(a) a probe in accordance with claim 5;

(b) reagent means for determining the reporter moiety of said probe, said probe and reagent means each being present in amounts effective to perform the determination.

25. A kit in accordance with claim 24 wherein said animal cells are human cells.

26. A compound in accordance with claim 1 linked to a macromolecular carrier.

27. A compound in accordance with claim 1 linked to agarose.

28. A method for inhibiting es transport activity in an animal by administering an es transporter activity inhibiting amount of the pharmaceutical composition of claim 22 or 23 to said animal.