Modified-galactosyl ceramides for staining and stimulating natural killer T cells

1. A compound represented by structural formula (I):

wherein:

R 1 is selected from:

(i) C(O)R 13 ;

(ii) C(R 13 )(R 14 )H, wherein R 14 is —H, or R 14 and R 2 taken together form a double bond between nitrogen to which R 2 is attached and the carbon to which R 14 is attached; or

(iii) SO 2 R 13 ;

wherein R 13 is halo; hydroxy, OR 9 ; OR 10 ; amino, NHR 9 ; N(R 9 ) 2 ; NHR 10 ; N(R 10 ) 2 aralkylamino; or C 1 -C 12 alkyl optionally substituted with halo, hydroxyl, oxo, nitro, OR 9 , OR 10 , acyloxy, amino, NHR 9 , N(R 9 ) 2 , NHR 10 , N(R 10 ) 2 , aralkylamino, mercapto, thioalkoxy, S(O)R 9 , S(O)R 10 , SO 2 R 9 , SO 2 R 10 , NHSO 2 R 9 , NHSO 2 R 10 , sulfate, phosphate, cyano, carboxyl, C(O)R 9 , C(O)R 10 , C(O)OR 9 , C(O)NH 2 , C(O)NHR 9 , C(O)N(R9) 2 , C 3 -C 10 cycloalkyl containing 0-3 R 11 , C 3 -C 10 heterocyclyl containing 0-3 R 11 , C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 5 -C 10 cycloalkenyl, C 5 -C 10 heterocycloalkenyl, C 6 -C 20 aryl containing 0-3 R 12 , or heteroaryl containing 0-3 R 12 ; or C 3 -C 10 cycloalkyl, C 3 -C 10 heterocyclyl, C 5 -C 10 cycloalkenyl, or C 5 -C 10 heterocycloalkenyl optionally substituted with one or more halo hydroxyl, oxo,OR 9 , OR 10 , acyloxy, nitro, amino, NHR 9 , N(R 9 ) 2 , NHR 10 , N(R 10 ) 2 , aralkylamino, mercapto, thioalkoxy, S(O)R 9 , S(O)R 10 , SO 2 R 9 , SO 2 R 10 , NHSO 2 R 9 , NHSO 2 R 10 , sulfate, phosphate, cyano, carboxyl, C(O)R 9 , C(O)R 10 , C(O)OR 9 , C(O)NH 2 , C(O)NHR 10 , C(O)N(R 10 ) 2 , alkyl, haloalkyl, C 3 -C 10 cycloalkyl containing 0-3 R 11 , C 3 -C 10 heterocyclyl containing 0-3 R 11 , C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 5 -C 10 cycloalkenyl, C 5 -C 10 heterocycloalkenyl, C 6 -C 20 aryl heteroaryl containing 0-3 R 12 , or C 6 -C 20 heteroaryl containing 0-3 R 12 ; or C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, aryl, or heteroaryl optionally substituted with one or more halo, hydroxyl, OR 9 , OR 10 , acyloxy, nitro, amino, NHR 9 , N(R 9 ) 2 , NHR 10 , N(R 10 ) 2 , aralkylamino, mercapto, thioalkoxy, S(O)R 9 , S(O)R 10 , SO 2 R 9 , SO 2 R 10 , NHSO 2 R 10 , sulfate, phosphate, cyano, carboxyl, C(O)R 9 , C(O)R 10 , C(O)OR 9 , C(O)NH 2 , C(O)NHR 9 , C(O)N(R 9 ) 2 , alkyl, haloalkyl, C 3 -C 10 cycloalkyl containing 0-3 R 11 , C 3 -C 10 heterocycyl containing 0-3 R 11 , C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 5 -C 10 cycloalkenyl, C 5 -C 10 heterocycloalkenyl, C 6 -C 20 aryl containing 0-3 R 12 , or C 6 -C 20 heteroaryl containing 0-3 R 12 ;

R 2 is —H or C 1 -C 6 alkyl, or R 2 and R 14 taken together form a double bond between nitrogen to which R 2 is attached and the carbon to which R 14 is attached;

R 3 is —H if R 4 is —OH, or R 3 is —OH if R 4 is —H;

R 4 is —H if R 3 is —OH, or R 4 is —OH if R 3 is —H;

R 5 is selected from:

(i) —(CH 2 ) x CH═CH(CH 2 ) y CH 3 ; or

(ii) —(CH 2 ) x CH═CH(CH 2 ) y CH═CH(CH 2 ) z CH 3 , wherein X, Y and Z are integers independently selected from 1 to about 14;

R 6 is —OH or forms a double bond with R 7 ;

R 7 is —H or forms a double bond with R 6 ;

R 8 is a saturated or unsaturated hydrocarbon having from about 5 to about 15 carbon atoms;

each R 9 is independently a C 1 -C 20 alkyl optionally substituted with halo, hydroxyl, alkoxy, amino, alkylamino, dialkylamino, sulfate, or phosphate;

each R 10 is independently an aryl optionally substituted with halo, haloalkyl, hydroxy, alkoxy, nitro, amino, alkylamino, dialkylamino, sulfate, or phosphate;

each R 11 is independently halo, haloalkyl, hydroxy, alkoxy, oxo, amino, alkylamino, dialkylamino, sulfate, or phosphate; and

each R 12 is independently halo, haloalkyl, hydroxy, alkoxy, nitro, amino, alkylamino, dialkylamino, sulfate, or phosphate.

2. The compound of claim 1 , wherein R 5 is (i), X is 13 and Y is 7.

3. The compound of claim 1 , wherein R 13 is —CH 3 .

4. The compound of claim 1 , wherein:

if R 1 is (i) then R 13 is not —CH 3 ;

if R 5 is (i), then x and y are not 13 and 7, respectively;

R 6 is not —OH;

R 7 is not —H; and

R 8 is not C 13 H 27 .

5. The compound of claim 1 , wherein the solubility of the compound is at least about 20 mg/mL in DMSO.

6. The compound of claim 1 , wherein the compound is capable of binding a CD1d monomer or tetramer.

7. The compound of claim 1 , wherein the compound is capable of activating an NKT cell.

8. A composition comprising the compound of claim 1 and a physiologically acceptable vehicle.

9. The composition of claim 8 , further comprising a CD1d monomer or tetramer, wherein the compound is bound to the CD1d monomer or tetramer.

10. The composition of claim 8 , further comprising an antigen.

11. A method of activating an NKT cell comprising contacting the NKT cell with the compound of claim 9 in the presence of a CD1d monomer or tetramer.

12. The method of claim 11 , wherein the compound is bound to the CD1d monomer or tetramer.

13. The method of claim 11 , wherein the CD1d monomer or tetramer is soluble.

14. The method of claim 11 , wherein the CD1d monomer is expressed on a cell surface.

15. The method of claim 14 , wherein the cell is an antigen presenting cell.

16. The method of claim 11 , wherein activation of the NKT cell is characterized by altered cytokine expression relative to a control.

17. The method of claim 11 , wherein the NKT cell is activated in vitro.

18. The method of claim 11 , wherein the NKT cell is activated in vivo.

19. A method of stimulating an immune response in a subject comprising administering to the subject an effective amount of: a) the compound of claim 1 wherein the compound is capable of activating an NKT cell; b) a population of NKT cells activated by contacting the cells with a compound according to a) in the presence of a CD1d monomer or tetramer; c) a population of CD1d+ antigen presenting cells contacted with the compound of claim 1 ; or d) any combination of a), b) and c).

20. An isolated cell for presenting a lipid antigen to NKT cells, the cell comprising CD1d molecules loaded with a compound of claim 1 .

21. The isolated cell of claim 20 , wherein in the compound:

R 1 is —C(O)R 13 , wherein R 13 is C1-C12 alkyl optionally substituted with carboxyl;

R 2 is —H;

R 3 , R 4 , and R 7 are —H or —OH, wherein R 3 is —H if R 4 is —OH and R 3 is —OH if R 4 is —H; and

R 6 is —OH.

22. The isolated cell of claim 20 , wherein the compound is:

23. The isolated cell of claim 20 , wherein the cell is an antigen presenting cell.

24. A composition comprising isolated cells for presenting a lipid antigen to NKT cells and a physiologically acceptable vehicle, wherein the cells comprise CD1d molecules loaded with the compound of claim 1 .

25. The composition of claim 24 , wherein the compound is:

26. The composition of claim 24 , wherein the cell is an antigen presenting cell.

27. The composition of claim 24 , further comprising an antigen.

28. A method of stimulating an NKT cell, comprising contacting the NKT cell with the antigen presenting cell according to claim 20 .

29. A method of stimulating an immune response in a subject in need thereof, comprising administering to the subject an effective amount of a composition according to claim 24 .

30. The method of claim 29 , wherein the cells are antigen presenting cells.

31. The method of claim 29 , wherein the compound is:

32. The method of claim 29 , wherein the composition comprises an antigen.

33. The composition of claim 9 , wherein the CD1d monomer is expressed on a cell surface.

34. The composition of claim 33 , wherein the cell is an antigen presenting cell.