Phospholipid conjugates and their preparation

• US 5,053,497 A
• Filed: 07/21/1988
• Issued: 10/01/1991
• Est. Priority Date: 12/28/1984
• Status: Expired due to Fees

• Current Assignee: Technicon Instruments Corporation
• Original Assignee: Technicon Instruments Corporation
• Inventors: Hedaya, Eddie, Scott, Mary E., Hwang, Deng R.
• Primary Examiner(s): Saunders, David A.
• Application Number:
  US07/224,195
• Time in Patent Office: 1,167 Days
• Field of Search: 260/403, 536/6.1, 536/6.2, 436/829
• US Class Current: 536/6.1
• CPC Class Codes: C07J 19/005 Glycosides
• CPC Class Codes: C07J 41/0055 the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives
• CPC Class Codes: C07J 51/00 Normal steroids with unmodified cyclopenta(a)hydrophenanthrene skeleton not provided for in groups C07J1/00 - C07J43/00
• CPC Class Codes: G01N 33/743 Steroid hormones
• CPC Class Codes: G01N 33/9453 Cardioregulators, e.g. antihypotensives, antiarrhythmics
• CPC Class Codes: Y10S 436/829 Liposomes, e.g. encapsulation

Abstract

Phospholipid conjugate compounds derived from cardiac glycosides and steroids useful in liposome immunoassays are disclosed as well as their method of preparation.

Claims

1. A phospholipid conjugate compound of the formulae:

##STR4## wherein R is derived from an organic compound containing 1,2-dihydroxy groups, which groups are subject to oxidative cleavage;

X and X'"'"' are side chain moiesties connecting the carbon atoms resulting from oxidative cleavage of the 1,2-dihydroxy groups with the R moiety;

P and P'"'"' are phospholipid ethanolamine moieties; and

R'"'"' is hydrogen.

2. The compound of claim 1, having formula I, wherein R is derived from digoxin, X is ##STR5## X'"'"' is --CH₂ -- and R'"'"' is hydrogen.

3. The compound of claim 2, digoxin-mono-DPPE, wherein P is derived from dipalmitoyl phosphatidyl ethanolamine (DPPE).

4. The compound of claim 2 wherein P is derived from dimyristyl phosphatidyl ethanolamine.

5. The compound of claim 1 having formula II wherein R is derived from digoxin, and X is ##STR6## and X'"'"' is --CH₂ --.

6. The compound of claim 5, digoxin-di-DPPE, wherein P and P'"'"' are each derived from dipalmitoyl phosphatidyl ethanolamine (DPPE).

7. The compound of claim 5 wherein P and P'"'"' are each derived from dimyristyl phosphatidyl ethanolamine.

8. The compound of claim 1, having formula I, wherein R is derived from digitoxin, X is ##STR7## X'"'"' is --CH₂ -- and R'"'"' is hydrogen.

9. The compound of claim 8 wherein P is derived from dipalmitoyl phosphatidyl ethanolamine (DPPE).

10. The compound of claim 1, having formula II, wherein R is derived from digitoxin, X is CH₃, ##STR8## and X'"'"' is --CH₂ --.

11. The compound of claim 10 wherein P and P'"'"' are each derived from dipalmitoyl phosphatidyl ethanolamine (DPPE).

12. The compound of claim 1, having formula I, wherein R is derived from gitoxin, X is ##STR9## X'"'"' is --CH₂ -- and R'"'"' is hydrogen.

13. The compound of claim 12 wherein P is derived from dipalmitoyl phosphatidyl ethanolamine (DPPE).

14. The compound of claim 1 having formula II wherein R is derived from gitoxin, and X is ##STR10## and X'"'"' is --CH₂ --.

15. The compound of claim 14 wherein P and P'"'"' are each derived from dipalmitoyl phosphatidyl ethanolamine (DPPE).

16. The compound of claim 1, having formula I, wherein R is derived from ouabain, X is ##STR11## ##STR12## or mixtures thereof, X'"'"' is ##STR13## and R'"'"' is hydrogen.

17. The compound of claim 16 wherein P is derived from dipalmitoyl phosphatidyl ethanolamine (DPPE).

18. The compound of claim 1 having formula II wherein R is derived from ouabain, X is ##STR14## or mixtures thereof, and X'"'"' is ##STR15## or zero or mixtures thereof.

19. The compound of claim 18 wherein P and P'"'"' are each derived from dipalmitoyl phosphatidyl ethanolamine (DPPE).

20. A method of preparing compounds of claim 1 having formula I or formula II, which comprises the steps of:

(a) oxidatively cleaving the 1,2-dihydroxy group,

(b) reacting the dialdehyde group resulting from step (a) with carboxy methoxylamine to form a corresponding dioxime intermediate,

(c) reacting the dioxime intermediate of step (b) with N-hydroxysuccinimide in the presence of dicyclohexycarbodiimide to form a dioxime active ester, and

(d) condensing the dioxime active ester with a phospholipid ethanolamine to form the phospholipid conjugate compound.

21. The method of claim 20, wherein the isolation of purified compounds of formula I in step (d) is performed by preparative thin layer chromatography.

22. The method of claim 20, wherein the isolation of purified compounds of formula II in step (d) is performed by preparative thin layer chromatography.

23. The method of claim 21 for isolating a phospholipid conjugate wherein R of the conjugate is derived from digoxin, R'"'"' is hydrogen, and P is derived from dipalmitoyl phosphatidyl ethanolamine (DPPE).

24. The method of claim 21 for isolating a phospholipid conjugate wherein R of the conjugate is derived from digitoxin, gitoxin, ouabain, or digitonin, R'"'"' is hydrogen, and P is derived from dipalmitoyl phosphatidyl ethanolamine (DPPE).

25. The method of claim 22 for isolating a phospholipid conjugate wherein R of the conjugate is derived from digoxin, and P and P'"'"' are each derived from dipalmitoyl phosphatidyl ethanolamine (DPPE).

26. The method of claim 22 for isolating a phospholipid conjugate wherein R of the conjugate is derived from digitoxin, gitoxin, ouabain, or digitonin, and P and P'"'"' are each derived from dipalmitoyl phosphatidyl ethanolamine.

27. The method of claim 20, wherein formation of reaction products of steps (a) through (d) is monitored by thin layer chromatography.