US2003036540A1PendingUtilityA1

Betulinol derivatives

Priority: Jun 4, 1997Filed: Aug 2, 2002Published: Feb 20, 2003

Est. expiryJun 4, 2017(expired)· nominal 20-yr term from priority

Inventors:Arkadiy Bomshteyn Premila Rathnam Brij B. Saxena

C07J 1/00C07J 53/00

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References

Claims

Abstract

The present invention is directed to betulinol derivatives and betulinol-antibody conjugates having the formulae: and HO-antibody-spacer-(A 2 ) n wherein A 1 is a moiety having the formula: A 2 is a moiety having the formula: n is an integer from 1 to 100; X and Y 1 are each independently selected from the group consisting of a hydroxy group, an alkoxy group, an alkanoyloxy group, and a-peptide-NHNH—C(O)-antibody-OH moiety; Y 2 is selected from the group consisting of a hydroxy group, an alkoxy group, and an alkanoyloxy group; and HO-antibody-H is an antibody and, in particular, betulinol dimethyl ether. Methods for making and using these derivatives and conjugates, as well as a method for making and using betulonic aldehyde, are also disclosed.

Claims

exact text as granted — not AI-modified

What is claimed:

1 . A diether having the formula:

wherein R is an alkyl group.

2 . A diether according to claim 1 , wherein R is methyl.

3 . A method of synthesizing a diether having the formula:

wherein R is alkyl,
said method comprising:
providing a dialcohol having the formula:

and
alkylating the dialcohol with a nitrile having the formula:
R—CηN
under conditions effective to form the diether.

4 . A method according to claim 3 , wherein R is methyl.

5 . A method according to claim 3 , wherein the dialcohol and the nitrile, respectively, are present in a mole ratio of from about 1:20 to about 1:60.

6 . A method according to claim 3 , wherein said alkylating is carried out at a temperature of from about 30° C. to about 70° C.

7 . A method of preparing betulonic aldehyde comprising:
oxidizing betulinol with chromium anhydride in acetone in the presence of sulfuric acid under conditions effective to produce betulonic aldehyde.

8 . A method according to claim 7 , wherein the betulinol and acetone, respectively, are present in a weight ratio of from about 1:100 to about 1:110.

9 . A method according to claim 7 , wherein the chromium anhydride and sulfuric acid, respectively, are present in a molar ratio of from 9:10 to about 10:9.

10 . A method according to claim 7 further comprising, after said oxidizing:
cooling the reaction mixture; and
adding water to the reaction mixture, whereby a sediment containing betulonic aldehyde forms.

11 . A method according to claim 10 further comprising:
recrystallizing the sediment.

12 . A compound having the formula:

wherein
X or Y is a -peptide-Q moiety and the other of X and Y is a hydroxy group, an alkoxy group, an alkanoyloxy group, or a -peptide-Q moiety;
Q is a hydroxy group, a —NHNH 2 moiety, an —NHNH—C(O)CH 2 Hal moiety, an -antibody-OH moiety, or an —NHNH—C(O)-antibody-OH moiety; and
Hal is a halogen.

13 . A compound according to claim 12 , wherein -peptide- is a pentapeptide.

14 . A compound according to claim 13 , wherein the pentapeptide is -Gly-Ala-Leu-Gly-Leu-.

15 . A compound according to claim 12 , wherein -peptide- is a tetrapeptide.

16 . A compound according to claim 15 , wherein the tetrapeptide is -Leu-Ala-Leu-Ala-.

17 . A method of producing a betulinol-antibody conjugate having the formula:

wherein
Y is a hydroxy group, an alkoxy group, or an alkanoyloxy group,
said method comprising:

providing a betulinol peptide having the formula:

and
converting the betulinol peptide with an antibody having the formula H-antibody-OH under conditions effective to produce the betulinol-antibody conjugate.

18 . A method according to claim 17 , wherein -peptide- is a pentapeptide.

19 . A method according to claim 18 , wherein the pentapeptide is -Gly-Ala-Leu-Gly-Leu-.

20 . A method according to claim 17 , wherein -peptide- is a tetrapeptide.

21 . A method according to claim 20 , wherein the tetrapeptide is -Leu-Ala-Leu-Ala-.

22 . A method according to claim 17 , wherein said providing the betulinol peptide comprises:
providing a compound having the formula: and converting the compound with a peptide having the formula H-peptide-OH under conditions effective to produce the betulinol peptide.

23 . A method of producing a betulinol-antibody conjugate having the formula:

wherein
Y is a hydroxy group, an alkoxy group, or an alkanoyloxy group,
said method comprising:

providing a haloacetylhydrazide having the formula:

wherein
Hal is a halogen
and

converting the haloacetylhydrazide with an antibody having the formula H-antibody-OH under conditions effective to produce the betulinol-antibody conjugate.

24 . A method according to claim 23 , wherein Hal is 1.

25 . A method according to claim 23 , wherein -peptide- is a pentapeptide.

26 . A method according to claim 25 , wherein the pentapeptide is -Gly-Ala-Leu-Gly-Leu-.

27 . A method according to claim 23 , wherein -peptide- is a tetrapeptide.

28 . A method according to claim 27 , wherein the tetrapeptide is -Leu-Ala-Leu-Ala-.

29 . A method according to claim 23 , wherein said providing a haloacetylhydrazide comprises:
providing a hydrazide having the formula: and converting the hydrazide with a p-nitrophenyl haloacetate under conditions effective to produce the haloacetylhydrazide.

30 . A method according to claim 29 , wherein said providing a hydrazide comprises:
providing a betulinol peptide having the formula: and converting the betulinol peptide with hydrazine hydrate under conditions effective to produce the hydrazide.

31 . A method according to claim 30 , wherein said providing the betulinol peptide comprises:
providing a compound having the formula: and converting the compound with a peptide having the formula H-peptide-OH under conditions effective to produce the betulinol peptide.

32 . A betulinol-antibody conjugate having the formula:

wherein
A are independently selected from a —CHO group or a moiety having the formula:

provided that at least one of A is not —CHO; and
Y is a hydroxy group, an alkoxy group, or an alkanoyloxy group.

33 . A method of producing a betulinol-antibody conjugate having the formula:

wherein
A are independently selected from a —CHO group or a moiety having the formula:

provided that at least one of A is not —CHO; and
Y is a hydroxy group, an alkoxy group, or an alkanoyloxy group,
said method comprising:

providing a carrier molecule having the formula:

and
converting the carrier molecule with a hydrazide having the formula:

and an antibody having the formula H-antibody-OH under conditions effective to produce the betulinol-antibody conjugate.

34 . A method according to claim 33 , wherein said converting the carrier molecule comprises:
reacting the carrier molecule with the antibody under conditions effective to produce an antibody-bound carrier molecule having the formula: and converting the antibody-bound carrier molecule with the hydrazide under conditions effective to produce the betulinol-antibody conjugate.

35 . A method according to claim 33 , wherein said converting the carrier molecule comprises:
reacting the carrier molecule with the hydrazide under conditions effective to produce a betulinol-bound carrier molecule having the formula: wherein
at least one A is a moiety having the formula:

and
converting the betulinol-bound carrier molecule with the antibody under conditions effective to produce the betulinol-antibody conjugate.

36 . A betulinol-antibody conjugate having the formula:
HO-antibody-spacer-(A) n wherein
A is a moiety having the formula:
an

Y is a hydroxy group, an alkoxy group, or an alkanoyloxy group; and
n is an integer from 1 to 100.

37 . A betulinol-antibody conjugate according to claim 36 , wherein -spacer-(A) n has the formula:

wherein
a is an integer from 1 to 100 and
b is an integer equal to n.

38 . A betulinol-antibody conjugate according to claim 36 , wherein spacer is a diamine derivative of polyethylene glycol having 2-(pyridyldithio)-propionyl and N-hydroxysuccinimide ester groups bonded thereto.

39 . A betulinol-antibody conjugate according to claim 36 , wherein spacer is a branched form of polyethylene glycol propionic acid N-hydroxysuccinimide ester.

40 . A betulinol-antibody conjugate according to claim 39 , wherein the branched form of polyethylene glycol propionic acid N-hydroxysuccinimide ester is a monomethoxypoly(ethylene glycol)-propionic acid N-hydroxysuccinimide ester.

41 . A method of producing a betulinol-antibody conjugate having the formula:
HO-antibody-spacer-(A) n wherein
A is a moiety having the formula:

Y is a hydroxy group, an alkoxy group, or an alkanoyloxy group; and
n is an integer from 1 to 100,
said method comprising:
providing a crosslinker having a first reactive terminus and one or more second reactive termini; reacting an antibody with the first reactive terminus; and reacting a hydrazide having the formula: with one or more of the one or more second reactive termini under conditions effective to produce the betulinol-antibody conjugate.

42 . A method according to claim 41 , wherein the first reactive terminus is selected from the group consisting of a hydroxy group, an aldehyde group, and a carboxyl group.

43 . A method according to claim 41 , wherein each of the one or more second reactive termini are independently selected from the group consisting of a hydroxy group, an aldehyde group, and a carboxyl group.

44 . A method according to claim 41 , wherein -spacer-(A) n has the formula:

wherein
a is an integer from 1 to 100 and
b is an integer equal to n.

45 . A method according to claim 41 , wherein spacer is a diamine derivative of polyethylene glycol having 2-(pyridyldithio)-propionyl and N-hydroxysuccinimide ester groups bonded thereto.

46 . A method according to claim 41 , wherein spacer is a branched form of polyethylene glycol propionic acid N-hydroxysuccinimide ester.

47 . A method according to claim 46 , wherein the branched form of polyethylene glycol propionic acid N-hydroxysuccinimide ester is a monomethoxypoly(ethylene glycol)-propionic acid N-hydroxysuccinimide ester.

48 . A method of treating cancer comprising:
administering to a cancer patient an effective amount of a compound selected from the group consisting of betulonic aldehyde and compounds having the formulae: and HO-antibody-spacer-(A 2 ) n wherein
A 1 is a moiety having the formula:

A 2 is a moiety having the formula:

n is an integer from 1 to 100;
X and Y 1 are each independently selected from the group consisting of a hydroxy group, an alkoxy group, an alkanoyloxy group, and a -peptide-NHNH—C(O)-antibody-OH moiety;
Y 2 is selected from the group consisting of a hydroxy group, an alkoxy group, and an alkanoyloxy group; and
HO-antibody-H is an antibody targeted to a site to be treated in the patient.

49 . A method according to claim 48 , wherein the compound is betulinol diacetate.

50 . A method according to claim 48 , wherein the compound is betulonic aldehyde.

51 . A method according to claim 50 , wherein the compound is betulinol dimethyl diether.

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