Stable analogs of bioactive peptides containing disulfide...

Details Stable analogs of bioactive peptides containing disulfide... Stable analogs of bioactive peptides containing disulfide...

1994-07-21

2003-12-16

Wessendorf, T. D. (Department: 1639)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

Somatostatin ; related peptides

C530S402000, C530S317000, C514S003100, C930S280000

Reexamination Certificate

active

06664367

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to conformationally and chemically stable analogs of bioactive peptides containing disulfide linkages. The present invention also relates to disulfide linkage analogs containing a bifunctional effector molecule, such as a bifunctional chelating agent, antineoplastic agent, enzyme, coenzyme, or chemotoxin, at the same site where the disulfide bond is modified.
2. Technology Background
There are many bioactive peptides containing disulfide linkages. Many naturally occurring cyclic (and conformationally restricted) peptides contain disulfide linkages. These disulfide bonds exist primarily to ensure conformational rigidity. Peptides containing disulfide bonds usually exist as 10 to 20 membered ring macrocycles, although much larger ring sizes are known and are common in large polypeptides or proteins. Such small cyclic peptides and small peptidomimetics are becoming extremely important in diagnosis and therapy. In particular, they are being actively considered in targeted delivery of radio- and chemotherapeutic agents. See, Fischman, A. J. et al., “A Ticket to Ride: Peptide Radiopharmaceuticals,”
The Journal of Nuclear Medicine
, Vol. 34, No. 12, December 1993.
A major limitation of these disulfide bridges is their instability to even mild reducing conditions; destruction of the —S—S— bond usually destroys the bioactivity of the peptide. For instance, due to the use of reducing agents, such as stannous ions and dithionite, the radiolabeling conditions required to prepare radiopharmaceuticals often destroy disulfide linkages. Similarly, blood stream constituents can degrade disulfide bonds. To be used in diagnosis and therapy, it is often necessary to attach a bifunctional effector molecule, such as a bifunctional chelating agents capable of complexing diagnostically and therapeutically useful metal ions, antineoplastic agents, enzymes, coenzymes, or chemotoxins, to the peptide. In the past, EDTA-like bifunctional chelating agents bearing side chains containing amino and carboxylic groups have been incorporated into the amino or carboxy terminal ends of bioactive peptides. See, Warshawsky and coworkers,
J. Chem. Soc. Chem. Comm
., 1133 (1985) and
Synthesis
, 825 (1989);
J. Chem. Soc., Perkin Trans. I
., 59 (1984); Arya and Gariépy
Bioconjugate Chem
., 2, 323 (1991).
Often the amino or carboxyl terminal of the peptide is required for biologically activity, so that conjugating a ligand to the terminal end of the peptide destroys the peptide's bioactivity. If the bioactivity of the peptide is destroyed, then the peptide subsequently labeled with a radioisotope will have little value in diagnostic or therapeutic application.
It, therefore, would be a significant advancement in the art to provide analogs of cyclic bioactive peptides containing disulfide linkages which have improved chemical and biological stability while substantially retaining the overall 3-dimensional peptide conformation and bioactivity. It would also be a significant advancement in the art to couple bifunctional effector molecules to such peptide analogs.
Such stable analogs of bioactive peptides are disclosed and claimed herein.
SUMMARY OF THE INVENTION
The present invention is directed to stable analogs of cyclic bioactive peptides containing disulfide linkages. According to the present invention, the —S—S— bond is modified by one of four methods: (a) sulfide contraction, (b) isosteric substitution, (c) thioketal expansion, or (d) alkylation expansion. These procedures are illustrated generally below:
Sulfide contraction according to the present invention is the replacement of the disulfide bond (—S—S—) with a monosulfide bond (—S—) in which a bifunctional effector molecule, such as a bifunctional chelate, antineoplastic agent, or chemotoxin, is bound to the new peptide at the modified site, as illustrated below:
Wherein at least one R
1
or R
2
is a bifunctional effector molecule and the other R
1
or R
2
is selected from H, alkyl, aryl, hydroxyalkyl, aminoalkyl, carboxyl, and carboxyalkyl in which the carbon containing portions contain from 1 to 10 carbon atoms.
Isosteric substitution as used herein is the replacement of one sulfur atom of the disulfide bond with a carbon atom. At least one of the carbon atoms in the modified site of the peptide bears a bifunctional effector molecule, as illustrated below:
Wherein at least one R
3
, R
4
, or R
5
is a bifunctional effector molecule and the other R
3
, R
4
, or R
5
is selected from H, alkyl, aryl, hydroxyalkyl, aminoalkyl, carboxyl, and carboxyalkyl in which the carbon containing portions contain from 1 to 10 carbon atoms.
Thioketal expansion according to the present invention involves inserting an alkylidene unit (—CR
6
R
7
—) between the two sulfur atoms. Thioketal expansion can be accomplished by reduction followed by reaction of the resulting dithiol with aldehydes or ketones as follows:
where R
6
and R
7
may be the same or different and are selected from hydrogen, alkyl, aryl, hydroxyalkyl, alkoxyl, alkoxyalkyl, aminoalkyl, carboxyl, carboxyalkyl, in which carbon containing portions contain from 1 to 10 carbon atoms, and —(CH
2
)
n
—Y, where Y is a bifunctional effector molecule and n is from 0 to 6; and DTT is dithiothreitol.
R
6
and R
7
may be joined together to form a ring having from 3 to 7 members. One method of forming such a ring system is illustrated below:
Where R is a substituent selected from hydrogen, alkyl, aryl, hydroxyalkyl, aminoalkyl, carboxyl, and carboxyalkyl in which the carbon containing portions contain from 1 to 10 carbon atoms, and —(CH
2
)
n
—Y, where Y is a bifunctional effector molecule and n is from 0 to 6.
Some peptides are either resistant to disulfide reduction or undergo reduction only under harsh conditions. In such cases, it would be preferable to synthesize the corresponding linear peptide containing protected thiol groups by standard peptide synthetic methods, remove the protecting group from sulfur, and immediately carry out the condensation of the dithiol with a suitable cyclic or acyclic carbonyl compound.
Alkylation expansion, as used herein, involves inserting an alkyl moiety of from C
2
to C
3
, between the two sulfur atoms. Although it is possible to insert larger alkyl moieties (greater than C
3
) between the sulfur atoms, it is likely that the peptide's structural conformation would be significantly altered with larger alkyl moieties thereby leading to loss of bioactivity if such modifications are made. Alkylation expansion is achieved by reduction followed by alkylation of the dithiol with electrophiles or alkylating agents as follows:
where R
8
and R
9
are defined in the same manner as R
6
and R
7
, above. X is a suitable leaving group such as halogen, tosyl, or mesyl groups.
R
8
and R
9
may be joined together to form a ring having from 3 to 7 members. Two methods of forming such a ring system are illustrated below:
Where R is a substituent selected from hydrogen, alkyl, aryl, hydroxyalkyl, aminoalkyl, carboxyl, and carboxyalkyl in which the carbon containing portions contain from 1 to 10 carbon atoms, and —(CH
2
)
n
—Y, where Y is a bifunctional effector molecule and n is from 0 to 6; and X is a suitable leaving group such as halogen, tosyl, or mesyl groups.


REFERENCES:
patent: 4272506 (1981-06-01), Schwarzberg
patent: 5225180 (1993-07-01), Dean et al.
patent: 5382654 (1995-01-01), Lyle et al.
patent: 5413778 (1995-05-01), Kunkel et al.
patent: 5431899 (1995-07-01), Redmond et al.
patent: 5439792 (1995-08-01), Blake et al.
Fischman et al., J. Nucl. Med., vol. 34, No. 12 (1993) “A Ticket to Ride: Peptide Radiopharmaceuticals”, pp 2253-2263.
Mather et al., Cell Biophysics, vol. 21 (1992) “Radiiolabeled Octreotide What Lessons for Antibody-Medicated Targeting?”, pp 93-107.
Thakur et al., Symposium Abstracts, IXth International Symposium on Radiopharmaceutical Chemistry (1992) “Tc-99M Labeled Sandostatin: Preparation and Preliminary Evaluation”, pp 365-366.
Edwards et al, BBRL, vol. 136, No. 2, pp

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