Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Peptides containing saccharide radicals – e.g. – bleomycins – etc.
Reexamination Certificate
2000-10-18
2003-04-22
Russel, Jeffrey E. (Department: 1654)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Peptides containing saccharide radicals, e.g., bleomycins, etc.
C530S338000, C530S345000
Reexamination Certificate
active
06552166
ABSTRACT:
BACKGROUND OF THE INVENTION
In 1994 cancer of the prostate gland is expected to be diagnosed in 200,000 men in the U.S. and 38,000 American males will die from this disease (Garnick, M. B. (1994). The Dilemmas of Prostate Cancer. Scientific American, April:72-81). Thus, prostate cancer is the most frequently diagnosed malignancy (other than that of the skin) in U.S. men and the second leading cause of cancer-related deaths (behind lung cancer) in that group.
Compositions useful in the treatment of prostatic cancer and related conditions are described in U.S. Pat. Nos. 5,599,686, 5,866,679 and 5,948,750, and U.S. patent applications Ser. No. 08/950,805, filed Oct. 14, 1997, now U.S. Pat. No. 8,948,750, (PCT Publ.No. WO 98/18493). Said compositions comprise chemical conjugates comprising known cytotoxic agents and oligopeptides having amino acid sequences that are selectively proteolytically cleaved by free prostate specific antigen and that include a cyclic amino acid having a hydrophilic substituent. The oligopeptide moieties are selected from oligomers that are selectively recognised by free prostate specific antigen (PSA) and are capable of being proteolytically cleaved by the enzymatic activity thereof.
Ideally, the cytotoxic activity of the cytotoxic agent is greatly reduced or absent when the intact oligopeptide containing the PSA proteolytic cleavage site is bonded directly, or through a chemical linker, to the cytotoxic agent. Also ideally, the cytotoxic activity of the cytotoxic agent increases significantly, or is restored completely, upon proteolytic cleavage of the attached oligopeptide at the cleavage site. Anthracycline antibiotics, in particular doxorubicin, are among the cytotoxic agents that were described in the published patent applications as preferably incorporated into such conjugates, which may be referred to as PSA conjugates.
It is the object of this invention to provide an efficient, scaleable and reproducible process for the preparation of PSA conjugates having an anthracycline antibiotic moiety as the cytotoxic agent and having the cleavable oligopeptide directly attached, via the C-terminus amino acid, to the glycosyl amine of the anthracycline antibiotic moiety.
Another object of this invention is to provide intermediate compounds useful in the preparation of such PSA conjugates.
SUMMARY OF THE INVENTION
A chemical process for the preparation of a PSA conjugate which comprises an anthracycline antibiotic and an oligopeptide, having an amino acid sequence that is selectively proteolytically cleaved by free prostate specific antigen (PSA) is disclosed. Such conjugates are useful in the treatment of prostatic cancer and benign prostatic hyperplasia (BPH).
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to a process for the preparation of compounds as illustrated by formula I:
or a pharmaceutically acceptable salt thereof
wherein
oligopeptide is an oligopeptide which is selectively recognized by the free prostate specific antigen (PSA) and is capable of being proteolytically cleaved by the enzymatic activity of the free prostate specific antigen,
R
a
is —CH
3
, —CH
2
OH, —CH
2
OCO(CH
2
)
3
CH
3
, or —CH
2
OCOCH(OC
2
H
5
)
2
;
R
b
is —OCH
3
, —OH or —H;
R
d
is —OH, —OTHP or —H; and
R
e
is —OH or —H, provided that R
e
is not —OH when R
d
is —OH or —OTHP;
R is selected from:
a) acetyl;
e) hydrogen;
R
1
and R
2
are independently selected from:
a) hydrogen,
b) unsubstituted or substituted aryl, unsubstituted or substituted heterocycle, C
3
-C
10
cycloalkyl, C
2
-C
6
alkenyl, C
2
-C
6
alkynyl, halogen, C
1
-C
6
perfluoroalkyl, R
3
O—, R
3
C(O)NR
3
—, (R
3
)
2
NC(O)—, R
3
2
N—C(NR
3
)—, R
4
S(O)
m
NH, CN, NO
2
, R
3
C(O)—, N
3
, —N(R
3
)
2
, or R
4
C(O)NR
3
—,
c) unsubstituted C
1
-C
6
alkyl,
d) substituted C
1
-C
6
alkyl wherein the substituent on the substituted C
1
-C
6
alkyl is selected from unsubstituted or substituted aryl, unsubstituted or substituted heterocyclic, C
3
-C
10
cycloalkyl, C
2
-C
6
alkenyl, C
2
-C
6
alkynyl, R
3
O—, R
4
S(O)
m
NH, R
3
C(O)NR
3
—, (R
3
)
2
NC(O)—, R
3
2
N—C(NR
3
)—, CN, R
3
C(O)—, N
3
, —N(R
3
)
2
, and R
4
OC(O)—NR
3
—; or
R
1
and R
2
are combined to form —(CH
2
)
s
— wherein one of the carbon atoms is optionally replaced by a moiety selected from: O, S(O)
m
, —NC(O)—, NH and —N(COR
4
)—;
R
3
is selected from: hydrogen, aryl, substituted aryl, heterocycle, substituted heterocycle, C
1
-C
6
alkyl and C
3
-C
10
cycloalkyl;
R
4
is selected from: aryl, substituted aryl, heterocycle, substituted heterocycle, C
1
-C
6
alkyl and C
3
-C
10
cycloalkyl;
m is 0, 1 or 2;
n is 1, 2, 3 or 4;
p is zero or an integer between 1 and 100;
q is 0 or 1, provided that if p is zero, q is 1;
r is an integer between 1 and 10; and
s is 3, 4 or 5.
The process comprises the step of preparing the compound of the formula Ia:
or a salt thereof;
wherein
oligopeptide, R, R
a
, R
b
, R
d
and R
e
are described as above
R′ is selected from:
a) R,
b) a protected precursor to R, and
c) an N-terminus protecting group;
by mixing an oligopeptide of the formula A:
wherein oligopeptide and R′ are described as above, or a salt thereof;
with an anthracycline antibiotic of the formula B:
or a salt thereof,
wherein R
a
, R
b
, R
d
and R
e
are as described hereinabove,
in the presence of a carboxyl activating agent and, optionally, in the presence of a base.
In an embodiment of the instant process, if R′ is a protected precursor to R or an N-terminus protecting group, the process further comprises the step of removing the protecting group to produce the compound of the formula I.
In an embodiment of the instant process, the oligopeptide of formula A is mixed with a salt of the anthracycline antibiotic of the formula B in the presence of a carboxyl activating agent and a base.
In an embodiment of the instant process, the carboxyl activating agent is selected from dicyclohexylcarbodiimide (DCC), N-ethyl-N-(3-dimethylaminopropyl)-carbodiimide (EDC), and 1,3-diisopropylcarbodiimide (DIC).
In a preferred embodiment, the carboxyl activating agent is EDC.
In an embodiment of the instant process, the base is selected from 2,4,6-collidine, lutidine, pyridine, triethyl amine and (iPr)
2
NEt.
In a preferred embodiment, the base is 2,4,6-collidine.
It has been surprisingly discovered that use 2,4,6-collidine as the base in the instant process for the formation of the compound of the formula Ia results in lower epimerization of the C-terminus amino acid moiety than when other bases previously described are utilized.
In a further embodiment of the instant process, the mixing of the compounds of formula A and formula B is additionally in the presence of an additive.
In a preferred further embodiment of the instant process the additive is selected from HOAt, HOBt, HOPO or a combination thereof.
In a preferred embodiment of the further embodiment of the instant process, the additive is HOAt.
In a second preferred embodiment of the further embodiment of the instant process, the additive is a combination of HOPO and HOAt.
In a further embodiment of the instant process, the oligopeptide of the formula A is mixed with the anthracycline antibiotic of the formula B in the presence of a carboxyl activating agent, an additive and a base.
In a preferred further embodiment of the instant process, the oligopeptide of the formula A is mixed with the anthracycline antibiotic of the formula B in the presence of an additive and a base, and a carboxyl activating agent is thereafter added to the mixture.
It has been surprisingly discovered that when the carboxyl activating agent is added last to a preformed mixture of the process components the amount of epimerization that occurs at the C-terminus amino acid of the oligopeptide moiety in the compound of formula Ia is less than when the base is added last to the reaction mixture.
In a more preferred further embodiment of the instant process, the oligopeptide of the formula A is mixed with the anthracycline antibiotic of the formula B in the presence of
Lieberman David R.
Lynch Joseph E.
Robbins Michael A.
Shi Yao-Jun
Daniel Mark R.
Merck & Co. , Inc.
Muthard David A.
Russel Jeffrey E.
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