Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Tripeptides – e.g. – tripeptide thyroliberin – etc.
Reexamination Certificate
2001-07-30
2004-07-27
Tate, Christopher (Department: 1654)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Tripeptides, e.g., tripeptide thyroliberin , etc.
C514S152000, C552S109000, C424S573000
Reexamination Certificate
active
06767992
ABSTRACT:
The present invention relates to a method for producing a pharmaceutically active peptide compound, which contains L-m-sarcolysine as the amino acid component. The active substance serves particularly for chemotherapy against cancers and is used especially for melanomas. In using a carrier substance on a cyclodextrin basis, the active substance is released delayed, which makes possible a sufficient bioavailability during a sufficiently long period of time.
A complex of six peptides containing m-L-sarcolysine has become known under the trade name “Peptichemio” (Insituto Sieroterapico Milanese S. Belfanti, Milan, Italy) for chemotherapy against cancer. It has been found that the activity of the individual peptides is different and that particularly one representative exhibits very high toxicity to melanoma cells. The peptides are a development which began with the product “Melphalan,” i.e., 4-[bis(2-chloroethyl)]-amino-L-phenylalanine. It has been found that this product has a cytostatic effect and can be utilized both for myeloma and for melanoma therapy. For the further development of the active substance, derivatives of the product were prepared. This also resulted in the L-m-sarcolysine [=m-{di-2-chlorethyl)amino}-L-phenylalanine], which was further derived in that peptides were prepared which contained the modified amino acid as a component. A combination of the six oligopeptides L-seryl-L-p-fluorophenylalanyl-L-m-sarcolysyl ethyl ester; L-prolyl-L-m-sarcolysyl-L-p-fluorophenylalanine ethyl ester; L-m-sarcolysyl-N-nitro-L-arginyl-L-norvaline ethyl ester; L-p-fluorophenylalanyl-L-m-sarcolysyl-L-asparagine ethyl ester; <sic. L-p->fluorophenylalanyl-glycyl-L-m-sarcolysyl-norvaline ethyl ester and L-m-sarcolysyl-L-arginyl-L-lysyl-L-m-sarcolysyl-L-histidine methyl ester formed the active principle of the antitumor agent “Peptichemio.” Of the six peptides, the L-prolyl-L-m-sarcolysyl-L-p-fluorophenylalanine (PSF) and its lower alkyl esters have proven particularly suitable.
It has been found that PSF showed considerably higher cytotoxicity compared with peptichemio itself (R. Levenson, et al., Radiumhemmet, Karolinska Hospital, Stockholm, Sweden, Eur. J. Cancer Clin. Oncol.; 23: 6, 783-788, 1987). According to these studies, it has been found that the peptide L-propyl-m-sarcolysyl-L-p-fluorophenylalanine (PSF) was 35 times and 28 times, respectively, more toxic to RPMI 8322 melanoma cells than melphalan and m-sarcolysine, respectively. Similar differences between the active substances have also been found for other melanoma cell lines.
It is the object of the present invention to make available a method of producing PSF that makes possible an economical and safe production of the active substance.
Production of such a compound is described in the printed publications BE-A-775775 and U.S. Pat. No. 3,814,746. The described production takes place according to the following schema 1:
The above schema shows, in step A, the condensation of the N-carbobenzoxy-L-proline with the ethyl ester of m-[di-(2-chlorethyl)-amino]-L-phenylalanine, the corresponding protected peptide resulting, as is shown in step B in schema 1.
In step C, the N-carbobenzoxy-L-prolyl-m-[di-(2-chlorethyl)-amino]-L-phenylalanine is obtained from the N-carbobenzoxy-L-prolyl-m-[di-(2-chlorethyl)-amino]-L-phenylalanine ethyl ester, and subsequently the condensation of this compound is carried out with p-fluoro-L-phenylalanine ethyl ester, while in step D of the schema 1 the carbobenzoxy-L-prolyl-m-[di-(2-chlorethyl)amino]-L-phenylalanine-p-chloro-L-phenylalanine ethyl ester is obtained.
The protection group is eliminated, whereby one arrives at step E of schema 1, the end product being the L-prolyl-m-[di-(2-chlorethyl)amino]-L-alanine-p-fluoro-L-phenylalanine ethyl ester.
The reaction conditions are such as are used generally with peptide syntheses. With the above method, the end product is obtained with a yield of 30%, with respect to the starting product m-[di-(2-chlorethyl)amino]-L-phenylalanine ethyl ester, it being necessary to carry out the purification of at least one intermediate product through column chromatography on silica gel.
The method can actually be applied industrially, but it is relatively complicated and leads to a rather insufficient yield.
If one takes into consideration the properties of the end product PSF-hydrochloride, the realization of another method of production, which can easily be applied industrially and which results in a better yield than that of the state of the art, is then an extraordinarily important and interesting object of the present invention.
It has been found that the method for producing PSF according to the invention, which uses another reaction sequence, is superior to the state-of-the-art method.
The subject matter of the present invention is thus the method defined in claim
1
for producing L-prolyl-L-m-sarcolysyl-L-p-fluorophenylalanine and esters and/or salts thereof.
The method according to the invention takes place according to the following schema 2:
The method comprises the following process steps, which are indicated in schema 2 above:
a) condensation of R-m-[di-(2-chlorethyl)amino]-L-phenylalanine with p-fluoro-L-phenylalanine ethyl ester, whereby R-m-[di-2-chlorethyl)amino]-L-phenylalanyl-p-fluoro-L-phenylalanine ethyl ester is obtained;
b) Removal of the protection group R;
c) condensation of the product obtained in step b) with R-L-proline, R-L-prolyl-m-[di-(2-chlorethyl)amino]-L-phenylalanyl-p-fluoro-L-phenylalanine ethyl ester being obtained;
d) Removal of the protection group R and synthesis of the hydrochloride; R can be benzyloxycarbonyl, t-butyloxicarbonyl or 9-fluorenylmethoxycabonyl. R is preferably a benzyloxycarbonyl group.
The method according to the present invention shows a total yield of 50% with respect to the starting product R-m-[di-(2-chlorethyl)amino]-L-phenylalanine.
The method according to the present invention has a great advantage in carrying out the synthesis of the end product since crystalline intermediate products are obtained which can be purified extraordinarily easily through crystallization.
The features and the advantages of the method according to the invention will be explained, for better comprehension, in the following description. The tripeptide, produced according to the inventive method, is produced according to schema 2 above. Therein R is a benzyloxicarbonyl or a t-butoxycarbonyl group (BOC) or a 9-fluorenylmethoxycarbonyl group (Fmoc).
As follows from schema 2, the method calls for, in step A, the condensation of R-m-[di-2-chlorethyl)amino]-L-phenylalanine with the ethyl ester of the p-fluorophenylalanine, the corresponding protected tripeptide being obtained in step B.
In step C, the benzyloxycarbonyl group is removed, and through a condensation of the R-L-proline with m-[di-(2-chlorethyl)amino]-L-phenylalanyl-p-fluoro-L-phenylalanine ethyl ester, the R-L-prolyl-m-[di-2-chlorethyl)amino]-L-phenylalanyl-p-fluoro-L-phenylalanine ethyl ester is obtained in step D of schema 2.
The protection group R is removed in step E of schema 2, the end product being L-prolyl-m-[di-(2-chlorethyl)amino]-phenylalanyl-p-fluoro-phenylalanine ethyl ester.
The reaction conditions are such as are generally standard with peptide synthesis.
The peptide L-prolyl-m-sarcolysyl-L-p-fluorophenylalanin (PSF) is produced preferably in the form of hydrochlorides or hydrobromides.
REFERENCES:
patent: 3814746 (1974-06-01), De Barbieri
patent: 775775 (1992-03-01), None
patent: 2094175 (1972-02-01), None
patent: 2101226 (1972-03-01), None
patent: 9420136 (1994-09-01), None
patent: WO 99/02177 (1999-01-01), None
Rajewski et al, “Pharmaceutical Applications of Cyclodextrins. 2. In Vivo Drug Delivery”, Journal of Pharmaceutical Sciences, vol. 85, No. 11, pp. 1142-1169, Nov. 1996.
Astaldi, “Peptichemio: A Multifaceted Antiblastic Drug”, Wadley Medical Bulletin, vol. 5, No.
Di Vittorio Pietro
Mehlem Francesco
PTC Pharma AG
Stevens Davis Miller & Mosher LLP
Tate Christopher
LandOfFree
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