carboxyl group in glutamic acid only by salt formation during acylation by 4-amino-4-deoxy-N10 methylpteroic acid (APA), thus simplifying the preparation to a ...
Proc. Natl. Acad. Sci. USA Vol. 90, pp. 6373-6376, July 1993 Medical Sciences
Selective coupling of methotrexate to peptide hormone carriers through a y-carboxamide linkage of its glutamic acid moiety: Benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate activation in salt coupling (drg targeting/cyttooic peptides/slte-seectve conJugation/chemotherapy agents)
A. NAGY*t, B. SZOKE*, AND A. V. SCHALLY**§ Endocrine, Polypeptide and Cancer Institute, tVeterans Affairs Medical Center, and *Department of Medicine, Tulane University School of Medicine, New
Orleans, LA 70146
Contributed by A. V. Schally, April 12, 1993
suitable for conjugation even to large "carriers" such as peptide hormones. These conjugates can preserve MTX-like antifolate activity and then be targeted to tumor cells that have specific receptors for the peptide portion of the hybrid molecule. In this paper, we describe a convenient synthesis of a selectively protected MTX derivative, utilizing benzotriazol1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent) in the salt-coupling method. The use of the salt-coupling method makes it possible to protect one carboxyl group in glutamic acid only by salt formation during acylation by 4-amino-4-deoxy-N10 methylpteroic acid (APA), thus simplifying the preparation to a one-vessel reaction (Fig. 1). This intermediate was used for synthesis ofpeptide-MTX conjugates, where MTX was coupled to a free amino group in the peptide carrier through a -carboxamide linkage of its glutamic acid moiety. Syntheses of a somatostatin-MTX conjugate (AN-51) and two conjugates of LH-RH analogs with MTX (AJ-04 and AJ-51) are presented as examples.
A convenient synthetic method is described ABSTRACT for the preparation of peptide-methotrexate (MTX) conjugates in which MTX is coupled selectively through the ycarboxyl group of its glutamic acid moiety to a free amino group in peptide analogs. The syntheses of a somatostatin analog-MTX conjugate (MTX-D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-ThrNH2) (AN-51) and two conjugates of analogs of luteinizing hormone-releasing hormone (LH-RH) with MTX [Glp-HisTrp-Ser-Tyr-D-Lys(MTX)-Leu-Arg-Pro-Gly-NH2I (AJ-04) and [Ac-Ser-Tyr-D-Lys(MTX)-Leu-Arg-Pro-NH-Et] AJ-51 are presented as examples. Benzotriazol-1-yloxyris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent) was used in the synthesis for activation of 4-amino-4-deoxy-N1°methylpteroic acid, which reacted with the potassium salt of glutamic acid a-teil-butyl ester in dimethyl sulfoxide to form the suitably protected MTX derivative. This synthesis provides an example of the high suitabilit of BOP reagent for the salt-coupling method. The selectively protected MTX derivative was then coupled to the different peptide carriers and deprotected under relatively mild conditions by trifluoroacetic acid. The conjugates of MTX with hormonal analogs are suitable for targeting to various tumors that possess receptors for the peptide moieties.
MATERIALS AND METHODS Synthesis. The LH-RH analogs and the somatostatin analog used as carriers were synthesized in our laboratory as described (7-10). APA was purchased from Aldrich; BOP reagent was purchased from Peptides International (Louisville, KY). Preparation of tert-butyl ester of the a-carboxyl group of the glutamic acid moiety in MTX (MTX-a-OtBu). APA hemihydrochloride dihydrate (38 mg; 100 ,umol) was dissolved in 300 A1 of dimethyl sulfoxide (DMSO) in the presence of 21 1d (150 ,umol) of triethylamine (TEA). To this solution 48.6 mg (110 ,mol) of BOP reagent was added. After stirring for 30 min, a yellow precipitate appeared. A solution of 22.4 mg (110 ,mol) of Glu-a-OtBu and 7.6 mg (55 ,umol) of K2CO3 in 300 j4 of DMSO was then added to the reaction mixture. After 5 min at 50°C, a clear solution was obtained. The reaction, monitored by analytical HPLC, was completed after stirring for 2 hr at room temperature. The product was isolated from DMSO either by evaporation in a SpeedVac centrifuge evaporator or by precipitation with ethyl acetate/diethyl ether (1:1) (vol/vol). This crude material was purified by reversed-
Drug targeting is a modem approach aimed at decreasing the undesired side effects of antineoplastic agents caused by their nonselective action on various human tissues. In view of extensive investigation of analogs of different peptide hormones such as luteinizing hormone-releasing hormone (LHRH), somatostatin, and, recently, bombesin (1-3) for the treatment of some cancers, we also endeavored to use them as carrier molecules for different chemotherapeutic agents in cases in which direct action of these peptides on the membrane receptors on tumors could be established (2, 4). Methotrexate (MTX) is a well-known and extensively characterized antineoplastic agent chemically suitable for conjugation to peptides. This compound is a potent inhibitor of the enzyme dihydrofolate reductase, which is responsible for recycling 7,8-dihydrofolate to its reduced, physiologically active 6(R)-tetrahydro form (5). Although not all types of tumors are affected by MTX, this cytotoxic agent has been used successfully in chemotherapy for more than 4 decades. While searching for more potent analogs of MTX, it was shown that certain alterations can be made within the side chain of its glutamic acid moiety without loss of activity (6). A "bulk tolerance" at the t-carboxyl position of its glutamic acid moiety has also been reported (6). This makes MTX
Abbreviations: APA, 4-amino-4-deoxy-N10-methylpteroic acid; BOP reagent, benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; LH-RH, luteinizing hormone-releasing hormone; MTX, methotrexate; OtBu, tert-butyl ester; TEA, triethylamine; TFA, trifluoroacetic acid. tOn leave from Institute for Drug Research, H-1325 Budapest P.O. Box 82, Hungary. §To whom reprint requests should be addressed at: Veteran's Affairs Medical Center, 1601 Perdido Street, New Orleans, LA 70146.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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2
CHf~N
N
COOH xTEA
~N H2N
N1.
BOP
reagent
DMSO,
15
min
C00- K+
2.
CH2 OH 2
H2N
OH- COOtBu
DMSO,
60
min
MTX-ax-OtBu
FIG.
Reaction scheme for synthesis of selectively protected 1. derivative, MTX-a-OtBu, by using BOP reagent in the saltcoupling method.
MTX
phase HPLC (see below). After lyophiiain, the purified end product weighed 48 mg, which represents a yield of 75%. MTX-y,.-OtBu was prepared the same way using Glu-y-OtBu instead of Glu-a-OtBu.
Preparation of AN-Si. MTX-at-OtBu (9.8 mg; 15.7 p.mol) dissolved in 100 of N,N-dimethylformamide (DMF) containing 4.2 p.1(30p.umol) of TEA, and 7.5 mg (17 p.mol) of BOP reagent was added. After stirring the reaction mixture for 15 min, a solution of 20 mg (14.5 p.mol) of RC-2170 [D-Phe-CyFs-Tyr-D-Trp-L ys(9-fluorenylmethoxycarbonyl)-
A.
was
Val-C~s-Thr-NHd
containing 4.2 p.1(30 p.mol) of acylation was completed within 1 hr. The reaction product was obtained by precipitation with ether and centrifugation. The dried precipitate was treated with 200 trifluoroacetic acid (TFA) containing 17.5 mg (100 p.mol) of indole-3-acetic acid for the protection of tryptophan. After standing for 3 hr at room temperature, the crude product was precipitated and washed with ether. After dryof 10%6 piperidine in DMF ing, it was dissolved in 200 (vol/vol). After 10 min, ether was used again for precipitation of the crude product, which was purified by reversed-phase HPLC. After isolation, 12 mg of >95% pure AN-Si was obtained in 52% overall yield. Synthesis of AJ-04. MTX-a-OtBu (15.6 mg; 25 p.mol) was dissolved in 200 of DMF containing 7 (50 pmnol) of TEA, and 12 mg (27.5 p.mol) of BOP reagent was added. After stirring for 15 min, a solution of 40 mg (25 p.mol) of [D-Lys6]LH-RH in DMF containing 7 p.1(50 p.mol) of TEA was added. The reaction was completed within 1 hr. Precipitation by ether was followed by centrifugation and the dried crude product was treated with 200 p.1 of TFA containing 35 mg (200 p.mol) of indole-3-acetic acid for protection of tryptophan. After 3 hr, the reaction mixture was precipitated with ether. Purification by HPLC resulted in 31 mg of >95% pure end product in 65% overall yield. Synthesis of AJ-51. MTX-a-OtBu (7.8 mg; 12.5 p.mol) was dissolved in 100 of DMF contiig3.5 p.1(25 p.mol) of TEA and 6 mg (13.7 p.mol) of BOP reagent was added. After 15 min, a solution of 13.8 mg (13 p.mol) of AJ-41 (Ac-SerTyr-D-Lys-Arg-Leu-Pro-NH-Et) in DMF containing 3.6 (26 p.mol) of TEA was added. The reaction mixture was stirred for 2 hr at room temperature and then precipitated with ether. The precipitate was dissolved in 100 p.1 of TFA. After 3 hr, ether was used again for precipitation of the crude peptide. After purification by HPLC, 9.2 mg of >98% pure TEA
Proc. NatL Acad Sci. USA 90 (1993)
Medical Sciences: Nagy et aL.Po.Nt.Aa.SiUA90(93
6374
was
in DMF
added. The
product was obtained in 66% overall yield. The -a-isomer of AJ-51 was obtained in a similar way by using MTX-ryOtBu. Purification. Purification of all the crude products was carried out on a Beckman model 342 semipreparative HPLC system using an Aquapore Octyl (250 x 10 mm; pore size, 300 A; particle size, 15 p.m) column. The solvent system consisted of two components-(i) 0.1% TFA in water, (ii) 0.1% TFA in 70%o aqueous acetonitrile-and was used in linear gradient mode. Analytical HPLC. A Beckman analytical HPLC system equipped with model 168 diode array detector and System Gold chromatography software (Beckman) was used to check purity and to monitor the chemical reactions. The column was Dynamax C18 (250 x 4.6 mm; pore size, 300 A; particle size, 12 p.m. 'Analysis. Mass spectrometry and 1H NMR analyses were
used for structure identification.
RESULTS A convenient method was developed for the site-selective conjugation of MTX to peptide carriers. The method makes it possible to couple selectively either the a- or the y-carboxyl group of the glutamic acid moiety in MTX to a free amino group in peptides. This was achieved by using two selectively protected MTX derivatives, MTX-a-OtBu and MTX-'yOtBu. These compounds were prepared by reacting an activated derivative of APA with the potassium salt of the corresponding glutamic acid OtBu. Before utilizing the BOP reagent in this reaction, we synthesized N-hydroxysuccinimide and p-nitrophenyl esters of APA and tried them in the salt-coupling procedure. These active esters were prepared by activating APA with 1,3-diisopropylcarbodiimide in DM50 and reacting overnight with N-hydroxysuccinmide or p-nitrophenol. A 5-fold excess of Glu-a-OtBu potassium salt and 8 hr of heating at 800C were necessary to complete the reaction between the two compounds. Even under these extreme conditions, the yield was only 40%6 after purification by HPLC. In contrast, use of BOP reagent for activation of the benzoic acid moiety in APA made this reaction complete within 2 hr at room temperature. Only a 10%' excess of the glutamic acid derivative was necessary to achieve a 75% yield and a purity >98% after HPLC purification. To determine whether MTX withstands the conditions necessary to remove the OtBu protecting group, MTX-a-OtBu was treated with TFA for 5 hr. Analytical HPLC of the reaction mixture showed the appearance of a product identical with the MTX standard, while no degradation products could be detected. This indicates that the general usefulness of this intermediate is limited only by the acid sensitivity of the peptide carrier. The somatostatin analog-MTX conjugate (AN-5i) was
designed as a chemotherapeutic agent targeted specifically to tumor cells that have somatostatin receptors. This compound
synthesized by activating MTX-a-OtBu with BOP reagent and coupling to the free N terminus of RC-2170, a derivative of a well-characterized somatostatin analog (RC121), with 9-fluorenylmethoxycarbonyl side chain protection. Analog RC-121 was made previously in our institute (7). A 3-hr TFA treatment to remove the OtBu group from the at-carboxyl Of L-glutamic acid in MTX was followed by cleavage of the 9-fluorenylmethoxycarbonyl group on the side chain of its Lys-5 residue by 10%' (vol/vol) piperidine in DMF. These deblocking procedures do not cause any damage to the peptide or MTX within the conjugate. It was reported earlier (8) that AN-Si had a greater effect than its carrier (RC-121) or free MTX in inhibiting the growth of MIA PaCa-2 human pancreatic cancer xenografts in nude mice while preserving MTX-like activity. AJ-04 and AJ-51, two LH-RH analog-MTX conjugates, were made previously in our laboratory (9, 10). Earlier, these was
Proc. Natl. Acad. Sci. USA 90 (1993)
Medical Sciences: Nagy et aL
E 0
a
0.1
0. L.
0
o
a0
0
10
2
0
4
0.0
Retention
20
time
30
40
(min)
FIG. 2. Chromatograms of the separations by HPLC of reaction products of AJ-51 formed during conjugation of MTX to LH-RH
analog AJ-41 by different methods: nonselective activation of MTX by 1,3-iisopropylcarbodiimide/1-hydroxybenzotriazole (trace a), selective activation of the a-carboxyl group in MTX by BOP reagent (trace b), selective activation of the y-carboxyl group in MTX by BOP reagent (trace c). Conditions of chromatography: column, Vydac C18 TP5 250 x 4.6 mm; eluent, 0.1% TFA in 15% (vol/vol) aqueous acetonitrile; flow rate, 1.2 ml/min.
compounds were synthesized by nonselective activation of the two carboxyl groups in MTX by the 1,3-diisopropylcarbodiimide/1-hydroxybenzotriazole method. This method leads to formation of two structural isomers, in which MTX is linked either through the a-carboxyl or the 'y-carboxyl group of its glutamic acid moiety. Analytical HPLC of the reaction mixture under optimized isocratic conditions (Fig. 2, trace a) shows the separation of two structural isomers and reveals the presence of two double peaks. Each pair of peaks most likely represents diastereomer peptide-MTX conjugates differing only in the configuration of the Ca atom of the glutamic acid moiety in MTX. Although this was not confirmed by optical rotation analysis, evaluation of the chromatograms in Fig. 2 strongly supports this assumption. A double peak appears in the chromatogram of the a structural isomer synthesized by our method (trace b) similar to the chromatogram in trace a, while only a single peak appears on the chromatogram of the y structural isomer (trace c). This demonstrates that our method makes it possible to conjugate MTX through the -t-carboxyl group of its glutamic acid moiety without racemization. It also clearly shows that nonspecific activation of the two carboxylic acid residues in MTX leads to a mixture of four products: two diastereomer forms ofthe a structural isomer and two diastereomers of the y
structural isomer.
The synthetic procedure described can serve as a general method for preparation of peptide-MTX conjugates. Synthesis of AN-51 illustrates the strategy for preserving unmasked a free amino group in the carrier molecule when it is necessary for binding. A variety of studies with the peptide-MTX conjugates synthesized by our method showed (8-11) that they are active in vivo and in vitro in inhibiting proliferation of mammary prostatic and pancreatic cancers.
DISCUSSION Various attempts have been made in our laboratory to use peptide hormone "carriers" for targeting different types of
6375
cytotoxic molecules to prostate, breast, and pancreatic tumors (8-13). MTX proved to be one of the most stable cytotoxic radicals suitable for coupling to peptides. The simplest way of coupling MTX to a free amino group of a carrier peptide would be through nonselective activation of the carboxyl groups in its glutamic acid moiety. Unfortunately, such activation leads to anhydride formation and, consequently, to formation of two structural isomers of the conjugate. The example of separation of synthetic products of AJ-51 (Fig. 2, trace a) shows that each of these structural isomers is a mixture of two diastereomer peptide conjugates formed by racemization of glutamic acid in MTX. It is well known that optically pure N-benzoyl a-amino acids undergo azlactone formation after activation of their a-carboxyl group, which is followed by racemization (14). Since the glutamic acid moiety in MTX is acylated by a benzoic acid derivative, it can also undergo racemization whenever its a-carboxyl group is activated. This accounts for the appearance of the double peaks of diastereomer peptide-MTX conjugates during coupling (Fig. 2, traces a and b). When the a-carboxyl group in MTX is protected by OtBu, and only the y-carboxyl group is activated, only one optically pure conjugate is formed (trace c). It is, however, worth noting that nonspecific activation of the a- and -t-carboxyl groups in MTX by 1,3-diisopropylcarbodiimide leads not only to formation of two diastereomers of the a structural isomer but also to production of two diastereomers of the y structural isomer. This can be explained by the assumption that the glutamic acid anhydride, which is formed after activation and is responsible for formation of the structural isomers, is in equilibrium with the azlactone, through which racemization takes place. It is also shown in the chromatogram (trace a) that the ratio of the two diastereomers in the a and in the y structural isomers is very similar. This indicates that racemization occurs before formation of the a or the y conjugate. The formation of several products during the previous synthesis (10) and the presence of a highly conserved arginine residue in dihydrofolate reductase, which binds specifically to the a-carboxyl group of the glutamic acid moiety in MTX (15), prompted us to develop a method for preparation of MTX-peptide conjugates that preserves the a-carboxyl group free. For this purpose, MTX-a-OtBu seemed to be the best choice, since the protecting group is stable and easily removed under mild conditions. The synthesis of MTX-aOtBu, by careful hydrolysis of the a-OtBu y-methyl ester of MTX, had already been reported (6), but we developed a more convenient and more efficient reaction for formation of this intermediate. BOP reagent was used for activation of APA. The activated product reacted, without isolation, with the potassium salt of Glu-a-OtBu in DMSO. Twenty minutes after activation, a yellow crystalline product precipitated, indicating completion of the first reaction step. These crystals redissolved after addition of the glutamic acid derivative and heating the reaction mixture to 50°C for 5 min. The reaction was complete within 2 hr. After purification by HPLC, the yield was 75% and the purity of the end product was >98%. These results clearly indicate the high efficacy of the BOP reagent in the salt-coupling procedure. The selectively protected MTX derivative thus obtained was used for synthesis of a somatostatin analog-MTX conjugate (AN-51). Effectiveness of this compound in vivo shows the usefulness of somatostatin analogs as carriers for targeted chemotherapy. This convenient and unambiguous synthetic procedure developed by us for selective coupling of MTX to peptides also made it possible to prepare two cytotoxic LH-RH analogs containing MTX (AJ-04 and AJ-51) in high yields and in high optical purity for in vivo studies, which require larger amounts of material.
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In vitro and in vivo investigations with these peptide-MTX conjugates reveal that MTX linked to peptide carriers can be used in targeted chemotherapy (8-11, 16). The convenient synthetic procedure presented in this paper is suitable for preparation of various peptide-MTX conjugates if the carrier is resistant to relatively mild TFA treatment. We thank Prof. J. Engel, Dr. M. Bernd, and Dr. E. Busker (Degussa AG and Asta Medica AG, Frankfurt/M, Germany) for MS and NMR analyses and for suggestions in the preparation of this manuscript. The work described in this paper was supported by National Institutes of Health Grants CA-4003 and CA-4004 (to A.V.S.) and by the Medical Research Service of the Veterans Affairs.
1. Schally, A. V., Srkalovic, G., Szende, B., Redding, T. W., Korkut, E., Szepeshazi, K., Bokser, L., Pinski, J., Groot, K., Serfozo, P., Comaru-Schally, A. M., Bajusz, S., GonzalesBarcena, D., Reissmann, T., Hilgard, P. & Engel, J. (1990) in Advances in the Study of GnRH Analogues, ed. Lunenfeld, B. (Parthenon, Park Ridge, NJ), Vol. 2, pp. 25-35. 2. Schally, A. V., Srkalovic, G., Szende, B., Redding, T. W., Janaky, T., Juhasz, A., Korkut, E., Cai, R.-Z., Szepeshazi, K., Radulovic, S., Bokser, L., Groot, K., Serfozo, P. & ComaruSchally, A. M. (1990) J. Steroid Biochem. Mol. Biol. 37, 1061-1067. 3. Radulovic, S., Cai, R.-Z., Serfozo, P., Groot, K., Redding, T. W., Pinski, J. & Schally, A. V. (1991) Int. J. Pep. Protein Res. 38, 593-600. 4. Srkalovic, G., Cai, R.-Z. & Schally, A. V. (1990) J. Clin. Endocrinol. Metab. 70, 661-669.
Proc. Natl. Acad Sci. USA 90 (1993) 5. Kralovec, G., Spencer, A. H., Blair, M., Mammen, M. & Ghose, T. (1989) J. Med. Chem. 32, 2426-2431. 6. Rosowsky, A., Fosch, R., Uren, J. & Wick, M. (1981) J. Med. Chem. 24, 1450-1456. 7. Cai, R.-Z., Szoke, B., Lu, R., Fu, D., Redding, T. W. & Schally, A. V. (1986) Proc. Natl. Acad. Sci. USA 83, 18961900. 8. Radulovic, S., Nagy, A., Szoke, B. & Schaily, A. V. (1992) Cancer Lett. 62, 263-271. 9. Janaky, T., Juhasz, A., Bajusz, S., Csernus, V., Srkalovic, G., Bokser, L., Milovanovic, S., Redding, T. W., Rekasi, Z., Nagy, A. & Schally, A. V. (1992) Proc. Natl. Acad. Sci. USA 89, 972-976. 10. Janaky, T., Juhasz, A., Rekasi, Z., Serfozo, P., Pinski, J., Bokser, L., Srkalovic, G., Milovanovic, S., Redding, T. W., Halmos, G., Nagy, A. & Schally, A. V. (1992) Proc. Natl. Acad. Sci. USA 89, 10203-10207. 11. Pinski, J., Schally, A. V., Yano, T., Szepeshazi, K., Halmos, G., Groot, K., Comaru-Schally, A. M., Radulovic, S. & Nagy, A. (1993) Prostate, in press. 12. Bajusz, S., Janaky, T., Csernus, V. J., Bokser, L., Fekete, M., Srkalovic, G., Redding, T. W. & Schally, A. V. (1989) Proc. Natl. Acad. Sci. USA 86, 6313-6317. 13. Bajusz, S., Janaky, T., Csernus, V. J., Bokser, L., Fekete, M., Srkalovic, G., Redding, T. W. & Schally, A. V. (1989) Proc. Natl. Acad. Sci. USA 86, 6318-6322. 14. Bodanszky, M. (1984) Principles of Peptide Synthesis (Springer, Heidelberg). 15. Bolin, J. T., Filman, D. J., Matthews, D. A., Hamlin, R. C. & Kraut, J. (1982) J. Biol. Chem. 257, 13650-13662. 16. Szepeshazi, K., Schally, A. V., Juhasz, A. & Janaky, T. (1992) Anti-Cancer Drug 3, 109-116.
