Antitumoral cyclic peptide analogues of chlamydocin

Peptides,Vol. 14, pp. 1091-1093, 1993

0196-9781/93 $6.00 + .00 Copyright© 1993PergamonPressLtd,

Printed in the USA.

Antitumoral Cyclic Peptide Analogues of Chlamydocin ELISABETH BERNARDI,* JEAN-LUC FAUCHERE,t GHANEM ATASSI,t P H I L I P P E V I A L L E F O N T * A N D R E N E L A Z A R O *l

*Laboratoire des Aminoacides et des Peptides, CNRS-URA 468, Universit£ Montpellier II, Place E. Bataillon, 34095 Montpellier Cedex 05, France and )~lnstitut de Recherche Servier, 11 Rue des Moulineaux, 92150 Suresnes, France Received 17 May 1993 BERNARDI, E., J.-L. FAUCHERE, G. ATASSI, P. V1ALLEFONTAND R. LAZARO. Antitumoral cyclicpeptide analogues ofchlamydocin. PEPTIDES 14(6) 1091-1093, 1993.--A series of cyclic tetrapeptides beating the bioactive alkylating group on an e-amino-lysylfunction have been examined for their antitumoral activity on L 1210 and P388 murine leukemia cell lines. One analogue belonging to the chlamydocin family and bearing a/3-chloroethylnitrosourea group was found to be potent at inhibiting L I210 cell proliferation and had a higher therapeutic index than the reference compound bis-/3-chloroethylnitrosourea(BCNU) on the in vivo P388-induced leukemia model. Chlamydocin

Antitumoral peptides

Cyclicpeptides

CHLAMYDOCIN, extracted first from Diheterospora chlamydosporia 20 years ago, exhibits potent cytotoxic activity in vitro (IC5o = 0.36 ng/ml) against P815 mastocytoma mouse cell proliferation (3). Unfortunately, this activity quickly fades under in vivo conditions and is destroyed within a few minutes when chlamydocin is incubated in blood serum [reported half-life 2.5 min (8)]. For example, the increase of life span (ILS%) of mice inoculated with L 1210 or P815 leukemias after being once (50400 mg/kg, IP) or daily injected (20-160 mg/kg, IP) with the fungus product was very low (ILS 19% and 10%, respectively) and showed no clear dose dependency (8). The authors concluded that "this drug produces only very moderate ILS if at all." Chlamydocin (Table 1) is the cyclic tetrapeptide c[Aib-PheI>-Pro-Aoe] containing the uncommon amino acid Aoe [(2S,9S)2-amino-8-oxo-9,10-epoxydecanoic acid]. The ketoepoxidic group of Aoe, which is required for biological activity, appears to be too reactive against nucleophilic attack, thus leading to epoxide opening before having reached its target. Other cyclic tetrapeptides, all from fungus origin, share the same structural features with chlamydocin, namely the presence of Aoe, of an imino acid such as proline or pipecolic acid, and of one ~ a m i n o acid. One of them, HC toxin from Helminsthosporium carbonum, a potent host-specific agent against sensitive maize hybrids, also has a considerable in vitro cytotoxic activity, although 10 times lower than chlamydocin (11). This result seems to imply that the cyclopeptide moiety is a part of the pharmacophore important for target recognition. Based on this assumption, we have prepared a series of cyclic peptides belonging either to the chlamydocin or to the HC toxin family. With the purpose of preserving the high in vivo activity by increasing the stability of the alkylating function, a lysine Requests for reprints should be addressed to Dr. Ren6 Lazaro. 1091

was introduced in the place of Aoe. This provided a free amino function on N' for the anchoring of one of the well-known alkylating ~-chloroethyl-nitrosourea, amidoepoxy, or N,N-di-(/3chloroethyl)-4-amino-benzoyl (nitrogen mustard) groups. METHOD

Peptide Synthesis The synthesis of these new analogues (Table 2) will be described in detail elsewhere. The cyclization step, which is always the bottleneck in the synthesis of small cyclic peptides, was carfled out with satisfactory yields (50% to 70%). For the two series of peptides, the best cyclization site among the four possibilities was located on the D-Pro- carboxylic function. Briefly, one of the two backbone peptides, Ala-D-Ala-Lys(Z)-D-Pro, was synthesized on solid phase, according to the general methodology of Barany and Merrifield (2) using tert-butyloxycarbonyl (Boc) for N ~ protection and benzyloxycarbonyl (Z) for blocking of the side chain of lysine. After cleavage of the peptide from the polystyrenic resin and Boc deprotection by trifluoroacetic acid (TFA), cyclization followed in dilute solution, using diethylphosphorocyanidate as the activating agent. Owing to the presence of Aib, a nonracemizable but bulky residue, the second backbone peptide, namely Lys(Z)-Aib-PheD-Pro, was prepared more conveniently in solution, using a 2 + 2 fragment coupling between Boc-Lys(Z)-Aib-OH and H-PhePro-OtBu under dicyciohexylcarbodiimide-dimethylaminopyridine (DCC-DMAP) activation. After TFA treatment leading to the deprotected main chain, cyclization was performed as above.

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BERNARDI ET AL. TABLE 1 NATURAL CYTOTOXIC CYCLIC PEPTIDES

Name

Structure

Reference

Chlamydocin HC toxin WF 3 1 6 1 Cyl I Trapoxin A

c[Aib-Phe-D-Pro-Aoe] c[D-Pro-Ala-D-Ala-Aoe] c[D-Phe-Leu-Pip-Aoe] c[o-Tyr(OMe)-lIe-Pip-Aoe] c[Phe-Phe-D-Pip-Aoe]

1 3 4 5 6

Aib = 2-amino-isobutyric acid; Aoe = (2S,9S)-2-amino-8-oxo-9,10epoxydecanoic acid; Pip = pipecolic acid.

Deprotection of the lateral amino function of lysine was achieved by catalytic hydrogenation, and substitution by one of the alkylating groups was carried out under specific activation conditions using, e.g., 2,4,5-trichlorophenyl-N-fl-chloroethyi-Nnitrosocarbamate for the synthesis of compounds l and 2. Nitrogen mustard derivatives 3 and 4 and amido-epoxy compounds 5, 6, 7 were prepared by in situ activation using the DCC-DMAP mixture as the activation agents. The final products were purified by silica gel column chromatography followed by reversed-phase HPLC and characterized by amino acid analysis and FAB-mass spectrometry. These analyses ascertained the integrity of the grafted lateral group and especially that of the epoxy compounds.

oratory by propagation in female DBA/2 mice by weekly intraperitoneal (IP) passages of ascitic fluid obtained from a 7-dayold ascitic tumor. The P388 tumor cells were harvested under aseptic conditions from the peritoneal cavity of a donor mouse. After dilution with saline, each animal received 1 × 106 leukemic cells in 0.1 ml by the IP route. The day of tumor cell inoculation was defined as day 0. The inoculated mice were randomized, with eight mice per treated group plus one control group of 20 animals. The peptide 1 was suspended in saline containing 5% DMSO while the reference compound BCNU and the positive control 5-fluoro-uracyi were dissolved in 0.9% NaC1 solution. Doses were expressed in mg per kg of body weight and dissolved in 0. I ml/kg body weight. The drugs were administered IP as a single injection on day 1. The control mice received the vehicle at 0.1 ml/10 g body weight by the same IP route. Mice were observed daily at the same time, and deaths were recorded. The antitumor activity was estimated according to the NCI protocol (4). Table 4 expresses the number of long-term survivals in each treated group on day 60 (final evaluation day), and the T/C% calculated from the median survival time of treated and control mice as follow:

T/C % = Med.S.T. (days) of treated mice (T)/ Med.S.T. (days)of control mice (C) × I00.

RESULTS

Biological Activity In in vitro experiments, the inhibitory concentration (IC50) of the inhibitor was determined after 48-h incubation of L 1210 cells at 37°C and photometrical determination of the viable cells in the microculture tetrazolium assay (I). Table 3 expresses this ICso (~M) for the most active compounds 1, 2, and 3. In vivo experiments were carried out on hybrid female mice B6D2Fj (C57 BI/6 × DBA/2) weighing 20 _+ 2.5 g. They were supplied by Iffa Credo (France) and kept under standard conditions (food and tap water ad lib, 12-h dark-light cycle). The P388 leukemia was obtained from the tumor Bank of the National Cancer Institute (NCI) and maintained in our lab-

A first screening of the in vitro activity on a L1210 cell line showed no effect on proliferation of compound 4 and of the epoxy-containing compounds 5 to 7 (results not shown). The cytotoxicity of compounds 1 to 3 is compared in the L1210 in vitro assay to that of the reference bis-~-chloroethylnitrosourea (BCNU) in Table 3. Cyclopeptide 1 exhibits a tenfold higher activity than BCNU and more than twentyfold higher than the best HC toxin analogue 2. Compound 1 was therefore investigated further in an in vivo P388 leukemia model (4). The results, summarized in Table 4, demonstrate a high in vivo activity of compound 1 (considerably higher than that of 5-fluoro-

TABLE 2 STRUCTURE OF THE SYNTHETIC ANALOGS R or X

ChlamydocinAnalog

Cpd R

I

R = --CO--N(NO)CH2CH2CI

c [Aib--Phe--D--Pro--Lys]

c [Aib--Phe--D--Pro--L~s]

!

l

R = --CO--CHmCH2

c [Aib--Phe--D--Pro--Lys]

\o / X= --CO--CH--CH--CO

\0 /

c [Aib--Phe--D--Pro--Lys]

I

X

I

c [Aib--Phe--D--Pro--Lys] Cpd = Compound number.

c [D--Pro--Ala--D--AlamLys] R

3

R

I

Cpd R

R R = --CO~"-N(CH2CH2C1)2

HC ToxinAnalog

c [D--Pro--Ala--D--Ala--L~s] R

5

I

c [D--Pro~Ala~D~Ala--Lys]

ANTITUMORAL CYCLIC PEPTIDE ANALOGUES OF CHLAMYDOCIN TABLE 4

TABLE 3 INHIBITORY POTENCY ON LI210 CELL LINE IN VITRO

IN VIVO ANTILEUKEMIC ACTIVITY OF 1 IN MICE INJECTED

WITH P388 CELLS Dose

IC5o+-SE (aM)

Compound

Compound 1

2 3 BCNU

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0.84 ___0.10 26.4 +-_5.2 43.2 +__8.2 9.2 +_ 1.0

1 1 1 1

1 1 BCNU 5-FU Control

BCNU = bis-~-chloroethyl nitrosourea.

uracil used as a positive control). At similar molarity (128 and 143 aM, respectively) compound 1 and BCNU have comparable activity with a T/C ratio of 600 and 500, respectively. No sign of toxicity is detectable for compound 1 under these conditions, since no loss of weight is seen and the number of survivals at day 60 is even higher (six out of eight) for 1 than for BCNU. However, the doses expressed in mg/kg are higher for 1 than for BCNU and further increase of the doses is limited by insolubility of 1.

DISCUSSION The lack of in vitro activity of a-amido-epoxydic compounds 5-7 could be explained by the loss of their alkylating property as compared to the a-keto-epoxydic natural compounds. Modification by reduction of chlamydocin leading to 8-dihydrochlamydocin also involves a drastic decrease of activity (IC50 = 5000 ng/ml) (3). In this case also, the epoxy-ring, which bears a weaker withdrawing a-substituent, would be less prone to nucleophilic attack by the biological target. From the in vitro experiments, it can be concluded that the peptide sequence of the nitrosourea analogues 1 and 2 plays an important role in target recognition, since the biological response is higher (about 20 times) for the chlamydocin compared to the HC toxin analogue, as this was the case for the natural parent

mg/kg

aM/kg

5 10 20

8 16 32

40 60 80 30 80 --

64 96 128 143 610 --

A Weight(g) D (1-4)

T/C (%)

+1.3 +0.9 +0.8 + 1.3

128 142 150 195

+0.4 0 -0.6 0 + 1.4

229 >600 500 164 100

Survivals at I)60 0/8 0/8 0/8

0/8 1/8 6/8 4/8 0/8 0/20

BCNU = bis-/~-chloroethylnitrosourea;5-FU = 5-fluoro-uracil. compounds [about 10 times (11)]. This marked effect is not likely to be due to a conformational difference of the backbone. According to NMR studies by Shute et al. (7), the two natural peptides exhibit the same conformation and the same solventinduced variation with respect to the amide bonds, going from aU-trans (T4) in CDCI3 to cis-(trans)3 in DMSO-Dr. Furthermore, the sequence imino acid Ape (D-Pro-APe, DPip-Ape, or Pip-Ape) appears to favor cytotoxic activity, in contrast to the corresponding sequence of HC toxin, which tends to confer phytotoxicity (11). The main result of this study was obtained in combining the favorable structure of a cyclotetrapeptide derived from chlamydocin with a classical nitrosourea alkylating substituent, giving rise to a potent antiproliferative agent showing important activity in vivo. Although a direct comparison with chlamydocin as used in the original work (8) was not possible, due to unavailability and instability of the keto-epoxide-containing compound, the in vivo results on P388-injected mice suggest a much higher efficiency (ILS 100% to 500%) of the new analogue 1. However, the fact that due to insolubility, the full dose-activity curve could not be measured means that efforts to enhance efficacy should not only concentrate on potency increase but also on better solubility of the analogues.

REFERENCES

1. Alley, M. C.; Scuderio, D. A.; Monks, A.; el al. Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res. 48:139-144; 1990. 2. Barany, G.; Merrifield, R. B. Solid-phase peptide synthesis. In: Gross, E.; Meienhofer, J., eds. The peptides, analysis, synthesis, biology, vol. 2. New York: Academic Press; 1980:1-284. 3. Close, A.; Huguenin, R. Isolation and structure elucidation ofchlamydocin. Helv. Chim. Acta 57:533-545; 1974. 4. Geran, R. I.; Greenberg, N. H.; McDonald, M. M.; Schumacher, A. M.; Abott, B. J. Protocols for screening chemical agents and natural products against animal tumors and other biological systems. Cancer Chemother. Rep. 3:1-100; 1972. 5. Itazaki, H.; Nagashima, K.; Sugita, K.; et at. Isolation and structural elucidation of new cyclotetrapeptides, trapoxins A and B, having detransformation activities as antitumor agents. J. Antibiot. 43:15241532; 1990. 6. Liesch,J. M.; Sweeley,C. C.; Staffeld,G. D.; Anderson, M. S.; Weber, D. J.; Scheffer, R. P. Structure of HC-toxin, a cyclic tetrapeptide from Helminthosporium carbonurn. Tetrahedron 38:45-48; 1982.

7. Shute, R. E.; Kawai, M.; Rich, D. H. Conformationally constrained biologically active peptides: Tentative identification of the antimitogenic bioactive conformer of the naturally occurring cyclic tetrapeptides. Tetrahedron 44:685-695; 1988. 8. Staehelin, H.; Trippmacher, A. Cytostatic activity ofchlamydocin, a rapidly inactivated cyclic tetrapeptide. Eur. J. Cancer 10:801-808; 1974. 9. Takayama, S.; Isogai, A.; Nakata, M.; Suzuki, H.; Suzuki, A. Structure of Cyl-l, a phytotoxic peptide produced by Cylindrocladiurn scoparium. Pept. Chem. 21:203-208; 1983. 10. Umehara, K.; Nakahara, K.; Kiyoto, S.; et at. Studies on W F-3161, a new antitumor antibiotic. J. Antibiot. 36:478-483; 1983. 11. Walton, J. D.; Earle, E. D.; Staehelin, H.; Grieder, A.; Hiroda, A.; Suzuki, A. Reciprocal biological activity of the cyclic tetrapeptides chlamydocin and HC-toxin. Experientia 41:348-350; 1985.