1366
J. M e d . Chem. 1987, 30, 1366-1373
Synthesis and Biological Activities of Pseudopeptide Analogues of the C-Terminal Heptapeptide of Cholecystokinin. On the Importance of the Peptide Bonds Marc Rodriguez, Marie-FranGoise Lignon, Marie-Christine Galas,? Pierre Fulcrand, Christiane Mendre, Andre Aumelas, Jeanine Laur, and Jean Martinez* Centre d e Pharmacologie-Endocrinologie,34094 Montpellier, Cbdex, France. Received December 22, 1986
A series of pseudopeptide analogues of the C-terminal heptapeptide of cholecystokinin in which each peptide bond, one a t a time, has been replaced by a CH2NH bond were synthesized: Z-Tyr(S03-)-Nle-Gly-Trp-Nle-Asp$(CHzNH)Phe-NH2 (11, Z-Tyr (SO,-) -Nle-Gly-Trp-Nle$( CH2NH)Asp-Phe-NH2 (2), Z-Tyr (SO,-)-Nle-Gly-Trp$(4), Z-Tyr(SO,-)-Nle$(CH2NH)Nle-Asp-Phe-NHz (3), Z-Tyr(SO3-)-Nle-Gly\(CH2NH)Trp-Nle-Asp-Phe-NH2 (CH2NH)Gly-Trp-Nle-Asp-Phe-NHz(5), Z-Tyr(SO3-)-Met-Gly-Trp-Nle-Asp$(CH2NH)Phe-NH2 (6), Z-Tyr(S03-)-Met-Gly-Trp-Nle$(CHzNH)Asp-Phe-NH2 (7), Z-Tyr(SO3-)-Met-Gly-Trp$(CH2NH)N1e-Asp-Phe-NH2 (8). These derivatives were studied for their ability to stimulate amylase release from rat pancreatic acini and to inhibit the binding of labeled CCK-9 to rat pancreatic acini and to guinea pig brain membrane CCK receptors. They were All of these pseucompared to the potent CCK-8 analogue Boc-Asp-Tyr(S03-)-N1e-Gly-Trp-Nle-Asp-Phe-NH2. dopeptides were able to stimulate amylase secretion with the same efficacy as CCK-8 but with varying potencies. These compounds were also potent in inhibiting the binding of labeled CCK-9 to CCK receptors from rat pancreatic acini and from guinea pig brain membranes.
Cholecystokinin is a 33 amino acid peptide first isolated peptides we have synthesized were protected on their from hog intestine,l where it stimulates gastrointestinal N-terminus by a benzyloxycarbonyl group (Z) because it motility and induces both gall bladder contraction and had already been demonstrated that analogues of the pancreatic amylase Structure-activity relaC-terminal heptapeptide of CCK bearing an Nu protecting tionship studies4 on cholecystokinin have shown that the group were somewhat more potent and more stable than full spectrum of CCK-like biological activities can be rethe heptapeptide analogues with a free a-amino group.14 produced by the C-terminal heptapeptide of CCK of The replacement of a peptide bond by a CH2NH bond structure Tyr(S03-)-Met-Gly-Trp-Met-Asp-Phe-NHz, In does not really affect the conformation of the pseudoaddition, CCK-8 was found in the brain: where it functions peptide as compared with that of the parent peptide.26 as a neurotransmitter and neurom~dulator.~~’ We recently showed the importance of the peptide bonds in the C-terminal tetrapeptide of gastrin for exhibiting Ivy, A. C.; Oldberg, E. A m . J. Physiol. 1928, 86, 599-613. biological activity on acid secretion and for binding to Jorpes, J. E.; Mutt, V. Handb. Exp. Pharmakol. 1972, 34, gastric mucosal cells.8 Replacing a peptide bond by a 1-179. CHzNH bond in the C-terminal tetrapeptide of gastrin led Harper, A. A.; Raper, H. S. J. Physiol. (London) 1943, 102, to compounds having a high apparent affinity for the 115-125. gastrin receptor but behaving either as gastrin agonists or Jensen, R. T.; Lemp, G. F.; Gardner, J. D. J. Biol. Chem. 1982, antagonists on acid secretion, depending on which peptide 257, 5554-5559. bond was replaced. Gastrin and cholecystokinin have the Dockray, G. J.; Gregory, R. A.; Hutchison, J. B.; Harris, J. I.; Runswick, M. J. Nature (London) 1978, 274, 711-713. same C-terminal pentapeptide amide fragment (-GlyTrp-Met-Asp-Phe-NH2),and they overlap in their bioEmson, P. C.; Marley, P. D. In Handbook of Psychopharmacology; Iversen, L. L., Iversen, S. D., Snyder, S. H., Eds.; Plelogical activities but are modulated in their potency by num: New York, 1983; Vol. 16, pp 255-306. different N-terminal extension^.^^^^ In order to evaluate Dodd, P. R.; Edwardson, J. A,; Dockray, G. J. Regul. Peptides the importance of the peptide bonds in the active fragment 1980, 1, 17-29. of CCK (e.g., the C-terminal heptapeptide, CCK-7), we Martinez, J.; Bali, J. P.; Rodriguez,M.; Castro, B.; Magous, R.; synthesized five pseudopeptide analogues of CCK-7 in Laur, J.; Lignon, M. F. J . Med. Chem. 1985, 28, 1874-1879. which each peptide bond, one at a time, has been replaced Holland, J.; Hirst, B. H.; Shaw, B. Peptides 1982, 3, 891-895. by a CHzNH bond, “reduced peptide bond”,ll e.g., ZMagous, R.; Martinez, J.; Lignon, M. F.; Bali, J. P. Regul. Tyr(S03-)-Nle-Gly-Trp-Nle-Asp$(CH2NH)Phe-NH2 (l), Peptides 1983,5, 327-332. Z-Tyr(S0,)-Nle-Gly-Trp-Nle$(CH2NH)Asp-Phe-NH2 (21, Szelke, M.; Leckie, B.; Hallett, A,; Jones, D. M.; Sueiras, J.; Z-Tyr(S03-)-Nle-Gly-Trp$(CH,”)Nle-Asp-Phe-NH2 (3), Atrash, B.; Lever, A. F. Nature (London)1982,299,555-557.
Z-Tyr(S0~)-Nle-Gly$(CH2NH)Trp-Nle-Asp-Phe-NH2 (4),
Gacel, G.; Ruiz-Gayo, M.; Durieux, C.; Charpentier, B.; Men-
and Z-Tyr(S03-)-Nle$(CH2NH)Gly-Trp-Nle-Asp-Phe-NH2 ant, I.; Begue, D.; Roques, B. P. In Forum Peptides; Castro, (5). In these pseudopeptide analogues of the C-terminal B., Martinez, J., Eds.; 1984; pp 137-139. heptapeptide of CCK, for easier syntheses and better Laur, J.; Rodriguez, M.; Aumelas, A.; Bali, J. P.; Martinez, J. I n t . J. Pept. Protein Res. 1986, 27, 386-393. stability of the ensuing compounds, the two methionine Bodanszky, M.; Natarajan, S.; Hahne, W.; Gardner, J. D. J. residues (Metz8and Met31) have been replaced by two Med. Chem. 1977,20, 1047-1050. norleucines. This change has been shown in many cases Rodriguez, M.; Lignon, M. F.; Galas, M. C.; Mendre, C.; Marnot to influence the biological activities of the anatinez, J. Pept., Proc. Eur. Pept. Symp., 19th, in press. 1 0 g u e s . ~ ~ As J ~ supplementary examples, we synthesized compounds Z-Tyr(S0,-)-Met-Gly-Trp-Nle-Asp+- Ferhentz, J. A.; Castro, B. Synthesis 1983, 676-678. (CHzNH)Phe-NH2(6), Z-Tyr(S03-)-Met-Gly-Trp-Nle$- Jacobson, K. A,; Marr-Leisy,D.; Rosenkrmz, R. P.; Verlander, M.; Melmon, K. L.; Goodman, M. J . Med. Chem. 1983, 26, (CH2NH)Asp-Phe-NH2(7), and Z-Tyr(S0,-)-Met-Gly492-499. Trp$(CHzNH)Nle-Asp-Phe-NHz(8), in which only Met Castro, B.; Dormoy, J. R.; Dourtoglou, B.; Evin, G.; Selve, C.; in position 31 has been replaced by Nle. The pseudoZiegler, C. Synthesis 1976, 751-752. + This work was partly supported by ARI Chimie-Biologie through a grant to M.C.G. (FPRCB).
0022-2623/87/1830-1366$01.50/0
Moroder, L.; Wilschowitz, L.; Jaeger, E.; Knof, S.; Thamm, P.; Wuensch, E. Hoppe-Seyler’s Z. Physiol. Chem. 1979, 360, 787-790. American Chemical Society
Journal of Medicinal Chemistry, 1987, Vol. 30, No. 8 1367
Analogues of the C-Terminal H e p t a p e p t i d e of CCK Scheme I Trp
X
Tyr
Nle
ASP 9
Boc
Phe
OBut
N( Me )OMe
Boc
"2
OH
osu
Y Y
Z
X-Nle 20 H so,, q z B a
- osu
X-Nle Z
X-Met 24
X=Met 6 =,A
Scheme I1
Boc
OB21 26 .OSU X-Nle,Y-Z 14 X=Met,Y-Bcc31 Y OH
z
X-Nle,Y=Z 17 X-Met,Y=Boc :!2 Y OSU
H
X-Nle,Y-Z 29 X-tdet,Y-Boc 31 Y -. X-Nle 30 Z+OSu
H-
4
H
/ "2
OB21 27 /
"2
28 "2
"E
X=Met 32
SO ,H
"2
"E
However, as a peptide bond induces some rigidity to the molecule, its replacement by a CHzNH bond induces some
motional freedom by the possibility of rotation around this "reduced bond". On the other hand, this modification
(20) Jensen, R. T.; Lemp, G. F.; Gardner, J. D. Proc. Natl. Acad. S C ~U.S.A. . 1980, 77, 2079-2083. (21) Pelaprat, D.; Zajac, J. M.; Gacel, G.; Durieux, C.; Morgat, J. L.; Sasaki, A.; Roques, B. P. Life Sci. 1985, 37, 2483-2490. (22) Lignon, M. F.; Galas, M. C.; Rodriguez, M.; Laur, J.; Martinez, J. J . B i d . Chem., in press.
(23) Chang, R. S. L.; Lotti, V. J.; Monaghan, R. L.; Birnbaum, J.; Stapley, E. 0.; Goetz, M. A.; Albers-Schonberg, G.; Patchett, A. A.; Liesch, J. M.; Hensens, 0. D.; Springer, J. P. Science (Washington,D.C.)1985, 230, 177-179. (24) Galas, M. C.; Lignon, M. F.; Rodriguez, M.; Mendre, C.; Fulcrand, P.; Law, J.; Martinez, J. Am. J . Physiol., submitted.
1368 Journal of
Medicinal Chemistry, 1987, Vol. 30, No. 8
Rodriguez et al.
Scheme 111 TYr
X
Gly
TrP
Nle
ASP
Phe
X=Nle,Y=Z 17 Y. X-Nle,Y-Z 37 Y
2
X-Nle 38 H.
- osu
SO,, i/20a X=Nle
X-Met 8
z SO ,I3
Scheme I V Tyr
z -r osu
introduces into the molecule a new possible charge that might be of some importance for the interactions with the receptor.
Chemistry The syntheses of the pseudopeptides prepared in this work are summarized in Schemes I-V. The aldehyde of the N-protected amino acid obtained from the corre~~
(25) Rodriguez, M.; Bali, J. P.; Magous, R.; Castro, B.; Martinez, J. Int. J . P e p t . Protein Res. 1986,27, 293-299. (26) Aumelas, A.; Rodriguez, M.; Heitz, A,; Castro, B.; Martinez, J. Int. J . P e p t . Protein Res., submitted.
sponding N,O-dimethylhydroxamateaccording to Ferhentz and Castro16 was allowed to react with the a-amino function of the protected amino acid or peptide, in the presence of NaBH,CN as reducing reagent in a methanol/acetic acid mixture,17 to yield the corresponding pseudopeptide with a CHzNH bond.8 Elongation of the peptide chains was performed stepwise or by fragment condensation using active ester derivatives as acylating agents and isobutyl chloroformate or BOP as coupling reagent.18 During the activation of pseudodipeptide derivatives (e.g., 42) for coupling or during condensation of dipeptide aldehydes (e.g., 11)) no racemization can occur by the oxazolone mechanism. N-(Benzyloxycarbonyl)-L-
Journal of Medicinal Chemistry, 1987, Vol. 30, No. 8 1369
Analogues of t h e C - Terminal Heptapeptide of CCK Scheme V N le
Tyr
I
I
-11
-10
I
-9
-
I
-8
TrP
G W
I
-7
1
I
-6
I
I
-5
Nle
I
ASP
I
Phe
I
1
-4
Agonist (log M 1
Agonist (tog M I
Figure 2. Ability of compounds 1-8 to inhibit binding of labeled Figure 1. The effects of B O C - [ N ~ ~ ~ ~ , N ~ ~( 0~ )~and ]-C of C K - ~1251-BH-CCK-9to rat pancreatic acini. Binding of Bolton-Hunter pseudopeptides Z-Tyr(S03-)-Nle-Gly-Trp-Nle-Asp$(CH2NH)-CCK-9 (that was identical to that of BH-CCK-8 from Amersham) Phe-NHz (1) (m),Z-Tyr(SO,-)-Nle-Gly-Trp-Nle$(CH,NH)Asp-was performed as described by Jensen et aLZ0Acini were incuPhe-NHz (2) (*), Z-Tyr(SO
