J. Med. Chem. 1992,35, 2007-2014
2007
Development of Potent and Selective CCK-A Receptor Agonists from Boc-CCK-4: Tetrapeptides Containing Lys(Nf)-AmideResidues Kazumi Shiosaki,* Chun We1 Lin, Hana Kopecka, Richard A. Craig, Bruce R. Bianchi, Thomas R. Miller, David G. Witte, Michael Stashko, and Alex M. Nadzan Neuroscience Research Division, Dept. 47H, Abbott Laboratories, Abbott Park, Illinois 60064. Received November 26,1991 A series of Boc-CCK-4 derivatives represented by the general structure Boc-Trp-Lys(N'-COR)-Asp-Phe-NH2, where R is an aromatic, heterocyclic, or aliphatic group, are potent and selective CCK-A receptor agonists. These amide-bearing compounds complement the previously described urea-based tetrapeptides (Shiosaki e t al. J. Med. Chem. 1991,34,2837-2842); structure-activity studies revealed parallel as well as divergent trends between these two series. A significant correlation was observed between pancreatic binding affinity and the resonance constant R of the phenyl substituent in one particular series of derivatives. Sulfation of phenolic amides appended onto the €-aminogroup of the lysine did not affect affinity for the CCK-A receptor in contrast to the 500-fold increase in binding potency observed upon sulfation of CCK-8, suggesting that the lysine appendage and the sulfated tyrosine in CCK-8, both key structural elements that impart high affinity for the CCK-A receptor, are interacting differently with the receptor. The amide-bearing tetrapeptides are full agonists relative to CCK-8 in stimulating pancreatic amylase release while being partial agonists in eliciting phosphoinositide (PI) hydrolysis. Both effects were blocked by selective CCK-A receptor antagonists. BOC-Trp-NH Cholecystokinin (CCK), originally isolated from porcine y CO-Asp(Nh4e)Phe-NH, intestine as a 33-amino acid peptide, is distributed widely in peripheral tissues and is responsible for a number of P Me I physiological actions including gallbladder contraction and secretion of pancreatic enzymes.'P2 CCK is found also in the central nervous system (CNS) and studies suggest that it may have a role there as a neurotransmitter or neuromodulators3 Although multiple molecular forms of CCK 1 have been identified,the full spectrum of biological activity In addition, many of the tetrapeptides in this series are can be elicited potently by the C-terminal octapeptide full agonists in stimulating pancreatic amylase secretion CCK-8 (AspTyr(S03H)-Met-Gly-Trp-Met-Asp-Phe-NHJ. Sulfation of tyrosine-27 (based on CCK-33 numbering) is a critical feature of the octapeptide for potent biological (1) Mutt, V. Cholecystokinin: Isolation, Structure and Functions. activity since the desulfated analogue CCK-8-DS (AspIn Gastrointestinal Hormones; Glass, G. B. J., Ed.; Raven Press: New York, 1980; pp 169-221. Tyr-Met-Gly-Trp-Met-Asp-Phe-NHz) is roughly 500-fold (2) Williams, J. A. Cholecystokinin: A Hormone and a Neuroweaker in binding to the peripheral receptor. In addition, transmitter. Biomed. Res. 1982, 3, 107-121. the C-terminal tetrapeptide Boc-CCK-4 (Boc-Trp-Met(3) Morley, J. E. The Ascent of Cholecystokinin (CCK) - From Asp-Phe-NH2) is able to elicit the peripheral actions of Gut to Brain. Life Sci. 1982,30,479-493. CCK-8 but is over 1000-fold less potent in binding to the (4) Lin, C. W.; Bianchi, B. R.; Grant, D.; Miller, T.; Danaher, E. A.; Tufano, M. D.; Kopecka, H.; Nadzan, A. M. Cholecystokperipheral r e ~ e p t o r . ~ inin Receptors: Relationships Among Phosphoinositide The predominant CCK receptor found in peripheral Breakdown, Amylase Release and Receptor Affinity in Pantissues such as the pancreas haa been labeled CCK-A? and creas. J. Pharmacol. Exp. Ther. 1986,236, 729-734. ita activation is responsible for the secretory and contractile (5) Dourish, C. T.; Hill, D. R. Classification and Function of CCK actions attributed to CCK as well as its anorexogenic efReceptors. Trends Pharmacol. Sci. 1987,8, 207-208. fects through activation of receptors on peripheral vagal (6) Smith, G. P.; Jerome, C.; Norgren, R. Afferent Axons in Abafferent fiberseB The CCK receptors in the CNS are ladominal Vagus Mediate Satiety Effect of Cholecystokinin in Rats. Am. J. Physiol. 1985,249, R638-R641. beled CCK-B6and possess similar ligand binding charac(7) Freidinger, R. M. Cholecystokinin and Gastrin Antagonists, teristics as the peripheral gastrin receptor. These two CCK Med. Res. Rev. 1989,9, 271-290. receptors can be differentiated by their relative affinity (8) Makovec, F.; Chiste, R.; Bani, M.; Pacini, M. A,; Setnikar, I.; for CCK and ita fragments as well as a number of synthetic Rovati, L. A. New Glutaramic Acid Derivatives with Potent ligands, both peptides and nonpeptides, that are highly Competitive and Specific Cholecystokinin-antagonistic Activity. Arrneim-Forsch. 1985, 35(Z0, 1048-1051. selective for the CCK-A receptor, including MK-329 (de(9) Lin, C. W.; Holladay, M. W.; Witte, D. G.; Miller, T. R.; vazepide),' CR-1409: and A-71378,9 and those that are Wolfram, C. A. W.; Bianchi, B. R.; Bennett, M. J.; Nadzan, A. selective for the CCK-B receptor, including CI-998,loLM. A-71378 a CCK Agonist with High Potency and Selectiv365,260," and A-63387." ity for CCK-A Receptors. Am. J. Physiol: Gastrointest. Liuer We recently reported a novel series of Boc-CCK-4 dePhysiol. 1990,258, G648-G651. rivatives in which the methionine was replaced with a (10) Horwell, D. C. Development of CCK-B Antagonists. Neuropeptides 1991,19, 57-64. lysine whose t-amino group had been incorporated into (11) Bock, M. G.; DiPardo, R. M.; Evans, B. E.; Rittle, K. E.; various aromatic and aliphatic ureas.13 In contrast to Whitter, W. L.; Veber, D. F.; Anderson, P. S.; Freidinger, R. Boc-CCK-4, which is 70-fold more selective for the CCK-B M. Benzodiazepine Gastrin and Brain Cholecystokinin Rereceptor, these tetrapeptides are potent and selective ceptor Ligands: L-365,260. J. Med. Chem. 1989,32, 13-16. agonists for the CCK-A receptor. A-71623 (I), a prototypic (12) Nadzan, A. M.; Garvey, D. S.; Tufano, M. D.; Holladay, M. W.; urea-bearing tetrapeptide, possesses high affinity (ICw = Shiosaki, K.; Shue, Y.K.; Chung, J. Y. L.; May, P. D.; May, C. S.; Lin, C. W.; Miller, T. R.; Witte, D. G.; Bianchi, B. R.; 3.7 nM) for the CCK-A receptor and is greater than Wolfram, C. A. W.; Burt, S.; Hutchins, C. W. Design of Cho1000-fold selective over the CCK-B receptor (Table I).14
HNyNHb
* Address for correspondence: Cardiovascular Research Division, Dept. 47V AP-10, Abbott Laboratories, Abbott Park, IL 60064. 0022-2623/92/1835-2007$03.00/0
lecystokinin Analogs with High Affinity and Selectivity for Brain CCK Receptors. In Peptides: Chemistry and Biology, Proceedings of the 12th American Peptide Symposium, Cambridge, MA, June 16-21,1991; Smith, J. A., Rivier, J. E., Eds.; Escom: Leiden, pp 100-102.
0 1992 American Chemical Society
Shiosaki et al.
2008 Journal of Medicinal Chemistry, 1992, Vol. 35, No. 11 Table I. Binding and Functional Data of Boc-CCK-4 and A-71623
ICw, nMapb ECw, nMaVc PId pancreas cortex amylase % max 2600 f 950 (5) 40 1800 f 630 (5) 25 f 4.5 (6) BN-CCK-4 0.39 f 0.06 (12) 100 3.7 f 0.85 (8) 4500 f 770 (4) A-71623 Number of determinations is indicated in parentheses. Each determination was conducted in duplicate with 10,000 3e 4-OMe 193-194 CGHs7N7O1o-0.5H~0 450 (1) 68 f 7.4 (3) 9900 (1) C,H,N 250 f 36 (6) 3f 4-F 114-124 CuHMN70$"HzO 19000 (1) agonist' 77 (1) C,H,N 390 f 76 (8) 1W110 CGHMN70$',.0.5HZO 3g 4-CF3 240 (1) 62 f 10 (3) 3h 4-Me C,H,N 290 f 100 (3) -10000 193-195 C45H67N708 C,H,N 160 f 48 (4) 4400 (2) 300 (1) 3i 3,4-di-OH 143-145 CaH,N7011.H20.AcOH 44 (2) Compounds gave satisfactory analyses within f0.4% of theoretical calculations. *Number of determinations is indicated in parentheses. Each determination was conducted in duplicate with 6%sample variability. Means f SE are indicated for those compounds with three or more determinations. cICMwas determined as the concentration of the peptide that inhibited 50% of the specific binding of [1251]BH-CCK-8 in each tissue. dEC, was determined in the amylase release assay as concentration of peptide that produce 50% of maximal response. 'Indicates percent response of peptide at concentration of 10"' M in PI hydrolysis relative to maximal response elicited by CCK-8. fIndicates the peptide elicited release of amylase at a concentration of 30 uM.
the receptor or the greater contributions for binding provided by the other residues in CCK-8 upon sulfation. Other substituents were incorporated at the 4-position of the phenyl ring in the hydrocinnamoyl compound. In general, these derivatives (3d-3h) exhibited comparable pancreatic receptor affinities as the parent compound 3a with the exception of the 4-methoxy analogue 3e whose potency was comparable to the 4-hydroxy example 3c. Results and Discussion Incorporation of disubstitution, exemplified by the 3,4dihydroxy compound 3i, yielded analogues of comparable As reported, the development of this novel series of binding potencies to 3c. All the hydrocinnamoyl derivaCCK-A selective tetrapeptides was initiated by our finding that a CCK-4 derivative, Boc-Trp-Lys(Cbz)-Asp-Phe-NHz, tives maintained selectivity for the CCK-A receptor and were demonstrated to exhibit full agonist activity in stimhad modest affinity (ICw = 510 nM) for the pancreatic ulating amylase release and partial efficacy in PI hyreceptor but, in contrast to Boc-CCK-4, was 10-fold sedrolysis. lective for the CCK-A receptor. Lack of biological activity Further improvement in CCK-A receptor affinity was by the free amino (Boc-Trp-Lys-Asp-Phe-NHz)and the obtained upon constraining the ethylene bridge in 3a by N-acetyl (Boc-Trp-Lys(Ac)-Asp-Phe-NHz) derivatives substituting with a cinnamoyl residue to produce 3j (Table suggested that an appropriate appendage extending from 111). A 4- to 6-fold increase in binding potency was rethe lysine camino group was necessary for CCK-A receptor alized in most of the cinnamoyl derivatives (31-3r) over recognition. The hydrocinnamoyl derivative 3a, prepared their saturated counterparts (3b-3h). As in the hydroas an isosteric replacement of the Cbz group, had similar cinnamoyl series, hydroxyl substitution at the 4-position potency for the CCK-A receptor as the original carbamoyl produced the most potent CCK-A receptor ligand 3m over compound (Table 11). Substitution at the 2- or 3-position the isomeric derivatives 3k and 31. Sulfation of the pheof the phenyl ring in the hydrocinnamoyl moiety with nolic derivatives 31 and 3m to produce 4c and 4d, rehydroxyl, e.g. 3b, had little effect on binding while the spectively, had little or no effect upon binding affinity, 4-hydroxyl derivative 3c improved pancreatic affinity over further supporting the notion that the phenolic group of 3a by 2-fold. This improvement in binding potency sugthe tetrapeptides and the tyrosine in the octapeptides are gested that the phenolic group extending from 3c might interacting differently with the CCK-A receptor. Other be interacting in a similar manner within the CCK-A resubstituents at the Cposition revealed compounds (3n-3u) ceptor as the phenolic group of tyrosine-29 in CCK-8. that were generally equipotent or slightly less potent than Should such a common means of interaction be involved, the parent cinnamoyl derivative 3j. In contrast, substithen sulfation of the phenolic group in the hydrocinnamoyl tution with methyl and chloro in the phenylurea series derivatives would be expected to significantly improve its resulted in a 7-fold improvement in binding affinity over affinity for the CCK-A receptor as had been clearly esthe unsubstituted derivative.13 A number of disubstituted tablished for CCK-8. Sulfation of the isomeric phenols cinnamoyl derivatives was also investigated. The binding produced 4a and 4b whose affinity for the peripheral reaffinities of most of the disubstituted compounds (3v-32) ceptor was improved by roughly 2-fold over their respective were comparable to that of the parent cinnamoyl derivative unsulfated analogues, far less than the 500-fold improve3j, although the 2,4-dichloroanalogue 3x yielded a 2-fold ment observed upon sulfation of CCK-8. This lack of a weaker compound. As in the previous series of compounds, significant enhancement in binding potency upon sulfation the cinnamoyl tetrapeptides exhibited binding selectivity suggests that the phenols of the lysine residue and the for the CCK-A receptor and showed agonist activity in tyrosine of CCK-8 are interacting differently with the reboth functional assays. ceptor, possibly through interactions at distinct sites on phosphoinositide (PI) breakdown in guinea pig pancreas have been de~cribed.~ The compounds were evaluated at concentrations between and lo4 M in the amylase assay and at lo-' M for the PI assay. Each determination was performed in duplicate with less than 5% sample variability. Additional determinations were conducted on selected compounds.
Shiosaki et al.
2010 Journal of Medicinal Chemistry, 1992, Vol. 35, No. 11
Table 111. Physical Properties and Biological Data of Cinnamoyl and Substituted Cinnamoyl Tetrapeptides 3 and 4 Boc-Trp-NH -CO-Asp-Phe-NH,
0
IC,, nMbvc ECw, nmbsd PIb,' no. R mp, "C formula anal: pancreas cortex amylase release % max 33 H 203-205 CUH~~N~O~*ACOH C,H,N 55 f 20 (3) 1800 f 190 (3) 30 (2) 52 (2) 135-138 CuH53N,010*1*5H20 3k 2-OH 210 (1) 210 (1) 54 (2) C,H,N 110 f 35 (7) 202-205 C44H53N7010 C,H,N 22 f 4.8 (8) 31 3-OH 970 (1) 59 (2) 54 f 10 (3) 203-204 CUH53N7010 3m 4-OH C,H,N 16 f 3.3 (6) 730 f 140 (4) 3.9 (2) 76 (2) 165-170 CUH53N7013S*NH,OH*2H20 C,H,N 39 f 13 (3) 4c 3-OSO3H 238 (2) 25 (2) 70 f 7.3 (4) 4a 4-OSO3H 470 f 54 (6) 7.2 f 2.2 (3) 72 4.7 (4) C,H,N 13 f 3.9 (7) 166-169 CuHaN70&NH,OH 3n 4 4 1 157-169 CuH62N,OgCl*HZO 2900 (2) 79 (2) 66 (2) C,H,N 71 f 49 (3) 125-130 C15H6SN7010'2H20 C,H,N 30 f 9.1 (3) 30 4-OMe 1700 f 96 (4) 20 (2) 72 f 8.7 (4) 132-149 C,H,N 65 f 24 (5) 1700 f 190 (5) 19 (2) 74 f 6.0 (3) 3P 4-F .. 220 f 56 (3) 131-139 60 (1) 3q 4-CF3 71 f 26 (4) 128-135 3r 4-Me 5300 1500 f (1)180 (3) 55 52 iij (2) 71 f 4.5 (3) 140 f 42 (4) 138-148 5100 f 440 (3) 38 (2) 72 f 10 (4) 3s 4-Br 120 f 36 (4) 2100 f 190 (3) 17 f 3.0 (3) 3t 4-N(Me) 181-185 49 (1) 145-152 57 f 19 (8) 4400 (1) 64 f 6.3 (4) 3u 4-NO2 27 (2) 3v 3,4-di-OH 164-166 33 f 6.9 (3) 66 f 5.8 (3) 750 (2) 45 (2) 49 f 7 (7) 3w 3,4-di-C1 145-156 5000 46 (2) 62 (2) 191-193 3x 2,4-di-C1 90 f 18 (3) 130 f 71 (6) -5000 86 3.5 (3) 40 f 19 (3) 61 f 5.8 (7) 4000 (2) 3Y 3.4-di-OMe 178-185 17 (2) 32 2,4-di-OMe 136-139 C,H,;N;O;;-0.6H20 C,H,N 38 f 26 (8) -3000 14 (2) 82 f 4.1 (3) Compounds gave satisfactory analyses within f0.4% of theoretical calculations. Number of determinations is indicated in parentheses. Each determination was conducted in duplicate with lo000
agonist
19 (2)
176-205
C44H61N709'H20
C,H,N
310f 86 (3)
-1OOOO
agonist
40 f 6.9 (7)
129-138 CaHaN709.0.5AcOH. HZO
C,H,N
57 f 17 (4)
4300 (1)
79 (2)
61 f 5.2 (6)
C,H63N709*0.5HzO
C,H,N
14 f 2.7 (7)
2700 (2)
4.0 f 1.4 (3)
36 (2)
CaH~N7010*1.5HZO
C,H,N
4.5 f 0.96 (4)
C ~ H ~ N 7 0 1 3 S ~ N H ~ OC,H,N H* 2H20
2.9 f 0.38 (2) 8.0 f 1.7 (5)
1200 f 130 (4) 3.5 f 0.81 (5) 50 f 7.9 (4) 300 (2)
3.5 (2)
49 f 6.1 (7)
1400 f 140 (4) 11 (2)
49 f 6.0 (8)
3ge
134-135 CIBHw"011*H20
C,H,N
3hh
143-145 C1SH6ZN809
C,H,N
33 f 2.2 (3)
200 (2)
39 (2)
46 f 4.7 (3)
3ii
158-161 C,SH61N8010'H20
C,H,N
7.7 f 2 (8)
670 (1)
2.3 (2)
45 f 6.2 (7)
155-170 C,HszN8Og.1.5HzO
C,H,N
39 f 10 (4)
2100 f 190 (4) 20 (2)
18 f 4.0 (3)
158-160 C~H61N8010.1.4AcOH. HZ0
C,H,N
14 f 4.1 (6)
OH
3jj
fl H
3kk
830 (1)
68 (2)
23 f 6.5 (6)
16 f 6.2 (3)
21 f 6.0 (5)
62 (2)
27 f 6.9 (6)
38 (2)
60 f 6.0 (5)
30 (2)
55 f 6.7 (7)
H
129-134
311
-3000
H
3mm
HC'
136-139 CUH61N8OgC1.2HzO
C,H,N
110 f 62 (5)
129-135 C,H52N80g*1.5HzO
C,H,N
21 f 6.5 (3)
147-151 CUHMN80g*1.5HzO
C,H,N
19 f 3.8 (8)
5200 (1)
H
3nn 300
3PP
H
204-206
87 f 17 (3) 4700 (1)
840 f 110 (3) 20 (2)
66 f 6.7 (5)
Compounds gave satisfactory analyses within +0.4% of theoretical calculations. Number of determinations is indicated in parentheses. Each determination was conducted in duplicate with
