J. Med. Chem. 1993,36, 166-172
166
CCK-B Agonist or Antagonist Activities of Structurally Hindered and Peptidase-Resistant Boc-CCK4 Derivatives P. J. Corringer, J. H. Weng, B. Ducos, C. Durieux, P. Boudeau,? A. Bohme,' and B. P. Roques* Unit6 de Pharmacochimie Mol4culaire et Structurale, U266 INSERM- URA 01500 CNRS, UniversitO R e d Descartes, UFR des Sciences Pharmaceutiques et Biologiques, 4, avenue de I'Observatoire, 75270 Paris Cedex OS, France, and Rhbne Poulenc Rorer S. A,, Centre de Recherche de Vitry-Alfortville, 13, quai Jules Guesde-BPl4, 94403 VitrylSeine Cedex France Received July 17, 1992
Replacement of Met31 by (N-Me)Nle in CCKa or CCK, has been shown to improve the affinity and selectivity for CCK-B receptors. In order to obtain molecules with enhanced bioavailability, two novel series of protected tetrapeptides of the general formula Boc-Trp30-X-A~p-Y~~ have been developed. Introduction of (N-Me)Nleand the bulky, aromatic naphthylalaninamide (Nal-NH2) in positions X and Y, respectively, does not greatly modify the affinity for guinea pig brain CCK-B receptors. In contrast, incorporation of hindering N-methyl amino acids such as (N-Me)Phe, (N-Me)Phg, or (N-Me)Chg, but not their non-methylated counterparts, in position X induced a large decrease in affinity for the CCK-B binding sites. Among the various peptides synthesized, Boc-[(N-Me)Nle31,1Nal-NH2331CCK, (2) (KI = 2.8 nM), B O C - [ P ~ ~ ~ ~ , ~ N ~ ~ (15) - N(KI H= ~~~ICC& 14 nM), and B o c - [ P ~ ~ ~ ~ , ~ N ~ ~ - N ( C H (17) ~ )(KI ~ W= C39KnM) , displayed good affinities for brain CCK-B receptors and had goodselectivity ratios. These pseudopeptides, in which the presence of unnatural and hydrophobic residues is expected to improve their penetration of the central nervous system, were shown to be very resistant to brain peptidases. Interestingly, whereas compounds2 and 15 proved to be full agonists for rat hippocampalCCK-B receptors when measured in an electrophysiological assay, compound 17 behaved as a potent antagonist in the same test and displayed a good affinity in rat brain KI(CCK-B) = 5 1 nM as compared to the Merck antagonist L365,260,KI(CCK-B)= 12 nM. This illustrates a simple means to obtain CCK-B antagonists and suggests that the free, CONH2 group plays a critical role in the recognition of the agonist state of brain CCK-B receptors. brain barrier as well as being resistant to enzymatic Introduction degradation. The peptide hormone cholecystokinin (CCK) first So far the best CCK-B agonists have been obtained by isolated from hog intestine' has been shown to stimulate modifying the peptide backbone of CCK8. We have many gastrointestinal functions such as gall bladder previously reported that N-methylation of Nle31in Boccontraction and pancreatic secretion.2 In 1975,the CCKa (BDNL), an analogue equiactive with C-terminal fragment, 26Asp-Tyr(S03H)-Met-Gly-Trp- [Nle28~3~]CCK7 CCK8,16did not modify the affinity for the CCK-A and Met-As~Phe-NH2~, was found to be present in the brain3v4 CCK-B receptors. Introduction of a retro-inverso bond where it seems to function as a neuromodulator and/or between residues 28 and 29 in this peptide led to BC 26417 neurotransmitter (for review see ref 5). Several studies (Boc-Tyr(S03H)-gNle-mGly-Trp-(N-Me)Nle-Asp-Phehave demonstrated that there are at least two classes of NH2) which behaves as a highly potent and selective CCK receptors,Bthe CCK-B receptor-type, which is very CCK-B agonist.18 Moreover, BC 264 was shown to be abundantin the central nervous system,7*8andthe CCK-A receptor-type, which is primarily present in the periphery highly resistant to peptidases17 and capable of entering but which is also found in some discrete brain region~.~J~ the brain in ita intact form after systemic administrati~n.~~ These receptor types can be differentiated by the relative Recently, Hruby et aL20described that the analogue Aspaffiiities of the tetrapeptide CC& which interacts with Tyr(S03H)-(N-Me)Nle-Gly-Trp-(N-Me)Nle-Asp-Pheabout loo0 times higher affinity for the CCK-B receptofl NH2 displays high affinities for both classes of receptor while the octapeptide CC& binds with nanomolar affinand that removal of the N-terminal part of this compound ities to both receptors. The biochemistry and pharmayields a pentapeptide (Gly-Trp-(N-Me)Nle-ASpPhe-NHz) cology of brain CCK are currently being intensively studied with nanomolar affinityand high selectivity for the CCK-B as this neuropeptide could have potential therapeutical receptors. Likewise, Nadzan et incorporated, transinterest in many fields such as analgesia,1l anxiety,12 or 3-n-propyl-~-proline, an even more constrained analogue neuropsychiatric disorders.l3 In particular, in humans of norleucine in Boc-CC& and showed that this modiCC& has been shown to induce panic attacks following fication has a similar beneficial effect on CCK-B receptor systemic inje~ti0n.l~ However, owing to the peptidase binding as the introduction of a N-methylated norleucine. susceptibility and the hydrophilicity of the tetrapeptide, Furthermore, several studies in our laboratory have shown its primary site of action, central or peripheral, remains that incorporation of a Phe31 residue in BDNL induces a an open question.l6 Further investigation of this phe55-fold selectivity for the CCK-B receptor22while intronomenon requires highly potent and selective CCK-B duction of an (Z)OmSlmoiety in BDNL or Boc-CC& leads agonists and antagonists, capable of crossing the bloodto CCK antagonists.22*23 In addition, while it appears difficult to modify the Trpm and Asp32 residues without To whom correspondence should be addressed. Tel: (33)43-26a loss of biological a~tivity,2~#~5 the C-terminal amino acid 60-46. FAX: (33)43-26-69-18. + Rh6ne Poulenc Rorer S.A. Phe-NH233has been successfully substituted by bulky ~~
~
~~
@ 1993 American Chemical Society
CCK-BReceptor Activity of Boc-CCK, Derivatives Scheme I
-
-
BOC A#p(OBd) + H-1Nd NH, , HCI
Journal of Medicinal Chembtry, 1993, Vol. 36,No.1 167
hydrogenation of Boc-(N-Me)Phg-OHwae very slow under the conditions used (45 psi, 46 OC, R02 catalyst). Therefore, Boc-(N-Me)Phg-OH was deprotected with trifluoroacetic acid, hydrogenated, and then protected using di-tert-butyl dicarbonate to give Boc-(N-Me)ChgOH. Resultr and Discussion
-
-
-
-
Boc (N-MdMc hp(OBr1) l N d NH,
- -
-
-
-
Boc Trp (N-Mc)Nlc hp(OBs1) 1Nd NH,
- -
- -
-
Boc Trp (N-MdNlc h p Mal NH,
amino acids." The removal of Phe-NHP in CC& and derivativesled to an antagonist for both classes of binding sitesn*28whereas removal of the CONH2 group was reported to induce a partial agonist activity for the peripheral CCK-A receptors.m Furthermore, a hydrazide analogue of CC& (Boc-TrpLeu-AspPhe-NH-NH2)was shown to behave as a full antagonist for CCK-A receptors but as a partial agonist for CCK-B receptors.go Taken together, these results show that voluminous amino acids can be introduced in positions 31 and 33 and that, depending on the nature of the side chains, an activation or blockade of CCK receptors can be obtained. In the preeent paper, we deacribe the synthesis and biological activity of Boc-CC& and Boc-[(N-Me)Nles11CC& analoguesmodified in position31and 33,which were designed with the aim of obtaining small CCK-B-selective ligands which are easy to synthesize and are endowed with good stabilities and bioavailabilities. Chemistry The synthesis of all analogues was performed as described for the preparation of compound 2 in Scheme I. Elongation of thepeptide chainswas performed stepwise using DCC/HOSu, BOP, or p-nitrophenol ester/HOBt methods. For the difficult coupling reactions, i.e., condensation of acids with N-methylamino peptides, either BOP or p-nitrophenol ester/HOBt methods were used. The amino protecting groupe Boc and Z were respectively removed with trifluoroacetic acid and by catalytic hydrogenolysis. The carboxylic acid protecting group benzylic ester was removed by catalytic hydrogenolysis. The naphthylalanine analogues H-1Nal-NH2, H-2NalN H 2 , and H-1Nal-NHCHs were obtained by esterification of the carboxylic acid function of the commercial producta H-3-(l-naphthyl)-Ala-OH(H-1Nal-OH) or H-3-(2-naphthy1)-Ala-OH (H-2Nal-OH) by thionyl chloride, followed by amidificationwith ammonia or methylamine. H-1NalN(CH& was synthesized by coupling Z-1Nal-OH with dimethylamine followed by hydrogenolysie of the Z protecting group. Boc-(N-Me)Phg-OH was prepared by the method described by McDermott and Benoiton,8l by reaction of Boc-Phg-OH with iodomethane in the presence of sodium hydride in THF. Boc-Chg-OH was prepared by hydrogenationof H-Phg-OH followed by protection of the amino group with di-tert-butyl dicarbonata. For the synthesis of Boc-(N-Me)Chg-OH,it was found that direct
The aim of thie study was .to develop new small CC&related ligands endowed with good affiiitiea and eelectivities for CCK-B receptors and improved bioavailabilities. The compoundssyntheeized were evaluated for their potency in displacing PH]pCC& from guinea pig pancreatic and brain membranes, and the results are shown in Table I. Ae expected, replacement of Gly in Gly-Trp-(N-Me)Nle-Asp-Phe-NH2 by a Boc group did not modify the higher preferential interaction of thie peptide for CCK-B sites.20 Compound 1 was at least 3 orders of magnitude more potent at B than at A biding sitee. The methylation of the peptide bond between residues 30 and 31 in compound 12 led to a 4fold increase in CCK-B binding affinity. In the c o w of structure-activity etudies on BDNL analogues, it was found that Phe-NHza3 can be replaced by convenient bulky residues, particularly l-NalNH2, without causing alteration in biological activity and specificity." Accordingly, l-Nal-NHa or 2-Nal-NH2 were introduced as C-terminal residues leading to peptidm 2 and 4 which retained the bindingprofile of their precursor, in spite of a slight decrease in affiiity for the CCK-B receptor (3.5- and 6.4-fold, respectively, as compared to compound 1). 1-Nal-NH2residue was chosen to replace Phe-NH2 in almost all the synthesizedcompounds, due to ita hydrophobicity and ita noncoded nature which was expectedto improve the resistance to degradingenzymes. Indeed, the peptidase sensitivity of CC& and compound 1 and 2 was inveetigated using crude rat brain membranea (Figure1). It was found that whereas CC& haa a half-life of only 5 min, introduction of a N-methyl residue in position 31 (compound 1) improves the resistance to peptidases, and additional incorporation of lNal in position 33 (compound 2) almost totally protecta the peptide from degradation. In our earlier studies on BDNL analogues,azwe showed that introduction of a Phe residue in position 31 induced a &fold selectivity for CCK-B receptors without great change in CCK-B affinity. In the case of compound 14, thb substitution led to a 22-fold decrease in affiity and a lower selectivity for central binding sites as compared to compound 12. However, introduction of othw cyclic amino acids in this position, such as phenylglycine in IS and cyclohexylglycine in 16, led to compounds displaying affiities in the nanomolar range for the CCK-B receptor (14 nM and 17 nM for 15 and 16, reepectively) and are therefore only about 5 times leae potent than their parent compound 12. Therefore, to further increase the affiity for CCK-B binding sites,the corresponding N-methylaminoacids (NMe)Phe, (N-Me)Phg,and (N-Me)Chgwere introduced in position 31. Surprisingly,these modifications induced a large decrease in CCK-B binding affinity since S is about 740-fold lese potent than 1. Similarresulta were obaerved with compounde7,8, and 9 when compared to 2. Moreover, all the N-methylated peptides are 10 timm lam potent than their unmethylated counterparta (compare 7,8, and
168 Journul of Medicinul Chemistry, 1993, Vol. 36, No. 1
Com'nger et al.
Table I. Apparent AtYhities (KI, M) of CCKe and Derivativee 1-18 on the Binding of PH1pCCK.gto the Brain (KD= 0.18 nM) and Pancreatic Manbranee (KD= 1.22 nM) of Guinea Pig KI, (MIa brain (CCK-B) pancream (CCK-A) 0.3 & 0.01 X 1o-B 0.7 & 0.04 X 10-0 0.8 0.1 x 1o-B >6 X 1od 1 2.8 & 0.6 X 10-0 1.2 0.1 x 1od 2 3.6 & 0.1 X 10-8 6.3 & 0.7 X lod 3 6.1 1.1 X 10-9 4 4.2 1.0 X 1od 5.9 0.6 x 10-7 3.2 0.3 X lod 6 3.0 0.6 X 10-8 8.0 0.5 X lod 6 7.7 1.7 x 10-7 3.6 & 0.8 X 1od 7 1.7 f 0.6 X lW7 8 8.3 0.8 x 10-7 1.4 & 0.3 X lW7 2.1 0.3 X 1od 9 6.8 0.1 X 10-8 1.2 0.1 x 1od 10 8.1 2.0 x 10-8 5.2 0.4 X 11 3.2 0.3 X 1o-B 2.9 & 0.1 X lod 12 1.6 0.1x 10-7 1.1 0.1 x lo+ 13 7.1 & 0.8 X 10-8 14 2.4 0.2 X 1od 1.4 & 0.1 X 10-8 16 9.9 0.6 x 10-7 1.7 0.3 X 10-8 1.4 0.3 X 1od 16 3.9 1.0 x 10-8 17 1.0 0.4 X 10-6 7.4 0.2 x 10-8 6.4 & 1.4 X 1od 18 a The KIvalues repreeent the meane & SEM of three mparata experimentaeach performed in triplicata. The values of the Hill coefficients were close to 1in all experiments. compoundn
no.
* ** * ** *
O
h 0
100
200
T.min
Figure 1. Time c o w for the hydrolysie of CC& analogues by rat brain homogenate. Substrates (WM)were incubated for varying timee at 35 O C with crude membrane fra&ione of rat brain (3 mg protein/mL). (W) CC&, (A) compound 1, ( 0 ) compound 2, (A)compound 17. Resulta are the mean of two separate experiments.
Swith 14,15, and 16,respectively). All these N-methylated analogues show the presence of the cis/trans isomerism around the Trp-(N-Me)Nle bond found in 1, excluding the poeeibility that the lose in affmity is due to the absence of the biologically active conformer. Furthermore, the proton of the amide bond between residues 30 and 31does not play a critical role for CCK-B receptor recognition as shown by the high affinity of compound 1. To further increase the lipophilicity of compounds 2 and 12, the C-terminal amide group w a ~replaced by a mono or dimethylamide function since, in the case of BDNL, this modification was shown to produce only a small loas in CCK-B receptor affinity (1.00 nM for Boc[Nle28*31,Phe-NH(CHa)331CCK, versus 0.13 nM for BDNL).= In the present series, the reduction in affmity was greater in compounds 10 and 11 (around 2630-fold as compared to that for compound 2), but remained in the same range in the corresponding non-methylated peptides 17 and 18 which are only 3-4 times less potent than their parent compounds for the CCK-B binding sites.
* **
** * **
The steric constraint caused by N-methylation of aromaticor cyclic residues in position 31, particularlywhen aesociated with a large C-terminalnaphthylalanineresidue, therefore seems to be responsible for the large reduction in CCK-B affinity, probably by driving the bulky phenyl or cyclohexyl rings towards the excluded volume of the receptor subsite. Inversion of the configuration of T r p m in the partial agonist Boc-Tyr(S03-)-Met-Gly-TrpNle-Aep-2-phenylethyl esteS2 led to a full CCK-A antagonist while in the peptoid antagonist PD 134308,99the presence of a Da-methyltryptophan residue inetead of the corresponding L amino acid led to a 100-fold higher affmity for the CCK-B receptor. In contrast, the intre p position 30 in compounds 2,6, and duction of a ~ T r in 12 led to an important decrease in affmity for the CCK-B receptor (13-, 5- and 50-fold decrease for compounds 3,6, and 13 as compared to their parent compounds). Among the peptides synthesized,compounds 2,15, and 17 display good affinities for the CCK-B receptors. However, these derivatives are modified in positione which are crucial for the activation of CCK binding sites. Their agonist/antagonist profile was thus investigated using a CCK-B selective electrophysiological test in which CC& and CCK-B agonista have been shown to stimulate the firing rate of rat CA1 hippocampal neurones in a doeedependent manner.18*s( As illustrated in Figure 2, compound 2 actedas a full agonist, showingthat N-methylation of Me3' and substitution of Phe-NHzWby 1Nal-NHam did not modify the activity of the peptides at the CCK-B receptors. Compound 1S also acted as a full CCK-B agonist, showing that Nle31 can be usefully replaced by Phg. In contrast, compound 17 waa unable to enhance the firing rate of the hippoca~npalneurones but antagonized, in a dose-dependent manner, the stimulation induced by CC& (Figures 3 and 4). In all caees the pharmacological potenciesof the peptides were relatively well-correlated with their affinities for CCK-B receptors. The opposing pharmacological activities of compounds 15 and 17 strongly suggest that bia-methylation of the NH2 terminus is responsible for the antagonist propertiea of 17, either through a steric hindrance or by eliminating possible hydrogen bonds between the CONHa group and appropriately located donor and acceptor groups in the
,/~urnalof Medicinal Chemistry, 1993, Vol. 36, NO.1 168
CCK-B Receptor Actiuity of Boc-CCK, Deriuotiuee I
Table 11. Apparent Affinities (KI, nM) of the Antdgonht 17 and L 586,280 on the Binding of [aHlpCC& to the Brain Membrana of Guinea Pig and Rat
In
‘0 c
~
KI(nMY compounds
guinea pig
rat 17 39 10 61 h 7 L366,280 2.2 & 0.6 12*3 “e KI values represent mean f SEM of three separate experimenta each performed in triplicate. The value of the Hill coefficients were close to 1 in all experiments.
-
CCK8
Cpnd. 2
Cpnd. 15
Figure 2. Agonist activity of compounds 2 and 15. Excitatory responses of hippocampal CCK-sensitive neurons to various concentrations of compound 2 (narrow-hatched bars) and compound 15 (wide-hatched bars) expressed relative to the response to 5 x 10-7 M CC& (cross-hatched bars). Note the concentrationdependent excitatory effect of both compounda.
Figure 3. ~ p o n s e ofs a hippocampal neuron to CCKe in the presence of increeeingconcentrations of compound 17. Segmenta of fvingrate recording to a CCK-sensitive CAl-neuron. (a) CC& (6x 10-8 M) alone (control); (b) CCKe + compound 17 (1o-B M); (c) CC& + compound 17 (10-8 M); (d) CCKe + compound 17 ( W M ) ;(e) CC%+ compound 17 (lWM). Note the progressive decline in excitatory responses to CCKe. Calibration bars: vertical, 20 spikesIs; horizontal, 5 min.
-
properties between peptide and non-peptide compounds. In addition,as observed for compound 2, the modificatione introduced in positions 31 and 33 in compound 17 almost fully protect this peptide from degradation (Figure 1). In conclusion, the modificationsperformed in position 31 and 33 in Boc-CC& provided potent and selective CCK-B agonists and antagonista. The noncoded nature of the residues incorporated significantly improves the protection of these peptides to degradation, while their high hydrophobicity is expected to improve their penetration into the brain. This feature is being studied in our laboratoryby in vivo binding techniques.gs One compound of the series proved to be a potent and specific CCK-B antagonist. The best CCK-B antagonists reported to date are the benzodiazepine derivativeL365,26OF7the peptoid PD 134,308t3 the ureidoacetamide RP 69758,= and the diphenylpyrazolidinone LY 262864.s9 The peptidomimetic CCK-B antagonist 17 developed in this work displays a good affmity for CCK-B receptors, especially in the rat where it is almost as potent as L365,260 and is highly protected from enzymatic degradation. A comparison of the structure of these molecules using NMR and molecular modeling could permit the characterizationof the essential structural components for the blockade of brain CCK-B binding sites. This work is now in progress in our laboratory.
Experimental Section
a“
9
8
7
6
5
4
- log [antagonist] (M) Figure 4. Antagonist activity of compound 17. Mean f SEM (n = 2-4 neurons per point) excitatory responses of rat hippocampal neurons to CC& (6 X 10-8 M) in the presence of various concentrations of compound 17 ( 0 )or L366,260 (B). Estimated half-maximal effective concentrations were 3.2 X 10-8M and 2.2 X M, reapectively.
receptor. This avoids, or at least strongly reduces, the possibility that the ligand w i l l fit the agonist state of the CCK-B receptor. The presence of both a Phg and a 1NalN(CHa)Z residue could also contribute to the potent antagonist activity of 17. This compound was also highly potent in displacing [?HlpCC& from rat brain membranes (Table II) with an affmity similar to that found in guinea pig brain. In contrast, the non-peptidic antagonist L365,260,s which is potent in the guinea pig, displays a 6-fold lower affinity for the rat CCK-B receptors. Therefore, 17 is only 5 times lees potent than L365,260 in the rat. However, this difference in potency appears slightlylarger in an electrophysiological assay since the EDm were 3.2 X 10-6 M for 17 and 2.2 X lom7M for L366,260 (Figure 4). This feature could be due to different pharmacokinetic
The following compounds were prepared as described in the literature: Boc-(N-Me)Nle-OH, H-Asp(OBzl)-Phe-NHz, H-NleAsp(OBzl)-Phe-NHrTFA, and Boc-Trp-(N-Me)Nle-Asp(OBzl)Phe-NH2;l7 H-1Nal-OMeHCl and H-1Nal-NHz.HC1.z6 All other amino acids were from Bachem. Solventa were from SDS. Chromatography wee carried out with Merck silica gel (230-400 meah). For thin-layer chromatugraphy (TLC), Merck platee precoated with F254 silica gel were used with the followingsolvent system (byvolume): A, CHzClzMeOH (956); B, CHlClrMeOH (9:1);C, EtOAcCHzClrMeOH-H!&AcOH (227:30.60.3); D, CHzClrMeOH-HzCbAcOH (R30.60.3). Plateswere developed with UV,iodine vapor, ninhydrin, or Ehrlich’s reagent.. The structure and the lack of racemization of the f i i compounds and all intermediatee were confiied by ‘H NMR spectroecopy (Brucker WH, 270 MHz) in DMSO-&. The purity of all final compounds wee checked by HPLC (Shimatzu apparatus) on a 250 X 4.6 m m Kromad C8 5-pm column with a mixture of CHaCN (solvent A) and HnOITFA 0.06% (solvent B) as eluent (flow rate, 0.8 mL/min) with W detection (214 nm). Molecular maas wee obtained by 262 Cf-plasma desorption maas spectroscopy on a Bio-Ion 20 time of flight instrument. Melting pointa were taken on a Kofler apparatus and are given uncorrected. T h e following abbreviations have been ueed: DMF, dimethylformamide: DCC, NJv’-dicyclohexylcarbodiimide; DCU, NN-dicydohexylurw HOSU, N-hydroxpuccinimide; HOBt, l-hydroxybenzotrhole; DIEA, N,”-diiaopropylethylamine; BOP, (benzotriezol-1-yloxy)trie(dimethylamino)phosphonium hexafluorophosphak, TFA, trifluoroacetic acid; H-1Nal-OH, 3-(1-naphthyl)alaniie; H-2NdOH, 3-(2-naphthyl)alanine. Other abbreviations used are thou, recommended by the IUPAC-IUB Commission (Biochem J. lSW, 219, 346).
Com'nger et al.
170 Journal of Medicinal Chemietry, 1993, Vol. 36,No. 1 Table 111. Physical and Analy&icalData of Peptides 1-18 Used in the Biological Testa
mp, "C 132-136 131-133 136-138 142-144 130-134 123-126 199-200 156-168 144-146 124-126 170-172 208-212 146147 202-206 116120 226226 143-146 130-132
Rf
HpLct~,min(A/B) 9.2 (54:46) 13.4 (M46) 12.6 (64:46) 13.6 (64:46) 9.3 (M46) 9.1 ( a 4 6 1 8.6 (80:40) 11.6 (64:46) 9.6 (60:40) 10.2 (80:40) lf7(80:40) 9.0(61:49) 8.8 (61:49) 13.9 (61:49) 9.6 (54:46) 7.8 (W40) 8.9 (80:40) 11.4 (60:40)
0.23 (C) 0.38 (C) 0.43(C) 0.31 (C) 0.36 (C) 0.42(C) 0.37 (C) 0.39 (C) 0.28 (C) 0.42 (C) 0.26 (C) 0.21 (C) 0.43(C) 0.34 (C) 0.20 (C) 0.31 (C) 0.43 (C) 0.30 (C)
Table IV. Physical and Analytical Data of the Synthetic Peptide Derivatives mp,OC 120-122 99-101 161-163 61-63 6647 78-81 101-103 84-86 Oil
75-77 89-91 67-59 68-61 50-62 199-201 166-168 Oil
201-203
FAB/MS(MH+) found 693 693 743 743 743 743 743 743 727 727 727 727 777 777 763 763 769 769 767 767 771 771 679 679 679 679 763 763 749 749 766 766 777 777 783 783
calcd
Rt
d.C,H,N
anal. C,H,N
0.50 (A)
0.30 (A) 0.28 (A) 0.50 (A)
0.27 (A)
0.54 (A) 0.73 (C)
0.63 (A) 0.71 (C) 0.48 (A)
0.61 (B) 0.63 (A) 0.61 (A) 0.46 (B) 0.63 (A) 0.69 (A) 0.28 (A) 0.59 (A) 0.80 (D) 0.50 (A) 0.54 (A)
72-74 Synthesis. The peptides described herein were synthesized according to the general procedures detailed in this section for the preparation of compound 2. Particular aspects of the syntheses could be obtained from the authors. Analytical and physical data of the synthesized compound are reported in Tables I11 and IV. Boc-Arp(OBzl)-lNal-NH2(19): To a solution of H-1NalNHrHCl(3 g, 11.9 "01) and Boc-Asp(OBzl)-OH (3.87g, 11.9 "01) in DMF (30mL), cooled to 0 OC, were added successively EtgN (1.68mL, 11.9 mmol), HOSU (1.38g, 11.9mmol), and DCC (2.95g, 14.3 "01). T h e mixture was stirred for 1 h at 0 OC and overnight at room temperature. After fiitration of DCU, the solvent was evaporated. T h e residue was dissolved in EtOAc, DCU was fiitered off, and the organic solution was washed with 10% aqueous citric acid, 1 N aqueous NaHCOs, water, and brine, dried over Nad304, and concentrated to yield 5.61 g (90%) of a white solid. Bos-(N-Me)NlaA8p(OB~l)-lNal-NH~ (20): Compound 19 (6.46g, 10.6 "01) was dissolved in TFA (20mL) and stirred for 1 h at 0 OC and for 3 h at room temperature. After evaporation of the solvent, the oily residue was precipitated with dry ether to yield 4.92 g Of H-Aep(OBzl)-lNal-NHP.TFA (88%). "hb compound (450mg, 0.84 mmol) was dissolved in DMF (4mL). The solution was cooled to 0 OC and treated successively with EtgN (117 pL, 0.92 mmol), Boc-(N-Me)Nle-OH (207 mg, 0.84 mmol), HOSU (100 mg, 0.84 mmol), and DCC (190 mg, 0.92 "01). T h e mixture was stirred for 1 h at 0 OC, overnight at
0.52 (A)
room temperature and was worked up as deecribed for the preparation of compound 19 to yield 373 mg (68%) of a white powder. Boc-Trp(N-Me)NlaAsp-1Nal-NHI(2): Compound 20(340 mg, 0.62 "01) was dissolved in T F A (4mL) and stirred for 1 h at 0 OC and for 1 h at room temperature. Aftar evaporation of the solvent, the oily residue was precipitated with dry ether to yield 290 mg (81%) of H-(N-Me)Nle-Asp(OBzl)-lNal-
NHTTFA. This compound (160mg,0.23 "01) was dissolved in DMF (2mL). The solution was cooled to 0 "C and treated successively with DIEA (88pl, 0.51 m o l ) , Boc-Trp-OH (70mg,0.23 mol), and BOP (112mg,0.26 "01). The mixture WM stirred for 1 h at 0 OC and overnight at room temperature. Aftar evaporation of the solvent, the residue was diesolved in EtOAc, washed sequentially with 10% aqueous citric acid, 1 N aqueous N a H C h water, and brine, dried over N 4 0 4 , concentrated, and p d e d by chromatography using CH2ClpMeOH (97:3)as eluent to yield 118 mg (61%) of Boc-Trp-(N-Me)Nle-Aep(OBzl)-lNal-~*. Thiscompound(86mg,O.11mmol) washydmgenatedinMeOH (6mL) in the presence of 10% Pd/C catalyst (10mg) for 10 h. After filtration and evaporation of the solvent, the d d w wm purified by chromatography using EtOAcCH&l&¶eOH-H& AcOH (20:7:3:0.60.3) as eluent to yield 56 mg (74%) of a white solid. Boc-(N-&)Phq-OH (26): Boc-Pk-OH (3g, 12 -01) and methyl iodide (6mL, 96 "01) were dissolved in dry THF (56 mL), and the solution was cooled to 0 OC in a flask protected
CCK-B Receptor Activity of Boc-CCK, Derivatives from moisture. Sodium hydride dipersion (1.08-mg, 36 "01) was added cautiously with gentle stirring. The suspension was stirred at room temperature for 16h. EtOAc (12 mL) was added, followed byH@ (12mL) dropwise. Thesolution was evaporated to dryness and the residue partitioned between ether and water. The ether layer was washed with 10% aqueous NaHco3, and the combined aqueous extracts were acidified to pH = 2 with 10% aqueous citric acid. The product was extracted into EtOAc and the extract was washed with water, 5% aqueous NaAOs, water, and brine, dried over HgSO4, and evaporated to yield 3 g (94%) of a pale yellow solid. Boc-(N-Me)Chg-OH (27): Compound 26 (2.1 g, 7.92 "01) was dissolved in "FA (10 mL) and stirred for 1h at 0 O C and 3 h at room temperature. After evaporation of the solvent, the residue was precipitated with dry ether to yield 2.05 g (91% ) of in H-(N-Me)Phg-OHSTFA. This compound (2.0 g, 7.17 "01) HoO (40mL) and AcOH (10 mL) was treated with platinum oxide (720 mg)and placed under an atmosphere of hydrogen at 50 psi and 45 "C for 24 h. After filtration of the catalyst, the filtrate was concentrated to yield 1.55 g (75%) of H-(N-Me)Chg-OH.TFA. To a solution of this compound (1.5 g, 6.26 "01) in HnO (5 mL) and dioxane (10 mL), cooled to 0 OC, were added E m (1.84mL, 13.15 "01) and di-tert-butyl dicarbonate (1.72 g, 7.89 "01). The mixture was stirred for 1 h at 0 OC and overnight at room temperature. After evaporation of the solvent, the residue was dissolved in 5%aqueous NaHCG, washed with ether, acidified to pH 2 with 10% aqueous citric acid, and extracted with EtOAc. The extract was washed with water, brine, dried over Nafio4, and concentrated to yield 600 mg (42%) of an oil. H-lNal-NH(CH,) (29): To a solution of H-1Nal-OMe-HCl (500mg,2.2 "01) in ethanol (10 mL) was added a solution of 33%NHzCHs in ethanol (2.2 mL). The mixture was refluxed for 24 h. After evaporation to dryness, the residue was purified by chromatography in CHzClrMeOH (956) to yield 150 mg (30%) of a white solid. H-lNal-N(CHs)t (32): To a solution of ZlNal-OH (4.55 g, 12.6 "01) in DMF' (25 mL), cooled to 0 O C , were added successively H-N(CHs)THCl(1.03g, 12.6 mmol), Et& (1.77 mL, 12.6 mmol), HOSU (1.45 g, 12.6 mmol), and DCC (2.86 g, 12.6 "01). The mixture was stirred for 1 h at 0 O C , overnight at room temperature and was worked up as described for the preparation of compound 19 to yield 3.66 g (72%) of ZlNalwas hydrogenated N(CH& This compound (4.55 g, 12.6 "01) in MeOH (20 mL) in the presence of 10% Pd/C catalyst (500mg) overnight at room temperature. After filtration and evaporation of the solvent, the residue was purified by chromatography with CH&lrMeOH (91) as eluent to yield 1.69 g (76%) of a white solid. Boc-Chg-OH (37): H-Phg-OH (400 mg, 2.65 mmol) in 40 mL of AcOH-EtOH-H20 (2:7:1) was treated with platinum oxide (260 mg) and placed under an atmosphere of hydrogen at 50 psi at 45 OC for 24 h, and then filtered and evaporated to yield 350 mg (84%) of H-Chg-OH. This compound (330 mg, 2.10 mmol) was treated as described for the preparation of 27 to yield 500 mg (94%) of an oil. Binding Assays. [3H]pCC&, 60 Ci/mmol, was purchased from Amersham. Membrane preparation and binding experiments with [sH]pCC& (0.2 nM) .were performed as described previ~usly.~~ Briefly, incubations (final volume 1 mL) were carried out in 50 mM Trii-HC1buffer, pH 7.4,5 mM MgC12, and 0.2 mg/mL bacitracin for 60 min at 26 OC in the presence of brain membranes (0.6 mg protein/mL) or in 10 mM Pipes-HC1buffer, pH 7.4, 30 mM MgC12, 0.2 mg/mL bacitracin, and 0.2 mg/mL soybean trypain inhibitor for 12Ominin the presence of pancreatic membranes (0.2 mg protein/&). Nonspecific binding was determined in the presence of 1 pM CC& Incubations were terminated by fdtration through Whatman CF/B filtersp r m t e d by incubation in buffer containing 0.15% bovine serum albumin. Filters were rinsed with 2 X 5 mL of ice cold buffer and dried and the radioactivity was counted. The& values were calculated using the Cheng-Prusoff equation. Peptide Degradation. Incubation with crude rat brain membranes (3 mg protein/mL) were carried out at 35 OC in 50 mM Trb-HC1, pH 7.4, with lo-' M of substrate in a volume of 500 pL. The reaction was stopped by boiling for 10 min, and
Journal of Medicinal Chemistry, 1999, Vol. 36,No. 1 171 &r addition of acetonitrile (25% v/v) and shnking for 46 s to extract substrates, samples were centrifuged for 10min at 13000g at 4 OC. The supernatants were analyzed by HPLC. Electrophysiology. Hippocampal slicesfrom male SpragueDawley rats were prepared for in vitro electrophysiological recordings as previously described.q In brief, 0.5 mm thick transverse hippocampal sliceswere maintained in a submereiontype recording chamber, and spontaneousaction-potential (AP) discharge frequencyof CAl-neurons was recorded extracelluhly. A fuing rate tracing was obtained by integration of the AP counta over2-48. Compounds2,15,and 17andL365,260weredieeolved in DMF and then diluted in the slice maintenance medium (composition: see ref 40) to the desired final concentration (DMF: 1%maximum). For the study of agonista, non-tachyphylactic CCK-sensitiveneurons were selected by repeated bath applicationsof 5 X lW7M sulfated CC& (CRB,Northwich, U.K.), and medium containing the compounds was then applied for 4 min. The excitatory responses were normalized relative to the last predrug response to C C b . For the study of antagonists,the compounds were applied alone for 10 min on neurone selected with a 10-fold lower concentration of CC&, and then coperfueed together with CC&. Antagonist activity was measured relative to the last CC& response as previously.
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