445
`
`J. Med. Chem. 1989, 32, 445-449
`Full Agonists of CCK8 Containing a Nonhydrolyzable Sulfated Tyrosine Residue
`I. Marseigne, P. Roy, A. Dor, C. Durieux, D. Pélaprat, M. Reibaud,7 J. C. Blanchard,7 and B. P. Roques*
`Département de Chimie Organique, U 266 INSERM, UA 498 CNRS, UER des Sciences Pharmaceutiques et Biologiques, 4
`de VObservatoire, 75006 Paris, France, and Laboratoire Rhone Poulenc Santé, 13 Quai Jules Guesde, B.P. 14,
`avenue
`94403 Vitry-Sur-Seine, France. Received May 16, 1988
`The sulfate ester of CCKje^ or CCK8 (Asp-Tyr(SG3H)-Met-Gly-Trp-Met-Asp-Phe-NH2) borne by the tyrosine residue
`In order to increase the stability of this molecule,
`is a critical determinant of the biological activity of this peptide.
`the sulfated tyrosine has been replaced by a synthetic amino acid (L,D)Phe(p-CH2S03Na) in which the 0S03H group
`was replaced by the nonhydrolyzable CH2S03H group. Both isomers were separated by chromatography and the
`stereochemistry of the Phe(p-CH2S03Na) residue in each peptide was established by NMR spectroscopy. The biological
`activities of the new derivatives Ac[X27,Nle28,Nle31]CCK27-33 were compared with those of Boc[Nle28,Nle31]CCK27_33,
`an equiactive analogue of CCK8 and Boc[D-Tyr(S03Na)27,Nle28,Nle31]CCK27_33. Besides their highly enhanced chemical
`stability, Ac[L-Phe(p-CH2S03Na)27,Nle28,Nle31]CCK27_33 and Ac[D-Phe(p-CH2S03Na)27,Nle28Nle31]CCK27_33 display
`high affinity for peripheral and central CCK receptors (Ki m 10"® M) and proved to be full agonists in the stimulation
`of pancreatic secretion as well as in the in vitro CCK8-induced contractions of the guinea pig ileum.
`Sulfation is the most abundant posttranslational cova-
`2), obtained by the liquid-phase method, were separated
`by chromatography and their structures established by
`lent modification of tyrosine residues found in animal
`NMR spectroscopy, following previously reported meth-
`The sulfate ester group is introduced by
`proteins.1,2
`transfer from S'-phosphoadenosine 5'-phosphosulfate
`ods.20 The binding properties to both guinea pig brain
`and pancreatic membranes and the peripheral activities
`(PAPS), a universal sulfate donor, onto tyrosine residues
`of proteins. This transfer is catalyzed by the enzyme
`(amylase secretion from pancreatic acini and contractile
`potency on ileum) of the new CCK-related peptides were
`Although sulfated
`tyrosylprotein sulfotransferase.3,4
`than 20 years ago to
`compared to those exhibited by the reference molecule
`tyrosine residues were known more
`in a few biological proteins such as fibrinopeptide
`Boc[Nle28,Nle31]CCK27_33 (compound 3) and its stereoiso-
`occur
`B,5 fibrinogens,6 gastrin,7 and cholecystokinin,8 their
`mer Boc[D-Tyr(S03Na)27,Nle28,Nle31]CCK27_33 (compound
`4), which was prepared for comparison.
`presence in a large number of proteins9"11 was not detected
`until recently.1
`Chemistry
`The biological role of protein tyrosine sulfation has been
`Ac(L,D)Phe(p-CH2S03Na) was prepared as already de-
`established only in the case of a few small peptides where
`it has been possible to compare the biological activity of
`the sulfated and the unsulfated forms. For example, the
`hormonal activity of cholecystokinin was shown to be
`dependent on the sulfation of tyrosine since the sulfated
`form was about 250 times more potent than the unsulfated
`one.12 Desulfation of tyrosine also caused a considerable
`decrease in the biological activity of the C-terminal octa-
`peptide of ceruletide.13
`Moreover, in the case of ceruletide, a large decrease in
`biological activity was observed when the 0S03H group
`was substituted by 0P03H2, N02, NH2, and S02NH2
`groups whereas a small activity was retained by compounds
`in which the 0S03H group was
`replaced by S03H and
`In CCK8, substitution of tyrosine
`NHS03H groups.13,14
`O-sulfate by hydroxynorleucine O-sulfate caused only a
`4-fold decrease in potency in the amylase release test
`whereas replacement by serine O-sulfate caused a 1000-fold
`decrease in potency,15"17 showing that the nature of the side
`chain seems to be of relatively minor importance provided
`that its structure allows the positioning of the sulfate ester
`group at a proper distance from the peptide backbone.17
`In order to study the functional importance of tyrosine
`sulfation, it appears to be essential to work with analogues
`of tyrosine which are similar enough to mimic sulfated
`tyrosine residues and which cannot be hydrolyzed at the
`level of the 0S03H group, even in acidic conditions.
`Recently, we described the synthesis of new amino acids
`in which the 0S03H group of the sulfated tyrosine was
`replaced by a CH2P03H2 or CH2S03H group.18
`In this work, we report the introduction of the new
`amino acid Ac(L,D)Phe(p-CH2S03Na) in the sequence of
`the equiactive analogue of CCK8:
`Boc[Nle28,Nle31]-
`CCK27_33.19 The mixture of the two isomers, Ac[L-Phe-
`(p-CH2S03Na)27,Nle28,Nle31]CCK27_33 (compound 1) and
`Ac[D-Phe(p-CH2S03Na)27,Nle28,Nle31]CCK27_33 (compound
`
`(1) Huttner, W. B. Nature (London) 1982, 299, 273-276.
`(2) Huttner, W. B.; Baeuerle, P. A.; Benedum, U. M.; Friederich,
`E.; Hille, A.; Lee, R. W. H.; Rosa, P.; Seydel, U.; Suchanek, C.
`In Hormones and Cell Regulation; Nunez, J., et al., Eds.;
`Colloque INSERM/John Libbey Eurotext Ltd., Paris, 1986;
`Vol. 139, pp 199-217.
`(3) Lee, R. W. H.; Huttner, W. B. J. Biol. Chem. 1983, 258,
`11326-11334.
`(4) Lee, R. W. H.; Huttner, W. B. Proc. Natl. Acad. Sci. U.S.A.
`1985, 82, 6143-6147.
`(5) Bettelheim, F. R. J. Am. Chem. Soc. 1954, 76, 2838-2839.
`( ) Jevons, F. R. Biochem. J. 1963, 89, 621-624.
`(7) Gregory, H.; Hardy, P. M.; Jones, D. S.; Kenner, G. W.;
`Sheppard, R. C. Nature (London) 1964, 204, 931-933.
`(8) Mutt, V.; Jorpes, J. E. Eur. J. Biochem. 1968, 6, 156-162.
`(9) Baeuerle, P. A.; Huttner, W. B. EMBO J. 1984, 3, 2209-2215.
`(10) Liu, M. C.; Lipmann, F. Proc. Natl. Acad. Sci. U.S.A. 1985,82,
`34-37.
`(11) Nachman, R. J.; Holman, G. M.; Haddon, W. F.; Ling, N.
`Science 1986, 234, 71-73.
`(12) Yajima, H.; Mori, Y.; Kiso, Y.; Koyama, K.; Tobe, T.; Setoya-
`ma, M.; Adachi, H.; Kanno, T.; Saito, A. Chem. Pharm. Bull.
`1976, 24, 1110.
`(13) Anastasi, A.; Bernardi, L.; Bertaccini, G.; Bosisio, G.; De Cas-
`tiglione, R.; Erspamer, V.; Goffredo, O.; Impicciatore, M. Ex-
`perientia 1968, 24, 771-773.
`(14) De Castiglione, R., First International Symposium on Hor-
`monal Receptors in Digestive Tract Physiology, INSERM
`Symposium No. 3, Bonfils, et al., Eds.; Elsevier/North-Holland
`Biomedical Press: Amsterdam, 1977.
`(15) Bodanszky, M.; Natarajan, S.; Hahne, W.; Gardner, J. D. J.
`Med. Chem. 1977, 20(8), 1047-1050.
`(16) Bodanszky, M.; Martinez, J.; Priestley, G. P.; Gardner, J. D.;
`Mutt, V. J. Med. Chem. 1978, 27(10), 1030-1035.
`(17) Gardner, J. D.; Walker, M. D.; Martinez, J.; Priestley, G. P.;
`Natarajan, S.; Bodanszky, M. Biochim. Biophys. Acta 1980,
`630, 323-329.
`(18) Marseigne, I.; Roques, B. P. J. Org. Chem. 1988, 53, 3621.
`(19) Ruiz-Gayo, M.; Daugé, V.; Menant, I.; Bégué, D.; Gacel, G.;
`Roques, B. P. Peptides 1985, 6, 415-420.
`(20) Fournié-Zaluski, M. C.; Coulaud, A.; Bouboutou, R.; Chaillet,
`P.; Devin, J.; Waksman, G.; Costentin, J.; Roques, B. P. J.
`Med. Chem. 1985, 28, 1158-1169.
`
`7 Laboratoire Rhone Poulenc Santé.
`
`0022-2623/89/1832-0445$01.50/0
`
`© 1989 American Chemical Society
`
`See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
`
`Downloaded via UNIV OF CALIFORNIA LOS ANGELES on July 11, 2018 at 20:37:56 (UTC).
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`
`
`-445-
`
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`MAIA Exhibit 1025
`MAIA V. BRACCO
`IPR PETITION
`
`

`

`446 Journal of Medicinal Chemistry, 1989, Vol. 32, No. 2
`
`Mars eigne et al.
`
`TyfSO^ie
`
`Boc-L-TyHSOÓ)- A
`
`1
`
`I
`4.5
`
`Figure 1. Scheme for the synthesis of compound 1 (Ac[L-Phe-
`(p-CHaSOsNa)27(Nle28(Nlesl]CCK27.33) and compound 2 (Ac[d-
`Phe(p-CH2-SOgNa)27,Nle28,Nle3l]CCK27-33), which were separated
`by chromatography on silica gel at the last step of the synthesis.
`scribed18 by base-catalyzed alkylation of diethyl acet-
`amidomalonate with -bromo-p-toluonitrile, followed by
`catalytic hydrogenation of the nitrile function and use of
`sodium nitrite in aqueous medium to obtain the hydrox-
`ymethyl derivative.
`Conversion to the desired amino acid was performed
`with first thionyl chloride and then sodium sulfite in a
`sodium hydroxide solution. A final acid treatment allowed
`decarboxylation.
`Compounds 1 and 2 were prepared in the liquid phase,
`according to Figure 1. Compound 4 was prepared like the
`equipotent analogue of CCK8: Boc [Nle28,Nle31] CCK27-33
`(compound 3).19
`The coupling steps were performed by using the
`DCC/HOBt condensation method, the active p-nitro-
`phenyl ester method, and the HONSu/DCC method.
`The amine protecting groups of these fragments were
`removed either with trifluoroacetic acid or by catalytic
`hydrogenolysis. The carboxylic acid functions were de-
`protected by saponification, by catalytic hydrogenolysis,
`or with trifluoroacetic acid. The S03-pyridine complex
`in a DMF-pyridine mixture was used to introduce a sulfate
`ester group into the tyrosine side chain, and the final
`product 4 was purified by silica gel column chromatogra-
`phy. The two stereoisomers, compounds 1 and 2, were
`separated by chromatography on silica gel, and the con-
`figuration of the -carbon of the modified tyrosine residue
`was determined by 1H NMR spectroscopy. The purity and
`the lack of racemization of each compound were checked
`by HPLC and NMR spectroscopy.
`NMR Studies
`. The studies were performed in aqueous medium in order
`to control precisely the pH of the solution. Unambiguous
`assignment of the spectra of compounds 1-4 (Figure 2) was
`carried out by classical COSY experiments.
`As compared to 3, the only significant change in the
`spectrum of compound 4 is the upfield shift (0.16 ppm)
`of one of the two Nle resonances
`(Figure 2). This shielded
`signal can be assigned to the residue Nle 28, which is close
`to the site of configurational change, Tyr(S03")27. On the
`other hand, the spectra of 3 and 1 differ only by the 0.25
`the Ha of
`ppm downfield shift of
`the Ac-Phe(p-
`CH2S03Na) residue in 1 as regards to the Ha of Boc-L-
`Tyr(SOsNa) in 3. Moreover, the position of the Ha signals
`of the two Nle residues are unchanged between 1 and 3.
`
`'
`
`1
`
`'
`
`1
`
`1
`
`r
`
`I
`PPM
`4.0
`Figure 2. Ha region of ID spectra of compounds 1-4: A =
`Nle-Gly-Trp-Nle-Asp-Phe-NH2, (·) full circle point the resonance
`of residue Nle28, ( ) full triangle point the resonance of residue
`[Phe(p-CH2S03Na) or Tyr(S03H)]27.
`Therefore it can be concluded that, in compound 1, the
`Phe(p-CH2S03Na) residue has the same L configuration
`as Tyr(S03Na) in 3.
`The same effects were observed with compounds 2 and
`in
`4: downfield shift of Ha of Boc-Tyr(S03Na) resonance
`Ac-Phe(p-CH2S03Na), and unchanged chemical shift of
`Ha of Nle.28 Therefore, the two peptides have a first
`amino acid in the D configuration.
`Finally the comparison of 1 and 2 confirms the previous
`remarks and attributions, i.e. an upfield shift (0.16 ppm)
`of Ha of Nle28 between 1 (L-Phe(p-CH2S08Na)) and 2
`(D-Phe(p-CH2S03Na)).
`The same behavior has already been observed with the
`dipeptides L-Phe-L-Ala and D-Phe-L-Ala, where the methyl
`of Ala was more shielded in the D,L than in the L,L dia-
`stereoisomer.20
`The small changes (including coupling constants of the
`glycine moiety) which occurred in the spectra of com-
`pounds 1-4 suggest slight modifications in the conforma-
`tional states of these peptides.
`The NMR attribution of the configuration of compound
`1 and 2 is in accordance with HPLC studies. Compound
`3 (L-Tyr(S03H) shows a higher retention time (tR = 4.5
`min) than that of compound 4 (D-Tyr(S03H)) (tR = 3.6
`min). A similar result was observed with the stereoisomers
`1 (tR = 9.0 min) and 2 (tR = 7.8 min). Thus, one can
`assume that both 3 and 1 have the same L configuration
`and compounds 4 and 2 the same D configuration at the
`level of Tyr(S03H) or Phe(p-CH2S03H).
`Figure 3 shows the increased stability of compound 1,
`a period of several days,
`which is in fact stable over
`whereas compound 3 is completely desulfated in 6 h in a
`solution of TFA (0.5 M) in DMSO.
`Biological Results
`Binding Experiments. The CCK-related analogues
`reported in this paper were evaluated for their potency in
`
`
`
`-446-
`
`
`
`
`

`

`CCKS with a Nonhydrolyzable Sulfated Tyr Residue
`
`Journal of Medicinal Chemistry, 1989, Vol. 32, No. 2
`
`447
`
`Table I. Potencies of CCK8 Analogues (1-4) in Inhibiting [3H]Propionyl-CCK8 Specific Binding in Guinea Pig Brain and Pancreatic
`Membranes
`
`compound
`
`brain
`pancreas
`(0.64 ± 0.04) X 10-9
`(0.28 ± 0.01) X 10"9
`CCK26-33 or CCKg
`(1.70 ± 0.20) X 10-9
`(3.20 ± 0.60) X 10-9
`1 (Ac(L-Phe(p-CH2S03Na)27,Nle28,Nle31)CCK27-33)
`(1.60 ± 0.20) X io-8
`(1.90 ± 0.40) X IO"8
`2 (Ac(D-Phe(p-CH2S03Na)27,Nle28,Nle31)CCK27.33)
`(0.93 ± 0.08) X 10-9
`(0.23 ± 0.05) X 10-9
`3 (Boc(L-Tyr(S03Na)27,Nle28,Nle31)CCK27.33)
`(3.00 ± 0.30) X 10-9
`(3.30 ± 0.70) X IO"8
`4 (Boc(D-Tyr(S03Na)27,Nle28,Nle31)CCK27.33)
`± SEM of three separate experiments performed in triplicate.
`6 [3H]Propionyl-CCKe was used at the concen-
`“ Values represent mean
`tration of 0.2 nM (KD = 0.2 nM) with brain tissue and at 0.1 nM (KD = 1.2 nM) with pancreatic tissue.
`
`binding Ki,a’b M
`
`compounds
`CCK28_33 or CCK8
`
`Table II. Pharmacological Potencies of CCK8 Analogues 1-4
`agonist act. : EC50.M
`contractile activities
`amylase secretion by
`of guinea pig ileum6
`guinea pig acini"
`(0.10 · 0.05) X 10"9
`(0.70 ± 0.02) X IO"9
`(0.33 ± 0.07) X IO"9
`(3.80 ± 0.30) X IO"9
`(1.60 ± 0.40) X IO"9
`(1.70 ± 0.30) X IO"8
`(0.40 ± 0.20) X 10"9
`(0.28 ± 0.11) X 10"9
`(4.50 ± 0.90) X IO"9
`(2.80 ± 0.40) X IO"8
`“Results are the mean ± SEM of three separate experiments,
`6 Results are the mean ± SEM of three
`each value in triplicate.
`separate experiments.
`performed on the sulfate ester group of the tyrosine of
`ceruletide—especially the replacement of the OS03H group
`by a NHS03H group—led to less active analogues.14
`In contrast, substitution of the OS03H group by a
`CH2S03H group does not affect CCKg activity in vitro,
`suggesting that (i) the oxygen atom linked to the tyrosine
`ring is not involved in a crucial hydrogen bound within the
`receptor and (ii) the methylene group is flexible enough
`to position the S03H moiety in the appropriate receptor
`subsite.
`The introduction of a D sulfated tyrosine residue in
`position 27 slightly modifies the affinity for central and
`peripheral receptors (13- and 35-fold decrease). Moreover,
`the analogue 4 is respectively 10 and 70 times less potent
`than Boc[Nle28,Nle31]CCK27_33 on amylase release and on
`the CCKg-induced contractions of guinea pig ileum. The
`same loss of affinity and peripheral activity is observed
`when comparing the biological properties of the two en-
`antiomeric peptides Ac[L-Phe(p-CH2S03Na)27,Nle28,
`Nle31]CCK27„33 and Ac[D-Phe(p-CE2S03Na)27,Nle28,Nl-
`e31]CCK27_33 (Tables I and II). Nevertheless, compounds
`2 and 4 with D-amino acids are of interest since they could
`resistant to aminopeptidase degradation.
`be more
`The use of tyrosine O-sulfate containing peptides is
`relatively limited as the sulfate ester bond is remarkably
`acid labile5 and is therefore likely to be hydrolyzed in some
`of the common protein-chemical procedures.1 The new
`CCK-related peptide 1 proved to be more stable in acid
`medium than the reference molecule Boc[Nle28,Nle31]-
`CCK27_33 (Figure 3). Besides its enhanced chemical sta-
`bility, compound 1 retains full in vitro biological activity
`and studies on its in vivo activity and stability are now in
`progress.
`Extensive in vivo pharmacological studies of CCK8-re-
`lated peptides require compounds able to recognize spe-
`cifically central receptors, protected from degrading en-
`zymes since in vitro CCKg is relatively quickly degraded21
`and chemically resistant to desulfation. The first
`two
`requirements have already been satisfied since cyclization
`in the N-terminal part of CCKg yielded peptides highly
`
`1234
`
`(21) Deschodt-Lanckman, M.; Bui, N. D.; Noyer, M.; Christophe,
`J. Reg. Peptides 1981, 2, 15-30.
`
`Figure 3. Stability of compounds 1 and 3 (2 mM) in a mixture
`of TFA (500 mM) and DMSO expressed in percentage of starting
`time in minutes; (v) 1, Ac-Phe(p-
`t
`(sulfated) product:
`(O) 3, Boc-Tyr-
`CH2S03H)-Nle-Gly-Trp-Nle-Asp-Phe-NH2;
`(S03H)-Nle-Gly-Trp-Nle-Asp-Phe-NH2.
`
`=
`
`displacing [3H] propionyl-CCK8 from guinea pig brain and
`pancreatic membranes. They all display high affinities (
`~ 1-30 nM) for brain and pancreatic binding sites (Table
`I).
`Amylase Release. The pancreozimin-like activities of
`the CCK-related peptides were assessed by measuring their
`effect on amylase secretion from guinea pig pancreatic
`acini. For compounds 1, 2, and 4, the shape of the dose-
`response curves was similar to that obtained with both
`CCK8 and Boc[Nle28,Nle31]CCK27„33. The efficiency of
`these CCKg analogues to stimulate amylase release is re-
`ported in Table II. Compound 1 proved to be the most
`active component with an EC50 value of 0.33 nM.
`Guinea Pig Ileum Contractions. The ability of com-
`pounds 1, 2, and 4 to stimulate the contraction of the
`isolated guinea pig ileum was used to evaluate cholecys-
`tokinin-like activity.
`All the analogues displayed agonist properties, com-
`pound 1 being the most active compound with an EC50
`value of 3.8 nM (Table II).
`Discussion
`Replacement of the sulfated tyrosine residue in the se-
`quence of CCKg by the new
`amino acid L-Phe(p-
`CH2S03Na) led to a compound which displays an affinity
`for pancreatic binding sites (Kj = 1.7 nM) as high as that
`of the reference molecule Boc[Nle28,Nle31]CCK27-33 ( =
`0.93 nM). Moreover, this compound is a full agonist of
`CCKg in the stimulation of the pancreatic secretion enzyme
`(ECgo = 0.33 nM) as well as in the induction of guinea pig
`In addition to its
`ileum contractions (EC50 = 3.8 nM).
`potent peripheral activity, this compound displays a high
`affinity for brain binding sites ( = 3.2 nM).
`Thus, compound 1 is the first described analogue of
`CCKg modified on the sulfate ester group which recognizes
`both central and peripheral CCK receptors and which
`Indeed, all the modifications
`retains peripheral activity.
`
`
`
`-447-
`
`
`
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`

`

`448 Journal of Medicinal Chemistry, 1989, Vol. 32, No. 2
`selective for central receptors22 and introduction of mod-
`ified amino acids in the sequence led to fully peptidase-
`resistant compounds.23
`As illustrated in this work, the third criteria, i.e.
`re-
`sistance to acid-induced desulfation, has now
`been
`achieved. The synthesis of CCK8 analogues containing all
`three of these modifications is now in progress.
`Experimental Section
`Synthesis. All protected amino acids were from Bachem AG.
`Solvents were of analytical grade from Prolabo. Chromatography
`was carried out with Merck silica gel (230-400 mesh). For
`thin-layer chromatography (TLC), Merck plates precoated with
`F 254 silica gel were used with the following solvent systems (by
`volume): A, CHCl3-Me0H-Ac0H-H20 (70:30:6:3); B, CHC13-
`MeOH-AcOH-H2G-EtOAc (35:15:3:1.5:1); C, CHCl3-MeOH
`(90:10); D, EtOAc-pyridine-AcOH-H20 (60:20:6:11); E, EtOAc-
`pyridine-AcOH-H20 (40:20:6:11); F, EtOAc-pyridine-AcOH-H20
`(50:20:6:11).
`Plates were developed with UV, iodine vapor, ninhydrin, or
`Ehrlich’s reagent. The structure of the compounds and of all the
`intermediates were confirmed by NMR spectroscopy (Bruker
`WH 270 MHz). The purity was checked by HPLC (Waters
`apparatus) on a 250 X 4.6 mm Prolabo ODS2 5-μ column with
`Et3N-H3P04 buffer (TEAP, 0.025 M, pH 6.5)/CH3CN system
`as eluent (flow rate, 1.5 mL/min) with UV (210 nm) or
`fluores-
`cence (290 nm) detection. At each step of the synthesis, the lack
`of significant racemization of a given peptide was checked by XH
`NMR spectroscopy and by HPLC. Amino acid analyses were
`carried out on a LKB biochrom 4400 analyzer after hydrolysis
`with 5.6 M HC1 containing 4% (v/v) thioglycolic acid, at 110 °C
`for 24 h. Mass spectra were recorded on a double-focusing VG
`70-250 instrument. The FAB (fast atom bombardment) ionization
`was obtained with a FAB saddle field source
`(Ion Tech Ltd.,
`Teddington, U.K.) operated with xenon
`at 8 kV and 1 mA.
`Glycerol or cesium iodide was used for calibration. Accelerating
`voltage was set at 6 kV and resolution was 1200. Mass spectra
`were obtained in different matrices and processed by means of
`the VG-250 software package.
`The following abbreviations were used: Z, benzyloxycarbonyl;
`Boc, tert-butyloxycarbonyl; MeOH, methanol; EtOH, ethanol;
`EtOAc, ethyl acetate; THF, tetrahydrofuran; AcOH, acetic acid;
`DMF, dimethylformamide; CHC13, chloroform; TFA, trifluoro-
`acetic acid; DCC, iV^ZV-dicyclohexylcarbodiimide; DCU, N,N'-
`dicyclohexylurea; HOBt, 1-hydroxybenzotriazole; HONSu, N-
`hydroxysuccinimide. Other abbreviations used were
`those rec-
`ommended by the IUPAC-IUB Commission (Biochem. J. 1984,
`219, 345). All the dipeptides and tripeptides were prepared in
`the liquid phase, as previously described.19,24
`Ac(L,D)Phe(p-CH2-S03Na)-Nle-Gly-Trp-0C2H5 (5). Ac-
`(L,D)Phe(p-CH2S03Na) (84.2 mg, 0.26 mmol) was dissolved in 6
`mL of DMF. Nle-Gly-Trp-OC2H6 (105 mg, 0.26 mmol), HOBt
`(41 mg, 0.27 mmol), and DCC (55 mg, 0.27 mmol) were successively
`added at 0 °C. The mixture was stirred for 1 h at 0 °C and
`overnight at room temperature. After evaporation of DMF, the
`residue was washed with EtOAc (10 mL). The product was
`isolated from the solid residue by two extractions with water (2
`X 25 mL) and lyophilization, to yield 147 mg (80%) of a white
`powder: Rf 0.24 (A), corresponding to a mixture of two stereo-
`isomers; FAB-MS (MH+) caled 708, found 708.
`Ac(L,D)Phe(p-CH2S03Na)-Nle-Gly-Trp-0H (6). Compound
`5 (100 mg, 0.14 mmol) was saponified with 1 M NaOH (0.3 mL)
`in the solvent mixture H20-MeOH (8 mL- mL) for 1 h at 0 °C
`and 3 h at room temperature. After evaporation of MeOH, the
`aqueous phase was acidified with 1 M HC1, washed with EtOAc,
`and lyophilized, to yield a white product: 90 mg (95%); R¡ 0.08
`(A) for the two stereoisomers.
`
`(22) Charpentier, B.; Pélaprat, D.; Durieux, C.; Dor, A.; Reibaud,
`M.; Blanchard, J. C.; Roques, B. P. Proc. Natl. Acad. Sci.
`U.S.A. 1988, 85, 1968-1972.
`(23) Charpentier, B.; Durieux, C.; Pélaprat, D.; Dor, A.; Reibaud,
`M.; Blanchard, J. C.; Roques, B. P. Peptides 1988, 9, 835-842.
`(24) Charpentier, B.; Durieux, C.; Menant, I.; Roques, B. P. J. Med.
`Chem. 1987, 30, 962-968.
`
`Marseigne et al.
`
`Ac(L,D)Phe(p -CH2S03Na)-Nle-Gly-Trp-Nle-Asp(0Bzl)-
`Phe-NH2 (7). Compound 6 (20 mg, 0.029 mmol) was dissolved
`in 1 mL of DMF. Nle-Asp(OBzl)-Phe-NH2-TFA (17.5 mg, 0.029
`mmol) and Et3N (5 pL) in DMF (1 mL), HOBt (6.1 mg, 0.04
`mmol), and DCC (8.2 mg, 0.04 mmol) were successively added
`at 0 °C. The reaction mixture was stirred for 30 min at 0 °C and
`overnight at room temperature.
`After filtration of DCU and evaporation of DMF, the residue
`was triturated with EtOAc-ether and washed several times with
`ether to yield a white solid: 30.1 mg (91%); Rfl 0.42 (B) and R/2
`0.39 (B) for the two stereoisomers.
`Ac(L^))Phe(p-CH2S03Na)-Nle-Gly-Trp-Nle-Asp-Phe-NH2
`(8). Compound 7 (22 mg, 0.019 mmol) in 5 mL of MeOH was
`hydrogenated in the presence of 10% Pd/C catalyst (15 mg) for
`3 h. After filtration of the catalyst, MeOH was evaporated to yield
`a white product: 19.5 mg (97%); Rfl 0.24 (B) and R¡2 0.21 (B) for
`the two stereoisomers, which were separated by chromatography
`on silica gel with CHCl3-Me0H-Ac0H-H20-Et0Ac (35:15:3:1.5:1)
`as eluent to yield 3.2 mg, Rf 0.24 (B), and 3.8 mg, R¡2 0.21 (B);
`HPLC, fRl HPLC 9.0 min and
`7.8 min (eluent: CH3CN-TEAP,
`29:71); the attribution of the configuration of the -carbon in the
`tR was attributed
`modified tyrosine is discussed in this paper.
`to compound 1 and tR to compound 2. FAB-MS (MH+) (com-
`pounds 1 and 2) calca 1054, found 1054.
`Boc-D-Tyr-Nle-Gly-Trp-OC2H5 (9). Nle-Gly-Trp-OC2H5
`(402.5 mg, 1 mmol) was dissolved in 30 mL of the solvent mixture
`THF-CH2C12 (75:25). Boc-D-Tyr-OH (281.3 mg, 1 mmol), HOBt
`(154 mg, 1 mmol), and DCC (227 mg, 1.1 mmol) were successively
`added at 0 °C. The mixture was stirred for 1 h at 0 °C and
`overnight at room temperature.
`After filtration of DCU, the solvents were evaporated. The
`residue was dissolved in EtOAc and washed with citric acid (10%),
`NaHC03 (10%), and brine, dried over Na2S04, concentrated in
`vacuo, and precipitated with anhydrous ether to yield a white
`powder: 550 mg (83%); Rf 0.30 (C). Anal.
`(C36H4708N5) C, H,
`N.
`Boc-D-Tyr-Nle-Gly-Trp-OH (10). Compound 9 (253.7 mg,
`O. 38 mmol) was saponified with 1 M NaOH (0.77 mL, 0.77 mmol)
`in EtOH (8 mL) for 1 h at 0 °C and 4 h at room temperature.
`After evaporation of EtOH, the residue was dissolved in water
`(20 mL). The unreacted product was extracted with EtOAc. The
`aqueous phase was acidified with cold 1 M HC1 and extracted
`with EtOAc, washed with brine, and dried over Na2S04 to yield
`after evaporation and precipitation with anhydrous ether a white
`powder: 202.4 mg (83%); Rf 0.83 (D). Anal.
`(C^H^OgNs) C, H,
`N.
`Boc-D-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH2 (11). Compound
`10 (0.199 g, 0.31 mmol) was dissolved in 8 mL of dry DMF.
`HONSu (0.036 g, 0.31 mmol) and DCC (0.065 g, 0.31 mmol) were
`then added at -10 °C. The reaction mixture was stirred for 30
`min at -10 °C and then for 1 h at 0 °C and overnight at room
`temperature.
`To the above mixture was added, at 0 °C, a solution of Nle-
`Asp-Phe-NH2-TFA (0.158 g, 0.31 mmol) and Et3N (44 pL, 0.31
`mmol) in DMF (3 mL). The resulting mixture was stirred ov-
`room temperature. After filtration of DCU and
`ernight at
`evaporation of DMF, the residue was triturated with EtOAc-ether
`and washed several times with ether to yield a white powder: 0.192
`(C62H69012N9) C, , N.
`g (61%); Rf 0.81 (E). Anal.
`Boc-D-Tyr(S03Na)-Nle-Gly-Trp-Nle-Asp-Phe-NH2 (4). A
`solution of compound 11 (0.185 g, 0.18 mmol) in dry DMF (4 mL)
`and dry pyridine (4 mL) was treated with S03-pyridine complex
`(1.11 g) and was stirred overnight under N2 at room temperature.
`After evaporation in vacuo,
`the residue was taken up in a cold
`saturated NaHC03 solution and the mixture was stirred at 0 °C
`for 1 h with the pH maintained at about 7. A solid residue was
`collected by centrifugation and dried in vacuo.
`The suspended product was isolated by lyophilization followed
`by precipitation of inorganic salts in MeOH, filtration, and
`evaporation of MeOH in vacuo. The two fractions were separately
`purified by chromatography on silica gel with EtOAc-pyridine-
`AcOH-H20 (50:20:6:11) as eluent, to yield 94 mg (48%) of com-
`pound 4: Rf 0.40 (F); HPLC tR 3.6 min (eluent: CH3CN-TEAP,
`35:65); FAB-MS (MH+) caled 1137, found 1137. Amino acid
`analysis: Asp 0.96, Nle 1.98, Gly 0.98, Tyr 0.94, Phe 1.02, Trp
`O. 86.
`
`
`
`-448-
`
`
`
`
`

`

`CCK8 with a Nonhydrolyzable Sulfated Tyr Residue
`NMR Measurements. Samples were prepared by dissolving
`of peptide in D20 (99.98% CEA). The pH was adjusted to 7.0
`by addition of DC1 or NaOD solutions (0.1 N in D20) and mea-
`sured with a microelectrod Ingold 405.M3 with a Tacussel pH
`meter PH N 75, without correction for deuterium effect. The
`final concentration was 0.8 mM for ID spectra and 2 mM for 2D
`experiments.
`in the Fourier transform mode at 270 MHz
`Spectra were run
`on a Bruker WH 270 instrument and at 400 MHz on a Bruker
`AM 400 instrument equipped with an Aspect 2000 and 3000
`computer, respectively, and with a Bruker temperature controller
`(±1 °C). Chemical shifts were given in ppm from DSS as external
`reference. The residual peak of HDD was suppressed by presa-
`turation. The COSY experiments were performed by using es-
`tablished techniques.26
`For stability in acidic conditions, the samples were dissolved
`in DMSO-d6 (99.8% CEA), and then 20 μ of TFA was added
`at room temperature. The final concentrations were
`1 mM in
`peptide and 0.5 N in acid. The proportion of sulfated and un-
`sulfated products was determined from characteristic peaks in-
`tensity in ID spectra (H, resonance
`for compound 3 and CH2S03H
`for compound 1).
`resonance
`Guinea Pig Brain Membrane Preparation. Male guinea
`pig brain cortex were dissected on ice and homogenized (12 mL/g
`of tissue, wet weight) in 50 mM Tris-HCl buffer, pH 7.4, containing
`5 mM MgCl2. The homogenate was incubated for 30 min at 35
`°C and centrifuged at 4 °C for 35 min at 100000g, and the resulting
`pellet was rehomogenized in a large excess of ice-cold buffer and
`centrifuged under the same conditions. The final pellet was
`resuspended in 50 mM Tris-HCl buffer, pH 7.4, supplemented
`with 0.2 mg/mL bacitracin and 5 mM MgCl2 (6-7 mg of pro-
`tein/mL). Protein concentration was determined by the method
`of Lowry et al.26 using bovine serum albumin standards.
`Guinea Pig Pancreatic Membrane Preparation. Male
`guinea pigs (250-350 g) were sacrificed by cervical dislocation,
`the pancreases were quickly dissected and placed into ice-cold
`10 mM Pipes-HCl buffer, pH 6.5, containing 30 mM MgCl2
`(Pipes-MgCl2 buffer). After careful removal of the fat, the
`pancreases of seven guinea pigs were homogenized in 25 volumes
`of Pipes-MgCl2 buffer at 4 °C with a Brinkmann Polytron PT10,
`the homogenate was filtered on gauze, and the filtrate was cen-
`trifuged twice at 50000g for 10 min with an intermediate reho-
`mogenization of the pellet in fresh buffer. The final pellet was
`resuspended in 2 vol of fresh buffer and stored frozen at -80 °C
`until used. This preparation usually led to 12-14 mL of a mem-
`brane suspension containing 25-30 mg of protein/mL.
`Binding Assays.
`[3H]Propionyl-CCK8 ([3H]pCCK8, 60 Ci/
`mmol) was purchased from Amersham. Binding experiments with
`
`(25) Ave, W. P.; Bartholdi, E.; Ernst, R. R. J. Chem. Phys. 1976,
`64, 2229-2246.
`(26) Lowry, O. H.; Rosebrough, N. J.; Fan, A. L.; Randall, R. J. J.
`Biol. Chem. 1951, 193, 265-275.
`
`Journal of Medicinal Chemistry, 1989, Vol. 32, No. 2 449
`[3H]pCCK8 were performed as described previously23 with some
`modifications. Briefly, incubations (final volume 1 mL) were
`carried out in 50 mM Tris-HCl buffer, pH 7.4, 5 mM MgCl2, 0.2
`mg/mL bacitracin, for 60 min at 25 °C, in the presence of brain
`membranes (0.6 mg of protein/tube), or
`in 10 mM Pipes-HCl
`buffer, pH 7.4, 30 mM MgCl2, 0.2 mg/mL bacitracin, 0.2 mg/mL
`soybean trypsin inhibitor, for 120 min at 25 °C in the presence
`of pancreatic membranes (0.2 mg of protein/tube). For dis-
`placement experiments, the radiolabeled probes (0.2 and 0.1 nM
`for brain and pancreatic membranes, respectively) were incubated
`in the presence of varying concentrations of the competitor.
`Nonspecific binding was determined in the presence of 1 μ CCK8
`in all cases. The incubation was terminated by filtration through
`Whatman GF/B filters precoated by incubation in buffer (brain
`membranes, 50 mM Tris-HCl, 5 mM MgCl2, pH 7.4; pancreatic
`membranes, 10 mM Pipes-HCl, pH 6.5) containing 0.1% bovine
`rinsed twice with 5 mL of ice-cold
`serum albumin; filters were
`buffer and dried, and the radioactivity was counted in 5 mL of
`ready-solvent EP scintillation cocktail (Beckman). The K\ values
`were calculated by using the Cheng-Prusoff equation.27
`In Vitro Bioassays. Acini were prepared as previously re-
`ported.28 Amylase release from pancreatic acini was measured
`after incubation for 30 min at 37 °C in the presence of CCK8 or
`CCK analogues as previously reported.22,29 Contractile activity
`of guinea pig ileum was measured according to Hutchinson and
`Dockray.30
`Acknowledgment. We are grateful to Dr. A. Beaumont
`for stylistic revision and A. Bouju for typing the manu-
`script.
`Registry No. 1,117942-22-0; 2,117942-23-1; 3,100654-12-4;
`4, 117942-24-2; (l)-5, 117942-32-2;
`(d)-5, 117942-33-3; (l)-6,
`(l)-7, 117942-36-6;
`(d)-6, 117942-35-5;
`(d)-7,
`117942-34-4;
`117942-37-7; 9, 117942-38-8; 10, 117942-39-9; 11, 117942-40-2;
`Z-Gly-OH, 1138-80-3; Trp-OEt, 7479-05-2; Z-Nle-OH, 39608-30-5;
`Boc-Nle-ONp, 21947-33-1; Boc-Asp(OBzl)-ONp, 26048-69-1;
`Phe-NH2, 5241-58-7; Ac-(L)-Phe(p-CH2S03Na)-0H, 117942-25-3;
`Ac-(D)-Phe(p-CH2S03H)-0H, 117942-26-4; Z-Gly-Trp-OEt,
`117942-27-5; Z-Nle-Gly-Trp-OEt, 117942-28-6; Gly-Trp-OEt,
`117942-29-7; Nle-Gly-Trp-OEt, 117942-30-0; Boc-Asp(OBzl)-
`Phe-NH2, 60058-69-7; Boc-Nle-Asp(OBzl)-Phe-NH2, 65864-24-6;
`TFA-Asp(OBzl)-Phe-NH2, 117942-31-1; TFA-Nle-Asp(OBzl)-
`Phe-NH2, 117959-03-2; Boc-(n)-Tyr-OH, 70642-86-3.
`
`(27) Cheng, Y. C.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22,
`3099-3108.
`(28) Peikin, S. R.; Rottman, A. J.; Batzri, S.; Gardner, J. D. Am. J.
`Physiol. 1978, 235(6), E 743-E 749.
`(29) Gardner, J. D.; Jackson, M. J. J. Physiol. London 1977, 270,
`439-454.
`(30) Hutchinson, J. B.; Dockray, G. J. Eur. J. Pharmacol. 1981, 69,
`87-93.
`
`
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`-449-
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