synthesis and biological activities of some cholecystokinin analogues substituted in position 29 by...

Ini. J . Pepiide Proiein Res. 34, 1989, 394404

Synthesis and biological activities of some cholecystokinin analogues substituted in position 29 by a p-alanine

MARC RODRIGUEZ, MARC ROLLAND, MARIE-FRANCOISE LIGNON, MARIE-CHRISTINE GALAS, JEANINE LAUR, ANDRE AUMELAS and JEAN MARTINEZ

CNRS-INSERM Centre for Pharmacology and Endocrinology, Montpellier. France

Received 1 December 1988, accepted for publication 17 April 1989

Syntheses of analogues of the C-terminal heptapeptide of cholecystokinin are described. These analogues were obtained by replacing glycine 29 by a /?-alanine. The C-terminal phenylalanine amide was in some cases substituted by 2-phenylethyl alcohol and/or residues of the C-terminal tetrapeptide by their D-enantiomers. These compounds were tested for their action on stimulation of amylase release from rat pancreatic acini and for their ability to inhibit binding of labeled CCK to rat pancreatic acini and guinea pig brain membranes. Some of these derivatives behaved as CCK receptor antagonists.

Key words: b-alanine; cholecystokinin analogues; 2-phenylethyl ester derivatives; receptor antagonists; syntheses

Cholecystokinin (CCK) is a 33-residue peptide hormone first isolated by Ivy & Oldberg (1) from hog intestine where it stimulates gastrointestinal motility and gall bladder contractions as well as pancreatic amylase secretion (2, 3). It has been demonstrated (4) that the C-terminal heptapeptide reproduces the entire range of biological activities of CCK and that the two methionines in positions 28 and 3 1 can be replaced by norleucines without loss of activity (5-8). In addition, CCK-8 has been found in the brain (9, 10) where it functions as a neuromodulator and neurotransmitter (1 1, 12). Among other physiological effects, cholecystokinin is involved in the control of food intake (13). We recently described a series of CCK analogues in which the C-terminal phenylalanine amide was substituted by a 2-phenylethylamine or a 2-phenylethyl alcohol (14). These compounds were able to re- produce only part of the biological response of CCK on isolated rat pancreatic acini, giving maximal stimulation of enzyme secretion with no decrease in the maximal response at supramaximal concentrations (1 5) which does not promote phosphoinositide break- down (16, 17). The same modification, combined with the reversal of configuration of the tryptophan residue, led to compounds behaving as CCK receptor antagonists (18). In order to increase enzymatic sta- bility of CCK and of the derivatives mentioned above, we synthesized a series of CCK analogues containing a fl-alanine in place of the glycine residue in position 29, in combination or not with the substitution of the

394

C-terminal phenylalanine amide by a 2-phenylethyl alcohol. Introduction of a fl-alanine residue might drastically change the general topochemistry of the molecule, as compared to the parent peptide, and thus might modify biological activity. In order to keep the amino acid side chains spatially correctly orientated, we decided to invert the configuration of the residues following the /?-alanine residue (1 9). The ensuing compounds were thus synthesized: Boc-Tyr(S0; )-Nle- flAla-Trp-Nle-Asp-Phe-NH, (l), Boc-Tyr( SO; )-Nle- PAla-D-Trp-D-Nle-D-Asp-D-Phe-NH, (2) (analogues of the parent peptide Boc-Tyr(S0; )-Nle-Gly-Trp- Nle-Asp-Phe-NH,, Boc-ple28, Nle3']-CCK-7) (20), Boc-Tyr(S0; )-Nle-PAla-Trp-Nle-Asp-O-CH, -CH, - c6 H, (3), Boc-Tyr(S0; )-Nle-flAla-D-Trp-D-Nle-D- Asp-O-CH, -CH2 c6 H, (4) (analogues of Boc- Tyr(S0; )-Nle-Gly-Trp-Nle-Asp-O-CH, -CH, -C6 H, (14), which is a partial agonist of CCK (15)), Boc- Tyr(S0; )-Nle-~Ala-~-Trp-Nle-Asp-O-CH, -CH, - C, H, (5) and Boc-Tyr(S0; )-NIe-PAla-Trp-D-Nle-D- Asp-O-CH, -CH2-C6H, (6) (analogues of Boc- Tyr(S0; )-Nle-Gly-D-Trp-Nle-Asp-O-CH, -CH2 - C,H, (14), which is a CCK receptor antagonist (18)).

RESULTS AND DISCUSSION

Chemistry Compounds 1 and 2 were synthesized in solution according to the general procedure illustrated in Scheme 1, compounds 3 to 6 according to the procedure de-

Cholecystokinin analogues

in the presence of 1,8-diazabicyclo[5,4,O]-undec-7-ene (DBU). Derivatives 1 to 6 were purified by silica gel chromatography (eluent: ethyl acetate/pyridine/acetic acid/water 80:20:5: 10) and HPLC (various mixtures of 0.05 M ammonium acetate pH 6.5 and methanol). As examples, syntheses of compounds 2 and 4 are detailed in the experimental section. Physical and analytical data of the final compounds are listed in Table 1, those of the synthetic intermediates in Table 2. Compounds 1 to 6 were identified by 'H n.m.r. spec- troscopy (Tables 3-8).

Biological evaluations All compounds were tested for their ability to stimulate in vitro amylase release from rat pancreatic acini (4), to inhibit the binding of I2'I-BH-CCK-8 to isolated rat pancreatic acini (25) and to guinea pig brain membranes (26). The results were compared to those of the potent CCK analogue Boc-[Nle2', Nle3']- CCK-7 (20) and are reported in Table 9. Replacement of the glycine residue in Boc[Nle2', Nle3']-CCK-7 by a p-alanine produced Boc-Tyr(S0; )-Nle-BAla-Trp- Nle-Asp-Phe-NH, (l), which was about 100 times less potent than Boc-[Nle2', Nle3']-CCK-7 in inhibiting binding of I2'I-BH-CCK-8 to isolated rat pancreatic acini (half maximal inhibition at 200 nM as compared to 1.5 nM), and in stimulating pancreatic amylase secretion (maximal stimulation at 20 nm as compared to 0.3 nM). These results seem to indicate that either the distance between the sulfate moiety and the C-terminal part of the molecule or the general topochemistry of the molecule have been dramatically modified by the sole homologation of the glycine residue, and that they are important both for binding to CCK-receptors and for activity on amylase secretion from rat pancreatic acini. These findings are somewhat different from the results already reported (9, although the species in which the study was carried out was not specified and could be different. However, the apparent affinity of compound 1 for central CCK receptors differed only by one order of magnitude from that of Boc-[Nle2', Nle3']-CCK-7 (half maximal inhibitions at 3 nM and 0.3 nM respectively). This value, very close to that of CCK-4 observed by Knight et al. (27) (1 .7n~) , is not surprising, since apparent affinity of CCK to its central receptors is mainly dependent on its C-terminal tetrapeptide. Inverting the configuration of the C-terminal tetrapeptide residues by replacing each of them by its D-enantiomer did not produce analogues with restored potency. On the contrary, maximal stimulation of pancreatic amylase release induced by compound 2 occurred only at doses higher than 0.1 mM (Table 9). Compounds 3 and 6 containing a 2-phenylethyl ester in place of the phenylalanine amide had poor apparent affinities for either central (half-maximal inhibitions at 20 nM and 100 nM respectively) or pancreatic (half-maximal inhibitions at 100 nM and 300 nM respectively) CCK-receptors and

395

SCHEME 1

scribed in Scheme 2. All syntheses were carried out by fragment condensation of the C-terminal tetrapeptide or the C-terminal tripeptide 2-phenylethyl ester with the N-terminal fragment Boc-Tyr-Nle-PAla-OSu 10. Condensation of Boc-Nle with BAla-OMe through a mixed anhydride afforded Boc-Nle-BAla-OMe 7, which led, after TFA deprotection and coupling to Boc-Tyr-OSu (21), to the tripeptide methyl ester Boc- Tyr-Nle-PAla-OMe 8. Saponification and esterification with N-hydroxysuccinimide in the presence of DCC yielded the activated intermediate 10. Dipeptide Boc-~-Asp(0Bzl)-~-Phe-NH, was obtained by mixed anhydride coupling of Boc-D-As~(OBZI) and D-Phe- NH, . Partial deprotection by TFA and coupling with Boc-D-Nle-OSu (2 1) afforded the tripeptide BOC-D- Nle-~-Asp(oBzl)-~-Phe-NH, 13, which led to Z-D- Trp-~-Nle-~-Asp(0Bzl)-~-Phe-NH, 15, after partial deprotection with FTA and coupling to Z - D - T ~ ~ with BOP (22). Hydrogenation of compound 15, coupling to Boc-Tyr-Nle-BAla-OSu 10 afforded the heptapeptide Boc-Tyr-Nle-BAla-D-Trp-D-Nle-D-Asp-D-Phe- NH, 19, which was sulfated with sulfur trioxide/ pyridine complex (1 4) to produce Boc-Tyr(S0; )-Nle- P A ~ ~ - D - T ~ ~ - D - N ~ ~ - D - A s ~ - D - P ~ ~ - N H , 2. Synthesis of peptide 1 was carried out in the same way from Z-Trp- Nle-Asp(OBz1)-Phe-NH, (23). Compounds 2 to 7 were similarly obtained (Scheme 2) from the corre- sponding C-terminal tripeptide ester. Esterification of the (D or L) aspartyl residue was carried out according to Ono et al. (24) by action of 2-phenylethyl bromide

M. Rodriguez et al.

TABLE 1 Physical and analytical daia of cholecystokinin analogues substituted in position 29 bv a j-alanine

Peptide derivatives M.p. "C [a]D, deg [(c). DMF] RP, min (A/B) RP ~

Boc-Tyr(S0; )-Nle-BAla-Trp-Nle- Asp-Phe-NH, 1 205 dec. - 23.0 (1.37) 5.97 (30/70) 0.23 Boc-Tyr(S0; )-Nle-/3A1a-D-Trp-D-Nle-D-Asp-D-Phe-NH2 2 200 dec. + 1 1 .O (0.69) 10.81 (35/65) 0.24 Boc-Tyr(S0; )-Nle-PAla-Trp-Nle-Asp-0-CH,-CH,-C,H, 3 140dec. - 14.0 (1.54) 8.36 (30/70) 0.31 Boc-Tyr(S0; )-Nle-PAla-D-Trp-D-Nle-D-Asp-O-CH,-CH, -C, H, 4 180 dec. + 10.6 (0.76) 8.03 (30/70) 0.36 Boc-Tyr(S0; )-Nle-BAla-D-Trp-Nle-Asp-0-CH ,-CH, -C, H, 5 150 dec. - 13.1 (1.25) 8.54 (30/70) 0.36 Boc-Tyr(S0; )-Nle-~Ala-Trp-D-Nle-D-Asp-O-CH,-CH, -C6 H, 6 135dec. + 9.2 (0.92) 10.82 (32/68) 0.37

a Solvents used in HPLC: (A) ammonium acetate 0.05 M, pH 6.5 (B) methanol, at a flow rate of 3 mL/min; Merck-Hitachi instrument; Beckman Ultrasphere@ ODS 10 x 250mm column (OSpm), with an U.V. detection at 279mm. bT.l.c. Solvent: ethyl acetate/pyridine/acetic acidiwater 80:20: 5: 10. All compounds showed the expected amino acid analyses.

TABLE 2 Physical and analytical data of synthetic peptide derivatives

Peptide derivatives M.p. "C [a]D, deg [(c), DMF] RP Anal. C, H, N.*

Boc-Nle-PAla-OMe 7 BocTyr-Nle-PAla-OMe 8 BocTyr-Nle-PAla-OH 9 BocTyr-Nle-PAla-OSu 10 Boc-D-Asp(OBzl)-D-Phe-NH2 1 1 Boc-D-As~(OB~I)-O-CH,-CH,-C,H, 12 Boc-D-Nle-o-Asp(OBzl)-D-Phe-NH, 13 Boc-D-Nle-D-Asp(0Bzl)-0-CH, CH, C, H5 14 Z-D-Trp-D-Nle-D-Asp(OBzl)-D-Phe-NH, 15 Z-D-Trp-D-Nle-D-Asp(0Bzl)-0-CH,-CH, -C, H, 16 Z-T~~-D-N~~-D-AS~(OB~I)-O-CH, -CH, -C,H, I7 BocTyr-Nle-PAla-Trp-Nle-Asp-Phe-NH, 18 BocTyr-Nle-PAla-o-Trp-D-Nle-D-Asp-D-Phe-NH, 19 BocTyr-Nle-BAla-Trp-Nle-Asp-0-CH, -CH,-C,H5 20 BocTyr-Nle-/?Ala-D-Trp-D-Nle-D-Asp-O-CH2 -CH, -C6 H5 21 BocTyr-Nle-PAla-D-Trp-Nle-Asp-0-CH, -CH2 -C, H, 22 BocTyr-Nle-BAla-Trp-D-Nle-D- Asp-O-CH, -CHI -C, H, 23

60-6 1 137- I40 85 dec.

165-168 138-140

57-59 153-1 55

65-68 225 dec.

152-155 118-120

2 10 dec. 2 10 dec. 165 dec. 190 dec.

180 dec. 160dec.

- 9.5 (1.56) -9.5 (1.13) -6.5 (1.01) -8.5 (1.33)

f28 .8 (0.91)

+33.1 (1.54) + 16.5 (1.05) +31.0 (1.11) f 2 4 . 3 (1.47)

+2.5 (1.23)

+ 17.1 (1.29)

+ 11.8 (1.43)

+20.1 (1.01)

- 20.6 (1.39)

-13.6 (1.17)

- 15.1 (1.46) +12.9 (1.18)

A0.59; B0.74 B 0.43; C 0.73

D0.75 BO.11; C0.64 B0.38; C0.70

A0.87 B0.30; C0.68 A0.81; B0.93 C0.46; D0.95 B 0.80; C 0.93 B 0.74; C 0.95 D0.25; E0.54 D0.26; E0.54 D0.66; E0.94 D0.72; E0.94

D0.74; E0.94 D0.78; E0.94

a T.1.c. solvent systems: A, AcOEt/hexane, 5:s ; B, AcOEtihexane, 7:3; C, AcOEt; D, chloroform/methanol/acetic acid, 85: 10:5; E, AcOEt/pyridine/acetic acid/water, 80:20:5: 10. 'Values of elemental analyses are within & 0.4% of the calculated values.

stimulated maximal pancreatic amylase release at doses higher than 0.1 mM. Compounds 4 and 5, both containing a D-tryptophan residue and a 2-phenylethyl ester in place of the phenylalanine amide did not stimulate amylase secretion from rat pancreatic acini, but were able to antagonize stimulation of amylase release by Boc[Nle2*, Nle3']-CCK-7 with about the same potency (half maximal inhibitions at 50 p ~ ) . Again, it can be mentioned that compounds 3 to 6, in which the C-terminal part largely differs from that of CCK, had weak apparent affinities for CCK central receptors.

These studies pointed out that replacement of glycine in position 29 by f3-alanine leads to CCK analogues of weaker potency, suggesting that the

length of the peptidic backbone around the glycine residue is important. As it has already been indicated that glycine plays a key role in the CCK-8 confor- mation (28), it is conceivable that its replacement by a fi-alanine somewhat disturbs the spatial orientation of the functional groups involved in the binding to the CCK-receptors. Replacing amino acid residues following the fl-alanine residue by their D-enantiomers, modifications which are supposed to restore changes induced by homologation of glycine (replacement by a 8-alanine), did not improve the potency of the resulting analogues. These results also showed and con- firmed that the binding capacity of CCK analogues to brain membranes is strongly dependent on the struc- ture of the C-terminal tetrapeptide.

396


TABLE 3 ’ H n.m.r. data (DMSO-d, ) of Boc- Tyr (SO; )-Nle-pAla- Trp-Nle-Asp-Phe- NH, (1)

NH Ha HB Others a(PPm) 3J(Hz) a(PPm) d(PPm) 3J(Hz) s(PPm) 3J(Hz)

~ ~~

~~

TY r d 6.88 8.6 4.11 j 2.92 o,md7.l1 p’2.69 10.7 d 7.05

25 14.4 Bocs 1.31 Nle d 7.81 8.0 4.19 /I 1.58 CHI t 0.83 6.7

or d 7.95 7.5 p’ 1.48 Or 0.82 6.7 yy‘ 1.22

PAla t 7.90 6.0 u 3.21 1 2.28 6.8-8.7 u’3.15 8’2.20 6.4-6.8

25 14.9 TrP d 8.06 7.9 4.53 83.12 4.6 N H d 10.75

p’2.93 9.4 s7.10 25 14.5 d 7.57

d 7.31 t 7.03 t 6.94

d7.81 8.0 4.19 p 1.58 CHlt0.83 or d 7.95 7.5 8’1.48 or 0.82

Nle

yy’ 1.22 ASP d8.14 7.8 4.48 p2.61 6.4

p’ 2.44 6.9 25 16.3

8’2.84 8.8 25 14.1

Phe d 7.88 8.2 4.34 p 3.04 4.9 Ar 7.3-7.0

TABLE 4 ‘H n.m.r. data (DMSO-d,) of Boc-Tyr(S0; )-Nle-BAla-o-Trp-D-Nle-D-Asp-D-Phe-NH, (2)

2.3

7.8 8.1 7.9 7.4 6.7 6.7

NH Hu HB Others a(PPm) 35(Hz) 4PPm) a(PPm) 3J(Hz) &PPm) 3J(Hz)

TY r d 6.90 8.5 4.11 j3 2.91 p’2.69

25 Nle d7.81 8.0 4.19 p 1.58

p‘ 1.47

pAla t 7.90 5.7 aa‘3.17 p 2.28

25 ~ T r p d 8.05 8.5 4.54 p 3.12

p’ 2.93 27

8’2.21

D-Nle d 7.94 7.8 4.19 B 1.58 8’1.47

D - A s ~ d8.11 7.6 4.46 p 2.54 (r. 2.39

25 D-Phe d 8.01 8.2 4.33 p 3.05

p’2.84 25

10.4 14.2

6.3-7.6 7.2-7.2

15.0 4.6 9.1

14.6

7.0 6.4

16.2 4.7 9.1

14.0

0, md7.12 d 7.05

Bocs 1.31 CH,t0.83

or 0.82 yS 1.23

NH d 10.79 s7.10 d7.57 d 7.3 1 t 7.03 t 6.95

CH,t 0.83 or 0.82 yS 1.23

Ar 7.3-7.0

6.4 7.0

2.0

7.8 8.0

7.3 6.4 7.0

-

397

M. Rodriguez et al.

TABLE 5 ‘H n.m.r. data (DMSO-d,) of Boc-Tyr(S0; )-Nle-8Ala-Trp-Nle-Asp-O-CH2-CH,-C, Hj (3)

Nle

pAla

Nle

d 6.87 8.5 4.12 8 2.92 8’2.69

25 d 7.80 8.0 4.23 8 1.62

8’ 1.49 t 7.90 a d 3. I9 8 2.27

8’2.18 25

d 8.02 8. I 4.55 3.12 8’2.93

25

d 7.92 8.3 4.30 8 1.62 8’ 1.49

d8.31 7.8 4.58 p 2.64 8’2.56

25 t24.22 t 2.86

7.3-7.0

10.9 14.0

6.4-8.5 6 . e8 .5

14.7 4.4 9.3

14.8

5.8 6.9

16.7

6.8 2.2-7.0

0, md7.11 d 7.05

Boc s 1.3 1 CH, t 0.83

y 6 1.23

NHd 10.73 s7.10 d 7.58 d7.30 t 7.03 t 6.95

CH, t 0.83 yb 1.23

2.2

7.8 8.0

7.4

TABLE 6 ‘ H n.m.r. data (DMSO-d, ) of Boc-Tyr(S0; )-Nle-8Ala-D-Trp-D-Nle-D-Asp-O-CH,-CH,-C,H, (4)

NH Ha HB Others b(PPm) 3J(Hz) b(PPm) b(PPm) 3J(Hz) d(PPm) 3J(Hz)

d 6.90 8.5 4.11 0 2.91 0, md7.12 8.4 p’2.69 10.6 d 7.04 8.4

TYr

Nle d 7.80 8.1 4.20 /I 1.62 CH, t 0.83 6.8 2J 13.5 Boc s 1.3 1

8’ 1.48 yb 1.22 8Ala t 7.90 rxa’3.17 p 2.28 6.7-7.9

8‘2.21 7.8-8.0 25 14.9

D-Trp d 8.03 8.1 4.55 8 3.11 4.9 NH d 10.79 2.3

25 14.5 d 7.57 7.9 d 7.31 7.9 t 7.03 - t 6.95 1.4

~ - N l e d 7.90 8.2 4.28 8 1.62 CH,t 0.83 6.8

D - A s ~ d 8.29 7.7 4.55 88‘2.51

CHI t 2.85

8’ 2.92 8.9 s 7.10

p‘ 1.48 y6 1.22

t24.20 2.2-1.2 7 .O

CH,

C,H, 7.3-1 .O

398


TABLE 7 ‘H n.m.r. data (DMSO-d,) of Boc-Tyr(S0; )-Nle-BAla-o-Trp-Nle-Asp-0-CH,-CH,-C, H, ( 5 )

NH Ha HB Others 6(PPm) 3J(HZ) G(PPm) &PPm) 3J(Hz) 4Ppm) 3J(Hz)

TY r d 6.89 8.3 4.11 B 2.91 0, md7.12 8’2.69 10.5 d 7.05

25 13.8 Bocs 1.31

8’1.48 y6 I .23

8’2.23 7.3-7.3

Nle d7.81 8.1 4.19 B 1.60 CH, t 0.83 6.7

BAla t 7.90 5.3 aa’ 3.18 B 2.29 6.3-7.6

25 15.0 D-Trp d8.13 7.5 4.56 B 3.08 6.6 NH d 10.75 2.3

27 14.4 d 7.57 7.6 8’2.91 7.9 s7.12

d7.30 - t 7.03 -

t 6.95 7.4 Nle d 8.05 8.3 4.19 B 1.54 CH, t 0.77 7.2

8’1.34 66‘ 1.14 yy‘ 1.02

ASP d 8.28 7.8 4.56 B 2.63 5.8 8’ 2.55 7.2

27 16.6 t 4.20 7.0 t 2.85 7.0

7.3-7.0

TABLE 8 ‘H n.m.r. data (DMSO-d,) of Boc-Tyr(S0; )-Nle-BAla-Trp-D-Nle-o-Asp-O-CH,CH, -C,H, (6 )

NH Ha HB Others 6(PPm) 3J(Hz) &PPm) 6(ppm) 3J(Hz) &PPm) 3J(Hz)

d 6.89 8.5 4.1 1 p 2.92 0, md7.12 8’ 2.69 10.3 d 7.05

25 13.7 Bocs 1.31 Nle d 7.84 7.8 4.20 B 1.59 CH, t 0.84

8’1.51 y6 1.24 BAla t 7.93 5.4 aa‘3.18 BP2.26

TY r

6.9

TrP d8.14 7.5 4.55 B 3.08 6.7 NH d 10.75 2.2

25 14.5 d 7.58 7.9 d 7.30 7.9 t 7.03 -

t 6.95 7.3

8’2.91 8.3 s7.12

D-Nle d 8.06 8.3 4.20 B 1.55 CH, t 0.77 7.3

D-Asp d 8.28 7.8 4.55 B 2.63 5.7

8’ 1.36 66’1.14 yy‘ 1.02

8’2.55 7.2 25 16.6

CH2 t24.20 1.6-7.0 CH, t 2.85 7.0 c6 H5 7.3-7.0

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M. Rodriguez et al.

TABLE 9 Biological activities of cholecystokinin analogues substituted in position 29 by a j-alanine on the binding to pancreatic and brain CCK receptors and on amylase release from rat pancreatic acini. In each experiment, each value was determined in duplicate and results given are the means

from at least three separate experiments

Peptide derivatives Rat pancreatic acini

Amylase stimulation Binding

Guinea pig brain membranes

Binding max ( ~ I M ) IC, GM) IC, (phi) IC, (PM)

~ ~ ~

Boc-[NleZ8, Nle3’]-CCK-7 0.0003 0.0020 0.0003 Boc-Tyr(S0; )-Nle-PAla-Trp-Nle-Asp-Phe-NH, 1 0.02 0.2 0.003 Boc-Tyr(S0; )-Nle-BAla-D-Trp-D-Nle-D-Asp-o-Phe-NH, 2 > 100 2.0 2.0 Boc-Tyr(S0; )-Nle-BAla-Trp-Nle-Asp-0-CH, -CH, -C,H, 3 > loo 0.1 0.02

Boc-Tyr(S0; )-Nle-PAla-D-Trp-Nle-Asp-0-CH, -CH, -C, H, 5 antagonist 50 0.5 0.8 Boc-Tyr(S0; )-Nle-BAIa-Trp-D-Nle-D-Asp-O-CH,-CH,-C,H, 6 > too 0.3 0.1

Boc-Tyr(S0; )-Nle-flAla-D-Trp-D-Nle-D-Asp-O-CH2 -CH, -C,H, 4 antagonist 50 0.4 1.5

EXPERIMENTAL PROCEDURES

Chemistry Melting points were taken on a Buchi apparatus in open capillary tubes. Optical rotations were determined with a Perkin-Elmer 141 polarimeter. Elemen- tal analyses were performed by “Le Service de Microanalyses de I’ENSCM” (Montpellier, France). Ascending t.1.c. was performed on precoated plates of silica gel 60 F 254 (Merck) using the following solvent systems (by volume): A, AcOEt/hexane, 5 : 5 ; B, AcOEt/hexane, 7: 3; C, AcOEt; D, chloroform/methanol/acetic acid, 85: 10:5; E, AcOEt/pyridine/acetic acid/water, 80:20:5: 10. Peptide derivatives were located with U.V. light (254nm), charring reagent or ninhydrin. Column chromatographies were performed with silica gel 60, 60-229 mesh, ASTM (Merck). HPLC purifications were run on a Merck/ Hitachi instrument on a Beckman Ultrasphere ODS (0.5 pm) 10 x 250 mm column, with an U.V. detection at 279 nm, at a flow rate of 3 mL/min of a mixture of A: ammonium acetate 0 . 0 5 ~ , pH6.5, and B: methanol. ‘ H n.m.r. were run on a Brucker 360 instrument at 305K. Amino acids and derivatives were purchased from Bachem (Switzerland) or Novabio- chem (Switzerland). All reagents and solvents were of analytical grade. The following abbreviations were used: DMF, dimethylformamide; HOBT, l-hydroxy- benzotriazole; DCC, N,N’-dicyclohexylcarbodiimide, HOSu, N-hydroxysuccinimide; DIEA, N , N - diisopropylethylamine; BOP, benzotriazolyloxytris (dimethy1amino)phosphonium hexafluorophosphate; NMM, N-methylmorpholine; IBCF, isobutyl- chloroformiate; TFA, trifluoroacetic acid. Other abbreviations used were those recommended by the IUPAC-IUB Commission (European J . Biochem.

Peptides and peptide derivatives described in this work were synthesized according to the general procedures detailed in this section for the preparation of

1984, 138, 9-37).

compounds 2 and 4. Some peptide derivatives rnen- tioned in this study have already been described (14, 23, 29, 30).

Boc-Nle-j3Ala-OMe 7 To a cold (0’) solution of Boc-Nle (2.49g, 10.76 mmol) in DMF (20 mL) were successively added NMM (1.2mL, 10.76mmol) and IBCF (1.46mL, 10.76mmol). After 5 min stirring, 8-alanine methyl ester hydrochloride (1.67 g, 12 mmol) was added, followed by DIEA (2.06 mL, 12 mmol), and the solution was stirred for 30min at 0” and a further 30min at room temperature. The solvent was evaporated under reduced pressure and the residue was dissolved in ethyl acetate (200 mL). This solution was washed with 1 M potassium hydrogen sulfate (3 x IOOmL), water, saturated aqueous sodium bicarbonate (3 x 100 mL), brine, dried over magnesium sulfate and concentrated under reduced pressure to leave a residue that crystallized upon trituration in hexane. Yield 2.86 g (84%). Physical and analytical data are given in Table 2.

BOC- Tyr-Nle-PAla-OMe 8 Compound 7 (2.74g, 8.66mmol) was partially deprotected with TFA (20 mL). After standing at room temperature for 30 min, the solvent was concentrated under reduced pressure at t < 40’and dried in vacuo over KOH pellets. It was added to a solution of Boc- Tyr (2.39 g, 8.5 mmol) in DMF (20mL) and BOP (22) (3.76 g, 8.5 mmol) followed by NMM (I .92 mL, 17.16 mmol), and the solution was stirred for 1 h at room temperature. The solvent was concentrated under reduced pressure and the residue was dissolved in ethyl acetate (200mL). The solution was washed with 1 M potassium hydrogen sulfate (3 x IOOmL), water, saturated aqueous sodium bicarbonate (3 x lOOmL), brine, dried over magnesium sulfate and concentrated under reduced pressure to leave a residue which crystallized upon trituration with

400


hexane. Yield 2.50 g (69%). Physical and analytical data are given in Table 2.

Boc- Tyr-Nle-PAla-OH 9 To a cold (0") solution of compound 8 (2.30g, 4.79mmol) in methanol (10mL) was added 1 N sodium hydroxide (IOmL, IOmmol) and the mixture was stirred at room temperature until t.1.c. showed disappearance of starting material. The reaction mixture was diluted with water (100 mL), extracted with ether (3 x 50mL), acidified to pH 3 with 1 M aqueous potassium hydrogen sulfate and extracted with ethyl acetate (3 x 80mL). The combined organic layers were washed with 1 M aqueous potassium hydrogen sulfate (100 mL), brine, dried over magnesium sulfate and concentrated under reduced pressure to leave a residue which crystallized upon trituration with a mixture of ether and hexane. Yield 2.13 g (95%). Physical and analytical data are given in Table 2.

Boc- Tyr-Nle-BAla-OSu 10 To a cold (0") solution of 9 (2.0g, 4.3mmol) in di- methoxyethane (80 mL) were successively added HOSu (0.69 g, 6.0 mmol), DCC (0.887g, 4.3 mmol). After 4 h stirring at room temperature, the precipitated DCU was filtered off, the solvent was concentrated under reduced pressure to leave a residue which was dissolved in AcOEt (100mL). The solution was washed with cold 2% aqueous sodium bicarbonate (3 x IOOmL), water, 1 M aqueous potassium hydrogen sulfate (100 mL), brine, dried over magnesium sulfate and concentrated under reduced pressure to leave a residue that crystallized upon trituration with ether. Yield 1.53 g (63%). Physical and analytical data are given in Table 2.

Boc- asp (OBzl) -D-Phe-NH2 1 1 To a cold (0") solution of Boc-~-Asp(OBd) (1.5 g, 4.64 mmol) in DMF (20 mL) were successively added NMM (0.52mL, 4.64mmol) and IBCF (0.63mL, 4.64 mmol). After 5 min stirring, D-Phe-NH, (0.821 g, 5mmol) was added and the solution was stirred for 30 min at 0" and further 30 min at room temperature. The expected compound precipitated upon addition of aqueous 1 M potassium hydrogen sulfate (200 mL). It was collected by filtration, washed with 1 M potassium hydrogen sulfate, water, saturated aqueous sodium bicarbonate, water and dried in vacuo over phosphorous pentoxide. Yield 2.15 g (98%). Physical and analytical data are given in Table 2.

Boc-D Nle-DAsp (OBzl) -I) Phe- NH, 13 Compound 11 (2.0g 4.26mmol) was partially deprotected with TFA (20mL). After standing at room temperature for 30 min, the solvent was concentrated under reduced pressure at t < 40". The residue crystallized upon trituration with ether. It was collected by

filtration, washed with ether and dried in vacuo over KOH pellets. It was added to a solution of Boc-D-Nle- OSu (21) (1.25 g, 3.8 mmol) in DMF (20mL), followed by DIEA (0.73 mL, 4.26 mmol), and the solution was stirred for 3 h at room temperature. The reaction mixture was treated as described for compound 11. Yield 1.9Og (86%). Physical and analytical data are given in Table 2.

Z-D Trp-D-Nle-DAsp (OBzl) -o-Phe-NH, 15 Compound 13 (1.45g, 2.49mmol) was partially deprotected with TFA as described above. The partially deprotected peptide was dissolved in DMF (20 mL) containing 2-D-Trp (0.842 g, 2.49 mmol) and BOP (22) ( ] . log, 2.49mmol). The solution was cooled down to 0" and NMM (0.56mL, 4.98mmol) was added. After 2h stirring at room temperature, the expected compound precipitated upon addition of saturated aqueous sodium bicarbonate (200 mL). It was collected by filtration, washed with saturated aqueous sodium bicarbonate, water, 1 M potassium hydrogen sulfate, water and dried in vacuo over phosphorous pentoxide. Yield I .85 g (92%). Physical and analytical data are given in Table 2.

Boc- Tyr- Nle-BAla-D Trp-D-Nle-&Asp-DPhe-NH, 19 Compound 15 (0.802 g, 1 .O mmol) was hydrogenated for 3 h in a mixture of DMF, acetic acid and water (25:5:5) (50mL) in the presence of a 10% PdjC catalyst at room temperature and atmospheric pressure. The catalyst was filtered off, and the filtrate concentrated under reduced pressure to leave a residue which crystallized upon trituration with ether. It was collected, washed with ether and dried in vucuo over KOH pellets. It was added to a solution of compound 10 (0.450g, 0.8mmol) in DMF (5mL), followed by DIEA (0.17mL, l.Ommol), and the solution was stirred for 3 h at room temperature. The expected compound precipitated upon addition of aqueous I M potassium hydrogen sulfate (100 mL). It was collected by filtration, washed with 1~ potassium hydrogen sulfate, water, and dried in vacuo over phosphorous pentoxide. Yield 0.792 g (96%). Physical and analytical data are given in Table 2.

Boc- Tyr (SO; ) - Nle-8Ala-D- T r p - ~ N l e - asp-~Phe- NH, 2 To a solution of compound 19 (0.250 g, 0.243 mmol) in a mixture of DMF (2 mL) and pyridine (2 mL) was added SO,-pyridine complex (1.5 g). After overnight stirring at room temperature, the solvents were concentrated under reduced pressure and the excess of complex was hydrolyzed with water (10mL) for 30min, while the pH was maintained around 7-8 by addition of 10% aqueous sodium carbonate. The solution was then acidified to pH 5 by addition of 1 M aqueous KHSO, and extracted with n-BuOH (3 x 20mL). The organic phases were washed with

40 1

M. Rodriguez et al.

water and concentrated under reduced pressure to leave a solid residue which was triturated with ether, collected and dried in vucuo. It was purified by silica gel chromatography (eluent (solvent E): 80:20: 5 : 10) to afford compound 2 as a white solid. Yield 0.075g (28%). Physical and analytical data are given in Table 1.

BOC-D- ASP ( OBZI) -0-CH2 -CH,-C, Hj 12 To a suspension of a Boc-~-Asp(OBzl) (3.23g, 10 mmol) in benzene (80 mL) was added DBU (1.49 mL, 10 mmol), followed by 2-phenylethyl bromide (1.36mL, IOmmol), and the mixture was stirred 2 h at reflux. Ethyl acetate (l00mL) was then added, and the resulting solution was washed with 1 M aqueous potassium hydrogen sulfate (3 x IOOmL), water, saturated aqueous sodium bicarbonate (3 x lOOmL), brine, dried over magnesium sulfate and concentrated under reduced pressure to afford 12, which crystallized on standing at 0" for several days. Yield 3.68g (86%). Physical and analytical data are given in Table 2.

Boc-D- Nle- asp ( OBzl) -0-CH, -CH, -C, H5 14 Compound 12 (3.68g, 8.61 mmol) was partially deprotected with TFA (20 mL). After standing at room temperature for 30 min, the solvent was concentrated under reduced pressure at t < 40". The residue was dried in vucuo over KOH pellets, added to a solution of Boc-D-Nle-OSu (21) (2.46g, 7.50mmol) in DMF (20mL), followed by DIEA (1.48 mL, 8.61 mmol), and the solution was stirred for 3 h at room temperature. The reaction mixture was treated as described for compound 7. Yield 3.52 g (87%). Physical and analytical data are given in Table 2.

Z-D- Trp-DN1e-o- Asp (0 Bzl) -0- CH, - CH, -C, H5 16 Compound 14 (l.Og, 1.85mmol) was treated with TFA (IOmL) as described above. The partially deprotected peptide was dissolved in DMF (20mL) containing Z-D-Trp (0.643 g, I .90 mmol) and BOP (22) (0.840 g, 1.90 mmol). The solution was cooled down to 0" and NMM (0.42 mL, 3.75 mmol) was added. After 2 h stirring at room temperature, the expected compound precipitated upon addition of saturated aqueous sodium bicarbonate (200 mL). It was collected by filtration, washed with saturated aqueous sodium bicarbonate, water, 1 M potassium hydrogen sulfate, water and dried in vucuo. Yield 1.34g (96%). Physical and analytical data are given in Table 2.

Boc- Tyr- Nle-BAla-5 Trp-D-Nle-DAsp-O-CH,-CH2-

Compound 16 (0.761 g, I .O mmol) was hydrogenated for 3 h in a mixture of DMF, acetic acid and water (25:3:3) (50mL) in the presence of a 10% PdjC catalyst at room temperature and atmospheric pressure. The catalyst was filtered off, and the filtrate con- 402

C,H, 21

centrated under reduced pressure to leave a residue which crystallized upon trituration with ether. It was collected, washed with ether and dried in vucuo over KOH pellets. It was added to a solution of compound 10 (0.450g, 0.8mmol) in DMF (5mL), followed by DIEA (0.17mL, I.Ommol), and the solution was stirred for 3 h at room temperature. The expected compound precipitated upon addition of aqueous I M potassium hydrogen sulfate (100 mL). It was collected by filtration, washed with 1 M potassium hydrogen sulfate, water, and dried in YUCUO over phosphorus pentoxide. Yield 0.577 g (73%). Physical and analytical data are given in Table 2.

Boc- T y r ( S 0 ; ) - N l e - ~ A l a - ~ T r p - ~ N l e - ~ A s p - O -

Synthesized as described for compound 2 from compound 21 (0.250 g, 0.254 mmol). Yield 0.1 10 g (41 YO). Physical and analytical data are given in Table 1.

CHJ-CH,-C, HJ 4

Biological evaluations Male guinea pigs (280-3OOg) were obtained from le Centre d'Elevage d'Animaux de Laboratoire (Ardenay, France); male Wistar rats (1 80-200 g) were from Effa-Credo (Saint Germain l'Arbresle, France). Hepes was from Boehringer-Mannheim; purified col- lagenase was from Serva (Garden City Park, NY); soybean trypsin inhibitor from Sigma (St Louis, MO); Eagle's basal amino acid medium (100 times concentrated) was from GIBCO (Grand Island, NY); essential vitamin mixture (100 times concentrated) was from Microbiological Associates (Bethesda, MD); Bovine Plasma Albumin (fraction V) was from Miles Laboratories Inc. (Elkhart, IN); Phadebas@ amylase test was from Pharmacia Diagnostics (Pis- cataway, NJ) and 12s1-labeled N-succinimidyl-3-(4- hydroxypheny1)propionyl-CCK-8 ('251-BH-CCK-8) was from Amersham Corp. (Buckinghamshire, UK). Unless otherwise stated, the standard incubation sol- utions contained 24.5 mM Hepes (pH 7.4), 98 mM NaCl, 6 m ~ KCI, 2 . 5 m ~ NaH,PO,, 5 m ~ sodium pyruvate, 5 mM sodium fumarate, 5 mM sodium gluta- mate, 2 m~ glutamine, 1 1.5 m~ glucose, 0.5 m~ CaCL, 1 mM MgCl,, 0.5mg/mL bacitracin, 0.2% (w/ v) albumin, 0.03% (w/v) soybean trypsin inhibitor, and 1 YO (v/v) essential vitamin mixture. The incubation solution was equilibrated with 95% 02, 5% CO, as the gas phase.

Dispersed acini from rat pancreas were prepared according to the previously described modifications (4) of the methods of Peikin et al. (31) described for isolated guinea pig pancreatic acini. Guinea pig brain membranes were prepared following the procedures described by Pelaprat e f al. (26).

Amylase release was measured using the procedure already described (25). Briefly, acini were resuspended in the standard incubation solution complemented with 1% bovine serum albumin, I mM calcium, and

5 mM theophylline containing about 1 mg protein/mL, and samples (1 mL) were incubated at 37" for 30min. Amylase activity was determined by the method of Ceska et al. (32) using the Phadebas reagent. Amylase release was measured as the difference of amylase activity at the end of incubation that was released into the extracellular medium, with and without secretagogue and expressed as the percentage of maximal stimulation obtained with Bo~- [Nle~~ ,~ ' ] - CCK-7 (40 f 5% of the total amylase contained in the acini) minus the basal amylase secretion (10 k 2% of the total amylase contained in the acini) obtained without secretagogue.

Binding of '251-CCK-8 to rat pancreatic acini was performed as previously described (25). Briefly, samples (0.5 mL containing z 1 mg/mL protein) were incubated with the appropriate peptide concentrations for 30min at 37" in the presence of lOpM of '251-CCK-8 plus various concentrations of Boc [Nle2833']CCK-7. After centrifugation at 10 000 g for 10 min and washings, the radioactivity associated with the acinar pellet was measured. Values are expressed as the percentage of the value obtained with labeled CCK-8 alone. The specific activity of the various preparations used in our experiments was 2000 Ci/ mmol. Acini from three rat pancreata were suspended in 100 mL of standard incubation solution. Specific binding in the absence of any unlabeled CCK-peptide was 13 f 3% of the total radioactivity present in the sample. Non-specific binding was determined in the presence of 1 ,DM B O C [ N ~ ~ ~ ~ ~ ~ ' ] C C K - ~ and was always less than 15% of the total binding.

Binding of 1251-CCK-8 to guinea pig or mouse brain membranes was performed according to Pelaprat et al. (26). The buffer used was 50 mM (Tris) HCl, 5 mM MgCl,, 0.1 mg/mL bacitracin, pH 7.4 (Tris-MgC1,- bacitracin buffer). Briefly, displacement experiments were performed by incubation of 1 mL of brain membranes (approximately 0.5 mg protein) in the presence of 15 PM '251-CCK-8 for 60 min at 25" in the presence of various concentrations of B o c [ N ~ ~ ~ ~ , ~ ' ] C C K - ~ or compounds to be tested in a total volume of 1 mL. Non-specific binding was determined in the presence of 1 PM B o c - [ N ~ ~ ~ ~ , ~ ' ] - C C K - ~ and was always less than 25% of the total binding. Total binding was about 10% of the total radioactivity contained in the sample.

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Lignon, M.F., Galas, M.C., Rodriguez, M., Guillon, G., Jard. S . & Martinez, J., (1987) in Gastrin and Cholecystokinin, Chemistry, Physiology and Pharmacology (Bali, J.P. & Mar- tinez, J., eds.), pp. 57-61, Elsevier Sciences Pub. Excerpta Medica, Amsterdam Martinez, J., Rodriguez, M., Lignon, M. & Galas, M.C. (1988) in Cholecystokinin Antagonists, Neurology and Neuro- biology (Wang, R.Y. & Schoenfeld, R., eds.), Vol. 41, pp.29-51, Alan R. Liss, New York

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5 .

8.

9.

10.

11.

257, 604-605

67-70

GI 76-GI82 16.

17.

7226-7231

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Address:

Jean Martinez Centre de Pharmacologie-Endocrinologie CNRS-INSERM Rue de la Cardonille 34094 Montpellier Cedex 2 France

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synthesis and biological activities of some cholecystokinin analogues substituted in position 29 by...

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