CCK peptides with combined features of hexa- and tetrapeptide CCK-A agonists

Article abstract of DOI:10.1021/jm990252e

Selective CCK-A agonist activity has been reported to induce satiety in a variety of animals, including man, and thereby suggests a therapeutic role for CCK in the management of obesity. To date, three general classes of CCK-A agonists have been reported,

Full text of DOI:10.1021/jm990252e

2350
J . Med. Chem. 2000, 43, 2350-2355
CCK P ep tid es w ith Com bin ed F ea tu r es of Hexa - a n d Tetr a p ep tid e CCK-A
Agon ists
M. Edward Pierson,* J eanne M. Comstock, Roy D. Simmons, Ronald J ulien, Frederick Kaiser, and
J ames D. Rosamond
AstraZeneca R&D Boston, Three Biotech, One Innovation Drive, Worcester, Massachusetts 01605
Received May 20, 1999
Selective CCK-A agonist activity has been reported to induce satiety in a variety of animals,
including man, and thereby suggests a therapeutic role for CCK in the management of obesity.
To date, three general classes of CCK-A agonists have been reported, the full-length, sulfated
hepta- and hexapeptides, a series of tetrapeptides, and most recently a series of benzodiazepines.
The SAR of the hexa- and tetrapeptide classes suggests that the Hpa(SO3H) and Tac groups
may not interact at the CCK-A receptor in the same location. However, the C-terminal dipeptide
part of the hexapeptides and tetrapeptides appear to interact at the CCK-A receptor in a similar
manner. Compound 7 (Hpa-Nle-Gly-Trp-Lys(Tac)-Asp-MePhe-NH2) derived from combining
the features of the hexapeptides and the tetrapeptides has subnanomolar affinity and 3500-
fold selectivity for CCK-A receptors. Compound 7 administered intraperitoneally produces
potent, long-lasting reduction in food intake in rats and a corresponding weight loss when
administered over nine consecutive days.
In tr od u ction
CCK-A receptor similarly to the hydroxy of Tyr7 in CCK-
8. However, sulfation of the 3- or 4-hydroxy groups only
led to a doubling of CCK-A affinity, suggesting that
these groups did not occupy the same space on the
CCK-A receptor as the sulfated tyrosine in CCK-8.14
Holladay methylated the amide nitrogen of each residue
of the tetrapeptide one residue at a time, finding that
the rank order of CCK-A affinities for the methylated
tetrapeptides paralleled exactly the rank order of the
CCK-A affinities of the corresponding methylated hep-
tapeptides.15 Here we report some of our findings on
comparison of the hexa- and tetrapeptides and studies
that led to hexapeptides incorporating the features of
the tetrapeptides.
Cholecystokinin (CCK), a peptide hormone found in
the central nervous system and gastrointestinal tract,
occurs in multiple biologically active forms (CCK-58,
CCK-39, CCK-33, CCK-8, and CCK-4) with CCK-8 (see
Table 1 for sequence) predominating.1,2 CCK mediates
many diverse hormonal and neuromodulatory functions
through the action of two CCK receptor subtypes,
CCK-A and CCK-B.3,4 Peripheral CCK-A receptors
mediate gall bladder contraction, pancreatic exocrine
secretion, and satiety.3,5
Three general classes of selective CCK-A agonists
have been identified; full-length, sulfated hepta- and
hexapeptides,6-8 tetrapeptides,9,10 and recently benzo-
diazapines.11 Very recently Bignon et al. reported a
thiazole with CCK-A agonist activity, but limited SAR
information is available.12,13 A hypothesis has been
proposed to describe how the related classes of peptide
agonists bind to the G-protein-coupled CCK-A recep-
tor.14,15 In general, a sulfated tyrosine or tyrosine mimic
at position 7 of CCK-8 is required for high affinity at
CCK-A receptors because nonsulfated peptides exhibit
100-1000-fold less affinity than the corresponding
sulfated peptides.
Meth od s
Tetrapeptides were prepared by standard solution
phase mixed anhydride coupling procedures using isobu-
tylchloroformate to form the mixed anhydride. Side
chain protecting groups of lysine and aspartic acid were
removed simultaneously with catalytic hydrogenation
to yield the N-terminal Boc protected peptides. The
ꢀ-amino group of lysine was allowed to react with
2-methylphenyl isocyanate to generate the final urea-
containing peptide. Unsulfated hexapeptide amides
were synthesized utilizing the Fmoc/tBu protection
strategy on RINKMBHA resin (Milligen/Biosearch).16
Cleavage from the resin was accomplished using reagent
K (TFA, H2O, thioanisole, phenol, ethanedithiol, 185:
5:5:5:2) for 2 h at room temperature.17 The crude
peptides were purified to homogeneity using preparative
reverse phase HPLC. The ꢀ-amino group of lysine was
allowed to react with either 2-methylphenyl isocyanate
or 4-hydroxycinnamic acid N-hydroxysuccinimide ester
to generate the final peptides. Sulfation was achieved
using pyridine‚SO3, and the final products were purified
to homogeneity using reverse phase HPLC and charac-
The suggestion that sulfated hexapeptides were re-
quired for high affinity at CCK-A receptors was brought
into question with the discovery of potent, CCK-A
selective, tetrapeptide agonists.10 An important issue
regarding this finding is, how do the tetrapeptides relate
to the hexapeptides? To address this question, Shiosa-
ki14 and Holladay15 generated sulfated and methylated
tetrapeptide compounds. Shiosaki14 found that the
4-hydroxyphenylpropionyl group attached to the ꢀ-amine
of lysine had twice the affinity of the unsubstituted
phenylpropionyl or the 3-hydroxyphenylpropionyl group.
This finding led them to speculate that the hydroxy
group of 4-hydroxyphenylpropionyl interacted at the
10.1021/jm990252e CCC: $19.00 © 2000 American Chemical Society
Published on Web 06/15/2000

Hexa- and Tetrapeptide CCK-A Agonists
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2351
Ta ble 1. Receptor Affinitya and Selectivityb
Ki (nM)c
CCK-A
no.
structure
CCK-A
CCK-B
selectivity
1
2
3
4
5
6
7
8
9
Hpa(SO3H)-Nle-Gly-Trp-Nle-MeAsp-Phe-NH2
Hpa-Nle-Gly-Trp-Nle-MeAsp-Phe-NH2
0.03 ( 0.005
224 ( 19
960
580
>1000
ND
36
200
123
84
6590
228
72
>2000
ND
11
4000
6150
86
4.2
Boc-Trp-Lys(Tac)-D-Asp-Phe-NH2
Boc-Trp-Lys(Tac)-D-Asp-MePhe-NH2
Hpa-Nle-Gly-Trp-Nle-Asp-MePhe-NH2
8.1
0.53
51
Hpa-Nle-Gly-Trp-Lys(Hci)-Asp-MePhe-NH2
Hpa-Nle-Gly-Trp-Lys(Tac)-Asp-MePhe-NH2
Hpa(SO3H)-Nle-Gly-Trp-Lys(Tac)-Asp-MePhe-NH2
Hpa(SO3H)-Met-Gly-Trp-Met-D-Asp-MePhe-NH2
Hpa(SO3H)-Met-Gly-Trp-Met-D-Asp-Phe-NH2
Boc-Trp-Lys(Tac)-Asp-MePhe-NH2
3.4
0.05 ( 0.007
0.02
0.98
19
10
11
CCK-8
150
250
0.41 ( 0.06
8
833
5
0.30 ( 0.05
0.09 ( 0.009
H-Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2
a
b
Assays and data analysis are summarized in the Experimental Section. CCK-A selectivity is the ratio of CCK-B to CCK-A binding
affinity. ND, not determined. c The values without standard errors are the results of one experiment performed in triplicate and the
individual determinations varied by less than 20%.
terized by analytical reverse phase HPLC, mass spec-
trometry, and amino acid analysis.
CCK-A receptor affinity was measured by displace-
ment of [125I]BH-CCK-8 from rat pancreatic tissue
according to the general procedure of Chang.18 CCK-B
receptor affinity was measured by displacement of
8.1 nM vs 0.53 nM). Combining these results with the
methylation study conducted by Holladay suggests that
the C-terminal portion of the hexa- and tetrapeptides
may occupy similar locations at the CCK-A receptor. It
may be that the CCK-A receptor requirements are less
stringent for the C-terminal portion of the peptides than
the CCK-B receptor.
[
125I]BH-CCK-8 from rat cortex synaptosomes according
to the general procedure of Chang.19 The ability of a
compound to inhibit food intake and to promote weight
loss in rats was measured by a modification of the
method described by Cox and Maickel.20
The studies described above led us to consider replac-
ing the Nle corresponding to Nle3 of 5 with the Lys(Tac)
(i.e., Lys(2-tolylaminocarbonyl)) group found in the
tetrapeptides. This change generates the hybrid pep-
tides 6-8. Generally, nonsulfated hexapeptides such as
5 have low affinity for CCK-A receptors; 2 is a notable
exception but still has 100-fold less affinity than 1, its
sulfated derivative. The hybrid hexapeptides 6 and 7
have very good affinity and selectivity for the CCK-A
receptor, even without a sulfate group. Sulfation of the
Hpa phenol of 7 to give 8 did not change the CCK-A
affinity; usually sulfation in the hexapeptides increase
affinity for CCK-A receptors 100-1000-fold. This is
similar to what Shiosaki et al.14 found when sulfating
a phenolic group attached to the side chain amine of
Lys in the tetrapeptide series. It is not completely clear
why sulfation of the hybrid compound 7 did not result
in an increase in affinity.
Resu lts a n d Discu ssion
AR-R 158496 (1) is a sulfated hexapeptide (see Table
1 for sequence) containing all of the elements classically
described as required for CCK-A agonism. Generally a
sulfated tyrosine or tyrosine mimic at position 7 of
CCK-8 is required for high affinity at CCK-A receptors
because nonsulfated peptides exhibit 100-1000-fold less
affinity than the corresponding sulfated peptides. The
selectivity of 1 results from N-methylation of Asp (see
ref 6), but the sulfate group on the hydroxyphenylacetic
acid (a tyrosine mimic) contributes 2 orders of magni-
tude to the CCK-A affinity (compare 1 with 2). The
nonsulfated compound 2, while still having affinity for
CCK-A receptors (Ki ) 4.2 nM vs 0.03 nM), has much
less than 1 and is much less selective for CCK-A
receptors than 1 (230-fold vs 6590-fold). Correspond-
ingly, compound 2 is also 52-fold less potent in inhibit-
ing food intake following ip administration in rats (ED50
3 h feeding ) 13 µg/kg vs 0.25 µg/kg). These results show
that sulfation is important for high receptor affinity and
potency while N-methylation of Asp is important for
CCK-A selectivity.
In another hexapeptide series, inversion of the ster-
eochemistry of Asp, to give D-Asp, in conjunction with
methylation of the amide nitrogen of Phe, to give
MePhe, leads to hexapeptides with similar CCK-A
affinity but reduced CCK-B affinity, resulting in in-
creased CCK-A selectivity.6 Thus, in the hexapeptide
series the combination of D-Asp, MePhe leads to potent
selective agonists, whereas the D-Asp, Phe analogue has
less affinity for both CCK-A and CCK-B receptors
(compounds 9 and 10 of Table 1 from ref 6). In the
tetrapeptide series, we also found that the D-Asp, Phe
analogue 3 had reduced affinity for CCK-A receptors,
when compared to the D-Asp, MePhe analogue 4 (Ki )
All of the peptides inhibited food intake in a dose
dependent manner. Figure 1 shows the inhibition of the
food intake dose-response curve for compound 7 in rats
following ip administration. Compound 7 more potently
inhibited food intake following intraperitoneal admin-
istration than the sulfated analogue 8 or the tetrapep-
tide 1121 (Table 2). To examine the weight loss produc-
ing effects of 7, the compound was administered to rats
for 9 consecutive days (Figure 2). Acutely, a 0.8 µg/kg
dose of 7 was found to inhibit food intake by 65%, while
a 0.3 µg/kg dose was found to inhibit feeding by 30%.
Over the 9-day study, 7 dose dependently induced
weight loss. At the higher dose (0.8 µg/kg) 7 produced a
3% weight loss after the first dose that was maintained
over the course of the study. The lower dose (0.3 µg/kg)
produced a slight decease in weight for the first 6 days
of the study, but as the study progressed, the animals
gained a small amount of weight, as compared to their
predose weights. However, even at the low dose the rats
weighed significantly less on day 9 than control animals
that had received saline injections for 9 days. It should

2352 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
Pierson et al.
hybrid peptide 7 was found to be a potent, selective, fully
efficacious CCK-A agonist even without sulfation. This
compound produced dose dependent weight loss over a
9-day study period, suggesting that CCK-A agonists
may be useful anorectic agents.
Exp er im en ta l Section
Abbr evia tion s. Peptide and amino acid abbreviations used
follow the guidelines of the IUPAC-IUB J oint Commission
on Biochemical Nomenclature22 and are of the L-configuration
unless otherwise noted. AAA, amino acid analysis; Boc, tert-
butyloxycarbonyl; Gly, glycine; Lys, lysine; MeAsp, N-methyl-
aspartic acid; MePhe, N-methylphenylalanine; Nle, norleucine
(2-aminohexanoic acid); Phe, phenylalanine; Trp, tryptophan.
Other abbreviations are as follows: CMA, chloroform-
methanol-acetic acid; DCM, methylene chloride; DIEA, N,N-
diisopropylethylamine; DMF, dimethylformamide; EtOAc, eth-
yl acetate; Hci, 4-hydroxycinnamoyl; HOBt, 1-hydroxybenzo-
triazole; Hpa, 4-hydroxylphenylacetyl; Hpa(SO3H), sulfated
4-hydroxyphenylacetyl; ip, intraperitoneal; OSu, succinimidyl-
oxy ester; Piv-Cl, pivaloyl chloride; Pyr, pyridine; Pyr‚SO3,
pyridine-sulfur trioxide complex; Tac, 2-tolylaminocarbonyl;
TBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tet-
rafluoroborate; TFA, trifluoroacetic acid; tR, HPLC retention
time.
F igu r e 1. Percent inhibition of food intake of 7 in rats 0.5
and 3 h following ip administration. Ten rats were tested per
dose (0.03, 0.3, 3.0, 30, 300 µg/kg). Half-hour and 3 h data were
taken from the same experiment. Assay and data analysis
summarized in the Experimental Section.
Ta ble 2. Feeding Inhibition Potencya
feeding inhibition (ED50, µg/kg)
P ep tid e P u r ifica tion . Preparative reverse phase HPLC
(Prep-LC) was conducted using an Amicon C18 column
(500 × 50 mm, 20 µm, 100 Α). Peptides were eluted using a
110 min gradient of 40-60% solvent B [0.025% TFA (solvent
A) and 0.025% TFA in CH3CN (solvent B)] at 25 mL/min. Pure
fractions were freeze-dried from H2O/CH3CN.
no.
0.5 h
3 h
1
2
7
8
0.11 ( 0.02
3.8 ( 0.7
0.25 ( 0.01
13 ( 2.7
0.81 ( 0.2
1.2 ( 0.2
3.2 ( 0.2
112 ( 3.8
0.27 ( 0.04
0.85 ( 0.07
0.9 ( 0.2
11
CCK-8
An a lytica l HP LC. Analytical reverse phase HPLC was
1.2 ( 0.1
performed on
a Hewlett-Packard 1050 system using the
a
Assays and data analysis are summarized in the Experimental
Section.
following columns and mobile phases. Columns: (I) Phenom-
enex Nucleosil C18 (150 × 3.2 mm, 5 µm, 100 Å) eluted at 0.5
mL/min, (II)) Phenomenex Columbus C18 (150 × 3.2 mm, 5
µm, 100 Å) eluted at 0.5 mL/min, or (III) Keystone Nucleosil
C18 (150 × 4.6 mm, 5 µm) heated at 60 °C and eluted at 1.0
mL/min. Mobile phases: (a) 20 min gradient of 20-80% solvent
B [0.05% TFA (solvent A) and 0.05% TFA in CH3CN (solvent
B)], (b) 30 min gradient of 0-100% solvent B [0.1% TFA
(solvent A) and 0.1% TFA in CH3CN (solvent B)], (c) 15 min
gradient of 35-60% solvent B (solvents same as in mobile
phase a), (d) 15 min gradient of 25-50% solvent B (solvents
same as in mobile phase a) By example, the term “HPLC (Ia)”
is used below to describe HPLC column I with mobile phase
a. All analytes were determined to be >95% pure when
monitored at 220 nm.
Gen er a l Meth od for Solid -P h a se P ep tid e Syn th esis.
The nonsulfated peptide amides were prepared on a MilliGen/
Biosearch 9600 synthesizer using standard solid-phase syn-
thesis techniques and the Fmoc/tBu protection strategy.16
Syntheses were performed starting with the RINKMBHA resin
(1.0 g, 0.30-0.43 mmol). Fmoc group removals were carried
out by treatment with piperidine/DMF. Double couplings were
performed using 6.67 equiv of Fmoc-amino acid with equiva-
lent amounts of N,N-diisopropylcarbodiimide and HOBt in
DMF (1-2 h). NH2-Terminal acylation was conducted using
6.67 equiv of Hpa-OSu in DMF (1-2 h). Completed peptidyl
resins were cleaved and deprotected using reagent K (TFA,
H2O, thioanisole, phenol, ethanedithiol, 185:5:5:5:2) for 2 h at
room temperature.17 The crude unsulfated peptide amides
were purified to homogeneity by Prep-LC. Fractions shown by
HPLC to be >95% pure were pooled and lyophilized to provide
unsulfated peptide amide products.
All peptides were found to have the expected composition
as determined by acid hydrolysis and amino acid analysis.
Peptide identity was further confirmed by mass spectral
analysis (MS, electrospray) with the expected (M - H) anionic
molecular ion peak observed for all peptides. Peptide purity
was determined by HPLC to be >98% when analyzed on two
different columns (Table 3).
F igu r e 2. Body weight change effect of 7 in rats following
daily ip dosing over 9 days. Values for day 2-9 for both doses
of 7 are significantly different from the control (P < 0.05,
ANOVA, Newman-Keuls). Assay and data analysis are sum-
marized in the Experimental Section.
be noted that the control group of rats continued to gain
weight in the expected manner throughout the study.
Previously, we have discussed our efforts to identify
and evaluate sulfated, hexapeptide-based, selective
CCK-A agonists. Compound 1 is such a compound that
produces weight loss following ip administration in a
9-day study. Abbott9,10 scientists have disclosed the
CCK-A agonist activity of a series of tetrapeptides. Here
we report that combining the features of the hexapep-
tides and the tetrapeptides successfully generates
hexapeptides that no longer require sulfation for high
affinity and good in vivo potency. The SAR of the hexa-
and tetrapeptides classes of molecules suggests that the
Hpa(SO3H) and Tac groups may not interact at the
CCK-A receptor in the same location, but the C-terminal
dipeptide part of the hexapeptides and tetrapeptides
interact at the CCK-A receptor in a similar manner. The
Meth od A: Boc-D-Asp (OBn )-MeP h e-NH 2 (11). Gen er a l
P iv-Cl Meth od . To a stirred, ice-cold solution of Boc-D-

Hexa- and Tetrapeptide CCK-A Agonists
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2353
Ta ble 3. Analytical Data
MS ES
HPLC tR
m/z
amino acid analysis
no.
Nle
Gly
Trp
Lys
Asp
Phe
MePhe
(M - H)
Ia
IIb
3
4
5
6
7
8
0.74
0.98
0.53
0.94
0.97
1.03
1.08
1.00
1.10
1.01
1.01
1.03
0.95
827
826
880
1056
1084
1122
14.08
14.22
12.86
10.88
13.20
20.35
20.44
18.92
17.74
19.18
2.11
0.91
1.04
1.10
1.01
1.07
1.04
1.04
0.89
0.91
0.98
0.92
0.96
0.84
0.94
0.93
Asp(OBn)-OH (10.87 g, 33.6 mmol.) in 300 mL of THF were
added Piv-Cl (4.14 mL, 33.6 mmol) and DIEA (16.7 mL, 96
mmol). After 30 min, a cold solution of H-MePhe-NH2‚HCl
(6.87 g, 32 mmol) was added. While the pH was maintained
at 7 using DIEA (2.8 mL, 16 mmol), the reaction was stirred
at room temperature for 3.5 h. The solvent was removed in
vacuo, and the resulting oil was dissolved in EtOAc; washed
with 1 M KHSO4, H2O, saturated NaHCO3, and saturated
NaCl; dried (MgSO4); and evaporated to a foam: TLC (CM,
9:1, NH3 plate) Rf 0.53.
Meth od B: H-D-Asp (OBn )-MeP h e-NH2‚HCl (12). Gen -
er a l Boc Dep r otection Meth od . To a stirred, -10 °C
solution of 11 (1.5 g, 3.1 mmol) in EtOAc (100 mL) was added
HCl gas to saturation (10 min). The solution was allowed to
warm to room temperature over 1 h. The solution was reduced
in vacuo to a small volume, and hexanes were added with
cooling to -20 °C. The white precipitated product was collected
by filtration to yield 1.30 g (3.1 mmol). TLC: (CMA 9:1:1) Rf
0.2. The product was used as is.
Meth od C: BocLys(Z)-D-Asp (OBn )-MeP h e-NH2 (13).
Gen er a l IBCF Meth od . To a stirred, iced solution of Boc-
Lys(Z)-OH (1.29 g, 3.4 mmol) in DMF (25 mL) were added
DIEA (0.59 mL, 3.4 mmol) and isobutylchloroformate (0.44 mL,
3.4 mmol). After 10 min, a cooled solution of H-D-Asp(OBn)-
MePhe-NH (0.54 mL, 3.1 mmol) in 25 mL of DMF was added.
After 2 h the reaction was filtered, the solvent was removed
in vacuo. The resulting oil was dissolved in EtOAc; washed
with 1 N NaHCO3, cold 1 N HCl, and saturated NaCl; dried
(MgSO4); and evaporated to an oil. The crude was purified on
a flash SiO2 column using CHCl3. Evaporation in vacuo of the
product fractions gave 2.3 g of 13 that was used as is: TLC
(CMA 9:1:1) Rf 0.72.
Biosearch 9600 peptide synthesizer.15 The synthesis was
performed using the program CNDIPCDI (Biosearch). Fmoc
group removals were carried out by treatment with piperidine/
DMF. Double couplings were performed using 6.67 equiv of
Fmoc-amino acid with equivalent amounts of N,N-diisopropyl-
carbodiimide and HOBt in DMF (1-2 h). NH2-Terminal
acylation was conducted using 6.67 equiv of Hpa-OSu in DMF
(1-2 h). Completed peptidyl resins were cleaved and depro-
tected using reagent K for 2 h at room temperature.16 The resin
was removed by filtration, the filtrate evaporated, and the fully
deprotected free peptide precipitated with ether. Collection by
filtration and drying under high vacuum gave 18 as a white
solid (234 mg, 65% yield, tR ) 8.87 min, 95% pure at 270 nm
and 75% pure at 220 nm by analytical HPLC (IIId).
Hp a -Nle-Gly-Tr p -Lys(Hci)-Asp -MeP h e-NH2 (6). Com-
pound 18 (246 mg, 0.29 mmol) was combined with 1 equiv of
DIEA (0.050 mL, 0.29 mmol) and 1.2 equiv of HciOSu (52 mg,
0.35 mmol) in 5 mL of DMF with stirring for 18 h, at which
time an additional 1 equiv of DIEA (0.050 mL) was added. The
reaction was then directly isolated by Prep-LC with a 110 min
gradient of 40-60% solvent B [10 mM NH4OAc (solvent A)
and 10 mM NH4OAc in MeOH (solvent B)] at 25 mL/min. The
pure factions were combined and 26 mg of product was
obtained. Analysis was by analytical HPLC (IIIc), tR ) 13.74
min.
Hp a -Nle-Gly-Tr p -Lys(Ta c)-Asp -MeP h e-NH2 (7). Com-
pound 18 (364 mg, 0.43 mmol) was combined with 1.25 equiv
of DIEA (0.1 mL, 0.58 mmol) and 1.0 equiv of 2-methylphenyl
isocyanate (0.53 mL, 0.43 mmol) in 10 mL of DMF with stirring
for 1 h at room temperature. The reaction was then directly
isolated by Prep-LC with a 180 min gradient of 50-100%
solvent B [10 mM NH4OAc (solvent A) and 10 mM NH4OAc
in MeOH (solvent B)] at 25 mL/min. The pure factions were
combined and lyophilized.
Hpa(SE)-Nle-Gly-Tr p-Lys(Tac)-Asp-MeP h e-NH2 (8). Pyr-
idine-sulfate complex (330 mg, 2.07 mmol, 23 equiv) was
added to thoroughly dried Hpa-Nle-Gly-Trp-Lys(Tac)-Asp-
MePhe-NH2 (7) (96 mg, 0.088 mmol) in 2 mL of pyridine with
stirring. After 6 h at room temperature the solution was
poured into 50 mL of 5% NH4OH, allowed to stir for 30 min,
and then evaporated to dryness. MeOH (50 mL) was added to
the resulting solid, the suspension filtered, and the filtrate
evaporated to give crude peptide.
H -Lys(Z)-D-Asp (OBn )-N-Me-P h e-NH 2‚H Cl (14). Com-
pound 13 (2.36 g, 3.16 mmol) was subjected to deprotection
using method B: yield 1.54 g (2.26 mmol); TLC (CMA 9:1:1)
Rf 0.2. The product was used as is.
Boc-Tr p -Lys(Z)-D-Asp (OBn )-N-Me-P h e-NH2 (15). Using
Method C, Boc-Trp-OH (0.83 g, 2.71 mmol) was coupled to 14;
yield 1.83 g (1.96 mmol, 87% yield); TLC (CMA 9:1:1) Rf 0.64.
Meth od D: Boc-Tr p -Lys-D-Asp -N-MeP h e-NH2(16). Gen -
er a l Sid e Ch a in Dep r otection Meth od . To a solution of 15
(1.83 g, 1.96 mmol) in 50 mL of glacial acetic acid was added
0.5 g of 10% Pd/C. The reaction was shaken under hydrogen
pressure on a hydrogenator for 16 h. The Pd/C was removed
by filtration through a layer of Celite and the solvent removed
in vacuo. The resulting oil was dissolved in MeOH and the
product precipitated with ether to yield 1.14 g (1.61 mmol, 82%
yield) of 16 as a white solid that was used directly in the next
reaction.
Meth od E. Boc-Tr p -Lys(Ta c)-D-Asp -MeP h e-NH2 (4).
Gen er a l Ta c Ad d ition Meth od . To a solution of 16 (0.4 g,
0.56 mmol) in 40 mL of DMF was added DIEA (0.25 mL, 1.4
mmol) and 2-tolyl isocyanate (0.07 mL, 0.56 mmol). After 1 h
the reaction was complete as measured by HPLC and the
solvent was removed in vacuo. The peptide was purified by
Prep-LC, to yield 195 mg of compound 4. See Table 3 for
analytical data.
The product was then isolated by Prep-LC with a 180 min
gradient of 50-90% solvent B [10 mM NH4OAc (solvent A)
and 10 mM NH4OAc in MeOH (solvent B)] at 25 mL/min. The
product fractions were combined and lyophilized to give 41 mg
of 8. Due to the acid sensitivity of this compound, analytical
HPLC was performed with the same solvent system used to
isolate the compound: HPLC (I) 35-100% solvent B in 30 min,
tR ) 8.9 min; HPLC (II) 40-100% solvent B in 30 min, tR
)
13.8 min [10 mM NH4OAc (solvent A) and 10 mM NH4OAc in
MeOH (solvent B)] at 0.5 mL/min.
Biologica l Eva lu a tion s. Affinity for CCK-A receptors in
rat pancreatic membranes was measured according to the
methods described by Chang et al.18 A pancreas from a
Sprague-Dawley rat was homogenized for 20 s with a Brink-
man Polytron (setting 5) in 50 vol of a 50 mM Tris buffer, at
pH 8.0 and 4 °C. The homogenate was centrifuged at 50000g
for 10 min and then the pellet was washed twice, recentrifuged,
and resuspended in 2000 vol of cold 5 mM Tris buffer (pH 7.4
at 37 °C), containing 5 mM MgCl2, 5 mM dithiothreitol, 2 mg/
mL BSA, and 0.14 mg/mL bacitracin. Affinity for CCK-A
Hp a -Nle-Gly-Tr p -Lys-Asp -MeP h e-NH2 (18). Hpa-Nle-
Gly-Trp-Lys-Asp-MePhe-NH2 was prepared by standard solid-
phase synthesis techniques using the Fmoc/tBu protection
strategy, with the lysine side chain amine protected with a
Boc group. Each amino acid was added sequentially to the
RINKMBHA resin (1 g, 0.3 mmol/g, lot A09655) using a

2354 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
Pierson et al.
receptors was measured using [125I]BH-CCK-8 (DuPont, 2000
Ci/mmol). One milliliter (0.5 mg original wet weight of tissue)
of membrane suspension, and 15 pmol of [125I]BH-CCK-8 was
incubated for 40 min at 37 °C. Bound ligand was collected onto
Whatman GF/B filters, and the filters were washed twice with
8 mL of ice-cold 50 mM Tris buffer, pH 7.4, and analyzed using
a Beckman liquid scintillation counter. Binding constants (Ki)
were determined (n ) 3) using ALLFIT, an iterative logistic
curve fitting program, as described by Cheng and Prusoff.23
The calculated Kd value for [125I]BH-CCK-8 was 0.12 nM.
Refer en ces
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Affinity for CCK-B receptors was measured in rat cerebral
cortex.19 Tissue was homogenized in 50 vol (vol/g of wet weight
of tissue) of 50 mM Tris (pH 7.5 at 25 °C). The homogenate
was centrifuged at 50000g for 10 min and the resulting pellet
was resuspended and recentrifuged. The final pellet was
resuspended in 80 vol of assay buffer (10 mM Hepes, 5 mM
MgCl2, 1 mM EGTA, bacitracin (0.25 mg/mL), and 130 mM
NaCl, pH 6.5). Then, 0.45 mL of resuspended membranes,
unlabeled compound, and 1 nmol of [125I]BH-CCK-8 in a final
volume of 0.5 mL was incubated at 25 °C for 2 h. Membrane-
bound 125I was collected by vacuum filtration on Whatman
GF/B filters presoaked in 50 mM Tris buffer, pH 7.7, contain-
ing 1 mg/mL bovine serum albumin (Sigma). Radioactivity was
determined with a Beckman γ-counter (efficiency of 45%). Ki
values (n ) 3) were determined using the Lundon AccuFit
Competition nonlinear curve fitting software program, which
is base in part on models described by Linden24 and Feldmen.25
(6) Pierson, M. E.; Comstock, J .; Simmons, R. D.; Kaiser, F. C.;
J ulien, R. P.; Zongrone, J .; Rosamond, J . D. Synthesis and
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Synthesis and Biological Activities of CCK Heptapeptide Ana-
logues. Effects on Conformational Constraints and Standard
Modifications on Receptor Subtype Selectivity and Functional
Activity in Vitro and Appetite Suppression In Vivo. J . Med.
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(9) Nadzan, A. M.; Kerwin, J r., J . F. Cholecystokinin Agonists and
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J . A., Ed., Academic: San Diego, CA, 1991; pp 191-200.
(10) Shiosaki, K.; Lin, C. W.; Kopecka, H.; Tufano, M. D.; Bianchi,
B. R.; Miller, T. R.; Witte, D. G.; Nadzan, A. M. Boc-CCK-4
Derivatives Containing Side-Chain Ureas as Potent and Selec-
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(11) Aquino, C.; Armour, D.; Berman, J .; Birkemo, L.; Carr, R.;
Croom, D.; Dezube, M.; Dougherty, R.; Ervin, G.; Grizzle, M.;
Head, J .; Hirst, G.; J ames, M.; J ohnson, M.; Miller, L.; Queen,
K.; Rimele, T.; Smith, D.; Sugg, E. Discovery of 1,5-Benzodi-
azepines with Peripheral Cholecystokinin (CCK-A) Receptor
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(12) Bignon, E.; Bachy, A.; Boigegrain, R.; Brodin, R.; Cottineau, M.;
Gully, D.; Hebert, J .-M.; Keane, P.; Labie, C.; Molimard, J .-C.;
Olliero, D.; Oury-Donat, F.; Petereau, C.; Prabonnaud, V.;
Rockstroh, M.-P.; Schaeffer, P.; Servant, O.; Thurneyssen, O.;
Soubrie, P.; Pascal, M.; Maffrand, J .-P.; Le Fur, G. SR146131:
A New Potent, Orally Active, and Selective Nonpeptide Chole-
cystokinin Subtype 1 Receptor Agonist. I: In Vitro Studies. J .
Pharmacol. Exp. Ther. 1999, 289, 742-751.
(13) Bignon, E.; Alonso, R.; Arnone, M.; Boigegrain, R.; Brodin, R.;
Gueudet, C.; He´aulme, M.; Keane, P.; Landi, M.; Molimard, J .-
C.; Olliero, D.; Poncelet, M.; Seban, E.; Simiand, J .; Soubrie, P.;
Pascal, M.; Maffrand, J .-P.; Le Fur, G. SR146131: A New Potent,
Orally Active, and Selective Nonpeptide Cholecystokinin Sub-
type 1 Receptor Agonist. II: In Vivo Pharmacological Charac-
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Food intake was measured by a modification of the method
described by Cox and Maickel.20 Individually caged male
Sprague-Dawley rats (300-400 g) were maintained on a 12
h light/dark cycle and were trained for at least 14 days to feed
(powdered Purina rat chow) during the initial 3 h period of
the dark cycle. To assess feeding inhibition potency, saline
control and peptide doses were administered intraperitoneally
(in 0.5 mL of 0.9% saline, pH 7.0-8.0) 10 min prior to food
availability, and cumulative food intake was then measured
after 0.5 and 3 h of feeding. Both 0.5 and 3 h time points were
determined in the same experiment. Dose-response curves
were constructed of pooled consumption data from five doses
(10 rats/dose) for each peptide, and ED50 (i.e., the dose that
inhibits feeding by 50%) values were determined using ALL-
FIT. A repeated-measures ANOVA followed by a Newman-
Keuls analysis was used to determine significant differences
in food intake between groups.
Weight loss was measured by a modification of the method
described by Cox and Maickel.20 To examine the weight loss
producing effects of 7, the compound was intraperitoneally
administered to rats for nine consecutive days (Figure 2). Male
Sprague-Dawley rats (300-400 g) trained to feed during the
initial 3 h of a 12 h dark cycle were weighed and dosed on a
daily basis 10 min prior to the 3 h feeding period. Cumulative
food intake was measured hourly during each feeding period.
Day 1 predosing body weights served as starting body weights
for each group of 10 animals. Each group was composed of
animals with similar body weight and similar feeding behavior.
Vehicle control and treatment groups were composed of the
following: (1) 0.9% saline, (2) 0.8 µg/kg of 7, and (3) 0.3 µg/kg
of 7. Vehicle and 7 were administered in a volume of 0.5 mL
of 0.9% saline. Mean and standard error (SEM) values for each
treatment group were calculated for food intake and body
weight each day. A repeated-measures ANOVA followed by a
Newman-Keuls analysis was used to determine significant
differences in food intake and body weight values across
treatment groups both within days and between days.
(15) Holladay, M.; Kopecka, H.; Miller, T.; Bednarz, L.; Nikkel, A.;
Bianchi, B.; Witte, D.; Shiosaki, K.; Lin, C. W.; Asin, K.; Nadzan,
A. Tetrapeptide CCK-A Agonists: Effect of Backbone N-Methyl-
ation on in Vitro and in Vivo CCK Activity. J . Med. Chem. 1994,
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(18) Chang, R. S. L.; Lotti, V. J .; Chen, T. B.; Kunkel, K. A.
Characterization of the Binding of [3H-(()-L-364,718: A New
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Ack n ow led gm en t. We would like to thank Tim
Ordway for the amino acid analysis, Bill Kuipers for the
NMR, David Coomber for the MS, and Dr. Nigel
Gensmantal for help with the molecular modeling. We
would also like to thank Dr. Ken Mattes for his helpful
discussions and critical reading of the manuscript.

Hexa- and Tetrapeptide CCK-A Agonists
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2355
(21) Asin, K.; Bendnarz, L.; Nikkel, A.; Gore, P.; Nadzan, A. A-71623,
A Selective CCK-A Receptor Agonist, Suppresses Food Intake
in the Mouse, Dog and Monkey. Pharmacol. Biochem. Behav.
1992, 42, 699-704.
(22) Peptide nomenclature follows the guidelines of the IUPAC-IUB
J oint Commission on Biochemical Nomenclature. Eur. J . Bio-
chem. 1984, 138, 9-37.
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Constant (Ki) and the Concentration of Inhibitor which Causes
50 Percent Inhibition (IC50) of an Enzyme Reaction. Biochem.
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J M990252E