Synthesis and biological evaluation of potent, selective, hexapeptide CCK-A agonist anorectic agents

Article abstract of DOI:10.1021/jm970477u

Cholecystokinin (CCK) is a 33-amino acid peptide with multiple functions in both the central nervous system (via CCK-B receptors) and the periphery (via CCK-A receptors). CCK mediation of satiety via the A-receptor subtype suggest a role for CCK in the ma

Full text of DOI:10.1021/jm970477u

4302
J . Med. Chem. 1997, 40, 4302-4307
Syn th esis a n d Biologica l Eva lu a tion of P oten t, Selective, Hexa p ep tid e CCK-A
Agon ist An or ectic Agen ts
M. Edward Pierson,* J eanne M. Comstock, Roy D. Simmons, Frederick Kaiser, Ronald J ulien,
J ohn Zongrone, and J ames D. Rosamond
Astra Arcus USA, P.O. Box 20890, Rochester, New York 14602
Received J uly 23, 1997X
Cholecystokinin (CCK) is a 33-amino acid peptide with multiple functions in both the central
nervous system (via CCK-B receptors) and the periphery (via CCK-A receptors). CCK mediation
of satiety via the A-receptor subtype suggest a role for CCK in the management of obesity.
The carboxy terminal octapeptide (CCK-8) is fully active in this regard, but is lacking in receptor
selectivity, metabolic stability, and oral bioavailability. Inversion of the chirality of Asp7 in
conjunction with N-methylation of Phe8 produces compound 5 which exhibits high affinity and
2100-fold selectivity for CCK-A receptors. Compound 6 (Hpa(SO3H)-Nle-Gly-Trp-Nle-MeAsp-
Phe-NH2), derived from moving the N-methyl group from Phe to Asp, decreased CCK-B affinity
substantially without affecting CCK-A affinity, giving a compound with 6600-fold selectivity
for CCK-A receptors. These compounds inhibit food intake with nanomolar potency following
intraperitoneal administration in fasted rats. In addition to greater potency, compound 6
produces weight loss in rats when administered over nine consecutive days. Intranasal
administration of 6 potently inhibits feeding in beagle dogs. Compound 6 produces potent
anorectic activity via the CCK-A receptor system.
In tr od u ction
tetrapeptides.12-14 Structure-activity relationships of
CCK-A agonists suggest that the anionic group of Asp7
in CCK-8 is important for agonist activity.15 We have
previously reported that inversion of the stereochemis-
try at Asp7 of 2 produced the CCK-A selective agonist
3.16 We report here further studies of CCK-A agonists
leading to the identification of the highly potent CCK-A
selective agonist AR-R 15849 (6).17
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 (1,
see Table 1 for sequence) predominating.1,2 CCK medi-
ates many diverse hormonal and neuromodulatory
functions through the action of two CCK receptor
subtypes, CCK-A and CCK-B.3,4 CCK-B receptors are
found mainly in the CNS where CCK-8 is proposed to
be a neurotransmitter or neuromodulator involved in
anxiety and pain modulation.4 Peripheral CCK-A re-
ceptors mediate gall bladder contraction, pancreatic
exocrine secretion, and satiety.3 These actions are
triggered by the release of CCK-8 from the small
intestine following nutrient ingestion.5,6 The satiety
signal is relayed to the brain by CCK-8-mediated
activation of CCK-A receptors on vagal afferents.5,6
Gibbs et al.7 observed that intraperitoneally (ip)
administered CCK-8 decreases meal size and produces
classical postprandial behaviors in rats. The satiety
effect of peripherally administered CCK-8 extends to a
number of species, including humans.8,9 Initial efforts
to develop a metabolically more stable analogue of
CCK-8 resulted in the identification of AR-R 14294 (2),
which potently inhibits feeding in rats following ip
administration and in dogs following intranasal admin-
istration.10
Meth od s
The unsulfated peptide amides were synthesized
utilizing the Fmoc/tBu protection strategy on Pal Resin
(Milligen/Biosearch).18 Cleavage from the resin was
accomplished using reagent K (TFA, 8.5; H2O, 0.5;
thioanisole, 0.5; phenol, 0.5; ethanedithiol, 0.2) for 2 h
at room temperature.19 The crude peptides were puri-
fied to homogeneity using preparative reverse phase
HPLC. Sulfation was achieved using Pyr‚SO3, and the
final products were purified to homogeneity using
reverse phase HPLC and characterized by analytical
reverse phase HPLC, mass spectrometry, and amino
acid analysis.
A fragment condensation solution synthesis of 6 was
developed to accommodate the larger quantity require-
ments for further biological investigation (Scheme 1).
Boc-MeAsp(OBn)-OH was prepared using the method
of Holladay.14 The C-terminal tetrapeptide H-Trp-Nle-
MeAsp(OBn)-Phe-NH2‚HCl and the N-terminal acyl
dipeptide, Hpa-Nle-Gly-OH (Hpa is 4-hydroxyphenyl-
acetyl), were assembled separately in a stepwise fashion
and coupled to give Hpa-Nle-Gly-Trp-Nle-MeAsp(OBn)-
Phe-NH2. After removal of the benzyl protecting group
by hydrogenation, the deprotected hexapeptide (Hpa-
Nle-Gly-Trp-Nle-MeAsp-Phe-NH2) was isolated by pre-
parative reverse phase HPLC, thoroughly dried, and
sulfated directly to give 6 which was isolated by
preparative reverse phase HPLC.
Using selective antagonists, it has been shown in rats
that CCK-A, and not CCK-B, receptors mediate the
anorectic effects of peripherally administered CCK-8.11
However, the therapeutic potential of CCK-8 is limited
by its short biological half-life and its poor bioavailabil-
ity, by other than parenteral administration. Only a
small number of compounds have been reported to be
CCK-A selective agonists, most notably a series of
CCK-A receptor affinity was measured by displace-
X Abstract published in Advance ACS Abstracts, December 1, 1997.
ment of [125I]BH-CCK-8 from rat pancreatic tissue
S0022-2623(97)00477-9 CCC: $14.00 © 1997 American Chemical Society

CCK-A Agonist Anorectic Agents
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26 4303
Ta ble 1. Receptor Affinity and Selectivity
Ki (nM)
no.
structurea
CCK-A
CCK-B
0.41
1.28
84
150
430
CCK-A selectivity
1
2
3
4
5
6
7
8
9
H-Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2
Hpa(SO3H)-Met-Gly-Trp-Met-Asp-MePhe-NH2
Hpa(SO3H)-Met-Gly-Trp-Met-D-Asp-MePhe-NH2
Hpa(SO3H)-Met-Gly-Trp-Met-D-Asp-Phe-NH2
Hpa(SO3H)-Nle-Gly-Trp-Nle-D-Asp-MePhe-NH2
Hpa(SO3H)-Nle-Gly-Trp-Nle-MeAsp-Phe-NH2
Hpa(SO3H)-Nle-Gly-Trp-Ile-MeAsp-MePhe-NH2
Hpa(SO3H)-Ile-Gly-Trp-Ile-MeAsp-Phe-NH2
Hpa(SO3H)-Nle-Gly-Trp-Nle-D-MeAsp-Phe-NH2
0.09
0.07
0.98
19
0.20
0.03
0.53
0.24
0.03
5
18
86
8
2100
6590
>2000
9200
48000
224
>1000
2200
1600
a
Hpa(SO3H) is sulfated 4-hydroxyphenylacetyl.
Sch em e 1. Solution Phase Synthesis of AR-R 15849
a
Reagents: (a) Piv-Cl, DIEA; (b) H-Phe-NH2; (c) HCl in EtOAc; (d) TBTU, HOBt, DIEA; (e) Boc-Nle-OH; (f) Boc-Trp-OH; (g) anisole;
(h) H-Gly-OMe‚HCl, DIEA; (i) Hpa-OSu, DIEA; (j) methanolic NaOH; (k) HCl; (l) H2 Pd/C; (m) Pyr‚SO3; (n) NH4OH.
according to the general procedure of Chang.20 CCK-B
receptor affinity was measured by displacement of [125I]-
BH-CCK-8 from rat cortex synaptosomes according to
the general procedure of Chang.21
that inhibits feeding by 50%, was calculated for the 0.5
and 3 h feeding interval. This ED50 was generated by
ALFIT analysis of pooled food consumption data from
five doses (10 rats/dose) of each test compound.
The ability of a compound to inhibit food intake in
rats was measured by a modification of the method
described by Cox and Maickel.22 Individually caged
male Sprague-Dawley rats (300-400 g) were main-
tained on a 12 h light/dark cycle and trained for at least
14 days to feed only during the initial 3 h period of the
dark cycle. Inhibition of food intake was measured
following intraperitoneal injection of saline or a solution
of the test compound. A preweighed food jar containing
powdered chow (Purina) was introduced 15 min after
injection. Cumulative food intake for each individual
animal was measured after 0.5 and 3 h by weighing the
food jar. From this information an ED50, i.e., the dose
Resu lts a n d Discu ssion
We previously reported that replacement of Asp-Tyr-
(SO3H) of CCK-8 with Hpa(SO3H) and N-methylation
of Phe as in 2 does not diminish CCK-A or CCK-B
affinity16 (Table 1). However, 2 inhibits feeding in rats
following ip administration much more potently than
CCK-8 (3 h ED50 0.56 and 112 µg/kg, respectively, Table
2).10 Inversion of the stereochemistry of Asp in 2 to give
3 has a modest effect on CCK-A affinity (Table 1).
However, the combination of D-Asp and MePhe in 3
appears to be more deleterious for CCK-B affinity,
producing a compound that is 86-fold CCK-A selective

4304 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26
Pierson et al.
Ta ble 2. Feeding Inhibition Potency in 21 h Fasted Rats (ip)
feeding inhibition ED50 (µg/kg)
no.
0.5 h period
3 h period
1
2
5
6
7
8
9
1.2 ( 0.1
112 ( 3.8
0.56 (0.11
2.5 ( 0.1
0.04 ( 0.01
0.45 ( 0.4
0.11 ( 0.02
2.0 ( 0.6
0.25 ( 0.01
2.6 ( 0.4
0.56 ( 0.1
0.06 ( 0.008
3.1 ( 0.3
0.21 ( 0.04
(Table 1). The nonmethylated analogue of 3, compound
4, has less affinity for CCK-A and CCK-B receptors,
suggesting that MePhe contributes to the CCK-A affin-
ity and therefore selectivity of 3 (Table 1). Replacing
Met with Nle as in 5 has little effect on CCK-A affinity
but removes sites of oxidation and presumably increases
chemical stability of the peptides. Compound 5 (Hpa-
(SO3H)-Nle-Gly-Trp-Nle-D-Asp-MePhe-NH2), the Nle
analogue of 3, is a selective CCK-A agonist that inhibits
feeding in rats after ip administration more potently
than CCK-8, especially for the 3 h feeding period (2.5
vs 112 µg/kg, Table 2). The finding that D-Asp-MePhe-
containing peptides were selective for CCK-A receptors
led to further manipulation of Asp.
F igu r e 1. Intranasal dose response of AR-R 15849 to inhibit
feeding in male beagles.
Methylation of the amide nitrogen of Asp in the
hexapeptides, regardless of the methylation status of
Phe, leads to a dramatic increase in CCK-A selectivity
as a result of substantial decrease in CCK-B affinity (6-
8). Compounds 6-8 all have subnanomolar affinity for
CCK-A receptors and are greater than 2000-fold selec-
tive for CCK-A over CCK-B receptors. The fact that
CCK-A receptors are highly tolerant of CCK analogues
containing MeAsp in the penultimate position correlates
well with a tetrapeptide series reported by Holladay.23
On the other hand, CCK-B receptors are far less tolerant
of this MeAsp modification, accounting for the high
CCK-A selectivity observed for compounds 6-8. While
6-8 are all potent inhibitors of food intake following ip
administration, 6 is the most potent at both the 0.5 and
3 h periods (Table 2). Replacing Asp7 with N-methy-
lated D-Asp gives 9, the most CCK-A selective agonist
in the series. Compound 9 also potently inhibits food
intake in rats following ip administration (Table 2).
Relative to CCK-8, all of the Hpa(SO3H) analogues
(2-9) showed greater feeding inhibition potency follow-
ing ip administration (Table 2). This increased potency
was particularly evident for the 3 h feeding period. The
increased feeding inhibition of these CCK analogues
may result from improved metabolic stability of these
compounds relative to CCK-8. Significantly, the highly
selective CCK-A ligands 6 and 9 are basically equipotent
in feeding inhibition at the 0.5 and 3 h time periods.
The apparent long-lasting effect on food intake by
compounds 6 and 9 is possibly due to an increased
metabolic stability resulting from methylation of Asp.
On the basis of their high affinity and selectivity for the
CCK-A receptor and potent, long-lasting feeding inhibi-
tion effects following ip administration, compounds 6
and 9 were chosen for further pharmacological profiling.
Both 6 and 9 decrease food intake following intranasal
administration to dogs. Fasted dogs were allowed to
consume 100 g of food over 15 min followed by a 20 min
delay. Each dog was then dosed intranasally with test
compound 10 min prior to the presentation of 400 g of
food. After 15 min the food containers were removed
F igu r e 2. Body weight change effect of AR-R 15849 in rats
following daily ip dosing over 9 days. *Values are significantly
different from controls (P < 0.05, ANOVA, Newman-Keuls).
and weighed. Comparison of food intake data with that
of vehicle-dosed controls gave the percent feeding inhi-
bition response. Compound 6 inhibits food intake with
an ED50 of 5.0 µg/kg following intranasal administration
(Figure 1). However, compound 9 inhibits food intake
less potently with an ED50 of 20 µg/kg following intra-
nasal administration. Because of its enhanced potency,
compound 6 was chosen for further evaluation.
To examine the weight loss producing effects of 6, the
compound was administered to rats for nine consecutive
days (Figure 2). Twenty 1 h fasted rats, trained to feed
at the beginning of a 12 h dark cycle, were weighed and
dosed on a daily basis 15 min prior to initiation of a 3
h feeding period. Food intakes were measured hourly
during each feeding period. Acutely, a 0.3 µg/kg dose
of 6 was found to inhibit food intake by 50%, while a
0.1 µg/kg dose was found to inhibit feeding by 30% (data
not shown). Over the 9 day study 6 dose dependently
induced weight loss. At the higher dose 6 continued to
decrease body weight over days 2-4; thereafter, a 4-5%
reduction in body weight was maintained (days 4-8)
which differed significantly from control’s weight gain
even on day 9. The upturn (Figure 2) in the low dose
(0.1 µg/kg) and high dose (0.3 µg/kg) curves at days 8
and 9, respectively, is most likely due to pharmacological
and behavioral adaptation to prolonged severe caloric
deprivation.24 Further pharmacological profiling of
compound 6 is reported elsewhere.17 Thus, 6 not only
reduces food intake but also significantly reduces body
weight in growing rats over a 9 day period.

CCK-A Agonist Anorectic Agents
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26 4305
Ta ble 3. Analytical Data
amino acid analysis
MS
adduct
HPLCa tR (min)
compd
Met
Nle
Ile
Gly
Trp
Asp
MeAsp
Phe
MePhe
1.11
m/z
Ia
IIb
3
4
5
6
7
1.81
1.83
1.03
1.11
1.04
1.04
1.00
1.04
0.59
1.00
0.61
NDb
1.06
1.03
1.00
1012
997
975
975
989
909
975
897
975
897
(M - H)-
38.1
37.6
41.7
41.8
42.4
4.93
4.95
6.76
7.11
7.42
1.04
0.98
(M - H)-
1.91
1.90
0.77
1.05
1.29
(M - H)-
1.07
0.92
(M - H)-
1.02
1.83
(M - H)-
(M - SO3H)-
(M - H)-
8
9
1.00
1.02
0.73
0.86
1.11
1.00
1.06
1.06
40.5
40.9
6.41
6.40
(M - SO3H)-
(M - H)-
1.91
(M - SO3H)-
a
b
See Experimental Section for description of HPLC Ia, IIb. Not determined.
with piperidine/DMF. Couplings were performed using 6.67
equiv of Fmoc-amino acid with equivalent amounts of N,N-
diisopropylcarbodiimide and HOBt in DMF (1-2 h). NH2-
terminal acylation was conducted with 6.67 equiv of Hpa-OSu
in DMF (1-2 h). Completed protected peptidyl resins were
cleaved and deprotected using reagent K19 (TFA, H2O, thio-
anisole, phenol, ethanedithiol, 185:5:5:5:2) for 2 h at room
temperature. 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.
Several other compounds have been reported to be
CCK-A selective agonists that inhibit food intake fol-
lowing ip administration. Most notable are a hexapep-
tide (A-71378) and a tetrapeptide (A-71623) reported by
the Abbott group.12-14,25 However, in our hands neither
compound shows any feeding inhibitory activity at 5.0
µg/kg following intranasal administration in dogs (data
not shown). In contrast, compound 6 following intra-
nasal administration in beagle dogs potently inhibits
food intake via a natural satiety mechanism.
The peptide amide products (dried overnight under high
vacuum) were sulfated with Pyr‚SO3 according to method D.27
The crude sulfated products were purified to homogeneity by
Prep-LC. Fractions shown by HPLC to be >95% pure were
pooled and lyophilized to provide sulfated peptide amide
products 3-9. 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) with the expected (M - H)- molecular
ion peaks observed for all peptides. The peptides were
determined by HPLC to be >98% pure when analyzed on two
different columns (Table 3).
Meth od A. Boc-MeAsp (OBn )-P h e-NH2 (10). Gen er a l
P iv-Cl Cou p lin g Meth od . To a stirred solution of Boc-
MeAsp(OBn)-OH (1.00 g, 2.97 mmol; prepared by the method
of Holladay14 from Fmoc-MeAsp(OBn)-OH supplied by Pep-
tides International) in DMF (15 mL) was added DIEA (0.52
mL, 2.97 mmol) and Piv-Cl (0.37 mL, 2.97 mmol). After 15
min a solution of H-Phe-NH2‚HCl (0.490 g, 2.97 mmol) and
DIEA (0.52 mL, 2.97 mmol) in DMF (10 mL) was added, and
the mixture was stirred at room temperature for 6 h. The
solvent was removed in vacuo, and the resulting oil was
dissolved in EtOAc; washed with 10% citric acid, 1 N NaHCO3,
and saturated NaCl; dried (MgSO4); and evaporated to an oil:
TLC (CMA, 9:1:1) Rf 0.69.
Meth od B. H-MeAsp (OBn )-P h e-NH2‚HCl (11). Gen -
er a l Boc Dep r otection Meth od . To a stirred, -10 °C
solution of 10 (1.44 g, 2.97 mmol) in EtOAc (100 mL) was
added HCl gas to saturation (10 min). The solution allowed
to warm to room temperature over 1 h. The solution was
reduced in vacuo to a small volume, and hexanes were added
with cooled to -20 °C. The white precipitated product was
collected by filtration: 1.13 g (99% from Boc-MeAsp(OBn)-OH);
TLC (CMA 9:1:1) Rf 0.38; mp 155-158 °C; MS (CI) m/z 384
(M + H)+; HPLC (IIIc) tR 12.7 min. Anal. (C21H25N3O4‚HCl)
C, H, N.
Meth od C. Boc-Nle-MeAsp (OBn )-P h e-NH2 (12). Gen -
er a l TBTU Cou p lin g Meth od . To a stirred solution of Boc-
Nle-OH (0.414 g, 1.79 mmol) in DMF (10 mL) were added
TBTU (0.570 g, 1.79 mmol), HOBt (0.270 g, 1.79 mmol), and
DIEA (0.62 mL, 3.58 mmol). After 5 min a solution of 11 (0.500
g, 1.19 mmol) and DIEA (0.207 mL, 1.19 mmol) in DMF (5
mL) was added, and the mixture was stirred at room temper-
ature for 2 h. The reaction was found to be incomplete by TLC.
Additional Boc-Nle-OH (0.414 g, 1.79 mmol) in DMF (10 mL)
with TBTU (0.570 g, 1.79 mmol), HOBt (0.270 g, 1.79 mmol),
and DIEA (0.62 mL, 3.58 mmol) was added, and stirring was
continued for 2 days. After removal of the solvent in vacuo,
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 Nomenclature.26 AAA, amino acid analysis;
Boc, tert-butyloxycarbonyl; Gly, glycine; MeAsp, N-methylas-
partic acid; Nle, norleucine (2-aminohexanoic acid); Phe,
phenylalanine; Trp, tryptophan. Other abbreviations are as
follows: CMA, chloroform, methanol, acetic acid; DCM, me-
thylene chloride; DIEA, N,N-diisopropylethylamine; DMF,
dimethylformamide; EtOAc, ethyl acetate; HOBt, 1-hydroxy-
benzotriazole; Hpa, 4-hydroxylphenylacetyl; OSu, succinim-
idyloxy ester; Piv-Cl, pivaloyl chloride; Pyr, pyridine; Pyr‚SO3,
pyridine sulfur trioxide complex; TBTU, 2-(1H-benzotriazol-
1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate; TFA, tri-
fluoroacetic acid; tR, HPLC retention time.
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 Å). Peptides were eluted with one of
two mobile phases. Prep-LC (A): 30 min gradient of 0-60%
solvent B (10 mM NH4OAc (solvent A) and 10 mM NH4OAc
in MeOH (solvent B)) at 75 mL/min. or Prep-LC (B): 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.
Analytical reverse phase HPLC was performed on a Hewlett-
Packard 1050 system using the following columns and mobile
phases. Columns: (I) Keystone Nucleosil C18 (150 × 3.2 mm,
5 µm) eluted at 0.5 mL/min, (II) Waters Symmetry C8 (50 ×
3.9 mm, 5 µm, 100 Å) eluted at 3.0 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) 60 min gradient of 0-60%
solvent B (0.05% TFA (solvent A) and 0.05% TFA in MeOH
(solvent B)); (b) 15 min gradient of 35-100% solvent B
(solvents same as in mobile phase a); (c) 30 min gradient of
25-75% solvent B (0.1% TFA (solvent A) and 0.1% TFA in
MeOH (solvent B)); (d) 30 min gradient of 20-75% solvent B
(solvents same as in mobile phase c); (e) 20 min gradient of
50-90% solvent B (solvents same as in mobile phase c); or (f)
55% MeOH in 0.1% TFA. 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 moni-
tored at 220 nm.
Gen er a l Meth od for Solid -P h a se P ep tid e Syn th esis.
The unsulfated peptide amides were prepared on a MilliGen/
Biosearch 9600 synthesizer using standard solid-phase syn-
thesis techniques and the Fmoc/tBu protection strategy.
Syntheses were performed starting with Pal Resin18 (1 g, 0.33
mmol). Fmoc group removals were carried out by treatment

4306 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26
Pierson et al.
the residue was dissolved in EtOAc; washed with 10% citric
acid, 1 N NaHCO3, and saturated NaCl; dried (MgSO4); and
evaporated to an oil (0.71 g). Further purification can be
performed using silica gel chromatography with EtOAc: TLC
(CMA 9:1:1) Rf 0.64; HPLC (IIId) tR 33.4 min.
H-Nle-MeAsp (OBn )-P h e-NH2‚HCl (13). Compound 12
(0.71 g, 1.28 mmol) was subjected to deprotection using method
B: yield 0.502 g (79% from 11); TLC (CMA 9:1:1) Rf 0.13; MS
(TS) m/z 497 (M + H)+; HPLC (IIId) tR 20.9 min.
Boc-Tr p -Nle-MeAsp (OBn )-P h e-NH2 (14). Using method
C, Boc-Trp-OH (0.960 g, 3.17 mmol) was coupled to 13 (1.21
g, 2.44 mmol). Following solvent removal, EtOAc (75 mL) and
hexane (100 mL) were added to the residue, precipitating a
white solid which was collected by filtration, 1.72 g (90% yield).
Further purification can be performed using silica gel chro-
matography with EtOAc or CHCl3: TLC (CMA 9:1:1) Rf 0.67;
mp 150-153 °C; HPLC (IIIe) tR 17.2 min. Anal. (C43H54N6O8‚
0.4EtOAc) C, H, N.
H-Tr p -Nle-MeAsp (OBn )-P h e-NH 2‚HCl (15). Compound
14 (2.93 g, 3.75 mmol) with anisole (1.63 mL, 15 mmol) was
deprotected using method B and lyophilized from glacial acetic
acid: yield 2.37 g (88%); TLC (CMA 9:1:1) Rf 0.2; MS (TS) m/z
783 (M + H)+; HPLC (IIIe) tR 8.6 min. Anal. (C38H46N6O6‚
0.12HCl‚0.02HOAc‚0.8H2O) C, H, N.
Pyr‚SO3 (60 mg, 0.38 mmol).24 After 1 h the reaction was
found to be incomplete by HPLC, and more Pyr‚SO3 (60 mg,
0.38 mmol) was added followed in 1 h with additional Pyr‚SO3
(360 mg, 2.26 mmol). After 2.5 h the reaction mixture was
diluted with 5% NH4OH (100 mL), stirred for 0.5 h, and
evaporated in vacuo. The residue was purified by preparative
reverse-phase chromatography, Prep-LC (B), 0.347 g (74%):
HPLC (Ia) tR 41.8 min; HPLC (Ia) tR 7.11 min; MS (TS)
m/z 975 (M - H)-; [R]23 -68.4° (c 0.1, MeOH). Anal.
D
(C47H63N9O13S‚NH3‚1.8H2O) C, H, N.
Ack n ow led gm en t. We would like to thank Tim
Ordway for the amino acid analysis, Bill Kuipers for the
NMR, and David Coombers for the MS. We would also
like to thank Dr. William Michne for his helpful dis-
cussions and critical reading of the manuscript.
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Boc-Nle-Gly-OMe (16). Using coupling method A, Boc-
Nle-OH (1.61 g, 11.16 mmol) was coupled with H-Gly-OMe‚
HCl (0.900 g, 10.1 mmol): yield 1.425 g (47%); TLC (CMA 9:1:
1) Rf 0.88; mp 97-99 °C; MS (CI) m/z 303 (M + H)+. Anal.
(C14H26N2O5) C, H, N.
H-Nle-Gly-OMe‚HCl (17). Compound 16 (3.77 g, 12.5
mmol) was deprotected using method B: yield 2.88 g (97%);
TLC (CMA, 9:1:1) Rf 0.16; mp 147-148 °C. Anal. (C9H18N2O3‚
HCl) C, H, N.
Hp a -Nle-Gly-OMe (18). To a stirred solution of Hpa-OSu
(3.32 g, 13.3 mmol; prepared by the method of Hankovszky27
)
and DIEA (2.32 mL, 13.3 mmol) in DMF (10 mL) was added a
solution of 17 (2.88 g, 12.1 mmol) and DIEA (2.11 mL, 12.1
mmol) in DMF (40 mL). After 18 h the solvent was removed
in vacuo; the residue was dissolved in EtOAc; washed with
10% citric acid, 1 N NaHCO3, and saturated NaCl; dried
(MgSO4); and evaporated to a small volume. Addition of
hexanes precipitated the product as a white solid, 3.02 g
(77%): TLC (CMA, 9:1:1) Rf 0.53; mp 146-147.5 °C; MS (CI)
m/z 337 (M + H)+. Anal. (C17H24N2O5) C, H, N.
Hp a -Nle-Gly-OH (19). To a stirred, -10 °C solution of
Hpa-Nle-Gly-OMe (3.00 g, 8.92 mmol) in MeOH (30 mL) was
added slowly 1 N NaOH (17.8 mL, 17.8 mmol). After the
solution was warmed to warm to room temperature over 1 h,
water (50 mL) was added. The aqueous solution was washed
with ether and acidified (1 N HCl) to pH 3. The product was
extracted into EtOAc, washed with saturated NaCl, dried
(MgSO4), and evaporated under reduced pressure. The residue
was recrystallized from EtOAc/hexanes, 1.47 g (51%): TLC
(CMA, 9:1:1) Rf 0.19; mp 136-138 °C; MS (CI) m/z 322 (M +
H)+. Anal. (C16H22N2O3) C, H, N.
Hp a -Nle-Gly-Tr p -Nle-MeAsp (OBn )-P h e-NH2 (20). Us-
ing coupling method C, 19 (0.538 g, 1.67 mmol) was coupled
to 15 (1.00 g, 1.39 mmol). To the product in EtOAc (50 mL)
was added ether, and the precipitate was collected by filtration,
1.04 g (76%): HPLC (IIId) tR 32.8 min.
Hp a -Nle-Gly-Tr p -Nle-MeAsp -P h e-NH2 (21). To a solu-
tion of 20 (1.07 g, 1.08 mmol) in MeOH (150 mL) was added
10% Pd/C (300 mg), and the suspension was shaken under a
hydrogen atmosphere for 6 h. The catalyst was removed by
filtration and the solvent evaporated to an oil. The product
was purified by preparative reverse-phase chromatography,
Prep-LC (A), tR 39 min (loading dependent). Product-contain-
ing fractions were evaporated to dryness, and the residue was
dissolved in EtOAc and precipitated with hexanes, 0.408 g
(42%): HPLC (IIIf) tR 9.8 min; MS (FAB) m/z 897 (M + H)+;
AAA, Gly 1.07, MeAsp 1.06, Nle 1.93, Phe 0.94. Anal.
(C47H60N8O10‚1.8H2O) C, H, N.
Meth od D. Hp a (SO3H)-Nle-Gly-Tr p -Nle-MeAsp -P h e-
NH2‚NH3 (6). Su lfa tion Meth od . To a stirred solution of
21 (242 mg, 0.27 mmol, dried overnight under high vacuum)
in anhydrous pyridine (4 mL) under nitrogen was added
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J M970477U