2-Oxopiperazine-based gamma-turn conformationally constrained peptides: synthesis of CCK-4 analogues.

Article abstract of DOI:10.1021/jo0256336

2-Oxopiperazine derivatives 1 have been designed as mimetics of gamma-turn conformationally constrained tripeptides. The synthetic pathway devised for the preparation of both epimers of 1 at C(5) involves a reductive amination of cyanomethyleneamino pseudopeptides with amino acid derivatives, followed by regiospecific lactamization of the resulting C-backbone branched pseudopeptides. The versatility of this methodology is illustrated in the synthesis of analogues of the tetrapeptides Boc-[Nle(31)]-CCK-4 and Boc-[Lys(o-tolylaminocarbonyl)(31)]-CCK-4. The introduction of the new conformational restriction into these Boc-CCK-4 analogues led to a loss of 2 or 3 orders of magnitude in the affinity at CCK receptors. These results suggest the absence of a gamma-turn in the bioactive conformation of the C-terminal tripeptide of CCK-4.

Full text of DOI:10.1021/jo0256336

3866
J . Org. Chem. 2002, 67, 3866-3873
2-Oxop ip er a zin e-Ba sed γ-Tu r n Con for m a tion a lly Con str a in ed
P ep tid es: Syn th esis of CCK-4 An a logu es
Susana Herrero,† M. Teresa Garc´ıa-Lo´pez,† Miriam Latorre,‡ Edurne Cenarruzabeitia,‡
J oaqu´ın Del R´ıo,‡ and Rosario Herranz*,†
Instituto de Quı´mica Me´dica (CSIC), J uan de la Cierva 3, 28006 Madrid, Spain, and Departamento de
Farmacologı´a, Universidad de Navarra, Irunlarrea 1, E-31080 Madrid, Spain
rosario@iqm.csic.es.
Received February 20, 2002
2-Oxopiperazine derivatives 1 have been designed as mimetics of γ-turn conformationally
constrained tripeptides. The synthetic pathway devised for the preparation of both epimers of 1 at
C5 involves a reductive amination of cyanomethyleneamino pseudopeptides with amino acid
derivatives, followed by regiospecific lactamization of the resulting C-backbone branched pseudo-
peptides. The versatility of this methodology is illustrated in the synthesis of analogues of the
tetrapeptides Boc-[Nle31]-CCK-4 and Boc-[Lys(o-tolylaminocarbonyl)31]-CCK-4. The introduction of
the new conformational restriction into these Boc-CCK-4 analogues led to a loss of 2 or 3 orders of
magnitude in the affinity at CCK receptors. These results suggest the absence of a γ-turn in the
bioactive conformation of the C-terminal tripeptide of CCK-4.
In tr od u ction
Turns, defined as regions where a peptide chain
reverses its overall direction, are important elements of
peptide secondary structure, often located at the protein
surface, where they have been implicated in molecular
recognition processes, such as receptor binding, antibody
recognition, and posttranslational modifications. In ad-
dition, structural studies have revealed the presence of
turns in the preferred solution conformations of biological
active peptides, which have been proposed to mediate
their biological activity. â-Turns, which are the most
frequent, involve four consecutive amino acid residues,
whereas γ-turns consist of three residues, which may or
may not be stabilized by an intramolecular hydrogen
bond between the CdO of the first residue (i) and the
NH of the third residue (i+2), forming a seven-membered
ring.1,2 γ-Turns have been classified into classic and
inverse (Figure 1) on the basis of the dihedral φ and ψ
angle values of the (i+1) residue. Classical γ-turns are
characterized by a φi+1 angle of 35° to 106° (mean value
75°) and a ψi+1 angle of -29° to -94° (mean value -64°),3
and the side chain of the i+1 residue assumes a pseudo-
axial orientation, having been called also C7 axial.4 In
contrast, inverse γ-turns are characterized by a φi+1 angle
of -9° to -110° (mean value -79°) and a ψi+1 angle of
14° to 131° (mean value 69°),5 and the side chain of the
i+1 residue in a pseudoequatorial orientation (C7 equato-
rial).4 Although γ-turns are less frequent than â-turns,
an analysis of 54 proteins from the Protein Data Bank
has shown the presence of 12 classic and 117 inverse
F igu r e 1. Schematic description of classic and inverse γ-turns
and the mimetic 1.
γ-turns, frequently forming part of higher secondary
structures such as loops, â-sheets, or R-helices.3,5 Fur-
thermore, it has been proposed that γ-turns are present
in the solution conformation of several peptides, including
vasopresin,6,7 cyclosporin,8 angiotensin II,9,10 bombesin,11
endothelin antagonists,12 bradykinin,13 and the RGD
tripeptide sequence in several fibrinogen and integrin
antagonists.14-19
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J . C. Science 1985, 227, 1309.
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Chem. Eur. J . 1999, 5, 2241.
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T. J .; Widmer, A. Helv. Chim Acta 1985, 68, 682.
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Lai, Z.; Tho¨rnwall, M.; Synnergren, B.; Nilsson, A.; Welch, C. J .;
Sohtell, M.; Westerlund, C.; Nyberg, F.; Karle´n, A.; Hallberg, A. J .
Med. Chem. 1997, 40, 903.
† Instituto de Qu´ımica Me´dica (CSIC).
‡ Universidad de Navarra.
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A. Bioorg. Med. Chem. 2000, 8, 2375.
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10.1021/jo0256336 CCC: $22.00 © 2002 American Chemical Society
Published on Web 04/30/2002

Synthesis of CCK-4 Analogues
J . Org. Chem., Vol. 67, No. 11, 2002 3867
Ta ble 1. Releva n t Top ogr a p h ic P a r a m eter s of th e
Low -En er gy Con for m er s for 3,5-cis- a n d
3,5-tr a n s-2-Oxop ip er a zin es 2 a n d Op tim ized γ-Tu r n s
Several structural motifs have been described to
stabilize or mimic γ-turn conformations, including 1-ami-
nocycloalkane carboxylic acids,20,21 cis-4-amino-L-proline
derivatives,22 cyclic â-enamino nitriles,23 tetrahydro-1H-
azepine derivatives,10,14,17,24 hexahydro-1,4-diazepines,25
cyclic hydrazide derivatives,26 morpholin-3-ones,7 (3-
aminomethyl-2-oxo-1-piperidyl)acetic acid,13 and 1,3,5-
trisubstituted-cyclohexanes.27 However, most of the syn-
thetic approaches described for these γ-turn mimetics do
not allow to introduce diversity of side chains at ap-
propriate positions or to control their stereochemistry.
Herein we describe the design and synthesis of 2-oxopip-
erazine derivatives 1 (Figure 1) as new γ-turn mimetics,
where a methylene bridge replaces the i f i+2 hydrogen
bond, locking the tripeptide into a six-membered ring.
The CdO of the γ-turn i residue is replaced by a chiral
center at C5 in the 2-oxopiperazines 1. Therefore, there
are two possible mimetics for each tripeptide, epimers
at C5. The synthetic pathway devised for mimetics 1
allows the introduction of a wide diversity of side chains
with controlled topography. To illustrate our approach
several analogues of the cholecystokinin (CCK) C-termi-
nal tetrapeptide CCK-4 (Trp-Met-Asp-Phe-NH2) have
been prepared. Although the conformational analysis of
CCK-4 does not show the presence of preferred turn
conformations, some authors have proposed the presence
of â- or γ-turns in its bioactive conformation.28,29 The
presence of a γ-turn has also been proposed in the
bioactive conformation of the CCK1 agonist Boc-Trp-Lys-
(o-tolylaminocarbonyl)-Asp-Phe-NH2 at the C-terminal
tripeptide.30 On the other hand, the piperazine skeleton
∆Ea
(Kcal/mol)
CRi-CRi+2
distance (Å)
conformer
φi+1
ψi+1
3,5-cis-2
-39.66
-54.33
-45.71
1
2
3
0.00
1.06
2.04
14.28
31.26
16.20
5.01
4.57
5.00
3,5-trans-2
-54.22
13.14
1
2
3
4
5
6
0.00
0.77
0.97
1.18
2.29
2.46
1.55
0.72
43.49
33.95
-40.68
-47.79
-46.70
29.58
4.69
5.01
4.90
4.71
4.84
5.00
5.74
5.63
52.41
-58.56
55.42
19.42
68.32
-76.02
classic γ-turn
inverse γ-turn
-56.41
76.40
a
Relative energy of the corresponding conformer.
is considered among the privileged scaffolds for peptido-
mimetic search.31 Particularly, the 2-oxopiperazine ring
has been included into several ligands for receptors of
neurokinins,32-34 CCK,34,35 and enkephalins,36 and into
fibrinogen antagonists.37
Resu lts a n d Discu ssion
Molecu la r Mod elin g. To study the ability of both
epimers 3,5-cis- and 3,5-trans-1 to mimic classic or
inverse γ-turns, a conformational search of the simplified
model compounds (3S,5R)- and (3S,5S)-1,3,5-trimethyl-
2-oxopiperazine (3,5-cis- and 3,5-trans-2, respectively)
was carried out by molecular dynamics at high temper-
ature, followed by minimization in vacuo (ꢀ ) 1) with the
Insight II program.38 The resulting unique minima were
fully optimized with the semi-ab-initio method SAM1,
included in the Ampac 5.0 program. The geometrical
parameters of the low-energy conformers of 3,5-cis- and
3,5-trans-2 are shown in Table 1, in comparison with
those of the optimized classic and inverse γ-turn confor-
mations for Ac-Ala-NHMe obtained with the SAM1
method, starting from the average values of the φi+1 and
ψi+1 dihedral angles for these conformations.38 The
calculated values of these angles for the three low-energy
conformers found for 3,5-cis-2 are in the range of those
reported for inverse γ-turns,5 indicated in Figure 1.
(14) Callahan, J . F.; Bean, J . W.; Burgess, J . L.; Eggleston, D. S.;
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Karash, C. B.; Keenan, R. M.; Kopple, K. D.; Miller, W. H.; Newlander,
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J . M.; Wong, A. S.; Yuan, C.-K.; Huffman, W. F. J . Med. Chem. 1997,
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E.; Mazza, F.; Pochetti, G. Tetrahedron 1995, 51, 2379.
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Tetrahedron Lett. 1996, 37, 1933.
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D. S.; Nichols, A.; Wong, A.; Huffman, W. F. Tetrahedron 1993, 49,
3479.
i
i+2
However, as it was expected, the CR -CR distance was
slightly shorter, as consequence of the closing of the
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35, 289.
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3868 J . Org. Chem., Vol. 67, No. 11, 2002
Herrero et al.
Sch em e 1. Retr osyn th esis of
2-Oxop ip er a zin e-Ba sed γ-Tu r n Mim etics
Sch em e 2. Red u ctive Am in a tion of Ψ[CH(CN)NH]
P seu d op ep tid es
seven-membered pseudocyclic γ-turn into a six-membered
ring. In the case of the epimer 3,5-trans-2, there is a
higher conformational variability, since although the φi+1
and ψi+1 dihedral angles of its lowest energy conformer
are also in the range of the reported values for inverse
γ-turns, conformers 3 and 5 have more similarity with
classic γ-turns.3 The electronic and geometric character-
istics of 3,5-cis- and 3,5-trans-2 have been compared with
those of model classic and inverse γ-turns, using the
SEAL program. Conformers 1 and 2 of 3,5-cis-2 and
conformer 1 of 3,5-trans-2 showed good similarity indices
for their superposition with the model inverse γ-turn,
whereas only the conformer 3 of 3,5-trans-2 gave a good
superposition with the classic γ-turn.38
Sch em e 3. La cta m iza tion of C-Ba ck bon e
Br a n ch ed P seu d op ep tid es
Syn th esis. As outlined in the retrosynthetic Scheme
1, the proposed pathway for 2-oxopiperazine derivatives
1 involves two key steps: reductive amination of cyano-
methyleneamino pseudopeptides39 4, with amino acid
derivatives, followed by regiospecific lactamization of the
resulting C-backbone branched peptides 3. For the study
of this route, we have first selected the pseudodipeptide
Boc-PheΨ[CH(CN)NH]Leu-OMe39 as starting material.
Next, this synthetic methodology has been extended to
the preparation of the proposed CCK-4 analogues.
Branched peptides 3a -d were prepared by applying
our recently described method for the synthesis of C-
backbone branched peptides, via catalytic hydrogenation
of Boc-protected Ψ[CH(CN)NH] pseudopeptides in the
presence of amino acid derivatives,40 shown in Scheme
2. The starting Ψ[CH(CN)NH] pseudopeptides 4a -d
were used as epimeric mixtures at the stereogenic center
of the peptide bond surrogate, which could not be
resolved, as well as the resulting branched peptides (R,S)-
3c and (R,S)-3d . Neither racemization at the inserted
amino acid derivatives 5 and 6 nor epimerization at any
of the stereogenic centers of the starting pseudodipep-
tides 4 were detected in this reductive amination. The
1-unsubstituted 2-oxopiperazines 7a -d , resulting from
the formation of the corresponding primary amine, fol-
lowed by in situ lactamization,40 were detected as minor
products in the RP-HPLC analysis of the crude reaction
mixtures.
xylene, obtaining in each case the 2-oxopiperazine de-
rivative (R)- and (S)-1a , along with their oxidation
product to the 2-oxo-1,2,5,6-tetrahydropyrazine deriva-
tive (R)- and (S)-8a , respectively (Scheme 3). Then, with
the aim of avoiding the formation of these oxidation
products, we lowered the temperature, heating at 100 °C
in toluene. In this way, the 2-oxopiperazines (R)- and (S)-
1a were obtained in good yield as the only reaction
products (Table 2). The study of the oxidation of these
compounds in refluxing xylene showed that the 3,5-cis
epimer (S)-1a oxidized faster (8 h for 100% oxidation)
than its 3,5-trans epimer (R)-1a (16 h for 70% oxidation).
The comparison of the 1H NMR spectra of the 2-oxo-
1,2,5,6-tetrahydropyrazines (R)- and (S)-8a with those of
their respective 2-oxopiperazine analogues (R)- and (S)-
1a showed the disappearance of the signals correspond-
The study of optimal reaction conditions for the lac-
tamization of branched pseudotripeptides 3 was carried
out in both resolved epimers (R)- and (S)-3a . Initially,
this cyclization was attempted by heating in refluxing
(39) Herranz, R.; Sua´rez-Gea, M. L.; Vinuesa, S.; Garc´ıa-Lo´pez, M.
T. J . Org. Chem. 1993, 58, 5186.
(40) Herrero, S.; Sua´rez-Gea, M. L.; Garc´ıa Lo´pez, M. T.; Herranz,
R. Tetrahedron Lett. 2002, 43, 1421.

Synthesis of CCK-4 Analogues
J . Org. Chem., Vol. 67, No. 11, 2002 3869
Ta ble 2. Resu lts of th e La cta m iza tion of Br a n ch ed
P seu d otr ip ep tid es 3
2-oxo-1,2,5,6-tetra-
2-oxopiperazine
yield
hydropyrazine
branched
tripeptide
T
(°C)
yield
t
compd
(%)
compd
(%)
(R)-3a
135 6 h
100 8 h
135 6 h
100 8 h
100 3 days (R)-1b
100 5 days (S)-1b
(R)-1a
(R)-1a
(S)-1a
(S)-1a
24
81
15
77
75
62
60
19
46
21
(R)-8a
(R)-8a
(S)-8a
(S)-8a
(R)-8b
(S)-9b
(R)-8c
(S)-8c
(R)-8d
(S)-8d
21
0
58
0
0
0
0
0
0
0
(R)-3a
(S)-3a
(S)-3a
(R)-3b
(S)-3b
(R,S)-3c (3:1)a 100 5 days (R)-1c
(S)-1c
(R,S)-3d (2:1)a 100 8 days (R)-1d
(S)-1d
F igu r e 2. Preferred conformations for the 2-oxopiperazine
ring in compounds 1.
a
(R)/(S)-Epimer ratio.
Sch em e 4. Con figu r a tion Assign m en t a t th e C5 of
th e 2-Oxop ip er a zin es 1a
ing to the 3-H of the 2-oxopiperazine ring and a deshield-
ing of 0.2-0.5 ppm for 5-H and of ≈1 ppm for the
methylene attached at C3. The 13C NMR spectra showed
the disappearance of the aliphatic C3 (54-58 ppm) and
its appearance in the iminic carbon range at 165-158
ppm.
As shown in Table 2, the lactamization of branched
pseudotripeptides 3b-d was also carried out at 100 °C,
although it required longer heating times than for
compounds 1a . We tried to decrease this lactamization
time by adding 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)
as basic catalyst. However, this addition did not reduce
the reaction time and led to dirtier reaction mixtures as
a result of the formation of decomposition products.
Similar unsuccessful results were obtained when the
lactamization of 3b-d was tried by heating in a micro-
wave oven.
The lactamization was regiospecific between the amino
group of the branching i+2 amino acid and the meth-
oxycarbonyl of the i+1 residue. The alternative lactam-
ization between the i+1 amino group and the carbonyl
of the i+2 residue was not observed in any case. The
disposition. Therefore, these 2-oxopiperazines could mimic
inverse γ-turns, in agreement with the previous molec-
ular modeling calculations. Also according to these
calculations, the 1H NMR parameters of 3,5-trans epimers
(R)-1a -d showed the existence of higher conformational
variability. Thus, the J 5,6 values for (R)-1a and (R)-1c
(4-5 and 9-10 Hz), and the weak NOE effect observed
in their DPFGSE-NOE spectra between 3-H and one of
the 6-H protons, indicative of a 1,4-cis diaxial disposition,
supported the presence of a preferred boat conformation
B, in which the Ri+1 substituent would also adopt an
equatorial disposition, as in inverse γ-turns. However,
1
comparison of the H NMR spectra of the 2-oxopipera-
zines 1a -d with those of their corresponding starting
branched pseudotripeptide 3a -d showed the disappear-
ance of the signal corresponding to the i+1 methoxy
group and a deshielding of 1.0-1.9 ppm for the R-H
proton of the i+2 residue and of 0.4-1.2 ppm for the 6-H
protons of the piperazine ring. The NOE effect between
the piperazine 3-H and 5-H protons, observed in the
DPFGSE-NOE spectra of epimers (S)-1a -d , indicative
of a relative 3,5-cis diaxial disposition, allowed the
assignment of the absolute configuration at C5 in the
2-oxopiperazine derivatives 1a -d and therefore at the
branching center in the starting pseudotripeptides 3a -
d . This assignment was confirmed in compounds (R)- and
(S)-1a , by conversion into their respective 3,6-dioxope-
hydroimidazo[1,5-a]pyrazine derivatives (R)- and (S)-9a
1
the H NMR spectra of (R)-1b and (R)-1d did not show
the presence of a preferred conformation between B, C
or D (Figure 2).
Syn t h esis a n d Biologica l E va lu a t ion of CCK-4
An a logu es. The saponification of the methyl ester of the
aspartic side chain in (R)- and (S)-1b led to the confor-
mationally constrained Boc-Trp-Asp-Phe-NH2 analogues
(R)- and (S)-10b (Scheme 5), which contain the most
significant side chains for the binding to CCK2 recep-
tors.41 On the other hand, as shown in Scheme 5, Boc-
removal in the tripeptide derivatives (R)- and (S)-1c,d ,
followed by coupling with Boc-Trp-OH, and final saponi-
fication, led to the Boc-CCK-4 analogues 12c and 12d .
As the replacement of Met by Nle has not a significant
effect on the biological activity of CCK-4 analogues,41 we
have included this replacement in the design of com-
1
(Scheme 4). The NOE effects observed in the H NMR
spectra of compounds 9a allowed the unequivocal assign-
ment of the absolute configuration at C8a in each epimer,
and at C5 in (R)- and (S)-1a , respectively.
The above-mentioned H3-H5 NOE effect observed in
the 3,5-cis epimers (S)-1a -d , along with their J 5,6
coupling constants (3-5 and 9-12 Hz, see Table 3),
indicated the presence of a preferred chair conformation
for the 2-oxopiperazine ring, (Figure 2, A), in which the
Ri+1 substituent (corresponding to the side chain of the
i+1 residue of a γ-turn) would adopt an equatorial
(41) Martinez, J . In Comprehensive Medicinal Chemistry; Hansch,
C., Sammes, P. G., Taylor, J . B., Eds.; Pergamon Press: Oxford, 1990;
Vol. 3, p 929.

3870 J . Org. Chem., Vol. 67, No. 11, 2002
Herrero et al.
Ta ble 3. Releva n t 1H NMR Da ta for th e Assign m en t of Con figu r a tion a n d Con for m a tion in th e 2-Oxop ip er a zin es 1a -d
compd
Ri
Ph-CH2
Ph-CH2
Ri+1
Ri+2
Me
Ri+3
J
5,6 (Hz)
NOE (%)
preferred conformation
(R)-1a
(S)-1a
(R)-1b
(S)-1b
(R)-1c
(S)-1c
(R)-1d
(S)-1d
iBu
iBu
OMe
OMe
NH2
NH2
NH2
NH2
NH2
NH2
5, 9
4, 11
4, 7
4, 10
4, 10
4, 11
4, 8
H3-H6 (4)a
B
A
Me
H3-H5 (12)b
c
In-CH2
In-CH2
Bu
CH2-CO2Me
CH2-CO2Me
CH2-CO2Me
CH2-CO2Me
CH2-CO2Me
CH2-CO2Me
CH2-Ph
CH2-Ph
CH2-Ph
CH2-Ph
CH2-Ph
CH2-Ph
B, C, D
A
B
A
B, C, D
A
c
H3-H5 (7)b
H3-H6 (1)a
H3-H5 (5)a
Bu
d
Tac-HN-(CH2)4
Tac-HN-(CH2)4
d
3, 9
H3-H5 (2)a
a
b
d
Determined in DPFGSE-NOE spectra. Determined in NOE difference spectra. c In ) Indol-3-yl. Tac ) o-tolylaminocarbonyl.
Sch em e 5. Syn th esis of CCK-4 An a logu es
Con clu sion s
In conclusion, the reductive amination of cyanometh-
yleneamino pseudodipeptides with amino acid deriva-
tives, followed by regiospecific lactamization affords
2-oxopiperazine derivatives, which could mimic γ-turn
conformations and in particular inverse γ-turns. The
application of the proposed synthetic methodology to the
synthesis of several Boc-CCK-4 analogues illustrates its
versatility for the preparation 2-oxopiperazine con-
strained tripeptide analogues with diverse amino acid
side chains. Finally, the loss of affinity at CCK receptors
produced by the introduction of this conformational
restriction into Boc-CCK-4 analogues discards the hy-
pothesis of the existence of a γ-turn in their bioactive
conformation.
Exp er im en ta l Section
Gen er a l. All reagents were of commercial quality. Solvents
were dried and purified by standard methods. Analytical TLC
was performed on aluminum sheets coated with a 0.2 mm layer
of silica gel 60 F254 and preparative TLC on 20 × 20 cm glass
plates coated with a 2-mm layer of silica gel PF254. Silica gel
60 (230-400 mesh) was used for flash chromatography.
Melting points were taken on a micro hot stage apparatus and
are uncorrected. 1H NMR spectra were recorded at 300 or 500
MHz, using TMS as reference, and 13C NMR spectra were
recorded at 50, 75, or 125 MHz. Elemental analyses were
obtained on a CH-O-RAPID apparatus. Analytical RP-HPLC
was performed on a C18 (3.9 × 300 mm, 10 mm) column, with
a flow rate of 1 mL/min, and using a tunable UV detector set
at 214 nm. Mixtures of CH3CN (solvent A) and 0.05% TFA in
H2O (solvent B) were used as mobile phases.
Molecu la r Mod elin g Meth od s. The conformational search
was carried out using the molecular dynamics (MD) technique
at high temperature and minimization in vacuo (ꢀ ) 1) with
the Insight II program.47 The MD procedures were carried out
by heating the molecules at 1500 K, increasing the tempera-
ture 10 K each 0.15 ps, and equilibrating at this temperature
during 20 ps. Finally, 75 ps of simulation was carried out,
storing 300 structures at equal intervals. Each structure was
minimized with the cff91 force field,48 using initially the
steepest descent minimization methods, followed by the con-
jugate gradient until the gradient was bellow 0.0001 kcal/Å.
The minima obtained were compared, and the repeated ones
were eliminated. The unique minima were fully optimized with
the SAM1 method.49 Again, the new minima were compared
in order to eliminate the repeated ones.
pounds 12c, to avoid the chemical instability of the Met
side chain.
Conformationally constrained tripeptides (R)- and (S)-
10b and tetrapeptides (R)- and (S)-12c and -12d were
evaluated as CCK1 and CCK2 receptor ligands, by mea-
suring the inhibition of the specific [3H]propionyl-CCK-
8 binding to rat pancreas and cerebral cortex homoge-
nates,42 respectively. For comparative purposes CCK-8
and the CCK1 and CCK2 selective antagonists
Devazepide43 and PD-135,15844 were also included in the
assay. Except for the Boc-[Lys(Tac)31]-CCK-4 analogue
(R)-12d , which showed a modest affinity at CCK1 recep-
tors (IC50 ) 6 × 10-7 M), none of the 2-oxopiperazine
derivatives 10b, 12c, and 12d showed significant affinity
for CCK1 or CCK2 receptors at concentrations below 10-6
M. The important loss of affinity, higher than 2 orders
of magnitude for compounds (R)- and (S)-12c at CCK2
receptors, with respect to Boc-[Nle31]-CCK-4,45 and 2 or
3 orders of magnitude at CCK1 receptors for compounds
(R)- and (S)-12d , with respect to Boc-[Lys(Tac)31]-CCK-
4,46 suggests the absence of a γ-turn, centered at the Asp
residue, in the bioactive conformation of both CCK-4
analogues.
Syn th esis of Br a n ch ed P seu d otr ip ep tid es (R)- a n d (S)-
3a . Et3N (0.14 mL, 1 mmol) was added to a suspension of
H-Ala-OMe‚HCl (140 mg, 1 mmol) in MeOH (10 mL), and the
mixture was stirred at room temperature for 15 min. Then,
(42) Dauge´, V.; Bohme, G. A.; Crawley, J . N.; Durieux, C.; Stutzman,
J . M.; Feger, J .; Blanchard, J . C.; Roques, B. P. Synapse 1990, 6, 73.
(43) Chang, R. S.; Lotti, V. J . Proc. Natl. Acad. Sci. U.S.A. 1986,
83, 4923.
(44) Hughes, J .; Boden, P.; Costall, B.; Domeney, A.; Kelly, E.;
Horwell, D. C.; Hunter, J . C.; Pinnock, R. D.; Woodruff, G. N. Proc.
Natl. Acad. Sci. U.S.A. 1990, 87, 6728.
(45) Holladay, M. W.; Lin, C. W.; May, C. S.; Garvey, D. S.; Witte,
D. G.; Miller, T. R.; Wolfram, C. A. W.; Nadzan, A. M. J . Med. Chem.
1991, 34, 455.
(46) Shiosaki, K.; Lin, C. W.; Kopecka, H.; Craig, R.; Wagenaar, F.
L.; Bianchi, B.; Miller, T.; Witte, D.; Nadzan, A. M. J . Med. Chem.
1990, 33, 2950.
(47) Biosym Technologies, Insight II (version 2.2.0), San Diego, CA.
(48) Maple, J . R.; Dinur, U.; Hagler, A. T. Proc. Natl. Acad. Sci.
U.S.A. 1988, 85, 5350.
(49) Dewar, M. J . S.; J ie, C.; Yu, J . Tetrahedron 1993, 49, 5003.

Synthesis of CCK-4 Analogues
J . Org. Chem., Vol. 67, No. 11, 2002 3871
Ta ble 4. Sign ifica n t An a lytica l a n d Sp ectr oscop ic Da ta of Br a n ch ed P seu d otr ip ep tid es 3
(R)-3a
(S)-3a
(R)-3b
In-CH2
CH2-CO2Me
Ph-CH2
NH2
(S)-3b
In-CH2
CH2-CO2Me
Ph-CH2
NH2
(R,S)-3ca
(R,S)-3d b
Ri
Ph-CH2
Ph-CH2
Bu
Tac-HN-(CH2)4
CH2-CO2Me
Ph-CH2
NH2
Ri+1
iBu
Me
OMe
(R)
34
iBu
Me
OMe
(S)
CH2-CO2Me
Ph-CH2
NH2
Ri+2
Ri+3
config (*)
yield (%)
formulac
tR (min) (A:B)d
(R)
46
(S)
22
(R,S)
71
(R,S)
54
32
C
26H43N3O6
C
26H43N3O6
C
32H43N5O7
C
32H43N5O7
C
27H44N4O7
C35H52N6O8
10.20, 9.65 (40:60)
25.41 (40:60)
23.33 (40:60)
16.00 (35:65)
14.80 (35:65)
16.90, 15.35 (35:65)
1H NMRe(δ)
Xaa
1-H
2-H
2.51, 2.71
2.36
2.55
2.55
2.39, 2.69
2.66
2.34, 2.52
2.52
2.30, 2.60
2.60
2.30, 2.62; 2.38, 2.52
2.50
3-H
4-H
2-H (Yaa)
2-H (Zaa)
3.87
2.59, 2.88
3.25
3.80
2.73, 2.81
3.37
4.15
2.78, 2.92
3.63
4.07
2.88, 2.96
3.70
3.60, 3.50
1.20
3.70
3.69, 3.48
1.25, 1.48
3.63, 3.60
3.28, 3.30
3.25
3.21
3.13
3.20
3.30, 3.35
13C NMRf
C1 (Xaa)
45.96
51.66
47.25
51.26
47.40
47.62, 50.98
a
b
d
(3:1) (R:S)-Epimer ratio. (2:1) (R:S)-Epimer ratio. c Compounds isolated as foams. Satisfactory analyses for C, H, N. µBondapak
18, A ) CH3CN, B ) 0.05% TFA in H2O. e Spectra registered at 300 or 500 MHz in CDCl3. Assignment carried out with the help of
C
DQCOSY spectra. f Spectra registered at 50 or 125 MHz in CDCl3. Assignment carried out with the help of HMQC spectra.
Ta ble 5. An a lytica l Da ta of 2-Oxop ip er a zin es 1a -d
compda
Ri
Ph-CH2
Ph-CH2
In-CH2
In-CH2
Bu
Ri+1
Ri+2
Me
Ri+3
config (*)
yield (%)
formulab
t
R (A:B)c
(R)-1a
(S)-1a
(R)-1b
(S)-1b
(R)-1c
(S)-1c
(R)-1d
(S)-1d
iBu
iBu
OMe
OMe
NH2
NH2
NH2
NH2
NH2
NH2
(5R)
(5S)
(5R)
(5S)
(5R)
(5S)
(5R)
(5S)
81
77
75
62
60
19
46
21
C25H39N3O5
C25H39N3O5
6.91 (50:50)
4.80 (50:50)
20.40 (30:70)
18.51 (30:70)
20.53 (30:70)
18.26 (30:70)
9.70 (40:60)
6.11 (40:60)
Me
CH2-CO2Me
CH2-CO2Me
CH2-CO2Me
CH2-CO2Me
CH2-CO2Me
CH2-CO2Me
CH2-Ph
CH2-Ph
CH2-Ph
CH2-Ph
CH2-Ph
CH2-Ph
C
C
C
C
C
C
31H39N5O6
31H39N5O6
26H40N4O6
26H40N4O6
34H48N6O7
34H48N6O7
Bu
Tac-HN-(CH2)4
Tac-HN-(CH2)4
a
b
Foams, except for (R)-1a , white solid, mp 142-144 °C (EtOAc-hexane). Satisfactory analyses for C, H, N. c µBondapak C18, A )
CH3CN, B ) 0.05% TFA in H2O.
the pseudodipeptide Boc-PheΨ[CH(CN)NH]Leu-OMe [(R,S)-
4a ] (200 mg, 0.5 mmol) and 10% Pd(C) (300 mg) were added,
and the mixture was hydrogenated at 1 atm of H2 and room
temperature for 8 h. Afterward, the catalyst was filtered off
and washed with MeOH, the solvent was evaporated, and the
reaction crude was dissolved in EtOAc (20 mL). This solution
was washed successively with H2O and brine, dried over Na2-
SO4, and evaporated to dryness. The residue was purified by
flash chromatography using 5-30% EtOAc in hexane gradient
as mobile phase. Significant analytical and spectroscopic data
of (R)- and (S)-3a are summarized in Table 4.
Gen er a l P r oced u r e for th e P r ep a r a tion of Br a n ch ed
P seu d otr ip ep tid es 3b-d . Pd(C) (10%, 300 mg) was added
to a solution of the corresponding Ψ[CH(CN)NH] pseudodipep-
tide (R,S)-4b-d (0.5 mmol) and H-Phe-NH2 (6) (164 mg, 1
mmol) in MeOH (10 mL), and the mixture was hydrogenated
at 1 atm of H2 and room temperature for 5 days. Afterward,
the reaction mixture was worked up as above indicated for
the synthesis of 3a . The resulting branched pseudotripeptides
3b-d were purified by flash chromatography, using 0-6%
MeOH in CH2Cl2 gradient as mobile phase. Significant ana-
lytical and spectroscopic data of these compounds are sum-
marized in Table 4.
Gen er a l P r oced u r e for th e Syn th esis of 2-Oxop ip er a -
zin es 1a -d . A solution of the corresponding branched pseudo-
tripeptide 3a -d (0.3 mmol) in toluene (50 mL) was stirred at
100 °C for 6 h to 8 days, depending on the starting pseudot-
ripeptide. Then, the solvent was evaporated under reduced
pressure, and the residue was purified by flash chromatogra-
phy, using 10-50% EtOAc in hexane (1a and 1c) or 1-5%
MeOH in CH2Cl2 (1b and 1d ) gradients as mobile phases. (R)-
Epimers and (S)-epimers of 1c and 1d were resolved in this
chromatographic purification. The significant analytical and
spectroscopic data of these conformationally constrained trip-
eptide analogues are summarized in Tables 5 and 6.
Syn th esis of th e 2-Oxo-1,2,5,6-tetr a h yd r op yr a zin es
(R)- a n d (S)-8a . A solution of the 2-oxopiperazines (R)- and
(S)-1a (69 mg, 0.15 mmol) in xylene (25 mL) was heated at
135 °C for 8 and 16 h, respectively. Afterward, the solvent was
removed under reduced pressure, and the residue was purified
by flash chromatography, using 10-50% EtOAc in hexane
gradient as mobile phase.

3872 J . Org. Chem., Vol. 67, No. 11, 2002
Herrero et al.
Ta ble 6. Releva n t 1H NMRa Da ta of 2-Oxop ip er a zin es
1H, J ) 7 Hz, 1-H (ethyl)], 7.21 (m, 5H, aromatics). Anal. Calcd
for C21H29N3O4: C, 65.09; H, 7.54; N, 10.84. Found: C, 65.42;
H, 7.87; N, 11.01.
1a -d
compd 3-H 5-H 6-Hax 6-Hec
J
5,6 (Hz)
3-CH2
5-CH 1-CH
(1S,5S,8aS)-5-Isobu tyl-7-[(1S)-1-m eth oxycar bon yleth yl]-
1-p h en ylm eth yl-3,6-d ioxop er h yd r o-im id a zo[1,5-a ]p yr a -
zin e [(S)-9a ]: foam (11 mg, 53%); 1H NMR (CDCl3, 500 MHz)
δ 0.90 and 0.92 [2d, 6H, J ) 6 Hz, 3-H (iBu)], 1.28 [d, 3H, J )
7 Hz, 2-H (ethyl)], 1.80 [m, 1H, 1-H (iBu)], 2.00 and 2.08 [2m,
2H, 2-H (iBu)], 2.80 (dd, 1H, J ) 3 and 11 Hz, 8-H), 2.86 (dd,
1H, J ) 7 and 13 Hz, CH2-Ph), 3.00 (dd, 1H, J ) 4 and 13 Hz,
CH2-Ph), 3.15 (dd, 1H, J ) 10 and 11 Hz, 8-H), 3.68 (m, 2H,
1-H and 8a-H), 3.73 (s, 3H, OCH3), 4.19 (dd, 1H, J ) 4 and 7
Hz, 5-H), 4.86 (s, 1H, 2-H), 5.18 [q, 1H, J ) 7 Hz, 1-H (ethyl)],
7.21 (m, 5H, aromatics). Anal. Calcd for C21H29N3O4: C, 65.09;
H, 7.54; N, 10.84. Found: C, 65.31; H, 7.65; N, 10.48.
Syn th esis of th e 2-Oxop ip er a zin e Der iva tives (R)- a n d
(S)-10b. NaOH (1 N, 0.5 mL, 50 mmol) was added to a solution
of the corresponding methyl ester (R)- and (S)-10b (23 mg, 40
µmol) in 10% H2O in MeOH (10 mL), and the mixture was
stirred at room temperature for 3 h. Afterward, the solvent
was removed under reduced pressure, the residue was dis-
solved in H2O, and the solution was washed with CH2Cl2. The
aqueous phase was acidified to pH 5-6, adding DOWEX 50×4
acid resin. After adding EtOAc, the resin was filtered off, and
the two phases were separated. The organic phase was dried
over Na2SO4 and evaporated to dryness. The residue was
triturated with diethyl ether, to yield the corresponding
carboxylic acid (R)- or (S)-10b quantitatively.
(R)-1a 3.53 3.20 3.26 3.35
(S)-1a 3.60 3.35 3.21 3.10
(R)-1b 3.84 2.76 3.12 3.36
(S)-1b 3.58 3.40 2.74 3.22
(R)-1c 3.75 2.78 3.20 3.44
(S)-1c 3.70 2.92 3.23 3.17
(R)-1d 3.84 2.79 3.16 3.37
(S)-1d 3.70 2.73 3.20 3.04
5, 9
4, 11
4, 7
4, 10
4, 10
4, 11
4, 8
1.55, 1.70 3.80 5.15
1.40, 1.86 3.88 5.06
2.43, 2.72 3.80 5.24
2.63, 2.86 3.80 4.60
2.20, 2.47 3.43 5.40
2.37, 2.76 3.51 5.10
2.40, 2.71 3.50 4.82
2.67, 2.85 3.50 4.90
3, 9
a
Spectra registered at 300 or 500 MHz in CDCl3, except for
(R)- and (S)-1c, registered in (CD3)2CO. Assignment carried out
with the help of DQCOSY spectra.
(5R)-3-Isobu tyl-5-[(1S)-1-(ter t-bu tyloxycar bon ylam in o)-
2-p h en ylet h yl]-1-[(1S)-1-m et h oxyca r b on ylet h yl]-2-oxo-
1,2,5,6-tetr a h yd r op yr a zin e [(R)-8a ]: white solid (35 mg,
51%); mp 42-45 °C (EtOAc-hexane); tR 12.13 (A:B ) 50:50);
1H NMR (CDCl3, 500 MHz) δ 0.94 and 0.97 [2d, 6H, J ) 7 Hz,
CH3 (iBu)],1.36 (s, 9H, Boc), 1.44 [d, 3H, J ) 7 Hz, CH3 (Ala)],
2.10 [m, 1H, CH (iBu)], 2.40 [dd, 1H, J ) 6 and 14 Hz, CH2
(iBu)], 2.66 [dd, 1H, J ) 5 and 14 Hz, CH2 (iBu)], 2.98 (d, 2H,
J ) 8 Hz, CH2Ph), 3.34 (dd, 1H, J ) 9.5 and 13 Hz, 6-H), 3.70
(m, 2H, 5-H and 6-H), 3.72 (s, 3H, OMe), 4.02 (m, 1H, 5-CH),
5.04 (d, 1H, J ) 9 Hz, NH), 5.16 [q, 1H, J ) 7 Hz, 1-H (Ala)],
7.08-7.20 (m, 5H, aromatics); 13C NMR (CDCl3, 125 MHz) δ
15.21 (CH3), 23.43 (CH3), 23.21 (CH3), 27.00 (CH), 29.03 (CH3),
30.46 (CH2), 39.99 (CH2), 42.34 (CH2), 52.40 (CH), 53.25 (CH3),
53.64 (CH), 59.81 (CH), 80.00 (C), 126.41, 128.81, 129.44 (CH),
137.82 (C), 155.30, 158.00, 165.22, 171.57 (C). Anal. Calcd for
(3S,5R)-5-[(1S)-1-(ter t-Bu tyloxyca r bon yla m in o)-2-(in -
d ol-3-yl)eth yl]-1-[(1S)-1-ca r ba m oyl-2-p h en yleth yl]-3-ca r -
boxym eth yl-2-oxop ip er a zin e [(R)-10b]: white solid (22 mg,
100%); mp 159-161 °C (CH2Cl2-MeOH); tR 7.73 (A:B ) 50:
1
C
25H37N3O5: C, 65.34; H, 8.11; N, 9.14. Found: C, 65.30; H,
50); H NMR [(CD3)2CO + D2O, 300 MHz] δ 1.30 [s, 9H, CH3
8.38; N, 8.98.
(Boc)], 2.10-2.40 [m, 2H, 2-H (Asp)], 2.80-3.50 [m, 8H, 5-H,
6-H, 2-H and 3-H (Trp), and 3-H (Phe)], 3.80-4.00 [m, 2H,
3-H and 2-H (Phe)], 5.30 (bs, 1H, NH-Boc), 6.90-7.30 [m, 8H,
Ph, and 2-, 5- and 6-H (In)], 7.35 [d, 1H, J ) 7 Hz, 7-H (In)],
7.53 [d, 1H, J ) 7 Hz, 4-H (In)], 10.15 [bs,1H, NH (In)]. Anal.
Calcd for C30H37N5O6: C, 63.93; H, 6.62; N, 12.43. Found: C,
63.84; H, 6.73; N, 12.16.
(5S)-3-Isobu tyl-5-[(1S)-1-(ter t-bu tyloxycar bon ylam in o)-
2-p h en ylet h yl]-1-[(1S)-1-m et h oxyca r b on ylet h yl]-2-oxo-
1,2,5,6-tetr a h yd r op yr a zin e [(S)-8a ]: foam (46 mg, 67%); tR
1
14.30 (A:B ) 50:50); H NMR (CDCl3, 500 MHz) δ 0.98 and
0.99 [2d, 6H, J ) 6 Hz, CH3 (iBu)],1.40 (s, 9H, Boc), 1.40 [d,
3H, J ) 8 Hz, CH3 (Ala)], 2.20 [m, 1H, CH (iBu)], 2.55 [m, 2H,
CH2 (iBu)], 3.07 (d, 2H, J ) 7 Hz, CH2Ph), 3.15 (dd, 1H, J )
4 and 12.5 Hz, 6-H), 3.28 (t, 1H, J ) 12.5 Hz, 6-H), 3.60 (m,
1H, 5-H), 3.57 (s, 3H, OMe), 4.00 (m, 1H, 5-CH), 4.71 (d, 1H,
J ) 10 Hz, NH), 5.20 [q, 1H, J ) 7 Hz, 1-H (Ala)], 7.10-7.40
(m, 5H, aromatics); 13C NMR (CDCl3, 125 MHz) δ 14.18 (CH3),
22.37 (CH3), 22.80 (CH3), 25.96 (CH), 28.29 (CH3), 29.71 (CH2),
39.29 (CH2), 42.59 (CH2), 51.92 (CH), 52.36 (CH3), 52.93 (CH),
56.93 (CH), 79.50 (C), 126.49, 128.55, 129.51 (CH), 136.00 (C),
(3S,5S)-5-[(1S)-1-(ter t-Bu t yloxyca r b on yla m in o)-2-(in -
d ol-3-yl)eth yl]-1-[(1S)-1-ca r ba m oyl-2-p h en yleth yl]-3-ca r -
boxym eth yl-2-oxop ip er a zin e [(S)-10b]: white solid (21 mg,
100%); mp 165-168 °C (CH2Cl2-MeOH); tR 9.33 (A:B ) 50:
1
50); H NMR [(CD3)2CO + D2O, 300 MHz] δ 1.30 [s, 9H, CH3
(Boc)], 2.40 [dd, 1H, J ) 8 and 16 Hz, 2-H (Asp)], 2.60-2.90
[m, 3H, 5-H and 3-H (Phe)], 2.80 [dd, 1H, J ) 5 and 16 Hz,
2-H (Asp)], 3.00-3.40 [m, 4H, 6-H, and 3-H (Trp)], 3.60 [m,
1H, 2-H (Trp)], 4.00 [m, 2H, 3-H, and 2-H (Phe)], 6.25 (d, 1H,
J ) 8 Hz, NH-Boc), 6.70-7.10 [m, 8H, Ph, and 2-, 5- and 6-H
(In)], 7.40 [d, 1H, J ) 7 Hz, 7-H (In)], 7.50 [d, 1H, J ) 7 Hz,
4-H (In)], 10.30 [bs,1H, NH (In)]. Anal. Calcd for C30H37N5O6:
C, 63.93; H, 6.62; N, 12.43. Found: C, 63.83; H, 6.72; N, 12.36.
Gen er a l P r oced u r e for th e Syn th esis th e 2-Oxop ip -
er a zin e-Ba sed Tetr a p ep tid e An a logu es 11c a n d 11d . A
solution of the corresponding 2-oxopiperazine derivative (R)-
and (S)-1c,d (50 mmol) in saturated solution of HCl in MeOH
(5 mL) was stirred at room temperature for 5 h. After the
solvent was removed under reduced pressure, the residue was
dissolved in water and lyophilized. The resulting residue was
dissolved in dry CH2Cl2 (5 mL); Boc-L-Trp-OH (22 mg, 75
µmol), benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate (BOP, 32 mg, 75 µmol), and TEA (13 µL,
100 µmol) were added successively to that solution; and
stirring was continued at room temperature for 16 h. The
reaction mixture was diluted with CH2Cl2, washed successively
with 10% citric acid, 10% NaHCO3, H2O, and brine, dried over
Na2SO4, and evaporated. The residue was purified by prepara-
tive TLC using 2% MeOH in CH2Cl2 as eluant. The relevant
analytical and spectroscopic data of compounds (R)- and (S)-
11c,d are summarized in Table 7.
152.41, 155.69, 166.00, 169.50 (C). Anal. Calcd for C25H37
N3O5: C, 65.34; H, 8.11; N, 9.14. Found: C, 65.07; H, 8.41; N,
8.85.
-
Syn t h esis of t h e 3,6-Dioxop er h yd r oim id a zo[1,5-a ]-
p yr a zin es (R)- a n d (S)-9a . A solution of the corresponding
2-oxopiperazine (R)- and (S)-1a (25 mg, 54 mmol) in saturated
solution of HCl in MeOH (5 mL) was stirred at room temper-
ature for 5 h. After the solvent was removed under reduced
pressure, the residue was dissolved in water and lyophilized.
The residue was dissolved in dry CH2Cl2 (5 mL), and the
solution was cooled to 0 °C. Then, TEA (15 µL, 108 µmol), bis-
(trichloromethyl) carbonate (8 mg, 27 µmol), and TEA (22 µL,
162 µmol) were added successively, and the reaction mixture
was stirred at 0 °C for 4 h. Afterward, this reaction mixture
was diluted with CH2Cl2 (5 mL), washed with water and brine,
dried over Na2SO4, and avaporated. The residue was purified
by preparative TLC using (1:1) EtOAc-hexane as eluant.
(1S,5S,8aR)-5-Isobu tyl-7-[(1S)-1-m eth oxycar bon yleth yl]-
1-p h en ylm et h yl-3,6-d ioxop er h yd r oim id a zo[1,5-a ]p yr a -
zin e [(R)-9a ]: foam (13 mg, 52%); 1H NMR (CDCl3, 500 MHz)
δ 0.86 and 0.93 [2d, 6H, J ) 6 Hz, 3-H (iBu)], 1.32 [d, 3H, J )
7 Hz, 2-H (ethyl)], 1.53 [m, 1H, 1-H (iBu)], 1.69 [m, 1H, 2-H
(iBu)], 1.73 [m, 1H, 1-H (iBu)], 2.71 (dd, 1H, J ) 10 and 13.5
Hz, CH2-Ph), 2.82 (dd, 1H, J ) 5 and 13.5 Hz, CH2-Ph), 3.10
(dd, 1H, J ) 4 and 11 Hz, 8-H), 3.68 (s, 3H, OCH3), 3.69 (t,
1H, J ) 11 Hz, 8-H), 3.86 (m, 1H, 8a-H), 4.08 (m, 1H, 1-H),
4.39 (dd, 1H, J ) 4 and 11 Hz, 5-H), 4.50 (s, 1H, 2-H), 5.08 [q,
Syn th esis of th e Boc-CCK-4 An a logu es (R)- a n d (S)-
12c,d . These compounds were prepared by saponification of
the methyl esters (R)- and (S)-11c,d (40 µmol), by applying
the methodology described above for the preparation of the

Synthesis of CCK-4 Analogues
J . Org. Chem., Vol. 67, No. 11, 2002 3873
Ta ble 7. Releva n t An a lytica l a n d Sp ectr oscop ic Da ta of 2-Oxop ip er a zin e-Ba sed Tetr a p ep tid e An a logu es 11c,d a
(R)-11c
(S)-11c
(R)-11d
(S)-11d
Ri
CH3(CH2)3
(R)
84
CH3(CH2)3
(S)
76
Tac-NH-(CH2)4
(R)
79
33.55 (32:68)
C45H59N8O
Tac-NH-(CH2)4
(S)
73
37.41 (32:68)
C45H59N8O
config (*)
yield (%)
tR (min) (A:B)b
formulac
9.90 (40:60)
9.98 (40:60)
C
37H50N6O7
C
37H50N6O7
1H NMRd (δ)
2-oxopiperazine
3-H
3.50
2.10
2.65
2.85
9
3.46
2.25
2.55
2.35
10
3.52
2.32
2.77
3.00
8.5
3.57
2.46
2.88
2.64
11
5-H
6-Hax
6-Hec
ax
J 5,6
J 5,6
ec
5
2
4
3
3-CH2
5-CH
1-CH
Trp
2.20, 2.50
3.65
5.22
2.38, 2.70
3.46
4.17
2.28, 2.58
3.58
5.18
2.54, 2.76
3.63
4.94
2-H
4.27
4.30
4.44
4.46
3-H
3.05, 3.13
3.05, 3.12
3.17, 3.20
3.14, 3.30
a
b
Foams, except for (R)-11d , mp 115-118 °C (EtOAc-hexane). µBondapak C18, A ) CH3CN, B ) 0.05% TFA in H2O. c Satisfactory
analyses for C, H, N. c Spectra registered at 500 MHz in CDCl3. Assignment carried out with the help of DQCOSY spectra.
Ta ble 8. Releva n t An a lytica l a n d Sp ectr oscop ic Da ta of th e Boc-CCK-4 An a logu es 12c,d a
(R)-12c
(S)-12c
(R)-12d
(S)-12d
Ri
CH3(CH2)3
(R)
151-154
13.65 (35:65)
CH3(CH2)3
(S)
110-113
15.60 (35:65)
Tac-NH-(CH2)4
(R)
120-123
15.23 (35:65)
Tac-NH-(CH2)4
(S)
130-132
16.61 (35:65)
config (*)
mp (°C)
tR (min) (A:B)b
formulac
C
36H48N6O7
C
36H48N6O7
C
44H57N8O8
C44H57N8O8
1H NMRd (δ)
2-oxopiperazine
3-H
3.60
2.65
2.85
3.20
10
3.24
2.24
2.88
2.12
11
3.67
2.74
3.42
3.48
14
3.34
2.37
3.04
3.07
14.5
7
5-H
6-Hax
6-Hec
ax
J 5,6
J 5,6
ec
3
1
9
3-CH2
5-CH
1-CH
Trp
2.08, 2.30
3.84
5.24
2.42, 2.73
3.67
4.30
2.07, 2.36
3.84
5.35
2.50, 2.76
3.40
4.40
2-H
3-H
4.42
3.04, 3.28
3.75
3.24
4.47
3.13, 3.27
3.99
3.27
a
b
Obtained in quantitative yields. µBondapak C18, A ) CH3CN, B ) 0.05% TFA in H2O. c Satisfactory analyses for C, H, N. c Spectra
registered at 500 MHz in (CD3)2CO + D2O. Assignment carried out with the help of DQCOSY spectra.
carboxylic acids 10b. The relevant analytical and spectroscopic
data of compounds (R)- and (S)-12c,d are summarized in Table
8.
100 000g. Brain membranes (0.45 mg protein/tube) were
incubated with 1 nM [3H]pCCK-8 in 50 mM Tris-HCl buffer,
pH 7.4, containing MgCl2 (5 mM) and bacitracin (0.2 mg/mL)
for 60 min at 25 °C. Final incubation volume was 0.5 mL in
both cases. Nonspecific binding was determined using CCK-8
1 µM as the cold displacer.
CCK1 a n d CCK2 Recep tor Bin d in g Assa ys. Binding
assays were performed using rat pancreas and cerebral cortex
homogenates, respectively, according to the method described
by Dauge´ et al.42 with minor modifications. Briefly, rat
pancreas tissue was carefully cleaned and homogenized in
Pipes HCl buffer, pH 6.5, containing 30 mM MgCl2 (15 mL/g
of wet tissue), and the homogenate was then centrifuged twice
at 4 °C for 10 min at 50 000g. For displacement assays,
pancreatic membranes (0.2 mg protein /tube) were incubated
with 0.5 nM [3H]pCCK-8 in Pipes HCl buffer, pH 6.5, contain-
ing MgCl2 (30 mM), bacitracin (0.2 mg/mL) and soybean
trypsin inhibitor (SBTI, 0.2 mg/mL) for 120 min at 25 °C. Rat
brain cortex was homogenized in 50 mM Tris-HCl buffer pH
7.4 containing 5 mM MgCl2 (20 mL/g of wet tissue), and the
homogenate was centrifuged twice at 4 °C for 35 min at
Ack n ow led gm en t. This work has been supported
by the CICYT (SAF00-0147), and Consejeria de Educa-
cio´n y Cultura de la Comunidad de Madrid (CAM-08.5/
0006/1998).
Su p p or tin g In for m a tion Ava ila ble: Complete combus-
tion elemental analytical date for the new described com-
pounds, and significant 13C NMR data for the 2-oxopiperazine
derivatives 1a -d , 11c,d , and 12c,d . This material is available
free of charge via the Internet at http://pubs.acs.org.
J O0256336