Biological activity of cholecystokinin (30-33) tetrapeptide analogues

Article abstract of DOI:10.1007/s11171-005-0016-6

Analogues of the endogenous peptide corresponding to the 30-33 sequence of cholecystokinin (Trp-Met-Asp-Phe-NH₂) were synthesized, and their biological activity was studied. It was shown that, in rats, the N-succinylated Nle² analogu

Full text of DOI:10.1007/s11171-005-0016-6

Russian Journal of Bioorganic Chemistry, Vol. 31, No. 2, 2005, pp. 119–127. Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 130–139.
Original Russian Text Copyright © 2005 by Proskuryakova, Bespalova, Pal’keeva, Petrichenko, Pankratova, Shokhonova, Anokhina.
Biological Activity of Cholecystokinin (30–33)
Tetrapeptide Analogues
1
T. V. Proskuryakova*, Zh. D. Bespalova**, M. E. Pal’keeva**, O. B. Petrichenko*,
N. V. Pankratova*, V. A. Shokhonova*, and I. P. Anokhina*
* National Research Center of Addiction, Ministry of Public Health of the Russian Federation,
Malyi Mogil’tsevskii per. 3, Moscow, 119002 Russia
** Russian Cardiological Research and Production Association, Ministry of Public Health of the Russian Federation,
Tret’ya Cherepkovskaya ul. 15a, Moscow, 121552 Russia
Received January 5, 2004; in final form, October 22, 2004
Abstract—Analogues of the endogenous peptide corresponding to the 30–33 sequence of cholecystokinin
(Trp-Met-Asp-Phe-NH2) were synthesized, and their biological activity was studied. It was shown that, in rats,
the N-succinylated Nle2 analogue of this tetrapeptide exhibits increased anxiolytic properties in the dark–light
chamber test and an enhanced alcohol intake by both the control animals and the alcohol-dependent animals
under the conditions of free choice. Introduction of an isopropyl residue into the C-terminal amide of the Nle2
analogue resulted in the appearance of anxiogenic and antialcohol activity and the ability to increase the mor-
phine analgesic effect in the tail-flick test on rats. The two synthesized analogues retained an affinity for chole-
cystokinin receptors.
Key words: cholecystokinin, 30–33 fragment, synthesis of analogues, biological activity
1
INTRODUCTION
affinity for CCR-1 and CCR-2, whereas CCK-4 has a
pronounced selectivity toward the CCR-2 [15].
The functional role of cholecystokinin is still not
completely elucidated, especially in the central nervous
system. However, it is now ascertained that the CCK
system is involved into the regulation of satiation [1],
gastrointestinal reactions [2], immune reactions [3],
motion activity [4], memory [5], learning [6], pain sen-
sitivity [7], and anxiety [8, 9]. One of the mechanisms
of participation of the CCK-system in all these pro-
cesses is associated with initiation of complex regula-
tory pathways by CCK peptides [10].
The ability of CCK peptides to modulate function-
ing of various neurotransmitter systems and a positive
clinical effect of some of them for the treatment of
anxiety [16, 17] prompted the search for new com-
pounds with affinity for CCRs. Compounds of various
chemical structures serve as a basis for the design of
new ligands of CCRs [18, 21]. We believe that new
ligands for CCRs created on the basis of endogenous
CCK peptides are the most promising [22–25].
2
The basic requirement to new structural analogues
of CCK peptides is the retention of their affinity for
CCRs and their selectivity to one of the two types of
CCRs, which allows the decrease in their unspecific
toxicity. This problem can be solved by the structure–
function analysis, which is however difficult for large
molecules. Therefore, most scientists use CCK-4 (Trp-
Met-Asp-Phe-NH2) as a minimal active fragment with
a pronounced selectivity toward CCR-2 [15] when
designing new structures.
CCK is synthesized in the pancreatic and brain tis-
sues. Proteolytic enzymes cleave CCK with the forma-
tion of a number of biologically active fragments (CCK
peptides), the octapeptide (CCK-8) and the tetrapeptide
(CCK-4) being the most studied among them [11]. It
was found that CCK-8 with the sulfated Tyr7 residue
[CCK-8(S)] is the most abundant CCK peptide in CNS
(its content is as high as 90%) [12, 13]. The activity of
biologically active CCK peptides is mediated by the
receptors of two types (CCR-1 and CCR-2) located
both in peripheral tissues and in CNS [14]. It is estab-
lished that CCK-8(S) exhibits practically the same
Our goal was the synthesis of CCK-4 peptide ana-
logues and the study of their biological activity in
experimental animal models.
1
Corresponding author; phone: +7 (095) 241-0243; e-mail: pros-
lab@mtu-net.ru
2
RESULTS AND DISCUSSION
Abbreviations: CCK, cholecystokinin; CCK peptide; CCR, CCK-
peptide receptor; CNS, central nervous system; EGTA, ethylene
glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid; HEPES,
N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid; ONSu,
N-hydroxysuccinimide; and Suc, succinyl.
It was shown that conformational changes in practi-
cally all the peptide bonds of CCK-4 affect both affinity
and selectivity of analogues for CCRs [26–28]. The
1068-1620/05/3102-0119 © 2005 Pleiades Publishing, Inc.

120
PROSKURYAKOVA et al.
Asp Phe
Trp
Nle
Z
ONp
NH3/MeOH (NH2–CH(CH3)2)
NH2 (I) (–NH–CH(CH3)2)
H2/Pd
Z
OBut
ONp H
Z
Z
NH2
OBut
NH2 (II)
H2/Pd
OBut
OBut
NH2 (III)
NH2 (IV)
ONp
H
Z
H
H2/Pd
OBut
Boc
Boc
ONp H
NH2 (V)
OBut
NH2 (VI) (–NH–CH(CH3)2)
TFA
H–Trp–Nle–Asp–Phe–NH2 (VII) H–Trp–Nle–Asp–Phe–NH–CH(CH3)2 (IX)
(CH2CO)2O
HOOC(CH2)COO–Trp–Nle–Asp–Phe–NH2 (VIII)
Scheme 1. Scheme of synthesis of analogue (VIII), Suc-Trp-Nle-Asp-Phe-NH .
2
group. Deprotection of both Boc- and But-protected tet-
rapeptides was achieved by the treatment with TFA
containing 5% water and 5% ethanedithiol. The target
products (VIII) and (IX) were identified by the meth-
ods of quantitative amino acid analysis and NMR spec-
troscopy. Their homogeneity was confirmed by TLC
and analytical reverse phase HPLC. The data of NMR
spectroscopy of the synthesized compounds and their
physicochemical characteristics are given in Tables 1
and 2, respectively.
structural modifications have been revealed that
resulted in the analogues with pronounced selectivity
toward CCR-1 [29]. Moreover, a possibility of prepara-
tion of agonists or antagonists of CCRs was demon-
strated by the simultaneous modification of N- and C-
terminal regions of CCK-4 [24, 25].
We carried out a conformational computer analysis
of CCK-4 using a commercial computer program Mod-
eller, proposed a number of structural modifications,
and synthesized two analogues: Suc-Trp-Nle-Asp-Phe-
NH2 (VIII) and Trp-Nle-Asp-PheNHCH(CH3)2 (IX)
(Scheme 1).
The affinity of analogues (VIII) and (IX) for the
CCK-receptors was examined in the competition
experiments on the binding of [125I]-ëëä-8(S) and the
analogues to the membrane preparations of cortex
(where CCR-2 are predominantly located) and to the
preparations of pancreas (where CCR-1 are localized).
Both peptides were synthesized by the conventional
method of peptide synthesis in solution using a step-
wise elongation of the peptide chains starting from the
corresponding C-terminal amino acid and active esters
of Z- and Boc-protected amino acid derivatives. The Both analogues of CCK-4 inhibited the [125I]-ëëä-8(S)-
side chain of aspartic acid was blocked by tert-butyl binding to the specific sites in the dose-dependent man-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 2 2005

BIOLOGICAL ACTIVITY OF CHOLECYSTOKININ (30–33) TETRAPEPTIDE ANALOGUES
121
Table 1. Chemical shifts (δ, ppm) of amide and aliphatic
times increased its affinity for CCR-1 (Ki 4770 against
11900 nM), whereas its affinity for CCR-2 remained
the same (Ki 34 against 42 nM). At the same time, the
introduction of isopropyl group into the C-terminal
amide of analogue (IX) further increased its affinity for
CCR-1 (Ki 234 nM) and simultaneously decreased its
affinity for CCR-2 by two orders of magnitude (Ki
3290 nM).
protons in the CCK-4 peptide analogues
Peptide
residue
NH CαH
CβH
Other signals
Suc–Trp–Nle–Asp–Phe–NH2 (VIII)
8.11 4.52 3.11; 2.94
Trp1
Nle2
Asp3
Phe4
–
–
–
7.93 4.16 1.47; 1.57
According to [26], succinylation of the N-terminal
8.13 4.49 2.65; 2.49
Trp
(Suc-Trp-Met-Asp-Phe-NH2,
Suc-CCK-4)
7.79 4.36 3.03; 2.84
Suc: CH2 3.35
increased selectivity of the peptide to CCR-2 by two
orders of magnitude in comparison with Boc-CCK-4. A
simultaneous substitution of Leu for Met2 slightly
decreased the selectivity to CCR-2, whereas the
replacement of Met2 by Gly, Ser, Phe, Pro, or Ala
resulted in a significant decrease in selectivity of the
corresponding analogues to CCR-2 (practically to the
level of that of the L-365,260 antagonist of CCR-2).
Other replacements of Met2 (in particular, by Asp, Lys,
and D-Pro) resulted in analogues devoid of a pro-
nounced selectivity to any of CCRs. A similar situation
was observed when simultaneously Ala for Trp1 and
Leu for Met2 were substituted or Leu for Met2 and Ala
for Phe4 were simultaneously substituted. On the con-
trary, other authors reported [31] that the replacement
of Met2 by Lys in Suc-CCK-4 considerably increased
the selectivity to CCR-2.
Trp–Nle–Asp–Phe–NH–CH(CH3)2 (IX)
Trp1
Nle2
Asp3
Phe4
–
3.92 3.19; 2.96
–
–
–
8.58 4.26 1.63; 1.50
8.40 4.49 2.45; 2.63
7.80 4.36 2.94; 2.83 –NH–CH(CH3)2 :
NH 7.60; CH 3.37;
CH3 1.02; 0.92
ner (Fig. 1) after their addition to the reaction medium.
The inhibition constants are given in Table 3.
These results demonstrated that both CCK-4 ana-
logues retained their affinity for CCRs; analogue (VIII)
exhibited a more pronounced selectivity to CCR-2,
whereas (IX) was predominantly bound by CCR-1.
A comparison of affinity of the synthesized peptides
with that of the endogenous CCK-4 [26, 30] revealed
Modification of both N- and C-terminal amino acid
the following characteristic features. The presence of residues affects the affinity and the selectivity of pep-
succinyl residue on the N-terminal Trp in (VIII) two tides toward CCRs. For example, the introduction of
Table 2. Characteristics of the compounds synthesized
Rf in systems
*
Rt ,
Compound
Mp, °C
Yield, %
min
A
B
C
D
E
F
Z–Phe–NH2 (I)
0.59 0.34
165–167
163–164
117–119
171–172
–
67
69
77
97
83
79
73
82
97
98
77
72
Z-Asp(OBut)–Phe–NH2 (II)
0.52 0.63
0.24
H–Asp(OBut)–Phe–NH2 (III)
Z-Nle–Asp(OBut)–Phe–NH2 (IV)
H–Nle–Asp(OBut)–Phe–NH2 (V)
Boc–Trp–Nle–Asp(OBut)–Phe–NH2 (VI)
H–Trp–Nle–Asp–Phe–NH2 (VII)
Suc–Trp–Nle–Asp–Phe–NH2 (VIII)
Z–Phe–NH–CH(CH3)2
0.54
0.37
0.39
0.19
0.51
0.61
179–181
0.25
20.6 125–127
22.4 121–123
154–155
0.56
0.86
0.94
Z-Asp(OBut)–Phe–NH–CH(CH3)2
Z-Nle–Asp(OBut)–Phe–NH–CH(CH3)2
Boc–Trp–Nle–Asp(OBut)–Phe–NH–CH(CH3)2 0.34
0.89 0.5
159–160
0.59
0.88
0.57
192–193
218–221
H–Trp–Nle–Asp–Phe–NH–CH(CH3)2 (IX)
0.28 0.55
19.5 132–137 24 (from the
starting
Z-Phe-ONp)
* Analytical reversed phase HPLC was carried out as described in the Experimental section under the following conditions: buffer A, 0.1%
TFA [for (VIII) and (IX)] or 0.05 M KH PO , pH 3.0 [for (VIII)]; buffer B, 80% acetonitrile [for (VIII) and (IX)] or 70% acetonitrile
2
4
[for (VIII)] in buffer A; gradient elution from 20 to 80% of buffer B in buffer A for 30 min.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 2 2005

122
PROSKURYAKOVA et al.
Specific binding, %
100
100
50
0
50
2
1
2
1
0
9
8
7
6
5
4
3
9
8
7
6
5
4
3
–log[analogue (VIII)]
–log[analogue(IX)]
125
Fig. 1. Specific binding of [ I]-CCK-8(S) to the membrane preparations from (1) cortex and (2) pancreas in the presence of ana-
logues (VIII) and (IX).
alkyl substituents into the amide of C-terminal Phe the time of animal presence in the light compartment
results in a decreased selectivity and affinity for CCRs and the number of the animal entrances into the light
of both types. The IC50 value of the analogue with one compartment of the chamber. Therefore, the modifica-
tion of N-terminal residue of the endogenous CCK-4
preserved and even increased the anxiogenic effect of
(VIII).
methyl substituent in amide of the C-terminal Phe was
170 nM for pancreas and 4250 nM for cortex in the
radioreceptor analysis. The presence of the second
diethyl substituent in the same amide decreases the IC50
value to 177 nM for pancreas and to more than
10000 nM for cortex [29]. The structure of CCR-2
seems to be more sensitive to the introduction of sub-
stituents into the C-terminal amide.
It was demonstrated above that the synthesized ana-
logues interacted with CCR-2 with various selectivi-
ties. Therefore, we initially examined their anxiogenic
activity (i.e., the ability to cause the anxiety) character-
istic of CCK-4, because CCR-2 are involved into the
development of this state [9].
One can see from Table 4 that the endogenous CCK-4
exhibited a pronounced anxiogenic effect in the test of
light–dark chamber (see the Experimental section): it
reliably decreased the time of presence of animals in
the light compartment of the chamber in the dose-
dependent manner. Analogue (VIII) did not reduce the
time of animal presence in the light compartment of the
chamber only at a minimum dose of 0.03 µg/kg of ani-
mal. A growth of dose was reliably accompanied with
Tests of analogue (IX) revealed reliable change nei-
ther in the time of animal presence in the light compart-
ment nor the number of entrances into the light com-
partment of the chamber in comparison with the control
animals. These results suggest the absence of anxio-
genic effect in (IX), which was achieved due to the
simultaneous modification of N- and C-terminal resi-
dues of the exogenous CCK-4.
Note that these properties of the synthesized ana-
logues of CCK-4 were found in the experiments on the
intact animals without consideration of their starting
level of anxiety.
Similar experiments were carried out on animals
with an increased level of anxiety. The anxiety was
induced by deprivation of ethanol for 24 h of animals
that got the ethanol solution as a sole liquid source for
7 months. The alcohol-dependent animals demon-
strated an enhanced anxiety in the period of abstinence,
which was evident from a decrease in the time of pres-
ence in the light compartment of chamber in compari-
son with the control animals (13.8 3.8 and 27.2 2.8 s,
p ≤ 0.05). Analogue (IX) increased the time of presence
of these animals in the light compartment to 42.3
10.7 s (p ≤ 0.05) and the number of entrances into the
light compartment from 1.5 0.3 to 2.4 0.2 s (p ≤
0.05). These results allow us to say on the selectivity of
the effect of (IX); in other words, this effect was exhib-
ited only in the case of the enhanced level of anxiety
and was not observed if the anxiety level was normal
(Table 4).
Table 3. Inhibition constants of the [125I]-CCK-8(S) bind-
ing to the receptors in cortex and pancreas* by the CCK-4
analogues
CCK-4
Ki, nM
Ki, nM
(cortex)
Ki(pancreas)/Ki(cortex)
analogue (pancreas)
(VIII) 4770 100
34
6
140.3
0.07
(IX)
234 150 3290 150
The effect of synthesized analogues of CCK-4 on
the ethanol consumption by the control animals and the
Notes: * CCR-1 and CCR-2 are predominantly located in pan-
creas and cortex, respectively.
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BIOLOGICAL ACTIVITY OF CHOLECYSTOKININ (30–33) TETRAPEPTIDE ANALOGUES
123
Table 4. Effect of endogenous CCK-4 and its analogues on
animals subjected to the ethanol intake for a long period
of time was studied in the test of the predominant intake
of alcohol under the condition of free choice between
water and 10% alcohol. The results given in Table 5
demonstrate that the endogenous CCK-4 has no signif-
icant effect on the use of the ethanol solution and water
by either control animals or alcohol-dependent ani-
mals. However, an attention is drawn to the tendency to
an increased consumption of ethanol solution by the
alcohol-dependent animals. The introduction of (VIII)
caused a reliable increase in the intake of ethanol solu-
tion and a decrease in the consumption of water by the
control and alcohol-dependent animals, and this effect
was continued after the end of introduction of (VIII).
Analogue (IX) did not change the ethanol consumption
by control animals, but reliably decreased it by the
alcohol-dependent animals. This decrease was
observed only during the analogue introduction. A reli-
able increase in the consumption of water was also
observed in this period; i.e., the proportion of ethanol
decreased in relation to the total volume of the con-
sumed liquid. A reliable increase in the consumption of
water was retained after the end of introduction of (IX)
during 10 days of observation.
the animal behavior in the test of dark–light chamber
Number of en-
trances to light
compartment
Compound
examined
Dose,
Time in light
µg/kg compartment, s
– (control)
CCK-4
–
27.2 2.8
27.9 1.9
21.9 4.5
13.7 3.5*
26.9 4.5
15.0 4.5*
9.8 3.1*
24.8 2.3
22.7 3.8
19.2 2.5
2.9 0.1
1.9 0.8
2.1 0.1
1.6 0.6
2.4 0.9
1.9 0.6
1.6 0.5
2.0 0.8
2.1 0.3
1.8 0.2
0.03
0.1
0.3
(VIII)
(IX)
0.03
0.1
0.3
0.03
0.1
0.3
* Statistically significant differences between the experimental and
the control groups were p < 0.05. Each group consisted of 10 ani-
mals.
Table 5. Effect of endogenous CCK-4 and its analogues on
the ethanol consumption by rats
The influence of synthesized analogues of CCK-4
on the morphine-induced analgesic effect was studied
in the tail-flick test. The results are given in Fig. 2.
Compound
examined
Injection
time
Alcohol
volume, ml
Water volume,
ml
Neither the endogenous CCK-4 nor its analogue
(VIII) changed the character and dynamics of the mor-
phine-induced analgesic effect (see Figs. 2a and 2b).
The introduction of (IX) increased the analgesic action
of morphine starting from the twentieth minute of the
observation. The maximum effect (410%) was regis-
tered after 60 min of observation (Fig. 2c) and duration
of the effect was 2.5–3 h. We should stress that none of
the CCK-4 analogues introduced separately exhibited
any analgesic effect (Fig. 2).
Thus, we can conclude that the structural modifica-
tions of the endogenous CCK-4 changed its biological
activity. Analogue (VIII) with Nle instead of Met and
with a succinyl residue attached to N-terminal Trp
exhibited an increased anxiogenic properties and the
ability to increase alcohol consumption. The introduc-
tion of isopropyl group into the C-terminal amide
resulted in appearance of anxiolytic and antialcohol
effects of analogue (IX) and its ability to increase the
analgesic effect of morphine.
According to literature, induction of contraction of
gall bladder, release of pancreatic enzymes, increase in
hydrolysis of phosphatidylinositol and amylase pro-
duction [32–34], and satiation [35] are characteristic of
agonists of CCR-1. These effects are not found for
antagonists of CCR-1.
Note that agonists of CCR-1, for example CCK-
8(S), can decrease the consumption of ethanol by ani-
mals under the conditions of free choice between etha-
nol and water [36–38]. However, no difference in the
effect of CCK-8(S) on the ethanol consumption by ani-
Control animals
CCK-4
(VIII)
(IX)
I
12.7 2.7
7.7 3.0
26.3 1.2
29.9 5.1
28.2 1.2
17.6 2.4
8.6 1.1*
5.8 0.7*
21.5 3.1
20.3 2.7
18.7 1.6
II
III
I
11.4 3.1
12.0 1.7
23.5 2.4*
27.1 2.8*
11.1 1.0
13.6 2.9
11.9 1.3
II
III
I
II
III
Alcohol-dependent animals
CCK-4
(VIII)
(IX)
I
15.4 3.1
21.4 3.7
19.4 1.6
17.4 2.9
29.2 3.0*
27.8 2.8*
14.4 1.7
4.9 1.6*
11.5 3.2
18.1 2.2
11.3 1.9
10.2 2.5
10.8 2.4
2.4 0.6*
1.7 0.4*
13.1 2.8
32.3 2.7*
26.3 2.6*
II
III
I
II
III
I
II
III
Note: I, before the injection of compounds (10 days); II, during the
injection (10 days); III, after the injection (10 days).
*Statistically significant differences in comparison with
group I for every compound were p < 0.05
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 2 2005

124
PROSKURYAKOVA et al.
Limit of the pain sensitivity,
% of the base value
300
pronounced anxiolytic effect [9] and the ability to
potentate the analgesic effect of morphine [42, 43]. In
addition, there is also an information [44] on the ability
of the CCR-1 antagonists to potentiate the morphine
analgesic effect.
(‡)
250
200
150
100
50
This literature information allows us the conclusion
that the physiological effects of analogue (IX) we
revealed (ability to reduce the ethanol consumption in
the alcohol-dependent animals, to reduce the anxiety,
and to potentiate the morphine analgesic effect) are
characteristic of the CCR-1 antagonists. The combina-
tion of such effects in one compound is promising for
practical medicine, because the creation of a therapeu-
tic agent on the basis of analogue (IX) can extend the
spectrum of pharmacological preparations used for the
treatment of alcoholism and drug dependence.
0
300
(b)
250
200
150
100
50
EXPERIMENTAL
0
700
600
500
400
300
200
100
0
L-Amino acids and their derivatives were from Rea-
nal (Hungary) and Bachem and Fluka (Switzerland);
isopropylamine, from Fluka. DMF was distilled over
ninhydrin and BaO. Solvents of domestic production of
os. ch. (high purity grade) were used for extraction,
crystallization, and chromatography. TLC was carried
out on precoated Kiesegel 60 plates (Merck, Germany) in
the following chromatographic systems: (A) 90 : 10 : 2
chloroform–methanol–50% acetic acid, (B) 1 : 1 ethyl
acetate–hexane, (C) 85 : 15 : 2 chloroform–methanol–
50% acetic acid, (D) 15 : 4 : 1 chloroform–methanol–
32% acetic acid, (E) 14 : 6 : 1 chloroform–methanol–
50% acetic acid, and (F) 6 : 3 : 1 chloroform–ethyl ace-
tate–methanol. Substance spots were detected on the
plates by the treatment with chlorine–benzidine or nin-
hydrin reagents. Hydrogenolysis of peptides was car-
ried out in the presence of 10% palladium on carbon
(Pd/C, Merck, Germany).
(c)
0
15
30
45
60
75
90
Time of observation, min
Control
Tetrapeptide
Morphine
Tetrapeptide + morphine
Fig. 2. Effect of (a) CCK-4 and its analogues (b) (VIII) and
(c) (IX) on the morphine analgesic effect. Limit of the pain
sensitivity measured in the adaptation period of animals to
the experimental conditions (three times for 30 min) was
taken as 100%. The dose of CCK-4 and morphine were
2.0 µg/kg and 5.0 mg/kg, respectively. The endogenous
CCK-4 and its analogues were injected 15 min before the
morphine injection. Statistically significant differences in
comparison with the group of rats injected only with mor-
phine were p < 0.05.
Analytical HPLC of (VII), (VIII), and (IX) was exe-
cuted on a Gilson chromatograph (France) equipped
with an Ultrasphere ODS column (4.6 × 250 mm, 5 µm,
Beckman, United States). The substances were eluted
from the column with gradients of buffer B [80% ace-
tonitrile in 01% TFA for (VII) and (IX) and 70% aceto-
nitrile in 01% TFA for (VIII)] in buffer A (0.1% TFA)
from 20 to 80% of buffer B at a flow rate of 1 ml/min.
The compounds were detected on a Gilson UV-detector
(France) at 220 nm. Preparative HPLC was carried out
on a Diasorb 130 C16-T column (10 µm) from ZAO
Biokhimmak CT (Russian Federation) eluted with a
concentration gradient of buffer B (80% acetonitrile in
0.1% TFA) in buffer A (0.1% TFA) from 20 to 80% of
buffer B for 30 min at a flow rate of 4 ml/min; detection
at 226 nm.
mals with high and low sensitivity to ethanol (mice of
the HAS and LAS lines, respectively) was found [39].
A decrease in the ethanol consumption by the alco-
hol-dependent animals was observed only on the cen-
tral introduction of CCK-8(S), and it did not change the
ethanol consumption by the alcohol-nonpreferring ani-
mals [40].
At the same time, it was reported that the CCR-1
antagonist (L364,718) decreases the ethanol consump-
tion by the ethanol-preferring animals at low doses and
had no effect on the alcohol-nonpreferring animals [41].
For amino acid analysis of (VII), a Biotronik LC
5001 (Germany) instrument was used after its hydroly-
The CCR-2 agonists induce the anxiety phobia, and sis with 6 N HCl containing 2% thioglycolic acid at
panic fear, whereas the CCR-2 antagonists exhibit a 110°C for 24 h in evacuated and sealed ampoules.
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BIOLOGICAL ACTIVITY OF CHOLECYSTOKININ (30–33) TETRAPEPTIDE ANALOGUES
125
Uncorrected melting points of the samples were oily residue was treated with cool anhydrous ether. The
measured on a Boetius device (Germany).
precipitated solid was filtered and washed with ether to
give (VII); yield 0.82 g (79%). The content of the target
product was 89% according to HPLC. The raw product
(0.15 g) was purified by the preparative reverse phase
HPLC under the conditions specified above. Fractions
containing the target product were collected and lyo-
philized; yield of peptide (VII) 0.11 g (73%); mp 125–
127°C; Rf 0.25 (E); amino acid analysis: Phe 1.0 (1),
Asp 1.04 (1), Nle 0.95 (1); Trp 0.33 (1).
Suc-Trp-Nle-Asp-Phe-NH2 (VIII). Succinic anhy-
dride (0.20 g, tenfold excess) and 1 N NaOH (2 ml)
were added to a stirred solution of (V) (0.11 g,
0.17 mmol) in 10% aqueous DMF (5 ml). The reaction
was monitored by TLC in system E (detection by the
ninhydrin color reaction). The target product was
extracted with ethyl acetate after acidifying the reaction
mixture with 2 N HCl to pH 3. The organic phase was
washed with water and evaporated. The residue was
suspended in water and lyophilized. The preparative
and analytical HPLC of peptide (VIII) was carried out
as described above. The yield of (VIII) 0.09 g (81.8%);
mp 121–123°C; Rf 0.56 (E).
1H NMR spectra were registered on a WH-500
Bruker spectrometer (500 MHz, Germany) in DMSO-
d6 at 300K at the peptide concentration of 2–3 mg/ml.
Chemical shifts (δ, ppm) were measured relative to tet-
ramethylsilane.
Z-Phe-NH2 (I). A saturated at 0°C methanolic solu-
tion of ammonia (20 ml) was added to a solution of Z-
Phe-ONp (10.0 g, 23.8 mmol) in methanol (20 ml). The
reaction mixture was kept for 12 h and evaporated to
dryness. The residue was reprecipitated from isopro-
panol with hexane to get (I); yield 4.8 g (66.7%); mp
165–167°C, Rf 0.59 (E), 0.34 (F).
Z-Asp(OBut)-Phe-NH2 (II). A catalytic hydro-
genolysis of (I) (2.0 g, 12.3 mmol) was carried out in
methanol (20 ml) containing 10% of DMF in the pres-
ence of Pd/C. The catalyst was filtered off and washed
with methanol. The filtrate was evaporated. The gel-
like residue was dissolved in DMF (50 ml), and Z-
Asp(OÇut)-ONp (5.3 g, 12.0 mmol) was added to the
solution. The reaction mixture was allowed to stay for
16 h, concentrated in a vacuum, and diluted with 5%
NaHCO3 (50 ml). The precipitated white gel-like solid
was filtered, washed with water to pH 7, dried, and
reprecipitated from isopropanol with hexane to get
crystalline (II); yield 3.8 g (69.2%); mp 163–164°C; Rf
0.63 (D), 0.52 (C).
H-Asp(OBut)-Phe-NH2 (III). A solution of (II)
(3.8 g, 8.3 mmol) in ethanol (25 ml) was subjected to a
catalytic hydrogenolysis as described for (I). The cata-
lyst was filtered off, the filtrate was evaporated, and the
residue was triturated in a 2 : 1 mixture of anhydrous
ether and hexane. The yield of crystalline (III) was
2.1 g (77.3%); mp 117–119°C; Rf 0.24 (C).
Z-Nle-Asp(OBut)-Phe-NH2 (IV). Z-Nle-ONp
(1.2 g, 3.0 mmol) was added to a solution of (III)
(1.0 g, 3.0 mmol) in DMF (15 ml). The reaction mix-
ture was kept for 26 h and treated as described for (II);
yield of (IV) 1.6 g (96.5%); mp 171–172°C, Rf 0.54
(C), 0.37 (F).
H-Trp-Nle-Asp-Phe-NH-CH(CH3)2 (IX). A 5%
solution of isopropylamine in chloroform (10 ml) was
added to a solution of Z-Phe-ONp (1.26 g, 3 mmol) in
methanol (10 ml). Further synthesis of the peptide was
carried out as described above for (VII). Characteris-
tics of the intermediates are given in Table 2. Yield of
(IX) was 0.11 g (24.4% from the starting Z-Phe-ONp
after the purification); mp 132–137°C, Rf 0.28 (C), 0.55
(D).
Characteristics of the compounds synthesized are
given in Table 2.
Biological activity of the peptides was studied on
150 Wistar rats.
The animals were examined for the preferable
alcohol consumption under the condition of free
choice between a 10% ethanol solution and water for
10 days by both alcohol-dependent rats (they drank
10% ethanol for 7 months) and the control animals that
drank water for the same period. After the tests, the
animals were randomly divided into subgroups with
10 rats in each. The examined analogue of CCK-4 (at a
dose of 2.0 µg/kg) or equivalent volumes of 0.9% NaCl
were introduced to the animals of different groups for
10 days under the conditions of free choice between the
10% ethanol solution and water. The volumes of the
consumed water and alcohol were daily measured. The
same animal subgroups were similarly tested for the
preferable consumption of alcohol during the next ten
days when the examined analogue of CCK-4 was not
introduced.
ç-Nle-Asp(OÇut)-Phe-NH2 (V). A catalytic hydro-
genolysis of (IV) (1.6 g) was in ethanol as described for
(I) and (II) led to (V) crystallized from a chloroform–
hexane mixture; yield 1.1 g (83.3%); Rf 0.19 (C), 0.39
(E).
Boc-Trp-Nle-Asp(OÇut)-Phe-NH2 (VI). Boc-Trp-
ONp (1.0 g, 1.4 mmol) was added to a solution of (V)
(1.1 g, 2.4 mmol) in DMF (10 ml). The reaction mix-
ture was kept for 16 h and treated as described for (II)
and (IV) to give (VI); yield 1.4 g (78.8%); mp 179–
181°C; Rf 0.51 (C), 0.61(E).
The dark–light chamber test was used as an exper-
imental model of anxiety [45].
The dark–light chamber was a box separated into
0°C was kept for 1 h and concentrated in a vacuum. The two compartments. The dark compartment with a cover
H-Trp-Nle-Asp-Phe-NH2 (VII). A solution of (VI)
(1.0 g, 1.4 mmol) in the mixture of TFA (9 ml), deion-
ized water (0.5 ml), and ethanedithiol (0.5 ml) cooled to
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 2 2005

126
PROSKURYAKOVA et al.
(20 × 20 × 20 cm) and the light compartment without a
cover (20 × 20 × 20 cm) were made from the dark and
light acrylic plastic, respectively. The light compart-
ment was illuminated from above with a lamp of 60 W
placed at a distance of 25 cm from the compartment
(4000 lux). The two compartments are connected with
a hole (7 × 7 cm), and the animals can easily move
between the compartments.
Radioreceptor analysis. The displacing radiore-
ceptor analysis was performed with the use of cell
membrane preparations of cortex and pancreas contain-
ing CCR-1 and CCR-2, respectively. The rats were
decapitated in the morning, and the brain and pancreas
were quickly isolated.
The brain and pancreas were homogenized in
50 mM Tris-HCl buffer (25 volumes, pH 7.4, 4°C) in a
Downs homogenizer. The suspension was centrifuged
at 30000 g for 15 min at 4°C. The supernatant was dis-
carded, and the pellet was resuspended in the starting
volume of 50 mM Tris-HCl buffer (pH 7.4) upon cool-
ing and centrifuged again. This procedure was repeated
once more.
The reaction mixture (0.5 ml) contained buffer
(250 µl, 20 mM HEPES, 5 mM MgCl2, 1 mM EDTA,
5 mM KCl, 120 mM NaCl, and 45 µM of bacitracin),
the labeled ligand (50 µl, 10 pM), a CCK-4 analogue
(50 µl) in the concentration range from 0.1 to 100 nM,
and a suspension of the membrane protein with the final
concentration from 1.2 to 1.8 mg/ml. The [125I]-ëëä-
8(S) (2000 Ci/mmol, Amersham, UK) was used as a
ligand for the CCK-receptors. The binding reaction was
carried out for 2 h at 21°C.
The specific binding of the label with the receptors
was determined as a difference between the radioactiv-
ity bound to the membranes in the absence of a unla-
beled ligand (the total binding) and that in the presence
of 1000-fold excess of unlabeled ligand (nonspecific
binding). The reaction was stopped by a fast filtration of
the samples on a filtration device (Millipore, France)
through GF/B fiberglass filters (Whatman, UK). The
vials were washed with Tris-HCl buffer (pH 7.4, 6 ml),
shortly dried in air, and placed in scintillation flasks.
The vials were filled with a standard dioxane scintilla-
tor (7 ml). The radioactivity was counted on a RackBeta
1219 scintillation spectrometer (LKB, Sweden) with
the efficiency of counting no less than 30%.
The dark–light test was made between 10.00 a.m.
and 16.00 p.m. in a poorly lighted room (4 lux). The
animals were adapted to the new conditions in individ-
ual cages for 30 min and randomly divided into sub-
groups with ten animals in each. The animals of one
group were intraperitoneally injected with a compound
studied. Animals of another group (control group) were
injected with the same volume of 0.9% NaCl. The
CCK-4 analogues were ip introduced at the doses of
0.03, 0.1, and 0.3 µg per kg of body weight 30 min
before testing. The animals were placed into the center
of dark compartment and observed for 5 min. The time
of presence in the light compartment and the number of
entrances into the light compartment were counted. We
considered that the examined analogue of CCK-4
exhibited anxiolytic activity if the time of animal pres-
ence in the light compartment and the number of
entrances into it were increased in comparison with the
control group. If these two parameters were decreased
in comparison with the control group, we considered
that the examined analogue of CCK-4 had an anxio-
genic activity. This experimental model of anxiety,
doses of the CCK-4 analogue, and time of its injection
were chosen on the basis of studies of other authors
[46–48].
Determination of the morphine analgesic effect
was carried out in the tail-flick test. The rats were
placed into individual cages, adapted for 30 min, and
thrice tested for the pain sensitivity by dipping their
tales into water heated to 56°C with 10-min intervals.
The time period from dipping of a tale to the taking it
The processing of results of the radioreceptor anal-
ysis was performed using a LIGAND program. The
out from the water was considered as the rate of pain inhibition constants (Ki) of the labeled ligand bound to
sensitivity. The starting rate of pain sensitivity was
measured in the adaptation period and taken as 100%.
The animals were randomly divided into groups (ten
animals in each). Morphine was intraperitoneally
injected at a dose of 5.0 mg/kg to the rats of the second
group, the examined CCK-4 analogue at a dose of
2.0 µg/kg to the third group, the examined CCK-4 ana-
logue at the same dose and with the solution of mor-
phine hydrochloride at the dose of 5.0 mg/kg 15 min
after the first injection to the fourth group. The animals
of the first (control) group received an equivalent vol-
ume of 0.9% NaCl. Then, the rate of pain sensitivity
were registered every 15 min within 1.5 h. The results
were presented as the ratio of the current value to the
base level. The average value of three measurements
made before the injections of preparations was taken as
the base level.
the cortex and pancreas membrane preparations by the
analogue were used as the assessment criteria. The sta-
tistic processing of the results was carried out using the
Student’s t-test.
ACKNOWLEDGMENTS
This study was partially supported by the Russian
Foundation for Basic Research, project no. 02-04-49902.
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