A study of the biologically active conformation of the cholecystokinin-4 dipeptide analogue GB-115

Article abstract of DOI:10.1134/S1068162013030060

The biologically active conformation of N-(6-phenylhexanoyl)glycyl- tryptophan amide (GB-115), a highly active cholecystokinin-4 retro dipeptide analogue with the anxiolytic activity, has been studied using the conformational analysis by ¹H NMR spectroscopy in solution and the method of sterically restricted analogues. A study of the relationship between the preferable conformation in solution and the anxiolytic activity in the series of GB-115 derivatives showed that the biologically active conformation of this compound is the β-turn. Based on the data on the nuclear Overhauser effect ¹H NMR spectroscopy, this structure was identified as the β-turn of type II. Subsequent synthesis and study of the pharmacological activity of novel sterically restricted analogues of dipeptide GB-115: (2S)-2-{(3R)-3-[(6- phenylhexanoyl)amino]-2-oxopyrrolidine-1-yl}-3-(1H-indole-3-yl)propionic acid ethyl ester, N-(6-phenylhexanoyl)glycyl-N ^α-methyltryptophan ethyl ester, (2S)-2-[(10,11-dihydro-5H-dibenzo[b, f]azepin-5-ylcarbonyl)amino]- 3-(1H-indole-3-yl)propionic acid methyl ester, and (2S)-2-[({3-[(ethoxycarbonyl) amino]-10,11-dihydro-5H-dibenzo[b, f]azepin-5-yl}carbonyl)amino]-3-(1H-indole-3- yl)propionic acid methyl ester confirmed that the β-turn of type II is the active conformation of GB-115.

Full text of DOI:10.1134/S1068162013030060

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2013, Vol. 39, No. 3, pp. 259–267. © Pleiades Publishing, Ltd., 2013.
Original Russian Text © T.A. Gudasheva, V.P. Lezina, E.P. Kir’yanova, O.A. Deeva, L.G. Kolik, S.B. Seredenin, 2013, published in Bioorganicheskaya Khimiya, 2013, Vol. 39,
No. 3, pp. 293–302.
A Study of the Biologically Active Conformation of the
Cholecystokininꢀ4 Dipeptide Analogue GBꢀ115
T. A. Gudasheva1, V. P. Lezina, E. P. Kir’yanova, O. A. Deeva, L. G. Kolik, and S. B. Seredenin
Zakusov Research Institute of Pharmacology, Russian Academy of Medical Sciences,
ul. Baltiiskaya 8, Moscow, 125315 Russia
Received June 29, 2012; in final form, October 30, 2012
Abstract—The biologically active conformation of
Nꢀ(6ꢀphenylhexanoyl)glycylꢀtryptophan amide (GBꢀ
115), a highly active cholecystokininꢀ4 retro dipeptide analogue with the anxiolytic activity, has been studied
1
using the conformational analysis by H NMR spectroscopy in solution and the method of sterically
restricted analogues. A study of the relationship between the preferable conformation in solution and the anxꢀ
iolytic activity in the series of GBꢀ115 derivatives showed that the biologically active conformation of this
1
compound is the
structure was identified as the
ity of novel sterically restricted analogues of dipeptide GBꢀ115: (2
noyl)amino]ꢀ2ꢀoxopyrrolidineꢀ1ꢀyl}ꢀ3ꢀ(1 ꢀindoleꢀ3ꢀyl)propionic acid ethyl ester,
ꢀmethyltryptophan ethyl ester, (2 )ꢀ2ꢀ[(10,11ꢀdihydroꢀ5Hꢀdibenzo[b, f]azepinꢀ5ꢀylcarboꢀ
ꢀindoleꢀ3ꢀyl)propionic acid methyl ester, and (2 )ꢀ2ꢀ[({3ꢀ[(ethoxycarbonyl)amino]ꢀ
ꢀdibenzo[b, f]azepinꢀ5ꢀyl}carbonyl)amino]ꢀ3ꢀ(1 ꢀindoleꢀ3ꢀyl)propionic acid methyl
ꢀturn of type II is the active conformation of GBꢀ115.
β
ꢀturn. Based on the data on the nuclear Overhauser effect H NMR spectroscopy, this
β
ꢀturn of type II. Subsequent synthesis and study of the pharmacological activꢀ
)ꢀ2ꢀ{(3 )ꢀ3ꢀ[(6ꢀphenylhexaꢀ
ꢀ(6ꢀphenylhexaꢀ
S
R
H
N
α
noyl)glycylꢀ
nyl)amino]ꢀ3ꢀ(1
10,11ꢀdihydroꢀ5
N
S
H
H
S
H
ester confirmed that the
β
Keywords: cholecystokininꢀ4, dipeptide analogue, GBꢀ115, 1H NMR spectroscopy, biologically active conformaꢀ
tion, sterically restricted analogues, conformational analysis
DOI: 10.1134/S1068162013030060
1
1
INTRODUCTION
tional analysis by H NMR spectroscopy in solution
and the method of spatially restricted analogues.
On the basis of the native anxiogenic tetrapeptide
cholecystokininꢀ4 (TrpꢀMetꢀAspꢀPheꢀNH2), its retro
dipeptide analogue
Nꢀ(6ꢀphenylhexanoyl)glycylꢀ
RESULTS AND DISCUSSION
tryptophan amide was constructed in the Zakusov
Research Institute of Pharmacology, Russian Acadꢀ
emy of Medical Sciences, using the topochemical
principle of Shemyakin–Ovchinnikov–Ivanov [1] [2].
In animal models, this dipeptide at doses of 0.0025–
0.25 mg/kg intraperitoneally and 0.1–0.8 mg/kg
orally exhibits anxiolytic activity, has no byꢀeffects
typical for tranquilizers of the benzodiazepine series,
and is almost nontoxic (LD50 > 6 g/kg for rats per os)
[3, 4]. GBꢀ115 can be a progenitor of a new group of
anxiolytics with the cholecystokinin mechanism of
action. In this connection, the question concerning
the biologically active conformation of GBꢀ115 is of
current interest.
To determine the biologically active conformation of
the dipeptide GBꢀ115, we studied the relationship
between the structure and the anxiolytic activity in the
series of GBꢀ115 analogues of both the previously
described
amide ( ) and
N
ꢀ(6ꢀphenylhexanoyl)prolylꢀtryptophan
I
Nꢀ(6ꢀphenylhexanoyl)prolylꢀtryptophan
ethyl ester (II) [2, 5] and the novel conformationally
mobile [ ꢀ(4ꢀphenylbutyryl)glycylꢀtryptophan methylꢀ
amide (III), ꢀ(5ꢀphenylpentanoyl)glycylꢀtryptophan
methylamide (IV), and ꢀ(6ꢀphenylhexanoyl)prolylꢀ
tryptophan metyhylamide ( )] and spatially restricted
analogues: )ꢀ2ꢀ{(3 )ꢀ3ꢀ[(6ꢀphenylhexaꢀ
noyl)amino]ꢀ2ꢀoxopyrrolidineꢀ1ꢀyl}ꢀ3ꢀ(1 ꢀindoleꢀ3ꢀ
yl)propionic acid ethyl ester (VI), ꢀ(6ꢀphenylhexꢀ
ꢀ(methyl)ꢀtryptophan ethyl ester (VII),
)ꢀ2ꢀ[(10,11ꢀdihydroꢀ ꢀdibenzo[b, ]azepinꢀ5ꢀ
ylcarbonyl)amino]ꢀ3ꢀ(1 ꢀindoleꢀ3ꢀyl)propionic acid
methyl ester (VIII), and ( )ꢀ2ꢀ[({3ꢀ[(ethoxycarboꢀ
nyl)amino]ꢀ10,11ꢀdihydroꢀ ꢀdibenzo[b, ]azepinꢀ5ꢀ
ꢀindoleꢀ3ꢀyl)propionic acid
N
N
N
V
(2S
R
H
N
In the present study, the biologically active conforꢀ
mation of GBꢀ115 was examined using the conformaꢀ
α
anoyl)glycylꢀ
N
(2S
5
Н
Н
f
Abbreviations: EPM, elevated plusꢀmaze; IHB, intramolecular
hydrogen bond; NOE, nuclear Overhauser effect; the configuraꢀ
2S
tion symbol in amino acids of the
L series is omitted; MeTrp,
α
5Н
f
N
ꢀmethyltryptophan.
Corresponding author: phone: (495) 601ꢀ22ꢀ46; fax: (499)ꢀ151ꢀ
12ꢀ61; eꢀmail: tataꢀsosnovka@mail.ru.
1
yl}ꢀcarbonyl)amino]ꢀ3ꢀ(1
Н
methyl ester (IX).
259

260
GUDASHEVA et al.
Substituted peptides were obtained by the method by means of methyl iodide to sulfonium salt and then
of mixed anhydrides as described earlier [2, 3, 5]. cyclized to lactam in the presence of sodium hydride.
Compound (VI), which contains an aminolactam Because ethyl ester was completely hydrolyzed under
fragment, was synthesized by the method of Freiꢀ the conditions of cyclization, the resulting acid was
dinger [6, 7] from
Nꢀ(6ꢀphenylhexanoyl)ꢀDꢀmethioꢀ esterified again by ethanol in the presence of thionyl
nylꢀtryptophan ethyl ester (VIA), which was converted chloride (Scheme 1).
I−
H3C
NH
CH3
NH
H3C
O
O
S
S+
O
O
CH3
CH3
CH3I
O
O
NH
NH
Ph(CH2)5
Ph(CH2)5
O
O
N
N
H
H
(
VIA
)
(VIB)
O
O
CH3
O
1. NaOH, 0°C
2. SOCl , C H OH
NH
2
2 5
(
VI)
NH
Ph(CH2)5
O
N
H
Scheme 1. Synthesis of the γꢀlactam analogue (VI).
Both dibenzoazepine analogues (VIII) and (IX
)
tophan by the corresponding
Nꢀchlorocarbonyldibenꢀ
were synthesized by acylating the methyl ester of trypꢀ zoazepine derivative (Scheme 2).
CH3
CH3
O
O
O
H2N
O
NH
N
N
Cl
H
ꢀmethyl morpholine
N
O
N
O
N
H
R
R
H
O
(
VIIIA
)
R
:
(
VIII
IX
)
(
IXA
)
(
)
NH
CH
3
O
Scheme 2. Synthesis of dibenzoazepine analogues (VIII) and (IX).
The pharmacological activity of the synthesized accompanied by an increase in activity, as is evident
compounds (Table 1) was studied using the EPM test, from Table 1, we concluded that the biologically active
which is the major model for assessing the anxiolytic conformation of GBꢀ115 is the turn.
and anxiogenic activities [8].
To determine the type of the turn structure, the
It is known that the proline residue contributes to a conformational analysis of compounds GBꢀ115 and
1
greater extent to the formation of the turn conformaꢀ
(I) in solution was carried out by H NMR spectroꢀ
1
tion of the peptide chain than the glycine residue [9– scopy. Oneꢀdimensional H NMR spectra and the
11]. Because the transition of the glycineꢀcontaining spectra of 1Нꢀ1Н double homonuclear resonance in
GBꢀ115 to its prolineꢀcontaining analogue (I) is DMSOꢀd6 and CDCl3 solutions were measured.
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A STUDY OF THE BIOLOGICALLY ACTIVE CONFORMATION
261
Table 1. Data of the pharmacological study of conformationally restricted analogues of GBꢀ115 in the EPM test on monꢀ
grel rats
Compound
Formula
Ph(CH ) COꢀGlyꢀTrpꢀNH2
Parameter
Dose,
mg/kg ip (n)
Code or
number
Runs into open Time of staying in open
arms, %
arms, %
GBꢀ115
0 (14)
13.0
27.2
34.1*
42.1*
31.0*
25.9
47.7*
42.7*
37.9
40.7
33.9
38.5
36.9
4.2
4.0
10.9
12.5*
13.0*
11.9*
2 5
0.01 (14)
0.05 (16)
0.1 (8)
0.2 (8)
0 (6)
0.01 (8)
0.05 (6)
0.1 (6)
0.5 (5)
0 (6)
0.01 (6)
0.05 (7)
Ph(CH ) COꢀProꢀTrpꢀNH2
(
I
)
10.6
2 5
30.4*
27.2*
20.1
20.5
Ph(CH ) COꢀProꢀTrpꢀOC2H5
(
II
)
7.7
10.7
9.3
0.1
1.2
0.5
5.4
6.7
2.5
2 5
Ph(CH ) COꢀGlyꢀTrpꢀNHCH3
(
III
)
0 (8)
2 3
0.8 (8)
1.6 (8)
3.2 (8)
0 (12)
0.1 (10)
7.0
6.3
16.6
18.4
6.4
Ph(CH ) COꢀGlyꢀTrpꢀNHCH3
(
IV
)
2 4
25.3
35.2
39.3*
Ph(CH ) COꢀProꢀTrpꢀNHCH3
(
V
)
0 (10)
0.01 (5)
0.05 (7)
6.5
12.4
24.3*
2 5
O
O
CH3
(VI)
0 (6)
0.01 (8)
0.05 (8)
32.7
35.8
53.5*
22.6
38.9
56.8*
O
N
NH
Ph(CH2)5
O
N
H
Ph(CH ) COꢀGlyꢀMeTrpꢀOC2H5
(
VII
)
0 (12)
0.01 (6)
0.05 (8)
25.8
28.2
36.5
15.1
12.0
23.8
2 5
CH3
O
O
NH
(
VIII
)
0 (6)
0.05 (6)
0.5 (6)
44.6
36.6
32.1
15.9
17.4
17.8
N
O
N
H
CH3
O
O
NH
(IX)
0 (6)
0.05 (6)
0.5 (6)
44.6
48.5*
35.3
15.9
51.5*
24.4
N
O
N
H
O
NH
CH3
O
Note:
n
, the number of animals; *
p < 0.05, statistical significance of the difference from the control according to the Mann–Whitney
Uꢀtest; ip, intraperitoneally.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 39
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262
GUDASHEVA et al.
Table 2. Δδ values and percentage of
β
ꢀturn conformation in a nonꢀpolar medium (0.027 M, 25°C)
Δδ, ppm
β
ꢀturn
Compound
free
amide
bound
NH
amide
fraction, % **
NH
NH
NH
Gly
NH
ind
Trp
GBꢀ115***
1.59
2.75
0.91
1.35
1.23
–
1.76
under Arom
0.57
0.27
65
90
(I)
Notes
*
**
Δδ values represent the difference of chemical shifts
ꢀturn fraction (%) in nonꢀpolar solvent was calculated as follows [14]: [
100 –39Δδ for compound (I).
*** Due to the poor solubility of the GBꢀ115 compound in pure CDCl , we used CDCl containing 8% DMSOꢀ
δ
in DMSOꢀd and CDCl .
6 3
β
β
ꢀturn] = 100–62Δδ for GB115 and [ ꢀturn] =
β
d
.
3
3
6
Table 3. Results of the conformational analysis of glycineꢀ and prolineꢀcontaining dipeptides with different
substitutes by 1H NMR spectroscopy in solution
C
ꢀterminal
Compound
Value of Δδ *, ppm
free
amide
NH
ind
NH
NHTrp
NHGly
Code
Formula
NHCH3
NH
bound
amide
NH
GBꢀ115 Ph(CH ) COꢀGlyꢀTrpꢀNH2 **
+
–
–
0.91
0.93
0.92
1.23
1.54
1.52
–
1.76 0.57
1.59
β
γ
γ
2 5
(
(
III
)
Ph(CH ) COꢀGlyꢀTrpꢀNHCH3
1.30
1.29
–
–
2.48
2.67
2 3
IV)
Ph(CH ) COꢀGlyꢀTrpꢀNHCH3
2 4
(
I)
Ph(CH ) COꢀProꢀTrpꢀNH2
+
1.35
–
–
under Arom
2.75
β
2 5
0.27
(
(
II
)
Ph(CH ) COꢀProꢀTrpꢀOC2H5
–
+
0.82
–
–
–
–
–
2.77
2.77
γ
2 5
V)
Ph(CH ) COꢀProꢀTrpꢀNHCH3
1.48
0.80
β
2 5
Notes: * Δδ values represent the difference of chemical shifts
** Due to the poor solubility of the GBꢀ115 compound in pure CDCl , we used CDCl containing 8% DMSOꢀd .
6
δ
in DMSOꢀd and CDCl .
6 3
3
3
In biologically active peptides,
structures occur most often [12, 13]. The
ture (
+ 1,
carbonyl group of the residue (
of the phenylhexanoyl group) and N Н proton of the
residue ( + 3) (in our case, the ꢀterminal amide
group). In the case of a ꢀturn, the IHB is formed
between the carbonyl group of the residue ( ) and the
+ 2) (in our case, trypꢀ
β
ꢀturn and ꢀturn GBꢀ115, this was also confirmed by the fact that the
γ
Δδ value for this proton did not depend on the concenꢀ
tration of the substance (data not shown). Because the
β
ꢀturn strucꢀ
β
ꢀturn) is formed by four amino acid residues (i,
+ 2, + 3) and is stabilized by IHB between the
) (in our case, carbonyl
Δδ values for NHTrp for GBꢀ115 and (I) were suffiꢀ
i
i
i
ciently great compared with those for other protons
(Table 2), it was assumed that the formation of the
ꢀturn stabilized by IHB in these compounds is
i
α
γ
i
С
unlikely.
γ
i
The calculation by the classical formula of Bousꢀ
sard and Marraud [14] showed that the share of the
putative ꢀturn is 65% in the pool of GBꢀ115 conforꢀ
mations and 90% in the case of the prolineꢀcontaining
GBꢀ115 analogue ( ) (Table 2). Thus, compound (
exhibits a higher activity than GBꢀ115 (Table 1) and
simultaneously is more prone to form ꢀturns.
α
N Н proton of the residue (
i
tophan). Thus, to determine the type of the turn structure
in solution, it would be sufficient to reveal the involveꢀ
ment of a particular proton in the formation of IHB.
β
I
I)
It is well known that the magnitude of the chemical
shift of the proton involved in IHB changes insignifiꢀ
β
1
cantly in the H NMR spectrum on going from the
The data obtained suggest that the biologically
active conformation of GBꢀ115 and its prolineꢀconꢀ
polar solvent DMSOꢀd6, which can participate in the
formation of IHB, to nonpolar CDCl3 [14]. It was
shown (Table 2) that the difference in chemical shifts
taining analogue (
consistent with the fact that compounds having the
ꢀturn stabilized by the IHB with the involvement of
I) is the
βꢀturn. These results are
Δδ in GBꢀ115 and (
small for one of the protons of the
group, which supposedly indicates the involvement of
I
) upon change of the solvents is
γ
α
Сꢀterminal amide
N H proton of the tryptophan residue are inactive
1
(Tables 1 and 3). According to the data of H NMR
this proton in the formation of IHB. In the case of spectroscopy in solution, among these are the methylꢀ
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 39
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2013

A STUDY OF THE BIOLOGICALLY ACTIVE CONFORMATION
H
263
N
N
OH
O
φ
3
HO
ψ
ψ
2
(i + 1)
φ
3 O(
N
N
2
i
+ 2)
O
N
O
O
N
H
N
HN
Ph(CH2)5
N
O
N
H
O
O
H (
i
+ 3)
OH
(i)
N
N
Fig. 1. βꢀTurn in the structure of GBꢀ115.
amides of glycineꢀcontaining dipeptides (III) and (IV
and the ester of prolineꢀcontaining dipeptide (II). At
the same time, the prolineꢀcontaining methylamide
)
Fig. 2. Superposition of molecules of (IX) and GBꢀ115
(solid lines) in Dreiding models.
(
V
), which forms the ꢀturn stabilized by the IHB with
β
the involvement of NH proton of the
methylamide group, is active.
Сꢀterminal Nꢀ
Finally, we examined the spatially restricted comꢀ
pounds (VIII) and (IX), which have the dibenꢀ
zoazepine structure. A pharmacological study showed
that compound (IX) at doses comparable with those of
GBꢀ115 possesses the anxiolytic activity, whereas
compound (VIII) is inactive (Table 1).
Three
type + 3)
choice between these types of
the data of NOE 1Нꢀ1Н double homonuclear resoꢀ
nance spectra of compounds GBꢀ115 and ( ). It is
known that, for ꢀmethylamides of ꢀacylꢀsubstiꢀ
β
ꢀconformations containing the IHB of the
) are known: I, II, and VI. The
ꢀturns was made from
(i
→
(i
β
β
β
β
I
The two dibenzoazepine derivatives differ in that
compound (IX) has a lateral substituent in the C3
position of the dibenzoazepine ring, which presumꢀ
ably participates in the interaction with the receptor
and is required for the manifestation of the anxiolytic
activity. According to the data of molecular simulation
using the Dreiding models (Fig. 2), it is this fragment
that imitates the hydrophobic phenyl group of GBꢀ
N
N
tuted prolineꢀcontaining dipeptides (RꢀProꢀXaaꢀ
NHCH3), which are structurally similar to our comꢀ
pounds [18, 19], the occurrence of the NOE between
α
α
the С Н proton of the residue (
the residue ( + 2) is evidence for the
the case of the prolineꢀcontaining compound (
i
+ 1) and N H proton of
ꢀturn of type II. In
) in
i
β
I
α
CDCl3, the NOE (7%) was detected between C HPro
115 in the IIꢀturn conformation. The superposition
β
α
and N HTrp protons; in the case of GBꢀ115, the NOE
of (VIII) and (IX) describes the threeꢀdimensional
structure of molecules in the unstrained state. In the
model, the structure of the dipeptide GBꢀ115 was
α
α
(4%) was detected between C HGly and N HTrp proꢀ
tons. In addition, a characteristic feature of II is the
proximity of NH( + 2)ꢀNH( + 3), which is also conꢀ
firmed by NOE: NH(Trp)ꢀNH( + 3) is 3% in GBꢀ115
and 5% in ( ). Therefore, there is reason to believe that
the ꢀturn of type II is also realized in these dipeptides
β
i
i
fixed in the type II ꢀturn conformation.
β
i
To summarize, it can be concluded that the bioꢀ
logically active conformation of GBꢀ115 is the type II
I
β
β
ꢀturn.
in solution (Fig. 1).
The biologically active conformation of GBꢀ115
was confirmed using the method of conformationally
restricted analogues.
EXPERIMENTAL
The peptide GBꢀ115 and its analogues
Ph(CH2)5COꢀProꢀTrpꢀNH2 ), Ph(CH2)5COꢀProꢀ
TrpꢀOC2H5 II , as well as the starting esters
Ph(CH2)3COꢀGlyꢀTrpꢀOC2H5 Ph(CH2)4COꢀGlyꢀ
TrpꢀOC2H5 and Ph(CH2)5COꢀGlyꢀTrpꢀOC2H5 were
obtained earlier [2, 5]. Commercial amino acids and
their derivatives were from Reanal (Hungary and
Acros Organics); reagents and solvents were from ZAO
Ekosꢀ1, Khimmed, Reakhim, Acros Organics, Fluka,
and Lancaster. Amino acid esters were obtained by
(
I
In analogue (VI), the
contributes to the formation of the type II
[6, 7], was used as a spatial restraint. Compound (VI
γ
ꢀlactam fragment, which
(
)
β
ꢀturn
,
)
,
was found to possess the anxiolytic activity, which
compares well in effective doses with that of the startꢀ
ing dipeptide GBꢀ115 (Table 1), indicating that the bioꢀ
logically active conformation of GBꢀ115 is the type II
β
ꢀturn.
On the other hand, the GBꢀ115
tuted analogue, compound (VII) (Table 1), did not were synthesized as described earlier [2].
N
ꢀmethylꢀsubstiꢀ standard methods.
N
ꢀacyl derivatives of amino acids
ꢀChloroꢀ
ꢀchlorocarbonylꢀ(3ꢀ
IIꢀturn in ethoxycarbonylamino)iminodibenzene were kindly
N
exhibit the anxiolytic activity. Because the
substitution counteracts the formation of
Nꢀmethylꢀ carbonyliminodibenzene and N
β
peptides [18–20], the lack of the activity in (VII) is provided by VEB Arzneimittelwerk (Dresden). The
also in favor of the IIꢀturn conformation of GBꢀ115. solvents were purified and dried by standard methods.
β
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264
GUDASHEVA et al.
1H NMR spectra (
δ, ppm,
J, Hz) were recorded on a
Ph(CH2)4C(O)ꢀGlyꢀTrpꢀNHCH3
(IV) was obtained
Bruker ACꢀ250 spectrometer (Germany) in DMSOꢀ
d6 and CDCl3 solutions using tetramethylsilane as an
internal standard. The melting point was determined
in open capillaries and was not corrected. The specific
optic rotation was measured on a PerkinꢀElmerꢀ241
polarimeter (England). Column chromatography was
carried out on Kieselgel 100 (Merck, Germany). TLC
was performed on Kieselgel 60 UV254 plates (Machꢀ
ereyꢀNagel, Germany) using the following systems of
solvents: chloroform–ethanol 9 : 1 (A); petroleum
ether–ethyl acetate–ethanol 9 : 9 : 2 (B); dioxane–
water 9 : 1 (C); butanol–acetic acid–water 5 : 1 : 2
(D); and isopropanol–ammonia 7 : 3 (E). Comꢀ
similarly to compound (III) from Ph(CH2)4C(O)
GlyꢀTrpꢀOC2H5 (0.42 g, 0.93 mmol). Yield 0.25 g
ꢀ
20
(62%); Rf 0.25 (A), mp 117–118
°
С, [α]D –6.56
°
(
c
0.1; ethanol); 1H NMR (DMSOꢀ
d6): 1.50 (4 H, m,
α
βγ
C H2 chain), 2.12 (2 H, t,
J 7.1, C H2 chain), 2.54 (3
δ
6.9, C H2
H, d, 7.1, NHCH3), 2.55 (2 H, t,
J
⎯
J
β
5.7, C H2
J 5.6, CH2
chain), 2.90 and 3.10 (2 H, two dd,
J
J
14.9,
16.5,
J
Trp), 3.56 and 3.73 (2 H, two dd,
α
Gly), 4.42 (1 H, m, C H Trp), 6.92–7.35 (10 H, m,
ArH, CH indole), 7.87 (1 H, q, –NHCH3), 7.97 (1 H,
d, J 8.1, NH Trp), 8.01 (1 H, t, J 6.1, NH Gly), 10.81
pounds were detected using iodine vapor and UV light. (1 H, s, NH indole); 1H NMR (CDCl3): 1.84 (4 H, m,
βγ
α
The elemental analysis was carried out on a device for
carbon and hydrogen determination with four electric
ovens (600–900°С, type MAꢀG/6P; LETO plant,
Russia) in a flow of oxygen and on a device for nitroꢀ
gen determination with three similar electric ovens in
a flow of carbon dioxide. The data of the elemental
analysis of compounds on the content of C, H, and N
deviate from the theoretical data by no more than
0.4%.
C H2 chain), 2.09 (2 H, t,
H, d, 7.1, –NHCH3), 2.65 (2 H, t,
chain), 2.76 and 3.82 (2 H, two dd,
Gly), 3.13 and 3.30 (2 H, two dd,
J 7.0, C H2 chain), 2.56 (3
δ
6.9, C H2
J
J
J
14.7,
J
5.7, CH2
β
5.7, C H2
J
14.9,
J
α
Trp), 4.70 (1 H, m, C H Trp), 6.49 (1 H, br s, NH
Gly), 6.58 (1 H, br s, –NHCH3), 6.90 (1 H, s, CH
indole), 7.05 (1 H, d,
m, ArH), 8.32 (1 H, s, NH indole).
Ph(CH2)5COꢀProꢀTrpꢀNHCH3 (V) was obtained in
J 8.1, NH, Trp), 7.00–7.61 (9 H,
Preparative HPLC was carried out using a Wellchrom
2001 chromatographic system (Knauer, Germany) on a the same way as compound (III) from compound (II
Diasorbꢀ130ꢀC16T column (15 250 mm, С16, 9 m; (0.50 g, 0.99 mmol) dissolved in methanol (10 mL)
ZAO BioKhimMak ST). The flow rate was 5 mL/min, saturated with methylamine, and the reaction mixture
)
×
μ
and the detection was at 214 nm.
Ph(CH2)3COꢀGlyꢀTrpꢀNHCH3 (III). A solution of
Ph(CH2)3COꢀGlyꢀTrpꢀOC2H5 (0.41 g, 0.95 mmol) in
was kept for 72 h (TLC control). Yield 0.35 g (72%),
20
1
oil; Rf 0.53 (А), [α]D –65
°
(c
0.4; methanol); H
NMR (DMSOꢀd6) (ratio of trans
/
cis isomers 7/3):
H2 chains, ꢀCH2 Pro),
2.44 (cis) and 2.55 (trans) (2 H, t, 6.8, C H2 chain),
2.55 (3 H, d, 6.8, –NHCH3), 2.94 cis) and 3.05
cis), 2.98 (trans) and 3.18 (trans) (2 H, two dd each,
methanol (10 mL) preliminarily saturated with gasꢀ
αβγδ
1.18–2.20 (12 H, m,
C
3,4
ε
eous methylamine at
0°С was allowed to stand at room
J
temperature for 36 h in a closed container at atmoꢀ
spheric pressure (TLC control). The solvent was
removed in vacuo, and the residue was triturated with
dry ether. The precipitated crystals were filtered and
dried over P2O5. Yield 0.28 g (70%); Rf 0.35 (A), mp
J
(
(
J
β
14.9,
J
5.7, C H2 Trp), 3.28–3.46 (2 H, m,
5ꢀCH2
Pro), 4.18 (trans) and 4.29 (cis) (1 H, m,
2
ꢀCH Pro),
α
20
4.40 (trans) and 4.55 (cis) (1 H, m each, C H Trp),
6.90–7.60 (10 H, m, ArH), 7.59 (trans) (1 H, q,
1
121–122
(DMSOꢀ
°
С, [α]D –8.8
°
(
c
0.1; ethanol); H NMR
β
d
6): 1.77 (2 H, m, C H2 chain), 2.12 (2 H, t,
⎯
NHCH3), 7.66
(
trans) (1 H, d,
J
8.2, NH Trp), 8.01
8.3, NH
α
J
7.1, C H2 chain), 2.54 (3 H, d,
J
7.1, –NHCH3),
(
cis) (1 H, m, –NHCH3), 8.27
(
cis) (1 H, d, J
γ
Trp), 10.81 (cis) and 10.83 (trans) (1 H, s, NH indole);
2.56 (2 H, t,
J 7.0, C H2 chain), 2.92 and 3.11 (2 H,
γ
1H NMR (CDCl3): 1.07 (2 H, m, C H2 chains), 1.26
β
two dd,
J
14.9,
16.6,
J
J
5.7, C H2 Trp), 3.58 and 3.75 (2 H,
5.6, CH2, Gly), 4.44 (1 H, m, C H
β
δ
α
(2 H, m C H2 chain), 1.58 (2 H, m, C H2 chains), 1.88
two dd,
J
α
(4 H, m, C H2 chain, 4ꢀCH2 Pro), 2.09 (2 H, m,
Trp), 6.92–7.59 (10 H, m, ArH, CH indole), 7.88
(1 H, m, –NHCH3), 7.98 (1 H, d, 8.3, NH Trp),
8.03 (1 H, t, 5.7, NH Gly), 10.80 (1 H, s, NH
ε
J
3
J
ꢀCH2, Pro), 2.62 (2 H, t, C H2 chain), 2.73 (3 H, d,
7.1, –NHCH3), 3.00 and 3.11 (2 H, two dd, ꢀCH2
14.8, 5.7, C H2
J
5
,
1
β
β
indole); H NMR (CDCl3): 1.84 (2 H, m, C H2
Pro), 3.12 and 3.56 (2 H, two dd,
J
J
α
α
chain), 2.09 (2 H, t, J 7.2, C H2 chain), 2.56 (2 H, t, J
Trp), 4.29 (1 H, dd,
Trp), 6.18 (1 H, d,
2
J
ꢀCH Pro), 4.75 (1 H, m, C H
8.1, NH Trp), 6.79 (1 H, m,
γ
7.1, C H2 chain), 2.65 (3 H, d,
J
7.0, –NHCH3), 3.13
β
⎯
NHCH3), 6.97 (1 H, s, CH indole), 7.07–7.59 (9 H,
m, ArH), 8.06 (1 H, s, NH indole).
Ph(CH2)5COꢀ ꢀMetꢀTrpꢀOC2H5
obtained in the same way as compound (VII) (see
8.1, NH Trp), 7.0–7.61 (9 H, m, ArH), 8.32 below) from ꢀ(6ꢀphenylhexanoyl)ꢀ ꢀmethionine
(1 H, s, NH indole). (1.0 g, 3.1 mmol). Chloroform was removed in vacuo,
and 3.30 (2 H, two dd,
J
J
14.9,
16.2,
J
J
5.7, C H2 Trp), 3.76
5.5, CH2 Gly), 4.70
and 3.82 (2 H, two dd,
α
(1 H, m, C H Trp), 6.49 (1 H, t,
(1 H, br s, –NHCH3), 6.98 (1 H, s, CH indole), 7.05
(1 H, d,
J
5.8, NH Gly), 6.58
D
(
VIA
)
was
J
N
D
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 39
No. 3
2013

A STUDY OF THE BIOLOGICALLY ACTIVE CONFORMATION
265
the resulting dark oil was kept at +5 С for 24 h and dropwise to the solution of oil in absolute ethanol
°
then triturated with diethyl ester, yielding a white crysꢀ
talline precipitate. Crystals were carefully and quickly
washed with ether and dried to obtain a white crystalꢀ
line product. Yield 0.38 g (23%); Rf 0.71 (A), mp
24
(3 mL) at –10
°
С. The reaction mixture was stirred for
2 h at –10 and kept for 48 h at room temperature,
°
С
after which dry ether (10 mL) and activated charcoal
(0.5 g) were added to the reaction mass under stirring.
The reaction mass was then filtered, and the filtrate
was evaporated in vacuo. The resulting ester in the
form of yellow oil was purified from contaminants by
HPLC using a gradient of acetonitrile concentrations
in 0.1% TFA (0–90%, 30 min). Yield of the product as
104
⎯
106°
С, [α]D +12.24
°
(c
0.4; methanol);
7.0, OCH2CH3),
1H NMR (CDCl3): 1.21 (3 H, t,
J
⎯
γ
βδ
1.29 (2 H, m, C H2, chain), 1.57 (4 H, m, C H2
γ
chain), 1.86 (2 H, m, C H2 Met), 2.07 (3 H, s, CH3S
α
24
Met), 2.09 (2 H, t, J 7.0, C H2 chain), 2.19 and 2.32
oil 0.100 g (50%, total yield 19%); Rf 0.81 (E), [α]D
β
ε
(2 H, two m, C H2 Met), 2.55 (2 H, t,
chain), 3.24 and 3.34 (2 H, two dd, 14.9,
Trp), 4.01 (2 H, q, 7.2,
C H Met), 4.81 (1 H, m, C H Trp), 6.37 (1 H, d,
9.1, NH Met), 6.88 (1 H, d, 8.1, NH Trp), 7.02
J
J
7.0, C H2
5.7, C H2
+32.08
(3 H, t,
chain), 1.50 (4 H, m, C H2 chain), 1.62 (2 H, m,
°
(
c
J
4.8, methanol); 1H NMR (DMSOꢀ
d6): 1.05
β
J
γ
7.1, –OCH2CH3), 1.23 (2 H, m, C H2
J
⎯OCH2CH3), 4.56 (1 H, m,
βδ
4ꢀ
α
α
α
CH2 lactam), 2.06 (2 H, t,
J
7.1, C H2 chains), 2.53 (2
6.8, C H2 chain), 3.02 and 3.21 (2 H, two t,
ꢀCH2 lactam), 3.08 and 3.19 (2 H, two dd, J
J
J
ε
H, t,
7.1,
14.9,
OCH2CH3), 4.23 (1 H, m,
J
(1 H, s, CH indole), 7.05–7.56 (9 H, m, ArH), 8.30 (1
H, s, NH indole).
J
5
β
J
5.7, C H2 Trp), 4.10 (2 H, q,
ꢀCH lactam), 4.70 (1 H,
m, C H Trp), 6.92–7.35 (10 H, m, ArH), 8.12 (1 H,
d, 8.6, NH pyrrolidine), 10.81 (1 H, s, NH indole).
J 7.0,
[Ph(CH2)5COꢀ
A solution of Ph(CH2)5COꢀ
D
ꢀMet(CH3)ꢀTrpꢀOC2H5] · I⎯
ꢀMetꢀTrpꢀOC2H5 (VIA
(VIB).
⎯
3
D
)
α
(0.75 g, 1.4 mmol) in methyl iodide (7.5 mL) was
stirred for 3 h at room temperature and left for 8 days.
After the termination of the reaction (TLC control),
the mixture was twoꢀphasic; it consisted of a solution
and a gelꢀlike mass. The removal of excessive methyl
iodide in vacuo followed by the drying over CaCl2 and
J
Ph(CH2)5COꢀGlyꢀMeTrpꢀOС2H5 VII . Isobutylꢀ
(
)
chloroformate (0.13 mL, 1.0 mmol) and ꢀethyl morꢀ
N
pholine (0.12 mL, 1.0 mmol) were simultaneously
added dropwise under stirring to a solution of ꢀ(6ꢀ
phenylhexanoyl)glycine (0.25 g, 1.0 mmol) in dry
DMF (12 mL) cooled to –10 . After stirring for 2–
3 min at this temperature, a solution of
tophan ethyl ester chlorohydrate (0.27 g, 1.0 mmol)
cooled to –10 and ꢀethyl morpholine (0.13 mL,
1.0 mmol) in dry DMF (5.0 mL) was added. The reacꢀ
tion mixture was stirred for another 30 min at –10
N
paraffin in vacuo (15 mmHg) yielded 0.825 g (87%) of
24
product (VIB) as oil; Rf 0.1 (А), [α D
]
+3.41
°
(
c
0.4
7.0,
–OCH2CH3), 1.24 (2 H, m, C H2 chain), 1.50 (4 H,
;
°C
methanol); 1H NMR (DMSOꢀ
d
6): 1.09 (3 H, t, J
α
γ
N
ꢀmethyltrypꢀ
βδ
γ
m, C H2 chain), 1.8 (2 H, m, C H2 Met), 2.12 (2 H,
α
ε
°С
N
t,
J
6.9, C H2 chain), 2.53 (2 H, t,
J
7.0, C H2 chain),
2.78 and 2.79 (6 H, two s, (CH3)2S+), 3.03 and 3.07 (2
β
°С
H, two m, C H2 Met), 3.08 and 3.17 (2 H, two dd,
J
7.1,
β
and for 1 h at room temperature, after which it was
poured into cold water (200 mL). After 24 h at room
temperature, oil formed, which was washed with water
14.9,
⎯
J
5.7 , C H2 Trp), 4.03 (2 H, q,
J
α
OCH2CH3), 4.45 (1 H, m, C H Met), 4.54 (1 H, m,
α
C H Trp), 6.91–7.54 (10 H, m, ArH), 8.08 (1 H, d,
J
(2 × 30 mL) and dissolved in chloroform (30 mL). The
9.0, NH Met), 8.49 (1 H, d, J 8.3, NH Trp), 10.31 (1
solution was dried over anhydrous magnesium sulfate,
the desiccant was filtered off, and the solvent was
removed in vacuo. The resulting oil (yield 93%) was
purified by HPLC using a gradient of acetonitrile conꢀ
centrations (0–90%, 30 min). The yield after purificaꢀ
H, s, NH indole).
(2
pyrrolidineꢀ1ꢀyl}ꢀ3ꢀ(1
ester (VI). Sodium hydride (0.087 g, 2.17 mmol) was
added to a solution of Ph(CH2)5COꢀ
ꢀMet(СH3)+
TrpꢀOC2H5 (0.735 g, 1.082 mmol) in 33.8 mL
I– (VIB
of the mixture DMF–CH2Cl2 1 : 1 cooled to and
placed into a nitrogen flow. The mixture was stirred at
for 2.5 h, after which ethyl acetate (7.3 mL) and
S)ꢀ2ꢀ{(3R)ꢀ3ꢀ[(6ꢀPhenylhexanoyl)amino]ꢀ2ꢀoxoꢀ
H
ꢀindoleꢀ3ꢀyl)propionic acid ethyl
D
ꢀ
tion by HPLC was 0.24 g (50.5%) as oil;
R
f 0.78 (А),
⋅
)
20
[α]D –23.3
°
(c
0.46; methanol); 1H NMR (DMSOꢀ
7.1, OCH2CH3), 1.33 (2 H, m,
C H2 chain), 1.62 (4 H, m, C H2 chain), 2.12 (cis
0°С
d
6): 1.27 (3 H, t,
J
⎯
γ
βδ
)
0°С
α
then water (2.8 mL) were added. The mixture was left
overnight at room temperature. The solvent was
removed in vacuo, after which water (5.64 mL) and
CH2Cl2 (5.64 mL) were added to the residue. The
organic phase was separated, the solvent was removed
in vacuo, and the residue was purified from contamiꢀ
nants by HPLC using a gradient of acetonitrile conꢀ
centrations in 0.1% TFA (0–90%, 30 min) to give 0.19 g
(0.41 mmol, 38%) of the derivative as oil. Then freshly
distilled SOCl2 (0.06 mL, 0.861 mmol) was added
and 2.20 (trans) (2 H, m, C H2 chain), 2.57 (cis) and
ε
2.59 (trans) (2 H, m, C H2 chain), 2.79 (trans) and
α
2.85 (cis) (3 H, s, N CH3), 3.25 and 3.47 (2 H, two dd,
β
J
J
⎯
14.6,
16.6,
J
J
5.8, C H2 Trp), 3.88 and 4.0 (2 H, two dd,
5.6, CH2 Gly), 4.21 (2 H, q, 7.3,
cis) and 5.30 (trans) (1 H, dd
J
OCH2CH3), 4.56
14.9, 8.1 and
Trp), 6.37 (cis) and 6.48 (trans) (1 H, t,
respectively, NH Gly), 6.80 (1 H, d,
(
α
each,
J
J
J
14.9,
J
7.6, respectively, C H
5.6 and 5.7
8.3, NH Trp),
J
J
,
J
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 39
No. 3
2013

266
GUDASHEVA et al.
tures. Spin–spin coupling constants 3J in the system
NHꢀC H were measured from the signal of NH proꢀ
tons. The magnitude of NOE in oneꢀdimensional
NMR spectra was determined by selective lowꢀintenꢀ
sity irradiation at the frequency of the resonance choꢀ
sen. The quantity NOEj(i) was defined as the ratio of
the change in the integral intensity of signal j in the
NMR spectrum upon the saturation of nucleus I (Ij) to
6.95–7.60 (10 H, m, ArH), 8.11 (trans) and 8.16 (cis
(1 , s, NH indole).
(2 )ꢀ2ꢀ[(10,11ꢀdihydroꢀ5
ylcarbonyl)amino]ꢀ3ꢀ(1
)
α
Н
S
H
ꢀdibenzo[b, f]azepinꢀ5ꢀ
H
ꢀindoleꢀ3ꢀyl)propionic
acid
methyl ester (VIII). A solution of tryptophan methyl
ether hydrochloride (0.43 g, 1.6 mmol) and ꢀethyl
N
morpholine (0.28 mL, 2.2 mmol) in DMF (5 mL) was
added under stirring to a solution of 10,11ꢀdihydrodiꢀ
benzo[b, f]azepinꢀ5ꢀcarbonylchloride (VIIIA) (0.39 g,
the integral intensity of signal
j in the absence of satuꢀ
ration (I0j ) : NOEj(i) = (Ij – Ij0 )/I0j
.
1.5 mmol) in DMF (5 mL) cooled to
tion mass was stirred for 30 min at room temperature
and for 3 h upon heating to 40 , poured into a conꢀ
tainer with distilled water (150 mL), and cooled to
. The precipitate was filtered, airꢀdried, and
recrystallized from toluene. Yield 0.20 g (30.3%); Rf
0.9 (A), mp 157–158 С, [ 0.4; methaꢀ
22 –5.43
nol); H NMR (DMSOꢀD
6): 2.92 (4 H, br s,
CH2CH2–), 3.12 (2 H, d, 5.7, C H2 Trp), 3.60 (3H,
s, –OCH3), 4.47 (1 H, m, C H Trp), 5.43 (1 H, d,
8.1, NH Trp), 6.84–7.42 (13 H, m, ArH), 10.89
(1 H, s, NH indole).
(2 )ꢀ2ꢀ[({3ꢀ[(ethoxycarbonyl)amino]ꢀ10,11ꢀdihyꢀ
droꢀ5 ꢀdibenzo[b, ]azepinꢀ5ꢀyl}carbonyl)amino]ꢀ3ꢀ
(1 ꢀindoleꢀ3ꢀyl)propionic acid methyl ester (IX).
solution of tryptophan methyl ether hydrochloride
(0.43 g, 1.6 mmol) and ꢀethyl morpholine (0.28 mL,
0°С. The reacꢀ
The biological activity of peptides in vivo was studied
on white mongrel male rats weighing 200–270 g. Stanꢀ
dard granulated feed and water were given ad libitum.
Ethical rules for the animal care formulated in the
directions of the Council of European Community
86/609/ЕЕС were followed. Rats were housed in a
°С
0°С
°
α]
°
(c
1
d
β
vivarium in standard plastic cages (60 × 40 × 20), eight
⎯
J
α
individuals in each. Each experimental rat was subꢀ
jected for three days preceding the experiment to a
“handling” procedure. Animals were placed in an
experimental room three to four hours prior to the
beginning of the experiment, which was carried out
between 16 and 20 h of the local time. Experiments
were performed in a darkened room; the light source
(60ꢀwatt bulb) was screened so that the device was illuꢀ
minated only with diffuse reflected light. Water–
Tween solutions of compounds were prepared ex temꢀ
pore and injected to rats intraperitoneally 15 min prior
to the testing in the maze. Control animals received an
equal volume of distilled water.
J
S
H
f
H
A
N
2.2 mmol) in DMF (7 mL) was added under stirring to
a solution of 10,11ꢀdihydrobenzo[b, f]azepinoꢀ[3ꢀ
ethoxycarbonyl)amino]ꢀ5ꢀcarbonylchloride
(IXA)
(0.52 g, 1.5 mmol) in DMF (5 mL). The reaction mass
was stirred for 30 min at room temperature and for 3 h
The EPM test in the basic modification [8] was
used as an experimental model. The maze had the folꢀ
lowing characteristics: the length of each of four maze
arms 50 cm, the width 15 cm, the height of the lightꢀ
proof skirtings of two opposite closed arms 15 cm; the
upon heating to 45–50 С after which it was allowed to
°
stand for 15 h at room temperature. Then the reaction
mass was poured into a container with distilled water
(150 mL), and cooled to
tered off, airꢀdried, and recrystallized from ethyl aceꢀ
tate. Yield 0.4 g (50.7%);
0
°
С
. The precipitate was filꢀ
f 0.75 (A), mp 155–157 С,
6):
dimensions of the central stage 15
× 15 cm; the maze
R
°
was elevated over the floor at a height of 75 cm. At the
beginning of the experiment, rats were placed in the
center of the maze and oriented randomly relative to
the entrance to the arm. The time of assessing the
behavior of animals was 5 min. The following paramꢀ
eters were recorded: the number of runs in all arms,
the number of runs into open arms, the time of stay in
all arms, and the time of stay in open arms.
22
[α]D +7.03
°
(c
0.4; methanol); 1H NMR (DMSOꢀ
7.0, –OCH2CH3), 2.85 (4 H, m,
d
1.22 (3 H, t,
CH2CH2–), 3.11 (2 H, d,
s, –OCH3), 4.10 (2 H, q,
J
β
⎯
J
J
5.7, C H2 Trp), 3.60 (3 H,
7.2, –OCH2CH3), 4.47
(1 H, m, C H Trp), 5.44 (1 H, d, 8.1, NH Trp),
α
J
6.85–7.47 (12 H, m, ArH), 9.61 (1 H, s, NH ethoxyꢀ
carbamoyl), 10.85 (1 H, s, NH indole).
The statistical processing of results was performed
using the nonparametric Mann–Whitney Uꢀtest
(software package Complete statistical system, version
B640, Statsoft, United States). The data are presented
as the means +/– error of the means. The results were
1H NMR study. 1H NMR spectra were recorded at
room temperature on a Bruker ACꢀ250 spectrometer
(Germany) in CDCl3 and DMSOꢀd6 solutions, using
tetramethylsilane as an internal standard. Solutions
with a concentration of 10–20 mg/mL were examꢀ
ined. Signals were assigned on the basis of literature
data on 1H NMR spectra of the corresponding amino
acids [21] and the method of homonuclear doubleꢀ
resonance, which enables the detection of groups of
spin–spinꢀcoupled protons. To determine IHBs, the
dependence of the chemical shifts of protons on the
concentration of a substance in CDCl3 and the nature
of solvent was studied using CDCl3/DMSOꢀd6 mixꢀ
considered as significant at p < 0.05.
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Translated by S. Sidorova
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 39
No. 3
2013