Stereoselectivity of the antidopamine effect of N-caproylprolyltyrosine methylate - A tripeptide neurotensin analog

Full text of DOI:10.1023/A:1005227325665

Pharmaceutical Chemistry Journal
Vol. 34, No. 8, 2000
STEREOSELECTIVITY OF THE ANTIDOPAMINE EFFECT
OF N-CAPROYLPROLYLTYROSINE METHYLATE – A TRIPEPTIDE
NEUROTENSIN ANALOG
N. I. Zaitseva,1 T. A. Gudasheva,1 V. K. Briling,1 L. S. Guzevatykh,1 R. U. Ostrovskaya,1
T. A. Voronina,1 and S. B. Seredenin1
Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 34, No. 8, pp. 20 – 22, August, 2000.
Original article submitted March 23, 2000.
Previously we described the synthesis of a series of
N-acylprolyltyrosines showing neuroleptic activity [1].
Prolyltyrosines were initially designed as analogs of sulpiri-
de – an atypical benzamide neuroleptic [2]. It was suggested
that both sulpiride and its dipeptide analogs represent exoge-
nous ligands for the receptors of neurotensin – a tridecapepti-
de possessing neuroleptic-like activity [3, 4].
obtained from isobutylformate and the tertiary base was rep-
resented by N-ethylmorpholine (II):
OH
O
N
+
i-BuOCOCl, II
OH
O
O
H2N
An analysis of the molecular conformations of sulpiride,
Pro-Tyr-NH2, and neurotensin (Fig. 1) showed that Leu13 – a
side radical of neurotensin that is important for its binding to
receptors – can be modeled by a hydrophobic terminal
N-radical of prolyltyrosine. Study of the structure – activity
relationship in the series of N-acylprolyltyrosines allowed
N-caproylprolyltyrosine methylate (I) to be selected as a
promising atypical neuroleptic for extended pharmacological
characterization [5].
CH3
O
CH3
O
N
HN
O
O
CH3
O
OH
CH3
I
It was established that this peptide (0.4 – 4.0 mg/kg, i.p.)
exhibited activity in dopamine-dependent tests (such as
apomorphine-induced verticalization and stereotypy and
L-DOPA-induced stereotypy and violation of the extrapola-
tion avoidance reflex), decreased the body temperature, and
potentiates hexenal action. It is also important to note that, at
these doses, the peptide does not affect motor activity, coor-
dination of movements, level of alertness, emotional state,
and pain sensitivity threshold. N-Caproylprolyltyrosine
methylate (I) is nontoxic and does not produce catalepsy at
doses below 500 mg/kg (i.p.) [6].
The purpose of this work was to study stereospecificity
of the antidopamine effect of peptide I in order to verify the
hypothesis concerning the receptor mechanism of its
neuroleptic action and estimate the effect of a diastereomer
admixture on the activity.
The diastereomer purity of the target peptides (above
1
98%) was confirmed by H NMR (250 MHz). A double set
of proton signals from CH3(CH2)4, CbH2Tyr, NHTyr, CaH
Pro, and CH3O observed in the spectra of all diastereomers is
manifested due to the trans – cis isomerism relative to the
tertiary imide bond –C(O)–N< [7].
The antidopamine activity of the synthesized peptides
was determined using the most selective and sensitive behav-
ioral test for neuroleptics – a decrease in the apomorphine-
induced verticalization in mice [8]. Investigation of the effect
of amino acid configuration in peptide I upon its antidopami-
ne activity showed that substitution of the optical isomers for
L-proline and L-tyrosine leads to the loss or reversal of activ-
ity (see Table 1). While the peptide CH3(CH2)4C(O)-L-Pro-
L-Tyr-OCH3, possessing a natural configuration of amino
acid residues, suppresses the apomorphine verticalization at
a dose of 0.4 and 0.8 mg/kg (by 21 and 30%, respectively),
the replacement of any amino acid by its D-isomer leads to
the loss of this effect: neither CH3(CH2)4C(O)-D-Pro-L-
Tyr-OCH3 nor CH3(CH2)4C(O)-L-Pro-D-Tyr-OCH3 are ac-
The diastereomers of compound I were synthesized by
the method of mixed anhydrides proceeding from N-caproyl-
proline and tyrosine methylate. The mixed anhydride was
1
Institute of Pharmacology, Russian Academy of Medical Sciences, Mos-
cow, Russia.
413
0091-150X/00/3408-0413$25.00 © 2001 Plenum Publishing Corporation

414
N. I. Zaitseva et al.
Thus, the action of N-caproylprolyltyrosine methylate (I)
is stereoselective and requires that both amino acid residues
occur in the L-configuration. The optical purity is an impor-
tant condition for manifestation of the antidopamine activity
of this promising atypical neuroleptic.
CH3
O
O
N
HN
S
NH2
H3C
O
O
(S)-Sulpiride
O
N
H
EXPERIMENTAL CHEMICAL PART
HN
OH
The melting temperatures were determined by the open
1
capillary technique without corrections. The H NMR spec-
O
H2N
L-Pro-L-Tyr-NH2
tra were measured on a Bruker AC-250 spectrometer (Ger-
many) using DMSO-d6 as the solvent and TMS as the inter-
nal standard. The specific optical rotation was measured on a
Perkin-Elmer Model 241 polarimeter. TLC was conducted
on Silufol Kavalier plates (Czech Republic) developed by
exposure to iodine vapors. The column chromatography was
effected in columns filled with Kieselgel 100 (Merck). The
solvents were purified and dehydrated by conventional meth-
ods. The data of elemental analyses for C, H, N agree with
the results of analytical calculations for C21H30N2O5.
O
N
pGlu1-Leu2-Tyr3-Glu4-Asn5-Lys6-Pro7-Arg8-Arg9
Leu13-Ile12
Neurotensin
HN
OH
O
Fig. 1. Structural formulas of sulpiride, L-Pro-L-Tyr-NH2, and
neurotensin.
General method for the synthesis of diastereomers of
N-caproylprolyltyrosine methylate (I). To a solution 10 ml of
the carboxyl component (N-caproylproline) in 50 ml of chlo-
roform are added by dropping from different funnels with
stirring and cooling to –15°C (on an ice – NaCl mixture)
10 mmole of N-ethylmorpholine and 10 mmole of isobutyl-
chloroformate, after which the mixture is stirred for
2 – 3 min. To this mixture are slowly added 10 mmole of
amino component (tyrosine methylate hydrochloride) in
10 ml of DMF containing 10 mmole of N-ethylmorpholine.
The reaction mixture is stirred for 30 min at –15°C and then
1 h at room temperature. The precipitate is separated by fil-
tration and the residual solvent evaporated in vacuum. The
precipitate is dissolved in chloroform and washed sequen-
tially with 5% NaHCO3, water, 1 N HCl, and water again un-
tive at these doses. Moreover, the enantiomer
CH3(CH2)4C(O)-D-Pro-D-Tyr-OCH3 at a dose of 0,4 mg/kg
inhibits the prodopamine effect (increases the apomorphine
verticalization by 50%). The latter fact indicates that
N-caproyl-L-prolyl-L-tyrosine methylate acts by a receptor
mechanism rather than through the inhibition of proteases
participating in the neuropeptide processing.
The results of our experiments are indicative of an im-
portant role of the Pro10 – Tyr11 fragment as an active center
of neurotensin responsible for the neuroleptic-like activity.
This conclusion agrees with the data of [9, 10], according to
which the L-configuration of the Pro10 and Tyr11 residues of
the neurotensin molecule are necessary for its binding to re-
ceptors.
TABLE 1. The Activity of Diastereomers of N-Caproylprolyltyrosine Methylate (I) in the Apomorphine-In-
duced Verticalization Test in Mice
Verticalization
Dose,
Activity,
%
Compound
mg/kg (i.p.)
Total rating
%
CH3(CH2)4CO-L-Pro-L-Tyr-OCH3
0.4
0.8
79*
70*
100
109
108
100
96
21*
30*
76 ± 4.6
67 ± 18.8
Control
96 ± 16.7
CH3(CH2)4CO-L-Pro-D-Tyr-OCH3
0.4
0.8
– 9
–8
90.5 ± 21.1
89.5 ± 15.5
82.7 ± 30.7
90.7 ± 21.4
82.3 ± 30.2
94 ± 24.8
Control
CH3(CH2)4CO-D-Pro-L-Tyr-OCH3
0.4
0.8
4
88
12
Control
100
149*
122
100
CH3(CH2)4CO-D-Pro-D-Tyr-OCH3
0.4
0.8
– 49*
– 22
105.2 ± 10.1
86.1 ± 20.8
70.5 ± 34.1
Control
*
Difference from control reliable for p < 0.05; each compound tested in a group of six mice.

Stereoselectivity of the Antidopamine Effect of N-Caproylprolyltyrosine Methylate
415
(nmax, cm – 1): 3310, 3230 (NH, OH); 1740 (COOMe); 1660,
1625, 1615 (CONH, CON<); 1H NMR spectrum in
DMSO-d6 (d, ppm): 0.84, 0.87 (tt, 3H, CH3(CH2)4), 1.0 – 2.3
(m, 12H, CbH2–CgH2Pro, CH3(CH2)4), 2.81, 2.98 (mm, 2H,
C3H2Tyr), 3.2 – 3.5 (m, 2H, CdH2Pro), 3.56, 3.63 (ss, 3H,
CH3O), 4.25 (m, 2H, CaHPro), 4.33, 4.48 (mm, 1H,
CaHTyr), 6.63, 6.65, 6.98, 6.99 (mm, 4H, AA¢BB¢ system,
C6H4Tyr), 8.10, 8.42 (dd, 1H, J 7.7 Hz, J 8.4 Hz, NHTyr),
9.21, 9.23 (ss, 1H, OHTyr); C21H30N2O5.
til reaching pH ~7. The the solution is dehydrated over
MgSO4, the solvent evaporated, and the product purified by
column chromatography using chloroform as eluent.
N-Caproyl-L-prolyl-L-tyrosine methylate [C3(CH2)4C(O)-
L-Pro-L-Tyr-OMe] was obtained from N-caproyl-L-
Pro-OH and L-Tyr-OMe × HCl with a 54% yield. The product
appears as a white crystalline substance; m.p., 115 – 116°C
(from ethyl acetate); [a]D20, – 58.4° (c, 0.4; chloroform); Rf ,
0.50 (chloroform – ethanol, 9 : 1); IR spectrum in KBr disks
(nmax, cm – 1): 3300 (NH, OH); 1735 (COOMe); 1660, 1638,
1
1620 (CONH, CON<); H NMR spectrum in DMSO-d6 (d,
EXPERIMENTAL PHARMACOLOGICAL PART
ppm): 0.84, 0.87 (tt, 3H, CH3(CH2)4), 1.02 – 2.20 (m, 12H,
CbH2–CgH2Pro, CH3(CH2)4), 2.7 – 3.1 (m, 2H, CbH2Tyr),
3.2 – 3.45 (m, 2H, CbH2Pro), 3.56, 3.63 (ss, 3H, CH3O),
4.26, 4.35 (mm, 2H, CaHPro), 4.35, 4.48 (mm, 1H, CaHTyr),
6.65, 6.66, 6.98, 7.00 (mm, 4H, AA¢BB¢ system, C6H4Tyr),
8.04, 8.42 (dd, 1H, NHTyr), 9.23, 9.26 (ss, 1H, OHTyr);
C21H30N2O5.
N-Caproyl-L-prolyl-D-tyrosine methylate [CH3(CH2)4C(O)-
L-Pro-D-Tyr-OMe] was obtained from N-caproyl-L-Pro
and D-Tyr-OMe × HCl with a 47% yield. The product ap-
pears as a white crystalline substance; m.p., 143 – 145°C
(upon ether treatment); [a]2D5, – 104.8° (c, 0.4; chloroform);
Apomorphine-induced verticalization test. The exper-
iments were performed on CC57/BL6 male mice weighing
22 – 25 g. The compounds to be tested were intraperitoneally
injected as suspensions in 0.9% NaCl solution containing
2.5% of Tween-80. Apomorphine (5 mg/kg, s.c.) was in-
jected 15 min after test compounds in 0.9% NaCl solution
containing 0.1% of ascorbic acid. Animals in the control
group in the first stage were injected with physiological solu-
tion. The intact (passive control) group received physiologi-
cal solution in both stages. After apomorphine injection,
each animal tested was placed in a wire cylinder with a diam-
eter of 12 cm and a height of 14 cm. The first verticalization
characteristics were taken 15 min after apomorphine injec-
tions. The observation was continued for 1 h with the charac-
teristics taken every 2 min according to the following scale:
all four paws on the floor, 0 (no verticalization); one paw on
the wall, 1; two paws on the wall, 2; three paws on the wall,
3; all paws on the wall, 4 (full verticalization). The total rat-
ing in the control was taken as 100%. The data were statisti-
cally processed in terms of the Wilcoxon – Mann – Whitney
criterion.
Rf, 0.47 (chloroform – ethanol, 9 : 1); IR spectrum in KBr
disks (nmax, cm – 1): 3230, 3220, 3070 (NH, OH); 1740
1
(COOMe); 1665, 1625, 1615 (CONH, CON<); H NMR
spectrum in DMSO-d6 (d, ppm): 0.85, 0.86 (tt, 3H,
CH3(CH2)4), 1.10 – 2.27 (m, 12H, CdH2–CgH2Pro,
CH3(CH2)4), 2.7 – 3.03 (m, 4H, CbH2Tyr, CdH2Tyr), 3.54,
3.61 (ss, 3H, CH3O), 4.26, 4.29 (mm, 2H, CaHPro), 4.39,
4.43 (mm, 1H, CaHTyr), 6.62, 6.65, 6.96, 6.99 (mm, 4H,
AA¢BB¢ system, C6H4Tyr), 8.11, 8.45 (dd, 1H, NHTyr), 9.23,
9.24 (ss, 1H, OHTyr); C21H30N2O5.
N-Caproyl-D-prolyl-L-tyrosine methylate [CH3(CH2)4C(O)-
D-Pro-L-Tyr-OMe] was obtained from N-caproyl-D-Pro
and L-Tyr-OMe × HCl with a 40% yield. The product appears
as a white crystalline substance; m.p., 139 – 142°C (upon
ether treatment); [a]2D5, + 95.8° (c, 0.4; chloroform); Rf, 0.48
This study was supported by the Russian Foundation for
Basic Research, project No. 99–04–48477.
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(chloroform – ethanol, 9 : 1); IR spectrum in KBr disks
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(chloroform – ethanol, 9 : 1); IR spectrum in KBr disks