The conformational features of some biologically active N-acylprolyltyrosinamides studied by 1H NMR spectroscopy

Full text of DOI:10.1023/A:1012707304680

Pharmaceutical Chemistry Journal
Vol. 35, No. 7, 2001
THE CONFORMATIONAL FEATURES OF SOME BIOLOGICALLY
ACTIVE N-ACYLPROLYLTYROSINAMIDES STUDIED
1
BY H NMR SPECTROSCOPY
N. I. Zaitseva,1 V. P. Lezina,1 A. N. Ignashin,1 V. K. Briling,1 and T. A. Gudasheva1
Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 35, No. 7, pp. 35 – 38, July, 2001.
Original article submitted January 11, 2001.
Previously [1], we described the design and synthesis of
a series of N-acylprolyltyrosinamides – potential neuroleptic
drugs. These substituted dipeptides were designed so as to be
analogous to the active site of neurotensin – a neuropeptide
known as an “endogenous neuroleptic” [2]. In experiment,
N-acylprolyltyrosinamides exhibited neuroleptic activity de-
pending on the nature and size of N-acyl radical (in the dose
range from 0.4 to 10.0 mg/kg, i.p.) and did not produce cata-
lepsy in amounts 100 – 1000 times the effective dose [3].
This therapeutic profile allowed us to suggest that N-acylpro-
lyltyrosinamides may represent a new group of potential neu-
roleptics free of neurologic side effects.
spectra of the synthesized N-acylprolyltyrosinamides
(I – VIII) were interpreted using published data on the spect-
ra of proline-containing peptides [5, 6] and double homonuc-
lear resonance. The chemical shifts in the 1H NMR spectra of
the substituted dipeptides are listed in Table 1. The spectra
contain a double set of proton signals from CbH2 Tyr, NH
Tyr, CaH Pro, and NH2, which reflects the difference in the
orientation (trans versus cis ) of substituents relative to the
quaternary amide bond –C(O)–N< (Fig. 1). By integrating
the NH resonance curves, it was established that the ro-
om-temperature trans to cis isomer ratio of N-acylprolyltyro-
sinamides dissolved in CDCl3 amounts approximately to
95 : 5 and is virtually independent of the N-acyl radical size.
Indeed, the content of trans isomers was the same
(95 – 98%) for peptide III with a branched tert-butoxycarbo-
nyl fragment and for peptides with linear valeryl (IV) and
caproyl (V – VIII) radicals (Table 2). The minimum content
of trans isomers (90%) was observed for N-pivaloyl-L-pro-
lyl-L-tyrosine (I).
Interest in the conformational features of N-acylprolylty-
rosinamides is related to the assumption that there exists a bi-
ologically active conformation of the dipeptide analogs of
neurotensin. Finding this conformation would facilitate the
search for new potential neuroleptics. In this context, we
have synthesized a series of N-acylprolyltyrosinamides
1
(I – VIII) and studied their conformation in solution by H
A trans isomer may acquire either an open form or a con-
formation stabilized by intramolecular hydrogen bonds. Ba-
sed on the published data [7 – 9] concerning the conformati-
ons of model dipeptides of the tert-BuCO-Pro-Y-NHCH3
(Y = Gly, L-Ala, D-Ala) type, we may suggest that trans iso-
mers of compounds I – III in solution represent an equilibri-
NMR spectroscopy.
OH
O
R1C(O)-X-Pro-Y-Tyr-NHR
O
N
H
N
NHR
1
R
O
I – VIII
I: R = CH3, R1 = (CH3)3C; II: R = CH3, R1 = (CH3)3CO; III: R = H,
R1 = (CH3)3CO; IV: R = H, R1 = CH3(CH2)3; V – VIII: R = H,
R1 = CH3(CH2)4; I – V: X = Y = L; VI: X = Y = D; VII: X = L, Y = D; VIII:
X = D, Y = L.
OH
OH
O
O
O
O
NH2
N
H
N
H
N
N
Peptides I – VIII were obtained by the method of mixed
anhydrides as described by Anderson et al. [4]. The 1H NMR
NH2
1
O
R1
R
O
cis
trans
Fig. 1. The structures of trans and cis conformations of
N-acylprolyltyrosinamides.
1
Institute of Pharmacology, Russian Academy of Medical Sciences, Mos-
cow, Russia.
382
0091-150X/01/3507-0382$25.00 © 2001 Plenum Publishing Corporation

The Conformational Features of Some Biologically Active N-Acylprolyltyrosinamides Studied
383
d, ppm
8.5
OH
1
OH
O
8.0
7.5
7.0
6.5
6.0
5.5
5.0
2
O
N
H
™
3
O
™
†
™
†
N
™
†
™
†
™
†
™
†
NH2
™
†
4
O
†
™
†
1
N
R
O
5
™
†
N
H
NH2
g-rotated
OH
1
open
R
O
™
6
O
†
O
N
H
0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
DMSO/ÑDCl3
N
N
R1
O
H
H
b-rotated
Fig. 3. Plots of the NH and NH2 proton chemical shifts in com-
pound V versus the DMSO-d6 /CDCl3 ratio in the solvent mixture:
(1 ) NH cis; (2 ) NH trans; (3, 6 ) NH2 cis; (4, 5 ) NH2 trans.
Fig. 2. The possible conformations of N-acylprolyltyrosinamides.
um mixture of three mutually converting forms: open, g-rota-
bonds. The signals from protons involved in such bonds are
characterized by smaller values of the parameter Dd =
dDMSO-d – dCDCl representing a difference between the
ted, and b-rotated (Fig. 2).
The results of 1H NMR investigations of N-acylprolylty-
rosinamides dissolved in CDCl3 or DMSO-d6 indicate that
6
3
proton chemical shifts in DMSO-d6 and CDCl3. As can be
seen from Fig. 3, the chemical shift for one of the NH2 pro-
tons in the trans conformation is virtually independent of the
nature of the solvent (unlike the signals from protons of the
NH group of tyrosine and NH2 group of the cis isomer). For
the NH proton of tyrosine, Dd = 1.13 ppm (Table 2), whereas
the value for one of the NH2 protons is b = 0.53 ppm, which
is evidence of the participation of this proton in the intramo-
b-rotated conformations with intramolecular hydrogen bonds
between a proton of the NH2 group and oxygen of the carbo-
nyl group of N-acyl radical predominate. Figure 3 shows
plots of the chemical shifts of the NH and NH2 proton signals
in most biologically active compound V versus the
DMSO-d6/CDCl3 ratio. An increase in the DMSO-d6 fracti-
on changes the ratio of intramolecular hydrogen bonds and,
hence, the chemical shifts of protons involved in these
TABLE 1. Proton Chemical Shifts (ppm) in the 1H NMR Spectra of N-Acylprolyltyrosinamides in DMSO-d6
Com- CbH2-CgH2 Pro CbH2 Tyr
CdH2 Pro
CaH Pro
CaH Tyr
C6H4 Tyr
m
NH Tyr
d
NH2 2s
NHCH3 q
OH Tyr
s
Other signals
pound
m
2dd
m
dd
m
I
1.35 – 1.95
2.60 – 3.10 3.20 – 3.50
3.61
4.22
6.60; 6.95 7.56, 8.28 7.64, 7.90 9.16, 9.17 1.15 (s, 9H, (CH3)3C),
2.51, 2.58 (2d, 3H,
NHCH3)
II
1.53 – 2.14
2.65 – 3.10 3.10 – 3.30 3.95, 4.01 4.30, 4.36 6.62; 6.99 7.81, 7.85 7.58, 7.82 9.13, 9.15 1.22, 1.40 (2s, 9H,
(CH3)3C), 2.50, 2.56
(2d, 3H, NHCH3)
III
1.56 – 2.10
2.60 – 3.00 3.20 – 3.40 3.95, 4.01 4.30, 4.41 6.62; 6.98, 7.70, 7.75 7.00; 7.28, 9.18, 9.20 1.22, 1.39 (2s, 9H,
6.62; 7.00
1.00 – 2.35* 2.60 – 2.98 3.13 – 3.59 4.16, 4.24 4.24, 4.42 6.63, 6.97 7.67, 8.12 7.03; 7.49, 9.17, 9.18 0.79, 0.89 (2t, 3H,
7.09; 7.12 CH3(CH2)3)
1.10 – 2.15** 2.66; 2.88, 3.22 – 3.54 4.22, 4.27 4.38, 4.53 6.60; 7.02, 7.66, 8.10 7.04; 7.47, 9.15, 9.17 0.83, 0.87 (2t, 3H,
2.70; 2.98 6.62; 6.97 7.08; 7.10 CH3(CH2)4)
1.05 – 2.30** 2.61; 3.02, 3.20 – 3.50 4.17, 4.24 4.28, 4.45 6.62; 6.98, 7.66, 8.11 7.08; 7.10, 9.15, 9.17 0.84, 0.85 (2t, 3H,
7.07; 7.15
(CH3)3C)
IV
V
VI
VII
VIII
2.64; 2.88
1.05 – 2.30** 2.70; 3.02, 3.20 – 3.50 4.17, 4.31 4.20, 4.41 6.62; 6.98, 8.11, 8.22 7.08; 7.48,
2.64; 2.88 6.62; 7.00 7.16; 7.29
1.10 – 2.30** 2.64; 3.02, 3.20 – 3.50 4.17, 4.31 4.21, 4.43 6.62; 6.98, 8.09, 8.20 7.07; 7.46,
2.63; 2.90 6.62; 7.00 7.15; 7.28
6.62; 7.00
7.04; 7.47
CH3(CH2)4)
9.19
9.17
0.84, 0.85 (2t, 3H,
CH3(CH2)4)
0.84, 0.86 (2t, 3H,
CH3(CH2)4)
*
Plus CH3(CH2)3 protons.
Plus CH3(CH2)4 protons.
**

384
N. I. Zaitseva et al.
lecular hydrogen bond stabilizing the b-rotated conformati-
on.
There are several types of b-rotation depending on the
peptide chain geometry [10]. The most frequently encounte-
red types are I and II. In order to determine the b-rotation
type, we have studied the spectra of the nuclear Overhauser
effect (NOE) and the spin – spin coupling constant of N-cap-
royl-L-prolyl-L-tyrosinamide (V) selected for broad pharma-
cological characterization [3].
In the spectra of compounds I – VIII, the Dd value of NH
protons of tyrosine in the trans isomer varies from 1.06 to
1.75 ppm, while the same difference for protons of the NHR
groups varies from 0.08 to 0.94 ppm (Table 2). The strongest
intramolecular hydrogen bonds stabilizing the b-rotated con-
formation are formed in heterochiral peptides VII and VIII
(Dd = 0.08 and 0.10 ppm). Among the homochiral peptides,
the minimum Dd value of NHR protons is observed for com-
pound III with tert-butoxycarbonyl radical.
For dipeptides of the tert-BuCO-Pro-Y-NHCH3 (Y = Gly,
L-Ala, D-Ala) type, Boussard and Marraud [8] established
the following relationship between the content of b-rotated
structures and the Dd value for amide proton of the NHCH3
group:
As is known [11, 12], the distance between CaH of the
(i + 1)th residue and NH of the (i + 2)th residue in the ideal
b-rotated conformation II is < 3 Å, and the NOE magnitude
is > 10%. For the ideal b-rotated conformation I, the above
distance between residues is ³ 3.5 Å and the NOE magnitude
is £ 10%. We have also established that NOE takes place for
NH Tyr and CaH Pro in the trans isomer V in CDCl3. Here,
the NOE magnitude is 7%, which is evidence of the b-rotated
conformation of type I. For the trans conformation of V, the
spin – spin coupling constants for CaH PRo and NH Tyr are
the same (on the average, 8.5 Hz) in CDCl3 and DMSO-d6.
Aubry et al. [9] studied the proton – proton coupling
H–N, Ca–H as a function of the torsion angle p and establis-
hed the following rule for substituted dipeptides of the
%[b-rotation] = 100 – 39Dd.
(1)
Taking into account the structural similarity of N-acylp-
rolyltyrosinamides and peptides studied in [8], we assumed
that the above relationship holds for our compounds as well.
As can be seen from Table 2, the value of percentage b-rota-
tion (63%) for (CH3)3CC(O)-Pro-Tyr-NHCH3 (I) agrees well
with that (65%) reported in [8]. This fact allowed us to use
relationship (1) for calculating the percentage content of
b-rotated structures in the compounds studied.
The data presented in Table 2 show that the fraction of
b-rotated conformation depends on the chirality of the pepti-
de amino acid residues. In the case of heterochiral compo-
unds VII and VIII, almost the entire trans isomer (~97%) oc-
curs in the the b-rotated form (in agreement with [8]). The
strength of intramolecular hydrogen bonds stabilizing this
conformation depends on the degree of amide substitution.
For example, unsubstituted amide III is characterized by a
higher content of b-rotated isomers (84%) than methylamide
II (70%).
3
L-Pro-L-Xaa type: the J(H–N, Ca–H) value of about 9 Hz
corresponds to the b-rotation of type I, while smaller values
(~ 4.5 Hz) correspond to the rotation of type II. A compari-
son of these criteria with our experimental value (8.5 Hz) le-
ads to a conclusion that compound V more likely exhibits
b-rotation of type I. The same conclusion follows from solu-
tion of the Karplus – Bystrov equation [13]
3J(H–N, Ca–H) = 9.4 cos2 (60 – j) – 1.1 cos (60 – j) + 0.4
(2)
for 3J(H–N, Ca–H) = 8.5, which yields jTyr = – 91° and 52°.
The conclusion that a b-rotation of type I is favorable for the
trans conformation of amide V is obvious, since the ideal
b-rotations of type
I and II are characterized by
j(i + 2) = – 90° and + 80°, respectively [10].
Thus, the 1H NMR spectroscopy allowed us to determine
the favorable conformations of N-acylprolyltyrosinamides
I – III in CDCl3 – DMSO-d6 solutions. Under these conditi-
ons, these compounds occur predominantly in the b-rotated
conformations. The potential neuroleptic N-caproyl-L-pro-
lyl-L-tyrosinamide (V) most favorably adopts the b-rotated
conformation of type I.
TABLE 2. Difference in Proton Chemical Shifts and Relative Con-
tent of trans Isomer and b-Rotated Conformation in Solutions of
Compounds I – VIII (CDCl3)
[b-rotation]*,
trans
NHTyr
trans
NHR
Dd
Dd
Compound
[trans], %
%
I
1.65
1.49
1.31
1.12
1.13
1.06
1.74
1.75
0.94
90
95
98
98
95
95
98
98
63
70
84
80
79
77
96
97
II
0.76
0.40
0.52
0.53
0.60
0.10
0.08
EXPERIMENTAL PART
III
IV
V
The melting temperatures (uncorrected) were determined
in open capillaries. The specific optical rotation was measu-
red on a Perkin-Elmer Model 241 polarimeter. TLC was con-
ducted on Kieselgel 60F-254 (Merck), Silufol UV-254 (Ser-
va), and Silufol UV-254 (Kavalier) plates. The spots were
developed by exposure to iodine vapor. Column chromatog-
raphy was performed on Kieselgel 100 columns. The data of
VI
VII
VIII
*
Calculated by formula (1) [8].

The Conformational Features of Some Biologically Active N-Acylprolyltyrosinamides Studied
385
white crystals; m.p., 205 – 207°C; [a]2D5, +57.2° (c, 0.3; met-
elemental analyses for C, H, and N correspond to the results
of analytical calculations.
hanol); Rf , 0.53 (Silufol Serva, chloroform – ethanol, 8 : 2);
C20H29N3O4.
General method for the synthesis of N-acylprolyltyro-
sinamides (I – III). A solution of 5 mmole of N-acyldipepti-
de ester in 50 ml of anhydrous methanol was saturated with
ammonia (or methylamine) at 0°C and allowed to stand for
two days at room temperature. Then the solvent was evapo-
rated in vacuum and the product was purified by column
chromatography (eluent, chloroform – ethanol, 9 : 1 v/v)
and crystallized by triturating with diethyl ether.
NMR measurements. The 1H NMR spectra were recor-
ded on a Bruker AC-250 spectrometer (Germany) using
CDCl3 and DMSO-d6 as solvents and TMS as the internal
standard. The sample solutions had a peptide concentration
of 5 – 10 mg/ml. The intramolecular hydrogen bond forma-
tion was established by measuring the NH proton chemical
shift depending on the solution concentration (in CDCl3) and
on the nature of the solvent (in CDCl3 – DMSO-d6 mixtu-
N-Pivaloyl-L-prolyl-L-tyrosine methylamide: (CH3)3C-
C(O)-L-Pro-L-Tyr-NHCH3 (I) was obtained from
(CH3)3C-C(O)-L-Pro-L-Tyr-OEt with a yield of 59% in the
3
res). The spin – spin coupling constants J in the NH–CaH
system was determined using the NH proton signal. The
NOE magnitude in the one-dimensional NMR spectra was
determined using low-power irradiation at a selected reso-
nance frequency. The NOEj (i ) value was determined as the
ratio of the change in the integral jth signal intensity in the
NMR spectrum under ith nucleus saturation conditions (I 0j )
form of
a
white crystalline substance with m.p.,
148 – 150°C; Rf , 0.64 (Silufol Kavalier, chloroform – etha-
nol, 8 : 2); C20H29N3O4.
tert-Butyloxycarbonyl-L-prolyl-L-tyrosine methyla-
mide: Boc-L-Pro-L-Tyr-NHCH3 (II) was obtained from
Boc-L-Pro-L-Tyr-OMe with a yield of 98%; white crystalline
substance; m.p., 90 – 93°C; [a]2D0, – 90° (c, 0.3; chloroform);
to the integral j-th signal intensity in the absence of saturati-
on (I 0j ):
Rf, 0.4 (Kieselgel, chloroform – ethanol, 9 : 1); C20H29N3O5.
tert-Butyloxycarbonyl-L-prolyl-L-tyrosine
amide:
NOE (i) = (I j - I 0j ) I 0j .
Boc-L-Pro-L-Tyr-NH2 (III) [1] was obtained from
Boc-L-Pro-L-Tyr-OMe with a yield of 60%; white crystalline
substance; m.p., 72 – 75°C; [a]2D0, – 49.7° (c, 0.38; chloro-
j
ACKNOWLEDGMENTS
form); Rf, 0.33 (Kieselgel, chloroform – ethanol, 9 : 1);
C19H27N3O5.
N-Valeryl-L-prolyl-L-tyrosine amide: CH3(CH2)3C(O)-
L-Pro-L-Tyr-NH2 (IV) [1] was obtained from
CH3(CH2)3C(O)-L-Pro-L-Tyr-OEt with a yield of 91%; whi-
te crystals; m.p., 202 – 204°C (treatment with ether); [a]2D5,
This study was supported by the Russian Foundation for
Basic Research, project No. 00-04-48477.
REFERENCES
– 60.7° (c, 0.3; methanol); Rf , 0.48 (Silufol Kavalier, chloro-
form – ethanol, 8 : 2); C19H27N3O4.
1. T. A. Gudasheva, T. A. Voronina, R. U. Ostrovskaya, et al., J.
Med. Chem., 41(3), 284 – 290 (1998).
N-Caproyl-L-prolyl-L-tyrosine amide: CH3(CH2)4C(O)-
L-Pro-L-Tyr-NH2 (V) [1] was obtained from
CH3(CH2)4C(O)-L-Pro-L-Tyr-OMe with a yield of 59%;
white crystals; m.p., 112 – 113°C; [a]2D0, –66.0° (c, 0.3; met-
2. C. V. Nemeroff, Biol. Psych., 15(2), 283 – 302 (1980).
3. S. B. Seredenin, T. A. Voronina, T. A. Gudasheva, et al., RF Pat-
ent No. 2 091 390 (1997); Chem. Abstr., 129, 453a (1998).
4. G. W. Anderson, J. E. Zimmerman, and F. M. Callahan,
J. Am. Chem. Soc., 89, 5012 – 5017 (1967).
5. C. M. Deber, F. A. Bovey, J. P. Carver, et al., J. Am. Chem. Soc.,
92(21), 6191 – 6198 (1970).
6. V. Madison and J. Schellman, Biopolymers, 9(5), 511 – 567
(1970).
hanol); Rf , 0.4 (Silufol Serva, chloroform – ethanol, 9 : 1);
C20H29N3O4.
N-Caproyl-D-prolyl-D-tyrosine amide: CH3(CH2)4C(O)-
D-Pro-D-Tyr-NH2
(VI)
was
obtained
from
7. G. Boussard, M. Marraud, and A. Aubry, Biopolymers, 18(6),
1297 – 1331 (1979).
8. G. Boussard and M. Marraud, J. Am. Chem. Soc., 107(7),
1825 – 1828 (1985).
9. A. Aubry, M. T. Cung, and M. Marraud, J. Am. Chem. Soc.,
107(7), 7640 – 7647 (1985).
CH3(CH2)4C(O)-D-Pro-D-Tyr-OMe with a yield of 73%;
white crystals; m.p., 145 – 147°C; [a]2D5, + 57.7° (c, 0.3; met-
hanol); Rf , 0.51 (Silufol Serva, chloroform – ethanol, 8 : 2);
C20H29N3O4.
N-Caproyl-L-prolyl-D-tyrosine amide: CH3(CH2)4C(O)-
L-Pro-D-Tyr-NH2
(VII)
was
obtained
from
10. J. B. Ball, R. A. Hughes, P. F. Alewood, et al., Tetrahedron,
49(17), 3479 – 3488 (1993).
11. B. N. Rao, A. Kumar, H. Balaram, et al., J. Am. Chem. Soc.,
105(25), 7423 – 7428 (1983).
12. G. Pietrzynski, Z. Kubica, and B. Rzeszotarska, “Conformatio-
nal propensities of model pegtides with a,b-dehydroamino
acids,” in: Peptides, E. Giralt and D. Andrew (eds.), ESCOM
Sci. Pub. B. V. (1991), pp. 462 – 464.
CH3(CH2)4C(O)-L-Pro-D-Tyr-OMe with a yield of 57%;
white crystals; m.p., 201 – 204°C (treatment with ether);
[a]2D5, –59.4° (c, 0.3; methanol); Rf , 0.53 (Silufol Serva,
chloroform – ethanol, 8 : 2); C20H29N3O4.
N-Caproyl-L-prolyl-D-tyrosine amide: CH3(CH2)4C(O)-
L-Pro-D-Tyr-NH2
(VIII)
was
obtained
from
CH3(CH2)4C(O)-L-Pro-D-Tyr-OMe with a yield of 57%;
13. V. F. Bystrov, Bioorg. Khim., 10(8), 997 – 1043 (1984).