Spin labeled hydroxyproline analogues

Article abstract of DOI:10.1016/S0014-827X(99)00083-X

The synthesis and properties of both epimers of 4-(N-hydroxyamino)-N-tosyl-L-proline methyl ester and derivatives thereof are reported. In the presence of air, the compounds bearing a free N-hydroxyamino group oxidized in solution to give the correspondin

Full text of DOI:10.1016/S0014-827X(99)00083-X

www.elsevier.nl/locate/farmac
Il Farmaco 54 (1999) 637–641
New compounds
Spin labeled hydroxyproline analogues
Jean M.J. Tronchet a,*, Chantal Jorand a, Guido Zosimo-Landolfo a,1,
Michel Geoffroy b
a Department of Pharmaceutical Chemistry, Uni6ersity of Gene6a, Sciences 2, CH-1211 Gene6a 4, Switzerland
b Department of Physical Chemistry, Uni6ersity of Gene6a, Sciences 2, CH-1211 Gene6a 4, Switzerland
Received 18 March 1999; accepted 19 July 1999
Abstract
The synthesis and properties of both epimers of 4-(N-hydroxyamino)-N-tosyl-
L
-proline methyl ester and derivatives thereof are
reported. In the presence of air, the compounds bearing a free N-hydroxyamino group oxidized in solution to give the
corresponding aminoxyl free radicals, EPR spectra of which were recorded. None of these compounds showed any interesting
biological activity as such, but they constitute potential synthetic building blocks towards spin labeled glycopeptide or
oligopeptide analogues. © 1999 Elsevier Science S.A. All rights reserved.
Keywords: L-Proline; N-hydroxyamino; EPR spectroscopy; Aminoxyls
mers of 4-hydroxyamino-L-proline. A preliminary ac-
count of this work has been published [6].
1. Introduction
Even if the alleged activity of thioproline against
certain lung cancers [1] was not confirmed, [2] the
interest of preparing other analogues of hydroxyproline
still remains. The conformational stability of collagen is
partially insured by hydrogen bonds, two molecules of
water acting as a bridge between the hydroxy groups of
two hydroxyproline moieties [3]. This entropically unfa-
vorable situation could be improved by replacing the
hydroxy group of hydroxyproline with a hydroxyamino
group. Such an elongation of the hydrogen bonding
groups could render unnecessary the second molecule
of water. Other interests in the 4-hydroxyaminoproline
derivatives reside in their spontaneous oxidation into
aminoxyl radicals allowing the monitoring of these
modified aminoacids by EPR spectroscopy [4] and their
possible use as building blocks for the synthesis of
oligopeptide or glycopeptide [5] analogues. We describe
here the synthesis of derivatives of both diastereoiso-
2. Experimental procedures
General techniques have been described [7]. Reduc-
tion of oxime 1 has been performed using NaBH3CN in
standard conditions [8] and nitrone 7 reduced by
NaBH3CN in acidic medium following a described
procedure [9]. Biological tests have been conducted as
previously described [10]. Complementary information
can be obtained from the authors (J.M.J. Tronchet).
3. Results and discussion
Upon cyanoborohydride reduction, oxime 1 [11] led
to a 2:1 mixture (83%) of 2 and 3 from which only 2
could be obtained in pure state by crystallization
(Scheme 1). The diastereoisomeric mixture of 2 and 3
was submitted to O-silylation and a chromatographic
separation of the O-silyl derivatives afforded pure 4
(39%) and 5 (15%). Pure 2 and 3 were obtained almost
quantitatively by de-O-silylation of 4 and 5 respec-
* Corresponding author. Fax: +41-22-702-6585.
E-mail address: jean.tronchet@pharm.unige.ch (J.M.J. Tronchet)
1 Present address: Elsevier Science S.A., 50 avenue de la Gare, 1001
Lausanne, Switzerland.
0014-827X/99/$ - see front matter © 1999 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(99)00083-X

638
J.M.J. Tronchet et al. / Il Farmaco 54 (1999) 637–641
tively. The (2S,4R) configuration of 3 was established
by X-ray diffraction [12]. Upon treatment with N-
methylhydroxylamine hydrochloride, the ketone 6 [11]
afforded an E/Z mixture of the unstable nitrone 7
which was directly reduced (NaBH3CN) to a 2:1 mix-
ture (94% from 6) of 8 and 9 from which only 8 could
be isolated in a pure state. From the acetylated mixture
of 8 and 9, the major isomer 10 (49%) was obtained by
recrystallization. The (2S,4S) configuration of the ma-
jor isomer 8 was assigned from the better accessibility
of the re face of the electrophilic carbon atom of 7 as
apparent from the issue of the reduction of 1. From
pure 2, some derivatives of the hydroxyamino function
were prepared. Thus, conjugate addition of 2 upon
acrylonitrile led to 12 (92.5%) whereas, upon acetyla-
tion (Ac2O), 2 gave the N,O-diacetylated derivative 13
(62%), selective deacetylation of which (one equivalent
of NaOEt in ether/MeOH) afforded the hydroxamic
acid 14 (75%). Treated with 4-phenylazobenzoyl chlo-
ride, 2 led to the hydroxamic acid 15 (67%). From 3,
16 (69%) and 17 (70%) were prepared using the same
procedures. Some properties of the novel compounds
are collected in Tables 1 and 2.
Either spontaneously in the air or upon addition of
traces of lead dioxide, compounds bearing a N-hy-
droxy group oxidized to the corresponding aminoxyl
free radical. Except for the primary hydroxylamines 2
and 3 giving rise to unstable aminoxyls which decom-
posed into at least two paramagnetic species of un-
known structure, other N-hydroxy derivatives afforded
Scheme 1.

J.M.J. Tronchet et al. / Il Farmaco 54 (1999) 637–641
639
Table 1
Some properties of modified proline derivatives
Compound
M.p. (°C)
[h]D (t°C, c)a
RF (solvent)
Anal.
EI MS
(C, H, N, S)b
+
+
’
’
2
3
4
93.6–98.5
112.0–113.5
Syrup
−71.4 [21, 0.3]
−40.1 [23, 1.0]
−30.4 [23, 1.3]
0.6 (5:1 AcOEt/MeOH)
0.7 (5:1 AcOEt/MeOH)
0.53 (10:1:11 Et2O/MeOH/
hexane)
0.45 (10:1:11 Et2O/MeOH/
hexane)
C13H18N2O5S
C13H18N2O5S
C29H36N2O5SSi
314 (8, M
314 (9, M
)
)
553 (1.2, M ++1),
’
495 (69, M +−Me3C)
’
’
5
Syrup
−40.8 [23, 0.5]
C29H36N2O5SSi
552 (2, M +),
495 (89, M +−Me3C)
’
+
’
’
8
10
111.0–112.5
79.0–88.0
−69.0 [26, 1.0]
−50.8 [25, 1.2]
0.5 (AcOEt)
0.4 (2:1 AcOEt/(Me2CH)2O
C14H20N2O5S
C16H22N2O6S
328 (0.6, M
)
370 (0.1, M +),
328 (11, M +−CH2CO)
’
+
367 (8, M ), 307 (M +−AcOH)
’
’
12
13
Syrup
119.8–123.2
−50.0 [22, 0.6]
−37.4 [22, 0.8]
0.45 (2:1 AcOEt/hexane)
0.70 (AcOEt)
C16H21N3O5S
C17H22N2O7S
398 (0.5, M +),
’
356 (36, M +−CH2CO)
’
14
148.0–150.0
−30.5 [25, 1.0]
0.5 (AcOEt)
C15H20N2O6S
356 (0.9, M +),
’
281 (1.2, M +−AcNOH)
’
’
’
15
16
17
195.2−198.0
Syrup
148.5−149.3
−20.8 [23, 1.1]
−45.3 [29, 1.2]
−26.6 [21, 0.8]
0.5 (2:1 AcOEt/hexane)
0.68 (AcOEt)
0.45 (AcOEt)
C26H26N4O6S
C17H22N2O7S
C15H20N2O6S
351 (3, M +−Ts−O)
356 (2, M +−CH2CO)
+
’
356 (1.1, M
)
a In CHCl3.
b Within90.3% of the calculated values.
Table 2
Selected spectroscopic data (UV, IR, 1H NMR, 13C NMR) of modified
L-proline derivatives
Compound
UV (EtOH)
umax (nm) (m)
IR
1H NMR (CDCl3)
lH J (Hz)
13C NMR (CDCl3)
l
wmax (cm−1 a
)
2
209 (3780)
230 (8970)
3460 (OH), 3150 (NH),
1725 (CO), 1590–1450 (CꢀC, Ar),
1340, 1160 (SO) [A]
Ha-3 2.07, J3a,4 5.5
C-3 33.02
C-4 60.77
C-5 51.12
Hb-3 2.17, J3b,4 3.0, J3a,3b 14.0
H-4 3.60, Ha–5 3.30 J4,5a 5.5
Hb-5 3.46 J4,5b 2.0, J5a,5b 10.5
3
4
5
8
202 (15900)
230 (11700)
3480, 3280 (OH, NH), 1750 (CO),
1600, 1495, 1455 (CꢀC, Ar), 1350,
1165 (SO) [B]
Ha-3 2.05, J3a,4 6.0
Hb-3 2.14, J3b,4 4.5, J3a,3b 14.0
H-4 3.73, J4,5 4.5, 2 H-5 3.48
C-3 33.72
C-4 60.28
C-5 50.81
202 (43800)
220 (24800)
265 (1840)
3275 (NH), 1747 (CO), 1601,
1500, 1474 (CꢀC, Ar), 1355,
1164 (SO) [A]
2 H-3 2.11, J3,4 4.0
H-4 3.43, Ha-5 3.20, J4,5a 5.5
Hb-5 3.75, J4,5b 2.0, J5a,5b 10.5
C-3 32.60
C-4 61.14
C-5 50.85
202 (46800)
220 (23400)
270 (11300)
3270 (NH), 1750 (CO), 1602,
1500, 1468 (CꢀC, Ar), 1355,
1168 (SO) [D]
Ha-3 1.93, J3a,4 7.0
Hb-3 2.05, J3b,4 4.0, J3a,3b 13.5
H-4 3.64, Ha-5 3.41, Hb-5 3.60
C-3 33.39
C-4 61.02
C-5 50.73
204 (13300)
230 (9900)
3140 (OH), 1746 (CO), 1598,
1499, 1449 (CꢀC, Ar), 1344,
1157 (SO) [A]
2 H-3 2.16, J3,4 7.5
H-4 3.00, Ha-5 3.40, J4,5a 6.0
Hb-5 3.57, J4,5b 6.8, J5a,5b 11.0
C-3 33.14
C-4 66.86
C-5 50.77
NꢁMe 46.47
10
12
202 (13500)
230 (9200)
1765 (CO), 1605, 1443 (CꢀC, Ar),
1360, 1166-1100 (SO), 1166-1100
(C-O-C) [C]
Ha-3 2.11, J3a,4 8.0
C-3 33.07
C-4 65.90
C-5 50.18
N-Me 45.05
NꢁCꢀO 168.84
MeꢁCON 19.26
Hb-3 2.35, J3b,4 6.0, J3a,3b 13.2
H-4 3.24, Ha-5 3.30, J4,5a 8.0
Hb-5 3.67, J5a,5b 8.0
202 (14700)
230 (11200)
3455 (OH), 2250 (CN), 1751 (CO),
1591, 1495, 1438 (CꢀC, Ar),
1346, 1159 (SO) [D]
Ha-3 2.12, J3a,4 7.0
Hb-3 2.22, J3b,4 6.0, J3a,3b 13.0
H-4 3.10, 2 H-5 3.50, J4,5 6.5
C-3 32.91
C-4 65.66
C-5 50.70
CH2CN 16.10
NꢁCH2CH2 53.02
CN 118.57
.

640
J.M.J. Tronchet et al. / Il Farmaco 54 (1999) 637–641
Table 2 (continued)
Compound
UV (EtOH)
umax (nm) (m)
IR
wmax (cm−1 a
1H NMR (CDCl3)
lH J (Hz)
13C NMR (CDCl3)
l
)
13
202 (18000)
230 (11900)
1810, 1760, 1685 (CO), 1605,
1500, 1450 (CꢀC, Ar), 1365,
1167 (SO) [C]
Ha-3 2.10, J3a,4 ca 7
Hb-3 2.43, J3b,4 ca 7
H-4 4.87, Ha-5 3.36, J4,5a 7.5
Hb-5 360, J4,5b 8.0, J5a,5b 13.0
C-3 32.15
C-4 53.99
C-5 48.73
N-CO 170.58
MeꢁCON 20.14
14
15
16
203 (21700)
229 (12700)
3280 (OH), 1727, 1640 (CO),
1595, 1440 (CꢀC, Ar), 1355,
1163 (SO) [C]
2 H-3 2.26, H-4 4.88
Ha-5 3.28, J4,5a 6.0
Hb-5 3.91, J4,5b 0.5, J5a,5b 10.5
C-3 35.09
C-4 54.80
C-5 49.16
202 (38700)
230 (24400)
324 (22100)
3130 (OH), 1750 (CO),
1600, 1585, 1500, 1460 (CꢀC, Ar),
1350, 1162 (SO) [A]
2 H-3 2.43, H-4 4.88
Ha-5 3.48, J4,5a 6.5
Hb-5 3.97, J4,5b 3.0, J5a,5b 11.0
C-3 34.76
C-4 56.98
C-5 59.04
203 (17700)
1800, 1750, 1685 (CO), 1600,
230 (12200)
2 H-3 2.26, H-4 5.21
1495, 1440 (CꢀC, Ar), 1370,
1175 (SO) [B]
Ha-5 3.49, J4,5a ca. 7
Hb-5 3.60, J4,5b ca. 7,
J5a,5b 10.0
17
202 (17700)
3300 (OH), 1755, 1655 (CO),
230 (12000)
2 H-3 2.26, H-4 4.88
1608, 1525, 1445 (CꢀC, Ar),
1350, 1170 (SO) [C]
Ha-5 3.28, J4,5a 6.0
Hb-5 3.91, J4,5b 0.5,
J5a,5b 10.5
a Solvent [A]=KBr, [B]=CCl4, [C]=CHCl3, [D]=film (no solvent).
Table 3
EPR spectra in diglyme of aminoxyl radicals generated from hydroxylamines or hydroxamic acidsa
Diamagnetic species
t (°C)
Oxidizing agent
g
aN
aH-4
aCH
aCH
Æb
2
3
8
12
14
15
17
45
11.5
25
50
25
None
None
None
PbO2
None
2.0061
2.0060
2.0064
2.0069
2.0065
14.95
14.15
7.55
7.45
7.5
7.7
7.1
4.35
4.7
4.8
12.25
1.2
1.5
2.0
1.2
1.3
10.6
a Hyperfine coupling constants in Gauss.
b Linewidth in Gauss.
free radicals giving rise to well resolved EPR spectra
(Table 3), exhibiting hyperfine coupling constants in
the expected range of values [4].
None of these compounds showed any significant
cytotoxic anticancer, or antiviral (HSV-1, RV-31, RV-
1B, influenza A) activity.
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and A. Zerial (Rhoˆne-Poulenc Research Center, 94407
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