Synthesis and pharmacological evaluation of new 3-aryl-3-hydroxyaminopropionic acids

Article abstract of DOI:10.1211/146080899128734668

This paper describes the synthesis and the behavioural evaluation in mice of various 3-aryl-3-hydroxylaminopropionic acids.

Full text of DOI:10.1211/146080899128734668

Pharm. Pharmacol. Commun. 1999, 5: 239±242
# 1999 Pharm. Pharmacol. Commun.
Synthesis and Pharmacological Evaluation of New
3-Aryl-3-hydroxyaminopropionic Acids
BERTRAND COUTURIER, MICHEL BOULOUARD*, PATRICK DALLEMAGNE AND SYLVAIN RAULT
Â
Centre d'Etudes et de Recherche sur le Medicament de Normandie, Laboratoire de Pharmacochimie and
and *Laboratoire de Pharmacologie, UFR des Sciences Pharmaceutiques, 1 Rue Vaubenard, 14032 Caen
Â
Cedex, France
Abstract
This paper describes the synthesis and the behavioural evaluation in mice of various 3-
aryl-3-hydroxylaminopropionic acids.
During the course of work on the synthesis of
heterocyclic systems of potential therapeutic inter-
est, we described the access to numerous new 3-
amino-3-arylpropionic acids (Dallemagne et al
1986, 1987, 1994; Rault et al 1987). The method
used was the Radionow-Johnson process. Benz-
aldehyde (1) was treated with malonic acid and
ammonium acetate to give in a one-step sequence
3-amino-3-phenylpropionic acid (2) (Figure 1).
Replacement of ammonia by hydroxyamine,
according to Posner's method (Posner 1903),
afforded the same product 2 via 3-hydroxyamino-3-
phenylpropionic acid (3) (Figure 1).
JNM-LA 400 spectrometer (400 MHz) using
CDCl3 or d6-DMSO as solvent. Chemical shifts
(d ppm) refer to tetramethylsilane which was used
as an internal reference. NH, NH2 and OH signals
appeared as broad singlets exchangeable with D2O.
Key: t ˆ triplet, s ˆ singlet, d ˆ doublet, dd ˆ
double doublet, m ˆ multiplet. Elemental analyses
(C, H, N) were performed by INSA, Rouen, France
and agreed with the proposed structures with-
inÆ 0Á3% of the theoretical values.
General procedure for preparation of 3-aryl-3-
hydroxyaminopropionic acids (3a±f)
We describe here the application of Posner's
method to various arylaldehydes to reinvestigate
this interesting access to rare compounds, such as
the title hydroxyamino acids, and to obtain homo-
logues of 3a with a view to evaluating them for
potential central pharmacological properties in
mice. We consider the 3-aryl-3-hydroxyamino-
propionic acids as lower hydroxyl analogues of
baclofen (4), the speci®c agonist of GABAB
receptor with muscle relaxant and analgesic
properties.
A hot solution of sodium ethoxide was prepared
from 0Á1 mol sodium and 100 mL ethanol. A solu-
tion of 0Á1 mol hydroxylamine hydrochloride in
10 mL hot water was added, with shaking. The
resulting suspension was cooled quickly by placing
the ¯ask in an ice-water mixture and then ®ltered
with suction through a Buchner. The residue of
sodium chloride was washed with small amounts of
ethanol. The ®ltrate was mixed with 0Á05 mol
acrylic acid. The mixture was re¯uxed on an oil
bath for 2 h. The ethanol was removed under
reduced pressure and the residue ®ltered and pur-
i®ed to give 3a±f.
Materials and Methods
3-Phenyl-3-hydroxyaminopropionic acid (3a). White
crystals (58%); mp 210ꢀC; IR (KBr) n: 3500 (OH),
3100 (NH), 1550 cm 1 (CO); 1H NMR (DMSO) d:
2Á41 (dd, J ˆ 16 and 7 Hz, H-2a), 2Á76 (dd, J ˆ 16
and 7 Hz, H-2b), 4Á16 (t, J ˆ 7 Hz, H-3), 7Á43 (m,
H-phenyl). Calculated for C9H11NO3: C, 59Á66; H,
6Á12; N, 7Á73. Found: C, 59Á68; H, 6Á16; N, 7Á67%.
Chemistry
Melting points were determined on a Ko¯er block
and are uncorrected. IR spectra were recorded on
an Unicam Mattson 1000 spectrometer. 1H, 13C and
1H-COSY NMR spectra were recorded on a Jeol
Correspondence: B. Couturier, Centre d'Etudes et de
Â
Recherche sur le Medicament de Normandie, Laboratoire de
Pharmacochimie, UFR des Sciences Pharmaceutiques, 1 Rue
3-(p,m -Dichlorophenyl)-3-hydroxyaminopropionic
acid (3b). White crystals (40Á2%); mp 214ꢀC; IR
1
Â
Vaubenard, 14032 Caen Cedex, France.
(KBr) n: 3500 (OH), 3000 (NH), 1600 cm (CO);

240
BERTRAND COUTURIER ET AL
Calculated for C7H9NO4: C, 49Á12; H, 5Á3; N, 8Á18.
Found: C, 48Á85; H, 5Á15; N, 8Á42%.
3-Furyl-3-hydroxyaminopropionic acid (3f). Beige
crystals (24%); mp 181ꢀC; IR (KBr) n: 3400 (OH),
3000 (NH), 1650 cm 1 (CO); 1H NMR (DMSO) d:
2Á40 (dd, J ˆ 16 and 7 Hz, H-2a), 2Á75 (dd, J ˆ 16
and 7 Hz, H-2b), 4Á10 (t, J ˆ 7 Hz, H-3), 6Á46 (s, H-
furyl), 7Á51 (s, H-furyl), 7Á53 (s, H-furyl). Calcu-
lated for C7H9NO4: C, 49Á12; H, 5Á3; N, 8Á18.
Found: C, 48Á85; H, 5Á15; N, 8Á42%.
Pharmacology
Pharmacological evaluation
OF-1 mice (France, Iffa Credo), 18±22 g, were
used. The animals were allowed free access to food
and water and were housed at room temperature
(20±22ꢀC). Derivatives 3a±f were suspended in an
aqueous solution of carboxymethylcellulose at
0Á5%. Compounds were administered by intraper-
itoneal injection. Except for the prescreening test,
ten mice were used in each treatment group.
Figure 1. 3-Aryl-3-hydroxylaminopropionic acids.
1H NMR (DMSO) d: 2Á43 (dd, J ˆ 16 and 7 Hz, H-
2a), 2Á69 (dd, J ˆ 16 and 7 Hz, H-2b), 4Á17 (t,
J ˆ 7 Hz, H-3), 7Á32 (d, J ˆ 8 Hz, H-phenyl),
7Á55 (d, J ˆ 8 Hz, H-phenyl), 7Á60 (s, H-phenyl).
Calculated for C9H9NO3Cl2: C, 43Á23; H, 3Á63; N,
5Á6. Found: C, 42Á26; H, 3Á35; N, 5Á25%.
Gross behavioural effects and acute toxicity in mice
Morpugo's modi®cation (Morpugo 1971) of Irwin's
multidimensional screening procedure was used on
groups of four mice to evaluate drug-induced
behavioural effects. The test compounds were
administered in log-spaced doses, and mice were
observed 30 min, 3 h and 24 h after treatment. The
approximate LD50 was obtained from the mortality
observed during a 48-h period.
3-(p-Methylphenyl)-3-hydroxylaminopropionic
acid (3c). White crystals (25Á6%); mp 220ꢀC; IR
1
(KBr) n: 3500 (OH), 3000 (NH), 1550 cm (CO);
1H NMR (DMSO) d: 2Á26 (s, CH3), 2Á39 (dd,
J ˆ 16 and 7 Hz, H-2a), 2Á76 (dd, J ˆ 16 and
7 Hz, H-2b), 4Á12 (t, J ˆ 7 Hz, H-3), 7Á08 (d,
J ˆ 8 Hz, H-phenyl), 7Á19 (d, J ˆ 8 Hz, H-phenyl).
Calculated for C10H13NO3: C, 61Á53; H, 6Á71 N,
7Á17. Found: C, 61Á3; H, 6Á62; N, 6Á97%.
Locomotor activity (Boissier & Simon 1965)
Locomotor activity was recorded using a photocell
activity cage for 15 min, beginning 30 min after
administration of each test compound. A counter
registered the number of interruptions of the photo-
beams. The approximate ED50 (dose which
induced a reduction of 50% of the locomotor
activity in comparison with the controls) was esti-
mated.
3-Pyridinyl-3-hydroxyaminopropionic acid
(3d). White crystals; IR (KBr) n: 3400 (OH), 3010
1
1
(NH), 1575 cm (CO); H NMR (DMSO) d: 2Á46
(dd, J ˆ 16 and 7 Hz, H-2a), 2Á75 (dd, J ˆ 16 and
7 Hz, H-2b), 4Á19 (t, J ˆ 7 Hz, H-3), 7Á32 (t,
J ˆ 5 Hz, H-pyridyl), 7Á73 (d, J ˆ 5 Hz, H-pyri-
dyl), 8Á42 (d, J ˆ 5 Hz, H-pyridyl), 8Á51 (s, H-
pyridyl).
Rotarod test (Dunham & Miya 1957)
3-Thienyl-3-hydroxyaminopropionic acid
This test was used to evaluate the activity of drugs
interfering with motor co-ordination. The apparatus
consisted of a horizontal wooden rod (3 cm diam.)
(3e). White crystals (38Á5%); mp 188ꢀC; IR (KBr)
n: 3500 (OH), 2500±3100 (NH2‡), 1550 cm
1
1
(CO); H NMR (DMSO) d: 2Á47 (dd, J ˆ 16 and
attached to
a
motor with
a
speed of
1
7 Hz, H-2a), 2Á77 (dd, J ˆ 16 and 7 Hz, H-2b), 4Á26
(t, J ˆ 7 Hz, H-3), 7Á11 (d, J ˆ 5 Hz, H-thienyl),
7Á31 (s, H-thienyl), 7Á42 (d, J ˆ 5 Hz, H-thienyl).
15 rotations min . Only those animals that were
capable of remaining on the revolving rod for at
least 1 min were used. Drugs were administered

NEW 3-ARYL-3-HYDROXYAMINOPROPIONIC ACIDS
Table 1. Pharmacological effects of compounds 3a±f.
241
30 min before the test. Mice were placed on the
rotating rod for 3 min, and the number of mice
falling off during this time was counted.
Compound
(LD50
Rotarod testa
(ED50
Actimeterb
(ED50
Traction testc
(ED50
1
)
1
1
1
)
mg kg
mg kg
)
mg kg
)
mg kg
Staircase test (Simiand et al 1984)
3a (>800)
3b (800)
3c (>800)
3e (>300)
3f (>400)
70
ND
60
50
ND
56Á3
ND
70
ND
60
50
ND
The experimental apparatus was similar to that
described by Simiand et al (1984). It consisted of
®ve steps (2Á5 cm high, 10 cm wide, 7Á5 cm deep)
surrounded by a 12Á5-cm wall. Drugs were admi-
nistered 30 min before the test. Mice were placed
singly on the ¯oor of the box, facing away from the
steps. The number of rears and the number of steps
climbed were measured; a step was considered
climbed only if the animal placed its four paws on
it. The duration of the test was 3 min.
23Á2
25Á3
115Á13
a
ED50 is de®ned as the dose causing 50% of mice to fall
b
from the rotarod; the dose that induced a 50% reduction in
locomotor activity compared with control; cthe dose that
induced the loss of catching re¯ex in 50% of mice. ND, not
determined.
hydroxylamine generated in-situ from hydro-
xylammonium chloride in presence of sodium
ethylate in hot ethanol. The reaction mixture
exhibited ®rst a thin precipitate, that was isolated
and identi®ed as hydroxylammonium acrylates 6a±
f. These salts were then converted, by maintaining
the ethanolic re¯ux for 2 h, into the expected
hydroxylamino acids 3a±f, which remained in
solution. The reaction was quenched by removing
the solvent, before reduction of 3a±f into the
aminoacids 2a±f. The products were isolated from
the oily residue by successive fractional crystal-
lizations from various solvents.
Grip strength (Boissier & Simon 1960)
The test was used to evaluate muscular strength or
neuromuscular function. Drugs were administered
30 min before the test. In a preliminary experiment
mice were tested for normal reactivity. They were
exposed to a horizontal thin thread or metallic wire
suspended approximately 30 cm into the air which
they immediately gripped with the forepaws. The
mice were then released to hang on with their
forelimbs. Normal animals catch the threat with the
hind limbs and climb up within 5 s. If they are not
able to touch the threat with the hind limbs within
5 s or fall off, they are considered to be impaired.
The percentage of animals loosing the catching
re¯ex was calculated.
Pharmacology
Results are given in Table 1. All the compounds
tested presented a pro®le of central depressants at
subtoxic doses, the major symptoms being
hypoactivity, myorelaxation and ataxy. The com-
pounds exhibited a low acute toxicity (LD50,
Results and Discussion
1
>300 mg kg , i.p).
Chemistry
The depressant central effect was con®rmed by
the actimetry test. All compounds tested induced
Posner's reaction, involved in the synthesis of the
title compounds, was applied to various acrylic
acids 5a±f (Figure 2). The latter were treated by
Table 2. Effects of 3c on the spontaneous locomotor activity
in the photocell-activity cage test.
1
)
Compound
Dose (mg kg
Photobeam
interruption (%)
3c
6Á25
12Á5
25
83Á33Æ 14Á58
61Á46Æ 18Á75
51Á04Æ 21Á87
27Á08Æ 22Á92*
9Á37Æ 20Á83**
18Á75Æ 16Á67***
28Á12Æ 26Á04**
50
100
200
20
Flunitrazepam
Results are expressed as percent photobeam interruptions
compared with control (100%). *P < 0Á05, **P < 0Á01,
***P < 0Á001 (analysis of variance, followed by Student's t-
test, using the method of Bonferroni).
Figure 2. Reagents: i. EtONa, NH2OH, HCl=EtOH

242
BERTRAND COUTURIER ET AL
Simiand et al (1984) non-anxiolytic substances do
not dissociate these behavioural patterns. Anxioly-
tics reduced rearing behaviour at doses which
produced no signi®cant reduction of, or increase in
the number of steps climbed.
This preliminary screening indicates the central
depressant activity of the 3-aryl-3-hydroxy-
aminopropionic acids. The results of the staircase
test seem to suggest that these compounds do not
have anxiolytic properties. Recently, Abbenante et
al (1997) found that compound 3a did not have
GABAB receptor antagonist activity at the guinea-
pig ileum. Further studies will be necessary to
characterize the mechanism of sedative activity of
these compounds, presumably in relation with
GABA interaction.
References
Abbenante, G., Hughes, R., Prager, R. (1997) Potential
GABAB receptor antagonists IX. Aust. J. Chem. 50: 523±
527
Boissier, J. R., Simon, P. (1960) L'utilisation du test de la
Â
traction, (test de Julou-Courvoisier) pour l'etude des psy-
Â
choleptiques. Therapie 15: 1170±1174
Boissier, J. R., Simon, P. (1965) Action de la Cafeine sur la
Â
motilite spontanee de la souris. Arch. Int. Pharmacodyn.
Â
158: 212±221
Â
Dallemagne, P., Rault, S., Cugnon de Sevricourt, M., Hassan,
M., Robba, M. (1986) Cyclisation de l'acide amino-3
Figure 3. Effects of 3a (A), 3b (B) and 3c (C) on the number
of steps climbed (j) and the number of rears (r) in the
staircase test in a 3-min period. Results are expressed as
percent change from controls. *P < 0Á05, **P < 0Á01,
***P < 0Á001 (analysis of variance, followed by Student's t-
test, using the method of Bonferroni).
Â
Â
(thienyl-3)-3 propionique en aminocyclopenta[b]thiophenes.
Tetrahedron Lett. 27: 2607±2610
Dallemagne, P., Rault, S., Gordaliza, M., Robba, M. (1987) A
convenient route to 6-aminocyclopenta[b]thiophene-6-one.
Heterocycles 26: 3233±3237
Dallemagne, P., Rault, S., Fabis, F., Dumoulin, H., Robba, M.
(1994) First Synthesis of 4-amino-4,5-dihydro-1-phenylcy-
clopenta[b]pyrrol-6(1H)-ones. Heterocyclic. Comm. 1:
1605±1608
Dunham, N., Miya, T. (1957) A note on a simple apparatus for
detecting neurological de®cit in rats and mice. J. Am.
Pharmaceut. Assoc. 46: 208±210
Morpugo, C. (1971) A new design for the screening of CNS-
active drugs in mice. Arzneim. Forsch. (Drug Res.) 21:
1727±1734
Posner, T. (1903) Zur Kenntniss der b-aminosauren. Ber.
Dtsch. Chem. Ges. 36: 4306
reduced locomotor activity (ED50 between 23Á2
1
and 115Á17 mg kg ). This effect is dose dependent
as show for compound 3c (Table 2).
The myorelaxation activity was con®rmed by the
traction test. However this effect appeared at doses
which induce high neurotoxocity that impairs
motor function.
Figure 3 shows the effects of 3a±c in the stair-
case test. All compounds signi®cantly decreased
both the number of rears and the number of steps
climbed, presumably due to the sedative and mus-
cle relaxant effects. The compounds did not dis-
sociate the two behavioural patterns. According to
Á
Rault, S., Dallemagne, P., Robba, M. (1987) Une synthese
simple des premieres amino-3 indanones-1. Bull. Soc. Chim.
Á
Fr. 6: 1079±1083
Simiand, J., Keane, P., Morre, M. (1984) The staircase test in
mice: a simple and ef®cient procedure for primary screening
of anxiolytic agents. Psychopharmacology 84: 48±53