Hydroxy-1,2,5-oxadiazolyl moiety as bioisoster of the carboxy function. Synthesis, ionization constants, and pharmacological characterization of γ-aminobutyric acid (GABA) related compounds

Article abstract of DOI:10.1021/jm051288b

Three 4-substituted 1,2,5-oxadiazol-3-ols containing aminoalkyl substituents (analogues and homologues of γ-aminobutyric acid (GABA)) were synthesized to investigate the hydroxy-1,2,5-oxadiazolyl moiety as a bioisoster for a carboxyl group at GABA recepto

Full text of DOI:10.1021/jm051288b

4442
J. Med. Chem. 2006, 49, 4442-4446
Hydroxy-1,2,5-oxadiazolyl Moiety as Bioisoster of the Carboxy Function. Synthesis, Ionization
Constants, and Pharmacological Characterization of γ-Aminobutyric Acid (GABA) Related
Compounds
Marco L. Lolli,† Suzanne L. Hansen,‡ Barbara Rolando,† Birgitte Nielsen,§ Petrine Wellendorph,§ Karsten Madsen,‡
Orla Miller Larsen,‡ Uffe Kristiansen,‡ Roberta Fruttero,† Alberto Gasco,*,† and Tommy N. Johansen*,§
Dipartimento di Scienza e Tecnologia del Farmaco, UniVersita` degli Studi di Torino, Via Pietro Giuria 9, 10125 Torino, Italy, and
Departments of Pharmacology and Medicinal Chemistry, The Danish UniVersity of Pharmaceutical Sciences, 2 UniVersitetsparken,
DK-2100 Copenhagen, Denmark
ReceiVed December 28, 2005
Three 4-substituted 1,2,5-oxadiazol-3-ols containing aminoalkyl substituents (analogues and homologues
of γ-aminobutyric acid (GABA)) were synthesized to investigate the hydroxy-1,2,5-oxadiazolyl moiety as
a bioisoster for a carboxyl group at GABA receptors. The pKa values of the target compounds were close
to those of GABA. At GABAA receptors of cultured cerebral cortical neurons, weak agonist and partial
agonist profiles were identified, demonstrating the 4-hydroxy-1,2,5-oxadiazol-3-yl unit to be a nonclassical
carboxyl group bioisoster.
Introduction
Isosteric replacement of functional groups in a lead compound
is a widely used approach to study receptor chemistry and to
develop new drugs with optimized behavior.1 When this
replacement affords products with broadly similar biological
properties, the groups are called bioisosters.2,3 A number of clear
bioisosteric relationships have been established for many
functional groups, in particular for the carboxyl group, which
successfully has been substituted by heterocycles such as
tetrazole, 3-hydroxyisoxazole, 3-hydroxyisothiazole, 3-hydroxy-
1,2,5-thiadiazole, and 3-cyclobutene-1,2-dione. These cyclic
systems have been used extensively to design amino acid
mimetics active at subtypes of central nervous system (CNS)
receptors.4-7 The 1,2,5-oxadiazole (furazan) system and its
Figure 1. Structures of the GABAA receptor agonists muscimol (1),
THIP (2), and 4-PIOL (4), the GABAA antagonist gabazine (3), and
the new 1,2,5-oxadiazol-4-ols (5-7).
2-oxide (furoxan) are heterocyclic rings whose pharmacochem-
istry some of us have been studying for many years.8 The former
is a classical isoster of the 1,2,5-thiadiazole ring. Similar to the
hydroxy-substituted 1,2,5-thiadiazoles, the hydroxy-1,2,5-oxa-
(3), have been developed over the years11 (Figure 1). THIP,
which has a particular partial agonist profile at cloned GABAA
diazoles are known to display marked acidic properties.9
Consequently, the 4-hydroxy-1,2,5-oxadiazol-3-yl moiety could
receptors, is currently undergoing phase III clinical investigation
reasonably behave as the bioisoster of the carboxy function. In
for treatment of sleep disorders.12 Whereas THIP shows very
this first paper we report the results of a work devoted to obtain
potent nonopioid analgesic effects and novel hypnotic effects
in human clinical studies, it seems likely that partial GABAA
potential biomimetics of the γ-aminobutyric acid (GABA), the
major inhibitory neurotransmitter in CNS.
In GABA neurotransmission, synaptically released GABA
exerts its effects through activation of ionotropic GABAA and
agonists showing lower levels of efficacy such as 5-(4-
piperidyl)-3-isoxazolol (4-PIOL, 4)13 may be of therapeutic
interest in certain CNS disorders such as schizophrenia.14
metabotropic GABAB receptors. After unbinding from the
Information about the mechanism of receptor-ligand interac-
tions resulting in partial agonism at the molecular level is still
not available, thus making the design of new GABAA agonists
receptor, GABA is taken up by GABA transporters of which
four subtypes have been cloned (GAT1-4).10 To pharmacologi-
cally characterize the GABAA receptors, a number of ligands
with a range of different efficacies relevant.
bioisosterically derived from GABA, such as the selective
In this paper we report the synthesis, the ionization constants,
agonists muscimol (1) and 4,5,6,7-tetrahydroisoxazolo[5,4-c]-
and the pharmacological characterization at GABA receptors
and GABA transporters of new analogues and homologues of
pyridine-3-ol (THIP, gaboxadol, 2) and the antagonist gabazine
GABA, 5-7 (Figure 1), in which the carboxyl group has been
* To whom correspondence should be addressed. For A.G.: phone, 0039
replaced by a 4-hydroxy-1,2,5-oxadiazol-3-yl moiety.
011 6707670; fax, 0039 011 6707286; e-mail, alberto.gasco@unito.it. For
T.N.J.: phone, +45 35306412; fax, +45 35306040; e-mail: tnj@dfuni.dk.
Results and Discussion
† Universita` degli Studi di Torino.
‡ Department of Pharmacology, The Danish University of Pharmaceutical
Chemistry. The synthetic pathway for preparing the final
products 6 and 7 is depicted in Scheme 1. The common starting
material was the 3,4-diphenylsulfonyl-1,2,5-oxadiazole 8. By
Sciences.
§ Department of Medicinal Chemistry, The Danish University of
Pharmaceutical Sciences.
10.1021/jm051288b CCC: $33.50 © 2006 American Chemical Society
Published on Web 06/21/2006

Brief Articles
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4443
Scheme 1. Synthetic Strategy for the Preparation of the GABA-Related Compounds 5-7 and the Reference Compound 23
Table 1. pKa Ionization Constants
action on this product of the Normant reagents 9 and 1015 in
compd
pKa1
pKa2
THF solution, alcohols 11 and 12 were obtained. These
substances were treated with a mixture of triphenylphosphine
(Ph3P) and diisopropyl azodicarboxylate (DIAD) in the presence
of phthalimide (Mitsunobu reaction) to give the related phthal-
imido derivatives 13 and 14. These products were converted
into analogues that are the Boc-protected derivatives 15 and
16, which were purified by flash chromatography. These
intermediates were transformed into the final hydrochlorides
of 6 and 7 by treatment with NaOH in DMSO and then with
HCl in ethyl acetate (EtOAc). For the synthesis of the strict
GABA analogue 5, a different synthetic procedure was explored
(Scheme 1). The furazan 8 was treated in THF with EtOAc in
the presence of lithium diisopropylamide (LDA) at low tem-
perature to give the acetate 17. The product results from the
nucleophilic displacement of one of the two phenylsulfonyl
groups of 8 under the action of the carbanion generated by action
of LDA on EtOAc. Reduction of 17 with NaBH4 in ethanol
afforded the related alcohol 18 that was transformed into the
corresponding bromide 19, for action of Ph3P and N-bromo-
succinimide (NBS). The reaction of 19 with tert-butyldimeth-
ylsilylphthalimide (TBDMSP) in the presence of tetrabutylam-
monium fluoride (TBAF) produced the expected phthalimido
derivative 20. This product was transformed into the final
GABA analogue 5, through the intermediate 21, following the
same procedure used to prepare 6 and 7 from 13 and 14,
respectively. The 4-propyl-1,2,5-oxadiazol-3-ol (23), used as a
reference in the ionization constant studies, was prepared as
reported in Scheme 1. The action of propylmagnesium chloride
on 8 gave the intermediate 1,2,5-oxadiazole derivative 22 that,
by action of NaOH in DMSO solution, afforded the expected
final compound 23.
23a
5b
3.77
3.12
3.37
3.56
4.04
9.28
9.87
10.22
10.71
6b
7b
GABA
a Titrations were performed using methanol as cosolvent (10-40 wt %).
pKa value was determined by extrapolation to zero content of cosolvent
according to the Yasuda-Shedlovsky procedure.16 SD ) 0.03. b Determined
by aqueous titration. SD e 0.02.
For neutral amino acidic compounds such as GABA and 5-7
the distribution across of the blood-brain barrier is expected
to be closely related to the I/U ratio of the compounds (the ratio
between the concentration of the ionized and the un-ionized
molecules), which is a function of the difference between the
individual compound’s two pKa values. On the basis of the
ionization constants (Table 1), the I/U ratio of GABA, which
does not cross the blood-brain barrier, is estimated to be 0.8
× 106. The I/U ratios of 5-7 are at least as high as that of
GABA, and these compounds are consequently not expected
to cross the blood-brain barrier. Therefore, only an in vitro
pharmacological characterization has been carried out.
In Vitro Pharmacology. Compounds 5-7 were characterized
in receptor binding studies using rat brain membrane prepara-
tions and electrophysiologically at GABAA receptors. Further-
more, the compounds were evaluated in GABA uptake studies
using cultured neurons and astrocytes and cloned GABA
transporters from mice (mGAT1-4) expressed in HEK-293
cells.
The affinities of the new compounds for GABAA and
GABAB receptor sites were determined using [3H]muscimol and
[3H]GABA in the presence of the selective GABAA receptor
agonist isoguvacine,18 respectively (Table 2). In [3H]muscimol
binding, 5 and 6 showed affinities more than a 100-fold lower
than those of GABA and THIP (3) but in the same range as
that of 4-PIOL (4), whereas the IC50 of 7 was found to be greater
than 100 µM. In the GABAB binding assay, 5 was the most
potent displacer of [3H]GABA binding (IC50 ) 2.0 µM) whereas
6 was 10-fold weaker and 7 appeared to be inactive. At 1 mM,
none of the new compounds, 5-7, were able to inhibit GABA
uptake in assays using neurons, astrocytes, or HEK293 cells
expressing the cloned transporters mGAT1-4.
Ionization Constants. The pKa ionization constants for the
three final compounds 5-7 and for the reference compound 23
were obtained by potentiometric titration using a GLpKa
apparatus. The pKa values are in Table 1 with those of GABA.17
Except for 23, all of the products show two dissociation
constants in the ranges 3.12-3.56 and 9.28-10.22 in accordance
with their chemical structure. The pKa obtained for 23 clearly
indicates that the lowest pKa of each product is related to the
dissociation of the acidic -OH function, while the highest is
related to the presence of the basic -NH2 function. Although
the observed ionization constants of 5-7 are slightly lower than
the corresponding constants of GABA, the three compounds
(similar to GABA) exist predominantly as zwitterions at
physiological pH.
The functional properties of 5-7 at GABAA receptors in
primary cultures of cerebral cortical neurons were investigated
using standard patch-clamp techniques. Because of extensive

4444 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
Brief Articles
Figure 2. Representative current traces from two different neurons comparing the responses evoked by 3 mM of each of the compounds 5-7 with
a close to maximum reponse of GABA (0.6 mM). Each compound was applied for approximately 5 s as shown by the thick bar above each trace.
The neurons have been chosen so that the amplitudes of the GABA responses are nearly identical, thereby making the traces for 5-7 directly
comparable.
Table 2. Affinities for GABA Receptor Sitesa
[3H]muscimol binding
[3H]GABA binding
compd
GABA
3 (THIP)
Ki (µM)b
IC50 (µM)b
0.049 (0.043, 0.056)
0.16c
0.013 (0.011, 0.014)
nde
4 (4-PIOL)
9.1 (8.2, 10)d
13 (11, 16)
27 (25, 30)
>100
>100d
5
6
7
2.0 (1.5, 2.6)
25 (19, 33)
>100
a Standard receptor binding on rat brain synaptic membranes, n ) 3.
b Mean and SEM were calculated assuming a normal distribution of the
logarithm of the IC50 and Ki values. Hence, numbers in parentheses (min,
max) indicate the antilog of the log of the mean ( SEM of IC50 and Ki.
c Data from ref 19. d Data from ref 4. e nd: not determined.
desensitization of GABAA receptors in receptor binding assays,
resulting in an increased apparent affinity,20 a marked discrep-
ancy between affinity in [3H]muscimol binding and the agonist
activity is normally seen for agonists and highly efficacious
partial agonists, whereas for antagonists and low efficacious
partial agonists, the affinity and the potency are in good
agreement. On the basis of this, it was decided to include 7 in
these studies despite the low affinity, primarily to look for
possible antagonist or partial agonist activities. Figure 2 shows
representative current traces from 5-7 obtained from two
different neurons and suggests that the compounds possess
agonist properties at the GABAA receptor. The currents were
evoked by high concentrations of each compound, i.e., 3 mM,
and compared to a close to maximal response of GABA (600
µM). The average peak currents induced by the compounds
relative to GABA were 40% ( 9% (N ) 7) for 5, 14% ( 2.6%
(N ) 7) for 6, and 0.8% ( 0.17% (N ) 3) for 7.
To further elucidate whether the currents elicited by 5-7 were
GABAA receptor mediated, antagonist experiments with gaba-
zine were conducted. Cells were pretreated with gabazine (100
µM) for 15 s followed by application of a mixture of gabazine
(100 µM) and 5, 6, or 7 (3 mM). Gabazine blocked the responses
elicited by 5, 6, or 7 nearly completely (5, 99.5% ( 0.5%, N )
7; 6, 100%, N ) 7; 7, 97% ( 3%, N ) 4). The close to complete
inhibition by the specific GABAA antagonist demonstrates that
the currents evoked by each of 5-7 are GABAA receptor
mediated.
Figure 3. Concentration response curves for 5-7. Currents are
normalized to maximum GABA response (refer to Supporting Informa-
tion for details). Data are presented as the mean ( SE from seven to
nine neurons for 5-7. For GABA and 4-PIOL the solid lines represent
the best fit to the Hill-type function shown in the Supporting
Information.
The agonist profiles of 5-7 were further characterized in
concentration-response experiments (Figure 3) in which con-
centration-response curves for GABA and 4-PIOL based on
previously reported data21 have been included for comparison.
Although exact estimates for potencies and efficacies of 5-7
cannot be extracted because maximum responses have not been
reached, 5 and 6 clearly possess efficacies greater than that of
the partial agonist 4-PIOL (Emax ≈ 5% 21) in cortical neurons,
whereas 4-PIOL is more efficacious than 7. In terms of
potencies, all three compounds 5-7 turned out to be less potent
than 4-PIOL (EC50 ) 139 µM 21).
To investigate whether 5-7 possess partial agonist properties,
the interactions between the full GABAA receptor agonist
isoguvacine at 20 µM (corresponding to E20) and 5-7 were
studied (Figure 4). In this type of experiment, a partial agonist
with a low efficacy is expected to competitively displace a full
agonist from the receptor, and at increasing concentration of
partial agonist the combined response will approach the
maximum response of the partial agonist alone. Compounds 5

Brief Articles
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4445
results demonstrate that the 4-hydroxy-1,2,5-oxadiazol-3-yl unit
can be used as a nonclassical carboxyl group bioisoster. The
use of 4-hydroxy-1,2,5-oxadiazol-3-yl in other receptor systems
in the design of new research tools with unique pharmacological
properties and potential drug candidates will be exploited further.
Experimental Section
General Procedure for the Synthesis of the Target Com-
pounds 5-7. A solution of appropriate Boc-protected derivatives
15, 16, and 21 (2.83 mmol) and 5 M NaOH (5 mL, 25 mmol) in
DMSO (8 mL) was heated at 70 °C for 2 h. The mixture was cooled
at room temperature and then diluted with water (8 mL) and washed
with ethyl ether (10 mL). The pH of the solution was adjusted to
3.6 by adding 6 M HCl, obtaining a milky mixture. The mixture
was extracted with diethyl ether (4 × 15 mL), maintaining the pH
of the aqueous layer constant by adding 6 M HCl. The collected
organic layers were washed with water (8 mL), dried, and
concentrated under reduced pressure, obtaining a white solid crude
material. The crude product was dissolved in dry EtOAc (4 mL),
and the resulting solution was mixed with a solution of HCl in dry
EtOAc (1.3 M, 10 mL). The white precipitate was filtered and
washed with dry EtOAc (2 mL) to afford the title compounds as
hydrochlorides.
4-(2-Aminoethyl)-1,2,5-oxadiazol-3-ol Hydrochloride (5). Yield
54%; mp 161-164 °C. 1H NMR (D2O) δ 3.00/3.33 (2H, t, J ) 6.8
Hz; -CH2NH2)/(2H, t, J ) 6.8 Hz, furazanCH2-). 13C NMR (D2O)
δ 20.7, 39.8, 145.7, 163.1. Anal. (C4H7N3O2‚HCl) C, H, N.
4-(3-Aminopropyl)-1,2,5-oxadiazol-3-ol Hydrochloride (6).
Yield 73%; mp 177-179 °C. 1H NMR (D2O) δ 2.00 (2H, m, J )
7.6 Hz, -CH2CH2CH2-), 2.70 (2H, t, J ) 7.4 Hz, furazanCH2-),
2.99 (2H, t, J ) 7.7 Hz, -CH2NH2). 13C NMR (D2O) δ 19.4, 24.1,
40.0, 148.0, 163.0. Anal. (C5H9N3O2‚HCl) C, H, N.
4-(4-Aminobutyl)-1,2,5-oxadiazol-3-ol Hydrochloride (7). Yield
68%; mp 176-178 °C. 1H NMR (D2O) δ 1.5-1.7 (4H, m,
-CH2CH2CH2CH2-), 2.58 (2H, t, J ) 7.0 Hz, furazanCH2), 2.86
(2H, t, J ) 7.0 Hz, -CH2NH2). 13C NMR (D2O) δ 21.5, 23.1,
26.4, 39.4, 148.8, 163.1. Anal. (C6H11N3O2‚HCl) C, H, N.
4-Propyl-1,2,5-oxadiazol-3-ol (23). A 5 M NaOH (5 mL, 25
mmol) sample was added to a stirred solution of 22 (1 g, 3.98 mmol)
in DMSO (8 mL). The mixture was heated at 70 °C for 1 h and
then allowed to reach room temperature. The reaction mixture was
diluted with water (8 mL), washed with ether (3 × 10 mL), and
acidified to pH 3.6 by adding 6 M HCl. The milky mixture was
extracted with ether (2 × 10 mL). The combined organic layers
were washed with water (8 mL), dried, and evaporated in vacuo to
give the title compound as a pale-yellow prismatic solid. Yield
Figure 4. Concentration-interaction plots for 5, 6, and 7 in panels
A, B, and C, respectively. Currents are normalized to the current induced
by 20 µM isoguvacine (IGU). Data are presented as the mean ( SE
from nine neurons for each compound. The concentrations are given
below panel C. Note the different y-axis scale for each compound.
and 6 concentration-dependently increased the currents elicited
by isoguvacine (parts A and B of Figure 4). This increased
response could be explained by a modulating effect mediated
by binding of 5 or 6 to distinct (allosteric) binding sites.
However, on the basis of the results shown in Figure 3 and the
sensitivity to gabazine, it is possible that 5 and 6 are GABAA
receptor partial agonists with maximum responses larger than
E20 of isoguvacine and GABA. The results obtained from the
experiments with 7 were more clear (Figure 4C). This compound
concentration-dependently inhibited the isoguvacine-induced
currents (IC50 ) 740 µM estimated by linear regression),
demonstrating that 7 is a partial GABAA receptor agonist mainly
possessing antagonist properties.
1
92%; mp 34.8-35.5 °C. MS (CI) m/z 129 (M + 1)+. H NMR
(CDCl3) δ 1.02 (3H, t, J ) 7.4 Hz, -CH2CH3), 1.79 (2H, se,
J ) 7.4 Hz, -CH2CH3), 2.70 (2H, t, J ) 7.4 Hz, furazanCH2). 13
C
NMR (CDCl3) δ 13.6, 20.1, 24.2, 148.2, 162.7. Anal. (C5H8N2O2)
C, H, N.
Acknowledgment. This work was supported by MIUR
(Grant COFIN 2003) and the Danish MRC (Grant 22-01-0291
to U.K.).
Supporting Information Available: Synthetic procedures for
the preparation of the intermediates, experimental details used for
receptor binding assays, uptake assays, in vitro electrophysiology,
table of elemental analysis results of intermediates and final
compounds. This material is available free of charge via the Internet
at http://pubs.acs.org.
Conclusion
In the present study we have shown the 4-hydroxy-1,2,5-
oxadiazol-3-yl moiety, when incorporated in GABA-related
compounds, to have protolytical properties slightly more acidic
than those of the carboxyl group of GABA. The GABA
analogues and homologues 5-7 all behaved as agonists with
lower potencies than those of GABA and 4-PIOL and displayed
a range of efficacy levels in the rank order 7 < 4-PIOL < 6 <
5 < GABA. Compound 7 behaved as a true low-efficacy partial
agonist and inhibited the currents induced by isoguvacine. A
similar conclusion could not be disclosed for 5 or 6, which may
be binding to distinct sites from the GABA binding site. The
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JM051288B