Cycloalkyl indole-2-carboxylates as useful tools for mapping the 'North- Eastern' region of the glycine binding site associated with the NMDA receptor

Article abstract of DOI:10.1002/(SICI)1521-4184(19993)332:3<73::AID-ARDP73>3.0.CO;2-5

A novel series of indole-2-carboxylate analogues of GV150526(1) in which the terminal phenyl ring belonging to the side chain present in the position C-3 has been replaced with a bridged cycloalkyl group was synthesized and evaluated for its pharmacologic

Full text of DOI:10.1002/(SICI)1521-4184(19993)332:3<73::AID-ARDP73>3.0.CO;2-5

Full Papers
Cycloalkyl Indole-2-Carboxylates as Useful Tools for Mapping the
“North-Eastern” Region of the Glycine Binding Site Associated with
the NMDA Receptor
Fabrizio Micheli*, Romano Di Fabio*, Anna M. Capelli, Alfredo Cugola, Ornella Curcuruto, Aldo Feriani, Paola Gastaldi,
Giovanni Gaviraghi, Carla Marchioro, Alessandra Orlandi, Alfonso Pozzan, Anna M. Quaglia, Angelo Reggiani,
Frank van Amsterdam
Glaxo Wellcome S.p.A., Medicines Research Centre, Via Fleming 4, 37100 Verona, Italy
Key Words: Neuroprotective agents; neurotransmitters; molecular modelling/mechanics
Summary
A novel series of indole-2-carboxylate analogues of GV150526(1)
in which the terminal phenyl ring belonging to the side chain
present in the position C-3 has been replaced with a bridged
cycloalkyl group was synthesized and evaluated for its pharma-
cological profile. Modelling studies on this class of novel glycine
antagonist allowed us to identify an asymmetric lipophilic pocket
present in the “North-Eastern” region of the pharmacophoric
Figure 1
modelof the glycine binding site associated to theNMDA receptor.
Among the derivatives prepared, 3-[2-(1-adamantylaminocar-
bonyl)ethenyl]-4,6-dichloroindole-2-carboxylic acid 6b and 3-[2-
(norbornylaminocarbonyl)ethenyl]-4,6-dichloroindole-2-carbox-
The indole-2-carboxylate derivative GV150526 1 (Fig-
[22–27]
ure 1) was identified by GlaxoWellcome
as a potent
ylic acid 6l were found to be antagonists acting at the strychnine-
insensitive glycine binding site, showing nanomolar affinity for
the glycine binding site (Ki = 63 and 19 nM, respectively), coupled
with high glutamate receptor selectivity (IC50 >10–5 M at the
NMDA, AMPA, KA binding sites) and high in vivo potency after
systemic administration by inhibition of convulsion induced by
NMDA in mice.
and selective glycine antagonist endowed with nanomolar
affinity in vitro and excellent in vivo activity in the MCAo
model in rats both pre and post-ischemia. The present paper
deals with the synthesis and the pharmacological charac-
terization of a novel series of analogues of compound 1, in
which the aromatic phenylamido moiety was replaced with
various cycloalkyl derivatives. This new series of indole-2-
carboxylates showed nanomolar affinity for the glycine bind-
ing site coupled with high receptor selectivity; moreover,
these molecules are endowed with a high in vivo potency in
the NMDA-induced convulsion model in mice, after systemic
administration.
Introduction
Glutamate is the most abundant excitatory neurotransmitter
present in the CNS. It is now widely recognized that in
[1–4]
pathological conditions
such as stroke, an abnormal
Results and Discussion
amount of glutamate is released into synaptic clefts. This is
due to an increase of release of glutamate presynaptically and
to the block of the re-uptake processes. This leads to the
over-stimulation of N-methyl-D-aspartate (NMDA) receptor
Synthesis
Compounds of general structure 6 have been prepared
(Scheme 1) starting from the indole-2-carboxylate 2 as pre-
++
raising significantly the intracellular level of Ca in the
post-synaptic neurons, causing the activation of several
[22]
viously reported . Chemoselective hydrolysis of the tert-
neurotoxic cascades responsible for irreversible neuronal
butyl ester protecting group in quantitative yield wasobtained
using formic acid at room temperature. The key intermediate
3 was transformed into the corresponding alkylamido deriva-
tives by activation of the carboxyl group via the formation of
[5]
damage
.
Based on the considerable body of experience gained in
animal models of stroke, therapeutic benefit can be obtained
++
by modulating the conductance of Ca through the ion
the corresponding 2-pyridyl thioester 4 using the well known
[6]
channel associated with the NMDA receptor using com-
[36]
“oxidation-reduction” procedure
in the presence of 2,2′-
[7,8]
petitive and non competitive NMDA antagonists
. Re-
dipyridyldisulphide and triphenylphosphine. The derivative
4 was found to be stable enough to be purified by standard
chromatographic methods and it was reacted with the desired
alkyl amines. Alternatively, a “one-pot” procedure was used:
the thiopyridyl ester was formed “in situ” and treated with the
desired amines. Amides 5 were smoothly obtained in high
cently, the glycine binding site has become one of the most
attractive targets for neuroprotection after stroke, in view of
[9–12]
the role of glycine as a co-agonist of glutamate
in the
activation of this ionotropic receptor complex and the favor-
[13–35]
able therapeutic index seen for glycine-antagonists
.
Arch. Pharm. Pharm. Med. Chem.
© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999
0365-6233/99/0303/0073 $17.50 +.50/0

74
Micheli, Di Fabio, and co-workers
[22]
yield following both the procedures. Finally, the basic hy-
In the paper
dealing with the discovery process of
drolysis of the ethyl ester present in position C-2 gave the GV150526 1, it was proved how the presence of the amidic
target compounds 6 in quantitative yields.
carbonyl group belonging to the α,β-unsaturated C-3 side
chain, in view of the suitable stereoelectronical features, was
crucial to maximize the affinity at the glycine binding site of
this series of ligands. Therefore, we decided to maintain this
key “pharmacophoric point” and to map in detail theso-called
“size-limited hydrophilic pocket” in order to gather informa-
tion both in terms of allowed space to the terminal substit-
uents on the C-3 side chain and on the recognition role of the
aromatic moiety. This exploration should allow us to under-
stand the structural requirements useful to design novel series
of glycine antagonists.
Biology
The biological evaluation of the new chemical entities
(NCE) was performed using the following screening se-
[22]:
1)
quence previously described
a) binding assay to evalu-
ate the affinity for the glycine site ; b) selectivity for the
glutamate receptors (NMDA/AMPA/KA); c) in vivo anticon-
vulsant activity in the NMDA induced convulsions model in
2)
mice (iv and po) .
To perform this task, the terminal aromatic ring was re-
placed initially with the corresponding cyclohexyl derivative.
As reported in Table 1, compound 6a showed only a slight
decrease in affinity at the glycine binding site associated with
Analysis of Experimental Findings
After the identification of GV150526 and the exploration
of the aromatic phenylamidic moiety present in the terminal
position of the C-3 side chain, our objective was to acquire
further SAR elements regarding the “North-Eastern” region
of the receptor, enhnacing the precision of the 3D pharmaco-
phoric model of the glycine binding site.
the NMDA-receptor with respect to compound 1 (K = 10 nM
i
vs. K = 3 nM, respectively). In view of this result, it was
i
realized that the “North-Eastern” region of the pharmaco-
phore was worth being further analyzed. A first series of
symmetric derivatives (6b–6f) bearing alkyl substituents with
increasing steric bulk with respect to cyclohexyl derivative
6a was synthesized and evaluated in terms of in vitro affinity
at the glycine binding site. The results obtained are shown in
Table 1. All these compounds were endowed with a reduced
affinity for the glycine binding site with respect to both
compounds 1 and 6a, confirming the limited space available
———
1)
Ki values for the products 6a–o were measured from at least six-point
inhibition curves and they are the geometric means of at least three inde-
pendent experiments. The standard error of the mean was less than 0.05 [22]
.
2) The research complied with national legislation and with company policy
on the Care and Use of Animals and with related codes of practice.
Arch. Pharm. Pharm. Med. Chem. 332, 73–80 (1999)

Mapping of Glycine Binding Site
75
within this region of the receptor. In particular, both the is worth marking as the norbornyl derivative 6i, despite the
higher steric bulk, showed an affinity comparable to the
cyclohexyl derivative 6a. This result was explained by hy-
pothesizing the presence of an asymmetric pocket of limited
size able to accept lipophilic substituents with a defined
adamantyl derivative 6b and its homologated derivative 6c
showed a significant decrease in terms of affinity (K = 63 and
i
200 nM, respectively vs. 10 nM for 6a). Conversely, a slight
reduction of the steric bulk (from adamantyl 6b to nor-
adamantyl 6d) caused a partial improvement of the affinity
(63 vs. 25 nM, respectively). Finally the same affinity at the
glycine binding site was observed for derivatives 6e and 6f
3)
molecular geometry .
Computational Chemistry Studies
(K = 31 nM): in view of this last result, the terminal alkyl
substituents should lie in the same “allowed area” of the
receptor.
After this preliminary exploration, a second series of un-
symmetrical cycloalkyl substituents (6g–6o) were carefully
chosen and synthesized to map this region of the receptor in
more detail. The affinity for the glycine binding site of this
subclass of derivatives is reported in Table 1. In particular, it
i
Based on the results described above, the different com-
pounds prepared were used as tools to map the “North-East-
ern” region of the pharmacophore model of the glycine
binding site. Modelling studies were performed using both
———
3) It is worth noting that the introduction of a basic heteroatom into the the
terminal lipophilic moiety seemed to be forbidden (derivative 6g, Ki = 1 µM).
Arch. Pharm. Pharm. Med. Chem. 332, 73–80 (1999)

76
Sybyl (TRIPOS Ass.) and Catalyst software (the methods
used for these analyses are described in footnote and ,
respectively).
Micheli, Di Fabio, and co-workers
[37]
4)
5)
“Excluded Volumes” Analysis
Indole derivatives 6a–o were arbitrarily divided into two
groups: the so called “higher affinity compounds” (K ≤
i
10 nM) and the so called “lower affinity molecules” (K ≥
i
100 nM).
Figure 2. “Excluded Volumes” Analysis. (A) GV 150526 1 is positioned
within the unsymmetrical binding region identified . From this figure, it is
evident that the ortho and meta positions of the terminal phenyl ring are
located nearby a disallowed region of the receptor (yellow area). Conversely,
the para position seems to be located in a region that could tolerate some
substituents of limited steric bulk within the same receptor (red dotted area).
(B) Both the lowest energy conformers of compound 6i (atom-type colored
structure E = 39.9 kcal/mol, magenta colored structure E = 40.7 kcal/mol)
are represented within the unsymmetrical binding region identified. Despite
the increasedstericbulkwithrespect tocompound1, thiscompoundperfectly
fits the allowed area in the described pocket.
As depicted in Figure 2A, subtracting the combined vol-
umes occupied by the “higher affinity compounds” from the
space occupied by the “lower affinity molecules” a partially
forbidden region of the receptor was identified, based on the
assumption that compound 1 and these cycloalkyl indole
derivatives bind in the same way to the glycine site of the
——
4) Computational methods: Sybyl (TRIPOS Ass.): The pharmacophore con-
former of compound 1 (GV150526) was used as reference structure of this
study regarding the values of its side chain rotable bonds, which define the
orientation of the side chain carbonyl group. As we aimed at keeping fixed
the orientation of this primary pharmacophore feature, we forced the corre-
sponding rotatable bonds of compounds 6a–n (Table 1) to assume almost
the same values of those of compound 1. At the same time, these “artificial”
conformations were relaxed by minimization to allow them to reach their
closer local minimum. Then, the amidic rotable bond (NH-Cycloalkyl) of
these structures were submitted to Systematic Search protocol implemented
within Sybyl using 10 deg. as resolution. In addition, an energy window of
25 kcal/mol was applied so as to discard very high energy structures. All the
conformations obtained were minimized using Powell algorithm, filtered for
duplicates and superimposed using as reference points the primary pharma-
cophore features previously described [22]. After that, the volumes occupied
by the conformers endowed with high affinity (pKi > 8) and lower affinity
compounds (pKi < 7) were calculated and combined using the algorithms
implemented within Sybyl. As far as the derivatives endowed with
10 nM ≤ Ki ≤ 100 nM (6b, 6d, 6e, 6f, and 6h) are concerned, the envelop of
their minimized conformations, being spherically shaped, did not add any
further information in terms of geometry and allowed space in the pocket.
Actually, the number of conformations which superimpose with the not
allowed area obtained is comparable to that of the conformations which are
completely enclosed in the allowed pocket. This fact should also explain the
intermediate affinity of these molecules.
NMDA receptor. This forbidden region should be located
nearby the ortho position of the aromatic ring of compound
6)
1, confirming what has been previously observed . Con-
versely, the para position should be able to accept substit-
uents with a limited steric bulk. Moreover, an additional size
limited region located behind this phenyl ring has been iden-
tified. Finally, as can be seen in Figure 2 (A and B), this
receptor pocket does not seem to possess a spherically shaped
profile. As depicted in Figure 2B, compound 6i (both the
lowest energy conformers are shown), despite its increased
bulk with respect to 1 or 6a, fits perfectly into this pocket.
This new receptor model could allow us to explain the re-
duced affinity observed for the less active derivatives with
respect to the “higher affinity compounds”.
Unbiased Pharmacophoric Evaluation
Based on the assumptions described above, the molecules
shown in Table 1 were used to generate a 3D chemical
5)
Computational methods: Catalyst[37]: The molecules for which stereo-
[37]
function based hypothesis using the Catalyst software
.
chemistry is known (1, 6a, 6b, 6c, 6d, 6e, 6f, 6h, 6m, and 6n) were imported
in Catalyst. The software then automatically generated conformational mod-
els[38] for each compound using the Poling Algorithm[39]. The models,
containing a representative set of conformers covering a 20 kcal/mol energy
range above the estimated global minimum, were submitted to Catalyst’s
hypothesis generation. The chemical functions used in this generation step
included hydrogen bond donor, hydrogen bond acceptor, hydrophobic and
negative ionizable. In generating a common features hypothesis, Catalyst
attempts to find all the possible combinations of chemical functions that fit
on all compounds and that share the same spatial position. The statistical
relevance of various hypotheses so obtained is assessed on the basis of their
cost relative to the null hypothesis and their correlation coefficients r. The
three lowest cost hypotheses obtained are listed in Table 3. The total fixed
cost is 28.98 and the cost of the null hypothesis is 67.10. The cost range over
the generated hypothesis is 20.37, while the cost range between best and null
hypothesis is 34.25. As expected, the small cost range and the small differ-
ence between the fixed cost and the best hypothesis suggest that molecules
in the model are fairly rigid, and share a high degree of structural similarity.
In order to test for chance correlation, 19 other hypotheses were generated
by scrambling, in a random way, the experimental activities in the training
set and then regenerating the set of hypotheses. None of the scrambled
hypotheses had a lower cost than our best hypothesis, indicating that there is
a 95% chance this hypothesis represents a true correlation in the data.
The aim of this approach was to further validate the pharma-
cophore model of the glycine binding site previously pro-
[22]
posed
via an unbiased method for pharmacophore
generation. A detailed description of the experimental proce-
6)
dure employed is reported in footnote
.
Table 3: Catalyst’s three best and null hypotheses for the indole-2-carboxyl-
ate analogues activity.
——————————————————————————————
Hypo HBA HBD LIP NegIoniz COST RMS
r
——————————————————————————————
1
1
1
1
–
1
1
1
–
2
2
2
–
1
1
1
–
32.85 1.30
50.36 2.10
53.22 2.32
0.94
0.70
0.61
2
3
null
67.10 2.95. 0.00
——————————————————————————————
———
6) The introduction of an isopropyl group in the ortho position on the terminal
phenyl of compound 1 caused a significant reduction of the affinity as can
be observed in Table 3 in ref.[22]
.
Arch. Pharm. Pharm. Med. Chem. 332, 73–80 (1999)

Mapping of Glycine Binding Site
77
potent than was observed. The higher affinity of this deriva-
tive cannot be explained on the basis of the pharmacophoric
features of this model only. Additional electronic features
(not implemented in Catalyst) typical of a phenyl ring can be
hypothesized. In particular the phenyl ring could be involved
in π-stacking interactions and/or the presence of the phenyl-
amidic moiety could modify considerably the stereoelec-
tronic properties of the carbonyl group (H-bond acceptor). In
conclusion, the results of this unbiased model do not only
confirm the validity of the five points pharmacophoric model,
but also shed new light on the role of the aromatic features of
Figure 3. A) Superimposition of GV 150526A (1) with compounds 6a and
6i (red and yellow, respectively) As described in the main text, there is a good
superimposition of the pharmacophoric points of these molecules with
compound 1. B) Superimposition of GV 150526A (1) with compound 6c
(pink). As described in the main text, it is clear from this figure that at least
one pharmacophoric point is missed with respect to compound 1; in the case
represented here, when the adamantyl substituent of compound 6c is forced
to superimpose with the aromatic moiety of compound 1, the crucial inter-
action with the carbonyl group is lost, causing a detrimental effect in terms
of affinity for the glycine binding site.
the C-3 side chain, explaining better the increase of pK s
i
obtained with the analogues of GV150526 1 on introducing
[22]
electron-donor substituents on the terminal phenyl ring
.
Obviously, a further refinement of the pharmacophoric model
described above will be possible once additional derivatives
are made and characterized.
As shown in Figure 3A, the most potent molecules (1, 6a,
6i) fit very well into the five pharmacophoric points of the
receptor model hypothesis . The terminal hydrophobic
Biological Characterization
[22]
The most in vitro active compounds (K < 100 nM) were
i
group of the side chain in the position C-3 and the chlorine
atom in position C-6 overlap with two hydrophobic features;
the indolic N-H maps with the hydrogen bond donor; the
COOH with the negative ionizable, and the C=O group
present within the C-3 side chain with the hydrogen bond
acceptor feature. Conversely, a poorer fit is observed with the
less potent molecules (6c, 6n). In particular, as depicted in
Figure 3B, when the compound 6c is forced to occupy only
the lipophilic “allowed” region of the receptor, the orientation
of the carbonyl group of its side chain changes dramatically,
leading to the disruption of a key hydrogen bond interaction;
as a result, its affinity for the receptor is greatly reduced.
Racemic compounds (6g, 6i, 6l, 6o) forced to fit this model
show a better fitting for molecules possessing the (R) stereo-
center with respect to those having the (S) one.
evaluated in terms of selectivity towards the glutamate recep-
[22–
tors (NMDA/AMPA/KA). As observed for compound 1
27]
, all the products tested were found to be highly selective
50
–5
(IC >10 M). Moreover, they were found to behave as
competitive antagonists at the glycine binding site.
Finally, these compounds were tested for their ability to
inhibit the convulsions caused by “in vivo” administration of
3
NMDA , a surrogate for stroke models, starting from the
basic assumption that NMDA receptor overactivation is the
key event in neurodegeneration following cerebral ischemia.
The ability ofthe new chemical entities to counteractNMDA-
induced convulsions was used as the end point of the model.
Some of the products described in Table 1 showed excellent
ED s in inhibiting convulsions in mice. In particular, com-
50
pound 6b and 6l, the 1-adamantyl and the norbornyl deriva-
tive, showed an outstanding anticonvulsant potency when
tested in the range of doses between 0.01–3 mg/kg iv and
Figure 4 shows the correlation existing between the ob-
served and estimated K values of the compounds listed in
i
Table 1. As predicted by the model, the affinity of these
compounds for the receptor decrease when increasing of the
steric bulk of the cycloalkyl substituents in the C-3 side chain;
compound 1, however, is predicted by this model to be less
1–100 mg/kgpo according to the general procedure described
[8a]
in ref.
. The estimated ED s obtained by intravenous
50
route were respectively 0.01 and 0.02 mg/kg (compared to
0.06 mg/kg for GV150526, 1), while the ED s obtained with
50
oral administration were 4.6 and 11 mg/kg respectively (com-
pared to 6 mg/kg for GV150526, 1).
Conclusions
A novel series of indole 2-carboxylates was explored with
the aim of identifying potent and selective glycine antagonists
both in vitro and in vivo. By replacing the terminal aromatic
ring belonging to the α,β-unsaturated C-3 side chain of the
indole nucleus of compound 1 with suitable cycloalkyl de-
rivatives, the “North-Eastern” portion of the receptor was
mapped, clarifying the key structural requirements necessary
to design novel classes of glycine antagonists. In particular,
an asymmetric region of limited size able to accept suitable
cycloalkyl substituents was identified. Moreover, the pres-
ence of an aromatic moiety was proved to be crucial to
maximize the affinity of this class of indole-2-carboxylates,
confirming the previous findings obtained for substituted
analogues of GV150526 1.
Figure 4. Correlation line (r = 0.94) displaying observed Kis vs. calculated
values using the statistically most significative hypothesis [Hypothesis no. 1
described in Table 3] derived by the compounds used in the Catalyst model.
Arch. Pharm. Pharm. Med. Chem. 332, 73–80 (1999)

78
Micheli, Di Fabio, and co-workers
and then the solution was concentrated in vacuo. The precipitate was filtered
and washed with cold water to give pure sodium salt derivative (65–95%).
Procedure B. To an aliquot of indole-2-carboxylic acid ethyl ester deriva-
Acknowledgments
We would like to thank Dr. M. Mugnaini for the in vitro experimental part
and Mr. S. Costa for the in vivo experiments. Moreover, we are indebted to
Dr. A. Garofalo for the elemental analyses performed.
.
tive 5a–o (100 mg, 0.27 mmoles) suspended in EtOH (6 mL), LiOH H2O
was added (34.3 mg, 0.82 mmol). The solution was heated at 60 °C for 1.5 h,
then concentrated and diluted with water and acidified with 1N HCl. The
resulting precipitate was filtered and washed with water to give the pure
carboxylic acid derivative (85–95%).
Experimental
Infrared spectra were recorded on a Bruker IFS 48 spectrometer. 1H NMR
spectrawererecordedon a VarianUnity400(400MHz); the data arereported
as follows: chemical shift in ppm from the Me4Si line as external standard,
multiplicity (b = broad, s = singlet, d = doublet, t = triplet, q = quartet, m =
multiplet) and coupling constants.
Chromatography was carried out by use of Merck Silica Gel 60 (230–400
mesh) as described by Still et al. Mass spectra were performed on a Triple
Quadrupole (VG-4 Fison Instrument, UK) equipped with Fast Atom Bom-
bardment (FAB) ionization. Elemental analyses were determined by a EA
1108 Carlo Erba elemental analyzer; C, H, N analyses were within 0.4% of
the theoretical values for the formulae given unless otherwise noted. Melting
points were determined on a Büchi 530 apparatus (scale 0 °C–250 °C) and
are uncorrected. All the reactions were carried out under a controlled atmos-
phere in flame dried glassware. Anhydrous DMF was purchased from
Aldrich; THF was used after distillation over K/benzophenone; CH2Cl2 and
CH3CN were used after distillation over P2O5. Reactions were monitored by
analytical thin-layer chromatography (TLC) usingMerckSilica Gel 60F-254
glass plates (0.25 mm).
3-[2-(Cyclohexylaminocarbonyl)ethenyl]-4,6-dichloroindole-2-carboxylic
Acid Ethyl Ester (5a)
Prepared from 3 following the general procedure described above: IR
(Nujol) ν = 3000 cm–1 (NH), 1674 (C=O).– 1H NMR ([D6]DMSO): δ = 12.47
(bs, 1H), 8.05 (d, 1H, J = 15.9 Hz), 7.97 (d, 1H), 7.47 (d, 1H), 7.27 (d, 1H),
6.50 (d, 1H, J = 15.9 Hz), 4.34 (m, 2H), 3.56 (m,1H), 1.85–1.05 (m, 11H),
1.35 (t, 3H).– MS m/z 409 [M+1]+.
3-[2-(1-Adamantylaminocarbonyl)ethenyl]-4,6-dichloroindole-2-carboxyl-
ic Acid Ethyl Ester (5b)
Prepared from 3 following the general procedure described above: mp =
151 °C. IR (Nujol) ν = 3335 cm–1 (NH), 1672 and 1657, (C=O).– 1H NMR
([D6]DMSO): δ = 11.6 (bs, 1H), 7.98 (d, 1H, J = 15.6 Hz), 7.57 (bs, 1H),
7.47 (d, 1H), 7.24 (d, 1H), 6.52 (d, 1H, J = 15.6 Hz), 4.34 (q, 2H), 2.10–1.64
(m, 15H), 1.34 (t, 3H).– MS m/z 461 [M+1]+.
3-[2-(1-Adamantylmethylaminocarbonyl)ethenyl]-4,6-dichloroindole-
2-carboxylic Acid Ethyl Ester (5c)
(E) 3-(2-tert-butylcarboxyethenyl)-4,6-dichloroindole-2-carboxylic Acid
Ethyl Ester (2)
Prepared from 3 following the general procedure described above. mp >
250 °C. IR (Nujol) ν = 3306 cm–1 (NH), 1680 (C=O).–1H NMR
([D6]DMSO): δ = 12.6 (bs, 1H), 8.00 (d, 1H, J = 15.6 Hz), 7.94 (bt, 1H),
7.47 (d, 1H), 7.27 (d, 1H), 6.56 (d, 1H, J = 15.6 Hz), 4.34 (q, 2H), 2.87 (d,
2H), 1.92 (m, 3H), 1.6 (m, 12H), 1.32 (t, 3H).– MS m/z 475 [M+1]+.
An aliquot of (tert-butoxycarbonylmethylene)triphenylphosphorane
(5.6 g, 15 mmol) and ethyl 3-formyl-4,6-dichloroindole-2-carboxylate
(3.3 g, 11.6 mmol) were dissolved in a 1:1 mixture CH3CN/dioxane (60 mL)
. The resulting solution was heated at 70 °C for 7 h under an atmosphere of
nitrogen. At the end of the reaction the solvent was evaporated under reduced
pressure and the crude residue purified by flash chromatography (cyclohex-
ane/AcOEt 1:1) to give 3.4 g of pure compound 2 (75%): mp 157–158 °C.
IR (Nujol) ν = 3302 cm–1 (NH), 1703 and 1674 (C=O).– 1H NMR
([D6]DMSO): δ = 9.20 (bs, 1H), 8.32 (d, 1H, J = 16Hz), 7.33 (d, 1H), 7.19
(d, 1H), 6.48 (d, 1H, J = 16Hz), 4.43 (q, 2H), 1.56 (s, 9H), 1.42 (t, 3H).– MS
m/z = 383 [M] +.
3-[2-(Noradamantyl-3-aminocarbonyl)ethenyl]-4,6-dichloroindole-
2-carboxylic Acid Ethyl Ester (5d)
Prepared from 3 following the general procedure described above: mp
140 °C . IR (Nujol) ν = 3368–3167 cm–1 (NH), 1718 and 1657 (C=O).–1H
NMR ([D6]DMSO): δ = 12.49 (bs, 1H), 8.15 (bs, 1H), 8.02 (d, 1H, J = 15.9
Hz), 7.49 (d, 1H), 7.29 (d, 1H), 6.56 (d, 1H, J = 15.9 Hz), 4.36 (q, 2H), 2.42
(m, 1H), 2.22 (m, 2H), 2.06 (m, 2H), 1.96 (m, 2H), 1.84 (dd, 2H), 1.62–1.46
(m, 4H), 1.34 (t, 3H).– MS m/z 447 [M+1]+.
(E) 3-(2-carboxyethenyl)-4,6-dichloroindole-2-carboxylic Acid Ethyl Ester
(3)
(E) Ethyl 3-[2-(tert-butoxycarbonyl)ethenyl]-4,6-dichloroindole-2-car-
boxylate (0.5 g, 1.3 mmol) was suspended in HCOOH (60 mL) and the
suspension was stirred at 23 °C for 2 h. The solvent was evaporatedin vacuo
to give 0.408 g of title compound as a white solid (95%): mp >250 °C. IR
(Nujol) ν = 3246–3128 cm–1 (NH), 1699 and 1670, (C=O).– 1H NMR
([D6]DMSO) δ =12.6 (bs, 2H), 8.28 (d, 1H, J = 16.2 Hz), 7.51 (d, 1H), 7.32
(d, 1H), 6.44 (d, 1H, J = 16.2 Hz), 4.37 (q, 2H), 1.35 (t, 3H).– MS m/z 329
[M] +.
3-[2-(Cyclopropylaminocarbonyl)ethenyl]-4,6-dichloroindole-2-carboxylic
Acid Ethyl Ester (5e)
Prepared from 3 following the general procedure described above: mp >
250 °C. IR (Nujol) ν = 3314 and 3260 cm–1 (NH), 1678 and 1659 (C=O).–1H
NMR ([D6]DMSO): δ = 12.50 (bs, 1H), 8.17 (d,1H, , J = 16.0 Hz), 8.04 (d,
1H), 7.45 (d, 1H), 7.26 (d, 1H, ), 6.44 (d, 1H, J = 16.0 Hz), 4.65 (q, 2H), 2.76
(m, 1H), 1.31 (t, 3H), 0.65 (m, 2H), 0.44 (m, 2H).– MS m/z 367 [M+1]+.
General Procedure for the Synthesis of Amides (5a–o)
3-[2-(Cyclopropylmethylaminocarbonyl)ethenyl]-4,6-dichloroindole-
2-carboxylic Acid Ethyl Ester (5f)
To an aliquot of 3-(2′-carboxyethenyl)-4,6-dichloroindole-2-carboxylic
acid ethyl ester 3 (300 mg, 0.91 mmol) dissolved in dry THF (18 mL),
Prepared from 3 following the general procedure described above: mp >
250 °C . IR (Nujol) ν = 3304 cm–1 (NH), 1676 and 1661(C=O).–1H NMR
([D6]DMSO): δ = 12.48 (bs, 1H), 8.18 (t, 1H), 8.03 (d, 1H, J = 16.0 Hz),7.47
(d, 1H), 7.27 (d, 1H), 6.53 (d, 1H, J = 16.0 Hz), 4.34 (q, 2H), 3.04 (t, 2H),
1.32 (t, 3H), 0.95 (m, 1H), 0.40 (m, 2H), 0.16 (m, 2H).– MS m/z 381 [M]+.
2,2′-dipyridyl disulfide (282 mg, 1.28 mmoles) and PPh3 (336 mg,
1.28 mmol), were added at room temperature under an atmosphere of nitro-
gen. The solution was stirred for 1.5 h, then the chosen amine derivative
(1.1 mmoles) was added. The reaction mixture was stirred at room tempera-
ture for 3 hours. The resultant precipitate was filtered to give pure title
compounds (45-90%).
3-[2-(3-Quinuclidineaminocarbonyl)ethenyl]-4,6-dichloroindole-2-carbox-
ylic Acid Ethyl Ester (5g)
General Procedure for the Basic Hydrolysis of the Ethyl Esters
Prepared from 3 following the general procedure described above: mp
160 °C. IR (Nujol) ν = 1718 and 1680 cm–1 (C=O).– 1H NMR ([D6]DMSO):
δ = 12.5 (bs, 1H), 8.2 (d, 1H), 8.00 (d, 1H, J = 15.7 Hz), 7.47 (d ,1H), 7.3(d,
1H), 6.6 (d, 1H, J = 15.7 Hz), 4.34 (q, 2H), 3.84 (m, 1H), 3.08 (m, 1H),
2.8–1.2(m, 10H), 1.32 (t, 3H).– MS m/z 436 [M+1]+.
Procedure A. To an aliquot of indole-2-carboxylic acid ethyl ester deriva-
tive 5a–o (312 mg, 0.68 mmol) suspended in isopropyl alcohol (20 mL),
NaOH was added (108 mg, 2.7 mmol). The solution was heated at 60 °C for
1.5 h. At the end of the reaction the solution was diluted with water (30 mL)
Arch. Pharm. Pharm. Med. Chem. 332, 73–80 (1999)

Mapping of Glycine Binding Site
79
3-[2-(2-Adamantylaminocarbonyl)ethenyl]-4,6-dichloroindole-2-carboxyl-
3-[2-(1-Adamantylaminocarbonyl)ethenyl]-4,6-dichloroindole-2-carboxyl-
ic Acid Ethyl Ester (5h)
ic Acid (6b)
Prepared from 5b according to procedure B: mp > 250°C. IR (Nujol) ν =
3418 cm–1 (NH), 1647 (C=O).– 1H NMR ([D6]DMSO): δ = 13.60 (bs, 1H),
12.40 (s, 1H), 8.02 (d, 1H, J = 15.6 Hz), 7.58 (bs, 1H), 7.45 (d, 1H), 7.25 (d,
1H), 6.55 (d, 1H, J = 15.6 Hz), 2.06–1.96 (m, 9H), 1.64 (m, 6H). MS m/z 455
[M+1]+.
Prepared from 3 following the general procedure described above: IR
(Nujol)ν = 3371cm–1 (NH), 1651 and 1607 (C=O).–1H NMR ([D6]DMSO):
δ = 12.5 (bs, 1H), 8.01 (d, 1H, J = 15.8 Hz), 8.00 (bs, 1H), 7.47 (d, 1H), 7.3
(d, 1H), 6.7 (d, 1H, J = 15.8 Hz), 4.34 (q, 2H), 4.00 (m, 1H), 2.29 (m, 1H),
2.11 (m, 1H), 1.9–0.9 (m, 4H) ; 1.34 (t, 3H).–MS m/z 461 [M+1]+.
3-[2-(1-Adamantylmethylaminocarbonyl)ethenyl]-4,6-dichloroindole-
2-carboxylic Acid Sodium Salt (6c)
3-{2-[(±)-endo-2-Norbornylaminocarbonyl]ethenyl}-4,6-dichloroindole-
2-carboxylic Acid Ethyl Ester (5i)
Prepared from 5c according to procedure A: mp > 250 °C . IR (Nujol) ν =
3429–3198cm–1 (NH), 1653–1612(C=O).–1H NMR ([D6]DMSO): δ = 11.7
(bs, 1H), 8.37 (d, 1H, J = 15.6 Hz), 7.72 (t, 1H), 7.38 (d, 1H), 7.07 (d, 1H),
6.94 (d, 1H, J = 15.6 Hz), 2.87 (d, 2H), 1.92 (m, 3H), 1.70–1.40 (m, 12H).–
MS m/z 469 [M+1]+.
Prepared from 3 following the general procedure described above:
mp 240 °C. IR (Nujol) ν =3312cm–1 (NH), 1680 and 1657 (C=O).– 1H NMR
([D6]DMSO): δ = 12.5 (bs, 1H), 8.06 (d, 1H, J = 15.8 Hz), 8.01 (d, 1H), 7.45
(d, 1H), 7.3 (d, 1H), 6.57 (d, 1H, J = 15.8 Hz), 4.34 (q, 2H),4.03 (m,1H), 2.31
(bs, 1H), 2.14 (bs, 1H), 1.88 (m, 1H), 1.6–0.9 (m, 8H),1.33 (t, 3H) .–MS m/z
421 [M+1]+.
3-[2-(Noradamantyl-3-aminocarbonyl)ethenyl]-4,6-dichloroindole-2-car-
boxylic Acid Sodium Salt (6d)
Prepared from 5d according to procedure A: mp > 250 °C . IR (Nujol) ν
= 1609 cm–1 (C=O).– 1H NMR ([D6]DMSO): δ = 11.50 (bs, 1H), 8.30 (d,
1H, J = 15.9 Hz), 7.83 (bs, 1H), 7.34 (d, 1H), 7.04 (d, 1H), 6.87 (d, 1H, J =
15.9 Hz), 2.42 (t, 1H), 2.19 (bs, 2H), 2.06 (d, 2H), 1.94 (m, 2H), 1.80 (m,
2H), 1.50 (m, 4H).– MS m/z 441 [M+1]+.
3-{2-[(±)-exo-2-Norbornylaminocarbonyl]ethenyl}-4,6-dichloroindole-
2-carboxylic acid Ethyl Ester (5l)
Prepared from 3 following the general procedure described above:
mp >250 °C. IR (Nujol) ν = 3302 cm–1 (NH), 1676 and 1659 (C=O).–1H
NMR ([D6]DMSO): δ = 11.9 (bs, 1H), 8.03 (d, 1H, J = 15.8 Hz), 7.95 (bd,
1H), 7.48 (d, 1H), 7.28 (d, 1H), 6.53 (d, 1H, J = 15.8 Hz), 4.35 (q, 2H), 3.62
(m, 1H), 2.30–2.10 (m, 2H), 1.62 (m, 1H), 1.50–1.30 (m, 3H), 1.30 (m, 1H),
1.20–1.00 (m, 3H), 1.33 (t, 3).– MS m/z 421 [M+1]+.
3-[2-(Cyclopropylaminocarbonyl)ethenyl]-4,6-dichloroindole-2-carboxylic
Acid (6e)
Prepared from 5e according to procedure B: mp > 250 °C. IR (Nujol) ν =
2950 cm–1 (NH),1647 (C=O).– 1H NMR ([D6]DMSO): δ = 13.62 (bs, 1H),
12.43 (s, 1H), 8.17 (d,1H, J = 16.0 Hz), 8.06 (d, 1H), 7.45 (d, 1H), 7.26 (d,
1H, ), 6.46 (d, 1H, J = 16.0 Hz), 2.76 (m, 1H), 0.64 (m, 2H), 0.44 (m, 2H).
MS m/z 339 [M+1]+.
3-[2-(1R-Bornyl-2-aminocarbonyl)ethenyl]-4,6-dichloroindole-2-carboxyl-
ic Acid Ethyl Ester (5m)
Prepared from 3 following the general procedure described above: mp
210 °C. IR (Nujol) ν = 3312 cm–1 (NH), 1682 and 1657 (C=O).– 1H NMR
([D6]DMSO): δ = 12.51 (bs, 1H), 8.03 (d, 1H, J = 15.8 Hz), 7.96 (bd, 1H),
7.49 (d, 1H), 7.29 (d, 1H), 6.64 (d, 1H, J = 15.8 Hz), 4.36–4.24 (m, 3H), 2.18
(m, 1H), 1.70–1.60 (m, 3H), 1.40–0.92 (m, 3H), 1.33 (t, 3H), 0.93 (s, 3H),
0.84 (s, 3H), 0.73 (s, 3H).– MS m/z 463 [M+1]+.
3-[2-(Cyclopropylmethylaminocarbonyl)ethenyl]-4,6-dichloroindole-
2-carboxylic Acid Sodium Salt (6f)
Prepared from 5f according to procedure A: mp > 250 °C. IR (Nujol) ν =
3437–3375cm–1 (NH), 1655–1641(C=O).–1H NMR ([D6]DMSO): δ = 11.7
(bs, 1H), 8.38 (d, 1H, J = 16.0 Hz), 7.96 (t, 1H),7.38 (d, 1H), 7.07 (d, 1H),
6.94 (d, 1H, J = 16.0 Hz), 3.02 (t, 2H), 0.95 (m, 1H), 0.40 (m, 2H), 0.18 (m,
2H). MS m/z 375 [M]+.
3-[2-(1R-isobornyl-2-aminocarbonyl) ethenyl]-4,6-dichloroindole-2-car-
boxylic Acid Ethyl Ester (5n)
3-[2-((±)-3-Quinuclidineaminocarbonyl)ethenyl]-4,6-dichloroindole-
2-carboxylic Acid (6g)
Prepared from 3 following the general procedure described above:
mp 210 °C.IR (Nujol) ν =3400cm-1 (NH), 1703 and 1682 (C=O),.– 1H NMR
([D6]DMSO): d = δ 12.48 (bs, 1H), 7.98 (d, 1H, J = 16.0 Hz), 7.47 (d, 2H),
7.27 (d, 1H), 6.61 (d, 1H, J = 16.0 Hz), 4.4–4.25 (m, 3H), 3.86 (m,1H),
1.50–1.15 (m, 6H), 1.31 (t, 3H), 0.91 (s, 3H), 0.78 (s, 6H).– MS m/z 463
[M+1]+.
Prepared from 5g according to procedure B: mp > 250 °C. IR (Nujol) ν =
3366 cm–1 (NH), 1661 and 1618 (C=O).– 1H NMR ([D6]DMSO): δ =
13.4–12.4 (bs, 1H), 11.96 (bs, 1H), 8.46 (d, 1H), 8.29 (d ,1H, J = 15.7 Hz),
7.41(d, 1H), 7.12 (d, 1H), 6.80 (d, 1H, J = 15.7 Hz), 4.14 (m, 1H), 3.6–3.0
(m, 6H), 2.2–1.6 (m, 5H).– MS m/z 408 [M+1]+.
3-[2-(1,2,3,4-tetrahydro-1-naphthylaminocarbonyl)ethenyl]-4,6-dichloro-
indole-2-carboxylic Acid Ethyl Ester (5o)
3-[2-(2-Adamantylaminocarbonyl)ethenyl]-4,6-dichloroindole-2-carboxyl-
ic Acid Sodium Salt (6h)
Prepared from 3 following the general procedure described above: IR
(Nujol)ν = 3302cm–1 (NH), 1676(C=O).– 1H NMR ([D6]DMSO): δ = 12.52
(bs, 1H), 8.53 (d, 1H), 8.14 (d, 1H, J = 16.0 Hz), 7.49 (d, 1H), 7.29 (d, 1H),
7.16 (m, 4H), 6.58 (d, 1H, J = 16.0 Hz), 5.13 (m, 1H), 4.35 (q, 2H), 2.75
(m,1H), 1.93–1.75 (m, 4H), 1.33 (t, 3H).– MS m/z 456 [M+1]+.
Prepared from 5h according to procedure A: mp > 250 °C. IR (Nujol) ν =
1653 cm–1 (C=O). 1H NMR ([D6]DMSO): δ = 11.3–11.6 (bs, 1H), 8.34 (d,
1H, J = 15.8 Hz), 7.74 (bd, 1H), 7.37 (d, 1H), 7.05 (d, 1H), 6.88 (d, 1H, J =
15.8 Hz), 3.95 (d, 1H), 2.03 (d, 2H), 1.8–1.6 (m, 10H), 1.46 (d, 1H).– MS
m/z 455 [M+1]+.
3-[2-(Cyclohexylaminocarbonyl)ethenyl]-4,6-dichloroindole-2-carboxylic
Acid (6a)
3-{2-[(±)-endo-2-Norbornylaminocarbonyl]ethenyl}-4,6-dichloroindole-
2-carboxylic Acid Sodium Salt (6i)
Prepared from 5a following the general procedure B: mp > 250 °C. IR
(Nujol) ν = 3418 and 3229 cm–1(NH), 1695–1684 (C=O).– 1H NMR
([D6]DMSO): δ = 13.64 (bs, 1H), 12.44 (s, 1H), 8.05 (d, 1H, J = 15.9 Hz),
7.99 (d, 1H), 7.46 (d, 1H), 7.27 (d, 1H), 6.53 (d, 1H, J = 15.9 Hz), 3.65 (dm,
1H), 1.82–1.55 (m, 5H), 1.38–1.10 (m, 5H).– MS m/z 381 [M+1]+.
Prepared from 5i according to procedure A: mp > 250 °C. IR (Nujol) ν =
3420 cm–1 (NH), 1651 (C=O.– 1H NMR ([D6]DMSO): δ = 11.69 (bs, 1H),
8.34 (d, 1H, J = 15.8 Hz), 7.84 (d, 1H), 7.37 (d, 1H), 7.05 (d, 1H), 6.87 (d,
1H, J = 15.8 Hz), 4.00 (m, 1H), 2.29 (m, 1H), 2.11 (m, 1H), 1.9–0.9 (m, 4H).–
MS m/z 415 [M+1]+.
Arch. Pharm. Pharm. Med. Chem. 332, 73–80 (1999)

80
Micheli, Di Fabio, and co-workers
[15] C. Chiamulera, S. Costa, A. Reggiani, Psycopharmacol. 1990, 112,
3-{2-[(±)-exo-2-Norbornylaminocarbonyl]ethenyl}-4,6-dichloroindole-
2-carboxylic Acid Sodium Salt (6l)
551–552.
[16] M. G. Palefreyman, M. B. Baron in: Excitatory Amino Acid Antagonists
(Ed.: B. Meldrum), Blackwell, Oxford (UK), 1991. Chapter 6
Prepared from 5l according to procedure A: mp > 250 °C. IR (Nujol) ν =
3296–3161 cm–1 (NH), 1682 and 1663(C=O).– 1H NMR ([D6]DMSO): δ =
13.62 (bs, 1H), 12.42 (s, 1H), 8.05 (d, 1H, J = 15.8 Hz), 7.95 (d, 1H), 7.45
(s, 1H), 7.25 (s, 1H), 6.53 (d, 1H, J = 15.8 Hz), 3.62 (m, 1H), 2.21–2.10 (m,
2H), 1.61 (m, 1H), 1.50–1.35 (m, 3H), 1.30 (m, 1H), 1.19–1.05 (m, 3H).–
MS m/z 415 [M+1]+.
[17] A. J. Carter, Drugs Future 1992, 17, 595–613.
[18] J. A. Kemp, P. D. Leeson, TiPS 1993, 14, 20–25.
[19] P.D. Leeson, L. L. Iversen, J. Med. Chem. 1994, 37, 4053–4067.
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Collingridge, J. C., Watkins), IRL Press, Oxford (UK), 1994, 469–486.
3-[2-(1R-Bornyl-2-aminocarbonyl)ethenyl]-4,6-dichloroindole-2-carboxyl-
ic Acid Sodium Salt (6m)
[21] R. Di Fabio, G. Gaviraghi, A. Reggiani, La Chimica e l’Industria 1996,
78, 283–289.
Prepared from 5m according to procedure A: mp > 250 °C. IR (Nujol) ν
= 3431 and 3377 cm–1 (NH), 1647–1610 (C=O).– 1H NMR ([D6]DMSO):
δ = 11.69 (bs, 1H), 8.37 (d, 1H, J = 15.8 Hz), 7.75 (bd, 1H), 7.39 (d, 1H),
7.07 (d, 1H), 6.98 (d, 1H, J = 15.8 Hz), 4.24 (m, 1H), 2.12 (m, 1H), 1.80–1.55
(m, 3H), 1.40–1.10 (m, 2H), 0.99 (m, 1H), 0.93 (s, 3H), 0.84 (s, 3H), 0.73 (s,
3H).– MS m/z 456 [M+1]+.
[22] R. Di Fabio, A.M. Capelli, N. Conti, A. Cugola, D. Donati, A. Feriani,
P. Gastaldi, G. Gaviraghi, C.T. Hewkin, F. Micheli, A. Missio, M.
Mugnaini, A. Pecunioso, A.M. Quaglia, E. Ratti, L. Rossi, G. Tedesco,
D.G. Trist, A. Reggiani, J. Med. Chem. 1997, 40, 841–850.
[23] R. Di Fabio, A. Cugola, D. Donati, A. Feriani, G. Gaviraghi, E. Ratti,
D. G. Trist, ACS Book of Abstracts, 211th ACS National Meeting, New
Orleans, LA, 24–28 March 1996, Abstract 107.
3-[2-(1R-Isobornyl-2-aminocarbonyl)ethenyl]-4,6-dichloroindole-
2-carboxylic Acid Sodium Salt (6n)
[24] A. Cugola, G. Gaviraghi, F. Micheli (Glaxo S.p.A.), PCT Int. Appl.,
WO 9420465 A1 940915
Prepared from 5n according to procedure A: mp >250 °C. IR (Nujol) ν =
3192 cm–1 (NH), 1609 (C=O).– 1H NMR ([D6]DMSO): δ = 11.6 (bs, 1H),
8.34 (d, 1H, J = 16.0 Hz), 7.35 (d, 1H), 7.21 (bd, 1H), 7.05 (d, 1H), 6.89 (d,
1H, J = 16.0 Hz), 1.8–1.6 (m, 4H), 1.48 (m, 1H), 1.2–1.06 (m, 2H), 0.91 (s,
3H), 0.76 (ss, 6H). MS m/z 457 [M]+.
[25] G. Gaviraghi, R. Di Fabio, A. Cugola, D. Donati, A. Feriani, E. Ratti,
D.G. Trist, A. Reggiani, Proceedings, XIVth International Symposium
on Medicinal Chemistry, (Ed.: F. Awouters), Maastricht, 1996
[26] A. Reggiani, S. Costa, C. Pietra, E. Ratti, D. G. Trist, L. Ziviani, G.
3-[2-((±)-1,2,3,4-Tetrahydro-1-naphthylaminocarbonyl)ethenyl]-
4,6-dichloroindole-2-carboxylic Acid Sodium Salt (6o)
Gaviraghi, Eur. J. Neurol. 1995, 2, 6.
[27] C. Pietra, L. Ziviani, E. Valerio, G. Tarter, A. Reggiani, Stroke, 1996,
27, 26.
Prepared from 5o according to procedure A: mp >250 °C. IR (Nujol) ν =
1607 cm–1 (C=O).– 1H NMR ([D6]DMSO): δ = 11.6 (bs, 1H), 8.47 (d,1H),
8.26 (d, 1H, J = 16.0 Hz), 7.36 (d, 1H), 7.05 (d, 1H), 7.22–7.04 (m, 4H), 6.93
(d, 1H, J = 16.0 Hz), 5.09 (m, 1H), 2.72 (m, 2H), 1.90 (m, 2H), 1.72 (m, 1H).–
MS m/z 450 [M]+.
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W. F. Hood, J. B. Monahan, J. Med. Chem. 1991, 34, 1283–1292.
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Received: October 16, 1998 [FP342]
Arch. Pharm. Pharm. Med. Chem. 332, 73–80 (1999)