Synthesis and biological evaluation of novel rigid 1,4-benzodiazepine-2,5- dione chimeric scaffolds

Article abstract of DOI:10.1002/ejoc.200700952

Novel, conformationally constrained 1,4-benzodiazepine-2,5-diones containing both a monosaccharide, fructose, and a proline moiety have been synthesized as chimeric scaffolds; preliminary biological evaluation as GABA_A receptor ligands is repor

Full text of DOI:10.1002/ejoc.200700952

SHORT COMMUNICATION
DOI: 10.1002/ejoc.200700952
Synthesis and Biological Evaluation of Novel Rigid 1,4-Benzodiazepine-
2,5-dione Chimeric Scaffolds
Ana C. Araújo,[a] Francesco Nicotra,[b] Cristina Airoldi,[b] Barbara Costa,[b]
Gabriella Giagnoni,[b] Pietro Fumagalli,[c] and Laura Cipolla*[b]
Keywords: Benzodiazepine / Carbohydrates / GABAA receptor / Lead generation / Biological activity studies
Novel, conformationally constrained 1,4-benzodiazepine-
2,5-diones containing both a monosaccharide, fructose, and
a proline moiety have been synthesized as chimeric scaffolds;
preliminary biological evaluation as GABAA receptor ligands
is reported.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2008)
Introduction
Benzodiazepines are among the most widely dispensed
drugs. Besides the well-known anxiolytic, sedative, anti-
convulsant, myorelaxant and hypnotic activities,[1] benzodi-
azepines have been shown to act as peripheral cholecystoki-
nin (CCK-A) receptor agonists,[2] α-thrombin inhibitors,[3]
antitubercular drugs,[4] endothelin receptor antagonists[5]
and can be cytotoxic against transformed T-cells.[6] Indeed,
several of these nitrogen-containing heterocycles have been
identified as antitumour antibiotics,[7] anti-HIV[8] and
antithrombotic agents.[9]
Moreover, because of both their structural motifs and
physicochemical properties, the benzodiazepine scaffold has
been considered among novel non-peptide peptidomimetics,
acting as a mimic of peptide secondary structures such as
γ- and β-turns.[10] Particularly useful in medicinal chemistry
are proline-derived benzodiazepines (pyrrolo[2,1-c][1,4]-
benzodiazepines),[11] showing promise as anxiolytic drug
candidates (i.e. 1; Figure 1) and as starting materials for
the synthesis of anthramycin-inspired anticancer drugs (e.g.
2),[11c] DNA-cross-linking agents (e.g. 3),[11d] and α5-selec-
tive GABAA receptor ligands (e.g. 4).[11e]
Figure 1. Chemical structures of some biologically active pyr-
rolo[2,1-c][1,4]benzodiazepines.
Recently, the design and synthesis of new chimeric scaf-
folds, that are hybrids of benzodiazepines with sugars
wherein the latent hydroxy groups of the carbohydrate moi-
ety permit diverse, controlled derivatization at different
sites, have been proposed (i.e. compounds 5 and 6,[12] 7 and
8,[13] 9;[14] Figure 2).
In addition, great efforts have been devoted to the syn-
thesis of conformationally constrained benzodiazepine de-
rivatives,[11a,15] since conformational changes in the benzo-
diazepine ring system have a strong effect on binding affin-
ities to the receptor complex.[16]
Herein we describe the synthesis and preliminary bio-
logical evaluation as GABAA receptor ligands of novel,
conformationally constrained 1,4-benzodiazepine-2,5-di-
ones as chimeric scaffolds, containing both a monosaccha-
ride, fructose, and a proline moiety. The free hydroxy
groups on the sugar moiety offer the possibility of further
[a] Departamento de Química e Bioquímica, Universidade de Lis-
boa e CQB,
C8, Campo Grande, 1749-016 Lisboa, Portugal
[b] Department of Biotechnology and Biosciences, University of
Milano-Bicocca,
P.za della Scienza 2, 20126 Milano, Italy
Fax: +39-02-64483565
E-mail: laura.cipolla@unimib.it
[c] Department of Environemental Sciences, University of Milano-
Bicocca,
P.za della Scienza 1, 20126 Milano, Italy
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
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L. Cipolla et al.
SHORT COMMUNICATION
Scheme 1. Synthesis of fructose-proline-containing 1,4-benzodia-
zepine-2,5-diones; (a) dry DMF, reflux; (b) H2, Pd(OH)2, MeOH/
AcOEt.
Table 1. Effects of test compounds in the [3H]Flunitrazepam spe-
cific binding to GABAA receptor performed on rat cortical mem-
branes.
Figure 2. Examples of glyco-benzodiazepines.
Compound % [3H]Flunitrazepam specific
binding[a]
Significance vs.
control[b]
functionalisation for tuning the pharmacokinetic properties
and the biological activity. The -proline moiety connected
to fructose through a spiro junction gives high conforma-
tional rigidity to the hybrid scaffolds, as confirmed by
NMR spectroscopic data (see below).
Control
11
100.00Ϯ2.00
111.81Ϯ2.33
96.44Ϯ8.85
101.01Ϯ2.10
74.89Ϯ1.50
78.22Ϯ7.43
93.04Ϯ12.77
80.17Ϯ9.43
70.63Ϯ2.36
n.s.
n.s.
n.s.
12
13
14
P Ͻ 0.05
P Ͻ 0.05
n.s.
n.s.
P Ͻ 0.01
15
16
17
Results and Discussion
18
[a] Values are meansϮSEM determined from at least three indepen-
dent experiments. [b] Statistical analysis is performed with Krus-
kal–Wallis ANOVA for non-parametric values followed by Dunns
test.
The synthesis of the 1,4-benzodiazepine-2,5-dione deriv-
atives 11–14 was accomplished by heating suitably function-
alised isatoic anydride in dry DMF, with the proline-fruc-
tose building block 10 (Scheme 1).[17] Subsequent hydro-
genolysis resulted in deprotection and reduction of the nitro
group to afford the water-soluble 1,4-benzodiazepine-2,5-
diones 15–18.
[(M) or (P)] of the benzodiazepine ring (Figure 3), which is
governed by the pseudoequatorial preference and absolute
stereochemistry of the C11a[18] substituent.[18,19]
Preliminary biological evaluation of compounds 11–18
as GABAA receptor ligands has been performed. In par-
ticular, we tested their ability to displace [3H]Flunitrazepam
from the receptor by using rat cortical membranes to per-
form a classical competition binding assay (Table 1). Data
show that compounds 14, 15 and 18 possess affinity for
GABAA receptor significantly inhibiting [3H]Flunitra-
zepam binding in the µ range. However, the biological ac-
tivity of compounds 14, 15 and 18 is actually far from that
of classical GABAA modulators, which falls in the n
range; nevertheless, this result indicates that compounds 14,
15 and 18 can be considered as lead compounds.
Figure 3. Conformational equilibrium present in the 1,4-benzodi-
azepine ring.
Hence, conformational analysis of sample benzodiazep-
It has been reported that binding affinities at the GA- ines 15 and 16 was performed by molecular modelling cal-
BAA receptor are exquisitely sensitive to the conformation culations and experimental NMR studies at variable tem-
636
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Novel Rigid 1,4-Benzodiazepine-2,5-dione Chimeric Scaffolds
Figure 4. Oxygen-substituent pseudo-axial (A) and pseudo-equatorial (B) conformers of pyrrolo ring in compound 15. Pseudo-axial
conformer: total energy = 25.028 kcal/mol, dihedral angle C5a–C9a–N10–C11 = +16.91°; pseudo-equatorial conformer: total energy =
22.319 kcal/mol, dihedral angle C5a–C9a–N10–C11 = +17.32°.
Figure 5. DNMR spectra (363–298K) of molecule 15 dissolved in D2O; spectrum 1: 298 K, 32 scans, spectrum 2: 313 K, 32 scans,
spectrum 3: 323 K, 32 scans, spectrum 4: 333 K, 32 scans, spectrum 5: 343 K, 32 scans, spectrum 6: 353 K, 32 scans, spectrum 7: 363 K,
32 scans.
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L. Cipolla et al.
SHORT COMMUNICATION
purged 3 times with Ar and then filled with H2. After 48 h, the
catalyst was removed by filtration, and the filtrate concentrated
under reduced pressure to afford compounds 15–18.
perature (dynamic NMR, DNMR). Molecular modelling
and NMR spectroscopic data show a unique preferential
conformation of the benzodiazepine ring, where 11a-H
adopts the pseudoaxial position (Figure 4 and Supporting GABAA Receptor Binding Assay: Membranes were prepared from
rat cortex as described by Ahboucha et al.[21] and were used for
Information). In addition, NMR experiments run at vari-
able temperature (Figure 5 and Supporting Information),
until a maximum value of 363 K indicate that even at high
temperature no conformational equilibrium of the benzodi-
displacement studies. [3H]Flunitrazepam (1 n) served as radioli-
gand and nonspecific binding was determined in the presence of
Flumazenil (100 µ) and represented about 1–2% of the total bind-
ing. Compounds 15–18 were diluted in TrisHCl buffer (50 m; pH
azepine ring is present, in contrast to quaternary 1,4-benzo-
= 7.4) to obtain the final concentration of 100 µ, whereas stock
diazepin-2-ones, which exist as mixtures of the (M) and (P)
solutions of compounds 11–14 were prepared in ethanol and then
conformers.[11a,15a] Actually, when sterically demanding
diluted in TrisHCl buffer to obtain the final concentration of
100 µ. The binding reaction consisted in 0.3 mg of membranes
substituents are present on the diazepine ring, they assume
pseudo-equatorial orientation, and in all reported
a
incubated with the radioligand in the presence or absence of the
cases,[20] the coalescence temperature for the pseudo-equa- test compounds and was stopped after 90 min at 4 °C by rapid
filtration under vacuum through a glass-fiber filter (GF/B; What-
man). The filters were washed twice with 2-mL portions of ice-cold
TrisHCl buffer and then dissolved in 8 mL of scintillation fluid
(UltimaGold). Filter-bound radioactivity was determined by a
scintillation counter. For all compounds three independent experi-
ments were performed in triplicate. Results are expressed in terms
of percentage of control [3H]Flunitrazepam specific binding and
analyzed by GraphPad Prism, using the Kruskal–Wallis ANOVA
for nonparametric data followed by Dunns test for specific com-
parisons.
torial/pseudo-axial equilibrium is always around 60 °C
(333 K). The dihedral angle C5a–C9a–N10–C11 was also
determined and was found to be +17° for compound 15
and +24° for compound 16, corresponding to helical chiral-
ity (P). However, conformers of the pyrrolo ring do exist,
as illustrated in Figures 4a and b, keeping the (P) confor-
mation of the diazepine. In order to best characterise this
equilibrium, DNMR studies were performed also at low
temperature for compound 15, down to 173 K (Supporting
Information). These studies indicate that the barrier for in-
version of the pyrrolo ring is very low, as suggested also by
molecular modelling (Figure 4).
Molecular Modelling: Conformational analysis of compounds 15
and 16 was performed by applying an MM2 force field in vacuo.
Each model was built with ChemDraw, the energy was minimized
(minimum RMS gradient = 0.010), and the dihedral angle C5a–
C9a–N10–C11 value calculated by using Chem3D. A Molecular
Dynamics Computation was performed (step interval = 2 fs; frame
interval = 1 fs; heating/cooling rate = 1.000 kcal/atomps; target
temperature = 300 K) by monitoring the same dihedral angle. The
average value was calculated; in both the cases it corresponded to
that found by MM calculations.
Conclusions
We can conclude that the synthesised hybrid fructose-
proline-benzodiazepines adopt a rigid conformation around
the benzodiazepine ring. Noteworthy, no conformational
equilibrium of the benzodiazepine ring is present, which
displays a rigid pseudoequatorial orientation of the C11a
substituent. However, the -proline ring induces a (P)-heli-
cal conformation, which is the opposite to that required for
best binding to the GABAA receptor.[18]
DNMR Experiments: 0.5 mg of compound 15 or 16 was dissolved
in 0.6 mL of D2O (363–293 K) or CD3CD2OD (293–173 K). 1H
and selective 1D-NOESY (data not shown) spectra were recorded
at different temperatures and than processed with MestreNova
(MestreLab Research s.r.l.). For spectra recorded in D2O, the HDO
Nevertheless, compounds 14, 15 and 18 show a signifi- chemical shift was calculated according to the equation δ = 5.060 –
0.0122T + (2.11ϫ10–5)T2.[22]
cant affinity for the GABAA receptor, hence they can be
considered as leads. Strategies that allow synthesis of -pro-
line analogues linked to fructose able to induce a rigid (M)
Supporting Information (see footnote on the first page of this arti-
cle): Detailed experimental procedures and spectroscopic data for
conformation, relevant for biological activity, are under in- the synthesized compounds.
vestigation and will be reported in due course.
Acknowledgments
Experimental Section
Work supported by Ministero dell’Università e della Ricerca under
contract MIUR-PRIN 2005 n. 2005037725 and Fundação para a
Ciência e Tecnologia (FCT) SFRH/BD/17815/2004.
General Procedure for Benzodiazepine Synthesis: To a solution of
10 in dry DMF, suitably functionalised isatoic anhydride was added
under argon, and the resulting mixture was stirred under reflux
until complete consumption of the starting material. The solvent
was then removed under reduced pressure and the residue dissolved
in dichloromethane and washed with 5% aqueous HCl. The or-
ganic layer was dried with sodium sulfate, filtered, and the solvents
were evaporated. The crude residue was purified by silica gel flash
chromatography to give the desired product.
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Received: October 8, 2007
Published Online: December 19, 2007
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