A simple polar deacetylated caloporoside derivative is a positive modulator of the GABA(A) chloride channel complex in cortical mammalian neurones

Article abstract of DOI:10.1016/S0960-894X(00)00330-9

Synthesis of octyl-O-β-D-mannopyranoside, a caloporoside analogue was achieved by the activation of 2,3,4,6-tetra-O-benzyl-1-O-1',3'2'-dioxaphosphacy clohexane-α,β-D-mannopyranosyl-2-oxide with TMSOTf (Trimethyl silyl triflate) and subsequent debenzylation. At 100 μM the molecule significantly and reversibly increased the magnitude of GABA(A) currents evoked in cultured rat pyramidal neurones whilst concomitantly reducing the incidence of spontaneous synaptic activity. These results contradict earlier proposals that such molecules bind to the TBPS (tert-Butylbicyclophosphorothionate) site to block the chloride channel. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00330-9

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1759±1761
A Simple Polar Deacetylated Caloporoside Derivative is a
Positive Modulator of the GABAA Chloride Channel
Complex in Cortical Mammalian Neurones
George Lees, Paul L. Chazot,* Hariprasad Vankayalapati and Gurdial Singh
Institute of Pharmacy and Chemistry, School of Sciences, University of Sunderland, Sunderland SR1 3SD, UK
Received 7 April 2000; accepted 6 June 2000
AbstractÐSynthesis of octyl-O-b-d-mannopyranoside, a caloporoside analogue was achieved by the activation of 2,3,4,6-tetra-O-
benzyl-1-O-10,3020-dioxaphosphacyclohexane-a,b-d-mannopyranosyl-2-oxide with TMSOTf (Trimethyl silyl tri¯ate) and subsequent
debenzylation. At 100 mM the molecule signi®cantly and reversibly increased the magnitude of GABAA currents evoked in cultured
rat pyramidal neurones whilst concomitantly reducing the incidence of spontaneous synaptic activity. These results contradict earlier
proposals that such molecules bind to the TBPS (tert-Butylbicyclophosphorothionate) site to block the chloride channel. # 2000
Elsevier Science Ltd. All rights reserved.
GABA is the most widespread inhibitory transmitter in
the brain where it is present in nerve terminals at ca. one
third of all CNS synapses.1 The GABAA receptor is a
ligand-gated chloride channel which is the target for a
variety of commercially important depressant drugs
including benzodiazepines, barbiturates, neurosteroids and
a variety of volatile anaesthetic agents.2,3 The clinically
useful congeners allosterically enhance the magnitude
and or duration of inhibitory synaptic currents to exert
their sedative, hypnotic or anaesthetic e€ects. However,
within each class of molecules certain congeners can
produce antagonistic actions resulting in reduction of
chloride currents, hyperexcitation in the CNS and con-
vulsions in whole animals.4 6 Flumazenil is an selective
antagonist of the benzodiazepine site on the channel
protein which competitively displaces both depressant
and sedative congeners from their recognition site without
exerting a physiological response.7 Halogenated insecti-
cides like the g isomer of hexachlorocylohexane (lindane)
block the GABA activated chloride channels expressed
on arthropod muscle or neurones but the non insecticidal
b analogue enhances chloride currents.8 The bi-functional
nature of this allosteric coupling means that it is dicult to
interpret simple radioligand binding studies in isolation6
(as this reveals only anity for the complex but gives no
indication of intrinsic ecacy to act as an agonist,
antagonist or inverse-agonist). The natural product dea-
cetyl-caloporiside has been reported to displace TBPS
from its site in the lumen of the chloride channel which has
been interpreted as evidence that it blocks the GABAA
receptor complex.9 11 Here, we have synthesised a very
simple polar caloporoside analogue, octyl-0-b-d-manno-
pyranoside 3, from 2,3,4,6-tetra-O-benzyl-1-O-10,3020 -
dioxaphosphacyclohexane-a,b-d-mannopyranosyl-2-oxide
112 and subsequent debenzylation of 2b with palladium
hydroxide, (Scheme 1)13 and characterised its functional
e€ects on inhibitory ion channels.
Details of cell-culture and electrophysiological methods
have been published in full.14 Brie¯y, whole cell patch
clamp recording was used with 70±80% series resistance
compensation applied at the axopatch. Extracellular
salines and the gluconate-based pipette solutions allow
us to resolve inhibitory synaptic currents (IPSCs) from
excitatory currents (EPSCs) and characterise sponta-
neous synaptic activity.15 Brie¯y we applied 2s pulses of
10 mM GABA (every 60s) to rat cortical pyramidal cells
maintained in culture for 14±28 days using a Y-tube
(rapid application across entire cell). The octyl-O-b-d-
mannopyranoside 3 was dissolved initially in dimethyl-
sulphoxide (DMSO) and diluted 1:1000 into test salines.
To control for solvent artefacts, 0.1% DMSO was
added routinely to drug free salines and had no e€ect on
any of the parameters reported here. The saccharide 3 was
applied to the cell by superfusion between pulses and also
added to the agonist solution to assess the modulatory
response under equilibrium conditions. Data were ana-
lysed using CED and Graphpad software: all data are
cited as mean Æ standard error of the mean.
*Corresponding author. Fax: +44-191-515-3077; e-mail: paul.chazot
@sunderland.ac.uk
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00330-9

1760
G. Lees et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1759±1761
Scheme 1. Reagents and Conditions: (i) HO(CH2)7CH3, TMSOTf (cat.), 78 ꢀC, 0.5 h; (ii) Pd (OH)2, MeOH, cyclohexene, 12 h.
100 mM 3 enhanced the response to GABA in all 10 cells
examined (Fig. 1a). Onset of the response was complete
within ca. 2±3 min and at equilibrium the currents
increased to 131 Æ 4.3% of the pretreatment responses
(Fig. 1b). Under identical conditions, in previous studies,
both the sleep modulator oleamide (at 20 mM) and the
inhalational anaesthetic iso¯urane (320 mM) enhanced
GABA currents by a similar magnitude (<2-fold
increase).14,16 A two-tailed paired t-test revealed that the
3 e€ect was highly signi®cant (p=0.0025). Like the
depressant drug and the sleep hormone above, 100 mM
3 also reversibly reduced the incidence of both sponta-
neous excitatory post-synaptic currents and inhibitory
post-synaptic currents (Fig. 1c).
can access its target site relatively quickly and reversibly.
Future studies will address whether the molecule exerts
an open-channel or use-dependent modulatory e€ect
(i.e., whether the molecule accesses its recognition site
via the channel lumen). Deacetyl-caloporoside (which
we were unable to procure for these studies) displaces
TBPS from its recognition site in the lumen of the
chloride channel site but has not been examined in a
functional assay to date. By inference the authors of
studies on the natural product and its synthetic coun-
terpart suggest that the molecules are ``inhibitors'' of
the GABAA receptor ion channel.9 The commercial
importance of the GABAA receptor as a drug and
insecticide target is well established. However, we still
do not have ideal anxiolytic, sedative, or hypnotic drugs
for chronic treatments. Our data highlights the impor-
tance of functional characterisation of channel-directed
ligands to indicate the physiological relevance of phar-
macophore modelling based solely on binding pro®les.
Caloporoside and octyl-O-b-d-mannoyranoside 3 may
occupy separate binding sites on the ligand-gated ion
channel complex but only further studies combining radio-
labelled probes with functional studies (micro-electrodes
or ion ¯uxing) can address this issue or resolve whether,
like benzodiazepines, a single receptor can explain the
actions of sedative and convulsant drugs. Such mole-
cules, if they can bypass the blood-brain barrier, war-
rant further study as potential drugs for important
pharmaceutical markets.17
These results strongly suggest that even the simple
amphiphilic alkylated mannose synthesised here has the
capacity to mimic caloporoside in binding to the GABAA
complex in mammalian brain membranes,9 albeit with
relatively low anity. However, our data demonstrate
that functionally the novel polar molecule is a positive
modulator of the channel complex and exerts similar
e€ects to those of commonly used depressant drugs and
a putative sleep hormone.
Many chloride channel antagonists (like picrotoxinin
and halogenated insecticides) are very apolar and block
the channel with high anity and reverse slowly if at
all.8 The saccharide 3 is a water-soluble ligand which
Figure 1. (a) Pairs of responses from the same cell to 10 mM GABA before (left), during (centre) and after (right) the administration of 100 mM 3.
Upper traces represent currents from the cell clamped at 45 mV; lower traces indicate timing and duration of GABA pulses. Scale bars: 400 pA,
24s; (b) Column graph depicting data from ten replicated experiments. The di€erence between the means was highly signi®cant (p=0.0025); (c)
compressed recording from another cell showing typical e€ects of 100 mM 3 (horizontal bar) on spontaneous synaptic trac. Note the reversible
depression in the incidence of both IPSCs (upward de¯ections) and EPSCs (small downward de®ections). Action currents (large downward de¯ec-
tions) represent action potential ®ring in the patch-clamped neurone were particularly sensitive in this cell. Scale bars: 400 pA, 2 min.

G. Lees et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1759±1761
1761
Acknowledgements
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We (GL and PLC) thank The Welcome Trust for
equipment support.
12. Singh, G.; Vankayalapati, H. Tetrahedron: Asymmetry
2000, 11, 125.
References and Notes
13. Data for 3: [a]d22 +29.5 (c 2.2, CHCl3); dH (270 MHz;
CDCl3) 0.84 (3H, t, J 6.59, 5.94), 1.25, (10H, bs), 1.52 (2H, s),
3.34 (1H, d, J 7.91), 3.46 (1H, d, J 7.26), 3.58 (1H, d, J, 6.60), 3.70
(1H, d, J 11.22), 3.83±3.89 (4H, m), 4.78 (1H, bs); dC, (67.8 MHz;
CDCl3) 14.03, 22.59, 26.05, 29.20, 29,37, 29.63, 31.80, 60.67,
65.93, 67.84, 70.99, 71.45, 72.23, 100.01; m/z (CI, NH3) Found:
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