Download A kalpain enzimrendszer idegrendszeri plaszticitást befolyásoló

Cell culture-based fluorometric assay techniques
in drug research
Thesis
Sándor Kolok
Ph.D. School of Biology
Neurobiology and humanbiology
Program leader: Dr. László Détári C.Sc.
Supervisor: Dr. István Tarnawa Ph.D.
Pharmacology and Drug afety Research
Gedeon Richter Ltd.
Pharmacology and Drug afety Research
Gedeon Richter Ltd.
2006
1.
Introduction
Several neurological diseases are characterized with abnormally increased excitatory tone
of certain central neurons. These include epilepsy, ischemic neuronal damage, chronic
neurodegenerative disorders, alcohol- and drug abuse, and chronic pain states.
No suitable pharmacotherapy of chronic pain exists. Large proportion of the patients does
not respond properly to the drugs presently available, or do not respond at all. These agents
typically have a variety of different side effects. Thus, there is a huge need for new drugs with
improved properties. A large body of evidence from basic and clinical research supports the view
that – besides being intimately involved in normal physiological functioning of the nervous
system – both voltage-gated sodium channels (NaV) and N-methyl-D-aspartate receptors
(NMDAR) play important role in chronic pain states (Amir et al., 2006; Gogas, 2006).
Furthermore, among other diseases, NMDARs may be involved in adaptive changes of neuronal
structures associated with alcohol- and drug abuse, and the expression of withdrawal symptoms
(Nagy, 2004).
Both NaV blockers and NMDAR antagonists proved to be efficacious in alleviating
symptoms in several in vitro and in vivo experimental models of the above diseases. However,
the use of presently available agents with these mechanisms of action is hindered by several
disadvantageous properties, such as low efficacy, lack of selectivity, side effects. However,
selection of new NaV blocking agents suitable for drug development has been greatly impeded by
the fact that (until very recently) no suitable methods that had sufficiently high throughput, and
supplied with clinically relevant information about the mechanism of the NaV blocking action of
the tested compounds were available. Of the NMDAR blocking compounds, it is those agents that
selectively inhibit NR2B subunit containing receptors, i.e. NR2B-selective agents, hold the
promise of being free from side effects, which prevent the use of non-selective agents in most
cases. Recognition of the peculiar molecular mode of action of these agents, and that of the
restricted expression pattern of the NR2B subunit allow some optimism (Gogas, 2006;.Loftis and
Janowsky, 2003)
This work consists of two main parts. I worked out and introduced an in vitro
pharmacological method suitable for supplying information about the state-dependence of the
action of the blocker on NaV. I used this method for an analysis of the mechanism of action of
tolperisone-like muscle relaxant agents. The second part of my work aimed at establishing
structure-activity relationship among new compounds with NR2B-selective NMDA antagonistic
action, characterizing their pharmacological properties, and verifying their selectivity.
Furthermore I investigated the action of some already known and newly synthesized NR2Bselective antagonists in an in vitro model of alcohol dependence and withdrawal.
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2. Aims of the study
1. One of the main goals was to establish and validate a novel, high capacity method, based
on fluorometric measurement of the membrane potential, which is suitable for
determination of the potency of voltage-gated sodium channel blocking agents.
2. We investigated whether our fluorometric method can supply suitable information about
possibly differential affinity of the blockers to different functional states of NaV.
3. We wanted to see if NaV inhibitory effect has a role in the spinal reflex inhibitory activity
of tolperisone.
4. Our second main goal was to work out and validate another high-throughput assay, based
on fluorometric measurement of cytoplasmic calcium changes, which is suitable for
supplying reliable data about the efficacy of new NR2B-selective antagonists for
establishing structure-activity relationship.
5. We intended to use this method for studying new compounds and establishing structureactivity relationships among them.
6. We investigated if old, or newly developed NR2B-selective antagonists can have an effect
in cell death elicited by alcohol withdrawal in neuronal cultures that had been chronically
treated with alcohol.
3.
Materials and methods
3.1.
Preparation and maintenance of cultures
Cortical cultures were prepared from 17-day-old rat embryos. Cells were plated into 96-
well plates pre-coated with poly-D-lysine in Dulbecco’s modified Eagle Medium (DMEM)
containing 10 % foetal bovine serum (FBS), antibiotics and vitamins.
Cerebellar cultures were prepared from 4-day old rats. Cells were plated into 96-well
plates pre-coated with poly-D-lysine in the same medium detailed above (+20 mM KCl).
EcR 293 cells stably and inducibly expressing recombinant NMDAR subunits were plated
into96-well plates pre-coated with poly-D-lysine in DMEM containing 10 % FBS and antibiotics.
Expression of the NMDAR subunits were induced by the addition of 1 μM muristerone A after
one day in culture. Cells were also treated with 500 µM ketamine to prevent the citotoxic effects
of the expression of NMDARs. All three types of cultures were kept in sterile incubators until
they were used for measurements (37 ºC. 5 % CO2).
3.2.
Membrane potential determination with fluorometry
Cerebellar cells cultures were loaded with a fluorescent membrane potential dye (FLIPR
membrane potential kit) after 6-9 days in culture. Fluorescence was measured byFlexStation II, a
plate reader fluorometer (excitation: 530 nm, emission: 565 nm) according to two types of
protocols. the preincubation protocol where cells were preincubated with the drugs before the
addition of the depolarizing agent veratridine (VER), and the reverse protocol, where the
depolarization of the cells by a supramaximal dose of VER preceded the administration of drugs.
3.2.1. Preincubation protocol
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After dye loading cells were treated with buffer containing vehicle or drugs and were
incubated for 10 min at 37 ºC. After monitoring baseline fluorescence for 20 s cells were
depolarized by the addition of KCl or VER using the pipettor of FlexStation and fluorescence
changes were followed for an additional 100-460 s. Data were expressed as ΔF/F-values as
follows: ΔF/F=(Fmax-Fmin)/Fmin, where Fmax was the maximum, Fmin was the minimum
fluorescence in the given well. In the dose-response experiments with VER data were expressed
as % of the maximum response. In the concentration-inhibition measurements effect of the
blockers was quantified as % inhibition of the control VER response.
3.2.2. Reverse protocol
After monitoring baseline fluorescence for 20 s cells were depolarized by the addition of a
supramaximal dose of VER and the fluorescence was monitored for 130 s. Then the blockers
were added to the cells the fluorescence was monitored for an additional 300 s. effect of the
blockers was quantified as % inhibition of the VER response.
3.3.
[3H]-BTX-B binding assay
Synaptosomes prepared from the cerebral cortices of 7-8 weel old rats were incubated in
the presence of 5 nM [3H]-BTX-B, 1 μM TTX, 40 μg/ml scorpion toxin and the test compounds
for 1 h at 37 ºC. Nonspecific binding was determined with 300 μM aconitine. After incubation
the synaptosomes were separated from the unbound radioligand by rapid filtration using 96-well
filter plates. Radioactivity traped on the filter plates was determined by liquid scintillation
spectrometry using Microscint 20 scintillation cocktail and a Topcount NXT counter.
3.4.
Sodium current measurements with whole-cell patch clamping
Whole-cell patch clamp measurements were carried on neurons of 6-12 day old cortical
cultures. Patch electrodes containing intracellular solution had resistances of 1.5–2.5 MΩ. Cells
were held at –55 mV holding potential. Sodium currents were evoked by 8-ms-long rectangular
step depolarizations to 0 mV at 10-s intervals. Drugs were applied onto the cells via multibarreled
ejection pipettes. Effects of drugs were quantified as % inhibition of the peak current evoked by
the depolarization in the absence of the drug.
3.5.
Spinal reflex study, in vitro, in the hemisected spinal cord preparation
Six-day old rats were anesthetized with ether and after the respiration stopped the spinal
chord was isolated and hemisected along the midline. Hemicords were transferred into a storage
chamber, and were superfused with buffer. Glass suction electrodes were used both for
stimulation (L5 dorsal root) and recording (L5 ventral root). Stimulation was carried out with
square-wave anodic current pulses (100 μs, 200 μA, at 30 s intervals). Effects of drugs were
quantified as % inhibition of the peak of the action potential evoked by the stimulus in the
absence of the drug.
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3.6.
Measurement of cytoplasmic Ca2+-concentration ([Ca2+]i) with fluorometry
Fluorometric measurements of [Ca2+]i were carried out using 3-7 day old cortical cultures
or EcR293 cells expressing recombinant NMDAR subunit combinations (NR1-1a/NR2B or NR13/NR2A.) 48-72 h after the induction of receptor expression by muristerone A. Cells were loaded
with a Ca2+-sensitive fluorescent dye, fluo-4/AM. After washing buffer containing vehicle or the
drugs was aded to the cells and fluorescence was monitored by a Fluoroskan Ascent plate reader
(excitation: 485 nm, emission: 538 nm). After monitoring baseline fluorescence for 5 min cells
were stimulated by the addition of NMDA (40-100 μM) using the dispenser of Ascent and
fluorescence changes were followed for an additional 5-15 min. Data were expressed as ΔF/Fvalues. Effects of the antagonists were quantified as % inhibition of the control NMDA response.
3.7.
Cytotoxicity elicited by alcohol withdrawal in cortical cultures
Cultures were treated with ethanol on the 10th day by replacing half of the culture
medium with fresh medium containing 100 mM ethanol. This treatment was repeated once daily
for three consecutive days. After the 3-day ethanol treatment, ethanol was eliminated from the
cells by replacing the ethanol-containing medium with fresh medium containing different
concentrations of the test compounds, and the cells were incubated for 24 h. Neuronal cell death
caused by the 24 h ethanol-withdrawal was quantified by measuring lactate dehydrogenaserelease into the culture medium.
4.
Results
1. We established a novel, high capacity method, based on fluorometric measurement of the
membrane potential, where VER was used to activate sodium channels in cerebellar cells, for
determination of the potency of voltage-gated sodium channel blocking agents. Using wellknown blockers we demonstrated that their potencies determined in our assay are comparable to
published results obtained by other methods.
2. The potency of certain agents was highly dependent on the sequence of application: they were
considerably more potent when applied after the depolarizing effect of VER had already
developed as compared with a pre-incubation protocol. Other blockers displayed only small
differences. This observation may be related to the higher affinity of some of the agents to the
inactivated state. Ours is the first non-electrophysiological method that is capable of supplying
relevant information in this regard.
3. We showed that tolperisone and all compounds with similar structure we have tested so far
specifically bind to NaVs and inhibit them. Furthermore, we demonstrated that - very probably –
this NaV blocking effect underlies their central muscle relaxant activity, and ability to depress
spinal reflexes.
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4. We worked out a high capacity method, based on fluorometric monitoring of cytoplasmic
calcium concentration, for measuring the in vitro potency of NR2B-selective blockers. We
showed that relevant data can be obtained about the potency of NR2B-selective, and nonselective NMDA antagonists by measuring changes in the intracellular calcium concentration,
evoked by NMDA application.
5. Using this fluorometric assay, with supplying potency data for a great-number of molecules,
we contributed to the establishment of structure-activity relationships among newly synthesized
compounds, and to the analysis of these data. As a result, several structures belonging to different
chemical classes have been identified. Several new compounds, such as RG-13579 and RG-1103,
have been selected. These compounds possess comparable, or better in vitro and in vivo potencies
selectivity, and pharmacokinetic properties than the presently known NR2B antagonists. RGH896 is a promising drug candidate at the clinical phase of development.
6. We have shown that NR2B-selective antagonists efficiently block neuronal death elicited by
alcohol withdrawal in cortical cultures treated chronically with alcohol. Their potency to inhibit
cell toxicity correlated well with their effect on NMDA induced intracellular calcium changes as
determined by fluorometry. RG-1103 was the most potent to inhibit alcohol withdrawal induced
cell death.
5.
Discussion
5.1.
Studies on NaV blockers with membrane potential fluorometry
The sodium channel activator veratridine (VER) induced concentration-dependent
depolarization in cultured rat cerebellar cells plated on 96-well plates, which was detected by a
membrane potential fluorescent dye. All NaV blocking agents tested inhibited VER-induced
depolarization concentration dependently. Molar efficacy of the blockers depended on VER
concentration and on the application order of VER and the blocker. Response to a higher
concentration of VER was more difficult to block. Furthermore, a subset of the blockers was
considerably more efficient when applied after the depolarizing action of VER had already
developed. This latter phenomenon may be related to the different affinities of various NaV
blockers to the different functional states of the channel. It is widely accepted, that this feature
serves as a basis of the advantageous therapeutic properties of NaV blocking drugs.
It is well known from earlier electrophysiological studies that many drugs with NaV
blocking properties, such as phenytoin, lamotrigine, lidocain, etc., display characteristic voltage-,
and/or use dependent inhibitory pattern. Such agents are typically 10-100 times more efficient
when the membrane is depolarized, or the cell is firing with high frequency (Hille, 1977;
Ragsdale at al., 1991). This phenomenon is generally explained with the “modulated receptor
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theory” (Hille 1977), according to which certain blockers bind to the inactivated channels with
much higher affinity than to the channels in the resting state. This implies that that these statedependently acting blockers do not affect normal neuronal functions at concentrations that
already cause a substantial inhibition of neurons that are in a sustained depolarized state, or
display high-frequency spiking activity (Clare et al., 2000). The development of such NaV
blocking new agents has been hindered by the fact that electrophysiology, which represent the
only method capable of giving information about the state-dependence of action of a drug, has
too low throughput for rapid testing and characterizing of sufficiently high number of compounds
for industrial drug research.
Several research groups reported experiments where membrane potential fluorometry was
used to determine the NaV blocking potency of drugs, in cell lines expressing recombinant NaVs
(Benjamin et al., 2006). Nevertheless, comparison of data obtained in expression systems with
those from native preparations frequently results in contradictions. Results from native
preparations, like our primary cerebellar cultures, have probably higher relevance regarding
human diseases.
In our experiments the EC50 of VER was 8 µM, which is good agreement with published
results obtained with various methods. VER was found a specific activator of NaV, since neither
excitatory amino acid receptor antagonists nor subtype-selective blockers of voltage-gated
calcium channels had notable effect on VER response. Glutamate and calcium channels may
depolarize cultured neurons, but according to our data they do not play a role in VER induced
depolarization under our experimental conditions.
All known NaV blockers that we tested inhibited VER induced depolarization
concentration-dependently. Low concentrations of TTX caused a complete inhibition of VER
responses, which is in agreement with literature data about the expression pattern of NaVs in the
cerebellum. Blocking potencies measured against EC80 of VER were comparable to those
determined in other functional assays and published in the literature. We tested a few known Na V
blockers (lamotrigine, carbamazepine, phenytoin) against different concentrations of VER, and
found that blocking potencies were invariably in inverse relationship with VER concentration, i.e.
the blockers were less potent to inhibit responses to higher VER concentrations. TTX behaved
similarly to these blockers in this regard. Further, lamotrigine caused a rightward shift of the
dose-response curve of VER, without changing maximal depolarization. In similar experiments,
TTX, as expected, displayed an inhibition pattern characteristic of non-competitively acting
drugs. These results of our own and some data coming from other laboratories suggest a
competitive-type interaction between VER and NaV blockers that act at the local anaesthetic (LA)
site, which we studied. Several point-mutation data, however, indicates that the LA site and the
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toxin-2 binding site, where VER binds to, are largely overlapping, but not identical (Nau and
Wang, 2004). The inverse relationship between the concentration of VER and the potency of
TTX, which we found, is in conflict with the observation of some laboratories, but in agreement
with those of others. Nevertheless, the dependence of the blocking potency of compounds on the
concentration of VER in our experiments, together with the findings of some other laboratories
(Felix et al., 2004), clearly show that setting VER concentration is a critical parameter in
functional studies, where NaV blocking potencies of drugs are measured and compared. In order
to compare potencies reliably, it is advisable to control this parameter exactly.
The major observation of this study was that phenytoin, carbamazepine, lamotrigine and
lidocaine displayed substantially higher potencies when applied after the maximal depolarizing
action of VER had already developed (post-treatment protocol), as compared with the situation
when their application preceded that of the toxin (pre-treatment protocol). Results obtained in the
post-treatment protocol can be explained by supposing that VER, initially binding just to a
relatively small portion of the channels, depolarizes the cells, thus the majority of NaVs (that do
not bind VER) becomes inactivated due to the depolarizing action of the toxin. These inactivated
channels bind state-dependently acting blockers with high affinity, thus their inactivated state is
stabilized. This decreases the number of channels available for VER binding, since VER binds to
the open state of the channels (Barnes and Hille, 1988). On the other hand, using the pretreatment protocol, the majority of channels are in the resting state at the time of drug application,
thus bind the blocker with low affinity, and thus higher proportion of the channels remains
available for VER activation. There may be alternative explanations besides ours. It is
conceivable that in experiments run according to the post-treatment protocol blockers bind with
high affinity to one of the states of the VER-bound channels rather than to the toxin-free,
inactivated ones. A further possible explanation of our observations may be that some blockers
specifically act on the open state of NaVs (“open channel block”). Our results with GBR-12909,
which binds to the resting and inactivated states of NaV with similar affinities (Mike et al., 2004),
however, clearly support our original hypothesis, since the difference between potencies of this
compound determined in the pre- and post-treatment protocols was much smaller than with the
other blockers. We suggest that the ratio of the potencies measured according to the two protocols
may be suitable for drawing conclusions regarding the extent of state-dependence of the blocking
action of a compound.
We showed that tolperisone and all the compounds with similar structure that we tested
bind to the voltage-gated sodium channels specifically, and block them. Furthermore, we
demonstrated that probably this NaV blocking action underlies the muscle relaxant and spinal
reflex inhibitory effects of these compounds.
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5.2.
Pharmacological characterization of new, NR2B-selective NMDA antagonists
A cell-based Ca2+-fluorometry assay was used for primary testing of the compounds.
This method was found suitable for identification and characterization of all compounds acting
on the NMDA receptors independently from their binding site, or mechanism of action. Using
both primary cortical cultures and cell lines expressing recombinant NR1-1a/NR2B NMDA
receptors, we demonstrated that known NMDA antagonists either non-selective, or selective to
NR2B, inhibited NMDA-evoked elevation of intracellular calcium in a concentration-dependent
manner, and the molar potencies we determined were similar to those published in the literature.
Besides preserving, or possibly increasing the high efficacy characterizing the known NR2B
antagonist compounds CP-101,606, or Ro-256981, the most important goal with planning new
compounds was to increase selectivity over other receptors and ion channels. The above blockers
are known to possess undesirably high affinity to adrenergic receptors and hERG potassium
channels.
As a result of structure-activity relationship data, based largely on the biological data
obtained with the calcium assay, several compounds with high in vitro and in vivo potencies have
been identified and selected for development, among others RG-13579 and RG-1103. We
demonstrated that these compounds are potent, subtype-selective and non-competitive inhibitors
of NMDA receptors in both native and recombinant systems (Farkas et al., 2002). Furthermore,
we have shown that NR2B-selective antagonists known from the literature, as well as RG-13579
and RG-1103 efficiently block neuronal death elicited by alcohol withdrawal in cortical cultures
treated chronically with alcohol. These results, together with the data indicating an enhanced
expression and function of NMDA receptors by chronic ethanol treatment (Nagy, 2004), and the
relatively low side-effect profile of the presently available NR2B selective antagonists (Gogas,
2006) suggest that NR2B-selective antagonists can be suitable pharmacological tools to cure
alcohol dependence.
Probably the most important result of this work is the contribution to the selection and
development of the oxamide compound RGH-896, which is at the clinical phase II of
development for neuropathic pain indication (Farkas et al., 2003). RGH-896 is regarded as one
of the most promising drug candidates presently under development in this category.
6.
References
Amir R et al. (2006) The role of sodium channels in chronic inflammatory and neuropathic pain. J Pain 7: S1-29.
Barnes S, Hille B (1988) Veratridine modifies open sodium channels. J Gen Physiol 91: 421-443.
Benjamin ER et al. (2006) State-dependent compound inhibition of Nav1.2 sodium channels using the FLIPR Vm
dye: on-target and off-target effects of diverse pharmacological agents. J Biomol Screen 11: 29-39.
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Clare JJ et al.(2000) Voltage-gated sodium channels as therapeutic targets. Drug Discov Today 5: 506-520.
Farkas S et al. (2002) NR2B-NMDA receptor antagonists for the treatment of chronic or neuropathic pain.
Pharmacologist 2002 44(2, Suppl. 1): Abst 37.13.
Gogas KR (2006) Glutamate-based therapeutic approaches: NR2B receptor antagonists. Current Opinion In
Pharmacology 6: 68-74.
Hille B (1977) Local anesthetics: hydrophilic and hydrophobic pathways for the drug-receptor reaction. J Gen
Physiol 69: 497-515.
Loftis JM, Janowsky A (2003) The N-methyl-D-aspartate receptor subunit NR2B: localization, functional properties,
regulation, and clinical implications. Pharmacol Ther 97: 55-85.
Mike A et al. (2004) A novel modulatory mechanism of sodium currents: frequency-dependence without statedependent binding. Neuroscience 125: 1019-1028.
Nagy J (2004) Renaissance of NMDA receptor antagonists: do they have a role in the pharmacotherapy for
alcoholism? IDrugs 7: 339-350.
Nau C, Wang GK (2004) Interactions of local anesthetics with voltage-gated Na+ channels. J Membr Biol 201: 1-8.
Ragsdale et al. (1991) Frequency and voltage-dependent inhibition of type IIA Na+ channels, expressed in a
mammalian cell line, by local anesthetic, antiarrhythmic, and anticonvulsant drugs. Mol Pharmacol 40: 756-765.
Papers of the author relevant to this study
Sándor Kolok, József Nagy, Zsolt Szombathelyi and István Tarnawa (2006) Functional characterization of sodium
channel blockers by membrane potential measurements in cerebellar neurons: Prediction of compound preference for
the open/inactivated state. Neurochemistry International [Epub ahead of print] doi:10.1016/j.neuint.2006.04.014
Pál Kocsis, Sándor Farkas, László Fodor, Norbert Bielik, Márta Thán, Sándor Kolok, Anikó Gere, Mónika Csejtei,
and István Tarnawa (2005) Tolperisone-type drugs inhibit spinal reflexes via blockade of voltage-gated sodium and
calcium channels. Journal of Pharmacology and Experimental Therapeutics 315:1237–1246.
József Nagy, Csilla Horváth, Sándor Farkas, Sándor Kolok and Zsolt Szombathelyi (2004) NR2B subunit selective
NMDA antagonists inhibit neurotoxic effect of alcohol-withdrawal in primary cultures of rat cortical neurones.
Neurochemistry International 44:17-23.
István Borza, Sándor Kolok, Anikó Gere, Éva Ágai-Csongor, Béla Ágai, Gábor Tárkányi, Csilla Horváth, Gizella
Barta-Szalai, Éva Bozó, Csilla Kiss, Attila Bielik, József Nagy, Sándor Farkas and György Domány (2003) Indole-2carboxamides as novel NR2B selective NMDA receptor antagonists. Bioorganic & Medicinal Chemistry Letters
13:3859–3861.
Gizella Barta-Szalai, István Borza, Éva Bozó, Csilla Kiss, Béla Ágai, Ágnes Proszenyák, György M. Keserű, Anikó
Gere, Sándor Kolok, Kornél Galgóczy, Csilla Horváth, Sándor Farkas and György Domány (2004) Oxamides as
novel NR2B selective NMDA receptor antagonists. Bioorganic & Medicinal Chemistry Letters 14:3953–3956.
István Borza, Sándor Kolok, Györgyi Ignácz-Szendrei, István Greiner, Gábor Tárkányi, Csilla Horváth, Sándor
Farkas and György Domány (2005) Indole-2-carboxamidines as novel NR2B selective NMDA receptor antagonists.
Bioorganic & Medicinal Chemistry Letters 15:5439–5441.
István Borza, Sándor Kolok, Anikó Gere, József Nagy, László Fodor, Kornél Galgóczy, József Fetter, Ferenc
Bertha, Béla Ágai, Csilla Horváth, Sándor Farkas and György Domány (2006) Benzimidazole-2-carboxamides as
novel NR2B selective NMDA receptor antagonists. Bioorganic & Medicinal Chemistry Letters 16:4638–4640.
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