Design, Synthesis, and Biological Evaluation of a New Series of Carvedilol Derivatives That Protect Sensory Hair Cells from Aminoglycoside-Induced Damage by Blocking the Mechanoelectrical Transducer Channel

Article abstract of DOI:10.1021/acs.jmedchem.8b01325

Aminoglycosides (AGs) are broad-spectrum antibiotics used for the treatment of serious bacterial infections but have use-limiting side effects including irreversible hearing loss. Here, we assessed the otoprotective profile of carvedilol in mouse cochlear cultures and in vivo zebrafish assays and investigated its mechanism of protection which, we found, may be mediated by a block of the hair cell's mechanoelectrical transducer (MET) channel, the major entry route for the AGs. To understand the full otoprotective potential of carvedilol, a series of 18 analogues were prepared and evaluated for their effect against AG-induced damage as well as their affinity for the MET channel. One derivative was found to confer greater protection than carvedilol itself in cochlear cultures and also to bind more tightly to the MET channel. At higher concentrations, both carvedilol and this derivative were toxic in cochlear cultures but not in zebrafish, suggesting a good therapeutic window under in vivo conditions.

Full text of DOI:10.1021/acs.jmedchem.8b01325

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Article
Design, synthesis and biological evaluation of a new series of carvedilol
derivatives that protect sensory hair cells from aminoglycoside-
induced damage by blocking the mechano-electrical transducer channel
Molly O'Reilly, Nerissa K Kirkwood, Emma J Kenyon, Rosemary Huckvale, Daire M Cantillon,
Simon J Waddell, Simon E Ward, Guy P Richardson, Corne J. Kros, and Marco Derudas
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01325 • Publication Date (Web): 14 May 2019
Downloaded from http://pubs.acs.org on May 14, 2019
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Design, synthesis and biological evaluation of a new series of carvedilol
derivatives that protect sensory hair cells from aminoglycoside-induced
damage by blocking the mechano-electrical transducer channel
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‡
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Molly O’Reilly , Nerissa K. Kirkwood , Emma J. Kenyon , Rosemary Huckvale , Daire
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⊥
‡§
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†
M. Cantillon , Simon J. Waddell , Simon E. Ward , Guy P. Richardson , Corné J. Kros
and Marco Derudas‡*
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†Sussex Neuroscience, School of Life Sciences, University of Sussex, Falmer, Brighton, BN1
9QJ, UK
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‡Sussex Drug Discovery Centre, School of Life Sciences, University of Sussex, Falmer,
Brighton, BN1 9QJ, UK
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⊥Wellcome Trust Centre for Global Health Research, Brighton and Sussex Medical School,
University of Sussex, Falmer, Brighton, BN1 9PX, UK
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§Medicines Discovery Institute, Cardiff University, Park Place, Cardiff, CF10 3AT, United
Kingdom
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* Email: m.derudas@sussex.ac.uk; Phone +44(0)1273876591
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Abstract:
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Aminoglycosides (AGs) are broad-spectrum antibiotics used for the treatment of serious
bacterial infections but have use-limiting side effects including irreversible hearing loss. Here,
we assessed the otoprotective profile of carvedilol in mouse cochlear cultures and in vivo
zebrafish assays and investigated its mechanism of protection which we found may be mediated
by block of the hair cell’s mechano-electrical transducer (MET) channel, the major entry route
for the AGs. To understand the full otoprotective potential of carvedilol, a series of 18 analogues
were prepared and evaluated for their effect against AG-induced damage as well as their affinity
for the MET channel. One derivative was found to confer greater protection than carvedilol itself
in cochlear cultures, and also to bind more tightly to the MET channel. At higher concentrations
both carvedilol and this derivative were toxic in cochlear cultures but not in zebrafish, suggesting
a good therapeutic window under in vivo conditions.
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Introduction:
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AGs are broad-spectrum antibiotics widely prescribed to treat severe bacterial infections.1-3
Despite being highly efficacious, they cause unfortunate side effects such as reversible
nephrotoxicity and irreversible hearing loss, with the latter occurring in up to 25% of treated
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patients. The AGs can enter the sensory hair cells of the inner ear through both endocytic
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processes and specialised cation channels, the MET channels that are located at the tips of the
stereocilia and are responsible for the detection of sounds and body movements.6-9 The
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mechanism of ototoxicity is not fully understood and it differs amongst the various AGs, with
neomycin and gentamicin for instance being shown to activate different cell-death pathways
once inside zebrafish lateral line hair cells.10-12 Once inside the cell they are thought to interact
with various targets such as ribosomes, the endoplasmic reticulum and mitochondria13, 14 leading
to the production of cytotoxic levels of reactive oxygen species (ROS) which, in turn, cause
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apoptosis. It is this AG-induced hair cell death that underlies the hearing loss associated with
clinical drug treatments. Aside from the redesign of novel AGs16, 17 or hair cell regeneration
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approaches, methods aimed at preventing this use-limiting side effect have primarily focussed
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on either preventing the entry of AGs into hair cells by identifying blockers of the MET
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channel or by reducing the cellular accumulation of ROS, often by application of antioxidants,
in an attempt to prevent the induction of apoptosis.14, 19 Recent efforts in this field have led to the
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identification of otoprotective agents able to reduce or prevent the AG-induced hearing loss both
in in vitro20-23 and in in-vivo models of AG ototoxicity.24
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Here we investigated the otoprotective potential of carvedilol, an FDA-approved non-selective
α1- and β-adrenergic blocker (Figure 1), used clinically for the treatment of hypertension, angina
and symptomatic chronic heart failure. Carvedilol has previously been reported to protect against
neomycin damage in lateral line hair cells of zebrafish when tested at 10 µM against 200 µM
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neomycin. In addition, carvedilol did not abrogate the antimicrobial properties of neomycin,
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making any interference with the bactericidal activity of AGs unlikely and making it an ideal
chemical starting point for further investigation.
H CO
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O
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Carvedilol
H
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Figure 1: structure of carvedilol, a non-selective α1- and β-adrenergic blocker.
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In this study, we report its protective properties against gentamicin-induced hair cell damage in
mouse cochlear cultures. We propose the mechanism by which it offers otoprotection with data
supporting the block of the MET channel as being responsible for its otoprotective effect. We
synthesised and evaluated a series of novel carvedilol derivatives aimed at improving the
protective efficacy and affinity for the MET channel, whilst concurrently reducing toxicity as
well as this compound as chemical starting point for future drug development
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Carvedilol protects mammalian sensory hair cells from gentamicin damage
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Mouse cochlear cultures were used to assess whether carvedilol can protect mammalian hair
cells from the death induced by exposure to 5 µM gentamicin for 48 h. A concentration of 5 µM
gentamicin is optimal, as it is close to the estimated concentration of 1 µM gentamicin reached in
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the endolymph in vivo at the onset of ototoxic symptoms and it kills >90% of the basal cells
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while sparing the apical cells, consistent with the predominantly high-frequency hearing loss
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observed in patients treated with aminoglycosides. On average, incubation with 5 µM
gentamicin caused a loss of 86% of outer hair cells (OHCs) from the mid-basal region of the
cochlea, with 110 ± 2.2 (n = 26) OHCs present in a 300 µm long segment of the control and only
15 ± 2.2 (n = 26) in the gentamicin-treated culture (p ˂ 0.001) (Figure 2A, B). Subsequently,
mouse cochlear cultures were co-incubated with 5 µM gentamicin together with escalating
concentrations of carvedilol with the aim to identify the minimal concentration required to
provide full protection. When tested at 5 µM, carvedilol provided partial protection, with OHC
survival differing significantly from that in both the control and gentamicin-only treated cultures
(p <0.001 in both cases), suggesting that this concentration is at the limit of its protective
efficacy (Figure 2C). When tested at 10 µM and 20 μM carvedilol provided complete protection
against the gentamicin-induced loss of OHCs (p ˂0.001) (Figure 2D, E respectively). Higher
concentrations of carvedilol (≥30 μM) proved to be generally cytotoxic, with widespread cell
death observed (Figure 2F).
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Figure 2. Protective effect of carvedilol against gentamicin-induced damage in a mouse cochlear
culture assay. Control cultures exposed to either 0.5% DMSO (A) or 5 μM gentamicin + 0.5%
DMSO (B) for 48 h. When co-incubated with 5 μM gentamicin, carvedilol was found to be
partially protective at 5 μM (C), fully protective at 10 and 20 μM (D and E respectively) and
generally cytotoxic at ≥30 μM (F). The asterix in (E) indicates an example of IHC hair bundle
disruption. Scale bar is 50 μm.
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When carvedilol was tested alone no OHC death was observed at either 10 µM or 20 μM, with
hair cell numbers similar to those observed in controls (Figure 3 A-D). Some degree of inner hair
cell (IHC) damage and disruption to hair bundle morphology in both IHCs and OHCs can be
observed when tested at 20 μM both alone and in combination with 5 μM gentamicin (Figure 2E,
Figure 3D). Figures 3E and F show confocal images of IHC stereociliary bundles in control
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conditions (Figure 3E) and after exposure to 20 μM carvedilol (Figure 3F), revealing the full
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extent of morphological disruption caused by carvedilol.
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At concentrations ≥30 µM, carvedilol is generally cytotoxic to all cell types in cochlear cultures,
both alone and in the presence of 5 µM gentamicin (Figure 2F, Figure 3G, H). Figure 3I
summarises the quantification of OHC survival through analysis of the mid-basal region of
cochlear cultures for both carvedilol alone and also co-exposed with gentamicin.
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Figure 3. Carvedilol disrupts mechanosensory hair bundles at 20 μM, and generally cytotoxic in
vitro at ≥30 μM. Cochlear cultures were exposed for 48 h to 0.5% DMSO (A and E), 5 μM
gentamicin + 0.5% DMSO (B), and escalating concentrations of carvedilol: 10 μM (C), 20 μM
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(D and F), 30 μM (G) and 40 μM (H). Scale bar is 10 μm (F) and 50 μm (H). Quantification of
hair cell survival in a 300 µm long segment of the mid-basal region of cochlear cultures is
reported in I.
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Carvedilol exerts its otoprotective effect by acting as a permeant blocker of the hair cell’s
MET channel
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Despite a narrow therapeutic window in vitro, we decided to investigate the potential mechanism
by which carvedilol provides its protective effect. It is well established that AGs can enter hair
cells via their MET channels7, 9 and that block of this channel reduces or prevents their entry into
the cells, thereby protecting them from any toxicity resulting from intracellular accumulation. To
determine whether carvedilol protects via an interaction with the MET channel, we recorded
MET currents from OHCs both before and during extracellular superfusion of carvedilol at
concentrations of 1, 3 and 10 µM.
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Figure 4A shows an example of the MET currents recorded before (black), during (red) and after
(blue) exposure to carvedilol (10 µM), at membrane potentials ranging from -164 mV to +96
mV. Carvedilol reduces the size of MET currents at all membrane potentials, with this reduction
particularly pronounced at intermediate and depolarized potentials. Upon re-exposure to the
control solution the currents recover, indicating a reversible block of the channel. The voltage-
dependent block and subsequent recovery of the currents can also be clearly seen from the
current-voltage relationships shown in Figure 4B.
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Figure 4. Extracellular exposure to carvedilol reduces OHC MET currents at all potentials, with
the reduction most pronounced at intermediate and depolarized potentials. (A) MET currents
recorded from a basal-coil OHC between -164 and +96 mV before, during and after exposure to
10 µM carvedilol. (B) Current-voltage relationships of the currents shown in A reveal the current
block at all potentials during carvedilol exposure and the reversibility of the block following
washout. The capacitance of the cell was 7.4 pF.
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Average normalized current voltage relationships derived from all cells recorded from at the
three different concentrations tested, normalized to the maximum control current at +96 mV for
each cell, demonstrate both the increase in the block with increasing compound concentration
and the voltage-dependence of the block, with the strongest block observed at the intermediate
and depolarized potentials (Figure 5A).
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Figure 5. Carvedilol acts as a relatively high-affinity permeant blocker of the MET channel. (A)
Average normalized current-voltage relationships for the peak MET currents recorded before and
during exposure to 1, 3 and 10 µM carvedilol with currents normalized to the peak control
current measured at +96 mV for each cell. (B) Fractional block curves of the current during
carvedilol exposure (1, 3 and 10 µM) relative to the control current at each membrane potential.
Data are fitted with a two-barrier, one binding site model (see Experimental Section).
Parameters for the fits are ΔE (the difference in free energy between the intracellular and
extracellular barrier) -0.63 kT; Eb (the free energy of the binding site) -15.4 kT; δb (the electrical
distance of the binding site from the extracellular side) 0.53; z (the electrical charge of the
blocker) 1.0; Hill coefficient 1.0. Maximum block occurs at +12.8 mV. (C) Dose-response
curves derived from the currents recorded at -164, +16 and +96 mV and fit with equation (1). -
164 mV: K 6.3 µM, Hill coefficient 1.1; +16 mV: K 0.1 µM, Hill coefficient 0.5; +96 mV: K
1
1
12
1
3
D
D
D
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
6
1
2
1.5 µM, Hill coefficient 0.9. (D) Variations in the half-blocking concentration and Hill
coefficient at each membrane potential.
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
5
6
7
8
9
0
Some recovery of the currents can be seen at the extreme depolarized potentials (+96 mV), with
even more pronounced recovery at the extreme hyperpolarized potentials (-164 mV). This
recovery with hyperpolarization is more evident from the average fractional block curves
showing the current during carvedilol superfusion relative to the control current at each
membrane potential (Figure 5B). Maximum block is seen at the intermediate membrane
potentials for each concentration of carvedilol with some recovery at depolarized potentials and
even greater recovery at the extreme hyperpolarized potentials. This recovery at the
hyperpolarized potentials is indicative of a permeant blocker with the compound, positively
charged at physiological pH (calculated pKa = 8.7), being drawn into the cell by the electrical
driving force and therefore reducing the block of the channel. Fitted curves are to a two-barrier,
one-binding site model of the MET channel permeation pathway. Dose-response curves for the
extracellular block of the MET channels by carvedilol were generated at each membrane
potential and fitted with equation (1) described in the experimental section. The dose-response
curves derived from the currents at -164 mV, +16 mV and +96 mV are shown in Figure 5C. The
1
11
1
1
2
3
14
1
1
1
1
1
2
5
6
7
8
9
0
K values range from 6.3 µM at -164 mV to 0.1 µM at +16 mV, close to the potential at which
D
the block was strongest (+12.8 mV). The K at -84 mV was 2.0 µM, lower than that previously
D
reported for the AG dihydrostreptomycin (DHS), which was found to have a K of 7.0 µM at -84
D
2
+ 7
21
mV in 1.3 mM extracellular Ca , indicating that carvedilol is a relatively high affinity blocker
2
2
2
3
of the MET channel. Figure 5D reports the K and Hill coefficient values for each dose-response
D
curve showing the strongest interaction near +16 mV. The Hill coefficients ranged from 0.5 to
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1.3 suggesting that two molecules may interact with the channel, potentially showing negative
cooperativity.26
2
6
7
8
3
4
5
These results demonstrate that carvedilol is a relatively high-affinity permeant blocker of the
MET channel and consistently protects OHCs from AGs damage at 10 and 20 µM. However, in
vitro, it appears to be cytotoxic at higher concentrations.
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
7
Chemistry
8
9
0
1
Driven by these results, we decided to investigate the potential of carvedilol as potential
chemical starting point for future drug development. We aimed at enhancing its protective effect
in mouse cochlear cultures, increasing its block of the MET channel current and reducing the
cytotoxicity observed in vitro. We decided to investigate its structure by modifying the carbazole
moiety, the anisole ring and the β-hydroxyl amino group. We firstly synthesised compound 2, by
substituting the carbazole with anisole. The synthesis involved the preparation of 1 by coupling
2-methoxyphenol and 2-(bromomethyl)oxirane followed by epoxide ring opening with
commercially available 2-(2-methoxyphenoxy)ethanamine in ethylene glycol dimethyl ether at
80 °C providing the desired compound in moderate yield.
1
1
12
1
1
3
4
15
1
1
6
7
Scheme 1
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
OCH3
OH
OCH3
OCH3
O
i
O
ii
O
O
N
H
OCH3
OH
2, 25%
1
, 62%
iii
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
OCH3
OCH3
OCH3
iv
O
O
O
Br
N
H
3, 22%
4, 40%
o
Reagents and conditions: (i) 2-(bromomethyl)oxirane, anhydrous K CO , DMF, 70 C, 6 h; (ii) 2-(2-
2
3
o
methoxyphenoxy)ethanamine, ethylene glycol dimethyl ether, 80 C, 6 h; (iii) 1,2-dibromoethane, NaOH,
o
water, reflux, 3 h; (iv) 2-(2-methoxyphenoxy)ethanamine, TEA, THF, 65 C, 6 h
1
2
In parallel, to simplify the structure of carvedilol and likely removing its interactions with
adrenergic receptors, we investigated the requirement for the β-hydroxyl amino group by
evaluating compound 4, which was prepared by coupling 2-(2-methoxyphenoxy)ethanamine and
3, which was previously made by reacting 2-methoxyphenol with 1,2-dibromoethane.
3
4
5
6
Scheme 2
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8
9
O
N
H
O
OH
CH3
N
H
O
OH
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H
6
, 54%
N
H
iii
7
, 35%
ii
O
HO
O
i
iv
N
N
N
O
N
H
OH
5, 55%
H
v
N
H
H CO
8, 80%
3
O
N
O
OH
O
H CO
3
N
H
9, 52%
o
Reagents and conditions: (i) 2-(bromomethyl)oxirane, NaOH, water, DMSO, 45 C, 16 h; (ii) 2-
o
phenoxyethanamine, ethylene glycol dimethyl ether, 80 C, 24 h; (iii) butan-1-amine, ethylene glycol
o
o
dimethyl ether, 80 C, sealed tube, 24 h; (iv) phenylpiperazine, ethanol, 65 C, 16 h; (v) 4, ethylene
o
glycol dimethyl ether, 80 C, sealed tube, 48 h.
1
2
3
4
5
6
7
8
We then investigated the role of the anisole ring by simplifying to a simple phenol 6 and an
aliphatic chain 7; the role of the basic centre by inserting a cyclic tertiary amine (phenyl-
piperazinyl) 8 and a tertiary amine bearing two 2-(2-methoxyphenoxy)ethanamine moieties 9.
These compounds are prepared starting from the common intermediate 5, which is made by
reacting 4-hydroxycarbazole with 2-(bromomethyl)oxirane in good yield. The epoxide ring is
then opened by the appropriate amine using ethylene glycol dimethyl ether as solvent for 6, 7
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
and 9 in moderate to good yield, while in the case of 8 ethanol was used as solvent affording the
desired compound in 80% yield.
3
4
We then investigated the role of the hydroxyl group by preparation of 13, the carbazole-amine
linker 14, removing the β-hydroxyl amine by linking the carbazole and the anisole rings via a six
carbon chain 15 and removing the basic centre by the introduction of an amidic bond 19. Based
on the biological data obtained with compounds 9 and 13, we combined these modifications to
make compound 16. For the synthesis, 4-hydroxycarbazole was reacted with either 1,3-
dibromopropane, 1,4-dibromobutane or 1,6-dibromohexane to give compounds 10-12
respectively. Compounds 10 and 11 were coupled with 2-(2-methoxyphenoxy)ethanamine
yielding 13 and 14, while compound 12 was coupled with 2-methoxyphenol to give 15 in
moderate yield. Compound 16 was obtained by coupling 10 with 4. For the preparation of amide
analogue 19, 4-hydroxycarbazole was reacted with ethyl chloroacetate to afford the ester 17,
which was hydrolysed with aqueous sodium hydroxide providing the acid 18. Coupling with 2-
(2-methoxyphenoxy)ethanamine was performed under standard amide coupling conditions using
1-hydroxybenzotriazole (HOBt) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC)
affording the desired compound 19 in good yield.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
6
7
8
9
10
1
1
1
1
2
3
14
1
1
5
6
1
7
18
1
2
2
2
2
9
0
1
2
3
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Scheme 3
4
5
6
7
8
9
OCH3
OCH3
O
O
O
( )n
N
O
H
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
N
H
H
1
5, 40%
1
1
3 n=1, 19%
4 n=2, 32%
ii
iii
H CO
3
O
Br
( )n
N
HO
O
O
i
iv
O
N
N
N
H CO
3
H
H
H
1
1
1
0 n=1, 59%
1 n=2, 52%
2 n=4, 51%
16, 3%
v
H
O
OEt
OH
N
OCH3
O
O
O
O
O
O
vi
vii
N
N
H
N
H
H
1
7
18, 23%
19, 64%
Reagents and conditions: (i) 1,3-dibromopropane for 10, 1,4-dibromobutane for 11, 1,6-dibromohexane
for 12, KOH, acetonitrile, rt, 5-18 h; (ii) 2-(2-methoxyphenoxy)ethanamine, anhydrous K CO , DMF, rt,
2
3
o
1
6 h for 13; 2-(2-methoxyphenoxy)ethanamine, TEA, THF, 65 C, 24 h for 14; (iii) 2-methoxyphenol,
KOH, acetonitrile, rt, 66 h; (iv) 4, K CO , DMF, rt, 36 h; (v) ethyl chloroacetate, K CO , acetone, reflux,
2
3
2
3
.
16 h; (vi) 1N aqueous NaOH, THF, rt, 16 h; (vii) 2-(2-methoxyphenoxy)ethanamine, HOBt, EDC HCl,
DIPEA, DMF, rt, 16 h.
2
3
4
5
6
7
8
9
We then investigated two isomers of carvedilol: the 2-hydroxycarvedilol derivative 21, which
was obtained by opening the epoxide ring of compound 20 with 2-(2-
methoxyphenoxy)ethanamine providing the desired compound 21 in moderate yield (Scheme 4);
and the 1-carbazole isomer by directly linking the side chain to the nitrogen of the carbazole
ring. Firstly, carbazole was reacted with epichlorohydrin to yield compound 22 followed by
epoxide ring opening using 2-phenylethylamine obtaining compound 23 in 49% yield.
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
Scheme 4
O
ii
N
i
O
H
H CO
3
OH
O
O
OH
N
H
N
N
H
H
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
20, 9%
21, 33%
iii
iv
N
H
N
N
N
O
H
OH
23, 49%
2
2, 68%
OCH3
O
OCH3
OCH3
O
O
N
v
vi
O
O
N
OH
Br
N
H
N
O
N
H
2
4, 9%
25, 29%
o
Reagents and conditions: (i) 2-(bromomethyl)oxirane, sodium hydroxide, water, DMSO, 45 C, 16 h; (ii) 2-
o
(2-methoxyphenoxy)ethanamine, ethanol, 65 C, 16 h; (iii) 2-(bromomethyl)oxirane, potassium hydroxide,
o
acetonitrile, rt, 20 h; (iv) 2-phenylethylamine, ethanol, 65 C, 16 h; (v) 4-(2-aminoethyl)morpholine, TEA,
o
o
THF, 65 C, 4 h; (vi) 4-(oxiran-2-ylmethoxy)-9H-carbazole, ethanol, 65 C, 16 h.
2
3
4
5
6
7
Finally, to improve the solubility of compound 9, we designed compound 25 in which one of the
2-methoxyphenoxy group was substituted with a morpholine ring. Compound 3 was reacted with
commercially available 4-(2-aminoethyl)morpholine to obtain compound 24 which was then
coupled with 5 to give the desired compound 25 in 29% yield.
8
9
Carvedilol derivatives: protective abilities and MET channel block
10
The newly synthesised compounds were screened to assess their protective ability against 5 µM
gentamicin using mouse cochlear cultures initially at 20 µM, a concentration at which carvedilol
1
1
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provided full protection and was not cytotoxic. Any compounds that showed full or partial
protection were then screened at lower concentrations to establish their minimal protective
concentration. We initially designed and synthesised 6 compounds aimed at evaluating the
chemical moiety essential for the interaction with the MET channel, these include: modification
at the carbazole (2 and 4) and anisole (6 and 7) rings, the removal of the hydroxyl group (13) and
the introduction of a second 2-phenoxyethyl moiety to form a tertiary amine (9). When tested at
20 µM (Figure 6), compounds 2 and 4 lacking the carbazole moiety provided no protection
(Figure 6C and D), while compound 6 with a phenyl group instead of the anisole proved to be
generally cytotoxic (Figure 6E). Compound 9, bearing two 2-methoxyphenoxyethyl moieties,
offered only partial protection (Figure 6G) while compounds 7 and 13 consistently protected
against OHC loss (Figure 6F and H). As a reference, control cochlear cultures exposed to 0.5%
DMSO (Figure 6A) and 5 µM gentamicin (Figure 6B) as well as quantification for both controls
and cultures exposed to compounds (Figure 6I) are included.
2
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
6
7
8
9
10
1
1
1
2
13
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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0
1
2
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1
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Figure 6. Otoprotective effect of compounds 2, 4, 6, 7, 9, and 13 in cochlear cultures against 5
µM gentamicin. (A) A control culture exposed to 0.5% DMSO for 48 h. (B) A culture exposed to
5 μM gentamicin + 0.5% DMSO for 48 h. (C-H) Cultures exposed to 5 μM gentamicin for 48 h
+ 20 µM: (C) 2, (D) 4, (E) 6, (F) 7, (G) 9, (H) 13. Scale bar is 50 µm. (I) Quantification of OHC
survival for the control and compound exposed cultures.
2
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
4
5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
7
8
9
0
The three derivatives (7, 9 and 13) that showed partial or full protection at 20 µM were
subsequently tested at 10 and 5 µM against 5 µM gentamicin. Only compound 13 provided full
protection at 5 µM (as well as at 10 µM), showing an improvement compared to the parent
compound carvedilol which was only partially protective at 5 µM (Figure 7A-E). As stated
before, in the control condition some 110 OHCs were present in each cochlear segment, reducing
to 15 OHCs following exposure to 5 µM gentamicin. Additional exposure to 5 µM carvedilol
increased the average number of surviving OHCs to 66 (54% protection), whereas 5 µM
compound 13 resulted in 99 surviving OHCs (88% protection) (Figure 7E).
1
11
1
1
2
3
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2
2
2
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Figure 7. When tested at 5 µM against 5 µM gentamicin, compound 13 showed a greater
otoprotective effect. (A) A control culture exposed to 0.5% DMSO for 48 h. (B) A culture
exposed to 5 μM gentamicin + 0.5% DMSO for 48 h. (C-D) Cultures exposed to 5 μM
gentamicin for 48 h + 5 µM: (C) carvedilol or (D) compound 13. Scale bar is 50 µm. (E)
Quantification of OHC survival for the control and compound-exposed cultures.
7
8
When tested alone 13 showed similar toxicity characteristics to carvedilol, proving toxic to
OHCs at concentrations ≥30 µM and affecting the IHC and OHC bundle morphology at 20 µM.
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Contrary to carvedilol, 13 was not toxic at 30 µM when tested together with 5 µM gentamicin,
2
but it did also affect IHC and OHC bundle morphology (data not shown).
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8
In conjunction with assessing the protective abilities of these 6 compounds, potential interactions
with the MET channel were investigated by recording MET currents from OHCs before and
during exposure to 10 µM of each compound. Figure 8 shows fractional block of the currents
during compound exposure relative to the control currents at each membrane potential revealing
that two compounds 4 (magenta) and 2 (blue) have limited interaction with the channel at this
concentration.
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Figure 8. Compounds 2, 4, 6, 7, 9 and 13 show varying degrees of inhibition of the MET
currents. Fractional block plots of the currents recorded during exposure to 10 µM of each
derivative relative to the control currents at each membrane potential.
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The poor interaction of compounds 2 and 4 with the MET channel corresponded to the lack of
protection observed for these compounds at 20 µM, providing further evidence that the
protection offered by carvedilol may come through a block of the MET channel. In addition,
these results indicate the need for the carbazole moiety to allow the interaction with the channel.
Compound 9 (bright green), which offered only a partial protection at 20 µM, showed
approximately 40% block of the MET current at all membrane potentials with no release at
extreme hyperpolarized potentials suggesting this compound may act as a non-permeant blocker.
Compounds 6 (red) and 7 (orange) were strong MET channel blockers at 10 µM with a blocking
profile similar to carvedilol giving approximately 50-60% block of the current at -164 mV, the
most relevant physiological potential. Interestingly, compound 13 (dark green) was the most
effective MET channel blocker of this series providing almost 100% block of the MET current at
all membrane potentials. This result is also in accordance with its protective effect, as 13 showed
protection of the OHCs from gentamicin damage at concentrations down to 5 µM. This result
again suggests that the protection observed is due to a block of the MET channels, reducing the
entry of gentamicin into the cells. Compounds 6, 7 and 13 appear to be permeant blockers of the
MET channel, indicated by the release of the block at the extreme hyperpolarized potentials, and
in the case of compound 13 the release of the block was less pronounced than with carvedilol
itself.
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Figure 9. Otoprotective effect of compounds 8, 14, 15, 16, 19, 21, 23 and 25 in cochlear cultures
against 5 µM gentamicin. (A) A control culture exposed to 0.5% DMSO for 48 h. (B) A culture
exposed to 5 μM gentamicin + 0.5% DMSO for 48 h. (C-J) Cultures exposed to 5 μM gentamicin
for 48 h + 20 µM: (C) 8, (D) 14, (E) 15, (F) 16, (G) 19, (H) 21, (I) 23, (J) 25. Scale bar is 50 µm.
(K) Quantification of hair cell survival for the control and cultures exposed to compounds.
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A further 7 compounds (8, 14, 15, 19, 21, 23 and 25) were then designed and synthesised in an
attempt to improve their protective abilities and physicochemical properties. Of these, 14, 15, 21
and 23 were generally cytotoxic when tested at 20 µM against 5 µM gentamicin (Figure 9D, E,
H and I respectively), 19 and 8 offered no protection (Figure 9C and G respectively), and 25 was
consistently protective (Figure 9J). When tested at 10 µM, 25 was protective in 2 out of 4 screens
but offered no protection at 5 µM (data not shown). MET channel interactions were subsequently
investigated for derivative 25 that showed protection at 20 µM and partial protection at 10 µM.
The resulting fractional block plot reveals that at a concentration of 10 µM, 25 blocks the MET
channel at all membrane potentials (Figure 10). However, the degree of block is far less than that
observed for carvedilol and 13 suggesting this derivative has a reduced affinity for the channel.
1
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Combining the desired characteristics of a non-permeant MET channel blocker (9) with a high-
affinity blocker (13), we designed, synthesised and tested a further derivative (16). This
compound was found fully protective at 20 µM against 5 µM gentamicin on only one out of
three occasions (Figure 9F), with no protection in the other two trials. The lack of consistency
with the data was probably caused by the poor solubility of the compound. When tested at 10
µM, compound 16 did not offer any protection (data not shown). When tested in our
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electrophysiology assay, compound 16 showed limited interaction with the MET channel at both
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3 and 10 µM (data not shown).
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Figure 10. The fractional block plot for compound 25 (10 µM). The size of the current during
exposure to the compound relative to the control current at each membrane potential reveals
compound 25 has a low affinity for the MET channel.
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Assessing the protective effect and MET channel properties of adrenergic receptor and
calcium channel blockers.
1
0
We then proceeded to investigate whether the adrenergic (α and β) receptors, primary
pharmacological targets of carvedilol, play a role in its otoprotective abilities. We tested the non-
selective β-blocker propranolol 26, the selective β -blocker CGP20712 27, the selective α -
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blocker naftopidil 28, and the non-selective adrenergic blocker and calcium channel-blocker
verapamil 29 (Figure 11).
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OH
NH2
OH
H
O
N
O
O
N
H
O
N
OH
F3C
N
CH3
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(Propanolol)
(CGP20712)
N
O
N
OCH3
H CO
3
N
OCH3
OCH3
OH
N
H CO
3
2
8
29
(Naftopidil)
(Verapamil)
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Figure 11. Chemical structure of other adrenergic blockers (26-28) and calcium channel-blocker
29 used in this study.
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Compounds 26, 27 and 29 did not offer any protection against 5 µM gentamicin when tested at
20 µM (Figure 12 C, D, and F respectively), while 28 proved to be partially effective under the
same conditions (Figure 12 E, G). However, when the test concentration was lowered to 10 µM,
compound 28 did not protect cochlear culture hair cells against gentamicin damage (data not
shown).
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Figure 12. Otoprotective effect of compounds 26, 27, 28, and 29 in cochlear cultures against 5
µM gentamicin. (A) A control culture exposed to 0.5% DMSO for 48 h. (B) A culture exposed to
5 μM gentamicin + 0.5% DMSO for 48 h. (C-F) Cultures exposed to 5 μM gentamicin for 48 h +
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