A (+)-Larixol Congener with High Affinity and Subtype Selectivity toward TRPC6

Article abstract of DOI:10.1002/cmdc.201800021

Natural products have many health benefits, and their application can improve the quality of life. Recently, the diterpene (+)-larixol and its acetylated congeners demonstrated selective inhibition of the second-messenger-gated cation channel transient receptor potential canonical 6 (TRPC6) over its close isoforms TRPC3 and TRPC7. Building on this knowledge, we expanded these findings by chemical diversification of (+)-larixol mostly at position C6. Implementing high-throughput Ca²⁺ FLIPR screening assays and electrophysiological patch-clamp recordings, we showcase larixyl N-methylcarbamate, termed SH045, as a compound with nanomolar affinity and 13-fold subtype selectivity over TRPC3 in stably expressing HEK293 cells. Expanding on this finding, TRPC6 inhibition was also observed in rat pulmonary smooth muscle cells. Furthermore, treatment of isolated perfused lung preparations with SH045 led to a decrease in lung ischemia-reperfusion edema (LIRE), a life-threatening condition associated with TRPC6 that may occur after organ transplantation. Taken together, and given the inexpensive, straightforward, and scalable preparation of SH045, we report a TRPC6 blocker that holds promise for the translational treatment of LIRE.

Full text of DOI:10.1002/cmdc.201800021

Accepted Article
Title: A (+)-larixol congener with high affinity and subtype selectivity
towards TRPC6
Authors: Stephanie Häfner, Finn Burg, Martina Kannler, Nicole Urban,
Peter Mayer, Alexander Dietrich, Dirk Trauner, Johannes
Broichhagen, and Michael Schaefer
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A (+)-larixol congener with high affinity and subtype selectivity
towards TRPC6
Stephanie Häfner[a], Finn Burg[b], Martina Kannler[c], Nicole Urban[a], Peter Mayer[b], Alexander Dietrich[c],
Dirk Trauner[b],[d] *, Johannes Broichhagen[b],[e] * and Michael Schaefer[a] *
[a]
Stephanie Häfner (0000-0003-0501-2193), Nicole Urban, and Prof. Dr. Michael Schaefer (0000-0003-3662-4387)
Rudolf-Boehm-Institute for Pharmacology and Toxicology, Leipzig University
Härtelstr. 16-18, 04107 Leipzig, Germany.
E-mail: michael.schaefer@medizin.uni-leipzig.de
[b]
Finn Burg (0000-0002-8038-5638), Peter Mayer (0000-0002-1847-8032), Prof. Dr. Dirk Trauner (0000-0002-6782-6056),
Dr. Johannes Broichhagen (0000-0003-3084-6595),
Department of Chemistry, Ludwig-Maximilians-University Munich and Center for Integrated Protein Science Munich
Butenandtstr. 5–13, 81377 Munich, Germany.
[c]
[d]
Martina Kannler (0000-0003-2152-3858), Prof. Dr. Alexander Dietrich (0000-0002-1168-8707)
Walther-Straub-Institute of Pharmacology and Toxicology, Ludwig-Maximilians-University Munich
Nußbaumstr. 26, 80336 Munich, Germany.
Prof. Dr. Dirk Trauner (0000-0002-6782-6056)
Present address: Department of Chemistry, New York University
Silver Center for Arts and Science, 100 Washington Square East, New York, NY 10003, USA.
E-mail: dirktrauner@nyu.edu
[e]
Dr. Johannes Broichhagen (0000-0003-3084-6595)
Present address: Department of Chemical Biology, Max Planck Institute for Medical Research
Jahnstr. 29, 69120 Heidelberg, Germany.
E-mail: johannes.broichhagen@mpimf-heidelberg.mpg.de
Supporting information for this article is given via a link at the end of the document.
Abstract: Natural products have many health benefits and their
application can improve the quality of life. Recently, the diterpene
(+)-larixol and its acetylated congeners demonstrated a selective
inhibition of the second messenger-gated cation channel transient
receptor potential canonical 6 (TRPC6) over its close isoforms
TRPC3 and TRPC7. Building on this knowledge, we expand these
findings by chemical diversification of (+)-larixol mostly at position
C6. Implementing High-Throughput Ca2+ FLIPR screening assays
and electrophysiological patch-clamp recordings, we showcase
larixyl N-methylcarbamate, dubbed SH045, as a compound with
nanomolar affinity and a 13-fold subtype selectivity over TRPC3 in
stably expressing HEK293 cells. Expanding on this finding, TRPC6
inhibition was also achieved in rat pulmonary smooth muscle cells.
Furthermore, treatment of isolated perfused lung preparations with
SH045 led to a reduction in lung ischaemia reperfusion oedema
(LIRE), a life-threatening condition associated with TRPC6 that may
occur after organ transplantation. Taken together, and given the
cheap, straightforward and scalable preparation of SH045, we report
a TRPC6 blocker that holds promise for translational treatment of
LIRE.
pseudogene in humans) forming a closely related subgroup
together with TRPC3 and TRPC7 (Figure 1a). TRPC6 is a non-
selective ion channel that, upon activation, mediates Ca2+ entry
and membrane depolarization (Figure 1b). Activated by
diacylglycerol (DAG),
a
second messenger produced
downstream of the GPCR signaling cascade by depletion of
[1]
PIP2
or by receptor mediated PLCγ-signaling[2], TRPC6
contributes to a variety of physiological and pathophysiological
responses. As such, high expression levels of TRPC6 are
observed in the lung, especially in vascular endothelial and
smooth muscle cells, where it is associated with hypoxic
vasoconstriction, idiopathic hypertension, lung ischaemia
reperfusion oedema (LIRE)[3,4] and idiopathic pulmonary arterial
hypertension (IPAH).[5] In addition, TRPC6 has also emerged as
a
potential target for the treatment of focal segmental
glomerulosclerosis that seems to be a phenotype of both gain-
and loss-of-function mutations in the TRPC6 gene.[6,7] Our latest
reports were based on the natural product (+)-larixol, a labdane
terpene occurring in the resin of the European larch, which
showed that channel inhibition can be achieved by
functionalizing the C6 alcohol to an acetyl or a carbamate
moiety.[8,9] Spurred by recent computational approaches
alongside with Biology-Oriented Synthesis (BIOS)[10], which have
ignited a renaissance in the use of natural products in drug
discovery and chemical biology, and in particular for TRP-
channel treatment[11,12], we intended to further explore the larixol
scaffold. Our aim was to utilize the vast chemical space in
natural products to obtain a channel blocker with unprecedented
affinity and selectivity (Figure 1c).
Introduction
The superfamily of transient receptor potential (TRP) channels
consists of more than 28 members, grouped into seven
subfamilies. The canonical transient receptor potential receptor
6 (TRPC6) is an isoform of seven encoded TRPC channels (that
were the first TRP channels to be described; TRPC2 being a
1
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(+)–Larixol (1) can be isolated from the oleoresin of Larix
decidua as a crystalline solid between 10–12 wt% together with
its congener epi-manool (2) that lacks the hydroxyl group at
position C6 (Figure 2; carbon numbering is depicted in centered
structure).[13] While 1 is a subtype-prevalent blocker of TRPC6
over TRPC3 and TRPC7, epi-manool shows lower affinity and
no selectivity, accounting for the importance of the C6 alcohol
function. The bioactivity of this alcohol can be further enhanced
by converting it to the corresponding acetate 3 and carbamate
4.[8,9] In accordance with previous studies, we aimed to expand
the knowledge about the structure-activity relationship of 1 by
i) further diversification of the alcohol at C6, ii) formation of a
third ring structure and oxidation chemistry and iii) chemical
engineering the side chain at C9. Detailed synthetic protocols
and full characterization are outlined in the SI, and final
structures are shown in Figure 2 for clarity.
performed on the exo-methylene group at C8 by ozonolysis
starting from methyl ketone 5 to obtain bisketone 8. Instead, by
monooxidation of 1 with mCPBA, we were able to convert the
exo-methylene group to its corresponding epoxide[14] that proved
to be unstable and was subsequently reacted with LAH to give
C8 alcohol 9 (Scheme 1b). Treatment of 5 with Tf2O initiated a
Prins reaction, resulting in ring formation between C13 and C17
with a double bond found between C8 and C9 to give 10. Its
congener 11, in which the double bond is between C8 and C14
and the stereochemistry of the tertiary alcohol differs, can be
obtained by Grubbs’ metathesis of 1, i.e. the fusion of the allylic
and the exomethylene groups. Interestingly, ozonolysis of 1
provided spiroacetal 12[15] but with unknown outcome of the
stereochemistry at C14, which we solve herein by X-ray
crystallography to be (R)-configured. Last but not least, the C6
alcohol was oxidized to ketone 13 (Scheme 1c), which could be
isomerized from its β,γ-unsaturated system to Michael system-
containing compound 14.[14]
Starting from ketone 13, DIBAL reduction gave access to epi-
larixol 15 with the C6 alcohol inverted, shown by X-ray
crystallography of glycol-cleaved compound 15b (see SI).
Activation of the alcohol with CDI gave larixyl imidazolo formate
16 (Scheme 1d) that could be isolated and further diversified to
a series of compounds, such as hydrazide 17, methyl carbonate
18, methyl carbamate 19 (termed SH045), n-butyl carbamate 20
and cyclohexyl carbamate 21. A completely different functional
group was installed using CS2 and MeI on 1 to yield xanthate 22
(Scheme 1e), which was unstable within days at –20 °C. Finally,
carbamate 4 was further derivatized on the tertiary alcohol
residing at C13, to get the mixed alcohol-modified structures 23
(biscarbamate) and 24 (carbamate and acetate) (Scheme 1f).
SAR for the inhibition of TRPC3, 6 and 7
After having generated a small labdane-based library, blocking
characteristics of all compounds were assessed by high
throughput fluorescence imaging plate reader (FLIPR) assay. To
determine subtype selectivity, the compounds were tested on
HEK293 cells stably overexpressing hTRPC3, hTRPC6 or
hTRPC7 (abbreviated as HEKhTRPC3, HEKhTRPC6, HEKhTRPC7
,
respectively). Serial dilutions were applied to fluo-4/AM-loaded
suspensions of HEKhTRPC6-YFP, HEKhTRPC3-YFP and HEKhTRPC7-YFP
Figure 1. Larixol and TRPC6. a) Phylogenetic analysis of the human TRPC
family shows close relation between TRPC3,
6 and 7. b) Schematic
cells that were subsequently stimulated with 50 µ
M 1-oleyl-2-
acetyl-sn-glycerol (OAG) to induce Ca2+ entry through TRPC
channels for the evaluation of channel inhibition (representative
traces and concentration-response curves of SH045 are
depicted in Figure 3a-c and 3d-e, respectively). A characteristic
of TRPC6 is its lower basal activity compared to TRPC3 and
TRPC7,[16] and treatment of HEKhTRPC3 and HEKhTRPC7 with
inhibitors results in a diminution of basal Ca2+ influx (of higher
significance for TRPC7). With this in mind and in order to obtain
comparable IC50 values, the ordinate of the calculated
concentration-response plots represents ∆F/Finh, where ∆F is
calculated as follows
representation of TRPC6 activation by DAG and inhibition by (+)-larixol.
c) (+)-Larixol derivatization for optimized blocking and selectivity towards
TRPC6.
Results and Discussion
Design of a labdane compound library
At first, we wondered how important the atomic arrangement of
the side chain at C9 is, which bears a tertiary allylic alcohol
linked by two methylene spacers. We therefore set out to
bishydroxylate and glycol cleave the corresponding trisalcohol to
obtain methyl ketone 5[14], which could either be reduced to
secondary alcohol 6 or treated with MeMgBr reagent to have
access to dimethyl tertiary alcohol 7 (Scheme 1a and Figure 2
for final compounds overview). The new stereocenter created in
6 leads to a d.r. = 5.4/4.6, as acquired by 1H NMR (see SI).
In line with the modification solely at the C9 chain, we were
further interested to oxidize the larixol scaffold and lock the side
chain by formation of a third ring structure. Oxidation was
∆F = Fstim – Finh
(Eq. 1)
with Fstim = fluorescence intensity after agonist stimulation and
Finh = fluorescence intensity after addition of inhibitor.
For all compounds tested, the calculated logP values range from
4 to 7 and for some derivatives pluronic F127 was added to
increase solubility, which had a positive effect on bioactivity (see
SI section 1.2). Results are referred to IC50 values in the text and
2
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Scheme 1. Synthetic route for (+)-larixol congeners. a) Modification of the C9 side chain. b) Modification of the exomethylene group. c) Oxidation and inversion of
C6 alcohol. d) Modification of the C6 alcohol via imidazoyl formate 16. e) Synthesis of xanthate 22. f) Derivatives at C13 alcohol with C6 carbamate.
to yield biscarbonyl 8 leads to complete loss of its bioactivity
(see SI), comparable with 9, where the exocyclic double bond is
oxidized. In a similar manner, strongly decreased activity can be
observed when a third ring system is installed by joining the two
exogenous double bonds via Prins reaction or Grubbs’
metathesis (10 and 11, respectively), which also applies for
represented as pIC50 values in Figure 4 for clarity (dose-
response curves for all compounds can be found in the SI).
In previous studies, we reported that potent and subtype-
selective TRPC6 inhibitors could be generated by acetylation
and carbamylation of (+)-larixol,[8,9] suggesting the high
importance of C6 substituents. Nonetheless, a more extensive
study of the effects of compound derivatization, e.g. the
influence of an altered C9 sidechain on TRPC6 inhibition and
selectivity, was still of interest. Hence, we first tested C9 side
chain modified compounds, such as ketone 5, secondary alcohol
6 and tertiary alcohol 7. These derivatives still acted as TRPC6
inhibitors, but showed significantly higher IC50 values (22, 13
spiroacetal 12 (IC50
= 26, >50 and >50 µM for TRPC6,
respectively). This is supported by previous studies, where larch
turpentine components with a three-ring system, such as abietic
acid or pimaric acid, displayed IC50 values higher than larixol.[8]
When testing 13 and 14, both bearing a sp2-hybridized carbonyl
at position C6, the spatial position and the different hydrogen
donor-acceptor capabilities of the oxygen atom seems to play a
minor role. This observation becomes more evident by
comparing the potency and the selectivity profile of alcohol
inverted epi-larixol 15 to its diastereomer 1. Despite this
intervention in the molecular scaffold, compounds 13, 14 and 15
still display lower IC50 values than unmodified 1 (0.49, 0.98 and
and 7 µ
M for 5, 6 and 7, respectively) than 1 (see SI, Figure 4).
The tertiary hydroxyl group in conjunction with the exact
stereochemical arrangement of the methyl and vinyl substituents
at C13 seems to be a necessary moiety for selective channel
binding. Given these results, we did not aim for higher
diastereometric excess of 6, for instance by CBS reduction. It
should be noted that oxidation of the exocyclic double bond of 5
0.89 µM on TRPC6, respectively).
3
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Figure 2. Overview of compounds derived from (+)-larixol (1). Carbon numbering is given in the centred structure. Obtained compounds were tested towards
TRPC6 inhibition, and X-ray structures have been solved for compounds 1, 3, 5, 8, 9, 10, 12, 16, 19 (SH045) and 23.
Further modifications of the C6 alcohol were achieved using the
imidazole-activated formate precursor 16, which allowed the
easy generation of the isosters 17, 18 and SH045. While 16, 17
demanding or rigid elements at position C6 reduced bioactivity in
all cases (Figure 4). This confirms the hypothesis that sterically
undemanding substituents at this site are crucial for binding and
blocking and hints towards the existence of a narrow binding
pocket that is suited best for the electronic environment of a
carbamate. The observation that an unbranched alkyl chain
substituent at this position (n-butyl, as in 20) was better tolerated
than a branched moiety (cyclohexyl as in 21), which is shown by
and 18 displayed IC50 values of 0.44, 0.45, and 0.17 µM,
respectively, the introduction of a N-methylated carbamate
function in compound SH045 yielded a 15-fold improvement in
potency over larixol (1) and a 3-fold improvement over the
previously described larixyl carbamate 4[9] with an IC50 = 63 n
M.
Furthermore, FLIPR measurements on TRPC3 and TRPC7
provided promising selectivity data for SH045 (IC50
0.84/0.22 µ , Figure 3d, e; for TRPC3 p<0.05, Figure 4) and
therefore outperformed every known congener regarding affinity
and selectivity to date. On the other hand, introducing sterically
the IC50 values of 0.55 and 1.54 µ
clearly supports this hypothesis. Interestingly, with an IC50 =
M on TRPC6, respectively,
a
M
=
13.6 µM, xanthate 22, which is isolobal to SH045, did not block
Ca2+ influx as potently. This shows that small alterations and fine
tuning of the electronic properties leads to pronounced effects
4
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Figure 3. Compound testing by Fluorescent Imaging Plate Reader (FLIPR). a-c) Representative FLIPR Ca2+ traces of OAG-stimulated TRPC currents blocked by
various concentrations of SH045. d-e) Corresponding concentration-response curves of SH045 were obtained from several experiments (n = 3-7) performed as in
(a-c).
on bioactivity. Previous studies revealed that a bisacetate is less
bioactive than the monoacetylated larixol at C6,[9] but the higher
subtype selectivity towards TRPC3 seemed to indicate the
benefit of modifying the secondary alcohol. While
biscarbamylation of larixol led to the barely active compound 23
these results in mind, we tested the most potent compounds
with a MTT assay in HEK293 cells for their influence on cell
viability (Figure S5). After 24-hour incubation with 5 or 15 µ
M of
the compounds, no significant reduction in metabolic activity was
detected when compared to vehicle alone. Taken together, and
given the high potency, high selectivity on direct and upstream
activation of TRPC6 and absent toxicity of SH045, this structure
emerged as the most promising candidate for further evaluation
in more physiologically relevant systems.
(IC50 = 20 µ
M), the mixed carbamate/acetate 24 is highly potent
(IC50 = 0.25 µ
M) and shows higher subtype selectivity over
TRPC7 than carbamate 4 (11-fold vs. 4-fold, Figure 4), albeit
with lower potency than SH045. In addition, SH045 was also
preliminary screened towards TRPC4 and TRPC5 among other
TRP isoforms from the ankyrin, vanilloid and the melastatin
family. Compared to TRPC6, SH045 showed high selectivity
over TRPA1 (630-fold), TRPV1 (400-fold), TRPM2 (1400-fold)
and did not have any notable effects on the other tested
channels including TRPC4, 5 and TRPV2, TRPV3, TRPM3,
TRPM8 (Figure S2). The high bioactivity of SH045 could also be
verified by stimulation of HEKhTRPC3, HEKhTRPC6 and HEKhTRPC7
Patch-clamp recordings in TRPC-stable HEK293 cells
In
a next step, we aimed to characterize SH045 more
conclusively by electrophysiological whole-cell recordings to
assess the respective IC50 values on TRPC3, TRPC6 and
TRPC7. Indeed, the high potency on TRPC6 could be confirmed
by patch-clamp measurements, which includes all cations that
are transmitted via the plasma membrane due to the unselective
pore properties of TRPC3, 6 and 7. More precisely, SH045
blocked the inward and outward currents through TRPC6 with
with a redundant mix of GPCR agonists (1 m
M carbachol,
300 µ adenosine-5’-triphosphate and 0.5 U/µL thrombin). This
M
IC50 values of 5.8 n
IC50 values of 22 n
determine a 3-fold selectivity over TRPC7 (Figure 5b), similar to
observations from fluorometric assays. The
[Ca2+]i
corresponding IC50 values of TRPC3 currents were 440 n
(inward) and 634 n (outward), and thereby ~2 orders of
magnitude higher compared to TRPC6 (Figure 5c). Original
traces from whole cell patch clamp recordings are shown in
Figure S6.
M
and 5.2 n
(inward) and 18 n
M
respectively (Figure 5a). With
mixture leads to the formation of DAGs via Gq/11, which
represents the endogenous way of TRPC3/6/7 activation (Figure
1b). Interestingly, SH045 is an even more potent blocker when
M
M
(outward), we could
TRPC6 is engaged via GPCR signalling (IC50 = 8.9 n
M
, p<0.05)
or via TRPC6 agonist GSK1702934A[17] (IC50 = 14.2 n
M, non-
M
M
significant). One could therefore speculate that SH045 induced
block is more potent when TRPC6 is activated from the cytosolic
site. Beneficially, the inhibitory performance of SH045 on
TRPC3 and TRPC7 was not considerably altered by using other
agonists than OAG (Figure S3, S4 and Table S2, S5). Having
5
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explanted perfused mouse lungs for 90 minutes, before
perfusion was re-initiated. Five µM of SH045 were applied for 15
minutes before stopping the perfusion and led to significantly
reduced oedema formation 60 and 90 minutes after reperfusion
compared to ischaemic lungs perfused and reperfused with
buffer (Figure 5f).
Conclusions
In the present study, we showcase the synthesis and structure-
activity relationship of several derivatives of the diterpene (+)-
larixol (1). The newly generated analogues are divided into three
categories: (i) alteration of the functionality at the C9 side chain,
(ii) third ring formation and (iii) functionalization of the hydroxyl
group at C6 with the introduction of different side chains.
Analogues generated by modification according to types i) and
ii) showed a diminished bioactivity in terms of TRPC6 inhibition.
However, the derivatization of 1 according to iii) led to potent
TRPC6 inhibitors if the modification was at the C6 position. This
validates the importance of this moiety for selectivity against
TRPC6’s closest relatives, TRPC3 and TRPC7, among more
distant relatives, such as TRPC4 and TRPC5 and others from
the ankyrin, vanilloid and melastatin family. Further testing
towards other transmembrane proteins, for instance GPCRs and
receptor-linked enzymes, can be considered for follow-up
studies. The introduction of small modifications could improve
the bioactivity and finally led to the highly potent and selective
TRPC6 inhibitor SH045, assessed both in Ca2+ FLIPR and
patch-clamp recordings. We note that in the concentration-
response curves generated by FLIPR-measurements steeper
Hill slopes were observed for some compounds (cf. larixol,
Figure 3e), especially those that display high logP and high IC50
values, which may point to colloidal aggregation. Nevertheless,
SH045 performs over a broad dynamic range for all tested
TRPC isoforms and we therefore conclude that it does not
Figure 4: Selectivity profiling for larixol derived compounds. TRPC6:TRPC3
and TRPC6:TRPC7 fold-selectivity of all blockers including their pIC50 (data
represents means ± S.E.M.; for SH045: one-way ANOVA with Tukey’s post
hoc test, * p<0.05). Label legend is given in the Figure.
aggregate and is
a suitable candidate for bioavailability.
Although the N-methyl carbamate function is just a small
alteration in the molecular framework, it led to a significant
improvement in potency compared to the non-methylated
carbamate. Interestingly, none of the compounds exhibited
higher affinity for TRPC3/7 over TRPC6 suggesting that the
TRPC binding site is conserved. Although our library covers <30
compounds, we derive a structure-activity relationship from our
results. It can be clearly stated that alterations anywhere but on
the C6 alcohol lead to decreased blocking capacity, thereby
suggesting that the C9 chain is well-fitted onto the protein
surface. By comparing the C6 derivatives, we observe that a
carbamate leads to the best results. This might be due to the
installed nitrogen being a hydrogen donor, which is absent for an
oxygen or sulfur atom. Furthermore, it is important for the
N-linked alkyl chain to be small in size; even by expansion to a
n-butyl group, a loss in affinity can be observed. This loss is
even more pronounced when a cyclohexyl ring is installed.
Given the fact that C6 and C9 is part of the same ring structure,
we therefore propose that indeed this part is the main
pharmacologic entity that is responsible for TRPC blocking.
After the description of several low potent TRPC6 channel
SH045 in rPASMCs and explanted lungs
In addition to the characterization of heterologously expressed
TRPC channels, we intended to test the TRPC6 inhibitor SH045
in its native, and therefore physiological environment. The
expression of TRPC6 is well-described in pulmonary smooth
muscle cells[18], thus we chose this system to validate the
inhibition of OAG-mediated Ca2+ influx by fura-2 Ca2+
measurements. By testing five different concentrations of SH045
we determined an IC50 of 339 n
M in rPASMCs, reflecting direct
TRPC6 blocking independent of detected signals from PKC
activation, which was ensured by preincubation with
bisindolylmaleimide-1 (BIM) (Figure 5d, e).
TRPC6 also contributes to severe pathophysiological effects in
the lung, such as oedema formation induced by excessive Ca2+
influx in endothelial cells and subsequent disruption of the
endothelial barrier.[19] This increase in endothelial permeability
induces the formation of lung ischaemia-reperfusion oedema
(LIRE), a primary graft failure, which causes alveolar damage
and hypoxemia in patients after lung transplantations.
Weissmann et al. reported a reduction of oedema formation in
TRPC6-/- mice after ischaemic conditions compared to wildtype
controls.[4] Hence, we tested if SH045 is able to reduce oedema
formation, which is correlated to increased lung weight in the
isolated perfused lung. Accordingly, oedema were provoked by
an interruption of lung perfusion, inducing ischaemia in
blockers like SKF96365[20], 8009-5364[21] or norgestimate[22]
,
more promising candidates for TRPC6 inhibition were discussed
in recent years. In 2013, anilino-thiazole based small molecules
were described to block Ca2+ influx through TRPC3 and TRPC6
in the low nanomolar range, however, most of them were not
6
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Figure 5. Compound SH045 was evaluated in cellulo (TRPC stably-expressing HEK and rPASMC cells) and ex vivo (murine lungs). a-c) IC50 determination of
SH045 in HEK293 cells stably expressing TRPC3, 6, or 7. Data represents means ± S.E.M. (n = 6-14). d) Concentration-response curves of SH045 in rPASMCs.
Data represents means ± S.E.M. (n = 6). e) Representative traces from fura-2 [Ca2+]i measurements of SH045 in rPASMC. f) LIRE in explanted mouse lungs
could significantly be reduced with 5 µM of SH045 (n = 6, two-way ANOVA).
suitable for in vivo exposure due to poor bioavailability.[23]
Another compound, termed SAR7334, has become
benchmark blocker since its synthesis in 2015 as it displays an
IC50 value of 7.9 n on TRPC6 with high selectivity over
TRPC3/7 (36-/29-fold with IC50 = 282/226 n
, respectively).[24]
CCDCs 1556986 (1), 1556990 (3), 1556984 (5), 1556987 (8), 1556991
(9), 1556982 (10), 1556985 (12), 1556988 (15b), 1556983 (16), 1572562
(SH045), 1556989 (24) contain the supplementary crystallographic data.
These data can be obtained free of charge from the Cambridge
a
M
Crystallographic
Data
Centre
under
M
https://www.ccdc.cam.ac.uk/structures/.
Therefore, our herein disseminated larixol congener SH045
expands the repertoire of TRPC6 inhibitors with an IC50 value of
5.8 n
M that compares favorably to SAR7334. Beneficially,
Acknowledgements
SH045 can be generated in a semisynthetic two-step protocol
from the abundant natural product (+)-larixol, whose isolation in
bulk quantities is straightforward and inexpensive, and indeed
SH045 has been obtained in gram quantities. Finally, with its
ability to reduce induced oedema formation in explanted murine
lungs, pharmacological profiling will be performed to set the
stage for studies in vivo in the near future.
D.T. and J.B. thank the Munich Centre for Integrated Protein
Science (CIPSM). This work was supported by the SFB TRR
152 (S.H., D.T., J.B. and M.S.). D.T. acknowledges support of
the European Research Council for an Advanced Grant
(268795). We thank Dominik Dosch for synthetic assistance and
Narges Pourmandi for proofreading.
Keywords: larixol • diterpene • labdane • TRPC6 • LIRE
Experimental Section
The Supporting Information contains synthetic protocols including
chemical and physical characterization of the described compounds and
biological methods. All compounds were determined to be >95% pure by
UHPLC (Shimadzu MS2020 connected to a Nexerra UHPLC system
equipped with a Waters ACQUITY UPLC BEH C8 1.7 µm 2.1x50 mm
column. Buffer A: 0.05% HCOOH in dH2O Buffer B: 0.05% HCOOH in
MeCN. Analytical gradient was from 10% to 90% B within 5.0 min with
1.0 ml/min flow, absorbance was monitored at λ = 200 nm), unless stated
otherwise in the synthetic procedure in the Supporting Information.
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Entry for the Table of Contents
The natural product (+)-larixol was derivatized to obtain SH045, a potent and selective TRPC6 channel blocker, comparable to synthetic
benchmark blockers such as SAR7334. We assessed SH045’s pharmacological properties by means of electrophysiology and FLIPR assays in
heterologously TRPC6 expressing HEK293 cells and in endogeneous pulmonary smooth muscle cells, and ultimately showcase a reduction of
oedema in explanted murine lungs.
9
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