The cation canonical transient receptor potential channels (TRPC3/6) are mechanosensitive,
receptor- and store-operated channels that mediate Ca2+ /Na+ influx into cells to govern cellular functions in response to stimulation of phospholipase
C-coupled membrane receptors. The TRPC3/6 channels are implicated in several human
pathologies – focal segmental glomerulosclerosis, pulmonary hypertension, ischaemia
reperfusion-induced lung oedema, myocardial hypertrophy, as well as in the regulation
of vascular tone, cell growth, proliferation, and inflammation.[1 ] Thus, since the discovery of the TRPC-channel subtypes in the 1990s, potent and
selective small-molecule TRPC3/6 agonist and antagonist tools are being sought to
study the functions of these lipid-sensitive channels and to evaluate the therapeutic
potential of such pharmacological modulators.[2 ] Recently, a small 1,3-dihydro-2H -benzo[d ]imidazol-2-one-based potent agonist (GSK1702934A, Figure [1 ]) has been briefly described by a team at GlaxoSmithKline-US as a tool to activate
lipid-sensitive TRPC channels directly, bypassing phospholipase-C signaling.[3 ]
Figure 1 GSK1702934A – a potent TRPC3/6-channel agonist
Whole-cell patch-clamp experiments demonstrated that GSK1702934A is able to induce
TRPC3/6-currents in HEK293 cells transduced with recombinant human TRPC3/6 with an
EC50 of ca. 0.08 mM and 0.44 mM, respectively. In a continuation of our attempts to evaluate
the potential value of TRPC3/6 channels as direct pharmacological targets, we sought
for the enhanced synthesis of novel 1,3-dihydro-2H -benzo[d ]imidazol-2-one-based synthetic activators in an intensified process from commercially
available starting materials.
Herein we disclose an improved, single-step synthetic protocol that provides a direct
access to novel agonists of TRPC3/6 channels by the means of microwave heating and
cyanuric chloride as an effective coupling reagent. Moreover, the agonist effects
of novel synthetic TRPC3 activators, generated by the means of the enhanced synthetic
protocol, were examined and few essential structure–activity relationships were established.
For the envisaged synthesis, an optimized N-acylation protocol was needed. Previously,
we gained access to small amounts of GSK1702934A by the means of a traditional amide-bond-generation
method using commercially available starting materials in a two-step procedure (Scheme
[1 ]) and for the purpose of uncovering (patho)physiological functions of the TRPC3/6
channels.[4 ] Thus, an acid chloride of a corresponding acid was generated and used in a following
coupling step. Moreover, an earlier microwave N-acylation procedure[5 ] successful in the synthesis of pyrazole-based selective inhibitors of TRPC3/6 channels
provided lower yields from a complex reaction mixture (data not shown).
Scheme 1 Previous synthesis of GSK1702934A. Reagents and conditions : a) oxalyl chloride, DMF, CH2 Cl2 , 0 °C to r.t., 1 h, 80%; b) 4-(2-keto-1-benzimidazolinyl)piperidine, DMAP, CH2 Cl2 , 0 °C to r.t., 15 min, 82%.
At this stage, with the available preliminary data at hand, and excluding further
standard room-temperature coupling reagents such as PyBOP/DIPEA (that typically require
rather long reaction times and are relatively expensive), we turned our attention
to a recent literature report by Rad et al. that describes an experimentally very
straightforward amidation process towards N-acylated nucleobases.[6 ] The described procedure involves simple heating of diverse nucleobases and carboxylic
acids (aromatic, aliphatic, heterocyclic) at 110 °C using cyanuric chloride in the
presence of NaH and Et3 N in DMF–MeCN (1:1) as a solvent mixture. Cyanuric chloride (2,4,6-trichloro-1,3,5-triazine,
TCT), besides other synthetic applications, is also a suitable activating reagent
for the in situ generation of acid halides that is a frequently used technique in
peptide synthesis.[7 ] It is a safe, cost-effective, and stable reagent that is amenable to large-scale
production.[7a ]
[i ] While working at elevated temperatures and with good yields, the reported reaction
needed at least six and up to 18 hours for some substrates to reach completion. For
these reasons, we sought for the intensification of the process by applying microwave
heating while at the same time looking for a milder base to work with. It was easy
to foresee that the application of microwave heating could potentially enhance the
process.[8 ]
In our optimization efforts we used 4-(2-keto-1-benzimidazolinyl)piperidine (Table
[1, ]
A ) and octanoic acid (Table [1, ]
B ) as reaction partners, envisaging our attempts to prepare TRPC3 agonists. In few
preliminary experiments (data not shown for simplicity), pyridine could be selected
as the sole organic base of choice to replace the NaH–Et3 N combination and MeCN as the preferred solvent. To access the optimal reaction conditions,
we elaborated the best reagent ratio and the reaction temperature with a reaction
time limit set at 5 minutes (Table [1 ]).
Table 1 Optimization of the TCT-Promoted Microwave N-Acylation
Entry
A /B /C /D (equiv)
Temp (°C)
Conv. (%)a
1b
1.0/1.2/0.42
90
traces
2
1.0/1.2/0.42/1.5
90
52
3
1.0/1.2/0.42/1.5
120
68
4
1.0/1.2/0.42/1.5
140
93 (54)c
5d
1.0/1.2/0.42
140
22
6
1.2/1.0/0.6/1.5
140
100 (63),c (47)e
a Determined by HPLC at 215 nm.
b Pyridine as a base was omitted.
c Isolated yield after flash chromatography given in parentheses.
d Pyridine used as a solvent.
e Isolated yield from a comparable microwave protocol using PCl3 for the in situ generation of an acid chloride instead of TCT.[5 ]
An exploratory room-temperature trial was terminated after six days, resulting in
only 22% conversion, as determined by HPLC. In a further experiment, we could confirm
the mechanistic necessity to use pyridine as a base (Table [1 ], entry 1).[9 ] As expected, the obtained result clearly demonstrated the crucial role of pyridine
since only trace amounts of amide 1a were detected in the reaction mixture. Fine tuning of the reaction temperature and
the reagent stoichiometry ultimately led to the final synthetic protocol (Table [1 ], entry 6; 1.2 equiv of amine, 1 equiv of acid, 0.6 equiv of TCT, and 1.5 equiv of
pyridine) providing full conversion and highest selectivity for the desired amide.[10 ] Isolation of the amide 1a by flash chromatography furnished 63% product yield, a significant improvement to
the 47% obtained in a PCl3 -mediated microwave procedure. Additionally, we wondered how robust the optimized
protocol was, and conducted an extended study with a set of various commercial amines
and acids (Table [2 ]). Gratifyingly and in all of the evaluated 20 examples, we could access the expected
amides via the tolerant microwave protocol and after a simple and direct chromatography
workup (for details see the Supporting Information). Interestingly, all tested examples
with heterocyclic amines or acids provided better yields as compared to a PCl3 -mediated microwave procedure (Figure [2 ]). Examples of primary, secondary, heteroaromatic, and heterocyclic amines and acids
were successfully investigated as well (Table [1 ] and Figure [2 ]).
a Isolated yield after flash chromatography.
Finally, we focused our attention on the synthesis of few selected 1,3-dihydro-2H -benzo[d ]imidazol-2-one based amides (Figure [2 ]). The newly synthesized compounds were easily obtained in up to 85% yields and fully
characterized by means of 1 H NMR, 13 C NMR, and high-resolution MS (APCI). Corresponding amounts were submitted to a semipreparative
HPLC purification after initial flash chromatography to assure highest purity (>99%)
prior the evaluation of the pharmacological effect on TRPC3 channels.
Figure 2 Library of selected 1,3-dihydro-2H -benzo[d ]imidazol-2-one-based amides. Isolated yields from a comparable microwave protocol
using PCl3 for the in situ generation of an acid chloride instead of TCT[5 ] are shown in parentheses.
To assess the activity of the synthesized GSK1702934A-analogues 1a –j in terms of TRPC channel activation, voltage-clamp technique in whole-cell mode on
TRPC3-overexpressing HEK293 cells was employed. The compound-induced current densities
at positive and negative test potentials were compared (Figure [3 ]). Patch clamp recordings were initiated in extracellular solution (ECS) free of
activators followed by continuous perfusion with ECS containing the test compound
of interest at 1 μM concentration (see the Supporting Information for full experimental
details). Satisfyingly, application of the various structural analogues of GSK1702934A
provoked a TRPC3 current, whereby the response to amides 1c , 1e , and 1i was almost negligible (Figure [3 ]). Interestingly, the outward current at 70 mV was comparable with the original TRPC3-agonist
for all other tested amides (1a ,b ,f ,h ). Furthermore, inward currents at –90 mV were slightly smaller or similar to those
observed with GSK1702934A. Inspection of current-to-voltage relations indicated no
significant difference in selectivity of the conductance generated by the different
compounds (data not shown). Importantly, the aliphatic ring adjacent to the thiophene
core appears crucial for the observed agonist properties. Reducing the ring size does
not significantly alter the activity profile. At the same time, oxidation of the sulfur
is detrimental (Figure [3 ], 1c ). The results obtained with compound 1e could be interpreted by low solubility in the standard solution used for the patch
clamp experiments (see the Supporting Information). Introducing substituents (Cl,
OCH3 , NO2 ) on the benzoimidazole ring decreases the observed activity (H > Cl > OCH3 > NO2 ). Installing a thiazole ring with adjacent short aliphatic chain provided a compound
(1h ) with identical activity to the originally reported GSK1702934A.
Figure 3 Screening of GSK1702934A analogues 1a –j : Comparison of current densities at –90/70 mV (n ≥ 5 for each condition) induced
by the individual amides at 1 μM concentration in TRPC3-overexpressing HEK293 cells.
Mean values ± SEM are depicted.
In conclusion, we present an intensified synthetic protocol for the generation of
various amides, including selected examples of 1,3-dihydro-2H -benzo[d ]imidazol-2-ones with promising agonist activity on mammalian TRPC3 channels. Our
method relies on the use of automated high-speed microwave synthesis and benefits
from the use of low-cost commercial cyanuric chloride as an effective coupling reagent
in a single-step protocol. Therefore, we believe that the reduction of reaction and
overall processing time and the higher yields as compared to other methods make our
procedure amenable for the rapid generation of related compound libraries on the search
for agonist-activity enhancement building on the collected structure–activity relationship
data. Finally, we could find a new and structurally simpler motif that provides identical
activation of TRPC3 channels as compared to GSK1702934A. Thus, the presented results
could lead to further improvement of the available tools to deepen the functional
studies of these lipid-sensitive channels.
