Intensified Microwave-Assisted N-Acylation Procedure - Synthesis and Activity Evaluation of TRPC3 Channel Agonists with a 1,3-Dihydro-2H-benzo[d]imidazol-2-one Core

Article abstract of DOI:10.1055/s-0036-1589472

Upon controlled microwave heating and using cyanuric chloride as a coupling reagent, an efficient amidation procedure for the synthesis of 1,3-dihydro-2H-benzo[d]imidazol-2-one-based agonists of TRPC3/6 ion channels has been developed. Compared to the few conventional protocols, a drastic reduction in processing time from ca. 2 days down to 10 minutes was achieved accompanied by significantly improved product yields. The robustness of the method was confirmed by 18 additional examples including aromatic, aliphatic, and heterocyclic amines and acids. The obtained agonists were screened for biological activity at 1 μM concentration and few structure-activity relations have been established.

Full text of DOI:10.1055/s-0036-1589472

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© Georg Thieme Verlag Stuttgart · New York
2016, 27, A–F
letter
A
G. Guedes de la Cruz et al.
Letter
Synlett
Intensified Microwave-Assisted N-Acylation Procedure – Synthesis
and Activity Evaluation of TRPC3 Channel Agonists with a 1,3-Di-
hydro-2H-benzo[d]imidazol-2-one Core
Gema Guedes de la Cruza
Barbora Svobodovab
Michaela Lichteneggerb
Oleksandra Tiapkob
Klaus Groschner*b
Toma Glasnov*a
a Institute of Chemistry, University of Graz and NAWI Graz,
Heinrichstrasse 28, 8010 Graz, Austria
toma.glasnov@uni-graz.at
b Institute of Biophysics, Medical University of Graz,
Harrachgasse 21/IV, 8010 Graz, Austria
klaus.groschner@medunigraz.at
Received: 23.09.2016
Accepted after revision: 14.11.2016
Published online: 08.12.2016
been briefly described by a team at GlaxoSmithKline-US as
a tool to activate lipid-sensitive TRPC channels directly, by-
passing phospholipase-C signaling.3
DOI: 10.1055/s-0036-1589472; Art ID: st-2016-b0634-l
H
N
O
Abstract Upon controlled microwave heating and using cyanuric chlo-
ride as a coupling reagent, an efficient amidation procedure for the syn-
thesis of 1,3-dihydro-2H-benzo[d]imidazol-2-one-based agonists of
TRPC3/6 ion channels has been developed. Compared to the few con-
ventional protocols, a drastic reduction in processing time from ca. 2
days down to 10 minutes was achieved accompanied by significantly
improved product yields. The robustness of the method was confirmed
by 18 additional examples including aromatic, aliphatic, and heterocy-
clic amines and acids. The obtained agonists were screened for biologi-
cal activity at 1 μM concentration and few structure–activity relations
have been established.
N
GSK1702934A
N
S
O
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, respec-
tively. In a continuation of our attempts to evaluate the po-
tential 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 acti-
vators 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. More-
over, the agonist effects of novel synthetic TRPC3 activators,
generated by the means of the enhanced synthetic protocol,
were examined and few essential structure–activity rela-
tionships were established.
Key words microwave synthesis, acylation, amides, Ca2+-signaling,
TRPC ion channels, agonist
The cation canonical transient receptor potential chan-
nels (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 patholo-
gies – focal segmental glomerulosclerosis, pulmonary hy-
pertension, ischaemia reperfusion-induced lung oedema,
myocardial hypertrophy, as well as in the regulation of vas-
cular 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 modula-
tors.2 Recently, a small 1,3-dihydro-2H-benzo[d]imidazol-
2-one-based potent agonist (GSK1702934A, Figure 1) has
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 avail-
Georg Thieme Verlag Stuttgart · New York — Synlett 2016, 27, A–F

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G. Guedes de la Cruz et al.
Letter
Synlett
able 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 follow-
ing coupling step. Moreover, an earlier microwave N-acyla-
tion procedure5 successful in the synthesis of pyrazole-
based selective inhibitors of TRPC3/6 channels provided
lower yields from a complex reaction mixture (data not
shown).
safe, cost-effective, and stable reagent that is amenable to
large-scale production.7a,i While working at elevated tem-
peratures and with good yields, the reported reaction need-
ed at least six and up to 18 hours for some substrates to
reach completion. For these reasons, we sought for the in-
tensification 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 micro-
wave heating could potentially enhance the process.8
In our optimization efforts we used 4-(2-keto-1-benz-
imidazolinyl)piperidine (Table 1, A) and octanoic acid (Ta-
ble 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–Et3N
combination and MeCN as the preferred solvent. To access
the optimal reaction conditions, we elaborated the best re-
agent ratio and the reaction temperature with a reaction
time limit set at 5 minutes (Table 1).
An exploratory room-temperature trial was terminated
after six days, resulting in only 22% conversion, as deter-
mined 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 demon-
strated 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 stoichi-
ometry 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
S
S
a
b
GSK1702934A
65%, 2 steps
HO2C
ClO2C
Scheme 1 Previous synthesis of GSK1702934A. Reagents and conditions:
a) oxalyl chloride, DMF, CH2Cl2, 0 °C to r.t., 1 h, 80%; b) 4-(2-keto-1-benz-
imidazolinyl)piperidine, DMAP, CH2Cl2, 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 re-
quire rather long reaction times and are relatively expen-
sive), we turned our attention to a recent literature report
by Rad et al. that describes an experimentally very straight-
forward amidation process towards N-acylated nucleobas-
es.6 The described procedure involves simple heating of di-
verse nucleobases and carboxylic acids (aromatic, aliphatic,
heterocyclic) at 110 °C using cyanuric chloride in the pres-
ence of NaH and Et3N in DMF–MeCN (1:1) as a solvent mix-
ture. Cyanuric chloride (2,4,6-trichloro-1,3,5-triazine, TCT),
besides other synthetic applications, is also a suitable acti-
vating reagent for the in situ generation of acid halides that
is a frequently used technique in peptide synthesis.7 It is a
Table 1 Optimization of the TCT-Promoted Microwave N-Acylation
H
N
H
N
Cl
O
Pyridine (D)
MeCN
O
N
O
N
N
N
+
+
C7H15
OH
MW, T °C, 5 min
Cl
N
Cl
1a
N
N
H
C7H15
O
Amine (A)
Octanoic acid (B)
TCT (C)
Entry
A/B/C/D (equiv)
Temp (°C)
Conv. (%)a
1b
2
1.0/1.2/0.42
90
90
traces
1.0/1.2/0.42/1.5
1.0/1.2/0.42/1.5
1.0/1.2/0.42/1.5
1.0/1.2/0.42
52
3
120
140
140
140
68
4
93 (54)c
22
5d
6
1.2/1.0/0.6/1.5
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
Georg Thieme Verlag Stuttgart · New York — Synlett 2016, 27, A–F

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G. Guedes de la Cruz et al.
Letter
Synlett
the amide 1a by flash chromatography furnished 63% prod-
uct yield, a significant improvement to the 47% obtained in
a PCl3-mediated microwave procedure. Additionally, we
wondered how robust the optimized protocol was, and con-
ducted 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 am-
ides via the tolerant microwave protocol and after a simple
and direct chromatography workup (for details see the Sup-
porting Information). Interestingly, all tested examples with
heterocyclic amines or acids provided better yields as com-
pared to a PCl3-mediated microwave procedure (Figure 2).
Examples of primary, secondary, heteroaromatic, and het-
erocyclic amines and acids were successfully investigated as
well (Table 1 and Figure 2).
Table 2 Confined Scope of the TCT-Promoted Microwave N-Acylation
Entry
1
Amine
Acid
Amidea
Yield (%)
71
NO2
O
O
NH2
CO2H
N
H
O2N
F
NH2
CO2H
CO2H
CO2H
2
3
63
75
N
H
F
CH3
H3C
H3C
NH2
CH3
CH3
O
O
N
H
CH3
NO2
NHCH3
4
5
6
7
83
55
72
57
N
O2N
CH3
F
O
O
NHCH3
CO2H
CO2H
CO2H
N
F
CH3
CF3
NHCH3
N
CH3 CF3
O
H
N
N
O
H
N
CO2H
N
8
9
28
68
H
N
CO2H
O
N
O
O
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G. Guedes de la Cruz et al.
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Table 2 (continued)
Entry
Amine
Acid
Amidea
Yield (%)
F
O
O
NH2
CO2H
10
11
45
82
N
H
F
NC
NC
NC
NO2
NHCH3
CO2H
CO2H
CO2H
N
O2N
CH3
NC
NO2
O
NHCH3
12
13
14
74
76
86
N
O2N
O2N
CH3
O2N
NO2
O
NHCH3
NHCH3
N
O2N
Cl
CH3
Cl
NO2
O
CO2H
N
O2N
N
CH3
N
CH3
NHCH3
CH3
CO2H
NO2
15
16
74
59
O2N
O
N
CH3
O
H
CO2H
N
N
N
N
CH3
O
CO2H
CO2H
CH3
17
18
87
82
CH3
N
H
CH3
NH2
O
N
H
NH2
NH2
Cl
CO2H
CO2H
H
N
19
20
79
39
N
O
N
Cl
O
N
S
N
S
NH2
NH
a Isolated yield after flash chromatography.
Georg Thieme Verlag Stuttgart · New York — Synlett 2016, 27, A–F

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G. Guedes de la Cruz et al.
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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 1H NMR, 13C NMR, and high-resolution MS
(APCI). Corresponding amounts were submitted to a semi-
preparative HPLC purification after initial flash chromatog-
raphy to assure highest purity (>99%) prior the evaluation
of the pharmacological effect on TRPC3 channels.
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 com-
pound (1h) with identical activity to the originally reported
GSK1702934A.
S
H
R1
85%
(73%)
1b, R1 = Cl, R2
=
N
O
N
O
S
O
34%
1c, R1 = Cl, R2
1d, R1 = H, R2
=
N
S
R2
96%
(82%)
O
=
S
89%
(50%)
1f, R1 = Cl, R2 = as 1e
85%
(58%)
1e, R1 = H, R2
=
1g, R1 = H, R2
1h, R1 = H, R2
=
=
1i, R1 = NO2, R2 = as 1b
1j, R1 = OCH3, R2 = as 1b
63%
72%
S
90%
N
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.
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 TCT5
are shown in parentheses.
In conclusion, we present an intensified synthetic pro-
tocol for the generation of various amides, including select-
ed 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 cou-
pling reagent in a single-step protocol. Therefore, we be-
lieve 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-activi-
ty enhancement building on the collected structure–activi-
ty relationship data. Finally, we could find a new and struc-
turally 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.
To assess the activity of the synthesized GSK1702934A-
analogues 1a–j in terms of TRPC channel activation, volt-
age-clamp technique in whole-cell mode on TRPC3-overex-
pressing HEK293 cells was employed. The compound-in-
duced current densities at positive and negative test poten-
tials were compared (Figure 3). Patch clamp recordings
were initiated in extracellular solution (ECS) free of activa-
tors 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 rela-
tions 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 al-
ter the activity profile. At the same time, oxidation of the
Acknowledgment
This work was supported by a grant (P28243-B27) from the Austrian
Science Funds (FWF). We thank to Mrs. Chiara Mayer for her help.
Georg Thieme Verlag Stuttgart · New York — Synlett 2016, 27, A–F

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G. Guedes de la Cruz et al.
Letter
Synlett
Supporting Information
Pattarawarapan, M. RSC Adv. 2015, 5, 52624. (i) Dunetz, J. R.;
Magano, J.; Weisenburger, G. A. Org. Process Res. Dev. 2016, 20,
140.
Supporting information for this article is available online at
http://dx.doi.org/10.1055/s-0036-1589472.
S
u
p
p
ortioInfgrmoaitn
S
u
p
p
ortiInfogrmoaitn
(8) Selected references: (a) Alterman, M.; Andersson, H. O.; Garg,
N.; Ahlsén, G.; Lövgren, S. B.; Classon, B.; Danielson, U. H.;
Kvarnström, I.; Vrang, L.; Unge, T.; Samuelsson, B. A.; Hallberg,
A. J. Med. Chem. 1999, 42, 3835. (b) Varma, R. S. Green. Chem.
1999, 1, 43. (c) Nöteberg, D.; Schaal, W.; Hamelink, E.; Vrang, L.;
Larhed, M. J. Comb. Chem. 2003, 5, 456. (d) Microwaves in
Organic Synthesis, 3rd ed., Vol. 1; de la Hoz, A.; Loupy, A., Eds.;
Wiley-VCH: Weinheim, 2012. (e) Microwaves in Organic Synthe-
sis, 3rd ed., Vol. 2; de la Hoz, A.; Loupy, A., Eds.; Wiley-VCH:
Weinheim, 2012.
References and Notes
(1) (a) Lichtenegger, M. In Mammalian Transient Receptor Potential
(TRP) Cation Channels, Vol. 1; Nilius, B.; Flockerzi, V., Eds.;
Springer: Berlin/Heidelberg, 2014, 67–84. (b) Dietrich, A. In
Mammalian Transient Receptor Potential (TRP) Cation Channels,
Vol. 1; Nilius, B.; Flockerzi, V., Eds.; Springer: Berlin/Heidelberg,
2014, 157–188. (c) Pathologies of Calcium Channels; Weiss, N.;
Koschak, A., Eds.; Springer: Berlin/Heidelberg, 2014.
(2) (a) Harteneck, C.; Gollasch, M. Curr. Pharm. Biotechnol. 2011, 12,
35. (b) Bon, R. S.; Beech, D. J. Br. J. Pharmacol. 2013, 170, 459.
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Korichneva, I.; Ouadid-Ahidouch, H. Br. J. Pharmacol. 2014, 171,
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(4) Doleschal, B.; Primessnig, U.; Wölkart, G.; Wolf, S.;
Schernthaner, M.; Lichtenegger, M.; Glasnov, T.; Kappe, C. O.;
Mayer, B.; Antoons, G.; Heinzel, F.; Poteser, M.; Groschner, K.
Cardiovasc. Res. 2015, 106, 163.
(5) (a) Colombo, M.; Bossolo, S.; Aramini, A. J. Comb. Chem. 2009,
11, 335. (b) Glasnov, T. N.; Groschner, K.; Kappe, C. O. ChemMed-
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(d) Bergman, K.; Elvingson, C.; Hilborn, J.; Svensk, G.; Bowden, T.
Biomacromolecules 2007, 8, 2190. (e) Chen, C.-Y.; Frey, L. F.;
Shultz, S.; Wallace, D. J.; Marcantonio, K.; Payack, J. F.; Vazquez,
E.; Springfield, S. A.; Zhou, G.; Liu, P.; Kieczykowski, G. R.; Chen,
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(10) General Experimental Procedure for Microwave-Assisted
TCT-Acylation
To a stirred mixture of a corresponding acid (0.819 mmol, 1
equiv), cyanuric chloride (90.5 mg, 0.491 mmol, 0.6 equiv), dry
pyridine (99.1 mg, 1.23 mmol, 1.5 equiv), and dry MeCN (4.5
mL) in a 5 mL Pyrex microwave vial, equipped with a magnetic
stir bar, the corresponding amine (0.983 mmol, 1.2 equiv) was
added after initial stirring at r.t. for 5 min. The reaction mixture
was then crimped with a Teflon septum, stirred for 10 s, and
subjected to microwave heating for 5 min (fixed hold time) at
140 °C and subsequently cooled down to 40 °C. The resulting
reaction mixture was concentrated under reduced pressure, and
the residue was purified by flash chromatography (with CHCl3
or a gradient of 0–10% MeOH in CHCl3) to afford amides 1a–g.
For pharmacological experiments, analytical-grade samples of
compounds 1a–g were additionally purified by the means of a
semipreparative HPLC (see the provided Supporting Informa-
tion).
1-(1-Octanoylpiperidin-4-yl)-1,3-dihydro-2H-benzo[d]imid-
azol-2-one (1a)
Transparent oil; yield 177 mg (63%). 1H NMR (300 MHz, CDCl3):
δ = 10.05 (br s, 1 H), 7.15–7.04 (m, 4 H), 4.90 (d, J = 13.1 Hz, 1 H),
4.60–4.52 (m, 1 H), 4.07 (d, J = 13.1 Hz, 1 H), 3.20 (t, J = 12.9 Hz,
1 H), 2.68 (t, J = 13.5 Hz, 1 H), 2.43–2.26 (m, 4 H), 1.93–1.88 (m,
2 H), 1.73–1.64 (m, 2 H), 1.40–1.25 (m, 8 H), 0.88 (t, J = 6.8 Hz, 3
H). 13C NMR (75 MHz, CDCl3): δ = 171.9, 155.1, 129.0, 128.2,
121.6, 121.3, 110.1, 109.4, 50.9, 45.4, 41.5, 33.6, 31.9, 29.6, 29.3,
25.6, 22.8, 14.2. HRMS (APCI): m/z calcd for C20H30N3O2 [M +
H]+: 344.2332; found: 344.2334.
Georg Thieme Verlag Stuttgart · New York — Synlett 2016, 27, A–F