Synthesis and biological evaluation of 4′-[(benzimidazol-1-yl) methyl]biphenyl-2-amides as dual angiotensin II and endothelin A receptor antagonists

Article abstract of DOI:10.1039/c5md00169b

By introducing the novel fragment [N-(1H-tetrazol-5-yl)-amide], a series of 4′-[(benzimidazol-1-yl)methyl]biphenyl-2-amides (1a-1w) was designed, synthesized and biologically evaluated. 1d, 1k and 1p showed potent antagonistic activities against the angiotensin II receptor (AT₁) and the endothelin A receptor (ET_A). The evaluation in spontaneous hypertensive rats indicated that the oral activity of compound 1p was more potent than Irbesartan. Structural biological studies of 1p revealed that strong interactions to the AT₁ and ET_A receptors were explicit and the tetrazol-5-ylamide could be an important moiety for the binding to the proteins.

Full text of DOI:10.1039/c5md00169b

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Synthesis and biological evaluation of 4′‐[(benzimidazol‐1‐yl)
methyl] biphenyl‐2‐amides as dual angiotensin II and endothelin
A receptor antagonists
Received 00th January 20xx,
Accepted 00th January 20xx
Xiao‐Feng Han, Wei‐Zhe Xue, Li‐Ping Hao and Zhi‐Ming Zhou∗
DOI: 10.1039/x0xx00000x
www.rsc.org/
By introduced a novel fragment [N‐(1H‐tetrazol‐5‐yl)‐amide], a series of 4′‐[(benzimidazol‐1‐yl)methyl]
biphenyl‐2‐amides (1a‐1w) was designed, synthesized, and biologically evaluated. 1d, 1k and 1p showed
potent antagonistic activities against angiotensin II receptor (AT1) and endothelin A receptor (ETA). The
evaluation in spontaneous hypertensive rats indicated that the oral activity of compound 1p was more
potent than Irbersartan. Structural biology studies of 1p exhibited that strong interactions were contributed
to the AT1 and ETA receptors and the tetrazol‐5‐ylamide could be an important moiety for the binding to the
proteins.
Introduction
Our research efforts over the last decade have concentrated on
Angiotensin II (Ang II) is an octapeptide and potent vasoconstrictor
that presents important functions in the pathophysiology of
hypertension. Ang II usually affects biological functions by activating
selective membrane‐bound receptors1. Two distinct subtypes of
Ang II receptors, namely, AT1 and AT2, have been identified, and
both are known belong to the G‐protein‐coupled receptor
superfamily (GPCRs)2. Numerous studies show that AT1 receptors
are located in various parts of the body and mediate all known
effects associated with Ang II, such as vasoconstriction, aldosterone
release and other functions that elevate blood pressure3. The use of
non‐peptide‐selective AT1 receptor antagonists has become a
globally accepted therapy for hypertension4,5. Endothelin‐1 (ET‐1) is
a potent vasoconstrictor that binds to two GPCR members6, namely,
identifying new types of AT1 receptor antagonists. The derivatives
of 6‐substituted carbonyl benzimidazoles were developed in recent
years12. To develop antihypertensive agents that are more potent
than their prototypes, we took advantage of the established
structure‐activity relationship 13 of 6‐substituted aminocarbonyl
benzimidazoles (Fig. 1, 2) and incorporated a new N‐(1H‐tetrazol‐5‐
yl)‐amide as the isostere of N‐(3,4‐dimethyl‐5‐isoxazolyl)‐
sulphonamide, which has been described as a key pharmacophore
for ETA receptor antagonists by Tellew 14 (Fig. 1, DARA‐3),
meanwhile, the biphenyl moiety with the simplest structure was
maintained as the common and crucial skeleton (Fig. 1, 1). The
present report describes the synthesis and evaluation of
benzimidazoles as DARAs, and further explains the development of
the pharmacophore model and docking study to better understand
the receptor‐ligand interaction.
7
ETA and ETB . Previous studies show that the selective blockade of
ETA receptor antagonists may also provide an alternative and
effective treatment for hypertension and heart failure8,9. Preclinical
studies on animals show that simultaneous antagonism of AT1 and
ETA receptors results in lower blood pressure and greater
therapeutic benefits than antagonising either of the receptors
individually10. Experimental results further show that dual AT1 and
ETA receptor antagonists (DARAs) are more effective than the
current standard therapies for antihypertension and other
cardiovascular diseases11.
R & D Center for Pharmaceticals, Beijing Institute of Technology, Beijing 100081,
China PR. E‐mail: zzm@bit.edu.cn; Fax: 86 10 68918982; Tel: 86 10 68918982
† Electronic Supplementary Informaꢀon (ESI) available: See
DOI: 10.1039/x0xx00000x
Fig. 1 Strategy for the design of target DARAs.
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The target compounds (1a to 1w) were synthesized according to
Results and discussion
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the route described in Scheme 1, andDOalIl: 1t0h.1e03b9/eCn5zMimDi0d0a1z6o9lBe
precursors (4 to 7) were prepared following our own methods
previously described12,17 with good yield. In the last step, 7a to 7w
were converted to the N‐(1H‐tetrazol‐5‐yl)‐amides (1a to 1w) with
anhydrous 5‐amino‐tetrazole in DMF. All final compounds were
identified by MS, 1H‐NMR, 13C‐NMR and elemental analysis.
Based on our study of DARA pharmacophore hypothesis15, the
structures of the test set of the target benzimidazoles (1a to 1w)
were constructed and their conformational models were generated.
By means of the selected DARA hypothesis (Fig. 2), the activities of
tested compounds were predicted during the Compare/Fit
process16, and the values of the best‐fitting conformers were
obtained and listed in Table 1. This molecular modelling simulation
revealed that compounds 1d (5.916), 1f (5.467), 1i (5.469), 1k,
(5.503), 1p (5.893) and 1t (5.571) could be considered as promising
candidates.
Scheme 1. Reagents and conditions: (a) t‐BuOK, DMF, r.t., 6h; (b)
KOH, CH3OH; 2h; (c) anhydrous 5‐amino‐tetrazole, HATU, DMF, 2h.
In vitro Ang II receptor 1 and endothelin receptor A binding assays18
were performed to evaluate the affinity of target compounds and
the results were expressed as IC50 (Table 1). Losartan and Bosentan
were taken as positive control drugs in the assays respectively.
From all the IC50 values in Table 1, 23 candidate compounds 1a to
1w display generally good activity to AT1 receptor. On the
contrary, their performance varies with that of the ETA receptor.
Unlike the relatively close fit values, there are significant activity
cliffs within the results of ETA activity, e.g. benzyl to Ph (1400×loss
of potency) or benzyl to 4‐methoxybenzyl (>2000×loss). It is
probably because the molecules with the reported fragment of N‐(3,
4‐dimethyl‐5‐isoxazolyl)‐sulphonamide were chosen for the training
set and test set of the DARA pharmacophore hypothesis15. However,
this new SAR data could enrich the structural diversity and the
fitting experiences necessary to improve this model in further study.
Some of the tested molecules containing saturated heterocyclic
structures fail to exhibit good binding affinity to ETA receptor,
except for 1d, which has a moderate value (AT1 IC50=44 nM, ETA
IC50=165 nM) possibly because of the introduced oxygen atom.
Aliphatic amides 1e to 1h cannot produce acceptable results. The
compounds 1i to 1w, which start with the AT1 receptor antagonist
core12, show good activity at the AT1 receptor and partly moderate
binding affinity at the ETA receptor. The compound 1k demonstrates
high ETA receptor affinity and good AT1 receptor binding. It suggests
that the tetrazol amide in 1k apparently substituted for the
pyrimidine sulphonamide in Bosentan. In addition, the
phenylethylamides 1p were more active (AT1 IC50=3.9 nM) than
Losartan was (AT1 IC50=16.2 nM). The methoxyl substituents of
amides maintain the AT1 receptor antagonism but decrease the ETA
receptor antagonism. This result may be attributed to the high
electron density on the benzene ring of the amide substituents that
might promote the binding with the AT1 receptor. Furthermore, the
replacement of the methoxyl group by fluorin does not improve the
activity (compound 1s vs. 1w). With regard to the ETA receptor, the
steric congestion affects the binding affinity between the
Fig.2 The selected pharmacophore hypothesis Hypo‐DARA.
Table 1 Fit values on the pharmacophore and in vitro experimental
data of compounds (1a‐1w).
Fit
AT1
ETA
IC50(nM)a
7100
R
value
5.180
5.274
IC50(nM)a
69.4±6.1
67.5±7.3
1a
1b
2‐(pyrrolidin‐1‐yl)ethyl
2‐(piperidin‐1‐yl)ethyl
2‐(4‐methylpiperazin‐1‐
yl)ethyl
>20000
1c
5.291
53.5±4.9
9100
1d
2‐morpholinoethyl
n‐propyl
5.916
5.266
5.467
5.327
5.235
5.469
5.151
5.503
5.305
5.324
5.439
5.274
5.893
5.346
5.296
5.142
5.571
5.234
5.138
5.281
‐‐‐‐
44±4.2
363±31
161±27
439±85
390±76
73.7±7.6
347±29
82±8.1
165±33
>20000
>20000
>20000
>20000
14000
1e
1f
isopropyl
1g
n‐butyl
1h
tert‐butyl
1i
phenyl
1j
2‐methoxyphenyl
benzyl
886±131
10±2.2
7200
1k
1l
1m
2‐methoxybenzyl
3‐methoxybenzyl
4‐methoxybenzyl
3,4‐dimethoxybenzyl
2‐phenylethyl
73.3±5.8
53.9±4.3
549.8±87
32.9±4.1
3.9±0.4
26.1±3.7
36.9±5.5
87.8±7.3
30.7±4.8
93±10.7
90±8.4
625±98
>20000
10000
1n
1o
1p
126±25
7000
1q
2‐methoxyphenethyl
3‐methoxyphenethyl
4‐methoxyphenethyl
2,5‐dimethoxyphenethyl
3,4‐dimethoxyphenethyl
2‐fluorophenethyl
4‐fluorophenethyl
‐‐‐‐
1r
>20000
13000
1s
1t
>20000
>20000
11000
1u
1v
1w
140±11.4
16.2±1.2
>20000
‐‐‐‐
Losartan
Bosentan
‐‐‐‐
‐‐‐‐
‐‐‐‐
8.4±1.1
a. Each value is the mean ± SEM of three determinations.
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compounds and the ETA receptor. When 6‐amide substituent the modelled ETA protein22,23. Hydrophobic groups are positioned in
View Article Online
groups on the benzimidazoles of the tested compounds are kept a a lipophilic cavity pocket formed by Phe20D8O,I:L1e0u.1101399/Can5MdDP0h0e12649B9
certain length, the groups display better antagonistic activity with (Fig. 4A). One H‐bond interaction was formed between the acidic
decrease in bulk, because the relatively small groups could easily tetrazole ring and O–H of Tyr184 (2.55 Å distance), then another
enter the cavity pocket of the ETA receptor and generate the was between the oxygen of tetrazol amide and N‐H of Asn200.
hydrophobic interaction. Compared to the isostere of isoxazolyl Compared with ETA model24, the single interaction caused by the
sulphonamides from our previous work15, though it appears that formation of an H‐bond between the tetrazole ring and the N‐H of
only several structures of the tetrazol amide provide a better ETA Gln165 (2.58 Å distance) could be observed in the hydrophilic
activity than the isoxazolyl sulphonamide, for example 2‐ pocket from Gln165, Lys166 and Gly170 (Fig. 4B). However, the
morpholinoethyl, phenyl and 2‐phenylethyl, the acylamino tetrazole effect of docking on the ETA model was not expected, given the
still provides a good new option and direction for deep study as the insufficient depth of the ligand pose into the corresponding protein
isostere of its traditional structure. To sum up, three of the cavity. Therefore, it seems that 1p docking to the AT1 receptor was
synthesised compounds 1d, 1k and 1p demonstrate obvious and more evident and had stronger interactions with the target sites
balanced dual antagonism to AT1 and ETA receptors in accord with compared with docking to the ETA receptor, and this finding was
the fit values, and 1p exhibits slightly better activity than 1d and 1k. consistent with the experimental binding result of 1p. More
important, the fragment of tetrazol amide played a key role both in
The most potent DARA 1p in in vitro binding assays was selected to
further evaluate its antihypertensive effects on in vivo models. As
shown in Fig. 3, after oral administration of 1p (20 mg/kg) and
AT1 and ETA antagonism.
Irbesartan (20 mg/kg) to spontaneous hypertensive rats (SHRs) 19
,
the maximal reduction of compound 1p obtained at 6h was
observed with 24mmHg on the mean blood pressure (BMP), while
the reductive effect of Irbesartan reached 20 mmHg at 4h. Although
both compound 1p and Irbesartan could show the antihypertensive
effect in 24h, the duration of lowering BMP over 20 mmHg was kept
for 4 hours by 1p, compared with only 1 hour by Irbesartan20.
Furthermore, the heart rate (HR) was as effectively controlled by 1p
as Irbesartan (Fig. 3). Therefore, compound 1p was evidently
superior to Irbesartan.
Fig. 4A The detailed representation of docking 1p to AT1.
Compound 1p are represented in sticks and colored by atom types
(carbon: green, oxygen: red, nitrogen: blue, H‐bond: sky‐blue).
Fig. 4B Model of the compound 1p bound to ETA. Compound 1p are
represented in sticks and colored by atom types (carbon: green,
oxygen: red, nitrogen: blue, H‐bond: sky‐blue).
Conclusions
By introducing the novel and potent N‐(1H‐tetrazol‐5‐yl)‐amide
fragment to the biphenyl ring, a benzimidazole‐based series of
DARAs was designed and synthesised. In the in vitro dual receptor
binding assay, 1d, 1k and 1p showed satisfactory antagonistic
biological activity towards AT1 and ETA receptors. Compound 1p,
which notably showed anti‐hypertensive activity in SHRs, proved to
be more efficacious than Irbesartan. Furthermore, the 1p docking
Fig. 3 Effects of 1p and Irbersartan (20 mg/kg po) on MAP and HR in
conscious SHRs after oral administration.
Docking studies were performed to evaluate the binding modes of
1p in a theoretical human AT1 receptor protein (PDB ID: 1ZV0)21 and
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study revealed that the relatively higher antihypertensive activity
was attributed to excellent fitting as a result of stronger lipophilic
and hydrophilic interactions between ligand 1p and AT1 receptor
pockets, whereas the relatively weaker activity to ETA resulted from
the single hydrophilic interaction of the tetrazole ring of the same
ligand with the ETA receptor residue.
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Acknowledgements
Financial support of this work by the National Technological Project
of the Manufacture and Innovation of Key New Drugs
(2009ZX09103‐143) is appreciated.
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