2-Amino- and 2-hydroxymethylbenzimidazolium bromides as protein tyrosine phosphatase 1В (PTP1В) inhibitors and other targets associated with diabetes mellitus

Article abstract of DOI:10.1007/s11172-020-2832-5

New 2-amino- and 2-hydroxymethylbenzimidazoles were synthesized and used to prepare the previously unknown 1,2,3-tri- and 1,2,3,5-tetrasubstituted benzimidazolium bromides with a biphenyl-containing substituent at the imidazole nitrogen atom. In some cases, these bromides exhibit activity against targets associated with diabetes mellitus. These compounds are strong protein tyrosine phosphatase 1B (PTP1B) inhibitors, exhibit chelating and antiglycation activity, but have no significant AT₁ receptor antagonist activity. Hence, biphenyl-containing benzimidazolium derivatives can be considered as a basis for the development of new promising agents for the treatment of type 2 diabetes mellitus (DM2) and other diseases mediated by high phosphatase PTP1B activity.

Full text of DOI:10.1007/s11172-020-2832-5

Russian Chemical Bulletin, International Edition, Vol. 69, No. 4, pp. 774—780, April, 2020
774
2-Amino- and 2-hydroxymethylbenzimidazolium bromides
as protein tyrosine phosphatase 1В (PTP1В) inhibitors and
other targets associated with diabetes mellitus*
a
b
a
a
a
A. A. Spasov, O. N. Zhukovskaya, D. A. Babkov, A. A. Brigadirova, V. A. Babkova,
b
a
a
A. S. Morkovnik, R. A. Litvinov, and E. V. Sokolova
aVolgograd State Medical University,
1 pl. Pavshikh Bortsov, 400131 Volgograd, Russian Federation.
E-mail: dababkov@volgmed.ru
bInstitute of Physical and Organic Chemistry, Southern Federal University,
194/2 prosp. Stachki, 344090 Rostov-on-Don, Russian Federation.
E-mail: asmork2@ipoc.rsu.ru
New 2-amino- and 2-hydroxymethylbenzimidazoles were synthesized and used to prepare
the previously unknown 1,2,3-tri- and 1,2,3,5-tetrasubstituted benzimidazolium bromides with
a biphenyl-containing substituent at the imidazole nitrogen atom. In some cases, these bromides
exhibit activity against targets associated with diabetes mellitus. These compounds are strong
protein tyrosine phosphatase 1В (PTP1В) inhibitors, exhibit chelating and antiglycation activity,
but have no significant AT1 receptor antagonist activity. Hence, biphenyl-containing benzimid-
azolium derivatives can be considered as a basis for the development of new promising agents
for the treatment of type 2 diabetes mellitus (DM2) and other diseases mediated by high phos-
phatase PTP1В activity.
Key words: biphenyl, 2-aminobenzimidazole, synthesis, benzimidazolium bromides, anti-
diabetic activity, PTP1B.
A modern strategy of pharmacotherapy of type 2 dia-
thereby requiring the use of combination therapy even in
the early-stage disease.3
betes mellitus (DM2) is to maintain the normal blood
glucose level by improving insulin secretion and reducing
peripheral insulin resistance and to prevent and decrease
macro- and microvascular complications of diabetes.1,2
There is a wide range of drugs, which can be used to
treat DM2. The main groups of these medications include
insulin secretagogues, i.e., compounds stimulating insulin
secretion (sulfonylurea derivatives), postprandial insulin
secretion stimulants, glucagon-like peptide-1 (GLP-1)
receptor agonists, dipeptidyl peptidase-4 (DPP-4) in-
hibitors, biguanides, peroxisome proliferator-activated
receptor γ (PPARγ) agonists, α-glucosidase inhibitors,
and sodium glucose cotransporter 2 inhibitors (glyphlo-
sins). However, available drugs have serious drawbacks,
including low efficacy and undesirable side effects, in
particular, the possibility of developing hypoglycemia up
to hypoglycemic coma, undesirable body weight gain, and
the unfavorable effect on the gastrointestinal tract. Besides,
the gradual development of drug resistance in patients with
DM subjected to monotherapy is a rather general problem,
Hence, the search for new more effective antidiabetic
agents with mechanisms of action that are different from
those of the known drugs is still a challenge. In this regard,
compounds, which are capable of interacting with the
insulin receptor (IR) and with enzymes involved in insu-
lin-receptor related signaling pathways, can be considered
as promising agents.
The insulin receptor belongs to the receptor tyrosine
kinase family,4 and its action is mediated by autophos-
phorylation of tyrosine residues in the intracellular part
of the receptor, which triggers a cascade of coupled events,
resulting in cellular glucose uptake.5 Hence, an important
mechanism of insulin resistance may be related to patholo-
gically high levels of intracellular dephosphorylation of
tyrosine residues of the receptor or even its substrates
catalyzed by protein tyrosine phosphatases (PTPs). The
cytoplasmic phosphatase PTP1B may play a key role in
this process. Consequently, the normal insulin receptor
function can be restored by providing cellular uptake of
PTP1B inhibitors, which can be used as antidiabetic agents.
These inhibitors can also be applied in the cancer treat-
ment6 taking into account that a series on malignant tumor
lines, for example, chronic myeloid leukemia (CML),
* Based on the materials of the IV Russian Conference on
Medicinal Chemistry with international participants (June 9—13,
2019, Ekaterinburg).
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 4, pp. 0774—0780, April, 2020.
1066-5285/20/6904-0774 © 2020 Springer Science+Business Media LLC

Benzimidazolium bromides
Russ. Chem. Bull., Int. Ed., Vol. 69, No. 4, April, 2020
775
breast cancer, ovarian cancer, and prostate cancer, are
characterized by increased PTP1B activity,7 and in the
treatment of metabolic syndrome, obesity, and dys-
lipidemia.
Agents based on PTP1B inhibitors are not used in
clinical practice. However, Russian and foreign research-
ers are currently giving considerable attention to this issue.
In particular, the bis-thiazole derivative JTT-551 was
found to have PTP1B inhibitory activity and is currently
in preclinical trials as an antidiabetic agent.
agent (9 months) leads to the reduction of the serum
HbA1c level and the body weight of patients.12
Reverting to the issue of PTP1B inhibitors, it is worth
noting that the use of compounds containing two privileged
scaffolds is a promising approach to the design of such
agents.13 Here, we applied this approach for biphenyl-
containing quaternary benzimidazolium salts bearing
benzimidazole and biphenyl moieties as privileged scaf-
folds. Previously, we showed that 1,2,3-substituted qua-
ternary benzimidazolium salts containing no biphenyl
groups can possess pharmacological effects essential for
the treatment of DM2, such as the improvement of
hemorheological properties,14 inhibition of platelet ag-
gregation combined with antioxidant and antiarrhythmic
activity,15 and even the rare ability to break crosslinks in
glycated proteins, which is exhibited by salts containing
the phenacyl and methyl groups at positions 1 and 2, re-
spectively, of the benzimidazolium cation.16
Results and Discussion
In this study, we report the synthesis of new biphenyl-
containing 1,2,3-tri- and 1,2,3,5-tetrasubstituted 2-amino-
and 2-hydroxymethylbenzimidazolium salts, i.e., salts
containing the privileged biphenyl and benzimidazole
substructures. We used biphenylmethyl or biphenoylmethyl
groups linked to the imidazolium ring through its nitrogen
atom as biphenyl-containing substituents (Scheme 1).
The two-step synthesis of amino-containing salts 2a—g
included the N-biphenylmethylation of 2-aminobenzimid-
azole at the endocyclic nitrogen atom giving the previously
unknown N-substituted amines 1a,b. The reaction was
performed in acetone in the presence of aqueous alkali.
Biphenylmethylation products 1a,b are colorless crystalline
compounds. It should be noted that amine 1a has low
solubility, which hinders, to some extent, its further trans-
formation into quaternary salts. The structures of amines
1a,b were confirmed by 1H NMR spectroscopy. The NMR
spectra show, apart from other signals, two-proton singlets
of NH2 (δ 4.61 and 6.53) and CH2 groups (δ 5.29 and 5.2).
Besides, the NMR spectrum of amine 1b shows a three-
proton singlet of the methoxy group at δ 3.65. The signals
of the biphenyl group and the benzimidazole nucleus ap-
pear as multiplets at δ 6.82—7.84. The IR spectra of two
amines contain two absorption bands of the primary amino
group at 3307—3387 cm–1; in addition, the IR spectrum
of amine 1b shows a band of the carbonyl group at
1725 cm–1. In the second (final) step, amines 1a,b were
quaternized with appropriate bromides.
The preliminary results obtained in the genetic animal
model of obesity (ob/ob mice) and the db/db mouse model
of type 2 diabetes mellitus showed that the the compound
JTT-551 improves glucose metabolism due to activation
of insulin and leptin signaling pathways.8 Besides, the
normalization of animal body weight and lipid metabolism
was reported.9
The 8-hydroxyquinoline derivative NSC-87877 con-
taining the sulfonated naphthylazo group at position 7 also
exhibits strong inhibitory activity.10 Biphenyls comprise
an important group of potential PTP1B inhibitors.11
The normal IR function can be maintained in a differ-
ent way using, instead of tyrosine phosphatase (PTP1B)
inhibitors, antisense oligonucleotides that arrest the ex-
pression of the gene responsible for PTP1B synthesis. Here,
the synthesis of the oligonucleotide IONIS-PTP1BRx was
the most significant advancement. This oligonucleotide
has successfully passed phase II clinical trials as a drug for
the treatment of DM2. The long-term treatment with this
Quaternary salts based on 2-hydroxymethyl-5-meth-
oxy-1-methylbenzimidazoles 2i,j were synthesized by
biphenoylmethylation of 2-hydroxymethyl-5-methoxy-1-
methylbenzimidazole (1c). Compound 1c was prepared
using the scheme, classical for benzimidazoles monosub-
stituted at the benzene ring, starting from the appropriate

Spasov et al.
776
Russ. Chem. Bull., Int. Ed., Vol. 69, No. 4, April, 2020
Scheme 1
Compound
1a, 2a—c
1b, 2d—g
1c, 2h
R1
NH2
NH2
CH2OH
CH2OH
R2
Biphenyl
4-(2-Carboxyphenyl)phenyl
R3
H
H
OMe
OMe
Me
H
1d, 2i,j
Reagents and conditions: i. 4-FC6H4COCH2Br, DMF; ii. 4-MeOC6H4COCH2Br, DMF; iii. 3,4-MeOC6H3COCH2Br, DMF;
iv. C6H5COCH2Br, DMF; v. 4-CNC6H4COCH2Br; vi. (CH2)4NC6H4COCH2Br, DMF; vii. 3,5-But2-4-OHC6H2COCH2Br, DMF;
viii. 4-C6H3C6H4COCH2Br, MeCN, ; ix. 4-BrC6H4C6H4COCH2Br, MeCN, ; x. (CH2)4NCC6H4COCH2Br, MeCN, .
methoxylated o-nitroaniline. The latter was initially con-
verted into the N-methyl derivative by the treatment with
iodomethane in DMSO in the presence of KOH. The
resulting product was reduced to the corresponding
o-phenylenediamine, which was subjected to cyclization
with glycolic acid in hydrochloric acid giving 2-hydroxy-

Benzimidazolium bromides
Russ. Chem. Bull., Int. Ed., Vol. 69, No. 4, April, 2020
777
methyl-5-methoxy-1-methylbenzimidazole (1d) as the
final product. 1-Ethyl-2-hydroxymethylbenzimidazole
(1c) was synthesized in a similar manner.
The quartenization of benzimidazoles was performed
by refluxing solutions of the substrate and the quartenizing
agent in acetonitrile or dimethylformamide on moderate
heating.
The structures of quaternary benzimidazolium brom-
ides 2a—j were confirmed by elemental analysis, IR and
1H NMR spectroscopy. The 1H NMR spectra of the brom-
ides show, apart from other signals, two-proton singlets
of N+CH2CO groups (δ 5.95—6.04), indicative of the
presence of phenacyl groups in compounds 2, singlets of
the primary amino group (δ 9.00—9.11), if the latter is
present, singlets of methylene protons of the NBzm—CH2—Biph
moiety at δ 5.57—6.46, and multiplets of aromatic protons
of the benzimidazole and biphenyl moieties at δ 7.14—8.04.
Besides, the spectra of benzimidazolium bromides 2b—g,
2i, and 2j display signals of methoxy groups (singlets at
δ 3.56—3.87). Hydroxy groups of these compounds appear
as singlets at δ 8.12 (2g) and 6.07—6.24 (2h—j).
that these compounds exhibit, to a greater or lesser extent,
inhibitory activity against phosphatase PTP1В. This type
of activity was previously unknown for benzimidazolium
salts. The amino-containing salts bearing the unsubstituted
biphenyl moiety proved to be the most active. The struc-
ture of the phenacyl substituent has a strong effect on
activity. The nearly complete inhibition of PTP1B at
a concentration of 100 μmol L–1 of the tested compounds
was observed for four salts, in which the phenacyl moiety
in the aromatic nucleus contains 4-F, 4-MeO, 3,4-di-
methoxy, or 3,5-di-tert-butyl-4-hydroxy groups as the
substituents. An investigation of the concentration depen-
dence of the inhibition demonstrated that only salt 2c
containing two methoxy groups has affinity to the enzyme
comparable to that of the reference agent NSC-87877.
Hence, the effect of methoxy groups is apparently attrib-
uted to significant electron density donation to the
phenacyl substituent. In the series of 2-hydroxymethyl-
benzimidazolium salts, 4´-bromobiphenyl-substituted salt
2i proved to be active.
Compound 2g showed strong inhibitory activity against
the formation of advanced glycation end-products (AGEs)
in an assay in the albumin—glucose system. This property
is useful for the treatment of DM2. In the assay, salt 2g
exhibited activity four times higher than that of the stan-
dard inhibitor aminoguanidine. Besides, compound 2g
proved to have quite strong antioxidant activity (the stron-
gest activity in the series of the salts under study), which
The solid-state IR spectra of compounds 2a—j show
absorption bands of the CO group at 1682—1736 cm–1
and, in the presence of an amino group, two stretching
bands of the latter at 2963—3240 cm–1. In the IR spectra
of compounds 2g and 2h—j, the hydroxy group appears as
absorption bands at 3184—3469 cm–1, and the nitrile group
in compound 2e gives a characteristic band at 2230 cm–1
.
The synthesized benzimidazolium salts were tested
is two times higher compared to losartan (the Cu2+
induced ascorbic acid oxidation assay). This suggests the
-
in vitro using antidiabetic assays (Table 1). It was shown
Table 1. Biological activity of benzimidazolium bromides 2a—j
Com-
pound
PTP1B
AT1
antagonistic
Antiglycation activity
Inhibition of
ascorbate
autoxidation,
per os)
LD50
/mg kg–1
(mice,
Inhibition
(%)
IC50
Inhibition (%)
(1 mmol L–1
IC50
activity
/μmol L–1
)
/μmol L–1
(10 μmol L–1
)
(100 μmol L–1
)
2a
2b
2c
2d
2e
2f
2g
2h
2i
92.73 63.79
>100
>32
14.68
—
7.98 2.27
0.0 0.0
0.0 0.0
5.20 7.37
4.5 6.43
0.0 0.0
0.0 0.0
3.3 4.64
0.0 0.0
40.10 5.45*1
50.58 3.45*1
3.40 6.74
—
—
—
—
—
—
193.36
—
—
—
>112.5*3
46.6
56.0
52.1
>50.0*3
78.3
43.4
105
57.2
>112.5*3
97.38 16.20*1
102.50 2.01*1
–7.27 8.74
39.74 3.12
58
56
0.27 1.91
—
–133.91 18.13*2
–86.38 3.20*2
70.12 2.51*1
18.84 1.54*1
18.09 4.73
67.38 6.46*1
116.96 10.33*1
9.61 5.21
100.93 0.20*1
65.45 8.4
>99
>60
—
19.36
—
2j
–15.8 3.83
5.96 2.54*1
NSC-87877 68.90 7.32
20.45
Losartan
Amino-
guanidine
99.0 1.27
91.2
57.83 0.58
765.0
*1 The significance level p < 0.05 with respect to the control (one-factor ANOVA with the Dunnett´s post-hoc test).
*2 Intrinsic fluorescence.
*3 Intrinsic absorption at the measured wavelength.

Spasov et al.
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Russ. Chem. Bull., Int. Ed., Vol. 69, No. 4, April, 2020
(NH2), 1725 (C=O). Found (%): C, 73.88; H, 5.36; N, 11.55
C22H19N3O2. Calculated (%): C, 73.94; H, 5.33; N, 11.77.
1H NMR (CDCl3), δ: 3.65 (s, 3 H, OMe); 4.61 (s, 2 H, NH2);
5.20 (s, 2 H, NHet—CH2); 7.09 (t, 1 H, Ar, J = 8.1 Hz); 7.14
(d, 1 H, Ar, J = 7.4 Hz); 7.17 (t, 1 H, Ar, J = 8.2 Hz); 7.21
(d, 2 H, Ar, J = 8.5 Hz); 7.28 (t, 2 H, Ar, J = 5.1 Hz); 7.32 (d,
1 H, Ar, J = 8.8 Hz); 7.41 (t, 1 H, Ar, J = 8.3 Hz); 7.49 (d, 1 H,
Ar, J = 7.9 Hz); 7.52 (t, 1 H, Ar, J = 8.3 Hz); 7.84 (d, 1 H, Ar,
J = 8.9 Hz).
Synthesis of 2-aminobenzimidazolium bromides 2a—g (gen-
eral procedure). The appropriate α-bromo ketone (5 mmol) was
added to a hot solution of amine (1a) in dimethylformanide or
of compound (1b) in acetonitirle (5 mmol). The mixture was
stirred until the reagent was dissolved, heated until the onset of
precipitation of the quaternary salt, and kept at room temperature
for 6—8 h. The precipitate was filtered off and thoroughly washed
with acetone. The chromatographically pure salts thus obtained
were used as-is without further purification.
2-Amino-3-[(4-fluorophenyl-4-yl)-2-oxoethyl]-1-biphenyl-
1H-benzimidazolium bromide (2a). Yield 80%, m.p. 226—226.5 C
(DMF). IR, /cm–1: 3128, 3105 (NH2), 1682 (C=O), 1665
(C=N), 1597 (C=C). Found (%): N, 8.01. C28H23BrFN3O.
Calculated (%): N, 8.13. 1H NMR (DMSO-d6), δ: 5.57 (s, 2 H,
NHet—CH2); 6.00 (s, 2 H, CH2CO); 7.26—7.35 (m, 3 H, Ar);
7.38—7.49 (m, 6 H, Ar); 7.54—7.56 (m, 1 H, C(7)HВzm);
7.62—7.68 (m, Ar); 8.16—8.18 (m, 2 H, Ar); 9.04 (s, 2 H, N+H2).
2-Amino-3-[(4-methoxyphenyl)-2-oxoethyl]-1-(biphenyl-4-yl)-
benzimidazolium bromide (2b). Yield 86.2%. M.p. 240—241.5 C
(MeCN). IR, /cm–1: 3149, 3112 (NH2), 1684 (C=O). Found (%):
C, 65.72; H, 5.08; Br, 14.93; N, 7.80. C29H26BrN3O2. Calculat-
ed (%): C, 65.90; H, 4.96; Br, 15.11; N, 7.95. 1H NMR
(DMSO-d6), δ: 3.87 (s, 3 H, OMe); 5.57 (s, 2 H, NBzm—CH2);
5.95 (s, 2 H, CH2CO); 7.14—7.16 (d, 2 H, Ar, J = 8.9 Hz);
7.27—7.35 (m, 3 H, Ar); 7.38—7.44 (m, 4 H, Ar); 7.53—7.68
(m, 6 H, Ar); 8.04—8.05 (d, 2 H, Ar, J = 8.9 Hz); 9.02 (s, 2 H,
N+H2).
2-Amino-3-[(3,4-dimethoxyphenyl)-2-oxoethyl]-1-(biphenyl-
4-yl)benzimidazolium bromide (2c). Yield 85%, m.p. 226—
226.5 C. IR, /cm–1: 3149, 3112 (NH2), 1684 (C=O). Found (%):
C, 64.39; H, 4.88; Br, 14.11; N, 7.33. C30H28BrN3O3. Calculat-
ed (%): C, 64.51; H, 5.06; Br, 14.30; N, 7.52. 1H NMR
(DMSO-d6), δ: 3.82 (s, 3 H, OMe); 3.88 (s, 3 H, OMe); 5.57
(s, 2 H, NBzm—CH2); 5.97 (s, 2 H, CH2CO); 7.19—7.20 (d, 1 H,
Ar, J = 8.58 Hz); 7.27—7.35 (m, 3 H, Ar); 7.38—7.44 (m, 4 H,
Ar); 7.52—7.55 (m, 2 H, Ar); 7.58—7.68 (m, 5 H, Ar); 7.78—7.79
(dd, 1 H, Ar, J = 2.07 Hz); 9.02 (s, 2 H, N+H2).
chelating mechanism of antiglycation action of 2g appar-
ently with the participation of amino and carbonyl groups.
In conclusion, it should be noted that none of the com-
pounds displayed statistically significant AT1 antagonistic
activity.
Experimental
The following reagents were purchased from Alfa Aesar (UK):
4-(bromoacetyl)benzonitrile, 2-bromo-4-(pyrrolidin-1-yl)ace-
tophenone, 4-bromomethylbiphenyl, methyl 2-[4-(bromo-
methyl)phenylbenzoate, 4-acetylbiphenyl, and 4-(4-bromophen-
yl)acetophenone. The starting compounds 2-amino-1-(biphenyl-
4-ylmethyl)-1H-benzimidazole (1a) and methyl 4-[(2-amino-
1H-benzimidazol-1-yl)methyl]biphenyl-2-carboxylate (1b) were
synthesized in a 85% KOH—acetone mixture. 2-Aminobenz-
imidazole was prepared by alkaline hydrolysis of technical grade
methyl N-(benzimidazol-2-yl)carbamate (BMC), used in the
synthesis of the fungicide Benomyl.17 2-Bromo-4-(phenyl)-
acetophenone and 2-bromo-4-(4-bromophenyl)acetophenone
were prepared by the bromination of 4-acetylbiphenyl and
4´-(4-bromophenyl)acetophenone, respectively, in a mixture of
dioxane and diethyl ether. Other α-bromo ketones were synthe-
sized by the bromination of the appropriate acetophenones in
ethanol. The melting points of the bromo ketones are in good
agreement with the literature data.
The IR spectra were recorded for powders on a Varian
Excalibur 3100 FT-IR spectrometer (Varian, USA) equipped
with an attenuated total reflectance accessory. The 1H NMR
spectra were measured on Varian Unity 300 (Varian, USA) and
Bruker Avance 600 N (USA) spectrometers using residual signals
of protons of the deuterated solvents (DMSO-d6 and CDCl3, δ 2.49
and 7.24, respectively) as the standard. The melting points were
determined on a Fisher Johns Melting Point Apparatus (Fisher
Scientific, USA). Elemental analysis was performed by the con-
ventional method.18 The course of the reactions was monitored
and the purity of the synthesized compounds was checked by
TLC on Al2O3 plates (CH2Cl2 as the eluent, visualization with
iodine vapor in a humid chamber).
2-Amino-1-(biphenyl-4-yl-methyl)-1H-benzimidazole (1a).
Acetone (20 mL) was added to a solution of 2-aminobenzimid-
azole (2.66 g, 20 mmol) and KOH (2.64 g, 40 mmol; with respect
to 85% KOH) in water (3 mL). The reaction mixture was stirred
for 20 min at room temperature, and then 4-bromomethylbi-
phenyl (4.94 g, 20 mmol) was added portionwise to the mixture.
The reaction mixture was stirred for 2 h at room temperature and
for 30 min at 45—50 C. Then the mixture was cooled and the
precipitate of the amine that formed was filtered off, succes-
sively washed with water and cold ethanol, and dried. The yield
of the amine was 5.80 g (97%), m.p. 245—246 C (DMF). IR,
/cm–1: 3987, 3287 (NH2), 1629, 1591, 1521, 1478, 1459 (C=C,
C=N). Found (%): C, 80.02; H, 5.55; N, 13.89. C20H17N3.
Calculated (%): C, 80.22; H, 5.73; N, 14.03. 1H NMR (CDCl3),
δ: 5.29 (s, 2 H, NHet—CH2); 6.53 (s, 2 H, NH2); 6.82 (t, 1 H,
Ar, J = 7.5 Hz); 6.92 (t, 1 H, J = 7.5 Hz); 7.13 (q, 2 H, Ar,
J = 7.8 Hz); 7.26—7.35 (m, 3 H, Ar); 7.42 (t, 2 H, Ar, J = 7.5 Hz);
7.59—7.61 (q, 4 H, Ar, J = 7.8 Hz).
2-Amino-3-[(phenyl-4-yl)-2-oxoethyl)]-1-{[2-(carboxylate)
biphenyl-4-yl]methyl}benzimidazolium bromide (2d). Yield 62%.
M.p. 223—225 C. IR, /cm–1: 3080, 3046 (NH2), 1721(COOMe),
1711(C=O). Found (%): C, 64.63; H, 4.83; Br, 14.22; N, 7.43.
C30H26BrN3O3. Calculated (%): C, 64.75; H, 4.71; Br, 14.36;
N, 7.56. 1H NMR (DMSO-d6), δ: 3.56 (s, 3 H, OMe); 5.60
(s, 2 H, N—CH2); 5.60 (s, 2 H, N—CH2); 6.04 (s, 2 H, CH2);
7.28—7.41 (m, 7 H, Ar); 7.58—7.76 (m, 8 H, Ar); 8.09—8.12
(d, 2 H, Ar, J = 8.4 Hz); 9.09 (s, 2 H, N+H2).
2-Amino-3-[2-(4-cyanophenyl-4-yl)-2-oxoethyl]-1-{[2-(carb-
oxylate)biphenyl-4-yl]methyl}benzimidazolium bromide (2e). Yield
73%. M.p. 210—212 C. IR, /cm–1: 3196, 3149 (NH2), 2230
(CN), 1725 (COOMe), 1695 (C=O). Found (%): C, 63.92;
H, 4.43; Br, 13.59; N, 9.51 C31H25N4BrO3. Calculated (%):
C, 64.04; H, 4.33; Br, 13.74; N, 9.64. 1H NMR (DMSO-d6), δ:
Methyl 4-[(2-amino-1H-benzimidazol-1-yl)methyl]biphenyl-
2-carboxylate (1b) was synthesized as described for 1a. Yield
63%, m.p. 150.0—150.5 C (MeCN). IR, /cm–1: 3423, 3307,

Benzimidazolium bromides
Russ. Chem. Bull., Int. Ed., Vol. 69, No. 4, April, 2020
779
3.56 (s, 3 H, OMe); 5.60 (s, 2 H, N—CH2); 6.07 (s, 2 H, CH2);
7.31—7.41 (m, 7 H, Ar); 7.46—7.61 (m, 3 H, Ar); 7.69—7.76
(m, 2 H, Ar); 8.14—8.17 (dd, 2 H, Ar, J = 8.1 Hz); 8.24—8.27
(d, 2 H, Ar, J = 8.4 Hz); 9.11 (s, 2 H, N+H2).
(DMSO-d6), δ: 1.95—1.98 (m, 4 H, (CH2)2); 3.33—3.36 (m, 4 H,
CH2NCH2); 3.79 (s, 3 H, N—CH3); 4.08 (s, 3 H, OCH3); 4.90
(s, 2 H, CH2); 6.07 (s, 1 H, OH); 6.21 (s, 2 H, N+—CH2);
6.63—6.65 (d, 2 H, Ar, J = 9.0 Hz); 7.24—7.26 (m, 1 H, Ar);
7.54 (s, 1 H, Ar); 7.89—7.92 (m, 3 H, Ar).
2-Amino-3-[2-(4-pyrrolidinophenyl-4-yl)-2-oxoethyl]-1-{[2-
(carboxylate)biphenyl-4-yl]methyl}benzimidazolium bromide (2f).
Yield 63%. M.p. 229—231 C. IR, /cm–1: 3240, 3089 (NH2),
1736 (COOMe), 1717 (C=O). Found (%): C, 65.15; H, 5.44;
Br, 12.63; N, 8.82. C34H33BrN4O3. Calculated (%): C, 65.28;
H, 5.32; Br, 12.77; N, 8.96. 1H NMR (DMSO-d6), δ: 1.98—2.00
(m, 4 H, (CH2)2); 3.35—3.37 (m, 4 H, CH2NCH2); 3.56 (s, 3 H,
OCH3); 5.62 (s, 2 H, CH2); 5.89 (s, 2 H, CH2); 6.66—6.67
(d, 2 H, Ar, J = 9.1 Hz); 7.31—7.32 (d, 2 H, Ar, J = 7.3 Hz);
7.33—7.39 (m, 1 H, Ar); 7.46—7.49 (m, 1 H, Ar); 7.53—7.56
(m, 2 H, Ar); 7.59—7.61 (m, 1 H, Ar); 7.73—7.75 (m, 1 H, Ar);
7.91—7.89 (d, 2 H, Ar, J = 9.0 Hz); 9.07 (s, 2 H, N+H2).
2-Amino-3-[2-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-oxo-
ethyl]-1-{[2-(carboxylate)biphenyl-4-yl]methyl}benzimidazolium
bromide (2g). Yield 80.3%. M.p. 223—225 C. IR, /cm–1: 3469
(OH), 3022, 2963 (NH2), 1714 (COOMe), 1675 (C=O). Found (%):
C, 66.54; H, 6.27; Br, 11.53; N, 6.01. C38H42N3O4Br. Calculat-
ed (%): C, 66.66; H, 6.18; Br, 11.67; N, 6.14. 1H NMR
(DMSO-d6), δ: 1.44 (s, 1 H, C(CH3)3); 3.56 (s, 3 H, OCH3);
5.59 (s, 2 H, CH2); 6.00 (s, 2 H, CH2); 7.27—7.42 (m, 7 H, Ar);
7.46—7.67 (m, 4 H, Ar); 7.73—7.76 (m, 1 H, Ar); 7.86 (s, 2 H,
Ar); 8.12 (s, 1 H, OH); 9.00 (s, 2 H, N+H2).
Synthesis of 2-hydroxymethylbenzimidazolium bromides 2h—j
(general procedure). A solution of substituted 2-hydroxymethyl-
benzimidazole 1c or 1d (10 mmol) and the appropriate α-bromo
ketone (10 mmol) in acetonitrile was refluxed for 16—20 h. Then
the reaction mixture was cooled in an ice bath. The precipitate
of the bromide that formed was filtered off, washed with acetone,
dried, and crystallized from ethanol.
1-Ethyl-3-[(4-biphenyl-4-yl)-2-oxoethyl]-2-hydroxymethyl-
benzimidazolium bromide (2h). Yield 82.5%, m.p. 205—205.5 C.
IR, /cm–1: 3190 (OH), 1689 (C=O). Found (%): C, 63.72;
H, 5.24; Br, 17.49; N, 5.06. C24H23BrN2O2. Calculated (%):
C, 63.87; H, 5.14; Br, 17.70; N, 6.05. 1H NMR (DMSO-d6), δ:
1.46—1.48 (t, 3 H, CH2CH3, J = 7.2 Hz); 4.67—4.70 (q, 2 H,
CH2CH3, J = 7.2 Hz); 5.07—5.08 (d, 2 H, CH2, J = 6.1 Hz);
6.22—6.24 (t, 1 H, OH, J = 6.1 Hz); 6.46 (s, 2 H, N+CH2);
7.43—7.53 (m, 3 H, Ar); 7.63—7.70 (m, 2 H, Ar); 7.78—7.79
(m, 2 H, HBzm); 7.94—8.02 (m, 3 H, C(7)HBzm+ Ar); 8.12—8.19
(m, 3 H, C(4)HBzm+ Ar).
3-[(4-Bromobiphenyl)-2-oxoethyl]-2-hydroxymethyl-5-
methoxy-1-methylbenzimidazolium bromide (2i). Yield 87%. M.p.
233—234 C. IR, /cm–1: 3184 (OH), 1689 (C=O). Found (%):
C, 52.58; H, 4.16; Br, 29.16; N, 4.99. C24H22Br2N2O3. Calculat-
ed (%): C, 52.75; H, 4.06; Br, 29.28; N, 5.13. 1H NMR
(DMSO-d6), δ: 3.83 (s, 3 H, N—Me); 4.12 (s, 3 H, OCH3);
5.00—5.02 (d, 2 H, CH2, J = 5.9 Hz); 6.10—6.14 (t, 1 H, OH,
J = 6.0 Hz); 6.41 (s, 2 H, N+—CH2); 7.28—7.32 (dd, 1 H, Ar,
J1 = 9.2 Hz, J2 = 2.3 Hz); 7.63—7.64 (d, 1 H, Ar, J = 2.1 Hz);
7.72—7.74 (d, 2 H, Ar, J = 7.9 Hz); 7.76—7.79 (d, 2 H, Ar,
J = 7.9 Hz); 7.96—7.99 (d, 3 H, Ar, Jav = 8.6 Hz); 8.19—8.22
(d, 2 H, Ar, J = 8.2 Hz).
2-Hydroxymethyl-5-methoxy-1-methyl-3-[(4-pyrrolidino-
phenyl-4-yl)-2-oxoethyl]benzimidazolium bromide (2j). Yield 96%,
m.p. 225—227 C. IR, /cm–1: 3200 (OH), 1690 (C=O).
Found (%): C, 57.27; H, 5.83; Br, 17.20; N, 9.03. C22H26BrN3O3.
Calculated (%): C, 57.39; H, 5.69; Br, 17.36; N, 9.13. 1H NMR
In vitro evaluation of antagonistic activity of compounds 2a—j
against angiotensin AT1 receptors was performed in an isolated
rat portal vein by known procedures1 using the modification
described in the study19 in Krebs buffer, pH 7.4, at 24 C under
constant oxygenation with a O2 (95%)/CO2 (5%) gas mixture.
Muscle contractions were detected using a 7006 isotonic trans-
ducer and a four-channel digital recorder at an isotonic load of
0.5 g in a single-chamber isolated organ and tissue bath set-up
(Ugo Basile S.R.L., Italy) with the LabScribe3.0™ software
(iWorx Systems, Inc., USA). The selective AT1 antagonist val-
sartan (≥98%, Sigma, USA) served as the reference compound.
In vitro evaluation of protein tyrosine phosphatase 1B (PTP1B)
activity was performed using full-length active human PTP1B
(Sigma, USA) and p-nitrophenyl phosphate as the substrate
(pNPP).20 An increase in absorbance of the reaction mixture at
405 nm was measured on an Infinite M200 PRO microplate
spectrophotometer (Tecan, Austria). The experimental inhibitor
NSC-87877 (8-hydroxy-7-[(6-sulfo-2-naphthyl)azo]-5-quino-
linesulfonic acid, >98%, Tocris Bioscience) served as the positive
control.21
In vitro evaluation of antiglycation activity was performed by
a method described in the study.22 The reaction mixture contained
glucose (500 mmol L–1) and bovine serum albumin (BSA)
(1 mg mL–1) in phosphate buffer, pH 7.4. After the completion
of incubation, the specific fluorescence of glycated BSA was
determined on a F-7000 spectrofluorometer (Hitachi, Japan) at
λex = 370 nm and λem = 440 nm. The non-enzymatic glycosylation
inhibitor aminoguanidine (≥98%, Sigma, USA) served as the
reference compound.23
Chelating activity was evaluated by a modified assay based
on metal-dependent ascorbic acid oxidation.24 Losartan (≥98%,
Sigma, USA) served as the reference compound. A 100 μmol L–1
ascorbic acid solution was utilized. A copper(II) sulfate solution
was used as an autoxidation catalyst. The complexation was
performed as follows. Solutions of the autoxidation catalyst
(final concentration was 150 μmol L–1) and the tested compound
(final concentration was 75 μmol L–1) or the solvent (99%
DMSO) were mixed, and the mixture was incubated in a water
bath for 5 min at 37 C. The reaction was initiated by the addition
of the pre-incubated mixture to the ascorbic acid solution. The
ascorbic acid oxidation was determined by spectrophotometry
on an APEL PD 303 UV spectrophotometer (Japan) based on
the absorbance at 265 nm at 5 s intervals.
Statistical processing of the results was performed using the
one-factor (one-way) ANOVA test with the Dunnett's post-hoc
test, the Microsoft Excel 2007 table editor, and the Prism 6
software (GraphPad Inc.). The level of statistical significance
was p < 0.05.
The compounds were synthesized and structurally
characterized by O. N. Zhukovskaya under the supervision
of A. S. Morkovnik with the financial support of the
Ministry of Education and Science of the Russian Federa-
tion within the framework of the basic part of the state
assignment in the sphere of scientific activities (Project

Spasov et al.
780
Russ. Chem. Bull., Int. Ed., Vol. 69, No. 4, April, 2020
14. V. A. Anisimova, I. E. Tolpygin, V. I. Minkin, A. A. Spasov,
A. V. Stepanov, N. A. Ar´kova, L. V. Naumenko, V. I. Petrov,
Pat. RF 2290404; Byul. Izobret. [Inventor Bull.], 2006, 36
(in Russian).
15. V. A. Anisimova, A. A. Spasov, V. A. Kosolapov, I. E.
Tolpygin, L. V. El´tsova, A. F. Kucheryavenko, L. V. Nau-
menko, N. A. Gurova, K. V. Lenskaya, D. S. Yakovlev, D. V.
Mal´tsev, T. M. Mitina, O. Yu. Grechko, Pharm. Chem. J.,
2012, 46, 526.
No. 4.5821.2017/8.9). Biological assays were performed
under the supervision of Academician of the Russian
Academy of Science A. A. Spasov with the financial
support of the Russian Science Foundation (Project
No. 14-25-00139).
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Received August 30, 2019;
accepted December 1, 2019