Synthesis and pharmacological activity of 3-phenoxybenzoic acid derivatives

Article abstract of DOI:10.1134/S1068162017020145

New esters of N-benzoyl-3-phenoxyphenylcarboxamide acid and N-benzoyl-N′-4-bromophenyl-3-phenoxybenzamidine were synthesized. Some of the synthesized compounds were shown to inhibit the activity of dipeptidyl peptidase-4 and nonenzymatic glycosylation of

Full text of DOI:10.1134/S1068162017020145

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2017, Vol. 43, No. 2, pp. 163–169. © Pleiades Publishing, Ltd., 2017.
Original Russian Text © A.A. Spasov, Yu.V. Popov, V.S. Lobasenko, T.K. Korchagina, P.M. Vassiliev, V.A. Kuznetsova, A.A. Brigadirova, A.I. Rashchenko, D.A. Babkov, A.N. Kochetkov,
A.I. Kovaleva, O.S. Efremova, 2017, published in Bioorganicheskaya Khimiya, 2017, Vol. 43, No. 2, pp. 189–196.
Synthesis and Pharmacological Activity of 3-Phenoxybenzoic
Acid Derivatives
A. A. Spasova, b, Yu. V. Popovc, V. S. Lobasenkoc, T. K. Korchaginac, P. M. Vassilievb, V. A. Kuznetsovab,
A. A. Brigadirovaa, b, 1, A. I. Rashchenkob, D. A. Babkovb, A. N. Kochetkovb,
A. I. Kovalevab, and O. S. Efremovac
aVolgograd Medical Research Center, Volgograd, 400131 Russia
bVolgograd State Medical University, Volgograd, 400131 Russia
cVolgograd State Technical University, Volgograd, 400005 Russia
Received April 11, 2016; in final form, May 5, 2016
Abstract—New esters of N-benzoyl-3-phenoxyphenylcarboxamide acid and N-benzoyl-N'-4-bromophenyl-
3-phenoxybenzamidine were synthesized. Some of the synthesized compounds were shown to inhibit the
activity of dipeptidyl peptidase-4 and nonenzymatic glycosylation of proteins and manifested antiplatelet and
antioxidant properties. The compounds tested did not display the antagonistic effect toward angiotensin II
type 1 receptor, did not influence the activity of glycogen phosphorylase and had very little ability to break
cross-links of the glycated proteins. The derivatives with the biological activity of two types were found, which
can serve as basic molecules in the search for new drug products.
Keywords: biologically active compounds, N-benzoyl-3-phenoxyphenylcarboxamide acid, diphenyl oxide,
ethers, synthesis
DOI: 10.1134/S1068162017020145
INTRODUCTION
tures come from their spatial and electron characteris-
tics, which support the binding to various targets
depending on the substituents introduced into the
basic structure and, thereby, support their polyfunc-
tional effects and a wide spectrum of pharmacological
activity [6]. Taking this into account, we believe that
the synthesis and studies of the pharmacological activ-
ity of new diphenyl oxide derivatives are promising.
Many of the diphenyl oxide derivatives are of inter-
est as important synthetic objects and potential bio-
logically active compounds, which are characterized
by certain pharmacological properties. Particularly,
nimesulide [N-(4-nitro-2-phenoxyphenyl)methane-
sulfonamide] displays anti-inflammatory, analgesic,
antipyretic, and antiplatelet effects and serves as a
starting compound for the synthesis of triazole deriva-
tives, effective agents against colon cancer [1]. Perme-
thrin [3-phenoxybenzyl 3-(2,2-dichlorovinyl)-2,2-
dimethylcyclopropanecarboxylate] and phenothrin
[(3-phenoxyphenyl)methyl 2,2-dimethyl-3-(2-meth-
ylprop-1-enyl)cyclopropane-1-carboxylate] are used
as drug products displaying antiparasitic properties
[2]. Diphenyl oxide derivatives are bioisosteric ana-
logues of diphenyl derivatives [3]. The latter are char-
acterized by antihypertensive, antimicrobial, antidia-
betic, diuretic, and antiplatelet activities; also, they
demonstrate psychotropic effects [4]. In addition,
diphenyl derivatives pertain to the so-called privileged
substructures [5], which can interact with biotargets of
several families. Specific properties of these substruc-
RESULTS AND DISCUSSION
In the Pinner reaction, aromatic nitriles interact
with alcohols and hydrogen chloride to give imidate
hydrochlorides.
The reaction of imidate hydrochlorides (IIa–e)
with benzoyl chloride resulted in N-(benzoyl)-3-phe-
noxyphenylcarboximidates (IVa–e) (Scheme 1). As
an acceptor of hydrogen chloride TEA was used. The reac-
tions were performed at equimolar ratios of the reagents
and a twofold excess of TEA in absolute 1,4-dioxane at
60–65°С for 2 h. The reactions proceeded selectively
in 85 to 96% yields.
The presence of a –С=NH ⋅ HCl functional group
in the imidate hydrochlorides synthesized makes it
possible to synthesize free imidates (IIIa, b). To this
end, imidate hydrochloride was treated with TEA.
1
Corresponding author: phone: +7(8442) 55-17-70; e-mail:
a.brigadirova@gmail.com.
Abbreviations: AT angiotensin type 1 receptor; DPP-4, dipep-
1,
With the goal of obtaining the target amidine (V)
containing a diphenyl oxide fragment, butyl N-(ben-
tidyl peptidase-4; GP, glycogen phosphorylase; LPO, lipid per-
oxidation; TBA, thiobarbituric acid; TEA, triethylamine.
163

164
SPASOV et al.
zoyl)-3-phenoxyphenylcarboximidate (IVа) was 3-phenoxybenzamidine (V) was purified by recrystal-
treated with p-bromoaniline in benzene at 65–70°C lization from a 1 : 1 ethanol–water mixture in 60%
for 2 h (Scheme 1). N-(Benzoyl)-N'-4-bromophenyl- yield.
+
NH2
Cl
C
O
O
C N
+ 2HCl
Cl
C
(I)
NH
OR
NH ⋅ HCl
OR
O
C
O
+ROH
+ (C2H5)3N
−HCl
·
−(C2H5)3N HCl
(II a−e)
(III a, b)
+2(C2H5)3N
−2(C H ) N ·
HCl
2
5 3
O
C
+
Cl
O
O
N C
OR
N
C
C
O
C
O
Br
+N2H
−ROH
Br
HN
(V)
(IV a−e)
where R = n-С4Н9 (IIa, IVa); sec-С4Н9 (IIb, IVb); iso-С4Н9 (IIc, IVc); С6Н5 (IId, IVd); С6Н5-CH2-С6Н4 (IIe, IVe); С2Н5
(IIIa), С3Н7 (IIIb).
Scheme 1. 1Preparation of 3-phenoxyphenylcarboximidates (IVa–e), (IIIa, b) and N-benzoyl-N'-4-bromophenyl-3-phe-
noxybenzamidine (V).
For the synthesis of 2-cyanopropyl 3-phenoxyben- stirring in absolute diethyl ether in the presence of
zoate (VII) 3-phenoxybenzoyl chloride (VI) was TEA for 1 h at 20–25°C (Scheme 2). The yield of
treated with 2-hydroxy-2-methylpropionitrile under nitrile (VII) was 95%.
O
O
CH3
CH3
C
O
C
O
+(C2H5)3N
Cl
C
C N
HO
C
CN
O
+
.
(C2H5)3N
HCl
CH3
CH3
(VI)
)
(VII
N
O
+
(C2H5)3N,
CHCl3
O
O
C
C
O
O
O
HCl, H2O
N
O
(VIII)
(IX)
Scheme 2. Preparation of 2-cyanoprop-2-yl 3-phenoxybenzoate (VII) and 2-(3-phenoxybenzoyl)cyclohexanone (IX).
Using acylation of enamines with acyl chlorides zoyl chloride (VI) at a reagent molar ratio of 1 : 1 in
as a reference [7] we performed acylation of 4- chloroform at 50–60°C for 7–8 h in the presence of
(cyclohexen-1-yl)morpholine with 3-phenoxyben- TEA. The treatment with hydrochloric acid of the
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SYNTHESIS AND PHARMACOLOGICAL ACTIVITY
165
product obtained (VIII) led to 1,3-diketone (IX) of 1-(3-phenoxyphenyl)-1-ethanone (X) with dimethyl
(Scheme 2).
amine hydrochloride and paraformaldehyde in ethanol
in the presence of concentrated hydrochloric acid gave
40% N,N-dimethyl-2-(3-phenoxybenzoyl)ethylamine
The Mannich bases were obtained in accordance with
the published procedure for the synthesis of β-dimethyl-
aminopropiophenone hydrochloride [8]. The interaction hydrochloride (XI) (Scheme 3).
O
O
·
(CH3)2NH HCl
C
O
C
O
CH2O
N(CH3)2 HCl
C
C
n
H2
H2
CH3
EtOH/HCl
(X)
(XI)
Scheme 3. Preparation of N,N-dimethyl-2-(3-phenoxybenzoyl)ethylamine hydrochloride (XI).
The structures and composition of the compounds observed, which can be explained by the presence of a
synthesized were confirmed by element analysis, IR rather bulky substituent. The efficacy of DPP-4 inhi-
and 1H NMR spectroscopy, and chromato-mass-
spectrometry.
bition statistically differed from the control only for
this derivative.
Compounds (IVe), (IIIb), and (V) were shown to
have moderate antioxidant activity, which was
3.5 times lower than that of the reference dibunol
(table). In addition, compound (XI) demonstrated a
low level of antioxidant activity.
All of the compounds tested were weak GP inhibi-
tors and low effective AT1 antagonists (table).
When studying the capacity to break cross-links of
glycated proteins, we found five compounds, whose
activity was statistically significantly different from the
control. However, the level of their activity did not
exceed that of the reference alagebrium.
To summarize, among the new derivatives of 3-
phenoxybenzoic acid we found the compounds dis-
playing antioxidant, antiplatelet, and antiglycation
properties, as well as the compounds with the DPP-4
inhibitory activity. Although the new derivatives did
not show the marked activity, even the low activity
makes them promising for further modifications. The
results demonstrated that 3-phenoxybenzoic acid
derivatives (IVb, e), (IIIb), (V), (VII), and (XI) can be
used as basic molecules for the directed design and
search for highly active polyfunctional compounds
with a combined activity of two types.
Pharmacological Properties
of Diphenyl Oxide Derivatives
AT1-antagonistic, antiplatelet, antioxidant, anti-
glycation, and inhibitory activities toward DPP-4 and
GP, as well as the capacity to break cross-links of the
glycated proteins, were studied for the 11 new deriva-
tives of 3-phenoxybenzoic acid (IIIa, b), (IVa–e), (V),
(VII), (IX), and (XI).
The most potent antiplatelet activity was found for
N-benzoyl substituted 3-phenoxyphenyl imidate
(IVe), which only slightly exceeded that of the refer-
ence acetyl salicylic acid. Other derivatives of 3-phe-
noxybenzoic acid, N-benzoyl substituted phenyl imi-
date (IVd), propyl imidate (IIIb), and 2-methyl-2-(3-
phenoxybenzoate)propionitrile (VII), manifested
moderate antiplatelet properties, which did not statis-
tically differ from the activity of the reference com-
pound (table). In total, five active compounds were
found, whose antiplatelet effect statistically signifi-
cantly exceeded that of the control preparation.
The experiments demonstrated that eight of the
tested derivatives of 3-phenoxybenzoic acid displayed
the antiglycation activity (table). Compounds
(IVb, e), (IIIa, b), (V), (VII), (X), and (XI) reliably
inhibited specific fluorescence of glycated BSA in the
range of 11.9 to 27.1%. The compounds studied dis-
played moderate and low antiglycation activity and
were inferior in this parameter to the reference amino-
guanidine.
The study of biological properties showed that the
derivatives of 3-phenoxybenzoic acid, N-benzoyl sub-
stituted n-butyl imidate (IVa) and N-benzoyl substi-
tuted imidates (IVb,c), inhibited DPP-4 although with
efficacy inferior to the reference vildagliptin (table).
For compound (IVb), the statistically significant mod-
EXPERIMENTAL
IR spectra (ν, cm–1) were registered on SPECORD
M 82 (Germany) and PERKIN-ELMER (United
States) in a film for liquid compounds and in vaseline
oil for solid forms.
1H NMR spectra (δ, ppm, J, Hz) were registered
on VarianMercury 300BB spectrometer (United
States) in DMSO-d6 with hexamethyldisiloxane as the
internal standard.
Chromatography mass spectra were obtained on a
erate level of the DDP-4 inhibitory activity was Varian МАТ-11 (United States) at an ionization volt-
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SPASOV et al.
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10oC. The mixture was stirred for 1.5–2 h at room
temperature. The precipitated TEA hydrochloride was
separated and the solvent was removed. While concen-
trating, the reaction mixture was crystallized. The tar-
get compound was purified by recrystallization from
absolute C6H6 to give 3.6 g (83%) of compound (IIIa);
age of 70 eV and cathode emission current of 240 μA.
The samples were directly injected into the ionization
chamber.
Butyl N-(benzoyl)-3-phenoxyphenylcarboximidate
(IVа). A solution of butyl phenoxyphenylcarboximi-
date hydrochloride (IIa) (6.4 g, 0.021 mol) in absolute
dioxane (10 mL) was placed in a four-neck reactor mp 130–131°C. IR: 3434, 3398, 1780-1690 (N–H);
1648 (C=N); 1240–1072 (С–O–C). 1H NMR: 1.14–
1.19 (t, 3Н, СН3); 3.59–3.65 (m, 2Н, СН2); 6.19 (s,
Н, NН); 6.93–7.45 (m, 9Н, С6Н5ОС6Н4). Found, %:
С 74.58; Н 5.93; N 5.31. (С15Н15O2N). Calc., %: С
74.69; Н 6.22; N 5.81.
Propyl 3-phenoxyphenylcarboximidate (IIIb) was
prepared similarly in a yield of 85%. 1H NMR: 0.81–
0.90 (m, 3Н, СН3); 1.64–1.87 (m, 2Н, СН2); 3.58–
3.73 (t, 2Н, СН2); 6.20 (s, Н, NН); 6.92–7.45 (m, 9Н,
Ar). Found, %: С 75.57; Н 6.31; N 5.08. (С16Н17O2N).
Calc., %: С 75.29; Н 6.67; N 5.49.
N-Benzoyl-N'-4-bromophenyl-3-phenoxybenzami-
dine (V). A solution of butyl 3-phenoxyphenylcarbox-
imidate hydrochloride (IVa) (2.1 g, 5.9 mmol) in abso-
lute benzene (10 mL) was placed in a four-neck reac-
tor equipped with a mechanical stirrer, thermometer,
and a refluxing system. p-Bromoaniline (1.22 g,
7.08 mmol) was added dropwise under cooling on an
ice bath at 5–10°C. The reaction mixture was kept for
55–60°C for 2 h and the solvent was removed. While
concentrating, the reaction mixture was crystallized.
The target compound was purified by recrystallization
from a 1 : 1 ethanol water–mixture to give 60% of the
product. IR: 3430–3394 (С–N); 1702 (C=O); 1648
(C=N); 1204 (N–H). 1H NMR: 6.89–7.76 (m, 18Н,
Ar). Found, %: С 66.57; Н 4.67; N 5.29; Br 16.38.
(С26Н19О2N2Br). Calc., %: С 66.25; Н 4.06; N 5.94;
Br 16.95.
equipped with a mechanical stirrer, thermometer, and a
refluxing system. Solutions of TEA (4.25 g, 0.042 mol) in
dioxane (10 mL) and benzoyl chloride (3 g, 0.021 mol)
in dioxane (5 mL) were successively added dropwise
under cooling on an ice bath at 5–10°C. The reaction
mixture was stirred for 30 min at room temperature
and kept at 60–65°C for 2 h. The precipitate of TEA
hydrochloride was separated and the solvent was
removed. During concentrating the reaction mixture
was crystallized. The target compound was purified by
recrystallization from anhydrous СCl4 to give 6.8 g
(94%) of the product. IR: 1702 (C=O); 1648 (C=N);
1
1246–1072 (С–O–C). H NMR: 1.08–1.13 (t, 3Н,
СН3); 2.38–2.51 (m, 2Н, СН2); 3.01–3.05 (m, 2Н,
СН2); 3.23–3.37 (t, 2Н, СН2); 6.96–7.97 (m, 9Н,
С6Н5ОС6Н4). Found, %: С 77.10; Н 5.85; N 3.46.
(С24Н23O3N). Calc., %: С 77.21; Н 6.17; N 3.75.
N-(Benzoyl)phenoxyphenylcarboximidates (IVb, c)
were prepared similarly.
(IVb): Yield 85%. IR: 1702 (C=O); 1650 (C=N);
1243–1072 (С–O–C). Found, %: С 77.20; Н 5.79; N
3.36. (С24Н23O3N). Calc., %: С 77.21; Н 6.17; N 3.75.
(IVc): Yield 89%. IR: 1702 (C=O); 1650 (C=N);
1240–1072 (С–O–C). Found, %: С 77.05; Н 5.72; N
3.58. (С24Н23O3N). Calc., %: С 77.21; Н 6.17; N 3.75.
Phenyl
N-(benzoyl)phenoxyphenylcarboximidate
(IVd). A solution of phenyl phenoxyphenylcarboximi-
date hydrochloride (IId) (2 g, 6.1 mmol) in absolute
dioxane (5 mL) was placed in a four-neck reactor
equipped with a mechanical stirrer, thermometer, and a
refluxing system. Solutions of TEA (1.245 g, 12.3 mmol)
in dioxane (5 mL) and benzoyl chloride (0.857 g,
6.1 mmol) in dioxane (3 mL) were successively added
dropwise under cooling on an ice bath at 5–10°C. The
reaction mixture was treated as described for (IVa).
The target compound was purified by recrystallization
2-Cyanoprop-2-yl-3-phenoxybenzoate (VII) was
obtained as described in [9].
2-(3-Phenoxybenzoyl)cyclohexanone (IX). A solu-
tion of 4-(cyclohexen-1-yl)morpholine (10.4 g,
62.5 mmol) and anhydrous TEA (6.3 g, 4.7 mL,
62.5 mmol) in anhydrous chloroform (32 mL) was
placed in a round-bottomed three-neck flask equipped
with a dropping funnel, a refluxing system, and a
mechanical stirrer. The reaction mixture was heated on a
glycerol bath at 35°C and a solution of 3-phenoxybenzo-
ate chloride (7.3 g. 31.25 mmol) (VI) in anhydrous chlo-
roform (12.5 mL) was added dropwise. The mixture
was stirred at 35°C for 3 h and diluted with 20% HCl
(32 mL). The mixture was refluxed under intensive
stirring for 3 h, cooled to room temperature, and the
aqueous layer was separated. The organic layer was
washed with water (6 × 10 mL), dried with magnesium
1
from absolute СCl4 to give 2.21 g (92%) of (IVd). H
NMR: 6.97–8.00 (m, 19Н, Ar). Found, %: С 79.68; Н
4.37; N 4.09. (С26Н19О3N). Calc., % С 79.37; Н 4.87;
N 3.56.
3-Phenoxyphenyl N-(benzoyl)phenoxyphenylcar-
boximidate (IVe) was obtained similarly in a yield of
85%. 1H NMR: 6.89–8.11 (m, 23Н, Ar). Found, %: С
79.05; Н 4.32; N 2.08. (С32Н23O4N). Calc., %: С
79.18; Н 4.74; N 2.89.
Ethyl 3-phenoxyphenylcarboximidate (IIIa). Ethyl sulfate, filtered, and evaporated. The residue was con-
3-phenoxyphenylcarboximidate hydrochloride (IIa) centrated in vacuum at 10–15 mm Hg. The resulting oil
(5 g, 0.018 mol) was placed in a reactor and a solution was slowly crystallized under cooling to give 2-(3-phe-
of TEA (2.02 g, 0.02 mol) in dioxane (10 mL) was noxybenzoyl)cyclohexanone (IX) (6.4 g, 21 mmol, 70%);
added dropwise under cooling on an ice bath at 5– mp 141–143°С/10 mm Hg. Mass (m/z): 197 (82%,
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SPASOV et al.
[C6H5OC6H4C(O)]+), 294 (100%, [M]+), 295 (25%,
[M + 1]+). Calc.: M 294 (С19Н18O3). 1H NMR: 1.81–
2.27 (s, 8Н, СН2); 3.73 (m, Н, СН); 7.14–7.79 (m,
9Н, С6Н5ОС6Н4).
acid was used as a reference compound (Sigma,
United States).
The properties of the compounds to break cross-
links of glycated proteins in vitro were determined as
described in [13]. The protein glycation reaction was
modeled in the reaction mixture containing glucose
(400 mM) and BSA (0.8 mg/mL) dissolved in 50 mM
phosphate buffer (рН 7.4). The mixture was incubated
at 60°C for 40 h and a mixture of BSA and glucose
(200 μL of each) was added into each of the Eppendorf
tubes followed by the addition of 100% TCA (20 μL).
The mixture was centrifuged at 15000 rpm for 4 min
and 50 mM phosphate buffer (pH 7.4) was added to
the precipitated glycated BSA to the volume of 300 μL.
A solution of the tested compound (30 μL) was added
(the volume of the reaction mixture was 330 μL) and
the mixture was incubated at 60°C for 40 h. In addi-
tion, a similar number of samples were prepared with
nonglycated nonprecipitated BSA (300 μL in each
sample), in which the tested compound was added,
and the mixture was incubated. All the compounds
were dissolved in DMSO (Fisher Scientific, United
States). The tested compounds at a final concentra-
tion of 1 mM were added to the samples and the
proper solvent volume, into the control solutions.
After the incubation was completed the precipitated
glycated and nonglycated BSA were dissolved in the
phosphate buffer (1 mL, pH 10.0) and specific fluo-
rescence of the samples was determined on a micro-
plate reader (Infinite M200, Tecan, Austria) at λex
370 nm and λem 440 nm.
N,N-Dimethyl-2-(3-phenoxybenzoyl)ethylamine
hydrochloride (XI). Morpholine hydrochloride
(2.84 g, 23 mmol) and paraform (1.065 g, 35.5 mmol)
were added to a solution of 3-phenoxyacetophenone
(5.19 g, 23 mmol) in isopropanol (30 mL). The reac-
tion mixture was saturated with dry hydrogen chloride
to pH 0–1.0 and refluxed for 20 h. The solvent was
removed at reduced pressure and the residue was dis-
solved in diluted hydrochloric acid and washed with
ether. The aqueous layer was alkalized with potassium
carbonate to pH 8.0–9.0 and extracted with ether. The
combined ether extracts were washed with water
(100 mL) and dried. After the drying agent was
removed a solution of dry hydrogen chloride in anhy-
drous isopropanol was added and kept at 0°C for 18 h.
The precipitate was filtered off, washed with an iso-
propanol–ether mixture, air-dried to a constant
weight, and recrystallized from isopropanol to give
25% of the product; mp 165–166°С. IR: 936–688,
1520–1496 (Ar); 1700 (С=О); 1496 (СН3, СН2);
1
1364–1340 (–C–N–); 1252–1192 (–С–О–С–). H
NMR: 2.39 (s, 6Н, СН3); 2.65–2.76 (m, 4Н, СН2);
6.84–8.0 (m, 9Н, С6Н5ОС6Н4).
Pharmacological Tests
The antagonistic activity toward angiotensin AT1
receptors was in vitro tested on isolated portal veins of
outbred rats of both sexes as described in [10] using a
modification with the Krebs buffer solution (pH 7.4)
at the constant oxygenation with 95% O2–5% CO2
and thermostating at 24°C [11].
The activity of the compounds was assessed by the
inhibition of the spasmogenic effect of the isolated
portal vein induced by 0.01 μM angiotensin II. The
contractions were registered with an isotonic 7006
transducer and a four-channel digital recorder at the
isotonic load of 0.5 g in a single-chamber organ bath
(UgoBasile, S.R.L., Italy) and a LabScribe3.0™ soft-
ware (iWorx Systems, Inc., United States). The com-
pounds were tested at a concentration of 10 μM. As a
reference, the valsartan preparation, an AT1 antago-
nist (Sigma, United States), was used.
The effects of the compounds at a concentration of
100 μM on the platelet aggregation of Chinchilla rab-
bits were determined in vitro as described in [12]. The
platelet aggregation was induced with 5 μM ADP
(Reanal, Hungary). The study was performed on a
two-channel laser 230 LA analyzer of platelet aggrega-
tion (NPF Biola, Russia). The activity was calculated
as a ratio of the reduction of ADP-induced platelet
The capacity of the tested compounds to break
cross-links of glycated proteins was calculated by for-
mula (1):
Ac (%) = ((Fc – Fb) – (Fs – Fsb)/(Fc – Fb)) × 10, (1)
where Fc is the fluorescence of incubated BSA, glu-
cose, and DMSO (control); Fb, the fluorescence of
incubated BSA (nonglycated); Fs, is the fluorescence
of incubated BSA, glucose, and the tested compound;
Fsb, the fluorescence of incubated BSA (nonglycated)
and the tested compound. Alagebrium was used as a
reference compound.
The capacity of the tested compounds to inhibit
DPP-4 was assessed as described in [14]. A solution of
the tested compound (0.1 mM, 10 μL), 0.1 M Tris-
HCl (pH 8.0, 50 μL), and human plasma (40 μL) were
preincubated for 5 min at 37°C. The reaction substrate
p-nitroanilide Gly-Pro (Sigma, United States)
(1 mM, 100 μL) was added and the mixture was incu-
bated for 15 min at 37°C. Intensification of yellow
color due to the release of 4-nitroaniline was deter-
mined at 405 nm using a microplate reader (Infinite
M200, Tecan, Austria). Vildagliptin (Sigma, United
States) was used as a reference compound.
For the evaluation of the inhibitory activity toward
aggregation in the presence of the compound tested to GP, a mixture of 50 mM HEPES buffer (pH 7.2,
that of the control compounds (as Δ%). Acetylsalicylic 100 μL) containing 100 mM potassium chloride,
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SYNTHESIS AND PHARMACOLOGICAL ACTIVITY
2.5 mM magnesium chloride, 0.5 mM glucose 1-phos- REFERENCES
169
phate (Sigma, United States), and 1 mg/mL glycogen
was preincubated for 30 min with muscular rabbit
α-GP (Sigma, United States) and the tested com-
pounds at 30°C. After preincubation 1 M HCl
(150 μL) containing 10 mg/mL ammonium molyb-
date and 0.38 mg/mL malachite green were added and
the released inorganic phosphate was measured in
20 min. The intensification of the color was deter-
mined at 620 nm with a microplate reader (Infinite
M200, Tecan, Austria) [15]. As a reference com-
pound, CP-316819 (Sigma, United States) was taken.
For assaying the activity of the compounds under
study and CP-316819 their solutions in 14% DMSO
were used, which were added to the reaction mixture
at a concentration of 0.1 mM. Into the control mix-
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ACKNOWLEDGMENTS
The work was supported by the Russian Scientific
Foundation, project no. 14-25-00139.
Translated by E. Shirokova
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 43 No. 2 2017