212
A. V. Semenov et al.
13C NMR spectra (Table 3) of Ia-k exhibited the pre-
tion in the studied samples as compared with a control. De-
rivatives Ib, Ii, and Ij exhibited statistically significant activ-
ity at concentrations ³ 2 mM.
dicted number of resonances in the ranges 150 – 160 ppm
(carbonyl) and 110 – 160 ppm (aromatic ring). A resonance
for the carboxylic acid C atom for Ig at 167.5 ppm and char-
acteristic resonances in the strong-field part of the spectrum
of alkoxy derivatives Ii-Ik were also identified.
Acute toxicity was assessed preliminarily for the most
active derivatives.
Acute toxicity. Compounds Ib, Ii, and Ij in
DMSO—H O (1:1, 0.2 mL) were administered once i.p. to
2
white laboratory mice. Survival was observed for two days
and after two weeks. Each group of mice contained an identi-
cal number of animals (6, 3 males and 3 females) to give
13 groups of animals (4 groups for Ib and Ii and 5 groups for
Ij at increasing doses from 100 to 400 or 800 mg/kg; higher
doses were not studied because of the low solubility of the
compounds). Lethal outcomes were not observed for all
EXPERIMENTAL BIOLOGICAL PART
Glucokinase activity was studied in fresh liver excised
from chinchilla rabbits and homogenized using an IKA T 18
basic homogenizer (Germany).
Glucose content was determined by the Samogyi—Nel-
son colorimetric method [12]. Optical density of the studied
solutions was measured on a UV-Vis UV2600 spectrometer
compounds at the maximum studied dose. Thus, the LD
50
values for Ib and Ii were >400 mg/kg; for Ij, >800 mg/kg.
This corresponded to a minimal class 3 hazard (moderately
toxic compounds).
(Shimadzu, Japan) at
l
502 nm in quartz cuvettes
(l = 10 mm) relative to a solution containing all components
except glucose. The glucose concentration was determined
from a calibration curve.
Thus, the synthesized asymmetric disubstituted urea de-
rivatives Ib, Ii, and Ij could provide a basis for further re-
search on the discovery of glucokinase activators to treat
type 2 diabetes mellitus.
Acute toxicity was assessed in 78 white laboratory mice
(39 males and 39 females, 18 – 20 g).
Preparation of liver homogenate. Fresh rabbit liver
(1 g) cooled to 5°C was ground in a homogenizer with cold
potassium-phosphate buffer (5 mL, pH 7.8). The content of
liver tissue in the resulting homogenate was 200 mg/mL.
Effect of studied compounds on glucokinase activity
[8]. Solutions (100 mL) of the following compounds in
REFERENCES
1. A. S. Grewal, B. S. Sekhon, and V. Lather, Mini-Rev. Med.
Chem., 14(7), 585 – 602 (2014).
2. F. M. Matschinsky, B. Zelent, N. M. Doliba, et al., Handb. Exp.
Pharmacol., 203, 357 – 401 (2011).
DMSO—H O (2:1) were placed into 1.5 mL centrifuge
2
tubes: glucose (1 mM), Na-ATP (5 mM), MgCl (5 mM),
2
3. A. Nakamura and Y. Terauchi, J. Diabetes Invest., 6(2),
124 – 132 (2015).
KCl (42 mM), and the studied compounds at various concen-
trations (1, 0.1, and 0.01 mM). The volumes were adjusted to
4. R. M. Borzilleri, J. S. Tokarski, et al., WO Pat. 2004001059,
Dec. 31, 2003.
0.3 mL using cold distilled H O and thermostatted at 37°C.
2
Homogenate (0.2 mL) thermostatted beforehand at the same
temperature was added to the solutions. The tubes and con-
tents were incubated at 37°C for 10 min, treated with
Ba(OH) (0.5, 0.3 N) and ZnSO solutions (5%) and centri-
5. J. Bloxham, M. C. T. Fyfe, et al., WO Pat. 2006018662, Feb. 23,
2006.
6. P. S. Petrov, A. V. Semenov, and I. V. Tarasova, in: Abstracts of
an International Congress on Organic Chemistry Dedicated to
the 150th Anniversary of Butler’s Theory of Chemical Structure
of Organic Compounds, Kazan (2011), p. 380.
7. B. Stanovnik, M. Tisler, V. Colob, et al., J. Heterocycl. Chem.,
17(4), 733 – 736 (1980).
2
4
fuged for 10 min at 2,000 rpm. The resulting supernatants
were separated. The glucose contents in them were deter-
mined. Results were processed statistically using Microsoft
Excel 2010 to determine the means and standard errors from
three experiments (M ± m). The results were compared using
one-way analysis of the variance (ANOVA). Differences
were considered statistically significant for p < 0.05. Ta-
ble 4 presents the results.
8. S. Long, Biochem. J., 50(3), 407 – 415 (1952).
9. Synthesis of Organic Preparations [in Russian], Collect. 5, Izd.
Inostr. Lit., Moscow (1954), pp. 44 – 45.
10. A. V. Semenov, I. V. Tarasova, et al., RU Pat. 2,559,889, Aug.
20, 2015.
The results indicated that all studied derivatives at con-
centrations ³ 20 mM activated glucokinase. This was seen in
the statistically significant reduction of glucose concentra-
11. C.-H. Chien, M.-K. Leung, J.-K. Su, et al., J. Org. Chem., 69(6),
1866 – 1871 (2004).
12. M. J. Somogyi, J. Biol. Chem., 195(1), 19 – 23 (1952).
Semenov
