Synthesis and evaluation of camphor and cytisine-based cyanopyrrolidines as DPP-IV inhibitors for the treatment of type 2 diabetes mellitus

Article abstract of DOI:10.1016/j.bmc.2018.07.018

In this study, bornyl- and cytisine-based cyanopyrrolidines as potent dipeptidyl peptidase-IV (DPP-IV) inhibitors were synthesised. The in vitro inhibiting activities of bornyl- and cytisine derivatives towards DPP-IV were evaluated. Bornyl-based cyanopyrrolidines were shown to have moderate inhibitory activity with regard to DPP-IV (1.27–15.78 μM). A docking study was performed to elucidate the structure-activity relationship of the obtained compounds. The in vivo hypoglycemic activities of the same compounds were evaluated with the oral glucose tolerance test (OGTT) in mice. Bornyl-based cyanopyrrolidines were shown to have good hypoglycemic activity.

Full text of DOI:10.1016/j.bmc.2018.07.018

Accepted Manuscript
Synthesis and evaluation of camphor and cytisine-based cyanopyrrolidines as
DPP-IV inhibitors for the treatment of type 2 diabetes mellitus
S.O. Kuranov, I.P. Tsypysheva, M.V. Khvostov, L.F. Zainullina, S.S.
Borisevich, Yu.V. Vakhitova, O.A. Luzina, N.F. Salakhutdinov
PII:
S0968-0896(18)30481-4
https://doi.org/10.1016/j.bmc.2018.07.018
BMC 14455
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
7 March 2018
25 June 2018
10 July 2018
Please cite this article as: Kuranov, S.O., Tsypysheva, I.P., Khvostov, M.V., Zainullina, L.F., Borisevich, S.S.,
Vakhitova, Yu.V., Luzina, O.A., Salakhutdinov, N.F., Synthesis and evaluation of camphor and cytisine-based
cyanopyrrolidines as DPP-IV inhibitors for the treatment of type 2 diabetes mellitus, Bioorganic & Medicinal
Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.07.018
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Synthesis and evaluation of camphor and cytisine-based cyanopyrrolidines as DPP-
IV inhibitors for the treatment of type 2 diabetes mellitus
S.O. Kuranova, I.P. Tsypyshevab, M.V. Khvostova,e, L.F. Zainullinac,d, S.S. Borisevichb,
Yu.V. Vakhitovac,d, O.A. Luzinaa, N.F. Salakhutdinova,e
aN. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch of the
Russian Academy of Sciences, Novosibirsk, 630090, Russian Federation
bUfa Institute of Chemistry of Russian Academy of Sciences, Ufa, 450054, Russian
Federation.
cFSBI «Zakusov Institute of Pharmacology», Moscow, 125315, Russian Federation
dIBG UFRC RAS, Ufa, 450054, Russian Federation
eNovosibirsk State University, Novosibirsk, 630090, Russian Federation
Abstract
In this study, bornyl- and cytisine-based cyanopyrollidines as potent dipeptidyl peptidase-
IV (DPP-IV) inhibitors were synthesised. The in vitro inhibiting activities of bornyl- and cytisine
derivatives towards DPP-IV were evaluated. Bornyl-based cyanopyrrolidines were shown to
have moderate inhibitory activity with regard to DPP-IV (1.27–15.78 µM). A docking study was
performed to elucidate the structure–activity relationship of the obtained compounds. The in vivo
hypoglycemic activities of the same compounds were evaluated with the oral glucose tolerance
test (OGTT) in mice. Bornyl-based cyanopyrrolidines were shown to have good hypoglicemic
activity.
1. Introduction
Diabetes mellitus is a chronic disease and a major healthcare problem that is estimated to
affect approximately 422 million people worldwide, of which more than 90% will be type 2
diabetes mellitus (T2DM). This number is likely to double in the next 20 years.1 T2DM is a
long-term metabolic disorder that is characterised by high blood sugar levels and insulin
resistance. An increase in the blood glucose levels leads to the development of nephropathy2,
retinopathy3, atherosclerosis4, and cardiovascular disease (CVD).5 Despite the clear advantages
of using insulin to treat diabetes, there is increasing evidence that other complications may limit
its benefits in the treatment of T2DM patients.6 Over the last few years, novel glucose-lowering
drugs with reduced risk of hypoglycemia and CVD have become available and may be
1

therapeutic alternatives bearing lower risk of serious adverse effects. Among antidiabetic drugs,
dipeptidyl peptidase-IV (DPP-IV) inhibitors occupy a significant portion of the market. Gliptins
(DPP-IV inhibitors), have been used in clinical practice since 2006 and are associated with a
lower risk (41%) of all-cause mortality compared with insulin treatment.7
Vildagliptin 1, saxagliptin 2 and sitagliptin 3 are marketed DPP-IV. The chemical
structures of 1 and 2 may be divided into three parts: the cyanopyrollidine fragment, which is an
important pharmacophore of this type of drug, the amino acid linker, and the bulky
hydroxyadamantane fragment (Figure 1).
Figure 1. DPP-IV inhibitors
A problem associated with this family of nitrile-based inhibitors is that they can be
chemically unstable because the cis-rotamer is prone to intramolecular cyclisation, which can
result in an inactive bicyclic compound.8 This has necessitated the development of compounds
that do not undergo this type of intramolecular cyclisation. One approach to avoid intramolecular
cyclisation is the synthesis of compounds containing bulky substituents at the amino group. Our
interest prompted us to synthesise gliptin analogues by replacing the adamantane fragment with
another bulky synthetic equivalents, derived from natural compounds.
Natural products have been a rich source of leads for drug discovery. Many clinical drugs
developed over the last 25 years are associated with natural compounds.9,10 There are some
examples in which replacement of the adamantyl moiety with bicyclic natural synthetic
equivalents leads to increased efficacy, e.g., the substitution of adamantyl fragments by bornyl
moieties enhances the antiviral activity of antiinfluenza agents 10-fold.11 One more perspective
approach consists in combining pharmacophore fragments with different targets in one
molecule.12 Our interest was pointed to cytisine, alkaloid isolated from the seeds or epigeal part
of the Fabaceae plant, due to the fact this quinolizidine alkaloid exhibits antidiabetic activity.13
This compound has a bulky bispidine moiety with a secondary amino group and pseudo-aromatic
2-pyridone core that can be modified.
So based on such suggestion our work was directed to synthesis of novel DPP-IV
inhibitors - cyanopyrrolidine compounds with bulky bornyl- or bispidine substituents starting
from commercially available natural compounds (+)-camphor 4a, (-)-camphor 4b and (-)-
cytisine 5.
2

Figure 2. Bulky nature compounds
2. Results and discussion
2.1. Chemistry
To search for new chemical classes of potential hypoglycemic agents, we designed and
synthesised a series of cyanopyrrolidine derivatives with bulky fragments of natural compounds.
First, we focused on the synthesis of a series of cyanopyrrolidine compounds with bornyl and
substituted bornyl moieties derived from (+)- and (-)-camphor.
Both enantiomers of the (R)- and (S)-exo-bornyl amines 6a and b were prepared using
the same procedure from the (1R)-(+)- and (1S)-(-)-camphor. Camphor 4a, b was reacted with
hydroxylamine in alcohol solution to generate a mixture of E- and Z-oximes of 6a, b. The
mixture obtained was reduced by sodium borohydride in methanol in the presence of nickel
chloride to give exo-bornylamine 7a, b.14
For a better understanding of structure-activity relationships (SARs), we introduced
additional functional groups in the bornyl moieties, which can contribute to the formation of
extra binding with an enzyme. (+)-Camphor 4a was oxidised by selenium dioxide in acetic
anhydride to camphoroquinone15 8, which was followed by reaction with hydroxyl amine to give
monosubstitued 3-oxime camphoroquinone16 9. Compound 9 was reduced in two different ways.
Treatment with zinc in alkali solution allowed us to reduce only the oxime group to give keto-
amine17 10, whereas lithium aluminium hydride reduced both the carbonyl and oxime groups to
amino alcohol 11 (Scheme 1).18
3

Scheme 1. Reagents and conditions: (a) NH2OH·HCl, AcONa·3H2O, MeOH, H2O,
reflux; (b) NaBH4, NiCl2·6H2O, MeOH, -250C; (c) SeO2, Ac2O, reflux; (d) NH2OH·HCl,
AcONa·3H2O, MeOH, H2O, 10 min, reflux; (e) Zn, NaOH, H2O; (f) LiAlH4, Et2O, reflux.
Derivatives of the quinolizidine alkaloid (-)-cytisine 12a-d with a modified 2-pyridone
core were synthesised as previously described (Scheme 2).19
Scheme 2. Reagents and conditions: (a) KCl, H2SO4 (50%), H2O2 (30%); (b) KCl, H2SO4
(50%), H2O2 (30%) (c) NaNO3, H2SO4, 20oC.
The general synthesis procedure of cyanopyrrolidine compounds is shown in Scheme 3.
The reaction of commercially available L-proline 13 with thionyl chloride in methanol produced
proline methyl ether hydrochloride 14, which was converted to proline amide 15 with saturated
4

ammonia solution in methanol.20 Proline amide 15 was acylated by chloroacetyl chloride in
dichloromethane (DCM) in the presence of triethylamine and dimethylaminopyridine (DMAP)
and the obtained compound 16 was dehydrated by trifluoroacetic anhydride to give 17.21 Title
compounds 18a–i were obtained by the reaction of 17 with the corresponding amines 6a and b,
9, 10, 11, and 12a–d in DCM in the presence of potassium carbonate at room temperature.
Scheme 3. Reagents and conditions: (a) SOCl2, MeOH, -50C to reflux; (b) NH3, MeOH, -
250C to r.t.; (c) ClCH2C(O)Cl, NEt3, DMAP, DCM; (d) TFAA, DCM; (e) RH, K2CO3, DCM, r.t.
2.2. DPP-IV inhibitory activity
The synthesised compounds were evaluated for inhibition of human recombinant DPP-IV
in an in vitro assay using sitagliptin 3 as a positive control and vildagliptin 1 as a reference
compound. Among all tested compounds only the bornyl derivatives 18a–d were found to inhibit
the activity of DPP-IV with IC50 values in the range of 1.27–15.78 µM (Table 1). We observed
that compound 18a obtained from (+)-camphor demonstrated the lowest IC50 value, whereas (-)-
camphor derivative (18b) was slightly less active than 18c and 18d. Replacement of the bornyl
moiety with a (-)-cytisine scaffold (compounds 18e–i) resulted in a loss of potency against DPP-
IV.
Table 1
DPP-IV inhibitory effect of bornyl- 18a-d and (-)-cytisine derivatives 18e-i
DPP-4
Compound
Structure
IC50, μM
1.27, (1.13 to 1.40) a
18a
5

1.78, (1.52 to 2.04) a
15.78, (13.51 to 17.52) a
7.58, (7.48 to 7.62) a
18b
18c
18d
b
-
18e-i
R1 = R2 = H
R1 = H; R2 = Cl
R1 = Br; R2 = Br
R1 = Cl; R2 = Cl
R1 = NO2; R2 = H
18e
18f
18g
18h
18i
a - Data presented as mean IC50, calculated from two independent experiments
performed in triplicate and 95% confidence interval (CI) (GraphPad Software Inc., San Diego,
CA).
b – No inhibition
2.3. DPP-IV docking study
To explore the binding modes of compounds 18a–i and vildagliptin 1 with the active site
of DPP-IV, molecular docking simulations were carried out using the Glide Docking application
of Schrödinger Suite 2018-1.22 Vildagliptin 1 is a potent cyanopyrrolidine-containing inhibitor
of DPP-IV, which resembles the compounds 18a–i. Therefore, vildagliptin 1 was selected as a
reference ligand for docking studies. The X-ray crystal structures of the DPP-IV complex with
vildagliptin were retrieved from the RCSB Protein Data Bank (PDB code: 3W2T).
6

A covalent docking of compounds 18a–i into the active site of DPP-IV was performed and
comparison of important parameters, such as covalent docking affinity, and binding energy
(GMMGBSA) with the analogous compound vildagliptin 1 (Table 2), was executed.
The structures of 18a–i can occur in the protonated form. According to quantum–
chemical calculations (optimisation by DFT method, see SI), the nitrogen atom of compounds
18a–d is protonated in the presence of water (e.g., calculated pKa value of 18a is 7.6) whereas
compounds 18e–i are non-protonated (pKa value of 18e is 4.1). Thus, the protonated molecules
of compounds 18a–d and non-protonated molecules of 18e–i were used for covalent docking.
The docking results of compounds 1 and 18a–i with DPP-IV are presented in Table 2.
Table 2
Docking results of compounds 1, 18a-i with DPP-IV
Covalent docking
GMM_GBSA
kcal/mol
-55.2
,
Compound
affinity, kcal/mol
Vildagliptin 1
18aa
-8.4
-6.7
-7.4
-6.8
-7.0
-3.5
-4.2
-3.7
-3.4
-3.8
-48.3
18ba
18ca
18da
18eb
18fb
18gb
18hb
18ib
-45.7
-40.7
-41.9
-32.5
-27.0
-18.8
-34.9
-16.9
a) protonated form
b) non-protonated form
Through molecular docking, we found that the resulting set of values of covalent docking
affinity of 18a-i falls within the range of -8.0 to -3.0 kcal/mol, while the resulting sets of values
of binding energy (GMM GBSA) fall within the range of -48.3 to -16.9 kcal/mol (Table 2). It
should be noted that compounds 18a-d demonstrated the efficiency of binding to the active site
of DPP-IV comparable to that of vildagliptin 1, and compound 18b manifested the superior
affinity value -7.4 kcal/mol (the affinity value of vildagliptin 1 is -8.4 kcal/mol). The calculated
7

GMM_GBSA values of reference ligand 1 and compounds 18a-d also were close: -55.2 and –48.3,
-45.7, -40.7, -41.9 kcal/mol (Table 2). ()-Cytisine derivatives (compounds 18e-i) showed the
insufficient covalent docking affinity and GMM_GBSA values.
Modelling studies have revealed that the nitrile group of compounds 18a–d (IC50 values
of 1.27–15.78 µM) covalently interacts with Ser630, while the secondary amino group forms salt
bridges with Glu205 and Glu206 (compounds 18a and 18d in the protonated form) or with
Glu206 (compounds 18b and 18c in the protonated form). The above-mentioned crucial
interactions are common for compounds 18a–d and the reference ligand 1. Importantly,
interactions with these two key recognition motifs are sufficient for low micromolar binding
affinities of DPP-IV inhibitors.23 In general, the lower DPP-IV inhibitory potential of the
compounds 18a–d could be due to the non-optimal location of bornyl-derivatives in the active
site of DPP-IV compared with the structurally similar adamantyl-containing vildagliptin. In
particular, the hydroxyl group on the adamantyl moiety of vildagliptin stabilises the location of
the ligand via hydrogen bonds with water molecules. The absence of polar atoms in the bornyl
moiety of compounds 18a and b and the position of the carbonyl oxygen behind 18c most likely
contributes to the instability of bornyl-containing derivatives relative to the N-terminal
recognition region of DPP-IV. (See SI)
It is worth noting that the docking mode of inactive (-)-cytisine derivatives 18e–i differs
significantly from that of active bornyl-containing compounds 18a–d. For example, the location
of compound 18e in the active site of DPP-IV is represented in Figure 3. The compounds 18e–i
does not exhibit the non-covalent interaction with key amino acids Glu205 and Glu206 and
despite the fact that the nitrile atom of preserves the covalent binding with Ser630, this binding
mode is likely insufficient to inhibit DPP-IV (Table 2).
8

A
B
Figure 3. Types of interactions of 18a and 18e with the amino acids of the active site of
DPP-IV (red lines depict covalent interactions, pink dotted lines depict salt bridges, and yellow
dotted lines depict hydrogen bonds). A, 18a; B, 18e. (For other see SI.)
2.4. Biological evaluation
All obtained compounds were tested in oral glucose tolerance test (OGTT) in male albino
mice (CD-1 line). Results obtained are shown in Table 3. All tested bornyl derivatives 18a-d at a
dose of 10 mg/kg do able to reduce blood glucose levels as good as vildagliptin in the same
dose. None of cytisine-derivative’s 18e-i possesses an ability to reduce blood glucose levels in
this test. Obtained difference between DPP-IV inhibitory activity in vitro and in vivo results is a
common case for such investigations and similar data could be found in the literature.24
Table 3. OGTT results (mmol/L, Mean ± SD, n = 5). *p<0.05 versus control
Time after the glucose load (min)
Group
0
30
60
90
Control
Vildagliptin 1
18a
18b
18c
3,06±0,16
2,63±0,17
3,37±0,24
4,04±0,52
2,88±0,29
3,78±0,25
15,45±0,45
10,58±0,62*
12,58±0,73*
10,88±1,29*
10,28±0,80*
10,62±0,24*
11,87±0,63
9,61±0,68*
9,18±0,79*
8,78±0,57*
10,32±0,49
9,60±0,61*
7,95±0,46
7,60±0,62
6,65±0,88
7,85±0,89
7,88±0,61
6,54±0,75
18d
9

3. Conclusions
In summary, two series of bornyl- and citisine-based cyanopyrrolidines have been
synthesized and evaluated in in vitro and in vivo tests. All bornyl compounds 11a-d showed
moderate inhibition activity against DPP-IV and comparable to Vildagliptin glucose lowering
activity in OGTT in mice. Cytisine’s derivatives showed no effect in in vitro and in vivo tests.
DPP-IV inhibition activity of synthesized compounds 18a-18i can be explained using molecular
docking results. It was found that more active bornyl compounds 18a-18d analogous to
vildagliptin 1 interacts with key amino acids of active enzyme site. In accordance with in vitro
and in silico results the more potent compounds 18a and 18b are characterized by the following
scores: IC50 equal to 1.27 and 1.76 µM, Glide Scores are -6.7 and -7.4 kcal/mol, GMMGBSA
values are -48.3 and 45.7 kcal/mol respectively.
4. Experimental
4.1. Chemistry
GC: 7820A gas chromatograph (Agilent Tech., USA); flame-ionisation detector; HP-5
capillary column, helium as carrier gas (flow rate 2 mL/min, flow division 99:1). Optical
1
13
rotation: polar 3005 spectrometer; CHCl3 soln. H and C NMR spectra: Bruker spectrometers
AV-400 at 400.13 MHz (1H) and 100.61 MHz (13C), AV-600 at 600.30 MHz (1H) and 150.95
MHz (13C) in CDCl3; chemical shifts δ in ppm relative to residual CHCl3[δ(CHCl3) 7.26,
δ(CHCl3) 77.00 ppm], J in Hz. The structure of the products was determined by analysing 1H and
13C NMR spectra. HR-MS: DFS Thermo Scientific spectrometer in a full scan mode (15–500
m/z, 70eV electron impact ionisation, direct sample administration). Column chromatography
was performed on silica gel (60–200 l, Macherey-Nagel. The purity of target compounds was
determined by gas chromatography. All the target compounds reported in this paper have the
purity of at least 95%. Spectral and analytical studies were carried out at the Collective Chemical
Service Center of the Siberian Branch of the Russian Academy of Sciences. All chemicals were
analytically pure and were used as received without purification prior to use. Dichloromethane
was distilled over phosphorus pentoxide before use.
4.1.1. (1R)-N-hydroxy-1,7,7-trimethylbicyclo[2.2.1]heptan-2-imine (6a)
A mixture of (+)-camphor (13 mmol, 1.98 g), hydroxylamine hydrochloride (215 mmol,
1.77 g), and water (7 ml) was heated to 80°C, and methanol (7 ml) was added to dissolve the
camphor. A solution of sodium acetate trihydrate (3.3 mmol, 4.5 g) in water (4 ml) was added,
and the reaction mixture was heated under reflux at 100°C for 12 h. Upon removal of the
10

methanol under reduced pressure, the white solid that precipitated was collected by filtration,
washed with water (3x10 ml), and dried in vacuo to afford 5a. White solid 1.95 g, 90 % yields.
1H NMR (400 MHz, CDCl3) δ 0.79 (s, 3 H), 0.91 (s, 3 H), 1.00 (s, 3 H), 1.19 - 1.27 (m, 1 H),
1.45 (ddd, J = 12.93, 9.17, 4.23 Hz, 1 H), 1.70 (td, J = 12.19, 4.10 Hz, 1 H), 1.78 - 1.88 (m, 1 H),
1.91 (t, J = 4.43 Hz, 1 H), 2.03 (s, 0.5 H), 2.07 (s, 0.5 H), 2.55 (dt, J = 17.86, 3.90 Hz, 1 H), 9.06
(br. s., 1 H).
4.1.2. (1S)-N-hydroxy-1,7,7-trimethylbicyclo[2.2.1]heptan-2-imine (6b)
Compound 5b was synthesized by analogous procedure from (-)-camphor as white solid.
95 % yields.
4.1.3. (1R,2R,4R)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-amine (7a)
The sodium borohydride (70.0 mmol , 2.66 g) was added portionwise to a solution of
(1S)-camphor oxime (12 mmol, 1.95 g) and nickel dichloride hexahydrate (23 mmol, 5.54 g) in
anhydrous methanol (40 ml) at –25°C over a period of 3 h. After completion of the addition, the
resulting black slurry was stirred at this temperature overnight. The reaction mixture was then
warmed to room temperature, and 25% ammonia solution (15 ml) in water (20 ml) was added
with vigorous stirring. The resulting slurry was extracted with diethyl ether (3x70 ml), and the
combined organic layers were washed with brine (20 ml), dried with anhydrous magnesium
sulfate, filtered, and concentrated in vacuo. The crude product was purified by column
chromatography (silica gel, dichloromethane/methanol 100:0–90:10) to afford exo-(–)-
bornylamine as a foamy white solid (0.682 g, 33% yield). 1H NMR (400 MHz, CDCl3) d 0.81 (s,
3 H), 0.86 (s, 3 H), 0.96 (s, 3 H), 0.97 - 1.08 (m, 2 H), 1.21 - 1.28 (m, 2 H), 1.48 - 1.57 (m, 2 H),
1.65 - 1.77 (m, 3 H), 2.69 (dd, J = 8.87, 5.10 Hz, 1 H).
4.1.4. (1S,2S,4R)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-amine (7b)
(+)-exo-bornylamine was synthesized by analogous procedure from 6b as white solid. 46
% yield.
4.1.5. (1R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptane-2,3-dione (8)
A mixture of (+)-camphor (0.16 mol, 20.0 g), selenium dioxide (0.07 mol, 8.0 g) in acetic
anhydride (14 ml) was stirred under reflux for 1h. Then the reaction mixture was cooled to room
temperature, and an additional portion of selenium dioxide (0.07 mol, 8.0 g) was added. The
mixture was again heated to reflux, and two further batches of selenium dioxide (8.0 g, 0.07 mol)
were added at 2.5-h and 6-h intervals. After the reaction mixture was refluxed for additional 4h,
during that time precipitation of selenium was observed, it was cooled to ambient temperature
11

and subsequently 200 ml of ethyl acetate were added. The gray precipitate was removed by
filtration, and the filtrate was diluted with 100mL of toluene. The filtrate was concentrated under
reduced pressure and that yielded crude camphorquinone as a yellow-orange solid which was
then dissolved in 200 mL of ethyl acetate, and the solution was filtered by vacuum filtration
through Celite. The filtrate was successively washed with 200 ml 10%. Sodium hydroxide
solution and 100 ml of brine. The organic layer was dried over anhydrous magnesium sulfate,
filtered, and concentrated in vacuo to afford pure (+)-camphorquinone which was used in the
1
following reactions without purification. Orange needle crystals, 85% 18.6 g. H NMR (400
MHz, CDCl3) δ 0.91 (s, 3 H), 1.04 (s, 3 H), 1.09 (s, 3 H), 1.57 - 1.67 (m, 2 H), 1.85 - 1.95 (m, 1
H), 2.10 - 2.20 (m, 1 H), 2.61 (d, J = 5.24 Hz, 1 H).
4.1.6. (1R,4S)-3-(hydroxyimino)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-one (9)
To a solution of hydroxylamine hydrochloride (11.76 mmol, 0.81 g) and sodium acetate
trihydrate (17.6 mmol, 2.39 g) in water (5 mL) was added 7 (5.9 mmol, 0.97 g) in absolute
ethanol (10 ml). The solution was refluxed gently for 10 min, after which time the solution was
cooled, ethanol was removed by rotary evaporation, and water (5 mL) was added to the
remaining aqueous slurry. The slurry was cooled in an ice bath, and the solid that precipitated
was filtered. The solid was dried under vacuum to yield an off-white solid, 0.50 g, 47% which
1
was used without further purification. H NMR (400 MHz, CDCl3) δ 0.86 (s, 3H), 1.00 (s, 3H),
1.03 (s, 3H), 1.50–1.63 (m, 2H), 1.70–1.84 (m, 1H), 1.99–2.12 (m, 1H), 3.25 (d, J = 4.5 Hz, 1H),
8.50 (s, 1H).
4.1.7. (1R,3S,4S)-3-amino-1,7,7-trimethylbicyclo[2.2.1]heptan-2-one (10)
Compound 8 (2.7 mmol, 0.48 g) was dispersed in 30% aqueous sodium hydroxide (3 ml)
and zinc powder (8 mmol, 0.52 g) was added. After 2 min, diethyl ether (10 ml) was added and
the mixture was stirred for 1 min. The organic layer was separated, and the residue was extracted
with diethyl ether (2x5 ml). The combined organic extracts were washed with water (3 ml), brine
(3 ml), and dried over anhydrous magnesium sulfate for 2 min. Drying agent was filtered off and
a solvent was evaporated under vacuo. Obtained residue was used in following step without
purification.
4.1.8. (1R,2S,3R,4S)-3-amino-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ol (11)
A solution of monooxime 8 (2.7 mmol, 0.49 g) in diethyl ether (5 ml) was added
dropwise to a solution of lithium aluminum hydride (13.5 mmol, 0.51 g) in diethyl ether (4 ml)
and the mixture was refluxed for 24 h. It was cooled in an ice bath, after which ethyl acetate (5
ml) was added dropwise with stirring, followed after 5 min with 10% aqueous sodium hydroxide
12

solution (4 ml), water (5 ml), diethyl ether (20 mL), and stirring was continued for 4 h at room
temperature. The mixture was filtrated, washed with saturated brine (10 mL) and dried with
anhydrous magnesium sulfate. Solvents were removed under reduced pressure and the product
was isolated by column chromatography (silica gel, hexane–ethyl acetate (1:1)), and then
1
recrystallized from hexane-ethyl acetate 0.27 g, 60%. H NMR (400 MHz, CDCl3) δ ppm 0.79
(s, 3 H), 0.90–0.87 (m, 1H), 0.95 (s, 3 H), 1.05–0.96 (m, 1H), 1.06 (s, 3H), 1.43–1.39 (m, 1H),
1.56 (d, J= 4.8 z, 1H), 1.74–1.67(m, 1H), 3.06 (d, J= 7.2 Hz, 1H), 3.38 (d, J= 7.2 Hz, 1H).
4.1.9. 5-Chlorocytisine (12a)
Compound was synthesized according to the literature procedure.19
4.1.10. 3,5-Dichlorocytisine (12b)
Compound was synthesized according to the literature procedure.19
4.1.11. 3,5-Dibromocytisine (12c)
Compound was synthesized according to the literature procedure.19
4.1.12. 3-Nitrocytisine (12d)
Compound was synthesized according to the literature procedure.19
4.1.13. Methyl (2S)-pyrrolidine-2-carboxylate hydrochloride (14)
To an ice-bath cooled stirring suspension of L-proline (46 mmol, 5.3 g) in methanol (25
ml) thionyl chloride (71 mmol, 5.2 ml) was added in 10 min. The reaction was stirring for 0.5
hours than hour at room temperature and reflux in 2 hours. Methanol and excess of thionyl
chloride were removed under reduced pressure. Obtained slightly yellow oil was used in the
following step without further purification.
4.1.14. (2S)-pyrrolidine-2-carboxamide (15)
A cooled to -25oC solution of compound 7 in methanol (40 ml) was saturated by gaseous
ammonia. A precipitation of ammonia hydrogenchloride was observed. After saturation, the
reaction mixture was resealed for 4 days. The reaction mixture was filtered, and the white
precipitate was washed by methanol (20 ml). Methanol was removed under vacuo and the
obtained residue was recrystallized from methanol-ethyl acetate. White solid, 2.9 g, 55% yield.
1H NMR (400 MHz, CDCl3) δ 1.58 - 1.79 (m, 2 H) 1.80 - 1.93 (m, 1 H) 2.03 - 2.14 (m, 1 H) 2.18
(s, 1 H) 2.82 - 3.01 (m, 2 H) 3.67 (dd, J=9.20, 5.57 Hz, 1 H) 6.25 (br. s., 1 H) 7.38 (br. s., 1 H)
13

4.1.15. (2S)-1-(chloroacetyl)pyrrolidine-2-carboxamide (16)
A solution of (2S)-pyrrolidine-2-carboxamide (2.6 mmol, 300 mg), triethylamine (2.8
mmol, 0.39 ml) and catalytic amount of dimethylaminopyridine (DMAP) in dichloromethane (3
ml) was added dropwise over 30 min to -100C cooled solution of chloroacetyl chloride (2.9
mmol, 0.23 ml) in dichloromethane (3 ml). Reaction mixture was stirring for 2 hours at -100C
and then 4 hours at 0oC. Reaction mixture was allowed to reach room temperature and poured
into ethyl acetate (25 ml). Formed white precipitate of triethyl amine hydrochloride was filtered
and washed with ethyl acetate (10 ml). The solvent was removed under reduced pressure and
residue was used in following reaction without further purification.
4.1.16. (2S)-1-(chloroacetyl)pyrrolidine-2-carbonitrile (17)
A solution of (2S)-1-(chloroacetyl)pyrrolidine-2-carboxamide 16 (0.26 mmol) in
dichloromethane (4 ml), trifluoroacetic anhydride (6.4 mmol, 0.9 ml) was added dropwise. After
2 hours the reaction mixture was poured into water (20 ml) and the product was extracted with
dichloromethane (3x10 ml). Combined extracts were washed with saturated sodium bicarbonate
solution (5 ml), brine (5 ml) and dried over magnesium sulfate. Solvent was evaporated under
reduced pressure and residue was purified by column chromatography using dichloromethane -
methanol (100:1 to 10:1) as eluent to afford 17. Light orange solid, 200 mg, 50% yield. 1H NMR
(400 MHz, CDCl3) δ 2.07 - 2.44 (m, 4 H), 3.51 (br. s., 1 H), 3.74 (br. s., 1 H), 4.00 - 4.25 (m, 2
H), 4.71 - 4.78 (m, 0.85 H), 4.85 (dd, J = 7.59, 2.08 Hz, 0.15 H).
4.1.17. General procedure of synthesis 18a-i
A solution of 17 (2.3 mmol) in dichloromethane (4 ml) was added dropwise into an
icewater-cooled mixture of the appropriate amine (1 equiv for secondary and 2 equiv for primary
amine) and potassium carbonate (6.9 mmol) in dichloromethane (1 ml). The resulting reaction
was stirred at ice-water temperature for 2 h and then at room temperature for several days until
disappearing of 17 (controlled by GC-MS). The resulting mixture was poured into water (10ml)
and extracted with dichloromethane (3x10ml). The combined organic layers were washed with
water (5 ml), brine (5 ml) dried over magnesium sulfate, concentrated in vacuo, and obtained
residue was purified by column chromatography using dichloromethane: methanol (100:1 to
10:1) as eluent to afford title compound.
14

4.1.17.1. (2S)-1-(2-{[(1R,2R,4R)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-
yl]amino}acetyl)pyrrolidine-2-carbonitrile (18a)
Colorless needle crystals, 71% yield. M.p. 54.20C. [α]D27 -136 (c 0.140, CHCl3). 1H NMR
(400 MHz, CDCl3) δ 0.80 (s, 3 H), 0.91 (s, 3 H), 1.03 (s, 5 H), 1.44 - 1.62 (m, 3 H), 1.63 - 1.72
(m, 2 H), 1.86 (br. s., 1 H), 2.05 - 2.32 (m, 4 H), 2.46 - 2.52 (m, 1 H), 3.24 - 3.52 (m, 3 H), 3.55 -
13
3.71 (m, 1 H), 4.75 (d, J = 10.21 Hz, 0.8 H), 4.87 (d, J = 9.54 Hz, 0.2 H). C NMR (151 MHz,
CDCl3) δ 12.0, 12.1, 20.4, 20.4, 20.5, 20.5, 22.7, 25.0, 27.2, 27.2, 29.8, 32.2, 36.7, 36.7, 38.2,
38.4, 45.1, 45.2, 45.4, 46.0, 46.3, 46.3, 46.7, 48.5, 50.6, 51.3, 66.9, 67.0, 118.2, 118.3, 170.2,
+
170.7. IR(KBr, cm-1): 1411.7, 1662.4, 2239.1. HRMS for C17H27ON3 calcd 289.2149, found
289.2151 [M]+.
4.1.17.2. (2S)-1-(2-{[(1S,2S,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-
yl]amino}acetyl)pyrrolidine-2-carbonitrile (18b)
Light yellow oil, 70 % yield. [α]D26 -26 (c 0.200, CHCl3). 1H NMR (400 MHz, CDCl3) 
0.80 (s, 3 H), 0.90 - 0.94 (m, 3 H), 1.00 - 1.09 (m, 5 H), 1.44 - 1.61 (m, 3 H), 1.68 (d, J = 1.61
Hz, 2 H), 1.84 (br. s., 1 H), 2.05 - 2.40 (m, 4 H), 2.51 (t, J = 6.51 Hz, 1 H), 3.22 - 3.54 (m, 3 H),
13
3.58 (br. s., 1 H), 4.76 (dd, J = 7.99, 2.08 Hz, 0.8 H), 4.84 - 4.89 (m, 0.2 H). C NMR (101
MHz, CDCl3)  12.1, 12.2, 20.4, 20.5, 22.7, 25.0, 27.2, 29.8, 32.2, 36.7, 38.3, 38.5, 45.2, 45.4,
46.1, 46.3, 46.4, 46.7, 48.5, 50.8, 51.7, 67.1, 67.3, 118.3, 170.6. IR(KBr, cm-1): 1407.9, 1662.4,
+
2239.1. HRMS for C17H27ON3 calcd 289.2149, found 289.2145 [M]+.
4.1.17.3. (2S)-1-(2-{[(1S,2S,4R)-4,7,7-trimethyl-3-oxobicyclo[2.2.1]heptan-2-
yl]amino}acetyl)pyrrolidine-2-carbonitrile (18c)
31
1
Light yellow oil, 41% yield. [α]D -74 (c 0.140, CHCl3). H NMR (400 MHz, CDCl3) δ
0.87 (d, J = 10.34 Hz, 6 H), 0.97 - 1.01 (m, 3 H), 1.32 - 1.40 (m, 1 H), 1.67 (d, J = 8.73 Hz, 2 H),
1.82 - 1.90 (m, 1 H), 2.31 (br. s., 6 H), 3.26 - 3.33 (m, 1 H), 3.29 (d, J = 4.57 Hz, 1 H), 3.37 -
3.75 (m, 4 H), 3.38 - 3.74 (m, 4 H), 4.75 (dd, J = 7.99, 2.08 Hz, 0.85 H), 4.83 (dd, J = 7.72, 1.81
13
Hz, 0.15 H). C NMR (101 MHz, CDCl3)  9.3, 19.0, 19.2, 19.8, 22.7, 25.0, 29.8, 31.7, 32.3,
44.1, 45.4, 46.1, 46.3, 47.4, 50.3, 51.3, 58.5, 64.1, 64.9, 118.2, 170.0, 219.0. IR(KBr, cm-1):
+
1411.7, 1664.3, 1741.5, 2242.9. HRMS for C17H25O2N3 calcd 303.1941, found 303.1947 [M]+.
4.1.17.4. (2S)-1-(2-{[(1S,2R,3S,4R)-3-hydroxy-4,7,7-trimethylbicyclo[2.2.1]heptan-2-
yl]amino}acetyl)pyrrolidine-2-carbonitrile (18d)
Light yellow oil, 35 % yield. [α]D29 -38 (c 0.178, CHCl3). 1H NMR (300 MHz, CDCl3) 
0.69 - 1.09 (m, 11 H), 1.37 - 1.52 (m, 1 H), 1.61 - 1.83 (m, 2 H), 2.05 - 2.37 (m, 4 H), 2.59 - 2.68
15

(m, 1 H), 3.27 - 3.60 (m, 5 H), 4.56 (dd, J = 7.32, 1.46 Hz, 0.1 H), 4.77 (d, J = 6.46 Hz, 0.9 H).
13C NMR (101 MHz, CDCl3) d 11.4, 21.3, 21.9, 25.1, 27.1, 29.8, 32.9, 45.4, 46.5, 49.0, 52.0,
+
52.7, 67.4, 79.0, 118.2, 170.1. IR(KBr, cm-1): 1409.8, 1660.5, 2241.0. HRMS for C17H27O2N3
calcd 305.2098, found 287.1995 [M-H2O]+.
4.1.17.5. (2S)-1-{2-[(1R,9R)-6-oxo-7,11-diazatricyclo[7.3.1.02,7]trideca-2,4-dien-11-
yl]acetyl}pyrrolidine-2-carbonitrile (18e)
White solid, 66 % yield. M.p. 209.5-211.20C. [α]D26 -268 (c 0.138, CHCl3). 1H NMR (400
MHz, CDCl3)  1.71 - 2.19 (m, 6 H), 2.35 - 2.67 (m, 3 H), 2.74 (dt, J = 10.75, 1.61 Hz, 0.5 H),
2.78 - 2.94 (m, 1.5 H), 2.92 - 3.10 (m, 2.5 H), 3.15 (d, J = 13.16 Hz, 0.5 H), 3.25 - 3.41 (m, 1.5
H), 3.54 (ddd, J = 11.65, 7.49, 4.23 Hz, 0.5 H), 3.74 - 3.95 (m, 1.5 H), 4.06 (d, J = 15.58 Hz, 0.5
H), 4.46 - 4.55 (m, 0.5 H), 4.66 (dd, J = 7.52, 2.69 Hz, 0.5 H), 5.93 (dd, J = 6.85, 1.07 Hz, 0.5
H), 6.09 (dd, J = 6.85, 1.07 Hz, 0.5 H), 6.41 (ddd, J = 13.80, 9.03, 1.34 Hz, 1 H), 7.16 - 7.29 (m,
0.5 H), 7.36 (dd, J = 9.07, 6.92 Hz, 0.5 H). 13C NMR (75 MHz, CDCl3)  22.6, 24.9, 25.0, 25.1,
27.3, 27.7, 29.7, 31.7, 35.0, 35.3, 45.1, 45.6, 46.2, 46.4, 50.0, 59.0, 59.9, 60.0, 61.7, 61.8, 62.4,
104.3, 105.0, 116.4, 116.6, 118.1, 118.2, 138.5, 139.2, 150.7, 151.2, 163.1, 163.2, 167.6, 168.2.
+
IR(KBr, cm-1): 1425.2, 1546.7, 1567.9, 1645.1, 2233.3. HRMS for C18H22O2N4 calcd 326.1737,
found 326.1735 [M]+.
4.1.17.6. (2S)-1-{2-[(1R,9R)-chloro-6-oxo-7,11-diazatricyclo[7.3.1.02,7]trideca-2,4-dien-11-
yl]acetyl}pyrrolidine-2-carbonitrile (18f)
White solid, 65 % yield. M.p. 171.60C. [α]D30 -144 (c 0.214, CHCl3). 1H NMR (400 MHz,
CDCl3)  1.88-2.21 (m, 5 H), 2.43 - 2.70 (m, 3 H), 2.80 - 3.22 (m, 4 H), 3.28 - 3.61 (m, 3 H),
3.79 - 4.09 (m, 2 H), 4.58 (d, J = 9.54 Hz, 0.65 H), 4.64 (d, J = 10.07 Hz, 0.35 H), 6.40 - 6.45
13
(m, 1 H), 7.29 (d, J = 9.54 Hz, 0.65 H), 7.41 (d, J = 9.54 Hz, 0.35 H). C NMR (75 MHz,
CDCl3)  22.6, 25.0, 25.0, 25.1, 27.0, 27.4, 29.7, 31.8, 32.2, 32.4, 45.2, 45.6, 46.3, 46.4, 51.0,
51.0, 56.4, 58.0, 58.8, 59.3, 61.5, 62.4, 109.6, 110.5, 117.2, 117.3, 117.9, 118.1, 139.8, 140.5,
146.7, 147.2, 161.8, 161.8, 167.6, 168.3.
IR(KBr, cm-1): 1425.2, 1529.3, 1567.9,
1652.8, 1745.3, 2239.1. HRMS for
35
C18H21O2N4 Cl + calcd 360.1348, found 360.1349 [M]+.
4.1.17.7. (2S)-1-{2-[(1R,9R)-3,5-dibromo-6-oxo-7,11-diazatricyclo[7.3.1.02,7]trideca-2,4-
dien-11-yl]acetyl}pyrrolidine-2-carbonitrile (18g)
White solid, 75 % yield. M.p. 204.60C. [α]D30 -119 (c 0.178, CHCl3). 1H NMR (400 MHz,
CDCl3)  1.84-2.24 (m, 6 H), 2.43 - 2.73 (m, 3 H), 2.81 - 3.20 (m, 4 H), 3.27 - 3.61 (m, 2 H),
16

13
3.85-4.13 (m, 2 H), 4.52 (m, 0.3 H), 4.60 (m, 0.7 H), 7.84 (s, 0.7 H), 7.95 (s, 0.3 H). C NMR
(75 MHz, CDCl3)  22.7, 25.0, 25.1, 25.1, 27.2, 27.5, 29.8, 31.7, 34.6, 34.8, 45.3, 45.6, 46.3,
46.4, 52.6, 56.3, 57.9, 58.7, 59.1, 61.2, 62.3, 96.7, 97.5, 112.5, 112.7, 117.9, 118.1, 143.4, 144.0,
147.5, 148.0, 158.4, 167.5, 168.3. IR(KBr, cm-1): 1429.1, 1519.7, 1569.8, 1650.8, 2233,3.
79
+
HRMS for C18H20O2N4 Br2 calcd 481.9948, found 481.9945 [M]+.
4.1.17.8. (2S)-1-{2-[(1R,9R)-3,5-dichloro-6-oxo-7,11-diazatricyclo[7.3.1.02,7]trideca-2,4-dien-
11-yl]acetyl}pyrrolidine-2-carbonitrile (18h)
30
1
White solid, 51 % yield. M.p. 192.90C. [α]D -172 (c 0.138, CHCl3). H NMR (400
MHz, CDCl3)  1.81 - 2.27 (m, 6 H), 2.45 - 2.98 (m, 5 H), 3.15 (m., 2 H), 3.29 - 3.67 (m, 2 H),
3.89 - 4.18 (m, 2 H), 4.53 - 4.57 (m, 0.35 H), 4.57 - 4.62 (m, 0.65 H), 7.55 (s, 0.65 H), 7.66 (s,
0.35 H). 13C NMR (126 MHz, CDCl3)  22.7, 24.9, 24.9, 27.0, 27.3, 29.7, 31.6, 32.1, 32.4, 45.3,
45.6, 46.3, 46.4, 52.1, 56.3, 57.9, 58.7, 59.0, 61.0, 62.2, 108.4, 109.3, 117.9, 118.1, 122.5, 122.6,
+
137.5, 138.1, 145.6, 146.1, 158.1, 158.1, 167.5, 168.3. HRMS for C18H20O2N4Cl2 calcd
394.0958, found 394.0962 [M]+.
4.1.17.9. (2S)-1-{2-[(1R,9R)-5-nitro-6-oxo-7,11-diazatricyclo[7.3.1.02,7]trideca-2,4-dien-11-
yl]acetyl}pyrrolidine-2-carbonitrile (18i)
30
1
Orange solid, 74 % yield. M.p. 204.60C. [α]D -178 (c 0.138, CHCl3). H NMR (400
MHz, CDCl3)  1.83 - 2.27 (m, 6 H), 2.50 - 2.65 (m, 1.3 H), 2.72 - 3.03 (m, 3.7 H), 3.03 - 3.26
(m, 3.4 H), 3.32 - 3.46 (m, 1.3 H), 3.51 - 3.62 (m, 0.3 H), 3.87 - 4.23 (m, 2 H), 4.52 (d, J = 10.48
Hz, 0.3 H), 4.55 - 4.60 (m, 0.7 H), 6.12 (d, J = 8.06 Hz, 0.7 H), 6.23 (d, J = 8.06 Hz, 0.3 H), 8.29
13
(d, J = 8.06 Hz, 0.7 H), 8.34 (d, J = 8.06 Hz, 0.3 H). C NMR (75 MHz, CDCl3) δ 22.7, 24.6,
25.0, 27.1, 27.4, 29.7, 31.7, 36.2, 36.3, 45.5, 45.7, 46.3, 51.3, 58.9, 59.1, 59.2, 60.3, 60.6, 61.8,
102.6, 103.1, 117.9, 118.1, 134.8, 135.1, 137.5, 137.7, 154.8, 154.9, 159.4, 160.1, 167.4, 168.1.
IR(KBr, cm-1): 1297.9, 1315.3, 1427.1, 1471.5, 1500.4, 1556.3, 1656.6, 2239. HRMS for
C18H21O4N5 + calcd 371.1588, found 371.1590 [M]+.
4.2. In vitro DPP-IV inhibition assay
The enzyme assay was performed using «DPP (IV) Inhibitor Screening Assay Kit» (KA
1311; Abnova, USA). The activity of test compounds was assayed using human recombinant
DPP IV enzyme, fluorogenic DPP substrate (H-Gly-Pro-AMC) followed by the manufacturer's
guidelines. Solutions of test compounds at varying concentrations (ranging from 1 nM to 100
µM) were prepared in 1% dimethyl sulfoxide (DMSO) and then diluted into assay buffer. 10 µl
of each compound’s solution was added in each reaction well. The final concentration of DMSO
17

in the assay well was 1% which was found to have no effect on DPP-IV enzyme activity.
Sitagliptin (provided by the assay kit) served as positive control inhibitor. The plate was
incubated at 37°C for 10 min to allow enzyme-inhibitor reaction and read continuously at
excitation wavelength of 360 nm and an emission wavelength of 460 nm with a microplate
reader (EnSpire 2300, Perkin Elmer, USA). Each sample was assayed in triplicate in two
independent experiments and the percentage of inhibition was calculated as the following
formula: % Inhibition = ((Initial activity- Inhibitor) / Initial activity) × 100.
IC50 values were determined by non-linear regression fitted by GraphPad Prizm V.5
(GraphPad Software Inc., San Diego, CA).
4.3. Molecular docking
Three-dimensional crystal structure of DPP-IV protein retrieved from RCBS PDB
database(PDB: 3W2T25) were used to model receptor by mean Schrödinger Suite Release 2018-1
software (Small-Molecule Drug Discovery Suite 2018-1, Schrödinger, LLC, New York, NY,
2018.). Geometrical parameters of protein were subjected to restrain minimization using the
OPLS3 force field. Hydrogen atoms have been added to amino acid residues and optimized at
pH 7.2±2.0. All molecules water was accounted. The 18a-i compounds were integrated into
common data base. All ionized states and molecular conformations at the pH 7.2±2.0 were
calculated by default OPLS3 methods.
We considered protonation of the nitrogen atom of 18a-i compounds in the presence of
water. The acidity constant (pKa) is directly related to the free energy of the deprotonation
reaction26:
Ga
2.303RT
pKa 
(1)
Here Ga is the free energy of the following reaction BH+  B + H+ in aqueous solution,
R – ideal gas constant and T – absolute temperature. BH+ and B correspond to protonated and
deprotonated forms of the molecule respectively. Geometries optimization of the 18a and 18i are
carried out with DFT method B3LYP with 6-31G* basis sets. Experimental value of the free
energy of solvation of the proton was used (G(H+) = -265.9 kcal/mol (J.Phys.Chem.A.
1998.102.7787)).
The docking was performed under the following conditions: a flexible ligand and protein,
standard prediction accuracy (Covalent Docking Protocol). The grid-box size of 15Å with the
native ligand being located at the center, we modeled reaction of the nucleophilic addition
hydroxyl group of the SER630 to a triple bond of the ligand. Free binging energy (GMM GBSA
)
of ligand-protein complex was estimated using the variable-dielectric generalized Born model,
18

which incorporates residue-dependent effects. The solvent is water. Flexible resides distance is
equal to 5 Å around the ligand.
4.4. In vivo studies
Oral glucose tolerance test.
Male albino mice (CD-1 line) weighing 25–35 g were used in the experiment. Animals
were obtained from the animal facility of the Institute of Cytology and Genetics SB RAS and
kept under standard conditions with free access to food and water. All experiments were
performed in accordance with the ‘‘European Convention for the Protection of Vertebrate
Animals Used for Experimental and other Scientific Purposes’’, 1986. After quarantine animals
were randomized by weight and divided into groups of 5 mice in each. All tested compounds
were mixed with few drops of Tween 80 or DMSO before dissolution in distilled water and then
were orally administered to 12 h fasting animals 0.5 h prior to oral glucose load (2.5 g/kg) at a
dose of 10 mg/kg. Animals in control group received just the vehicle (water with Tween 80 or
DMSO). Blood glucose values were measured with ONE TOUCH Select blood glucose meter
(LIFESCAN Inc., USA) at 0, 30, 60, 90 min after the glucose load. Vildagliptin (Novartis) was
used as the positive control at a dose of 10 mg/kg. The statistical analysis was performed using
Mann–Whitney U test. P <0.05 was considered to be statistically significance.
Acknowledgements
Authors would like to acknowledge the Multi-Access Chemical Service Center SB RAS
for spectral and analytical measurements.
Supplementary data
Supplementary data associated with this article can be found, in the online version.
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