Synthesis and Nootropic Activity of new 3-Amino-12-N-Methylcytisine Derivatives

Article abstract of DOI:10.1007/s10600-015-1446-x

Reductive alkylation of 3-amino-12-N-methylcytisine by aromatic aldehydes synthesized a series of secondary amines. The nootropic activity of the synthesized compounds was studied in vivo (mnestic and antihypoxic properties) and in vitro (antiradical prop

Full text of DOI:10.1007/s10600-015-1446-x

DOI 10.1007/s10600-015-1446-x
Chemistry of Natural Compounds, Vol. 51, No. 5, September, 2015
SYNTHESIS AND NOOTROPIC ACTIVITY OF NEW
3-AMINO-12-N-METHYLCYTISINE DERIVATIVES
1*
1
1
I. P. Tsypysheva, A. V. Kovalꢀskaya, A. N. Lobov,
1
1,3
2
N. S. Makara, P. R. Petrova, E. I. Farafontova,
2
2
1,4
L. F. Zainullina, Yu. V. Vakhitova, and F. S. Zarudii
Reductive alkylation of 3-amino-12-N-methylcytisine by aromatic aldehydes synthesized a series of secondary
amines. The nootropic activity of the synthesized compounds was studied in vivo (mnestic and antihypoxic
properties) and in vitro (antiradical properties and ability to affect transcription factor HIF-1 DNA-binding
activity). The cytotoxicity of the synthesized compounds was assessed. The lead compound was identified.
Keywords: (–)-cytisine, nootropic activity, mnestic effect, antihypoxic properties, antiradical properties, AOS, TF HIF-1.
Nootropic agents are a significant component in the therapy of neurodegenerative diseases, consequences of injuries
and concussions, and brain dysfunctions caused by alcoholism and acute neural infections owing their broad spectrum of
activity and proven positive clinical outcomes. Nootropic agents for human health are applied mainly to the prevention of
stress and maintenance of functionality in critical situations and with heightened physical and intellectual exertion [1–4].
The mechanism of action of many nootropic agents is due to nootropic and mnestic properties themselves in addition
to antioxidant and antihypoxic activity [5]. Therefore, both in vivo screening tests [conditioned passive-avoidance response
(CPAR), normobaric hypercapnic hypoxia] and the potential protective activity of tested compounds in in vitro cell models for
antiradical properties and the ability to influence the hypoxia-induced DNA-binding activity of transcription factor HIF-1
should be assessed [6, 7].
We demonstrated earlier that several carboxamides obtained from 3-amino-12-N-methylcytisine (1) and
the corresponding isocyanates had nootropic properties that were comparable or superior to those of the reference drug piracetam
[8]. A library of secondary amines 2–11 was prepared via one-pot syntheses using reductive alkylation of 1 with hetero- and
aromatic aldehydes (the azomethines formed in the first step were reduced by NaBH without isolation as before [9]) in order
4
to determine nootropic activity as a function of the 2-pyridone substituent. Compounds 2–11 were obtained in 56–97% yields.
1
13 15
Their structures were established by NMR ( H, C, N) spectroscopy.
11
14
5
7
N
N
a
13
16
15
N
N
9
3
H N
2
R
N
1
10
H
O
O
1
2 - 11
OH
17
2
R =
HO
19
21
MeO
HO
3
4
5
6
N
N
O
S
S
7
8
9
11
10
a. RCHO, benzene, 80°C; then NaBH , ÌåÎÍ, 0ꢁ20°C
4
1) Ufa Institute of Chemistry, Russian Academy of Sciences, 450054, Ufa, Prosp. Oktyabrya, 71, e-mail:
tsipisheva@anrb.ru; 2) Institute of Biochemistry and Genetics, Ufa Scientific Center, Russian Academy of Sciences, Ufa;
3) Bashkir State University, Ufa; 4) Bashkir State Medical University, Ufa. Translated from Khimiya Prirodnykh Soedinenii,
No. 5, September–October, 2015, pp. 780–785. Original article submitted February 27, 2015.
©
910
0009-3130/15/5105-0910 2015 Springer Science+Business Media New York

TABLE 1. Nootropic and Antihypoxic Activity of 2–11*
Compounda
Mnestic activity, %
Antihypoxic activity, %
Compounda
Mnestic activity, %
Antihypoxic activity, %
Piracetamb
71.7
72.3
71.8
89.9
99.7
38.5
11.2
–
9.7
–
–
–
40.3
87.4
35.5
50.1
13.3
40.1
9.0
11.6
–
7
8
2
3
4
5
6
9
10
11
10.2
______
*Piracetam: LD , 2000 mg/kg; ED , 400; LD /ED , 26.5 mg/kg.
50 ꢂmol/kg dose; 400 mg/kg dose.
50
50
50
50
a
b
TABLE 2. Effect of 2–11 on AOS and Transcription Factor HIF-1 Activity in vitro
AOSa
Compound
HIF-1, EC50b, mM
1, ꢂM
10, ꢂM
100, ꢂM
0.23
2
3
121.6 ꢃ 9.9
89.3 ꢃ 5.4
114 ꢃ 8.7
100.5 ꢃ 6.1
86.5 ꢃ 8.4
165.3 ꢃ 6.9
117.4 ꢃ 9.9
93.7 ꢃ 7.3
119.5 ꢃ 5.4
104.15 ꢃ 3.7
222.4 ꢃ 16.2*
105.4 ꢃ 7.1
69.4 ꢃ 5.5
–
0.07
–
–
–
–
0.15
–
4
91.9 ꢃ 10.7
101.1 ꢃ 7.7
103.84 ꢃ 6.1
102.3 ꢃ 11.2
103.9 ꢃ 9.6
85.2 ꢃ 1.1
5
95.3 ꢃ 6.0
6
101.45 ꢃ 2.8
131.2 ꢃ 12.4
78.9 ꢃ 9.4
60.9 ꢃ 12.2
95.2 ꢃ 9.8
7
8
9
10
11
98.4 ꢃ 5.8
106.25 ꢃ 5.6
151.9 ꢃ 8.9*
108.3 ꢃ 1.5
–
102.95 ꢃ 7.3
______
a
b
Experimental groups were compared using the Student t-criterion for independent sets (n = 3, *p < 0.05); arithmetic aver-
ages ꢃ standard deviation of the mean are given. The EC values (compound concentration increasing luciferase activity by
50%) were calculated using nonlinear regression of the logarithm of the compound concentrations and normalized percent
luciferase activation (GraphPad Prism v.5.0, GraphPad Software Inc., USA).
50
The specific nootropic activity of 2–11 was studied previously [10–12] using a basic CPAR model that enabled the
mnestic effect to be assessed. Antihypoxic activity was determined using a normobaric hypercapnic hypoxia model [10].
Table 1 presents the results.
According to the results, 6, 7, and 9–11 without oxygen-containing substituents in the aromatic amine exhibited weak
mnestic activity. However, the activities of secondary amines 2 and 3, derivatives of p-methoxy- and benzaldehyde, were
comparable to that of the reference drug piracetam at 72.3 and 71.8%. The lead compounds in this group were secondary
amines 8, 4, and 5. Thus, introducing a hydroxyl into the ortho-position of the benzyl substituent on the 3-amine increased this
parameter to 89.9%; in the para-position, to 99.7%. The lethal and effective median doses for lead compound 5 were
LD (1800 mg/kg) and ED (9.5 mg/kg) with therapeutic index LD /ED 191.5. This exceeded significantly that for the
50
50
50
50
reference drug piracetam [8].
Parallel in vivo screening of the antihypoxic activity showed that only amines 7 and 9, reaction products of
cinnamaldehyde and furfurol with 1, had pronounced antihypoxic properties in the series of secondary amines 2–11. Thus,
the lifespan of laboratory animals under hypoxia conditions was increased relative to the control in the first case by 40%; in
the second, by 11.6%, which was comparable to the antihypoxic activity of piracetam (11.2%).
The potential cytotoxicity of synthesized 2–11 was assessed preliminarily in vitro using standard MTT-assay methods
[13]. As it turned out, none of these compounds exhibited cytotoxicity against the conditionally normal cell line HEK293
(because IC > 1 mM, data not presented).
50
The antiradical properties of 2–11 were studied using HEK293 cells under oxidative stress induced by injection of
PMA (4-phorbol-12-myristate-13-acetate). Table 2 shows that the majority of the tested 12-N-methylcytisine derivatives
(3–6, 8, and 11) had no effect on the intracellular active-oxygen-species (AOS) level in the HEK293 cell cytoplasm. However,
secondary amines 2 and 7 and thiophene derivative 10 produced a dose-dependent increase of the AOS level, which argued in
911

favor of a possible pro-oxidant effect for these compounds. Compound 9 with a furan moiety probably had moderate antioxidant
properties and was capable of reducing AOS accumulation stimulated by PMA.
Next, the in vitro effects of 2–11 were compared using the DNA-binding activity of TF HIF-1 and luciferase reporter
constructs. The results (Table 2) showed that only a few of the experimental compounds (4, 9, and 2) exhibited moderate
stimulation of HIF-1 (EC values of 0.07, 0.15, and 0.23 mM, respectively).
50
Acomparison of results from the in vivo tests (CPAR, normobaric hypercapnic hypoxia model) and in vitro experiments
(effect onAOS level and HIF-1 basal activity) showed that 9 exhibited a pronounced antihypoxic effect analogous to piracetam
and also increased the DNA-binding activity of TF HIF-1. Furthermore, it reduced the accumulation ofAOS under conditions
modeling oxidative stress in vitro by demonstrating antiradical properties. It could be assumed that the antihypoxic properties
of 9 were due to an increased resistance to hypoxia because of the induction of HIF-1, a dependent signal pathway, and
the antioxidant activity. The positive effects of 2 and 4 on mnestic processes (assessed in the CPAR test) may have been due
at least partially to activation of TF HIF-1, which requires further research.
Thus, N-benzyl and –hetarylmethylene derivatives of 12-N-methylcytisine-3-amine were synthesized via reductive
alkylation of 3-amino-12-N-methylcytisine by aromatic and heteroaromatic aldehydes. Their mnestic, antihypoxic, and
antiradical properties in addition to their effects on TF HIF-1 DNA-binding activity were studied. N-(4-Hydroxybenzyl)-12-
N-methylcytisine-3-amine was identified as the lead compound. Its lethal and effective median doses and therapeutic index
were determined and exceeded those of the reference drug piracetam.
EXPERIMENTAL
General. Commercially available (–)-cytisine (CAS 485-35-8) and NaBH (CAS 16940-66-2) were used as starting
4
compounds. N-Methylcytisine was prepared according to the literature [14]. The course of reactions was monitored by TLC
®
on ALUGRAM plates. Column chromatography (CC) was performed over standard silica gel 60 (0.05–0.1 mm) (Macherey-
Nagel, Germany). Melting points were determined on a Boetius apparatus. Optical rotation angles were measured on a
13
PerkinElmer 341 LC polarimeter. PMR and C NMR spectra were recorded on equipment at the Khimya CCU, UfIC, RAS
1
13
(Bruker Avance III pulsed spectrometer at operating frequency 500.13 MHz for H and 125.47 MHz for C relative to TMS
internal standard [15]).
Preparation of Substituted N-Benzyl-(12-N-methylcytisin)-3-amines (2). A mixture of 1 (260 mg, 0.9 mmol) and
benzaldehyde (116 mg, 1.1 mmol) in benzene (10 mL) was refluxed. When the reaction was finished (TLC monitoring),
the mixture was evaporated. The residue was dissolved in MeOH (10 mL), cooled to 0°C, treated with NaBH (450 mg,
4
10.0 mmol), stirred on a magnetic stirrer for 1 h, and concentrated. The residue was dissolved in H O (2 mL) and extracted
2
with CHCl (5 ꢄ 10 mL). The extracts were combined, dried over Na SO , and evaporated. The residue was chromatographed
3
2
4
20
over SiO (EtOAc eluent) to afford N-benzyl-(12-N-methylcytisine)-3-amine (2) (290 mg, 80% yield), [ꢅ] –28.0° (c 1.1,
2
D
CHCl ).
3
1
2
3
3
4
Í NMR spectrum (CDCl , ꢆ, ppm, J/Hz): 1.66 (1H, dtd, J = 12.7, J
= 3.2, J
= 3.2, J
= 1.3,
8anti-10endo
3
8anti-7
4
8anti-9
2
3
3
4
H
-8), 1.78 (1H, dtt, J = 12.7, J
= 3.4, J
= 3.4, J
= 1.7, J
= 1.7, H -8), 2.08 (3H, s, H-14),
anti
8syn-7
8syn-9
8syn-11endo
8syn-13endo
syn
2
3
4
2
3
2.17 (1H, ddd, J = 11.3, J
(1H, m, H-9), 2.74 (1H, ddt, J = 10.5, J
= 2.5, J
= 1.0, H -11), 2.16 (1H, dd, J = 10.5, J
= 2.4, H -13), 2.35
11exo-9
2
11exo-10exo
exo
13exo-7 exo
3
4
4
= 3.2, J
= 1.7, J
= 1.7, H
-13), 2.80 (1H, m, H-7),
-11), 3.94 (1H, ddd, J = 15.1,
13endo-7
13endo-11endo
13endo-8syn
= 1.7, H
endo
2
3
4
4
2
2.85 (1H, ddt, J = 11.3, J
= 3.3, J
= 1.7, J
11endo-9
11endo-13endo
2
11endo-8syn
endo
3
4
3
4
J
= 7.00, J
= 1.0, H -10), 4.12 (1H, dt, J = 15.1, J
= 1.0, J
= 1.0, H
-10), 4.28 (2H, s,
10exo-9
10exo-11exo
exo
10endo-9
3
10endo-8ànti
endo
3
3
H-16), 5.36 (1H, s, N-H), 5.85 (1H, d, J = 7.4, H-5), 6.13 (1H, d, J = 7.4, H-4), 7.22 (2Í, t, J
= 7.7,
5-4
4-5
19(21)-20
3
3
3
J
= 7.7, H-19, 21), 7.29 (1H, d, J
= 7.7, H-20), 7.33 (2Í, d, J
= 7.7, H-18, 22).
19(21)-18(22)
20-19(21)
18(22)-19(21)
13
C NMR spectrum (CDCl , ꢆ, ppm): 26.1 (C-8), 27.9 (C-9), 34.6 (C-7), 46.3 (C-14), 47.6 (C-16), 50.2 (C-10), 62.2 (C-11), 63.2
3
(C-13), 104.7 (C-5), 107.5 (C-4), 126.9 (C-20), 127.2 (C-18, 22), 128.4 (C-19, 21), 136.0 (C-3), 136.0 (C-6); 139.0 (C-17),
15
157.9 (C-2). N NMR spectrum (CDCl , ꢆ, ppm): 28.9 (N12), 60.3 (N15), 167.6 (N1).
3
N-(4-Methoxybenzyl)-12-N-methylcytisin-3-amine (3) was prepared from 1 (260 mg), yield 300 mg (75%),
20
1
2
3
3
[ꢅ] –13.0° (c 2.5, CHCl ). Í NMR spectrum (CDCl , ꢆ, ppm, J/Hz): 1.67 (1H, dt, J = 12.7, J = 3.2, J = 3.2, H -8), 1.79
D
3
3
3
anti
2
2
3
2
3
(1H, dt, J = 12.7, J = 3.4, J = 3.4, H -8), 2.08 (3H, s, H-14), 2.16 (1H, dd, J = 10.5, J = 2.4, H -13), 2.17 (1H, dd, J = 10.8,
syn
exo
3
3
2
3
2
J = 2.5, H -11), 2.36 (1H, m, H-9), 2.74 (1H, dd, J = 10.5, J = 3.2, H
-13), 2.82 (1H, m, H-7), 2.85 (1H, dd, J = 10.8,
exo
endo
2
3
2
J = 3.3, H
-11), 3.77 (3H, s, CH -O), 3.92 (1H, dd, J = 15.1, J = 7.0, H -10), 4.11 (1H, d, J = 15.1, H
-10), 4.21
endo
3
exo
endo
912

3
3
2
(2H, s, H-16), 5.22 (1H, s, N-H), 5.86 (1H, d, J = 7.4, H-5), 6.16 (1H, d, J = 7.4, H-4), 6.85 (2Í, d, J = 8.7, H-19, 21), 7.26
2
13
(2Í, d, J = 8.7, H-18, 22). C NMR spectrum (CDCl , ꢆ, ppm): 26.1 (C-8), 27.9 (C-9), 34.6 (C-7), 46.3 (C-14), 47.1 (C-16),
3
50.1 (C-10), 55.2 (C-24), 62.3 (C-11), 63.2 (C-13), 104.7 (C-5), 107.4 (C-4), 113.9 (C-19, 21), 128.5 (C-18, 22), 131.0 (C-17),
15
136.0 (C-3), 136.0 (C-6), 158.0 (C-2), 158.7 (C-20). N NMR spectrum (CDCl , ꢆ, ppm): 28.6 (N12), 62.3 (N15), 167.7
3
(N1).
N-(2-Hydroxybenzyl)-12-N-methylcytisin-3-amine (4) was prepared from 1 (260 mg), yield 308 mg (80%),
20
1
2
3
3
[ꢅ] –49.0° (c 3.1, CHCl ). Í NMR spectrum (CDCl , ꢆ, ppm, J/Hz): 1.68 (1H, dt, J = 12.7, J = 3.2, J = 3.2, H -8), 1.79
D
3
3
anti
2
3
3
2
3
(1H, dt, J = 12.7, J = 3.4, J = 3.4, H -8), 2.10 (3H, s, H-14), 2.18 (1H, dd, J = 11.7, J = 2.5, H -11), 2.19 (1H, dd,
J = 10.7, J = 2.1, H -13), 2.37 (1H, m, H-9), 2.77 (1H, dd, J = 10.7, J = 3.1, H
-11), 3.93 (1H, dd, J = 15.1, J = 7.1, H -10), 4.12 (1H, d, J = 15.1, H
5.24 (1H, br.s, N-H), 5.90 (1H, d, J = 7.6, H-5), 6.41 (1H, d, J = 7.6, H-4), 6.80 (1H, td, J = 7.6, J = 7.6, J = 1.5, H-21), 6.87
syn
exo
2
2
3
2
3
-13), 2.85 (1H, m, H-7), 2.87 (1H, dd,
exo
endo
2
3
2
3
J = 11.7, J = 3.3, H
-10), 4.29 (2H, s, H-16),
endo
exo
endo
3
3
3
3
4
3
4
3
3
3
13
(dd, J = 8.2, J = 1.5, H-19), 7.11 (1H, dd, J = 8.2, J = 7.6, H-20), 7.14 (1H, dd, J = 7.6, H-22). C NMR spectrum (CDCl ,
3
ꢆ, ppm): 25.9 (C-8), 27.9 (C-9), 34.6 (C-7), 46.3 (C-14), 50.4 (C-16), 50.4 (C-10), 62.2 (C-11), 63.0 (C-13), 105.3 (C-5), 111.5
(C-4), 116.1 (C-19), 119.6 (C-21), 123.9 (C-18), 128.6 (C-22), 128.6 (C-20), 136.0 (C-3), 137.9 (C-6), 156.3 (C-17), 158.4
15
(C-2). N NMR spectrum (CDCl , ꢆ, ppm): 29.0 (N12), 57.8 (N15), 167.9 (N1).
3
N-(4-Hydroxybenzyl)-12-N-methylcytisin-3-amine (5) was prepared from 1 (260 mg), yield 310 mg (80%),
20
1
2
3
3
[ꢅ] –48.0° (c 1.71, CHCl ). Í NMR spectrum (CDCl , ꢆ, ppm, J/Hz): 1.71 (1H, dt, J = 12.7, J = 3.2, J = 3.2, H -8), 1.82
D
3
3
anti
2
3
3
2
3
(1H, dt, J = 12.7, J = 3.4, J = 3.4, H -8), 2.11 (3H, s, H-14), 2.20 (1H, dd, J = 11.1, J = 2.5, H -11), 2.21 (1H, dd,
J = 10.5, J = 2.4, H -13), 2.39 (1H, m, H-9), 2.81 (1H, dd, J = 10.5, J = 3.2, H
-11), 3.98 (1H, dd, J = 15.1, J = 7.0, H -10), 4.13 (2H, s, H-16), 4.18 (1H, d, J = 15.1, H
5.20 (1H, br.s, N-H), 6.01 (1H, d, J = 7.4, H-5), 6.29 (1H, d, J = 7.4, H-4), 6.81 (2H, d, J = 8.6, H-19, 21), 7.09 (2H, d,
syn
exo
2
2
3
2
3
-13), 2.89 (1H, m, H-7), 2.91 (1H, dd,
exo
endo
3
2
3
2
J = 11.1, J = 3.3, H
-10),
endo
exo
endo
3
3
3
3
13
J = 8.6, H-18, 22). C NMR spectrum (CDCl , ꢆ, ppm): 26.0 (C-8), 27.8 (C-9), 34.5 (C-7), 46.3 (C-14), 47.1 (C-16), 50.4
3
(C-10), 62.0 (C-11), 63.0 (C-13), 106.3 (C-5), 108.5 (C-4), 115.6 (C-19, 21), 128.7 (C18, 22), 129.2 (C-17), 135.7 (C-6),
15
136.1 (C-3), 156.3 (C-20), 157.9 (C-2). N NMR spectrum (CDCl , ꢆ, ppm): 29.7 (N12), 64.1 (N15), 169.5 (N1).
3
N-(4-Hydroxymethylbenzyl)-12-N-methylcytisin-3-amine (6) was prepared from 1 (260 mg), yield 225 mg (56%),
20
1
2
3
3
[ꢅ] –22.0° (c 2.49, CHCl ). Í NMR spectrum (CDCl , ꢆ, ppm, J/Hz): 1.67 (1H, dt, J = 12.7, J = 3.2, J = 3.2, H -8),
D
3
3
anti
2
3
3
2
3
1.78 (1H, dt, J = 12.7, J = 3.4, J = 3.4, H -8), 2.08 (3H, s, H-14), 2.16 (1H, dd, J = 10.7, J = 2.1, H -13), 2.18 (1H, dd,
J = 11.7, J = 2.5, H -11), 2.35 (1H, m, H-9), 2.74 (1H, dd, J = 10.7, J = 3.1, H
-11), 3.36 (1H, br.s, OH), 3.90 (1H, dd, J = 15.1, J = 7.00, H -10), 4.09 (1H, d, J = 15.1, H
4.23 (2H, m, H-16), 4.60 (2H, m, H-23), 5.20 (1H, s, N-H), 5.86 (1H, d, J = 7.4, H-5), 6.12 (1H, d, J = 7.4, H-4), 7.27 (2H,
m, H-18, 22), 7.28 (2H, m, H-19, 21). C NMR spectrum (CDCl , ꢆ, ppm): 26.0 (C-8), 27.9 (C-9), 34.6 (C-7), 46.3 (C-14),
47.3 (C-16), 50.2 (C-10), 62.2 (C-11), 63.2 (C-13), 64.7 (C-23), 105.1 (C-5), 107.8 (C-4), 127.2 (C-18, 22), 127.2 (C-19, 21),
136.0 (C-3), 136.0 (C-6), 138.0 (C-17), 140.3 (C-20), 158.0 (C-2). N NMR spectrum (CDCl , ꢆ, ppm): 28.9 (N12), 60.4
syn
exo
2
2
3
2
3
-13), 2.82 (1H, m, H-7), 2.85 (1H, dd,
exo
endo
3
2
3
2
J = 11.7, J = 3.3, H
-10),
endo
exo
endo
3
3
13
3
15
3
(N15), 168.1 (N1).
N-(3-Phenylprop-2-en-1-yl)-12-N-methylcytisin-3-amine (7) was prepared from 1 (260 mg), yield 278 mg (70%),
20
1
2
3
3
[ꢅ] –36.0° (c 0.78, CHCl ). Í NMR spectrum (CDCl , ꢆ, ppm, J/Hz): 1.68 (1H, dt, J = 12.7, J = 3.2, J = 3.2, H -8),
D
3
3
anti
2
3
3
2
3
1.80 (1H, dt, J = 12.7, J = 3.4, J = 3.4, H -8), 2.10 (3H, s, H-14), 2.18 (1H, dd, J = 10.7, J = 2.1, H -13), 2.18 (1H, dd,
J = 11.7, J = 2.5, H -11), 2.37 (1H, m, H-9), 2.77 (1H, dd, J = 10.7, J = 3.1, H
-11), 3.89 (2H, dd, J = 5.5, J = 1.8, H-16), 3.94 (1H, dd, J = 15.1, J = 7.00, H -10), 4.12 (1H, d,
-10), 5.09 (1H, s, N-H), 5.92 (1H, d, J = 7.4, H-5), 6.26 (1H, d, J = 7.4, H-4), 6.27 (1H, dt, J = 15.9, J = 5.6,
H-17), 6.59 (1H, dt, J = 15.9, J = 1.8, H-18), 7.20 (1H, d, J = 7.7, H-22), 7.29 (2H, t, J = 7.7, H-21, 23), 7.35 (2H, d,
J = 7.7, H-20, 24). C NMR spectrum (CDCl , ꢆ, ppm): 26.1 (C-8), 27.9 (C-9), 34.6 (C-7), 45.5 (C-16). 46.3 (C-14), 50.2
(C-10), 62.2 (C-11), 63.2 (C-13), 104.9 (C-5), 107.7 (C-4), 126.3 (C-20, 24), 126.7 (C-17), 127.3 (C-22), 128.5 (C-21, 23),
131.0 (C-18), 136.0 (C-3), 136.0 (C-6), 136.9 (C-19), 158.1 (C-2). N NMR spectrum (CDCl , ꢆ, ppm): 29.2 (N12), 59.2
syn
exo
2
2
2
3
2
3
-13), 2.84 (1H, m, H-7), 2.88 (1H, dd,
exo
endo
3
3
4
2
3
J = 11.7, J = 3.3, H
endo
exo
3
3
3
3
J = 15.1, H
endo
3
4
3
3
3
13
3
15
3
(N15), 167.8 (N1).
N-(Pyridin-3-ylmethyl)-12-N-methylcytisin-3-amine (8) was prepared from 1 (260 mg), yield 353 mg (96%),
20
1
2
3
3
[ꢅ] –35.5° (c 1.3, CHCl ). Í NMR spectrum (CDCl , ꢆ, ppm, J/Hz): 1.75 (1H, dt, J = 12.7, J = 3.3, J = 3.3, H -8), 1.80
D
3
3
anti
2
3
3
2
3
(1H, dt, J = 12.7, J = 3.4, J = 3.4, H -8), 2.10 (3H, s, H-14), 2.25 (1H, dd, J = 11.3, J = 2.5, H -11), 2.25 (1H, dd,
J = 10.8, J = 2.5, H -13), 2.40 (1H, m, H-9), 2.79 (1H, dd, J = 10.8, J = 3.1, H
-11), 2.93 (1H, m, H-7), 3.92 (1H, dd, J = 15.3, J = 6.5, H -10), 4.08 (1H, d, J = 15.3, H
6.06 (1H, d, J = 7.6, H-5), 6.28 (1H, d, J = 7.6, H-4), 7.36 (1H, dd, J = 7.8, J = 4.9, H-21), 7.80 (1H, dt, J = 7.8, J = 1.7,
syn
exo
2
3
2
3
2
3
-13). 2.92 (1H, dd, J = 11.3, J = 3.3,
exo
endo
2
2
3
H
-10), 4.40 (2H, s, H-16),
endo
exo
endo
3
3
3
3
3
4
4
3
4
4
13
J = 1.7, H-22), 8.40 (1H, dd, J = 4.9, J = 1.7, H-20), 8.53 (1H, d, J = 1.7, H-18). C NMR spectrum (MeOD, ꢆ, ppm): 26.6
913

(C-8), 29.1 (C-9), 35.5 (C-7), 45.2 (C-16), 46.6 (C-14), 51.4 (C-10), 63.0 (C-11), 64.1 (C-13), 107.3 (C-5), 110.5 (C-4), 125.1
15
(C-21), 136.6 (C-3), 137.1 (C-22), 137.1 (C-17), 137.7 (C-6), 148.5 (C-20), 149.1 (C-18), 159.1 (C-2). N NMR spectrum
(MeOD, ꢆ, ppm): 29.2 (N12), 56.6 (N15), 169.6 (N1), 300.1 (N19).
N-(2-Furylmethyl)-12-N-methylcytisin-3-amine (9) was prepared from 1 (260 mg), yield 308 mg (87%),
20
1
2
3
3
[ꢅ] –39.7° (c 3.18, CHCl ). Í NMR spectrum (CDCl , ꢆ, ppm, J/Hz): 1.66 (1H, dt, J = 12.7, J = 3.2, J = 3.2, H -8), 1.77
D
3
3
anti
2
3
3
2
3
(1H, dt, J = 12.7, J = 3.4, J = 3.4, H -8), 2.06 (3H, s, H-14), 2.14 (1H, dd, J = 13.2, J = 2.4, H -13), 2.15 (1H, dd,
J = 11.4, J = 2.5, H -11), 2.34 (1H, m, H-9), 2.73 (1H, dd, J = 13.2, J = 3.2, H
-11), 3.89 (1H, dd, J = 15.1, J = 7.00, H -10), 4.07 (1H, d, J = 15.1, H
5.17 (1H, s, N-H), 5.89 (1H, d, J = 7.4, H-5), 6.19 (1H, d, J = 3.1, H-18), 6.26 (1H, d, J = 7.4, H-4), 6.27 (1H, dd, J = 3.1,
J = 1.8, H-19), 7.31 (1H, d, J = 1.8, H-20). C NMR spectrum (CDCl , ꢆ, ppm): 26.0 (C-8), 27.9 (C-9), 34.6 (C-7), 40.8
(C-16), 46.3 (C-14), 50.15 (C-10), 62.2 (C-11), 63.2 (C-13), 104.6 (C-5), 106.8 (C-18), 107.7 (C-4), 110.2 (C-19), 135.6 (C-3),
136.5 (C-6), 141.7 (C-20), 152.5 (C-17), 158.0 (C-2). N NMR spectrum (CDCl , ꢆ, ppm): 29.0 (N12), 57.8 (N15), 167.9 (N1).
syn
exo
2
2
3
2
3
-13), 2.82 (1H, m, H-7), 2.83 (1H, dd,
exo
endo
2
3
2
3
J = 11.4, J = 3.3, H
-10), 4.24 (2H, s, H-16),
endo
exo
endo
3
3
3
3
3
3
13
3
15
3
N-(2-Thienylmethyl)-12-N-methylcytisin-3-amine (10) was prepared from 1 (260 mg), yield 360 mg (97%),
20
1
2
3
3
[ꢅ] –90.0° (c 1.09, CHCl ). Í NMR spectrum (CDCl , ꢆ, ppm, J/Hz): 1.67 (1H, dt, J = 12.7, J = 3.2, J = 3.2, H -8),
D
3
3
anti
2
3
3
2
3
1.79 (1Í, dd, J = 12.7, J = 3.4, J = 3.4, H -8), 2.08 (3H, s, H-14), 2.16 (1H, dd, J = 11.3, J = 2.5, H -11), 2.16 (1H, dd,
J = 10.3, J = 2.4, H -13), 2.35 (1H, m, H-9), 2.75 (1H, dd, J = 10.3, J = 3.2, H
-11), 3.90 (1H, dd, J = 15.1, J = 7.00, H -10), 4.10 (1H, d, J = 15.1, H
J = 5.5, H-16), 5.29 (1H, t, J = 5.5, N-H), 5.89 (1H, d, J = 7.4, H-5), 6.26 (1H, d, J = 7.4, H-4), 6.93 (1H, dd, J = 5.0,
J = 3.5, H-20), 6.98 (1H, d, J = 3.5, H-21), 7.17 (1H, d, J = 5.0, H-19). C NMR spectrum (CDCl , ꢆ, ppm): 26.0 (C-8),
27.9 (C-9), 34.6 (C-7), 42.9 (C-16), 46.3 (C-14), 50.1 (C-10), 62.2 (C-11), 63.1 (C-13), 104.6 (C-5), 108.0 (C-4), 124.3
(C-19), 124.7 (C-21), 126.7 (C-20), 135.6 (C-3), 136.6 (C-6), 142.8 (C-17), 157.9 (C-2). N NMR spectrum (CDCl , ꢆ,
syn
exo
2
2
3
3
3
2
3
-13), 2.83 (1H, m, H-7), 2.85 (1H, dd,
exo
endo
3
2
3
2
J = 11.3, J = 3.3, H
-10), 4.45 (1H, d,
endo
exo
endo
3
3
3
3
3
3
13
3
15
3
ppm): 28.6 (N12), 63.1 (N15), 167.5 (N1).
N-(1,3-Thiazol-2-ylmethyl)-12-N-methylcytisin-3-amine (11) was prepared from 1 (260 mg), yield 330 mg (88%),
20
1
2
3
3
[ꢅ] –17.5° (c 1.53, CHCl ). Í NMR spectrum (CDCl , ꢆ, ppm, J/Hz): 1.70 (1H, dt, J = 12.7, J = 3.2, J = 3.2, H -8),
D
3
3
anti
2
3
3
2
3
1.80 (1H, dd, J = 12.7, J = 3.4, J = 3.4, H -8), 2.10 (3H, s, H-14), 2.16 (1H, dd, J = 10.4, J = 2.2, H -13), 2.19 (1H, dd,
J = 11.3, J = 2.5, H -11), 2.39 (1H, m, H-9), 2.76 (1H, dd, J = 10.4, J = 3.5, H
syn
exo
2
2
3
3
2
3
-13), 2.84 (1H, m, H-7), 2.88 (1H, dd,
exo
endo
3
2
3
2
J = 11.3, J = 3.3, H
-11), 3.95 (1H, dd, J = 15.1, J = 7.00, H -10), 4.12 (1H, d, J = 15.1, H
-10), 4.64 (2H, d,
endo
exo
endo
3
3
3
3
J = 6.0, H-16), 5.64 (1H, t, J = 6.0, N-H), 5.87 (1H, d, J = 7.4, H-5), 6.24 (1H, d, J = 7.4, H-4), 7.25 (1H, d, J = 3.3, H-20),
3
13
7.72 (1H, d, J = 3.3, H-19). C NMR spectrum (CDCl , ꢆ, ppm): 26.0 (C-8), 27.9 (C-9), 34.6 (C-7), 46.1 (C-16), 46.3 (C-14),
50.2 (C-10), 62.2 (C-11), 63.1 (C-13), 104.5 (C-5), 108.7 (C-4), 119.2 (C-20), 135.2 (C-3), 137.4 (C-6), 142.5 (C-19). 158.0
(C-2), 171.8 (C-17). N NMR spectrum (CDCl , ꢆ, ppm): 28.6 (N12), 58.0 (N15), 168.1 (N1), 310.5 (N21).
3
15
3
Specific nootropic activity of the synthesized compounds was studied using a basic CPAR model [10] and Wistar
white rats (180–200 g). Tested compounds were administered orally at a dose of 50 ꢂmol/kg; piracetam, 400 mg/kg;
distilled H O (control group), various volumes 1 h before training. The test protocol was described by us earlier [8].
2
Mnestic activity (M ) (memory improvement under the influence of the tested compounds) was calculated using the formula
t
M = [(t – t )/t ]4100%, where M is the mnestic activity (%), t , the average residence time in the dark section of control
t
c
t
c
t
c
animals; t , the average residence time in the dark section of test animals [11, 12].
t
Antihypoxic properties of the synthesized compounds were studied using laboratory white mice of both sexes
(20–25 g). Distilled H O (control group) and tested compounds were administered orally at a dose of 50 ꢂmol/kg 1 h before
2
the start of the experiment. Hypercapnic hypoxia was modeled as before [10]. The lifespan of an animal under hypoxia was
recorded in minutes. The efficacy of the tested compounds was assessed using the formula [(t – t )/t ]4100%, where t is the
t
c
k
t
average lifespan of test animals; t , the average lifespan of control animals [16].
c
Acute toxicity was determined using laboratory white mice (18–22 g) of both sexes with a single i.p. injection at
doses of 100, 200, 1000, 2000, 3000, 4000, and 5000 mg/kg. During the acute toxicity study, the animals were observed, times
of death were estimated, number of dead animals was recorded, and the clinical intoxication condition was noted for 14 d. The
acute toxicity parameters were calculated using the Litchfield–Wilcoxon method [17].
In vitro experiments used cell line HEK293 (Russian Collection of Cell Cultures, Institute of Pathology, Russian
Academy of Sciences, St. Petersburg) that was cultivated at 37°C with 5% CO in DMEM medium containing fetal bovine
2
serum (10%), L-glutamine (2 mM), and gentamicin sulfate (50 ꢂg/mL). Stock solutions of the compounds (1 M in DMSO)
were diluted in complete growth medium immediately before the experiments to final concentrations of active ingredients 1,
10, 100, and 1000 ꢂM. The final DMSO concentration in the growth medium was 0.1%.
914

Cytotoxic properties of the compounds were studied using the MTT-assay according to the manufacturer’s protocol [13].
Antiradical Properties. The intracellular level of AOS in HEK293 cell cytoplasm was measured by the fluorescent
probe 2,7-dichlorodihydrofluorescein diacetate (CM-H2DCFDA, Invitrogen, USA) according to the manufacturer’s protocol.
HEK293 cells were seeded into 96-well plates in DMEM growth medium in order to evaluate the effects of the compounds on
the AOS level. After 24 h, they were treated with 2–11 (final concentrations 1, 10, and 100 ꢂM). Oxidative stress was induced
®
by adding PMA (Sigma, USA) to a final concentration of 1 ꢂM. Measurements were made on a 2300 EnSpire Multimode
Plate Reader (PerkinElmer, USA) at 488 and 530 nm.
Quantitative assessment and comparison of the abilities of the 12-N-methylcytisine derivatives 2–11 to regulate
TF HIF-1 activity used a luciferase reporter construct containing the HIF-1 binding site [18]. Cells were incubated in the
presence of the compounds (final concentrations 1, 10, 100, and 1000 ꢂM) for 24 h. Cells treated with solvent (DMSO, 0.1%)
served as the controls. Luciferase activity in cell lysates was detected using a Dual-Luciferase Reporter Assay System kit and
®
a 2300 EnSpire Multimode Plate Reader.
ACKNOWLEDGMENT
The work used equipment at the Biomika CCU (Department of Biochemical Research Methods and Nanobiotechnology,
RTsKP Agidel, Ufa) and UNU KODINK. The work was sponsored by the RAS Presidium Basic Research Program “Basic
Sciences – Medicine” for 2014; grants of the RF President for leading scientific schools NSh-5923.2014.4; and RFBR Grant
No. 14-04-97035-povolzhꢀe-a.
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915