Synthesis and biological activity of N-benzyl derivatives of cytisine

Article abstract of DOI:10.1007/s10600-006-0181-8

The reductive alkylation of cytisine by various aromatic aldehydes was studied. Preliminary pharmacological investigations of the synthesized compounds were performed.

Full text of DOI:10.1007/s10600-006-0181-8

Chemistry of Natural Compounds, Vol. 42, No. 4, 2006
SYNTHESIS AND BIOLOGICAL ACTIVITY OF -BENZYL
N
DERIVATIVES OF CYTISINE
Sh. B. Rakhimov,1 V. I. Vinogradova,1 Yu. R. Mirzaev,2
N. L. Vypova,2 and D. S. Kazantseva2
UDC 547.94.571;615.015
Thereductivealkylationofcytisinebyvariousaromaticaldehydeswasstudied. Preliminarypharmacological
investigations of the synthesized compounds were performed.
Key words:
cytisine, substitutedbenzaldehydes, reductivealkylation, -benzylcytisinederivatives, acutetoxicity, effect
N
on H-choline receptors, hemostatic properties, spasmolytic activity.
More than 2000 derivatives of cytisine are known today. However, the reaction of cytisine with substituted
benzaldehydes has not been reported.
1
It seemed interesting to investigate the reductive alkylation of cytisine ( ), which contains a secondary N atom, with
aromatic aldehydes.
Secondary amines are known to react with aliphatic aldehydes in a 1:2 mole ratio to form geminal diamines that give
enamines during distillation [1]. The condensing agent is K CO .
2
3
Nucleophiles add slowlyto aromatic aldehydes because the aromatic ring and electron-donating substitutents increase
the electron density on the carbonyl and, as a rule, the reaction occurs under general base- or acid-catalysis conditions [1].
OH
C
N
CH
2
NH
t°
NaBH
4
ArCHO
N
+
N
R
N
H
2
R
O
O
A
1
3 - 24
X
OH
X
OH
X
X
X
OH
OCH
OH
OCH
3
OCH
3
3
4, 5
3
6, 7
8, 9
10, 11
12, 13
4: X = H
5: X = Br
6: X = H
7: X = Br
8: X = H
9: X = Br
10: X = H
11: X = Br
12: X = Br
13: X = OCH
3
X
X
O
X
OCH
OCH
OCH C H
2 6 5
3
3
O
OH
14, 15
OCH
OCH
O
3
3
16 - 18
19 - 22
19: X = H; 20: X = Cl
21: X = Br; 22: X = NO
23
24
14: X = H
15: X = Br
16: X = H
17: X = Br
18: X = NO
2
2
1) S. Yu. Yunusov Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan,
700170, Tashkent, fax (99871) 120 64 75; 2) A. S. Sadykov Institute of Bioorganic Chemistry, Academy of Sciences of the
Republic of Uzbekistan, 700143, Tashkent, ul. Kh. Abdullaeva, 83. Translated from Khimiya Prirodnykh Soedinenii, No. 4,
pp. 373-378, July-August, 2006. Original article submitted April 26, 2006.
0009-3130/06/4204-0462 ©2006 Springer Science+Business Media, Inc.
462

TABLE 1. Physicochemical Properties of 3-24
Compound
Formula
mp, °C
150-151
Yield, %
3
C18H20N2O
80
50
56
70
42
46
58
46
86
74
39
61
65
40
65
80
36
47
49
24
60
50
4
5
6
7
8
9
C18H20N2O2
C18H19N2O2Br
C18H20N2O2
C18H19N2O2Br
C19H22N2O2
C19H21N2O2Br
C19H22N2O3
C19H21N2O3Br
C19H21N2O3Br
C20H24N2O4
C19H22N2O3
C19H21N2O3Br
C20H24N2O3
C20H23N2O3Br
C20H23N3O5
C19H20N2O3 HCl
C19H19N2O3Cl
C18H19N2O3Br
C19H19N3O5
C25H28N2O3
C16H18N2O2
225-226
145-146
115-116
191-192
259-260
amorph.
150-151
235-236
180-181
140-141
180-183
200-201
amorph.
170-171
150-151
275-276
amorph.
amorph.
amorph.
139-140
213-215
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
We reacted various aldehydes 2 with 1. The reactions were carried out in benzene with boiling. In the first step,
geminal α-hydroxyamines (A) were formed that, as a rule, were unstable. This reaction was reversible. The course of the
reaction was monitored using TLC. For most aldehydes, the product A was soluble in benzene (toluene). Only for
2-bromoisovanillin did the product precipitate, thereby making the process irreversible. In the second step, A was reduced
without isolation by NaBH , which gave the expected N-benzyl derivatives of cytisine (3-24).
4
The yields of 3-24 in the reductive amination depended on the nature of the substituent on the aryl ring. Hydroxyls
and methoxyls gave products in yields of 38-61%. The product 6 from reaction of 1 and salicylaldehyde was obtained in 70%
yield. The o-hydroxyl group may have had an effect. As a rule, introducing Br, Cl, and NO groups increased the yield. The
2
exceptions were 7 and 22. As expected, good yields were obtained with benzaldehyde (3, 80%), 6-nitroveratraldehyde (18,
80%), 2-bromoisovanillin (11, 86%), and 6-bromoisovanillin (12, 74%). Replacing OH and OCH by a methylenedioxy
3
decreased the yields to 24-49% (19-22) (Table 1).
The structures of the prepared compounds were confirmed by PMR spectra. Chemical shifts of the cytisine protons
depended little on the nature of the substitution in the benzene ring (see Experimental).
The high analeptic activity of cytisine is well known.
Before investigating 3-24 for certain types of pharmacological activity, we used a computer prediction ofthe biological
activity, PASS, performed at the V. N. Orekhovich Institute of Biomedicinal Chemistry (Moscow).
According tothePASSsystem, a broad spectrum ofvariouspharmacological activitieswaspredictedfor thesynthesized
cytisine derivatives 3-24. These included cholinomimetic, cholinolytic, spasmolytic, sedative, nootropic, pressor, hypotensive,
etc.
Herein resultsofpreliminarypharmacological investigationsofthe17N-benzyl cytisinederivativesarereported. These
include acute toxicity of the compounds in white mice with ip administration, the effect on H-choline receptors of isolated frog
stomach sphincter, and spasmolytic activity and hemostatic properties. Therefore, Table 2 lists the probability (Pa, from 0 to
1) of finding three types of activityamong all those predicted bythe PASS program: H-choline-stimulating, H-cholinolytic, and
smasmolytic (papaverine-like).
463

TABLE 2. Acute Toxicity, H-Cholinolytic (on Isolated Frog Stomach Sphincter) and Spasmolytic (Rat Ileum) Activity of 3-24
PASS data, Pa
Activity, EC50, g/mL
cholinotropic
LD50, mg/kg, ip,
white mice
Compound
1
2
spasmolytic
stimulation
blockade
3
4
5
6
7
8
10
11
12
14
15
16
17
19
21
23
24
0.766
0.692
0.623
0.682
0.612
0.675
0.627
0.589
0.640
0.627
0.574
0.665
0.676
0.643
0.661
0.623
0.658
-
0.623
0.581
0.526
0.603
0.533
0.599
0.546
0.791
0.862*
0.546
0.522
0.603
0.880*
0.406
0.823*
0.593
0.408
-
162 (140÷180)
112 (97÷128)
-
2.5 10-6
5 10-5
6 10-6
7 10-6
3 10-6
1.3 10-5
6 10-6
8 10-6
1 10-5
2 10-6
4 10-6
7 10-6
5 10-6
3 10-5
2 10-6
7 10-6
3 10-6
2 10-6
8 10-6
-
5 10-6
102 (91÷115)
162 (140÷180)
120 (90÷160)
224 (200÷250)
350 (270÷450)
286 (220÷360)
180 (140÷230)
224 (180÷250)
171 (150÷200)
6 10-5
-
-
-
-
3.3 10-6
4 10-5
2.5 10-6
5 10-5
1.5 10-5
-
-
10-6
2 10-6
-
-
1.3 10-6
4 10-6
129 (110÷150)
-
7 10-6
129 (110÷150)
282 (200÷390)
-
4 10-6
5 10-6
10-6
Carbacholine
Tubocurarine
Papaverine
-
-
-
3 10-7
-
-
-
-
-
-
6 10-7
3 10-6
______
Pa is the probability of activity (from 0 to 1); 1, nicotine receptor agonists; 2, spasmolytic activity (bladder); *predicted
spasmolytic and papaverine-like activity (Pa from 0.426 to 0.480).
According to the results (Table 2), the synthesized compounds are moderatelytoxic. These compounds can be divided
according to toxicity into CNS stimulating as tremors and cramps (4, 12, 14) and sedating in combination with general muscle
relaxation (7, 8, 15, 16, 23, 24). With respect to the effect on stomach muscle, some of the compounds (4, 12, 14, 24), like
carbacholine, elicited contraction, i.e., wereH-choline-receptor agonists. Their activityreached10-33%ofcarbacholineacitivty.
The other part of the compounds relieved contraction induced by carbacholine, i.e., had H-cholinolytic activity. With respect
to H-cholinolytic activity, the synthesized compounds can be placed in the following order: 15 > 10 ≥ 3 > 7 > 23 ≥ 16 > 24 >
19 > 12 > 8 > 11 ≥ 4, where their activity varies from 1 to 30% of the tubocurarine activity.
Thus, structurally similar cytisine derivatives had various activities for H-choline-receptors of isolated frog stomach
sphincter. Some ofthem stimulated H-choline receptors and reached 33% ofthe carbacholine activity. The others blocked them
and reached 33% of the tubocurarine activity.
The high spasmolytic activity of 11, 12, 17, and 21 agrees well with the PASS data (Table 2), despite the fact that the
PASS data are given for the bladder and our results were obtained for rat ileum. The experimental activities of 5 and 23 were
significantly greater than that predicted.
It is known from the literature that the production and functioning of thrombocytes and the hemostasis system can be
activated or suppressed depending on the stimulation or blocking of H-choline receptors [2].
The effect of the cytisine derivatives on the blood clotting system was studied in in vitro experiments. The time for
blood clotting, the circulation time, and the amount of blood loss were studied by thromboelastography and in vivo in rats [3].
Table 3 lists the results of the in vitro experiments and shows that the cytisine derivatives containing methoxyls
possessed mainlydistinct hemostatic properties whereas those with one phenol hydroxyl decreased blood clotting. The presence
of both substituents produced hypo- or hypercoagulation that was dependent on the dose.
464

TABLE 3. Effect of Cytisine Derivatives on Hemostasis in vitro and in vivo (M ± m; n = 6)
in vitro
, 0.001 mg/mL
in vivo
, dose 1 mg/kg, ip
C
Compound
thromboeleastogram parameters, mm
A
B
R
K
R+K
MA
Ci
K
3
4
6
7
247±22
180±18
140±15
150±16
87±8.5*
96±10*
145±15
130±13
180±18
140±14
70±7*
56±4
57±5
60±5
20±2*
20±2*
35±3*
57±4
30±2*
52±4
50±4
57±3
28±2*
32±3*
25±2*
35±2
27±3
31±3
27±2*
80±6
29±3*
32±3
25±2*
33±3
33±3
92±6
85±6
91±8
52±4*
100±9
64±5*
89±7
55±4*
85±6
36±2
36±2
30±2
36±2
25±1
40±2
37±2
36±2
33±2
36±2
29±2
37±2
37±2
36±2
0.4±0.02
0.42±0.03
0.3±0.02
285±32
205±20
220±22
282±23
100±8.9*
108±11*
245±20
195±13.7
300±27
255±20
76±7.0*
120±9.5*
270±21
115±11*
300±27
200±19
33±32
315±31
15±1.2*
75±8*
200±18
100±9*
250±20
289±22
20±2.5
48±5*
0.69±0.04
0.25±0.02
0.63±0.05
0.42±0.03
0.66±0.05
0.39±0.02
0.43±0.03
0.28±0.02
0.74±0.05
0.6±0.05*
0.76±0.04
8
10
11
12
14
15
16
19
23
83±7
48±3
105±9
50±4*
62±5*
48±3*
105±10
160±16
95±10*
22±2*
29±2*
22±2*
280±23
86±8.6*
______
*P ′≤ 0.01 relative to the control; K, control; A, blood clotting time, s; B, circulation time, s; C, blood loss, mg.
The most active hemostatics among the studied compounds were 7, 8, 15, 16, and 23, which possessed H-cholinolytic
activity. In in vitro experiments at concentrations of 0.1, 0.01, and 0.001 mg/mL, theydecreased the blood clotting time by 58-
73% [2, 4]. We observed in thromboelastograms a decrease of the reaction time (R) by 53-60%; of the clot formation time (K),
by 20-57%; and an increase of MA from 25 ± 1 to 40 ± 2 mm and of the hypercoagulation index Ci, by 75-117%. The data
indicate that the studied derivatives influence the I and II phases of blood clotting, accelerating thromboplastin formation and,
probably, activating the functional activity of the thrombocytes. The vascular-thrombocytic mechanism of action of these
derivatives was confirmed in in vivo experiments where the circulation time and amount of blood loss decreased by 58-73% [5].
On the other hand, H-cholinomimetics suppress thrombocytopoeia and elicit hypocoagulation. Only 4 of the
H-cholinomimetics studied by us exhibited mild hypocoagulation. Thus, the R+K index increased by 20%. The
hypercoagulation index Ci decreased by 25%.
The most promising compounds as hemostatic agents were 7, 8, 15, 16, and 23; as anticoagulants, 4.
EXPERIMENTAL
General Comments. Column chromatography used KSK silica gel; TLC, silica gel of the same grade and
CHCl :CH OH (4:1). The developer was iodine and Dragendorff's reagent.
3
3
PMR spectra were recorded on a Tesla BS-567A spectrometer at working frequency 100 MHz in DMSO-d , CDCl ,
6
3
and C D N-d .
5
5
5
Condensation of Cytisine and Aromatic Aldehydes. A mixture of cytisine (0.01 mol) and the appropriate aldehyde
(0.012 mol) in benzene (30 mL) was refluxed for 4-6 h. The solvent was distilled off. The solid was treated with CH OH
3
(20 mL) and reduced with NaBH (5 g) with cooling over 1 h. The solvent was distilled off. The solid was dissolved in water
4
(20 mL) and extracted exhaustivelywith CHCl . The crude product was purified over a silica-gel column using CHCl :CH OH
3
3
3
(100:0-100:10). The products were crystallized from acetone or methanol.
N-Benzylcytisine (3). Prepared from cytisine (1.9 g) and benzaldehyde (1.2 g), R 0.78.
f
PMR spectrum (CCl , δ, ppm, J/Hz): 7.15 (1H, dd, J = 9, 7, H-4), 7.10-6.94 (5H, m, Ar-H), 6.25 (1H, d, J = 9, H-3),
4
5.59 (1H, d, J = 7, H-5), 3.88 (1H, d, J = 15.7, H -10), 3.62 (1H, dd, J = 15.7, 6.4, H -10), 3.38 (2H, s, N–CH ), 2.88 (1H,
eq
ax
2
m, H-7), 2.80 (1H, d, J = 13, H -11), 2.71 (1H, d, J = 13, H -13), 2.22 (1H, m, H-9), 2.20-2.12 (2H, d, H -11, H -13), 1.69
eq
eq
ax
ax
(2H, m, H-8).
465

N-(4-Hydroxybenzyl)cytisine (4). Prepared from cytisine (1.9 g) and p-hydroxybenzaldehyde (1.4 g), R 0.6.
f
PMR spectrum (C D N, δ, ppm, J/Hz): 7.06 (1H, dd, J = 6.8, 9, H-4), 6.40 (4H, br.s, H-2′, H-3′, H-5′, H-6′), 6.41 (1H,
5
5
dd, J = 9, 1.6, H-3), 5.70 (1H, dd, J = 6.8, 1.6, H-5), 4.09 (1H, d, J = 15.7, H -10), 3.74 (1H, ddd, J = 15.7, 6.4, 1.0, H -10),
eq
ax
3.16 (2H, s, N–CH ), 2.73, 2.61 (3H each, H-7, H -11, H -13), 2.03 (2H, H -11, H -13), 1.93 (1H, m, H-9), 1.40 (2H, m,
2
eq
eq
ax
ax
H-8).
N-(3-Bromo-4-hydroxybenzyl)cytisine (5). Prepared from cytisine (1.9 g) and 3-bromo-4-hydroxybenzaldehyde
(2.1 g) [6], R 0.78.
f
PMR spectrum (DMSO-d , δ, ppm, J/Hz): 9.90 (1H, s, OH-4′), 7.26 (1H, dd, J = 9, 7, H-4), 6.99 (1H, d, J = 5.3, H-5′),
6
6.71 (2H, s, H-6′, H-2′), 6.18 (1H, dd, J = 9, 1.4, H-3), 5.94 (1H, dd, J = 7, 1.4, H-5), 3.81 (1H, d, J = 15.7, H -10), 3.69 (1H,
eq
dd, J = 15.7, 6.4, H -10), 3.26 (2H, d, J = 5, N–CH ), 2.90 (1H, m, H-7), 2.75 (1H, d, J = 12, H -11), 2.59 (1H, d, J = 13,
ax
2
eq
H -13), 2.29 (1H, m, H-9), 2.19 (1H, d, J = 12, H -13), 2.08 (1H, d, J = 13, H -11), 1.70 (1H, m, H-8).
eq
ax
ax
N-(2-Hydroxybenzyl)cytisine (6). Prepared analogously from cytisine (1.9 g) and salicylaldehyde (1.18 g), R 0.6.
f
+
Mass spectrum (m/z, I , %): 296 (10) [M] , 279 (11), 233 (11), 232 (48), 218 (50), 217 (100), 191 (10), 190 (45), 167
rel
(21), 158 (41), 146 (57), 145 (81), 145 (98), 122 (88), 107 (37), 95 (21), 94 (28), 92 (33), 86 (87), 69 (64).
*
PMR spectrum (300 MHz, DMSO-d + CCl , δ, ppm, J/Hz): 8.96 (1H, s, OH-4′), 7.22 (1H, dd, J = 9, 6.8, H-4), 6.95
6
4
*
(1H, dd, J = 8, 1.2, H-6′), 6.81 (1H, d, J = 8, H-3′), 6.60 (2H, dd, J = 8, 1.2, H-4′, H-5′), 6.24 (1H, d, J = 9, H-3), 5.91 (1H, d,
**
J = 6.8, H-5), 3.85 (1H, d, J = 15.7, H -10), 3.71 (1H, dd, J = 15.7, 6.4, H -10), 3.56 (2H, s, N–CH ), 3.06 (1H, d, J = 13,
eq
ax
2
**
H -13), 3.00 (1H, s, H-7), 2.95 (1H, d, H -11), 2.49 (H-9), 2.37, 2.35 (1H each, d, J = 13, H -11, H -13), 1.85 (2H, m,
eq
eq
ax
ax
H-8).
N-(2-Hydroxy-5-bromobenzyl)cytisine (7). Prepared from cytisine (1.9 g) and 5-bromosalicylaldehyde (2.1 g) [7],
PMR spectrum (C D N, δ, ppm, J/Hz): 7.14 (1H, dd, J = 9, 7, H-4), 7.08 (1H, s, H-6′), 7.06 (1H, d, J = 8, H-3′), 6.65
R 0.76.
f
5
5
(1H, d, J = 8, H-4′), 6.50 (1H, dd, J = 9, 1.4, H-3), 5.75 (1H, dd, J = 7, 1.4, H-4), 4.16 (1H, d, J = 15.7, H -10), 3.76 (1H, dd,
eq
J = 15.7, 6.4, H -10), 3.36 (2H, d, J = 5, N–CH ), 2.77 (1H, m, H-7), 2.69 (2H, m, H -11, H -13), 2.02-2.13 (3H, m, H-9,
ax
2
eq
eq
H -11, H -13), 1.45 (1H, m, H-8).
ax
ax
N-(4-Methoxybenzyl)cytisine (8). Synthesized from cytisine (1.73 g) and anisaldehyde (1.3 g), R 0.8.
f
*
*
PMR spectrum (DMSO-d , δ, ppm, J/Hz): 7.26 (1H, dd, J = 9, 6.8, H-4), 6.85 (2H, d, J = 8.2, H-2′, H-6′), 6.69 (2H,
6
d, J = 8.3, H-3′, H-5′), 6.18 (1H, d, J = 9, H-3), 5.96 (1H, d, J = 6.8, H-5), 3.63 (3H, s, OCH -4′), 3.65 (2H, m, 2H-10), 3.28
3
(2H, s, N–CH ), 2.91 (1H, m, H-7), 2.74 (1H, m, H -11), 2.65 (1H, m, H -13), 2.44 (1H, m, H -13), 2.23 (1H, m, H-9), 2.12
2
eq
eq
ax
(1H, m, H -11), 1.68 (2H, m, H-8).
ax
N-(3-Bromo-4-methoxybenzyl)cytisine (9). Prepared from cytisine (2 g) and 3-bromo-4-methoxybenzaldehyde
(2.26 g) [6], R 0.76.
f
PMR spectrum (DMSO-d , δ, ppm, J/Hz): 7.23 (1H, dd, J = 9, 7, H-4), 7.02 (1H, s, H-5′), 6.87 (1H, s, H-2′), 6.76 (1H,
6
d, J = 8, H-6′), 6.19 (1H, d, J = 9, H-3), 5.93 (1H, d, J = 7, H-5), 4.36 (2H, d, J = 15.7, H -10, H -10), 3.67 (3H, s, OCH -4′),
eq
ax
3
3.26 (2H, s, NCH ), 2.89, 2.76, 2.62 (3H, m, H-7, H -11, H -13), 2.29, 2.19, 2.10 (3H, m, H-9, H -11, H -13), 1.69 (2H,
2
eq
eq
ax
ax
m, H-8).
N-(3-Hydroxy-4-methoxybenzyl)cytisine (10). Prepared from cytisine (1.9 g) and isovanillin (1.7 g) [8], R 0.76.
f
PMR spectrum (C D N, δ, ppm, J/Hz): 7.09 (1H, d, J = 8, H-6′), 7.07 (1H, dd, J = 9, 7, H-4), 6.82 (1H, br.s, H-2′), 6.70
5
5
(1H, d, J = 8, H-5′), 6.46 (1H, d, J = 9, H-3), 5.69 (1H, d, J = 6.8, H-5), 4.13 (1H, d, J = 15.7, H -10), 3.76 (1H, dd, J = 15.7,
eq
6.4, H -10), 3.55 (3H, d, J = 1, OCH -4′), 3.17 (2H, s, N–CH ), 2.62 (3H, m, H-7, H -11, H -13), 2.00 (3H, m, H-9, H -11,
ax
3
2
eq
eq
ax
H -13), 1.42 (2H, m, H-8).
ax
N-(3-Hydroxy-4-methoxy-2-bromobenzyl)cytisine (11). Prepared from cytisine (1.9 g) and 2-bromoisovanillin
(2.6 g) [9], R 0.8.
f
+
Mass spectrum (m/z, I , %): 405 (8) [M] , 325 (22), 214 (33), 190 (12), 189 (32), 158 (12), 146 (16), 145 (29), 137
rel
(100), 95 (13), 93 (16), 84 (20), 69 (28).
PMR spectrum (DMSO-d , δ, ppm, J/Hz): 7.25 (1H, dd, J = 8.9, 6.8, H-4), 6.67 (1H, d, J = 8.4, H-2′), 6.63 (1H, d,
6
J = 8.4, H-3′), 6.16 (1H, dd, J = 8.9, 1.4, H-3), 5.96 (1H, dd, J = 6.8, 1.4, H-5), 3.70 (5H, br.s, 2H-10, OCH -4′), 3.29 (2H, s,
3
N–CH ), 2.86 (1H, m, H-7), 2.76, 2.70 (1H each, m, H -11, H -13), 2.31, 2.21 (3H, m, H-9, H -11, H -13), 1.66 (2H, m,
2
eq
eq
ax
ax
H-8).
466

N-(3-Hydroxy-4-methoxy-6-bromobenzyl)cytisine (12). Preparedfrom cytisine(1.9g)and3-hydroxy-4-methoxy-6-
bromobenzaldehyde (2.6 g) [10], R 0.63.
f
PMR spectrum (DMSO-d , δ, ppm, J/Hz): 8.94 (1H, s, OH-3′), 7.22 (1H, dd, J = 9, 7, H-4), 6.92 (1H, s, H-5′), 6.44
6
(1H, s, H-2′), 6.14 (1H, dd, J = 9, 1.4, H-3), 5.94 (1H, dd, J = 1.4, 7, H-5), 3.67 (4H, br.s, 2H-10, N–CH ), 2.38 (3H, OCH -4′),
2
3
2.92, 2.77-2.69 (3H, H-7, H -11, H -13), 2.20-2.28 (3H, H-9, H -11, H -13), 1.68 (2H, m, H-8).
eq
eq
ax
ax
Preparation of 6-Methoxyisovanillin. Sodium methoxide (70 mL absolute CH OH, 4.5 g metallic Na) was treated
3
with freshlydried CuCl (anhydrous, 0.75 g), 6-bromoisovanillin (13 g), and dryDMF (56 mL). The reddish-brown suspension
2
was stirred at room temperature and treated dropwise with HCl solution (31 mL, 20%) until the pH was 2-3. When the pH of
the suspension reached 7, it became a dark red solution. The resulting solution was extracted with CHCl (3 × 70 mL). The
3
CHCl extract was washed with KOH (4%, 50 mL, 5 × 25 mL). The alkaline solution was acidified with conc. HCl (with
3
cooling). The resulting yellow precipitate was filtered off to afford a mixture of three compounds (7.02 g). The mother liquor
was extracted with ether and CHCl . Recrystallization from benzene produced the compound (5 g), mp 139-141°C.
3
N-(3-Hydroxy-4,6-dimethoxybenzyl)cytisine (13). Prepared from cytisine (1.7 g) and 6-methoxyisovanillin (1.8 g),
R 0.5.
f
+
Mass spectrum (m/z, I , %): 355 (6) [M] , 320 (17), 319 (77), 288 (9), 231 (13), 217 (24), 190 (69), 168 (22), 167
rel
(56), 160 (36), 146 (50), 145 (83), 145 (100), 117 (32), 96 (12), 95 (14), 88 (24).
PMR spectrum (DMSO-d , δ, ppm, J/Hz): 8.94 (1H, s, OH-3′), 7.22 (1H, dd, J = 9, 7, H-4), 6.92 (1H, s, H-5′), 6.44
6
(1H, s, H-2′), 6.13 (1H, dd, J = 9, 1.2, H-3), 5.95 (1H, dd, J = 7, 1.2, H-5), 3.68 (4H, br.s, 2H-10, N–CH ), 3.27 (6H, s,
2
OCH -4′, OCH -6′), 2.69-2.93 (3H, m, H-7, H -11, H -13), 2.28-2.44 (3H, m, H-9, H -11, H -13), 1.69 (2H, m, H-8).
3
3
eq
eq
ax
ax
N-(3-Methoxy-4-hydroxybenzyl)cytisine (14). Prepared from cytisine (1.9 g) and vanillin (1.7 g), R 0.76.
f
PMR spectrum (DMSO-d , δ, ppm, J/Hz): 8.64 (1H, s, OH), 7.28 (1H, dd, J = 9, 7, H-7), 6.56 (1H, d, J = 8, H-5), 6.42
6
(1H, s, H-2′), 6.27 (1H, dd, J = 8, 1.2, H-6′), 6.17 (1H, dd, J = 9, 1.4, H-3), 5.96 (1H, dd, J = 7, 1.4, H-5), 3.84, 3.64 (2H, dd,
J = 15.7, 6.4, 2H-10), 3.50 (3H, s, OCH -3′), 3.29 (2H, s, N–CH ), 2.90 (1H, m, H-7), 2.76 (1H, m, H -11), 2.65 (1H, m,
3
2
eq
H -13), 2.22 (2H, m, H-9, H -13), 2.10 (1H, m, H -11), 1.68 (1H, m, H-8).
eq
ax
ax
N-(3-Methoxy-4-hydroxy-5-bromobenzyl)cytisine (15). Prepared from cytisine (1.9 g) and 5-bromovanillin (2.6 g)
[11], R 0.6.
f
*
PMR spectrum (DMSO-d , δ, ppm, J/Hz): 9.4 (1H, s, OH-4′), 7.27 (1H, dd, J = 9, 7, H-4), 6.65 (1H, d, J = 1.2, H-2′),
6
*
6.45 (1H, d, J = 1.2, H-6′), 6.16 (1H, dd, J = 1.2, 9, H-3), 5.97 (1H, dd, J = 7, 1.2, H-5), 3.87 (1H, d, J = 15.7, H -10), 3.71
eq
(1H, dd, J = 15.7, 6.4, H -10), 3.56 (3H, s, OCH -3′), 3.29 (2H, s, N–CH ), 2.93 (1H, m, H-7), 2.76, 2.64 (1H each, m, H -11,
ax
3
2
eq
H -13), 2.28, 2.21, 2.11 (3H, m, H-9, H -11, H -13).
eq
ax
ax
N-(3,4-Dimethoxybenzyl)cytisine (16). Prepared from cytisine (1.9 g) and 3,4-dimethoxybenzaldehyde (1.8 g),
R 0.8.
f
PMR spectrum (DMSO-d , δ, ppm, J/Hz): 7.26 (1H, dd, J = 8.9, 6.8, H-4), 6.83 (1H, d, J = 7, H-5′), 6.67 (1H, s, H-2′),
6
6.49 (1H, dd, J = 7, 1.9, H-6′), 6.18 (1H, dd, J = 1.5, 8.9, H-3), 5.97 (1H, dd, J = 1.5, 6.8, H-5), 3.70 (1H, d, J = 15.6, H -10),
eq
3.63 (1H, dd, J = 15.6, 6.4, H -10), 3.67 (3H, s, OCH ), 3.63 (3H, s, OCH ), 3.49 (2H, s, N–CH ), 2.75, 2.67 (1H each, m,
ax
3
3
2
H -11, H -13), 2.92 (1H, H-7), 2.12 (2H, m, H-9, H -11, H -13), 1.68 (2H, m, H-8).
eq
eq
ax
ax
N-(3,4-Dimethoxy-6-bromobenzyl)cytisine (17).
bromobenzaldehyde (2.6 g) [6], R 0.33.
Prepared from cytisine (1.9 g) and 3,4-dimethoxy-6-
f
PMR spectrum (C D N, δ, ppm, J/Hz): 7.11 (1H, dd, J = 9, 7, H-4), 7.02 (1H, s, H-5′), 6.66 (1H, s, H-2′), 6.45 (1H,
5
5
dd, J = 9, 1.2, H-3), 5.77 (1H, dd, J = 7, 1.2, H-5), 4.12 (1H, d, J = 15.7, H -10), 3.76 (1H, dd, J = 15.7, 6.4, H -10), 3.58 (6H,
eq
ax
s, OCH -3′, OCH -4′), 3.38 (2H, s, N–CH ), 2.62-2.71 (1H, m, H-7, H -11, H -13), 2.00-2.18 (1H, m, H-9, H -11, H -13),
3
3
2
eq
eq
ax
ax
1.45 (2H, m, H-8).
N-(3,4-Dimethoxy-6-nitrobenzyl)cytisine (18). Preparedfromcytisine(1.5g)and3,4-dimethoxy-6-nitrobenzaldehyde
(1.64 g) [12], R 0.57.
f
PMR spectrum (C D N, δ, ppm, J/Hz): 7.52 (1H, s, H-5′), 7.14 (1H, dd, J = 9, 6.8, H-4), 6.80 (1H, s, H-2′), 6.44 (1H,
5
5
dd, J = 9, 1.6, H-3), 5.78 (1H, dd, J = 7, 1.6, H-5), 4.13 (1H, d, J = 15.6, H -10), 3.81 (1H, dd, J = 15.6, 6.4, H -10), 3.65 (3H,
eq
ax
s, OCH -3′ or OCH -4′), 3.62 (5H, s, OCH -4′ or OCH -3′ and N–CH ), 2.66 (3H, m, H-7, H -11, H -13), 2.09 (3H, m, H-9,
3
3
3
3
2
eq
eq
H -11, H -13), 1.42 (2H, br.s, H-8).
ax
ax
N-(3,4-Methylenedioxybenzyl)cytisine (19). Prepared from cytisine (1.9 g) and 3,4-methylenedioxybenzaldehyde
(1.7 g), R 0.85.
f
467

PMR spectrum (C D N, δ, ppm, J/Hz): 7.16 (1H, dd, J = 9, 7, H-4), 6.64 (1H, d, J = 8, H-5′), 6.47 (1H, d, J = 8, H-6′),
5
5
6.50 (1H, s, H-2′), 6.44 (1H, dd, J = 9, 1.2, H-3), 5.77 (2H, s, OCH O), 5.74 (1H, dd, J = 7, 1.2, H-5), 4.12 (1H, d, J = 15.7,
2
H -10), 3.77 (1H, dd, J = 15.7, 6.4, H -10), 3.14 (2H, s, N–CH ), 2.70 (1H, m, H-7), 2.63 (2H, br.s, H -11, H -13), 2.08
eq
ax
2
eq
eq
(1H, d, J = 13, H -13), 2.01 (1H, d, J = 13, H -11), 1.90 (1H, m, H-9), 1.42 (2H, m, H-8).
ax
ax
N-(3,4-Methylenedioxy-6-chlorobenzyl)cytisine (20). Prepared from cytisine (1.9 g) and 3,4-methylenedioxy-6-
chlorobenzaldehyde (2.0 g) [13], R 0.6.
f
PMR spectrum (DMSO-d , δ, ppm, J/Hz): 7.25 (1H, dd, J = 9, 7, H-4), 6.87 (1H, s, H-5′), 6.34 (1H, s, H-2′), 6.19 (1H,
6
dd, J = 9, 1.4, H-3), 5.93 (2H, s, OCH O), 5.95 (1H, dd, J = 7, 1.4, H-5), 3.82 (1H, d, J = 15.7, H -10), 3.60 (1H, dd, J = 15.7,
2
eq
6.4, H -10), 3.30 (2H, s, N–CH ), 2.93 (1H, m, H-7), 2.97 (1H, d, J = 13, H -11), 2.65 (1H, d, J = 13, H -13), 2.20-2.37 (3H,
ax
2
eq
eq
m, H-9, H -11, H -13), 1.72 (2H, m, H-8).
ax
ax
N-(3,4-Methylenedioxy-6-bromobenzyl)cytisine (21). Synthesized from cytisine (1.9 g) and 3,4-methylenedioxy-6-
bromobenzaldehyde (2.5 g) [6], R 0.8.
f
PMR spectrum (DMSO-d , δ, ppm, J/Hz): 7.25 (1H, dd, J = 9, 7, H-4), 7.00 (1H, s, H-5′), 6.34 (1H, s, H-2′), 6.18 (1H,
6
dd, J = 9, 1.4, H-3), 5.95 (1H, dd, J = 7, 1.4, H-5), 5.93 (2H, s, OCH O), 3.81 (1H, d, J = 15.6, H -10), 3.55 (1H, dd, J = 15.6,
2
eq
6.4, H -10), 3,27 (2H, d, J = 5, N–CH ), 2.91 (1H, m, H-7), 2.75 (1H, br.s, H -11), 2.63 (1H, br.s, H -13), 2.20-2.35 (3H,
ax
2
eq
eq
m, H-9, H -11, H -13), 1.71 (2H, m, H-8).
ax
ax
N-(3,4-Methylenedioxy-6-nitrobenzyl)cytisine (22). Prepared from cytisine (1.7 g) and 3,4-methylenedioxy-6-
nitrobenzaldehyde (1.9 g) [14], R 0.7.
f
PMR spectrum (DMSO-d , δ, ppm, J/Hz): 7.26 (1H, dd, J = 9, 7, H-4), 6.62 (1H, s, H-5′), 6.38 (1H, s, H-2′), 6.19 (1H,
6
dd, J = 9, 1.4, H-3), 5.94 (1H, dd, J = 7, 1.4, H-5), 5.86 (2H, s, OCH O), 3.81 (1H, d, J = 15.7, H -10), 3.61 (1H, dd, J = 15.7,
2
eq
6.4, H -10), 3.24 (2H, s, N–CH ), 2.90 (1H, m, H-7), 2.75 (1H, d, H -11), 2.63 (1H, d, H -13), 2.24 (2H, H-9, H -13), 2.11
ax
2
eq
eq
ax
(1H, H -11), 1.67 (2H, m, H-8).
ax
N-(4-Methoxy-3-benzyloxybenzyl)cytisine (23).
methoxybenzaldehyde (2.64 g) [15], R 0.7.
Prepared from cytisine (1.9 g) and 3-benzyloxy-4-
f
PMR spectrum (DMSO-d , δ, ppm, J/Hz): 7.34-7.15 (7H, m, H-5′, H-6′), 7.26 (1H, dd, J = 9, 7, H-4), 6.83 (1H, s,
6
H-2′), 6.16 (1H, dd, J = 9, 1.4, H-3), 5.96 (1H, dd, J = 7, 1.4, H-5), 4.35 (2H, d, 2H-10), 3.67 (3H, s, OCH ), 3.64 (2H, s, OCH ),
3
2
3.36 (2H, s, N–CH ), 2.69-2.88 (3H, H-7, H -11, H -13), 2.06-2.19 (3H, m, H-9, H -11, H -13), 1.65 (2H, m, H-8).
2
eq
eq
ax
ax
N-Furfurylcytisine (24). Prepared from cytisine (1.9 g) and furfurol (1.15 g), R 0.8.
f
*
PMR spectrum (C D N, δ, ppm, J/Hz): 7.18 (1H, dd, J = 7, 8.5, H-3′), 7.16 (1H, dd, J = 9, 7, H-4), 6.49 (1H, d,
5
5
*
J = 8.5, H-2′), 6.41 (1H, dd, J = 9, 1.4, H-3), 5.88 (1H, d, J = 7, H-4′), 5.81 (1H, dd, J = 7, 1.4, H-5), 4.12 (1H, d, J = 15.7,
H -10), 3.77 (3H, dd, J = 15.7, 6.4, H -10, N–CH ), 2.5-2.8 (3H, m, H-7, H -11, H -13), 2.02-2.4 (3H, m, H-9, H -11,
eq
ax
2
eq
eq
ax
H -13), 1.40 (2H, m, H-8).
ax
Acute toxicity of the studied compounds was determined in white mongrel mice of mass 20 ± 2 g of both sexes with
ip injection. The Litchfield and Wilcoxson method was used to determine the acute toxicity parameters [16].
The influence of the synthesized compounds on blood clotting was investigated on a Tromb-2 thromboelastograph.
Blood was drained dropwise into a cuvette from a rabbit ear vein. The cuvette was charged beforehand with the preparation
at a final concentration of 0.1, 0.01, and 0.001 mg/mL (1:9).
The following parameters were considered on the thromboelastograms: R, blood reaction time, which characterizes
phases I and II of blood clotting; K, clot formation time or thromboelastographic constant of thrombin, which depends on the
concentration of the thrombin formed and the amount of fibrinogen; R + K, coagulation constant that expresses the overall
duration of blood clotting; MA, maximum amplitude, which is influenced by the fibrinogen concentration, the amount and
quality of thrombocytes; and Ci, index of hypercoagulation [3].
The effect of the preparations on hemostasis was studied in in vivo experiments for the screening model "circulation
time and blood-loss value" [5]. Rats of mass 130 ± 10 g were administered once ip preparations at a dose of 1 mg/kg. The
circulation time and blood-loss value were studied 60 min after administering the preparations.
The effect of the compounds on H-choline receptors was studied using isolated frog stomach sphincter by the De Elio
method [17].
468

REFERENCES
1.
N. K. Kochetkov and A. I. Usov, eds., General Organic Chemistry, Vol. 2 [in Russian], Khimiya, Moscow (1982), pp.
522 and 722.
2.
3.
4.
N. L. Vypova, Candidate Dissertation in Medical Sciences (1991).
V. P. Baluda, Clinical Methods of Hemostasis Investigation [in Russian], Tomsk (1980), p. 422.
P. P. Denisenko, S. Kh. Nasirov, and D. S. Kazantseva, Pharamcological Regulation of Thrombocytopoeia [in
Russian], Tashkent (1989).
5.
6.
I. E. Akopov, ed., Problems in Blood Coagulation and Hemostasis [in Russian], Scientific Works, XXXIII, (1974).
N. U. Baratov, E. G. Mil′grom, V. I. Vinogradova, Ya. V. Rashkes, and M. S. Yunusov, Khim. Prir. Soedin., 839
(1993).
7.
8.
9.
Beilsteins Handbuch der organischen Chemie, E II, 8, p. 54.
A. Brossi, H. Gurien, A. J. Rachlin, and S. Teitel, J. Org. Chem., 32, No. 4, 1435 (1967).
T. Kametani, K. Fukumoto, S. Shibuya, H. Nemoto, T. Nakano, T. Sugahara, T. Takahashi, Y. Aizawa, and
M. Toriyana, J. Chem. Soc., Perkin Trans. I, 1435 (1972).
10.
11.
S. E. Hazlet and R. J. Brotherton, J. Org. Chem., 27, 3253 (1962); T. A. Henry and T. M. Shart, J. Chem. Soc., 2285
(1930).
V. I. Vinogradova, T. I. Golodnyuk, N. Tulyaganov, M. S. Yunusov, and N. U. Baratov, Khim. Prir. Soedin., 404
(1993).
12.
13.
14.
15.
16.
17.
A. F. Plate, ed., Synthesis of Organic Preparations, Coll. 5, IL, Moscow (1954), p. 9.
I. Guben, Methods of Organic Chemistry, Vol. 3, ONTI, Moscow (1935), No. 3, p. 350.
I. Guben, Methods of Organic Chemistry, Vol. 4, ONTI, Moscow (1949), No. 1, p. 240.
V. I. Vinogradova, M. S. Yunusov, I. Khamdamov, and F. Sadritdinov, Khim. Prir. Soedin., 343 (1979).
M. A. Belen′kii, Methods of Quantitative Analysis in Biology [in Russian], Moscow (1963).
V. V. Gatsura, Methods of Fundamental Pharmacological Investigations of Biologically Active Substances [in
Russian], Meditsina, Moscow (1974).
469