Synthesis and biological activity of amination products of the alkaloid securinine

Article abstract of DOI:10.1007/s10600-008-9013-3

A method for stereospecific synthesis of allomargaritarin and other amination products of the natural alkaloid securinine was developed. Stereoselective nucleophilic addition of an amine at the double bond of the azobicyclooctane moiety of securinin was achieved by using ytterbium triflate as the catalyst. The biological activity of securining caused by adding pharmacophores to the molecule was shown to change experimentally.

Full text of DOI:10.1007/s10600-008-9013-3

Chemistry of Natural Compounds, Vol. 44, No. 2, 2008
SYNTHESIS AND BIOLOGICAL ACTIVITY OF AMINATION
PRODUCTS OF THE ALKALOID SECURININE
S. G. Klochkov, S. V. Afanas¢eva, and V. V. Grigor¢ev
UDC 577.112
A method for stereospecific synthesis of allomargaritarin and other amination products of the natural
alkaloid securinine was developed. Stereoselective nucleophilic addition of an amine at the double bond of
the azobicyclooctane moiety of securinin was achieved by using ytterbium triflate as the catalyst. The
biological activity of securining caused by adding pharmacophores to the molecule was shown to change
experimentally.
Key words: alkaloids, securinine, allomargaritarin, ytterbium triflate, stereospecific synthesis.
Securininealkaloidsform a groupoftetracycliccompoundswith 6-azobicyclo[3.2.1]octaneasthekeystructuretowhich
an a ,b-unsaturated-g-lactone moietyand a piperidine ring are fused. Such heterocycles areinterestingnot onlyduetothe unique
structure but also because of their biological activity. Securinine (1) is the most well-known alkaloid.
Securinine is a stereospecific competitive antagonist ofthe GABA -receptor (IC about 50 mM [1]) with proven CNS-
A
50
activity in animals [2, 3]. Furthermore, this compound exhibits neuroprotective properties, enhances learning and memory [4,
5], and was used previously in medical practice [6].
Securinine and its analogs have rigidly fixed structures. Margaritarine, which was isolated from Margaritaria indica
(Dalz.) G. L. Webster, is the onlynatural securinine-type alkaloid that contains an N-tryptamine peripheral substituent [7]. The
3
replacement of the multiple bond by sp -hybridized C atoms and the presence of an additional asymmetric center imparts a
definite conformational flexibility to margaritarine, which increases its ability to interact with active receptor centers.
Margaritarine is a minor constituent of alkaloid mixtures and is difficultly isolated in quantities sufficient to investigate it. We
attempted to develop convenient synthetic methods for this compound, to prepare other derivatives of securinine alkaloids, and
to test them for certain types of biological activity.
Studies on the synthesis of margaritarine or modification of securinine alkaloids by introducing new pharmacophores
have not yet been reported. The system of conjugated double bonds of the azobicyclooctane and lactone rings are most
interesting for preparing modified securinine derivatives. Because this system is activated by the carbonyl group, it is readily
attacked by nucleophiles. Nucleophilic addition of amines to securinin can produce the alkaloid allomargaritarine (3)
(margaritarine epimer at C-2¢) and other amino derivatives.
Yb(OTf)
3
CH OH
3
O
O
O
5
H N
2
O
O
O
12'
14'
3'
7
H
N
H
N
H
N
CH OH
3
3a
a
7a
+
+
8'
5'
N
H
N
H
b
2
7'
N
H
N
H
N
H
H
H
2
3
4
1
Sheme 1.
Institute of PhysiologicallyActive Substances, Russian Academyof Sciences, 142432, Moscow Oblast, Nogin Region,
Chernogolovka, Severnyi proezd, Bldg. 1, fax 7-(496) 524 95 08, e-mail: klochkov@ipac.ac.ru. Translated from Khimiya
Prirodnykh Soedinenii, No. 2, pp. 156-160, March-April, 2008. Original article submitted July 16, 2007.
0009-3130/08/4402-0197 ©2008 Springer Science+Business Media, Inc.
197

TABLE 1. Chemical Shifts (d, ppm) and Spin-Spin Coupling Constants (J/Hz) in PMR and 13C NMR Spectra of
Allomargaritarine (3) and its Epimer 4
3
4
C atom
dC, ppm
dH, ppm
dC, ppm
dH, ppm
2¢
3¢
4¢
5¢
6¢
62.18
24.16
21.53
26.11
49.76
2.32, (dd, J1 = 2.7, J2 = 11.8)
1.4 (br.s, dd, J = 11.8), 2.2 m
1.2 m, 1.6 m
63.51
24.15
21.9
26.12
49.44
2.6 (dd, J1 = 2.5, J2 = 10.8)
1.36 (br.s, dd, J = 10.8), 2.8 m
1.2 - 1.4 m (2H)
1.2 - 1.4 m (2H)
2.43 (br.s, d, J = 12.0; 2.9 3.0 m
1.1 - 1.2 m (2H)
2.52 (br.s, d, J = 10.7);
2.95 (ddd, J1 = J2 = 10.7, J3 = 3.7)
3.1 (d, J = 6.7)
7¢
8¢
60.84
35.94
59.96
32.82
3.08 (t, J1 = 9.5, J2 = 4.6)
1.7 (d, J = 10.9);
1.16 (d, J = 11.1);
2.58 (dd, J1 = 11.1, J2 = 6.7)
2.75 (dd, J1 = 10.9, J2 = 4.6)
9¢
91.68
173.92
111.75
174.25
31.72
91.85
173.77
111.78
174.95
26.24
11¢
12¢
13¢
14¢
5.5 (d, J = 2.1)
5.3 (d, J = 2.3)
2.98 m, 14¢a -H;
2.25 (ddd, J1 = 2.1, J2 = 8.9, J3 = 16)
2.8 (ddd, J1 = 8.9, J2 = 6.5, J3 = 1.5)
6.9 (d, J = 2.1)
2.9 m, 14¢a -H
2.35 (ddd, J1 = 2.3, J2 = 6.01, J3 = 15)
2.9 (dd, J1 = 9.5, J2 = 6.01)
6.9 (d, J = 2.2)
15¢
2
3
a
b
60.48
122.70
113.84
26.51
60.18
122.55
113.91
26.25
2.7 - 2.98 m (2H)
2.7 - 2.98 m (2H)
2.7 - 2.9 m (2H)
2.7 - 2.9 m (2H)
47.11
47.99
3a
4
5
6
7
127.67
119.14
119.69
122.46
110.14
136.97
127.71
119.18
119.76
122.55
111.28
136.91
7.48 (br.s, d, J = 7.6)
7.07 (ddd, J1 = 7.6, J2 = 7.0, J3 = 1.0)
7.09 (ddd, J1 = 8.1, J2 = 7.0, J3 = 1.0)
7.22 (dd, J = 8.1)
7.53 (br.s, d, J = 7.8)
7.09 (ddd, J1 = 7.8, J2 = 7.0, J3 = 1.0)
7.12 (ddd, J1 = 7.6, J2 = 1.3, J3 = 1.0)
7.3 (dd, J = 7.6)
7a
1-NH
8.3 br.s
8.06
br.s
O
O
O
O
H
H
N
N
+
N
H
NH
2
H
H
CH
2
N
m/z 234
m/z 247
m/z 83
O
O
NH
2
H
N
H
H
N
N
+
NH
m/z 160
N
H
H
m/z 56
m/z 377
+
+
N
N
H
H
m/z 144
m/z 130
Fig. 1. Mass spectral fragmentation of allomargaritarine.
198

O
H
2.3
O
4.6
H
N
10.9
H
15.0
H
H
N
H
6.01
H
9.5
Fig. 2. Spin—spin coupling constants (Hz) of protons
of the azobicyclooctane and lactone rings of
allomargaritarine epimer 4.
We developed a stereospecific synthetic method for allomargaritarine (3) from 1 and tryptamine (2) using ytterbium
triflate as the catalyst. We also produced several products of amination of securinin by various pharmacophoric amines.
A mixture of two compounds in equal amounts that we isolated using HPLC and characterized by spectral methods
was formed under non-catalytic conditions as a result of the reaction of 1 and 2. Spectral data established that these were
allomargaritarine (3) and its epimer 4. The presence of an , -unsaturated lactone ring was confirmed by the presence in IR
a b
1
13
spectra of 3 and 4 of absorption bands at 1745 and 1630 cm- in addition to the corresponding resonances in C NMR spectra
(ppm): 173.92 and 173.77 for the carbonyl C atom and 111.75 and 111.78 for the C-12 atom (Table 1). The PMR spectrum
¢
showed doublets for the olefinic proton on C-12 ( = 5.5, J = 2.1 Hz and 5.3 ppm, J = 2.3 Hz) that had allyl spin—spin
¢ d
H
coupling constants (SSCC) with the -proton on C-14 ( = 2.25 and 2.35 ppm). Resonances of the last, in turn, are related
¢ d
b
H
to resonances of the C-15 proton ( = 2.8 and 2.9 ppm, respectively). The NMR spectra also contained resonances for protons
¢
d
H
1
of a -substituted indole; the IR spectrum, absorption bands for NH vibrations at 3380 cm- . The elucidated structure 3 and
b
the presence of tryptamine were confirmed by electron-impact fragmentation (Fig. 1).
The position and stereochemistry of adding tryptamine to the conjugated system of double bonds of securinine were
determined using PMR spectra. The tryptamine moiety in 3 had the 15 -orientation. In this instance the -proton on C-15
¢a
b
¢
bisects the angle between the - and -protons on C-14 . This is evident from their SSCC (8.9 and 6.5 Hz) and the small
a
b
¢
constant with the C-7 proton. The resonance for the C-14 -proton also had a constant with a well resolved resonance for the
¢ b
¢
C-12 proton.
¢
The tryptamine in 4 had the 15 -orientation. This was confirmed byanalyzing the PMR spectra. The main difference
¢b
in the spectra of the two epimers was seen in the position and shape of the resonance for the C-7 proton. Although a slightly
¢
broadened doublet for this proton was observed for 3 at 3.1 ppm, the resonance for the C-7 proton of 4 was a triplet with
¢
coupling constants with the -protons on C-14 and C-15 . The value of the last constant was much greater than for
a
¢
¢
allomargaritarin and reached 9.5 Hz (Fig. 2).
Weusedthemethoddevelopedfor tryptamine toprepare amination products ofsecurinin. Compounds5-8wereformed
in good yields by the reactions of securinin with pharmacophoric arylpiperazines and pyrrolidine.
O
R =
N
N
N
N
N
N
N
OCH
3
O
H
N
6
5
7
N
R
H
8
5 - 8
The structures of the prepared compounds were elucidated using PMR and mass spectrometry.
Compounds 3 and 5 in addition to securinine (1) were tested using the patch-clamp method for the ability to modify
currents in rat brain Purkinje neurons.
199

180
140
180
140
100
60
100
60
3
1
1
2
20
20
2
-8
-7
-6
-5
-8
-7
-6
-5
C om pound concentration, log, M
C om pound concentration, log, M
Fig. 3
Fig. 4
Fig. 3. Activity of securinine (1) and 5 toward cainate-induced currents in Purkinje neurons of rat cerebellum; securinine (1)
and 5 (2).
Fig. 4. Activity of securinine (1), 5, and allomargaritarine (3) toward GABA-activated currents in Purkinje neurons of rat
cerebellum; securinine (1), 5 (2), and allomargaritarine (3).
The electrohysiological investigations showed that the biological activityof securinin and its derivatives changed after
adding the new fragments to the molecule. For example, 1 had a substantial effect on currents in rat cerebellum Purkinje
neurons that were due to cainic acid (CA) (Fig. 3). Low doses typically increased currents whereas large ones blocked them.
Thus, currents at a concentration of 10 nM were 133 ± 7% of the control; at 100 nM, 150 ± 20%; at 1-10 mM, 65 ± 7 and
45 ± 6%, respectively. Compound 5 was even more active for CA-induced currents. Thus, at a concentration of 10 nM the
currents were 140 ± 7% of the control; at 100 nM, 165 ± 8%; at 1 mM, 50 ± 4%; and at 10 mM, 0%. Compound 3 had no effect
on the amplitude of CA-induced currents in the studied (from 10 nM to 10 mM) dose range.
Compound 1 also affected GABA-induced currents in rat cerebellum Purkinje neurons (Fig. 4). Like for CA-induced
currents, low doses of the compound increased the currents whereas large ones blocked them. This agreed well with the
literature [8]. Thus, currents at a concentration of 10 nM were 135 ± 7% of the control; at 100 nM, 125 ± 7%; at 1-10 mM,
42 ± 3 and 30 ± 3%, respectively. Compound 5 was more active toward GABA-induced currents than 1. Thus, the currents
at a concentration of 10 nM were 165 ± 12%; at 100 nM, 150 ± 8%; at 1 mM, 20%; at 10 mM, 0% of the control. Compound
3 at low doses had no effect on the amplitude of GABA-induced currents. It decreased them at higher concentrations of
100 nM to 70%; 1 mM, 65%; and 10 mM, 50% of the control.
The investigations conducted by us confirmed that 1 was active toward the rat CNS. The amination products of 1 were
also CNS-active but differed from the starting alkaloid.
Thus, a method ofstereoselective addition ofamines tothe natural alkaloid securinin using ytterbium triflate as catalyst
was developed. Several newaddition products of securinin and secondaryand tertiaryamines were prepared and characterized.
It was shown that adding new pharmacophoric fragments to the rigid securinin molecule is a promising route for synthesizing
new biologically active compounds.
EXPERIMENTAL
NMR spectra were recorded on a Bruker DPX-200 spectrometer at operating frequency 200 MHz (Table 1). Mass
spectra were recorded in a Finnigan 4021 GC—MS at ionizing potential 70 eV; IR spectra (KBr disks); on a Bruker ZFS-113v
instrument.
Chromatographicanalysisandpreparativeisolation ofthe compounds wereperformedon a Gilson chromatograph with
UV detection (280 nm) using an analytical column (4 × 100 mm) with Diasorb 130 C16/T (5 mm) and a preparative column
(16 × 250 mm) with the same stationary phase (BioChemMack, Russia). The mobile phase was a gradient of CH CN and
3
distilled water with added trifluoroacetic acid (0.1%) from 5 to 75%. The flow rate was 1.5 mL/min for the analytical column;
5 mL/min for the preparative column. The sample volume was 20 mL for the analytical column; 1 mL for the preparative
200

column. Alkaloid 1 was supplied by InterBioScreen (Moscow). Elemental analysis of all newly prepared compounds agreed
with those calculated.
3-{N-[(2b,15a )-14,15-Dihydro-11-oxosecurinan-15-yl]-2-aminoethyl}1H-indole (3) and 3-{N-[(2b,15b)-14,15-
Dihydro-11-oxosecurinan-15-yl]-2-aminoethyl}1H-indole (4). A mixture of 1 (0.217 g, 1 mmol) and tryptamine (2, 0.24 g,
1.5 mmol) in methanol (5 mL) was stirred at room temperature. The course of the reaction was monitored by TLC on Silufol
UV 254 plates with elution by acetone:benzene (1:3). After the spot of the starting lactone disappeared (after several days), the
reaction mixture was evaporated in vacuo to form an oil that was dissolved in CH CN and analyzed by reversed-phase HPLC.
3
The pure components were separated bypreparative HPLC. Fractions were evaporated in vacuo to afford 3 and 4 as amorphous
powders (~1:1 ratio), mp 65-67°C (3), 75-77°C (4), yields 30% (for both). Table 1 gives the NMR spectra.
+
+
Compound 3. Mass spectrum (m/z, I, %): 377 (5) [M] , 333 (8) [M - CO ] , 247 (22), 234 (6), 218 (14), 203 (29),
2
187 (6), 174 (44), 160 (40), 144 (52), 130 (78), 108 (80), 83 (100), 77 (70), 56 (55), 43 (27). IR spectrum (cm- 1): 3335 br.
(NH), 2922 m, 2845 m, 1745 vs (C=O), 1636 m, 1455 m, 1351 w, 1200 m, 1069 m, 970 w, 737 m.
+
+
Compound 4. Mass spectrum (m/z, I, %): 377 (4) [M] , 333 (8) [M - CO ] , 247 (4), 217 (11), 203 (8), 175 (15), 160
2
(27), 143 (15), 130 (100), 108 (56), 83 (95), 56 (55), 43 (31). IR spectrum (cm- 1): 3300 br.s (NH), 2930 m, 2849 m, 2362 w,
1748 vs (C=O), 1640 m, 1455 m, 1339 m, 1250 m, 1200 m, 1073 m, 907 w, 741 s.
Stereospecific Amination of Securinine (general method). A mixture of 1 (1 mmol) and the amine (1.5 mmol) in
methanol (5mL)with ytterbium(III) trifluoromethanesulfonate hydrate (0.1 mmol) was stirred at room temperature. The course
of the reaction was monitored by TLC (Silufol UV 254; benzene:acetone, 3:1; R 0.55, 1; R ~0.30, amine derivative) and by
f
f
the disappearance of resonances of olefinic protons on 14¢C and 15¢C in the PMR spectrum. After the reaction was complete
(after several days), the mixture was evaporated in a rotaryevaporator. The dry solid was dissolved in benzene and passed over
a column of neutral Al O (5 g) to remove ytterbium salts. The product was purified by column chromatography over SiO
2
3
2
(benzene:acetone, 10:1).
The following compounds were prepared by this method.
3-{N-[(2b,15a )-14,15-Dihydro-11-oxosecurinan-15-yl]-2-aminoethyl}1H-indole (3). Yield 80%, mp 65-67°C.
(2b,15a )-14,15-Dihydro-15-(4-phenylpiperazin-1-yl)securinan-11-one (5). Yield 62%, mp 227-228°C. PMR
spectrum (CDCl , d, ppm, J/Hz): 2.07 (2H, d, J = 10.2, 8¢-H), 3.18 (4H, m, piperazine), 3.48 (1H, d, J = 6.5, H-7¢), 5.66 (1H,
3
+
d, J = 2.0, H-12¢), 6.85 (2H, m, o-H), 6.95 (2H, m, m-H), 7.30 (2H, m, p-H). Mass spectrum (m/z, I, %): 379 (6) [M] , 335 (11),
218 (8), 175 (100), 160 (38), 132 (64), 120 (85), 106 (45), 83 (62), 70 (55), 56 (54), 43 (38). IR spectrum (cm- 1): 2934 m,
2841 m, 1745 vs (C=O), 1640 m, 1598 m, 1501 m, 1447 m, 1231 s, 1200 m, 1150 m, 1069 m, 907 m, 752 m, 686 w.
(2b,15a )-14,15-Dihydro-15-[(4-(4-methoxyphenyl)piperazin-1-yl)]securinan-11-one (6). Yield 44%, mp 152-
154°C. PMR spectrum (CDCl , d, ppm, J/Hz): 1.97 (2H, d, J = 10.0, 8¢-H), 3.06 (4H, m, piperazine), 3.48 (1H, d, J = 6.8,
3
+
H-7¢), 3.75 (3H, s, OMe), 5.61 (1H, d, J = 2.0, H-12¢), 6.83 (4H, m, Ph). Mass spectrum (m/z, I, %): 409 (11) [M] , 365 (6),
288 (4), 217 (15), 205 (91), 192 (63), 174 (15), 162 (31), 150 (100), 134 (29), 120 (35), 106 (42), 83 (85), 70 (95), 55 (74), 45
(45), 43 (44). IR spectrum (cm- 1): 2930 m, 2814 m, 1745 vs (C=O), 1636 m, 1509 vs, 1451 m, 1246 s, 1030 m, 907 m,
822 m.
(2b,15a )-14,15-Dihydro-15-[4-(2-pyridyl)piperazin-1-yl)]securinan-11-one (7). Yield 89%, mp 267-268°C.
PMR spectrum (CDCl , d, ppm, J/Hz): 1.99 (2H, d, J = 10.4, H-8¢), 3.33 (2H, m, H-4¢), 3.45 (4H, m, piperazine), 5.60 (1H, d,
3
J = 1.8, H-12¢), 6.56 (1H, t, J = 5.5, J = 7.2, b-H), 6.67 (1H, d, J = 9.2, b¢-H), 7.44 (1H, ddd, J = 2.0, J = 7.23, J = 9.2, g-H),
1
2
1
2
3
+
8.15 (H, dd, J - 2.0, J = 5.5, a -H). Mass spectrum (m/z, I, %): 380 (9) [M] , 336 (11), 308 (4), 218 (14), 176 (100), 163 (18),
1
2
147 (70), 133 (26), 121 (78), 107 (69), 95 (67), 83 (74), 77 (60), 55 (56), 43 (42). IR spectrum (cm- 1): 2930 m, 2837 m, 1745
vs (C=O), 1640 m, 1590 s, 1482 s, 1439 s, 1200 m, 1154 m, 976 m, 907 m, 829 m, 771 s.
(2b,15a )-14,15-Dihydro-15-(pyrrolidin-1-yl)securinan-11-one (8). Yield 20%, viscous yellow oil. PMR spectrum
(CDCl , d, ppm, J/Hz): 1.99 (2H, d, J = 10.4, H-8¢), 2.87 (4H, m, pyrrolidine), 3.36 (1H, d, J = 6.5, H-14¢b), 3.46 (1H, d,
3
J = 6.5, H-14¢a ), 3.76 (1H, t, J = J = 7.0, 2¢-H), 4.27 (1H, dd, J = 4.0, J = 6.5, H-15¢), 5.73 (1H, d, J = 1.2, H-12¢). Mass
1
2
1
2
+
spectrum (m/z, I, %): 288 (3) [M] , 233 (44), 218 (45), 204 (38), 172 (35), 160 (42), 134 (45), 120 (37), 84 (100), 55 (57), 43
(31). IR spectrum (cm- 1): 2945 m, 2856 w, 2250 s, 1752 vs (C=O), 1632 w, 1454 w, 1250 w, 1901 w.
Biological activity of the compounds was investigated by an electrophysiological method on freshly isolated Purkinje
neurons from cerebellum of rats that were 12-15 days old. A modified method was used for the isolation. Slices of cerebellum
400-600 mm thick were placed in a 10-mL thermostatted chamber. The isolation solution contained (mM) NaCl (150.0), KCl
(5.0), CaCl (2.0), MgSO ·7H O (2.0), HEPES (10.0), and glucose (15.0) at pH 7.42. Slices were incubated in the solution for
2
4
2
201

60 min. Then, the solution was replaced by an analogous solution containing pronase (2 mg/mL, Sigma) and collagenase
(1 mg/mL, Sigma) and incubated for 70 min. After rinsing with the initial solution for 20 min, slices were placed in Petri dishes
and joined mechanicallyusing Pasteur pipettes. Solutions were continuouslypurged with O (100%) at 34°C. Purkinje neurons
2
were placed in the working chamber (0.6 mL) that contained (mM) NaCl (150.0), KCl (5.0), CaCl (2.6), MgSO ·7H O (2.0),
2
4
2
HEPES (10.0), and glucose (15.0) at pH 7.36.
Transmembrane currents were induced by activation of AMPA receptors through application of solutions of their
agonist cainic acid (CA). GABA-activated currents were induced by application of 5 or 10 mM g-aminobutyric acid using fast
superfusion. Currentswererecordedusingborosilicatemicroelectrodes(resistance2.0-4.5 mW) filled with a solution containing
(mM) KCl (100.0), EGTA (11.0), CaCl (1.0), MgCl (1.0), HEPES (10.0), and ATP (5.0) at pH 7.2.
2
2
The studied compounds at various concentrations wereplacedintotheexperimental chamber. Then, the corresponding
receptor agonists were applied and transmembrane currents were recorded. Mediators were applied repeatedly at a given
concentration in order to obtain a steady response. An EPC-9 instrument (HEKA, Germany) was used for the recording.
Currents were plotted using the Pulse program (HEKA, Germany). Results were processed by the Pulsefit program (HEKA,
Germany). The reliability of the differences was calculated using the Student criterion.
ACKNOWLEDGMENT
The work was supported financially by the Russian Foundation for Basic Research (Project 07-03-00669) and the
Program Basic Sciences, Medicine.
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