Synthesis of the first dihydroquercetin-cytisine conjugates

Article abstract of DOI:10.1007/s10600-014-0982-0

The first mono- and disubstituted conjugates of the natural biologically active flavonoid dihydroquercetin and alkaloid cytisine were synthesized using a Mannich reaction.

Full text of DOI:10.1007/s10600-014-0982-0

Chemistry of Natural Compounds, Vol. 50, No. 3, July, 2014 [Russian original No. 3, May–June, 2014]
SYNTHESIS OF THE FIRST DIHYDROQUERCETIN–CYTISINE CONJUGATES
1*
1
1
N. V. Kosheleva, E. I. Chernyak, S. V. Morozov,
2
2
V. I. Vinogradova, Sh. Sh. Sagdullaev,
2
1
N. D. Abdullaev, and I. A. Grigorꢀev
The first mono- and disubstituted conjugates of the natural biologically active flavonoid dihydroquercetin
and alkaloid cytisine were synthesized using a Mannich reaction.
Keywords: dihydroquercetin, cytisine, Mannich reaction.
Chemical modification of natural biologically active compounds can expand their spectrum of activity and is a promising
direction in new drug discovery [1]. Flavonoids are a large group of natural biologically active compounds, among which
dihydroquercetin (DHQ) occupies a special place. It is isolated from Larix sibirica wood and exhibits powerful antioxidant,
hepatoprotective, antitumor, immunomodulating, and other properties [2]. The broad spectrum of biological activity and low
toxicity [2] classify DHQ as a lead compound for chemical modification in order to synthesize new hybrid polyfunctional
pharmacologically active compounds.
Alkaloids such as cytisine occupy a special place among numerous heterocyclic natural compounds. The
pharmacological properties of cytisine, like nicotine, are typical of ganglionic toxins that excite the CNS and autonomic
nervous system ganglia, reflexively strengthen breathing [3], and exhibit hypolipidemic activity [4]. Cytisine derivatives with
isoflavones and coumarins were obtained earlier [5]. The reaction of DHQ and its close structural analogs with primary and
secondary amines was used as an example to demonstrate [6, 7] that the Mannich reaction occurred at the 6-position of ring A
and produced primarily the mono-substituted derivative if equimolar amounts of the reagents were used; the disubstituted
derivative at the 6- and 8-positions, with a two-fold excess of amine.
The goal of the present work was to synthesize conjugates of DHQ (1) and cytisine (2) that were promising for
discovering new pharmacologically active polyfunctional drugs.
O
N
OH
5'
N
OH
OH
OH
OH
6'
2
HO
N
O
B
2'
8
11
1
OH
HO
O
O
2
HO
N
O
O
NH
H CO
2
13
A
+
+
4
OH
6
N
9
5
3
OH
OH
OH
O
6
OH
OH
O
1
2
3
4
N
N
O
O
Scheme 1
1) N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch, Russian Academy of Sciences,
630090, Novosibirsk, Prosp. Akad. Lavrentꢀeva, 9, e-mail: kosheleva@nioch.nsc.ru; 2) S. Yu. Yunusov Institute of the Chemistry
of Plant Substances, Academy of Sciences of the Republic of Uzbekistan, 100170, Tashkent. Translated from Khimiya Prirodnykh
Soedinenii, No. 3, May–June, 2014, pp. 383–385. Original article submitted December 29, 2013.
©
0009-3130/14/5003-0443 2014 Springer Science+Business Media New York
443

The reaction of 1, 2, and formaldehyde in a 1:1.4:1.4 mol ratio was carried out by adding a mixture of the reagents to
the substrate to produce a mixture of mono- and disubstituted 3 and 4 (Scheme 1) in an ~2:1 ratio (with an equimolar ratio of
reagents and ~10–15% DHQ). The product ratio was found using HPLC. Amixture of mono- and disubstituted DHQ derivatives
in an ~1:3 ratio was obtained if a two-fold excess of the reagents was used and the order of addition was reversed. Apparently,
disubstituted 4 formed because of the greater basicity of 2 compared with the previously used amines [6].
13
Compounds 3 and 4 were isolated from the reaction mixtures and characterized by IR, UV, PMR, and C NMR
spectroscopy and mass spectrometry.
–1
IR spectra of the synthesized compounds showed a strong broad band at 1643 cm that was due to cytisine and DHQ
carbonyls. The UV spectrum of monosubstituted 3 retained the absorption maximum (ꢁ ) at 290 nm that was characteristic
max
of DHQ. The characteristic DHQ band for disubstituted 4 shifted by 24 nm to longer wavelength. Mass spectra of 3 and 4
+
+
exhibited molecular ions [M + H] with m/z 507.185 and 709.285 and fragments [M + H – 190] with m/z 317.076 and
519.176 that corresponded to loss of cytisine. We observed earlier an analogous fragmentation of spin-labeled aminomethyl
13
DHQ derivatives [7]. Resonances in PMR and C NMR spectra were assigned taking into account the corresponding literature
data [6–8].
Thus, we synthesized for the first time mono- and disubstituted aminomethyl derivatives of DHQ with the alkaloid
cytisine.
EXPERIMENTAL
NMR spectra of DMSO-d solutions were recorded (relative to TMS) on a Bruker AV-400 spectrometer. The course
6
of reactions was monitored by TLC on Sorbfil 254 plates using EtOAc–MeOH–H O (10:2:1). IR spectra were recorded in
2
KBr pellets on a Vector-22 instrument. UV spectra were obtained during HPLC analysis. HPLC-UV analysis of the products
was performed on an Agilent 1100 chromatograph with a diode-matrix detector and a Zorbax XDB-C8 column (4.6 ꢂ 150 mm)
using MeOH–TFA (0.1%) mobile phase and a MeOH gradient from 10% to 90% over 30 min. HPLC-MS analysis of the
products was performed on an Agilent 1200 LC with a micrOTOF-Q hybrid quadrupole-TOF mass spectrometer (Bruker) and
a Zorbax XDB-C8 column (2.1 ꢂ 50 mm) using MeOH–HCOOH (2%) mobile phase and a MeOH gradient from 20% to 100%
over 20 min. The mass detector used electrospray at atmospheric pressure (API-ES) and scanning of negative and positive
ions in the range m/z 100–1200. The drying gas was N .
2
DHQ of purity 96–97% was purchased (Sibirskii Kedr Ltd.).
–4
Preparation of 3 and 4. Formaldehyde (as paraformaldehyde, 3.8 mg, 1.2 ꢂ 10 mol) and ZnCl (1.4 mg,
2
–6
8.5 ꢂ 10 mol) were refluxed in EtOH (1 mL) for 4–6 h until the paraformaldehyde dissolved, cooled to ~20–25°C, treated
–4
with cytisine (22.5 mg, 1.2 ꢂ 10 mol), and stirred until the cytisine dissolved. The resulting mixture was added dropwise
–5
with stirring over 15 min to a solution of DHQ (25 mg, 8.2 ꢂ 10 mol) in EtOH (0.6 mL) and stirred for 3.5 h. The solvent was
distilled off to afford a mixture of 3 and 4 in an ~2:1 ratio according to HPLC. The yields of 3 and 4 after separation and
purification were 55 and 35%, respectively.
–4
–6
Formaldehyde (as paraformaldehyde, 5.3 mg, 1.7 ꢂ 10 mol) and ZnCl (1.4 mg, 8.5 ꢂ 10 mol) were refluxed in
2
EtOH (1 mL) for 4–6 h until the paraformaldehyde dissolved, cooled to ~20–25°C, treated with cytisine (32 mg,
–4
1.7 ꢂ 10 mol), and stirred until the cytisine dissolved. The resulting mixture was treated dropwise with stirring over 15 min
–5
with a solution of DHQ (25 mg, 8.2 ꢂ 10 mol) in EtOH (0.6 mL) and stirred for 3.5 h. The solvent was distilled off. The
residue was dried to afford a mixture of 3 and 4 in an ~1:3 ratio according to HPLC. The yields of 3 and 4 after separation and
purification were 25 and 65%, respectively.
Separation and Purification of 3 and 4. A mixture of 3 and 4 (10 mg) was treated with EtOAc (5 mL), stirred at
~20–25°C, treated dropwise with HCl-saturated MeOH (0.2 mL, pH 2), stirred for 10 min, and centrifuged. The solid was
separated, dried, dissolved in H O (4 mL), treated with NaHCO solution (1%) until the pH was 7–8, treated with EtOAc
2
3
(4 mL), and shaken. The organic layer was separated. The aqueous layer was extracted with EtOAc (2 ꢂ 4 mL). The organic
extracts were combined and dried over MgSO (anhydr.). The solvent was distilled off to afford 3. The aqueous layer was
4
extracted with CHCl (2 ꢂ 4 mL). The organic layer was separated, dried over MgSO (anhydr.), and evaporated to afford 4.
3
4
3-{[3,5,7-Trihydroxy-2-(3,4-dihydroxyphenyl)chroman-4-on-6-yl]methyl}-1,2,3,4,5,6-hexahydro-1,5-methano-
8H-pyrido[1,2-ꢃ]diazocin-8-one (3). Pale-yellow powder, R 0.5 (EtÎAc–ÌåÎÍ–H O, 10:2:1), mp 199–202ꢄÑ. Mass
f
2
+
+
+
spectrum m/z 507.185 [Ì + H] , calcd for Ñ Í N Î
507.176 [Ì + H] . UV spectrum (ꢁ , nm): 290. IR spectrum
27 27
2
8 ,
max
444

–1
1
(KBr, ꢅ, cm ): 3406, 3256, 2939, 1643, 1547, 1479, 1450, 1391, 1358, 1283, 1163, 1126, 1090, 966, 804, 737, 685. Í NMR
spectrum (400 MHz, DMSO-d , ꢆ, ppm, J/Hz): à) cytisine: 1.72–1.83 (2Í, m, Í-13), 2.46 (1Í, br.s, Í-5), 2.42, 2.47, 2.90,
6
2.99 (each 1Í, d, J = 10.8, H -2, 4), 3.08 (1Í, br.s, Í-1), 3.68–3.80 (2Í, m, H-6), 6.09 (1H, dd, J = 7.0, 1.0, H-11), 6.22 (1H,
2
dd, J = 8.7, 1.2, H-9), 7.32 (1H, dd, J = 8.9, 7.1, H-10); b) 3.57 (2Í, br.s) – CH -6 methylene protons); c) DHQ: 4.50 (1H, dd,
2
J = 11.6, 4.0, H-3), 4.94 (1H, d, J = 11.4, H-2), 5.72 (1H, s, H-8), 5.75 (1H, d, J = 5.6, 3-ÎÍ), 6.73 (2H, s, H-5ꢀ, 6ꢀ), 6.85 (1H,
13
s, H-2ꢀ), 8.98, 9.05 (each 1H, s, 3ꢀ, 4ꢀ-OH), 11.92 (1H, s, 7-OH), 12.34 (1H, s, 5-OH). C NMR spectrum (100 MHz,
DMSO-d , ꢆ, ppm): à) cytisine: 24.68 (C-13), 26.99 (C-5), 34.12 (C-1), 49.23 (C-6), 58.59, 59.59 (C-2, 4), 104.17 (C-11),
6
115.91 (C-9), 138.80 (C-10), 150.90 (C-12), 162.19 (C-8); b) 50.87 – C-6 methylene C atom; c) DHQ: 71.49 (C-3), 83.03
(C-2), 94.83 (C-8), 99.97, 100.91 (C-4a, 6), 115.07 (C-2ꢀ), 115.40 (C-5ꢀ), 119.42 (C-6ꢀ), 128.02 (C-1ꢀ), 144.91 (C-3ꢀ), 145.78
(C-4ꢀ), 160.68 (C-8a), 161.41 (C-5), 167.18 (C-7), 198.14 (C-4).
3-{3-[(3,5,7-Trihydroxy)-2-(3,4-dihydroxyphenyl)chroman-4-on-8-ylmethyl]-(1,2,3,4,5,6-hexahydro-1,5-
methano-8H-pyrido[1,2-ꢃ]diazocin-8-one)-6-yl}methyl-1,2,3,4,5,6-hexahydro-1,5-methano-8H-pyrido[1,2-ꢃ]diazocin-
+
8-one (4). White powder, R 0.25 (EtÎAc–ÌåÎÍ–H O, 10:2:1), mp 205–208ꢄÑ. Mass spectrum m/z 709.285 [Ì + H] , calcd
f
2
+
+
–1
for Ñ Í N Î , 709.287 [Ì + H] . UV spectrum (ꢁ , nm): 314. IR spectrum (KBr, ꢅ, cm ): 3408, 3233, 2928, 1643,
1547, 1480, 1450, 1358, 1285, 1136, 1124, 1084, 993, 802, 752, 665. Í NMR spectrum (400 MHz, DMSO-d , ꢆ, ppm, J/Hz):
39 41
4
9
max
1
6
à) two cytisines: 1.64–1.81 (4Í, m, Í-13, 13ꢀ), 2.39 (2Í, br.s, Í-5, 5ꢀ), 2.25–2.33, 2.78–2.86 (each 4Í, br.m, Í -2, 2ꢀ, 4, 4ꢀ),
2
3.10 (2Í, br.s, Í-1, 1ꢀ), 3.73–3.83 (4Í, m, Í-6, 6ꢀ), 6.15, 6.20 (each 1Í, dd, J = 7.0, 1.0, Í-11, 11ꢀ), 6.24 (2H, m, Í-9, 9ꢀ), 7.34
(1Í, dd, J = 9, 6.5), 7.35 (2H, dd, J = 9, 6.5) – (2Í, Í-10, 10ꢀ); b) 3.44 (2Í, br.s), 3.48 (2Í, br.s) – ÑÍ -6, ÑÍ -8 methylene
2
2
protons; c) DHQ: 4.36 (1H, dd, J =11.3, 6.1, H-3), 4.86 (1H, d, J = 11.4, H-2), 5.73 (1H, d, J = 6.2, 3-ÎÍ), 6.69 (1Í, dd,
J = 7.4, 1.8, H-5ꢀ), 6.74 (1H, d, J = 8.1, Í-6ꢀ), 6.81 (1Í, d, J = 1.8, H-2ꢀ), 9.01, 9.11 (each 1H, br.s, 3ꢀ, 4ꢀ-OH), 12.36 (1H, s,
13
5-OH). C NMR spectrum (100 MHz, DMSO-d , ꢆ, ppm): à) two sets of cytisine resonances: 24.96, 25.02 (C-13, 13ꢀ), 27.32,
6
27.40 (C-5, 5ꢀ), 34.42, 34.47 (C-1, 1ꢀ), 49.66, 49.73 (C-6, 6ꢀ), 58.68, 58.87, 59.50, 59.69 (C-2, 2ꢀ, 4, 4ꢀ), 104.44, 104.61 (C-11,
11ꢀ), 115.66, 115.90 (C-9, 9ꢀ), 139.16, 139.31 (C-10, 10ꢀ), 151.63, 152.04 (C-12, 12ꢀ), 162.57, 162.62 (C-8, 8ꢀ); á) 49.77,
49.87 – C-6 and C-8 methylene C atoms; c) DHQ: 71.90 (C-3), 83.28 (C-2), 99.71, 100.92, 101.02 (C-6, 4a, 8), 115.09 (C-2ꢀ),
115.36 (C-5ꢀ), 119.66 (C-6ꢀ), 128.39 (C-1ꢀ), 145.27 (C-3ꢀ), 146.04 (C-4ꢀ), 159.50 (C-8à), 160.25 (C-5), 167.22 (C-7), 198.57 (C-4).
The spectral characteristics of the synthesized compounds were obtained at the Chemical Service, CCU, SB, RAS.
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