Synthesis of α,ω-Diketodiesters from Betulin

Article abstract of DOI:10.1007/s10600-021-03454-3

A new synthesis of 3-oxo-28-hydroxylup-20(29)-ene from the available natural triterpenoid betulin was developed. α,ω-Diketodiesters were prepared for the first time by different methods from 3-oxo-28-hydroxylup-20(29)-ene and a series of natural dicarboxylic acids. Steglich reaction conditions gave the highest yields. One of the synthesized α,ω-diketodiesters was moderately active in vitro against A-549 lung carcinoma.

Full text of DOI:10.1007/s10600-021-03454-3

DOI 10.1007/s10600-021-03454-3
Chemistry of Natural Compounds, Vol. 57, No. 4, July, 2021
SYNTHESIS OF α,ω-DIKETODIESTERS FROM BETULIN
1
1
1*
V. A. Vydrina, R. R. Sayakhov, M. P. Yakovleva,
2
3
1
Z. R. Zileeva, R. F. Talipov, and G. Yu. Ishmuratov
A new synthesis of 3-oxo-28-hydroxylup-20(29)-ene from the available natural triterpenoid betulin was
developed. α,ω-Diketodiesters were prepared for the first time by different methods from 3-oxo-28-hydroxylup-
2
0(29)-ene and a series of natural dicarboxylic acids. Steglich reaction conditions gave the highest yields.
One of the synthesized α,ω-diketodiesters was moderately active in vitro against A-549 lung carcinoma.
Keywords: betulin, 3-oxo-28-hydroxylup-20(29)-ene, dicarboxylic acids, [2+1]-reaction, α,ω-diketodiesters, synthesis.
Betulin esters are known to exhibit broad spectra of biological activity [1–3]. For example, betulin diacetate exhibits
antitumor, antiviral, and antioxidant properties while betulin mono- and diacetate and disuccinate exhibit hypolipidemic and
expectorant properties and possess hepatoprotective activity.
2
8-O-Esters of betulin can be prepared via acylation of 1 mol of betulin by 2 mol of 3,3-dimethyl-5-oxohexanoic or
-[1-(2-oxopropyl)cyclopentyl]acetic acid chlorides in Py at 40°C in the presence of 1 mol of 4-dimethylaminopyridine (DMAP)
4]. Although the esters are formed primarily at the primary hydroxyl (71 and 68% yields), the reaction mixtures contain
2
[
unreacted betulin and products from acylation at both hydroxyls (14 and 12%).
Reactions of betulin with dicarboxylic acid derivatives are limited to a few examples aimed mainly at preparing
3
,28-O-diesters. The reactions were conducted in Py in the presence of DMAP catalyst with a two-fold molar amount of
phthalic anhydride (76% yield) [5] and with ten-fold excesses of succinic, glutaric, or phthalic anhydrides (60–70% yields)
6–8] and by fusion of betulin with three equivalents of phthalic acid (87% yield) [9]. 28-O-Derivatives are selectively formed
by reacting betulin with a five-fold molar excess of maleic or succinic anhydride in CH Cl in the presence of DMAP catalyst
[
2
2
(
66 and 73% yields, respectively) [10] and by reacting 1 mol of betulin with 1.2 mol of succinic anhydride in N-methyl-2-
pyrrolidone in the presence of imidazole (73% yield) [11]. Products from acylation of both hydroxyls were also present in
these instances. This was probably related to the presence of DMAP as an esterification catalyst for secondary hydroxyls.
We hypothesized that acylation of betulin (1) by acid dichlorides without a catalyst would occur exclusively at the
primary OH. However, the reaction of 1 with sebacic acid dichloride in a 2:1 ratio formed a chromatographically inseparable
mixture of esterification products at the primary and secondary OH groups. The intensity ratio in PMR spectra of the doublet
of doublets for primary (3.18 ppm) and secondary (3.82 ppm) esters was 5:3. Therefore, the alcohols of 1 were differentiated
by converting it to the hydroxyketone 3-oxo-28-hydroxylup-20(29)-ene (2), which possesses valuable biologically active
properties such as antitumor, anti-inflammatory, antiparasitic, and antiviral, including anti-HIV, and cytotoxic activity against
various cancer cell lines [12].
Previously, 3-oxo-28-hydroxylup-20(29)-ene (2) was prepared from 1 via oxidation by oxygen over Au–TiO , La O
2
2
3
6
catalyst in mesitylene at 140°C (42% yield) [12]; CrO on SiO in CH Cl (9%) [13]; or K Cr O , H SO , Bu NBr in C H
3
2
2
2
2
2
7
2
4
4
6
(
12%) [14] and via hydrolysis of its ethers {tetrahydropyranyl, by pyridinium p-toluenesulfonate (PPTS) in EtOH (95%) [15]
and trityl, by TsOH in THF (58%) [16]} and the 28-O-acetate by LiOH in THF–H O (80%) [17], KOH in MeOH–THF–H O
2
2
mixtures (90%) [18], or C H –EtOH–H O (94%) [19], and NaOH in MeOH–THF–H O (50%) [11].
6
6
2
2
1
) Ufa Institute of Chemistry, Ufa Federal Research Center, RussianAcademy of Sciences, 71 Prosp. Oktyabrya, Ufa,
4
50054, e-mail: insect@anrb.ru; 2) Institute of Biochemistry and Genetics, Ufa Federal Research Center, RussianAcademy of
Sciences, 71 Prosp. Oktyabrya, Ufa, 450054; 3) Bashkir State University, 32 Zaki Validi St., Ufa, 450076. Translated from
Khimiya Prirodnykh Soedinenii, No. 4, July–August, 2021, pp. 603–607. Original article submitted January 22, 2021.
706
0009-3130/21/5704-0706 ©2021 Springer Science+Business Media, LLC

OH
O
a
b
9
5%
65%
HO
O
n
1
3
Cl
Cl
O
O
O
O
OH
c
n
A
O
O
O
O
HO
OH
B
n
O
O
O
n = 4, 6, 8, 10
2
4 - 7
d
4: n = 2 (40%, A; 58%, B); 5: n = 4 (42%, A; 86%, B); 6: n = 6 (43%, A; 80%, B); 7: n = 8 (50%, A; 88%, B)
a. PCC, CH Cl ; b. NaBH(OAc) , CH Cl ; c. Et N, CH Cl , DMAP, 40%; d. DCC, CHCl , DMAP
2
2
3
2
2
3
2
2
3
HO
OH
n
OTs
OTs
4
5
, 60%
O
O
, 68%
a
0%
b
1 7
6, 63%
, 68%
c
90%
7
HO
O
8
9
a. TsCl, Py; b. PCC; c. MeCN, K CO
2
3
A new method for preparing hydroxyketone 2 was proposed by us and consisted of Corey transformation of 1 into
betulonic aldehyde (3) (95% yield) and chemoselective reduction of its aldehyde in the presence of the ketone by sodium
tris-acetoxyborohydride [20, 21] (65% yield). The relatively low yield was probably explained by steric hindrance to approach
of the bulky reagent to the aldehyde.
Betulin α,ω-diketodiesters were prepared using several natural dicarboxylic acids, i.e., adipic, which occurs in sugar
cane and sugar beet juice; suberic, which occurs in the cork tree; sebacic, found in common castor oil; and lauric, a constituent
of coconut and palm oils. The [2+1]-condensation reaction of hydroxyketone 2 with these carboxylic acid dichlorides produced
the corresponding α,ω-diketodiesters, i.e., the di-[3-oxolup-20(29)-en-28-yl] esters of the acids (4–7), in yields up to 50%.
These derivatives of 3-oxo-28-hydroxylup-20(29)-ene (2) had not been obtained until now.
Higher yields (up to 88%) of the α,ω-diketodiesters (4–7) were obtained in [2+1]-condensation reactions of
hydroxyketone 2 with the above acids under Steglich reaction conditions [22].
Also, another synthetic pathway to the α,ω-diketodiesters (4–7) used mono-tosylation of starting betulin (1), Corey
oxidation of the intermediate mono-substituted diol 28-tosyloxy-20(29)-lupen-3β-ol (8), and [2+1]-condensation of the resulting
2
8-tosyloxy-20(29)-lupen-3-one (9) with these same acids in MeCN in the presence of potash.
All synthesized compounds, hydroxyketone 2 and α,ω-diketodiesters 4–7, were tested for cytotoxicity against
conditionally normal cells (HEK 293 human embryonic kidney cells) and tumor cell lines (A-549 lung carcinoma, MCF-7
breast adenocarcinoma, and SH-SY5Y neuroblastoma). The di-[3-oxolup-20(29)-en-28-yl] ester of decanedioic acid (6) was
moderately active against lung carcinoma cells (A-549) (IC50 49.1 ± 4.78 μM, p = 0.002). The other compounds were slightly
active or inactive.
Thus, an effective synthetic pathway to 3-oxo-28-hydroxylup-20(29)-ene starting from the available natural triterpenoid
betulin that was used to produce for the first time α,ω-diketodiesters with a series of natural dicarboxylic acids, one of which
was moderately active against A-549 lung carcinoma cells, was developed.
707

EXPERIMENTAL
Equipment of the Khimiya Center for Collective Use, UfIC, UFRC, RAS, was used in the work. IR spectra were
recorded from thin layers on an IR-Prestige-21 FTIR instrument (Shimadzu). NMR spectra were recorded in CDCl [CD(H)Cl
3
3
resonances as internal standards: δ 7.27 ppm, δC 77.00 ppm] on a Bruker AM-500 spectrometer (operating frequency
H
1
13
5
00.13 MHz for H and 125.76 MHz for C). TLC monitoring used Sorbfil SiO (Russia). APCI mass spectra were taken on
2
a 2010 EV LCMS (Shimadzu) (syringe injection, sample solution in MeCN at flow rate 100 μL/min) in positive- and
negative-ion modes. The interface temperature was 250°C, CDL 230°C, heater 200°C, nebulizer-gas (dried N ) flow
2
rate 1.5 L/min. Melting points were measured on a Kofler apparatus; optical rotation, on a PerkinElmer 141-MC polarimeter.
Elemental analyses agreed with those calculated. Compounds were synthesized and separated using 4-diemthylaminopyridine
(
DMAP). Solvents were prepared by well-known methods [23]. Pyridinium chlorochromate (PCC) was prepared by
the literature method [24].
-Oxolup-20(29)-en-28-al (3). A suspension of PCC (14.00 g, 65.0 mmol) in anhydrous CH Cl (200 mL)
3
2
2
was stirred (20°C, Ar), treated with 1 (5.00 g, 11.3 mmol), stirred for 5 h, diluted with anhydrous Et O (100 mL), and
2
filtered through a layer of Al O (5 cm). The precipitate was rinsed with anhydrous Et O (100 mL) and evaporated to afford
2
3
2
3
[
(4.71 g, 95%), mp 162–163°Ñ, lit.: 162–164°Ñ [25], 163–166°Ñ [26], 165–166°Ñ [27]. R 0.3 (PE–MTBE, 1:1);
f
2
1
25
25
α] +42.0° (ñ 1.50, CHCl ), lit.: [α] +47.7° (c 4.1, CHCl ) [25], [α] +33.8° (c 0.45, CHCl ) [4], [α] +52.4° (c 1.3,
D 3 D 3 D 3 D
1
3
CHCl ) [26]. IR, PMR, and C NMR spectra were analogous to those in the literature [25, 27, 28]; the mass spectrum, to the
3
reported one [25].
3
-Oxo-28-hydroxylup-20(29)-ene (2). A solution of glacial AcOH (7.56 g, 126.0 mmol) in anhydrous CH Cl
2 2
(
13 mL) was added to a suspension of NaBH (1.43 g, 42.0 mmol) in anhydrous CH Cl (64 mL). The mixture was stirred for
4 2 2
2
3
h, stirred at 10°C, and treated with 3 (4.00 g, 9.2 mmol). The reaction mixture was warmed to room temperature, stirred for
h, cooled to 10°C, and treated with a solution of NaOH (2.88 g, 72.0 mmol) in H O (64 mL). The organic layer was
2
separated, washed successively with saturated NH Cl solution and H O, dried over Na SO , and evaporated. The solid was
4
2
2
4
2
5
chromatographed (SiO , PE–MTBE, 5:1) to afford 2 (2.63 g, 65%), mp 95–96°Ñ, lit.: 94°Ñ [29]; [α] +52.9° (c 0.5, CHCl3),
lit.: [α] +52.7° (c 0.3, CHCl ) [16]. IR, PMR, C NMR, and mass spectra were analogous to those in the literature [15, 16].
2
D
2
5
13
D
3
2
8-Tosyloxy-20(29)-lupen-3β-ol (8). A solution of 1 (5.00 g, 11.3 mmol) in anhydrous Py (44 mL) was treated in
portions with TsCl (2.55 g, 13.4 mmol). The reaction mixture was stirred for 10 h, diluted with cold H O (20 mL), and
2
extracted with CHCl (3 × 100 mL). The organic layer was washed successively with HCl (2%) and saturated NaCl solution,
3
dried over MgSO , and evaporated. The solid was chromatographed (SiO , PE–MTBE, 5:1) to afford 8 (4.69 g, 70%),
4
2
2
5
25
13
[
α] +8.8° (c 0.98, CHCl ), lit. [α] +8.7° (c 1.08, CHCl ) [30]. IR, PMR, C NMR, and mass spectra were analogous to
D 3 D 3
those given before [30].
8-Tosyloxy-20(29)-lupen-3-one (9). A suspension of PCC (4.69 g, 21.8 mmol) in anhydrous CH Cl (67 mL) was
2
2
2
stirred (20°C, Ar), treated with 8 (4.50 g, 7.6 mmol), stirred for 2 h, diluted with anhydrous Et O (100 mL), and filtered
2
through a layer of Al O (5 cm). The solid was rinsed with anhydrous Et O (100 mL) and evaporated to afford 9 (4.08 g,
2
3
2
2
1
9
0%), [α] +30.5° (c 1.00, CHCl ), lit. [α] +30° (c 0.65, CHCl ) [31]. The IR spectrum was identical to the one in the
D 3 D 3
1
literature [31]. Í NMR spectrum (ÑDCl , δ, ppm, J/Hz): 0.80, 0.87, 0.89, 1.00, 1.05 (3Í each, s, ÑÍ ), 1.62 (3H, s, ÑÍ -30),
3
3
3
3
.73, 4.06 (2Í each, d, J = 9.4, Í-28), 4.55, 4.63 (2Í each, s, Í-29), OTs: 2.46 (3Í, s, ÑÍ ), 7.35 (2Í, d, J = 8.1, H-3′, 5′), 7.86
3
1
3
(
2Í, d, J = 8.1, H-2′, 6′). C NMR spectrum (ÑDCl , δ, ppm): 14.6 (C-27), 15.5 (C-26), 15.8 (C-25), 18.9, 21.0 (C-8′, 30),
3
1
9.5 (C-6), 21.1 (C-11), 21.6 (C-23), 24.9 (C-12), 26.4 (C-15), 26.5 (C-24), 29.0 (C-6), 29.1 (C-16), 29.7 (C-21), 33.3, 34.0
(
(
(
C-22, 7, 2), 36.7 (C-10), 37.7 (C-13), 39.5 (C-1), 40.5 (C-8), 42.6 (C-14), 46.6, 47.3 (C-4, 17), 47.5 (C-18), 48.5 (C-19), 49.5
C-9), 54.8 (C-5), 69.2 (C-28), 110.1 (C-29), 128.0 (C-3′, 7′), 130.0 (C-4′, 6′), 132.7 (C-2′), 144.7 (C-5′), 149.5 (C-20), 218.0
C-3).
General Method for Preparing Diketodiesters 4–7. a) A solution of 2 (2.50 g, 5.7 mmol) and anhydrous Py (3 mL,
2
.94 g, 37.2 mmol) in anhydrous CH Cl (25 mL) was stirred, treated with 2.8 mmol of dicarboxylic acid dichloride obtained
2 2
via reaction of adipic (0.41 g), suberic (0.49 g), sebacic (0.57 g), or lauric acid (0.64 g) with thionyl chloride (0.4 mL, 0.60 g,
5
4
.0 mmol) in anhydrous CH Cl (2 mL) followed by evaporation of the excess of thionyl chloride. The mixture was stirred for
8 h (TLC monitoring), diluted with MTBE (100 mL), washed successively with HCl (5%, 3 × 5 mL) and saturated NaCl
2 2
solution (3 × 5 mL), dried over MgSO , and evaporated. The solid was chromatographed (SiO , PE–Et O, 5:2).
4
2
2
b) A solution of 2.8 mmol of adipic (0.41 g), suberic (0.4 9g), sebacic (0.57 g), or lauric acid (0.64 g) and K CO
2
3
(
0.79 g, 5.7 mmol) in anhydrous MeCN (42 mL) was refluxed (Ar) for 1 h, stirred, treated dropwise with 9 (3.39 g, 5.7 mmol)
708

in anhydrous MeCN (42 mL), refluxed for 3 d, and evaporated. The resulting solid was dissolved in CH Cl , washed with
2
2
saturated NaCl solution (3 × 5 mL), dried over Na SO , and evaporated. The solid was chromatographed (SiO , PE–MTBE, 5:1).
2
4
2
c) A solution of 2.8 mmol of adipic (0.41 g), suberic (0.49 g), sebacic (0.57 g), or lauric acid (0.64 g) in anhydrous
CH Cl (5 mL) was stirred, treated with DMAP (0.04 g, 0.3 mmol) and hydroxyketone 2 (2.50 g, 5.7 mmol), cooled to 0°C,
2
2
and treated with DCC (0.63 g, 3.1 mmol). The reaction mixture was stirred for 5 min at 0°C and for 3 h at 20°C and filtered.
The filtrate was diluted with CH Cl (100 mL), washed successively with HCl (0.5 N, 2 × 5 mL) and saturated NaHCO
2
2
3
solution, dried over MgSO , and evaporated. The solid was chromatographed (SiO , PE–Et O, 5:2).
4
2
2
bis[3-Oxo-20(29)-lupen-28-oyl]hexanedioate (4), yield 2.25 g (40%) by method a; 3.38 g (60%), method b; 3.28 g
1
(
(
58%), method c; R 0.46 (PE–MTBE, 1:1). Í NMR spectrum (δ, ppm, J/Hz): 0.90 (3Í, s, Í-25), 0.95 (3Í, s, Í-27), 1.00
f
3Í, s, Í-24), 1.04, 1.05 (3Í each, s, Í-26, 23), 1.65 (3Í, s, Í-30), 2.35 (2Í, t, J = 7.5, H-2′), 3.82, 4.25 (1Í each, d,
J = 11.0, Í-28), 4.56, 4.66 (1Í each, br.s, Í-29). C NMR spectrum (ÑDCl , δ, ppm): 14.6 (Ñ-27), 15.8 (Ñ-26), 15.9 (Ñ-25),
1
3
3
1
3
4
9.1 (Ñ-6), 19.5 (Ñ-30), 21.0 (Ñ-23), 21.2 (Ñ-11), 24.4 (Ñ-3′), 25.1 (Ñ-12), 26.5 (Ñ-24), 26.9 (Ñ-15), 29.5 (Ñ-21), 29.7 (Ñ-16),
3.4 (Ñ-7), 34.0 (Ñ-2), 34.1 (Ñ-22), 34.5 (Ñ-2′), 36.8 (Ñ-10), 37.6 (Ñ-13), 39.5 (Ñ-1), 40.7 (Ñ-8), 42.7 (Ñ-14), 46.3 (Ñ-17),
7.3 (Ñ-4), 47.6 (Ñ-19), 48.6 (Ñ-18), 49.6 (Ñ-9), 54.9 (Ñ-5), 62.6 (Ñ-28), 109.9 (Ñ-30), 150.0 (Ñ-20), 173.7 (Ñ-1′), 218.0
+
+
(
Ñ-3). Mass spectrum (APCI, 20 eV), m/z (I , %) 991.78 (50) [M + H] , 1009.79 [M + H + H O] . Calcd for Ñ Í Î ,
rel 2 66 102 6
9
90.77.
bis[3-Oxo-20(29)-lupen-28-oyl]octanedioate (5), yield 2.44 g (42%) by method a; 3.95 g (68%), method b; 5.00 g
1
(
86%), method c. R 0.5 (PE–MTBE, 1:1). Í NMR spectrum (δ, ppm, J/Hz): 0.91 (3Í, s, Í-25), 0.96 (3Í, s, Í-27), 1.00 (3Í,
f
s, Í-24), 1.04, 1.05 (3Í each, s, Í-26, 23), 1.66 (3Í, s, Í-30), 2.30 (2Í, t, J = 7.5, H-2′), 3.82, 4.25 (1Í each, d, J = 11.0,
1
3
Í-28), 4.57, 4.67 (1Í each, br.s, Í-29). C NMR spectrum (ÑDCl , δ, ppm): 14.7 (Ñ-27), 15.8 (Ñ-26), 15.9 (Ñ-25), 19.1
3
(
1
(
2
Ñ-30), 19.6 (Ñ-6), 21.0 (Ñ-23), 21.3 (Ñ-11), 24.8 (Ñ-3′), 25.2 (Ñ-12), 26.6 (Ñ-24), 27.0 (Ñ-15), 28.8 (Ñ-4′), 29.6, 29.8 (Ñ-21,
6), 33.5 (Ñ-7), 34.1 (Ñ-2′), 34.4 (Ñ-2), 34.6 (Ñ-22), 36.8 (Ñ-10), 37.7 (Ñ-13), 39.6 (Ñ-1), 40.8 (Ñ-8), 42.7 (Ñ-14), 46.4
Ñ-17), 47.3 (Ñ-4), 47.7 (Ñ-19), 48.7 (Ñ-18), 49.7 (Ñ-9), 55.0 (Ñ-5), 62.5 (Ñ-28), 109.9 (Ñ-30), 150.0 (Ñ-20), 174.1 (Ñ-1′),
+
+
17.9 (Ñ-3). Mass spectrum (APCI, 20 eV), m/z (I , %) 1019.82 (50) [M + H] , 1037.83 [M + H + H O] . Calcd for
r
e
l
2
Ñ Í Î , 1018.80.
6
8 106 6
bis[3-Oxo-20(29)-lupen-28-oyl]decanedioate (6), yield 2.57 g (43%) by method a; 3.76 g (63%), method b, 4.77 g
1
(
80%), method c. R 0.5 (PE–MTBE, 1:1), Í NMR spectrum (δ, ppm, J/Hz): 0.86 (3Í, s, Í-25), 0.92 (3Í, s, Í-27), 0.96 (3Í,
f
s, Í-24), 1.00 (3Í, s, Í-26), 1.01 (3Í, s, Í-23), 1.63 (3Í, s, Í-30), 2.27 (2Í, t, J = 7.5, H-2′), 3.75, 4.20 (1Í each, d, J = 10.9,
1
3
Í-28), 4.52, 4.62 (1Í each, br.s, Í-29). C NMR spectrum (ÑDCl , δ, ppm): 14.7 (Ñ-27), 15.8 (Ñ-26), 15.9 (Ñ-25), 19.1
3
(
(
Ñ-29), 19.6 (Ñ-6), 21.0 (Ñ-23), 21.3 (Ñ-11), 25.0 (Ñ-3′), 25.2 (Ñ-12), 26.5 (Ñ-24), 27.0 (Ñ-15), 29.1 (Ñ-35), 29.5 (Ñ-34), 29.7
Ñ-21, 16), 33.4 (Ñ-7), 34.4 (Ñ-2), 34.5 (Ñ-22), 36.8 (Ñ-10), 37.6 (Ñ-13), 39.6 (Ñ-1), 40.8 (Ñ-8), 42.7 (Ñ-14), 46.4 (Ñ-17),
4
7.3 (Ñ-4), 47.6 (Ñ-19), 48.7 (Ñ-18), 49.7 (Ñ-9), 54.9 (Ñ-5), 62.4 (Ñ-28), 109.9 (Ñ-30), 150.0 (Ñ-20), 174.2 (Ñ-31), 218.0
+
+
(Ñ-3). Mass spectrum (APCI, 20 eV), m/z (I , %) 1047.84 (50) [M + H] , 1065.86 (100) [M + H + H O] . Calcd for Ñ Í Î ,
rel 2 70 110 6
1
046.83.
bis[3-Oxo-20(29)-lupen-28-oyl]dodecanedioate (7), yield 3.06 g (50%) by method a; 4.17 g (68%), method b;
1
5
1
.39 g (88%), method c. R 0.5 (PE–MTBE, 1:1), Í NMR spectrum (δ, ppm, J/Hz): 0.92 (3Í, s, Í-25), 0.97 (3Í, s, Í-27),
f
.00 (3Í, s, Í-23), 1.06 (6Í, s, Í-23, 26), 1.67 (3Í, s, Í-30), 2.30 (2Í, t, J = 7.5, H-2′), 3.83, 4.26 (1Í each, d, J = 11.0,
Í-28), 4.58, 4.67 (1Í each, br.s, Í-29). C NMR spectrum (ÑDCl , δ, ppm): 14.7 (Ñ-27), 15.8 (Ñ-26), 15.9 (Ñ-25), 19.2
1
3
3
(
2
(
2
Ñ-30), 19.6 (Ñ-6), 21.1 (Ñ-23), 21.3 (Ñ-11), 25.1, 25.2 (Ñ-3′, 12), 26.6 (Ñ-24), 27.1 (Ñ-15), 29.2, 29.2, 29.4 (Ñ-34, 36, 35),
9.7, 29.8 (Ñ-21, 16), 33.5 (Ñ-7), 34.1 (Ñ-2), 34.6 (Ñ-22), 36.9 (Ñ-10), 37.7 (Ñ-13), 39.6 (Ñ-1), 40.8 (Ñ-8), 42.8 (Ñ-14), 46.4
Ñ-17), 47.3 (Ñ-4), 47.7 (Ñ-19), 48.7 (Ñ-18), 49.7 (Ñ-9), 55.0 (Ñ-5), 62.4 (Ñ-28), 109.9 (Ñ-29), 150.1 (Ñ-20), 174.3 (Ñ-31),
+
+
18.0 (Ñ-3). Mass spectrum (APCI, 20 eV), m/z (I , %) 1075.87 (50) [M + H] , 1093.89 (100) [M + H + H O] . Calcd for
r
e
l
2
Ñ Í Î , 1074.86.
7
2 114 6
Biological Part. The cytotoxic properties of the compounds were determined in vitro using the MTT assay in
6-well plates [32]. Conditionally normal (HEK293, human enbryonic kidney) and tumor cells (A549 human lung carcinoma,
9
MCF-7 human breast adenocarcinoma, SH-SY5Y human neuroblastoma) were used in the work. All cell lines were obtained
from the Russian Cell Culture Collection, Institute of Cytology, Russian Academy of Sciences, St. Petersburg. HEK293
4
3
4
4
(
2.5 × 10 cells/well), A549 (5.0 × 10 cells/well), MCF-7 (1.2 × 10 cells/well), and SH-SY5Y cells (5 × 10 cells/well) were
cultivated in DMEM medium (Biolot, Russia) for 24 h in the presence of fetal bovine serum (10%, Invitrogen, USA),
L-glutamine (2 mM), and gentamycin sulfate (50 mg/mL). Wells were treated with the tested compound at final concentrations
of 1, 10, and 100 μM (in 0.1% DMSO), incubated for 48 h, and treated with MTT reagent. Optical density at 540 nm with the
709

measured background absorption at 600 nm subtracted was determined using a plate reader (2300 EnSpire® Multimode Plate
Reader, PerkinElmer, USA). The positive control for cytotoxicity studies was 5-fluorouracil, for which the IC50 value was
determined for each cell line (IC50 6.3 M for HEK293; 0.8,A549; 1.6, MCF-7; and 2.4, SH-SY5Y). The compound concentration
causing 50% suppression of cell viability (IC ) was determined by constructing dose-dependent curves using Graph Pad
5
0
Prism v.5.02 software (Graph Pad Software Inc., USA). Data obtained in two independent experiments were expressed as the
mean of three measurements for each concentration ± standard error of the mean vs. the control (0.1% DMSO) values taken as 100%.
ACKNOWLEDGMENT
The studies were financially supported by the RFBR in the framework of Science Project No. 20-33-90200.
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