Synthesis of conjugates of lupane triterpenoids with chromane antioxidants and in vitro study of their influence on the production of nitrogen monoxide and on the arginase activity in activated macrophages

Article abstract of DOI:10.1007/s11172-010-0382-y

Conjugates of lupane triterpenoids (betulin and betulonic and betulinic acids) with synthetic analogs of α-tocopherol were obtained via ester bond formation and tested in vitro. Showing low cytotoxicity, some of them suppress nitrogen monoxide production

Full text of DOI:10.1007/s11172-010-0382-y

Russian Chemical Bulletin, International Edition, Vol. 59, No. 12, pp. 2219—2229, December, 2010
2219
Synthesis of conjugates of lupane triterpenoids with chromane antioxidants
and in vitro study of their influence on the production of nitrogen monoxide
and on the arginase activity in activated macrophages
A. Yu. Spivak,a R. R. Khalitova,a Yu. P. Bel´skii,b A. N. Ivanova,b E. R. Shakurova,a N. V. Bel´skaya,b
V. N. Odinokov,a M. G. Danilets,b and A. A. Ligachevab
aInstitute of Petrochemistry and Catalysis, Russian Academy of Sciences,
141 prosp. Oktyabrya, 450075 Ufa, Russian Federation.
Fax: +7 (347) 284 2750. Eꢀmail: ink@anrb.ru
bResearch Institute for Pharmacology,
Siberian Branch of the Russian Academy of Medical Sciences,
3 prosp. Lenina, 634028 Tomsk, Russian Federation.
Fax: +7 (382) 241 8379
Conjugates of lupane triterpenoids (betulin and betulonic and betulinic acids) with synthetꢀ
ic analogs of αꢀtocopherol were obtained via ester bond formation and tested in vitro. Showing
low cytotoxicity, some of them suppress nitrogen monoxide production without affecting the
activity of arginase, which suggests their antiinflammatory and immunomodulating properties.
Key words: lupane triterpenoids, antioxidants, chromanes, tocopherols, bioconjugates, nitroꢀ
gen monoxide, activated macrophages, antiinflammatory activity.
Nitrogen monoxide (NO) is generated enzymatically
in mammalian cells from Lꢀarginine under the action of
constitutive or inducible NO synthases (cNOS and iNOS,
respectively). This diatomic molecule fulfills very imporꢀ
tant physiological functions and contributes greatly to the
immune protection of living organisms from pathogenic
microorganisms and transformed cells.1 Under normal
physiological conditions, the concentration of NO in cells
and tissues is low. However, oxidative and toxic stresses
caused by macrophage activators such as microbial agents
and cytokines of T helpers (Th) increase the expression of
the inducible NO synthase in macrophages. This type of
macrophage activation is termed "classical activation" and
the macrophage phenotype is denoted M1. Cells M1 exꢀ
hibit proꢀinflammatory properties, support Th1ꢀdepenꢀ
dent immunologic reactions, produce proꢀinflammatory
cytokines (such as interleukinsꢀ1, ꢀ6, ꢀ12 and tumor neꢀ
crosis factor α), and very actively devour microorganisms,
destroy the extracellular matrix, and promote the apoptoꢀ
sis of infected and transformed cells.2—5
ry substances (interleukinꢀ10, an interleukinꢀ1 receptor
antagonist, which transforming the growth factor β), faꢀ
vor angiogenesis and remodeling, and produce chemoatꢀ
tractants for Th2 cells.2—5
It has been shown that the uptake of Lꢀarginine follows
only one pathway promoted by either NO synthase (giving
nitrogen monoxide and citrulline) or arginase (giving
polyamines and Lꢀornithine). This allowed one to considꢀ
er the arginine metabolism to be an index of the macroꢀ
phage activation type.6,7 Regulation of the activity balꢀ
ance for these enzymes is of great importance for an orꢀ
ganism since pathological excesses of nitrogen monoxide
(the concentration of NO increases ca. 103 times),8,9
a superoxide radical anion, and their reaction product (the
potent oxidant peroxynitrite) initiate oxidation of the lipids
of biological membranes, breaks of DNA chains, and other
mutagenic phenomena, which are believed to cause seriꢀ
ous diseases such as arthritis, diabetes, stroke, aseptic
shock, autoimmune diseases, and chronic inflammation.
In the last decade, active research has been undertaken
to find medicinal agents based on native substances that
can inhibit the production of nitrogen monoxide.10—15 In
this respect, lupane triterpenoids and some of their synꢀ
thetic derivatives are very promising.16,17
LꢀArginine is also a substrate for another enzyme (arꢀ
ginase 1) expressing on activated macrophages. The type
of macrophage activation that imparts functionally oppoꢀ
site (with respect to M1) properties to macrophages is
termed "alternative activation"; its inductors are glucoꢀ
corticoids and cytokines Th2 (see Ref. 2). Macrophages
M2 exhibit antiinflammatory activity, suppress Th1ꢀ and
enhance Th2ꢀimmune response, secrete antiinflammatoꢀ
We assumed that synthetic combinations of betulin,
betulinic acid, or betulonic acid with antioxidant moleꢀ
cules containing the pharmacophore chromane fragment
of αꢀtocopherol will help to discover novel broadꢀspecꢀ
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 12, pp. 2164—2174, December, 2010.
1066ꢀ5285/10/5912ꢀ2219 © 2010 Springer Science+Business Media, Inc.

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Russ.Chem.Bull., Int.Ed., Vol. 59, No. 12, December, 2010
Spivak et al.
trum compounds with NOꢀmodulating and antiradical
activities. Hybrid compounds with a trimethylated chroꢀ
mane fragment that showed themselves as efficient polyꢀ
functional agents have been reported earlier.18—20
In the present work, we obtained a number of acylates
of betulin and betulinic and betulonic acids with pharmaꢀ
cologically significant analogs of αꢀtocopherol and studꢀ
ied in vitro the influence of some of the hybrid compounds
on the NO production stimulated by lipopolysaccharides
(LPS) and on the activity of arginase in mouse peritoneal
macrophages. We also estimated their cytotoxic effects on
macrophages. In addition, we tested as NO production
inhibitors the earlier obtained conjugates of triterpene
acids with Trolox acid, in which a hydrazine residue acts
as a spacer.
4 in the presence of dicyclohexylcarbodiimide (DCC) and
dimethylaminopyridine (DMAP) as activators regioselecꢀ
tively gave compounds 6 and 7, respectively, in high yields
(Scheme 1). Acylate 8 was obtained by prolonged reflux
(48 h) of betulonyl chloride 1d with chromanol 5 in CH2Cl2
(see Scheme 1). In this case, NꢀethylꢀN´ꢀ(3ꢀdimethylꢀ
aminopropyl)carbodiimide (EDC) used in a double molar
excess with respect to the alcohol is preferred as a condenꢀ
sation agent.
However, the carbodiimide method failed in condenꢀ
sation reactions of chromane acids 2 and 3 with betulin or
betulinic acid. These reactions in the presence of various
activators (DCC, EDC, and diisopropylcarbodiimide
(DIC)) were substantially complicated by side processes
leading to complex mixtures of products.
Our attempted syntheses of acid chlorides from acids 2
and 3 were also unsuccessful: the action of COCl2 or SOCl2
on compounds 2 and 3 in various solvents (benzene, diꢀ
ethyl ether, and pyridine) resulted in decomposition of the
substrate molecules. The target acylates 9—11 were obꢀ
tained using 6ꢀOꢀprotected acids 2 and 3 and 28ꢀOꢀether
derivatives of betulin or betulinic acid. The protecting
groups were selected so that they could be removed from
acylates 9—11 in one step. Silyl ethers 12 and 13a were
obtained in three steps by transforming acids 2 and 3 into
the corresponding methyl esters, which reacted with tertꢀ
butyl(dimethyl)silyl chloride (TBSꢀCl) and imidazole to
give compounds 14 and 15a in good yields (Scheme 2).
The acid function in methyl carboxylates 14 and 15a
was deblocked by halogenolysis with LiBr in DMF as deꢀ
scribed earlier.13 Benzyl ether 13b was obtained from
acid 3 in a similar way (methanolysis of the acid funcꢀ
tion, benzylation of the phenolic hydroxyl, and halogenoꢀ
lysis of the ester group). Condensation of TBS ethers 12
and 13a with TBS ether of betulin (1e) and condensaꢀ
tion of 6ꢀOꢀbenzyl derivative 13b with benzyl betulinate 1f
(in the presence of DCC and DMAP as activators) affordꢀ
ed acylates 9—11, respectively, in 30—62% yields (see
Scheme 2).
Results and Discussion
For esterification of the functional groups at the C(3)
and C(28) atoms of lupane triterpenoids 1a—f, we used
the following chromaneꢀcontaining acids: 3ꢀ(6ꢀhydroxyꢀ
2,5,7,8ꢀtetramethylchromanꢀ2ꢀyl)propionic acid (2)
(αꢀCEHC), a waterꢀsoluble metabolite of αꢀtocopherol;
the accessible hydrophilic antioxidant 6ꢀhydroxyꢀ2,5,7,8ꢀ
tetramethylchromaneꢀ2ꢀcarboxylic acid (3) (Trolox acid);
and a nonhydrolyzable ether analog of αꢀtocopherol with
a redoxꢀpassive chromane fragment, referred to as αꢀtocoꢀ
pheryloxyacetic (2,5,7,8ꢀtetramethylꢀ2ꢀ(4,8,12ꢀtrimethylꢀ
tridecyl)chromanꢀ6ꢀyloxyethanoic) acid (4). The acid
function in betulonic acid was esterified with chromanylꢀ
methanol 5 prepared in three steps from acid 3 as deꢀ
scribed earlier.21
R1 = OH, R2 = H:
R3 = CH2OH (a),
COOH (b), COCl (c);
R
R
1 + R2 = O, R3 = COCl (d);
1 = OH, R2 = H:
R3 = CH2OTBS (e),
COOBn (f)
Compounds 8 and 11 were smoothly debenzylated by
their hydrogenation over Pd/C in diethyl ether for 2 and
3 h, respectively, yielding products 16 and 17 (Scheme 3). It
was found that hydrogenation of acylate 8 for 8 h results in
the reduction of the C(20)=C(29) double bond (cf. Ref. 22).
The conditions for removal of the TBS protection from
Acylation of the OH group at the C(28) atom in beꢀ
tulin 1a and at the C(3) atom in betulinic acid 1b with acid
n = 2 (2), 0 (3)

Conjugates of lupane and tocopherol
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 12, December, 2010
2221
Scheme 1
Reagents and conditions: a. DCC, DMAP (10 mol.%), CH2Cl2, 20 °C; b. EDC, CH2Cl2, reflux, 48 h.
acylates 9 and 10 were studied with compound 10 as an
example. Deblocking of the phenolic OH group in acylate
10 under the action of tetrabutylammonium fluoride
(TBAF) in THF at 0 °C gave silyl monoether 18a in 0.5 h.
The next TBS group at the C(28) atom in compound 18a
was removed at ~20 °C for 24 h, yielding compound 18b
(see Scheme 3).
The syntheses of conjugates 19 and 20 by reactions of
Trolox hydrazide 21 with betulinyl (1c) and betulonyl chloꢀ
rides (1d), respectively (Scheme 4), have been described
earlier.23
The structures of all the compounds obtained were
identified from spectroscopic data. Their 13C NMR specꢀ
tra show signals for all the C atoms belonging to the triterꢀ
penoid and chromanol residues. Since acid 3 and chromaꢀ
nylmethanol 5 were used as racemic mixtures, their
adducts with terpenoids were mixtures of two diastereoꢀ
mers. In some cases, this was evident from the presence of
1
a double set of signals in the H and 13C NMR spectra
(see, e.g., the 1H NMR spectra of compound 16b and the
13C NMR spectra of compound 18a).
Pharmacological studies were carried out in vitro for
compounds 6, 7, 19, and 20 versus betulinic (1b) and
αꢀtocopheryloxyacetic acids (4). The MTT assay data on
the cytotoxic effects of betulinic acid and hybrid comꢀ
pounds 19 and 20 are summarized in Table 1. Betulinic
acid was toxic in concentrations of 25 and 50 μg mL–1
,
while hybrid compounds 19 and 20 in the same concenꢀ
trations showed no cytotoxicity.

2222
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 12, December, 2010
Spivak et al.
Scheme 2
e
R = TBS (12, 13a, 14, 15a), Bn (13b, 15b)
n = 2 (2, 9, 12, 14); 0 (3, 10, 13a,b, 15a,b)
Reagents and conditions: a. TsOH—MeOH, CH2Cl2, reflux; b. TBSꢀCl—imidazole, DMF, 85 °C; c. BnCl—K2CO3, DMF, 20 °C;
d. LiBr, DMF, reflux; e. DCC, DMAP (10 mol.%), CH2Cl2, 20 °C.
Table 1. Cytotoxic effects of betulinic acid 1b and hybrid comꢀ
pounds 19 and 20 on peritoneal macrophages (X m)a
The influence of conjugates 6, 7, 19, and 20 and acids
1b and 4 on the functional state of macrophages was estiꢀ
mated from the NO production by LPSꢀactivated macꢀ
rophages and from the activity of macrophagal arginase.
To do this, we incubated the peritoneal macrophages of
mice of the line C57Bl/6 with LPS and the above compounds
in the concentrations specified in Table 2 for 48 h. After
the incubation was completed, we estimated the NO producꢀ
tion in the supernatants of cell cultures (from the niꢀ
trite content) and the activity of arginase in homogeꢀ
nate cells.
These studies showed that all the compounds tested
have an effect on the NO production by LPSꢀstimulated
macrophages (see Table 2). Betulinic (1b) and αꢀtocopherylꢀ
oxyacetic acids (4) decreased the NO production in a conꢀ
centration range from 0.1 to 50 μg mL–1, depending on the
dose used. Hybrid compounds 6, 7, 19, and 20 had an inꢀ
hibitive effect in higher concentrations (25 and 50 μg mL–1).
Concentration
/μg mL–1
Optical densityb
1b
19
20
c
—
—
0.403 0.031
0.448 0.034
0.448 0.021
0.459 0.047
0.458 0.045
0.478 0.028
0.484 0.044
—
0.01
0.447 0.029
0.445 0.019
0.452 0.030
0.332 0.049
0.237 0.020c
0.273 0.046c
0.411 0.017
0.357 0.014
0.455 0.047
0.467 0.058
0.477 0.042
0.503 0.012d
0.10
1.00
10.00
25.00
50.00
a Here and in Tables 2 and 3, X is the mean of experimental data
and m is the standard mean error.
b The optical density in the wells containing only macrophages
(without LPS) was 0.425 0.049.
c Blank entry.
d The differences from the blank entry are reliable for p < 0.05.

Conjugates of lupane and tocopherol
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 12, December, 2010
2223
Scheme 3
a
b
R = C(Me)=CH2 (16a), CHMe2 (16b), CH2OTBS (18a), CH2OH (18b)
Reagents and conditions: a. H2, Pd/C, Et2O; b. TBAF, THF.
The study of the influence of these compounds on the
arginase activity (Table 3) revealed that betulinic (1b) and
αꢀtocopheryloxyacetic acids (4) decrease the arginase acꢀ
tivity in concentrations of 10, 25, and 50 μg mL–1. Conjuꢀ
Table 2. Influence of betulinic acid 1b, αꢀtocopheryloxyacetic acid (4), and hybrid compounds 6, 7, 19, and 20 on
the NO production by peritoneal macrophages (X m)a
Concentration
/μg mL–1
Concentration of nitrites (μmol L–1) in the presence of compound
1b
4
6
7
19
20
b
—
47.8 1.0c
0.1
1.0
10.0
25.0
50.0
44.8 0.3d
41.2 0.9d
29.2 1.2d
5.9 0.5d
1.4 0.8d
43.50 1.2
41.35 1.9d
14.81 0.6d
4.44 0.6d
0.38 0.3d
54.20 2.5
48.46 1.7
38.59 2.1d
28.66 0.3d
26.41 3.1d
46.39 1.0
44.22 0.6
43.71 1.8
42.36 0.7d
38.35 0.6d
47.7 0.7
47.8 0.4
42.8 1.4d
34.4 1.1d
13.1 1.3d
45.0 1.4
47.7 0.8
44.1 1.7
40.6 2.1d
19.8 1.2d
a The concentration of nitrites in the wells containing only macrophages (without LPS) was 7.5 1.8 μmol L–1
b Blank entry.
c The differences from the entry with LPSꢀfree culture for p < 0.05.
d The differences from the blank entry are reliable for p < 0.05.
.

2224
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 12, December, 2010
Spivak et al.
Scheme 4
Reagents and conditions: EDC, CH2Cl2, reflux, 48 h.
gates 6, 7, 19, and 20 did not affect the arginase activity in
these concentrations.
immune response by suppressing M1. Because the comꢀ
pounds obtained have lower cytotoxicity (compared to the
starting acids) and selectivity toward activated macroꢀ
phages, these compounds can probably serve as a basis in
the design of antiinflammatory and immunocorrecting
drugs for efficient control of various autoimmune diseases
(rheumatoid arthritis and type I diabetes).
Thus, in contrast to betulinic acid, the hybrid comꢀ
pounds showed no cytotoxicity in the MTT assay toward
mouce peritoneal macrophages. Betulinic and αꢀtocoꢀ
pheryloxyacetic acids suppressed both the NO production
(feature M1) and the arginase activity (feature M2), which
can also be attributed to their direct cytotoxic effects. Unꢀ
like the starting acids 1b and 4, the hybrid compounds
were not cytotoxic; they selectively influenced macroꢀ
phages, suppressing the NO production without changing
the arginase activity (in concentrations below 50 μg mL–1).
Based on the results obtained, one can state that comꢀ
pounds 6, 7, 19, and 20 exhibit antiinflammatory effect.
Therefore, these compounds can weaken the Th1ꢀtype of
Experimental
IR spectra were recorded on a Specord IRꢀ75 spectrometer
(thin films or solutions in CHCl3). UV spectra were recorded on
a Specord Mꢀ40 spectrometer. 1H and 13C NMR spectra were
recorded on a Bruker AVANCEꢀ400 instrument (400.13 (1H)
and 100.62 MHz (13C)) in CDCl3 with Me4Si as the internal
Table 3. Influence of betulinic acid 1b, αꢀtocopheryloxyacetic acid (4), and hybrid compounds 6, 7, 19, and
20 on the arginase activity in peritoneal macrophages (X m)a
Concentration
/μg mL–1
Arginase activity (in conventional units) in the presence of compound
1b
4
6
7
19
20
b
—
31.2 1.6
1
32.0 1.0
21.7 1.2c
2.4 0.8c
0.3 0.3c
31.1 1.0
19.0 0.3c
4.3 0.9c
1.1 0.6c
33.7 1.0
32.7 1.2
27.1 1.7
20.5 1.6c
31.6 1.7
29.2 1.0
29.5 0.4
25.5 1.2c
31.8 2.7
31.3 0.7
31.3 1.1
29.5 0.7
32.1 1.6
30.2 1.0
27.9 1.2
13.5 0.7c
10
25
50
a The arginase activity in the wells containing only macrophages (without LPS) was 29.7 1.28.
b Blank entry.
c The differences from the blank entry are reliable for p < 0.05.

Conjugates of lupane and tocopherol
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 12, December, 2010
2225
standard. Mass spectra were measured on a BrukerꢀAutoflex III
spectrometer (MALDI TOF, positive ion mode, 2,5ꢀdihydroxyꢀ
benzoic and αꢀcyanoꢀ4ꢀhydroxycinnamic acids as the matrices).
Optical rotation was measured on a Perkin—Elmerꢀ141 polaꢀ
λ
max/nm (ε): 286 (2092). 1H NMR, δ: 1.69 (m, 3 H, Me—C(2));
1.94—2.00 (m, 1 H, H(3)); 2.23, 2.26, 2.32 (all s, 3 H each,
Me—Ar); 2.47—2.50 (m, 1 H, H(3)); 2.80—2.95 (m, 2 H, H(4));
4.81 (s, 2 H, OCH2Ph); 7.28—7.64 (m, 5 H, Ph); 8.13 (s, 1 H,
COOH). 13C NMR, δ: 11.96, 12.08, 12.97 (Me—Ar); 20.75
(C(4)); 25.21 (Me—C(2)); 30.16 (C(3)); 74.74 (OCH2Ph); 76.94
(C(2)); 117.28 (C(5)); 123.02 (C(7)); 126.20 (C(8)); 127.12,
127.81, 128.55, 137.92 (Ph); 127.71 (C(4a)); 147.52 (C(8a));
149.13 (C(6)); 179.62 (COOH).
rimeter. Specific rotation was expressed in (deg mL) (g dm)–1
the concentration of the solution was expressed in g (100 mL)–1
;
.
Elemental analysis was carried out on a Carlo Erba 1106 analyzꢀ
er. TLC was carried out on Sorbfil plates (Sorbpolimer, Krasnoꢀ
dar, Russia) in hexane—ethyl acetate (3 : 1) (A) and chloroform
(B); spots were visualized in a solution of anisaldehyde in ethaꢀ
nol. For column chromatography, silica gel L (KSKG grade,
50—160 μm) was employed. Racemic αꢀtocopherol, DCC, EDC,
DMAP, TBAF, TBSꢀCl, imidazole, Trolox, 10% Pd/C, and
oxalyl chloride (Fluka) were used. Betulin was isolated in a known
way.24 Betulonic acid, betulinic acid, and betulonyl chloride
were prepared as described earlier.25—27 Chromanylpropionic
acid 2 was prepared according to a known procedure.28 Chroꢀ
manylmethanol 5 was prepared from Trolox acid in three steps;21
TBS ethers 1e (see Ref. 29), 14, and 15a (see Ref. 30), αꢀtocoꢀ
pheryloxyacetic acid (4),31 and benzyl betulinate 1f (see Ref. 22)
were prepared according to the corresponding procedures.
Halogenolysis of methyl carboxylates 14 and 15a,b with LiBr
(general procedure). Compound 14 or 15a,b (0.3 mmol) and
LiBr (4.4 mmol) were dissolved in dry DMF (2.5 mL) and reꢀ
fluxed with stirring for 6 h (monitoring by TLC in system A). The
reaction mixture was cooled, diluted with water (2.5 mL), and
neutralized with 5% HCl; the product was extracted with AcOEt.
The extract was concentrated and the residue was chromatoꢀ
graphed on SiO2 with hexane—AcOEt (3 : 1) as an eluent to give
compounds 12 and 13a,b, respectively.
3ꢀ[6ꢀtertꢀButyl(dimethyl)silyloxyꢀ2,5,7,8ꢀtetramethylꢀ3,4ꢀ
dihydroꢀ2Hꢀ1ꢀbenzopyranꢀ2ꢀyl]propionic acid (12). Yield 82%,
amorphous powder. Found (%): C, 67.13; H, 9.31; Si, 7.22.
C22H36O4Si. Calculated (%): C, 67.30; H, 9.24; Si, 7.15. IR,
ν/cm–1: 1710 (COOH). UV (CHCl3), λmax/nm (ε): 294 (2588).
1H NMR, δ: 0.12 (s, 6 H, Me—Si); 1.06 (s, 9 H, Me in But); 1.24
(s, 3 H, Me—C(2)); 1.77—1.92 (m, 2 H, H(1´) + 2 H, H(3));
2.04, 2.06, 2.10 (all s, 3 H each, Me—Ar); 2.52—2.61 (m, 2 H,
H(2´) + 2 H, H(4)); 8.04 (s, 1 H, COOH). 13C NMR, δ: –3.36
(Me—Si); 11.92, 13.39, 14.31 (Me—Ar); 18.59 (But—Si); 20.71
(C(4)); 23.32 (Me—C(2)); 26.09 (Me in But); 28.58 (C(3)); 31.66
(C(2´)); 34.34 (C(1´)); 73.34 (C(2)); 117.16 (C(5)); 122.72 (C(7));
123.60 (C(8)); 126.01 (C(4a)); 144.32 (C(8a)); 145.44 (C(6));
179.07 (COOH).
6ꢀtertꢀButyl(dimethyl)silyloxyꢀ2,5,7,8ꢀtetramethylꢀ3,4ꢀdiꢀ
hydroꢀ2Hꢀ1ꢀbenzopyranꢀ2ꢀcarboxylic acid (13a). Yield 72%,
amorphous powder. Found (%): C, 65.74; H, 8.92; Si, 7.78.
C20H32O4Si. Calculated (%): C, 65.89; H, 8.85; Si, 7.70. IR,
ν/cm–1: 1720 (COOH). UV (CHCl3), λmax/nm (ε): 292 (2700).
1H NMR, δ: 0.13 (s, 6 H, Me—Si); 1.06 (s, 9 H, Me in But); 1.62
(s, 3 H, Me—C(2)); 1.85—1.93 (m, 1 H, H(3)); 2.04, 2.12, 2.15
(all s, 3 H each, Me—Ar); 2.36—2.42 (m, 1 H, H(3)); 2.60
(m, 2 H, H(4)); 8.05 (m, 1 H, COOH). 13C NMR, δ: –3.45,
–3.30 (Me—Si); 12.01, 13.39, 14.31 (Me—Ar); 18.59 (But—Si);
20.96 (C(4)); 25.10 (Me—C(2)); 26.09 (Me in But); 30.28 (C(3));
76.74 (C(2)); 117.18 (C(5)); 122.61 (C(7)); 123.53 (C(8)); 126.12
(C(4a)); 144.87 (C(8a)); 145.70 (C(6)); 177.65 (COOH).
6ꢀBenzyloxyꢀ2,5,7,8ꢀtetramethylꢀ3,4ꢀdihydroꢀ2Hꢀ1ꢀbenzoꢀ
pyranꢀ2ꢀcarboxylic acid (13b). Yield 73%, amorphous powder.
Found (%): C, 74.18; H, 7.06. C21H24O4. Calculated (%):
C, 74.09; H, 7.11. IR, ν/cm–1: 1718 (COOH). UV (CHCl3),
(6ꢀBenzyloxyꢀ2,5,7,8ꢀtetramethylꢀ3,4ꢀdihydroꢀ2Hꢀ1ꢀbenzoꢀ
pyranꢀ2ꢀyl)methyl 3ꢀoxolupꢀ20(29)ꢀenꢀ28ꢀoate (8). NꢀEthylꢀN´ꢀ
(3ꢀdimethylaminopropyl)carbodiimide (0.23 mL, 1.28 mmol)
and freshly prepared betulonyl chloride 1d (0.48 g, 1.04 mmol)
in dry CH2Cl2 (5 mL) were added dropwise to a stirred solution
of benzylated chromanylmethanol (5) (0.21 g, 0.64 mmol) in dry
CH2Cl2 (5 mL). The reaction mixture was refluxed with stirring
for 48 h, diluted with CH2Cl2 (10 mL), washed with water, dried
over MgSO4, and concentrated in vacuo. The residue was chroꢀ
matographed on SiO2 with CHCl3 as an eluent. The yield was
20
57%, white crystals, m.p. 96—98 °C, [α]D +18.5° (c 0.27,
CHCl3). Found (%): C, 80.19; H, 9.31. C51H70O5. Calculatꢀ
ed (%): C, 80.27; H, 9.25. IR, ν/cm–1: 1725 (C=O). UV (CHCl3),
λ
max/nm (ε): 288 (2000). 1H NMR, δ: 0.92, 1.00, 1.02, 1.06, 1.11
(all s, 3 H each, H(23´), H(24´), H(25´), H(26´), H(27´));
1.25—2.60 (m, 24 H, CH2, CH in the betulonic acid residue
+ 5 H, Me—C(2), H(3) in the chromanol residue); 1.73 (s, 3 H,
H(30´)); 2.14, 2.21, 2.26 (all s, 3 H each, Me—Ar); 2.68 (t, 2 H,
H(4), J = 6.4 Hz); 3.08 (m, 1 H, H(19´)); 4.08, 4.20 (both d,
1 H each, CH2—O, J = 12.2 Hz); 4.62, 4.75 (both s, 1 H each,
H(29´)); 4.70 (s, 2 H, OCH2Ph); 7.28—7.54 (m, 5 H, Ph).
13C NMR, δ: 11.90, 12.04, 12.90 (Me—Ar); 14.68 (C(27´)); 15.84
(C(25´)); 15.99 (C(26´)); 19.41 (C(6´)); 19.68 (C(30´)); 20.26
(C(4)); 21.07 (C(24´)); 21.47 (C(11´)); 22.40 (Me—C(2)); 25.59
(C(12´)); 26.65 (C(23´)); 28.85, 28.95 (C(3)); 29.73 (C(15´));
30.66 (C(21´)); 32.21 (C(16´)); 33.67 (C(7´)); 34.15 (C(2´));
36.92 (C(10´)); 37.08 (C(22´)); 38.49 (C(13´)); 39.66 (C(1´));
40.68 (C(8´)); 42.52 (C(14´)); 47.08 (C(19´)); 47.34 (C(4´));
49.39 (C(18´)); 49.93 (C(9´)); 55.00 (C(5´)); 56.77 (C(17´));
68.44, 68.63 (CH2—O); 74.76 (CH2—Ph); 76.81 (C(2)); 109.79
(C(29´)); 117.19 (C(5)); 123.04, 126.04, 128.28 (C(4a), C(7),
C(8)); 127.71, 127.84, 128.50, 137.94 (Ph); 147.31 (C(8a));
148.63 (C(6)); 150.38 (C(20´)); 175.96 (COO); 217.98 (C(3´)).
MS, m/z: 786.151 [M + Na]+.
Synthesis of acylates 6, 7, and 9—11 (general procedure).
Dimethylaminopyridine (0.03 mmol), an appropriate compound
4, 12, or 13a,b (0.27 mmol) (see Schemes 1 and 2), and DCC
(0.27 mmol) were added with stirring to compound 1a,b,e,f
(0.24 mmol) in dry CH2Cl2 (5 mL). The reaction mixture was
stirred at ~20 °C for 12—20 h, filtered, and concentrated in vacuo.
The residue was chromatographed on SiO2 with CHCl3 as an
eluent to give compounds 6, 7, and 9—11, respectively.
28ꢀ{[2,5,7,8ꢀTetramethylꢀ2ꢀ(4,8,12ꢀtrimethyltridecyl)ꢀ3,4ꢀ
dihydroꢀ2Hꢀ1ꢀbenzopyranꢀ6ꢀyloxy]acetoxy}lupꢀ20(29)ꢀenꢀ3βꢀol
(6). Yield 63%, amorphous powder, [α]D20 +6.9° (c 4.97, CHCl3).
Found (%): C, 80.15; H, 11.09. C61H100O5. Calculated (%):
C, 80.21; H, 11.03. IR, ν/cm–1: 1759 (O—C=O). UV (CHCl3),
λ
max/nm (ε): 289 (2438). 1H NMR, δ: 0.84—0.98 (m, 12 H,
Me—C(4″), Me—C(8″), Me—C(12″)); 0.98, 1.00, 1.12 (all s,
3 H each, H(23´), H(24´), H(25´), H(26´), H(27´)); 1.20—2.17
(m, 24 H, CH2, CH in the betulin residue + 26 H, Me—C(2),
CH2, CH in the tocopherol residue); 1.70 (s, 3 H, H(30´)); 2.09,

2226
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 12, December, 2010
Spivak et al.
2.16, 2.18 (all s, 3 H each, Me—Ar); 2.50 (m, 1 H, H(19´)); 2.52
(t, 2 H, H(4), J = 6.5 Hz); 3.20 (dd, 1 H, H(3´)); 4.12—4.45
(both d, 1 H each, C(28´), J = 11.2 Hz); 4.32 (d, 2 H, CH2—O,
J = 3.2 Hz); 4.45 (s, 1 H, OH); 4.62, 4.75 (both s, 1 H each,
H(29´)). 13C NMR, δ: 11.78, 11.92, 12.79 (Me—Ar); 14.80
(C(27´)); 15.40 (C(24´)); 16.11 (C(25´)); 16.12 (C(26´)); 18.30
(C(6´)); 19.63 (C(30´)); 19.70, 19.77 (Me—C(4″), Me—C(8″));
20.63 (C(11´)); 20.80, 21.03 (C(4)); 22.65, 22.74 (Me—C(12´));
23.87 (C(2″)); 24.44 (C(6″), C(10″)); 24.82 (C(12´)); 25.21
(Me—C(2)); 27.10 (C(15´)); 27.42 (C(2´)); 27.98 (C(12″)); 28.00
(C(23´)); 29.59 (C(16´)); 29.78 (C(21´)); 31.21 (C(3)); 32.69,
32.77 (C(4″), C(8″)); 34.21 (C(22´)); 34.55 (C(7´)); 37.15 (C(3″));
37.29 (C(10´), C(5″)); 37.41 (C(7″)); 37.46 (C(9″), C(13´)); 38.73
(C(1´)); 38.86 (C(4´)); 40.02 (C(11″)); 40.90 (C(8´), C(1″)); 42.72
(C(14´)); 46.52 (C(17´)); 47.70 (C(18´)); 48.84 (C(19´)); 50.39
(C(9´)); 55.32 (C(5´)); 63.37 (C(28)); 70.07 (CH2—O); 74.87
(C(2)); 78.93 (C(3´)); 109.95 (C(29´)); 117.62 (C(5)); 123.02
(C(7)); 125.63 (C(8)); 127.56 (C(4a)); 147.93 (C(8a)); 148.19
(C(6)); 150.01 (C(20´)); 169.85 (COO). MS, m/z: 913.591 [M]+,
936.599 [M + Na]+, 952.549 [M + K]+.
(all s, 3 H each, Me—Ar); 2.40 (m, 2 H, H(2″)); 2.50 (m, 1 H,
H(19´)); 2.62 (t, 2 H, H(4), J = 6.5 Hz); 3.26, 3.70 (both d, 1 H
each, CH2—O, J = 9.2 Hz); 4.50 (m, 1 H, H(3´)); 4.60, 4.71
(both s, 1 H each, H(29´)). 13C NMR, δ: –5.43, –3.34 (Me—Si);
11.99, 13.40, 14.33 (Me—Ar); 14.74 (C(27´)); 15.90 (C(24´));
16.16 (C(25´)); 16.57 (C(26´)); 18.17, 18.33 (But—Si); 18.60
(C(6´)); 19.11 (C(30´)); 20.76, 20.88 (C(4)); 23.69 (C(11´)); 23.70
(Me—C(2)); 25.22 (C(12´)); 25.99, 26.12 (Me in But); 27.03
(C(2´)); 27.44 (C(15´)); 28.01 (C(23´), (C(3)); 29.20 (C(16´));
29.93 (C(21´)); 31.66 (C(2″)); 34.13 (C(22´)); 34.33 (C(7´));
34.67 (C(1″)); 37.07 (C(10´)); 37.37 (C(13´)); 37.87 (C(1´));
38.37 (C(4´)); 40.94 (C(8´)); 42.68 (C(14´)); 48.06 (C(18´),
C(17´)); 48.39 (C(19´)); 50.32 (C(9´)); 55.38 (C(5´)); 60.45
(C(28´)); 73.48 (C(2)); 80.85 (C(3´)); 109.43 (C(29´)); 117.23
(C(5)); 122.71 (C(7)); 123.59 (C(8)); 125.99 (C(4a)); 144.29
(C(8a)); 145.52 (C(6)); 150.89 (C(20´)); 173.77 (COO). MS,
m/z: 931.632 [M]+, 954.659 [M + Na]+.
28ꢀ[tertꢀButyl(dimethyl)silyloxy]ꢀ3ꢀ({6ꢀ[tertꢀbutyl(diꢀ
methyl)silyloxy]ꢀ2,5,7,8ꢀtetramethylꢀ3,4ꢀdihydroꢀ2Hꢀ1ꢀbenzoꢀ
pyranꢀ2ꢀyl}carbonyloxy)lupꢀ20(29)ꢀene (10). Yield 62%, white
20
3βꢀ{[2,5,7,8ꢀTetramethylꢀ2ꢀ(4,8,12ꢀtrimethyltridecyl)ꢀ3,4ꢀ
dihydroꢀ2Hꢀ1ꢀbenzopyranꢀ6ꢀyloxy]acetoxy}lupꢀ20(29)ꢀenꢀ28ꢀ
oic acid (7). Yield 81%, amorphous powder, [α]D20 +9.35° (c 1.07,
CHCl3). Found (%): C, 78.83; H, 10.69. C61H98O6. Calculatꢀ
ed (%): C, 79.00; H, 10.65. IR, ν/cm–1: 1732 (CO—OH). UV
(CHCl3), λmax/nm (ε): 289 (2649). 1H NMR, δ: 0.84—0.98
(m, 12 H, Me—C(4″), Me—C(8″), Me—C(12″)); 0.88, 0.90,
0.95, 1.03, 1.10 (all s, 3 H each, H(23´), H(24´), H(25´), H(26´),
H(27´)); 1.10—2.05 (m, 24 H, CH2, CH in the betulinic acid
residue + 26 H, Me—C(2), CH2, CH in the tocopherol residue);
1.70 (s, 3 H, H(30´)); 2.09, 2.16, 2.20 (all s, 3 H each, Me—Ar);
2.57 (t, 2 H, H(4), J = 6.4 Hz); 3.04 (m, 1 H, H(19´)); 4.29
(br.s, 2 H, CH2—O); 4.63, 4.76 (both s, 1 H each, H(29´)); 4.67
(dd, 1 H, H(3´)); 7.20 (s, 1 H, COOH). 13C NMR, δ: 11.79,
11.91, 12.78 (Me—Ar); 14.68 (C(27´)); 16.03 (C(24´)); 16.19
(C(25´)); 16.49 (C(26´)); 18.16 (C(6´)); 19.36 (C(30´)); 19.70,
19.77 (Me—C(4″), Me—C(8″)); 20.87 (C(4)); 21.03 (C(11´));
22.65, 22.74 (Me—C(12´)); 23.86 (Me—C(2), C(2″)); 24.45
(C(10″)); 25.45 (C(12´)); 27.60 (C(2´)); 27.99 (C(12″), C(23´));
29.71 (C(15´)); 30.58 (C(21´)); 31.26 (C(3)); 32.18 (C(16´));
32.70, 32.79 (C(4″), C(8″)); 34.24 (C(7´)); 37.13 (C(10´), C(22´),
C(3″)); 37.29 (C(5″)); 32.79 (C(6″)); 37.41 (C(7″)); 37.46 (C(9″));
38.42 (C(1´), C(13´)); 39.38 (C(4´)); 40.01 (C(1″), C(11″)); 40.69
(C(8´)); 42.43 (C(14´)); 47.80 (C(18´)); 48.90 (C(19´)); 50.39
(C(9´)); 55.50 (C(5´)); 56.42 (C(17´)); 70.02 (CH2—O); 74.85
(C(2)); 81.77 (C(3´)); 109.78 (C(29´)); 117.59 (C(5)); 122.99
(C(7)); 125.66 (C(8)); 127.60 (C(4a)); 148.03 (C(8a)); 148.14
(C(6)); 150.35 (C(20´)); 169.30 (COO); 182.54 (C(28´)). MS,
m/z: 927.565 [M]+, 950.575 [M + Na]+, 966.499 [M + K]+.
28ꢀ[tertꢀButyl(dimethyl)silyloxy]ꢀ3βꢀ(3ꢀ{6ꢀ[tertꢀbutyl(diꢀ
methyl)silyloxy]ꢀ2,5,7,8ꢀtetramethylꢀ3,4ꢀdihydroꢀ2Hꢀ1ꢀbenzoꢀ
pyranꢀ2ꢀyl}propionyloxy)lupꢀ20(29)ꢀene (9). Yield 62%, white
crystals, m.p. 118—120 °C, [α]D +11.2° (c 0.75, CHCl3).
Found (%): C, 74.36; H, 10.53; Si, 6.28. C56H94O5Si2. Calculatꢀ
ed (%): C, 74.44; H, 10.49; Si, 6.22. IR, ν/cm–1: 1723 (O—C=O).
UV (CHCl3), λmax/nm (ε): 293 (2743). 1H NMR, δ: 0.06 (s, 6 H,
Me—Si); 0.09, 0.10 (both s, 3 H each, Me—Si); 0.91 (s, 9 H,
Me in But); 0.70, 0.76, 0.84, 0.96, 1.01 (all s, 3 H each, H(23´),
H(24´), H(25´), H(26´), H(27´)); 1.05, 1.06 (both s, 9 H, Me in
But); 1.10—1.90 (m, 24 H, CH2, CH in the betulin residue + 5 H,
C(2)Me, H(3) in the Trolox acid residue); 1.69 (s, 3 H, H(30´));
2.01, 2.09, 2.15 (all s, 3 H each, Me—Ar); 2.39—2.60 (m, 3 H,
H(4), H(19´)); 3.25, 3.68 (both d, 1 H each, CH2—O, J = 9.6 Hz);
4.39 (m, 1 H, H(3´)); 4.58, 4.68 (both s, 1 H each, H(29´)).
13C NMR, δ: –5.45, –3.52, –3.44 (Me—Si); 11.98, 13.31, 14.33
(Me—Ar); 14.73 (C(27´)); 15.87 (C(24´)); 16.07 (C(25´)); 16.37
(C(26´)); 18.07, 18.31 (But—Si); 18.58 (C(6´)); 19.08 (C(30´));
20.84 (C(4)); 21.22 (C(11´)); 25.54 (Me—C(2)); 25.19 (C(12´));
25.97, 26.08 (Me in But); 27.02 (C(2´)); 27.21 (C(15´));
27.95 (C(23´)); 29.44 (C(16´)); 29.71 (C(21´)); 29.91 (C(3));
34.07 (C(22´)); 34.32 (C(7´)); 37.02 (C(10´)); 37.36 (C(13´));
37.73 (C(1´)); 38.28 (C(4´)); 40.91 (C(8´)); 42.66 (C(14´));
48.06 (C(17´), C(18´)); 48.37 (C(19´)); 50.24 (C(9´)); 55.25
(C(5´)); 60.47 (C(28´)); 73.50 (C(2)); 81.60, 81.69 (C(3´)); 109.39
(C(29´)); 117.05, 117.23 (C(5)); 122.47, 122.56 (C(7)); 123.41,
123.58 (C(8)); 125.86, 126.04 (C(4a)); 144.62 (C(8a));
146.49 (C(6)); 150.90 (C(20´)); 173.96, 174.15 (COO). MS,
m/z: 903.598 [M]+.
Benzyl 3βꢀ[(6ꢀbenzyloxyꢀ2,5,7,8ꢀtetramethylꢀ3,4ꢀdihydroꢀ
2Hꢀ1ꢀbenzopyranꢀ2ꢀyl)carbonyloxy]lupꢀ20(29)ꢀenꢀ28ꢀoate (11).
Yield 30%, amorphous powder, [α]D20 +12.0° (c 0.22, CH2Cl2).
Found (%): C, 80.07; H, 8.86. C58H76O6. Calculated (%):
C, 80.14; H, 8.81. IR, ν/cm–1: 1725 (O—C=O). UV (CHCl3),
λ
max/nm (ε): 287 (2052). 1H NMR, δ: 0.74, 0.79, 0.90, 0.97, 0.98
20
crystals, m.p. 122—124 °C, [α]D +9.3° (c 0.55, CHCl3).
(all s, 3 H each, H(23´), H(24´), H(25´), H(26´), H(27´));
1.15—2.70 (m, 24 H, CH2, CH in the betulinic acid residue
+ 7 H, Me—C(2), (3), H(4) in the Trolox acid residue); 1.71
(s, 3 H, H(30´)); 2.15, 2.22, 2.28 (all s, 3 H each, Me—Ar); 3.05
(m, 1 H, H(19´)); 4.40 (dd, 1 H, H(3´), J = 11.2 Hz, J = 4.0 Hz);
4.62, 4.75 (both s, 1 H each, H(29´)); 4.70 (s, 2 H, OCH2Ph in
the Trolox acid residue); 5.10—5.16 (m, 2 H, OCH2Ph in the
betulinic acid residue); 7.28—7.51 (m, 10 H, Ph). 13C NMR, δ:
11.92, 11.96, 12.88 (Me—Ar); 14.70 (C(27´)); 15.85 (C(24´));
Found (%): C, 74.63; H, 10.64; Si, 6.09. C58H98O5Si2. Calculatꢀ
ed (%): C, 74.78; H, 10.60; Si, 6.03. IR, ν/cm–1: 1721 (O—C=O).
1
UV (CHCl3), λmax/nm (ε): 294 (2440). H NMR, δ: 0.07, 0.14
(both s, 12 H, Me—Si); 0.93, 1.07 (both s, 18 H, Me in But);
0.85, 0.86, 0.87, 0.99, 1.05 (all s, 3 H each, H(23´), H(24´),
H(25´), H(26´), H(27´)); 1.20—2.05 (m, 24 H, CH2, CH in the
betulin residue + 7 H, Me—C(2), H(3), H(1″) in the chromanylꢀ
propionic acid residue); 1.70 (s, 3 H, H(30´)); 2.08, 2.10, 2.13

Conjugates of lupane and tocopherol
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 12, December, 2010
2227
16.14 (C(25´)); 16.36 (C(26´)); 18.14 (C(6´)); 19.37 (C(30´));
20.90 (C(11´)); 21.10 (C(4)); 25.50 (C(12´)); 25.86 (Me—C(2));
27.17 (C(2´), C(15´)); 28.02 (C(23´)); 29.74 (C(21´)); 30.77
(C(3)); 32.13 (C(16´)); 34.21 (C(7´)); 37.00 (C(22´)); 37.08
(C(10´)); 38.19 (C(13´)); 38.34 (C(1´), C(4´)); 40.68 (C(8´));
42.41 (C(14´)); 46.98 (C(18´)); 49.47 (C(19´)); 50.43 (C(9´));
55.38 (C(5´)); 56.38 (C(17´)); 65.74, 75.00 (OCH2Ph); 78.90
(C(2)); 82.00 (C(3´)); 109.60 (C(29´)); 117.36 (C(5)); 122.92
(C(7)); 125.86 (C(8)); 127.79 (C(4a)); 127.89, 128.07, 128.27,
128.51, 136.53, 137.94 (Ph); 147.32 (C(8a)); 148.77 (C(6));
150.52 (C(20´)); 173.68 (COO); 175.80 (C(28´)). MS, m/z:
892.052 [M + Na]+, 908.014 [M + K]+.
Debenzylation of compounds 8 and 11 (general procedure).
A catalyst (10% Pd/C; 20 wt.% of compound 8 or 11) was added
to a solution of compound 8 or 11 (0.1 mmol) in anhydrous Et2O
(7 mL). The mixture was stirred in a hydrogen atmosphere for
2—8 h. After the reaction was completed (monitoring by TLC in
system B), the catalyst was filtered off and washed with Et2O.
The filtrate was concentrated and the residue was chromatoꢀ
graphed on SiO2 with CHCl3 as an eluent to give compound
16a,b or 17, respectively.
(C(15´)); 32.02 (C(21´)); 33.71 (C(16´)); 34.15 (C(2´), C(7´));
36.88 (C(10´), C(22´)); 37.36 (CH in Pri); 38.40 (C(13´)); 39.62
(C(1´)); 40.66 (C(8´)); 42.65 (C(14´)); 44.28 (C(19´)); 47.35
(C(4´)); 49.84 (C(18´)); 49.67 (C(9´)); 54.97 (C(5´)); 57.21
(C(17´)); 73.32 (CH2—O); 73.36 (C(2)); 116.92 (C(5)); 118.53,
121.32, 122.71 (C(4a), C(7), C(8)); 144.91 (C(8a)); 145.02
(C(6)); 176.26 (COO); 218.16 (C(3´)). MS, m/z: 675.077 [M]+,
698.064 [M + Na]+.
3βꢀ[(6ꢀHydroxyꢀ2,5,7,8ꢀtetramethylꢀ3,4ꢀdihydroꢀ2Hꢀ1ꢀ
benzopyranꢀ2ꢀyl)carbonyloxy]lupꢀ20(29)ꢀenꢀ28ꢀoic acid (17).
20
Yield 75%, amorphous powder, [α]D +14.8° (c 0.58, CHCl3).
Found (%): C, 76.57; H, 9.40. C44H64O6. Calculated (%):
C, 76.70; H, 9.36. IR, ν/cm–1: 1717 (O—C=O). UV (CH2Cl2),
λ
max/nm (ε): 296 (2858). 1H NMR, δ: 0.78, 0.81, 0.86, 0.89, 0.96
(all s, 3 H each, H(23´), H(24´), H(25´), H(26´), H(27´));
1.29—2.31 (m, 24 H, CH2, CH in the betulinic acid residue
+ 7 H, Me—C(2), H(3), H(4) in the Trolox acid residue); 1.71
(s, 3 H, H(30´)); 2.08, 2.16, 2.21 (all s, 3 H each, Me—Ar); 3.02
(m, 1 H, H(19´)); 4.29 (dd, 1 H, H(3´), J = 11.6 Hz, J = 4.4 Hz);
4.63, 4.76 (both s, 1 H each, H(29´)); 7.28 (s, 1 H, OH); 7.39
(s, 1 H, COOH). 13C NMR, δ: 11.18, 11.84, 12.92 (Me—Ar);
14.71 (C(27´)); 15.90 (C(24´)); 16.10 (C(25´)); 16.39 (C(26´));
18.10 (C(6´)); 19.34 (C(30´)); 20.84 (C(11´)); 21.18 (C(4)); 25.42
(C(12´)); 25.70 (Me—C(2)); 27.23 (C(2´)); 28.04 (C(23´)); 29.69
(C(21´)); 30.57 (C(15´)); 30.74 (C(3)); 32.16 (C(16´)); 34.19
(C(7´)); 37.09 (C(22´), C(10´)); 37.82 (C(13´)); 38.28 (C(1´));
38.41 (C(4´)); 40.68 (C(8´)); 42.43 (C(14´)); 46.95 (C(18´));
49.26 (C(19´)); 50.33 (C(9´)); 55.36 (C(5´)); 56.42 (C(17´));
77.23 (C(2)); 81.81 (C(3´)); 109.75 (C(29´)); 116.90 (C(5));
118.51 (C(7)); 121.25 (C(8)); 122.48 (C(4a)); 145.11 (C(8a));
146.03 (C(6)); 150.35 (C(20´)); 173.73 (COO); 182.47 (C(28´)).
MS, m/z: 689.145 [M]+, 712.136 [M + Na]+, 728.099 [M + K]+.
28ꢀ[tertꢀButyl(dimethyl)silyloxy]ꢀ3βꢀ[(6ꢀhydroxyꢀ2,5,7,8ꢀ
tetramethylꢀ3,4ꢀdihydroꢀ2Hꢀ1ꢀbenzopyranꢀ2ꢀyl)carbonyloxy]ꢀ
lupꢀ20(29)ꢀene (18a). A solution of TBAF (0.03 g, 0.11 mmol) in
THF (0.11 mL) was added dropwise under argon at 0 °C to
a solution of compound 10 (0.09 g, 0.10 mmol) in dry THF
(2 mL). The reaction mixture was stirred at 0 °C for 0.5 h and
diluted with water. The product was extracted with AcOEt and
the extract was washed with brine, dried over MgSO4, and conꢀ
centrated. The residue was chromatographed on SiO2 with
CHCl3 as an eluent. The yield of compound 18a was 62%, amorꢀ
(6ꢀHydroxyꢀ2,5,7,8ꢀtetramethylꢀ3,4ꢀdihydroꢀ2Hꢀ1ꢀbenzoꢀ
pyranꢀ2ꢀyl)methyl 3ꢀoxolupꢀ20(29)ꢀenꢀ28ꢀoate (16a). Yield 59%,
white crystals, m.p. 98—100 °C, [α]D20 +11.3° (c 0.15, CH2Cl2).
Found (%): C, 78.41; H, 9.62. C44H64O5. Calculated (%):
C, 78.53; H, 9.59. IR, ν/cm–1: 1724 (C=O). UV (CHCl3),
λ
max/nm (ε): 295 (3123). 1H NMR, δ: 0.89, 0.93, 0.99, 1.04, 1.09
(all s, 3 H each, H(23´), H(24´), H(25´), H(26´), H(27´));
1.20—2.60 (m, 24 H, CH2, CH in the betulonic acid residue
+ 5 H, Me—C(2), H(3) in the chromanol residue); 1.71 (s, 3 H,
H(30´)); 2.12, 2.14, 2.20 (all s, 3 H each, Me—Ar); 2.68 (t, 2 H,
H(4), J = 6.4 Hz); 3.08 (m, 1 H, H(19´)); 4.15 (m, 2 H, CH2—O);
4.40, 4.61 (both s, 1 H each, H(29´)); 4.75 (s, 1 H, OH).
13C NMR, δ: 11.30, 11.82, 12.23 (Me—Ar); 14.65 (C(27´)); 15.85
(C(25´)); 15.94 (C(26´)); 19.37 (C(6´)); 19.64 (C(30´)); 20.29
(C(4)); 21.04 (C(24´)); 21.43 (C (11´)); 22.06 (Me—C(2)); 25.57
(C(12´)); 26.62 (C(23´)); 28.96, 29.06 (C(3)); 29.67 (C(15´));
30.63 (C(21´)); 32.19 (C(16´)); 33.63 (C(7´)); 34.15 (C(2´));
36.90 (C(10´)); 37.09 (C(22´)); 38.46 (C(13´)); 39.64 (C(1´));
40.64 (C(8´)); 42.50 (C(14´)); 47.04 (C(19´)); 47.35 (C(4´));
49.34 (C(18´)); 49.90 (C(9´)); 54.99 (C(5´)); 56.76 (C(17´));
68.00 (CH2—O); 73.36 (C(2)); 109.72 (C(29´)); 116.90 (C(5));
118.55, 121.35, 122.69 (C(4a), C(7), C(8)); 144.89 (C(8a));
145.04 (C(6)); 150.42 (C(20´)); 175.98 (COO); 218.18 (C(3´)).
MS, m/z: 673.189 [M]+.
20
phous powder, [α]D +11.7° (c 1.52, CHCl3). Found (%):
C, 75.99; H, 10.25; Si, 3.59. C50H80O5Si. Calculated (%):
C, 76.09; H, 10.22; Si, 3.56. IR, ν/cm–1: 1727 (O—C=O). UV
(CHCl3), λmax/nm (ε): 295 (2814). 1H NMR, δ: 0.07 (s, 6 H,
Me—Si); 0.92 (s, 9 H, Me in But); 0.91, 0.95, 0.97, 1.00, 1.03
(all s, 3 H each, H(23´), H(24´), H(25´), H(26´), H(27´));
1.10—1.92 (m, 24 H, CH2, CH in the betulin residue + 5 H,
Me—C(2), H(3) in the Trolox acid residue); 1.70 (s, 3 H, H(30´));
2.08, 2.17, 2.19 (all s, 3 H each, Me—Ar); 2.37—2.68 (m, 3 H,
H(4), H(19´)); 3.26, 3.70 (both d, 1 H each, CH2—O, J = 9.6 Hz);
4.27 (s, 1 H, OH); 4.40 (m, 1 H, H(3´)); 4.58, 4.68 (both s,
1 H each, H(29´)). 13C NMR, δ: –5.36 (Me—Si); 11.22, 11.84,
12.18 (Me—Ar); 14.76 (C(27´)); 15.88 (C(24´)); 16.09 (C(26´));
16.41 (C(25´)); 18.10 (But—Si); 18.32 (C(6´)); 19.09 (C(30´));
20.86 (C(11´)); 21.10 (C(4)); 25.41 (Me—C(2)); 25.20 (C(12´));
25.98 (Me in But); 27.03 (C(2´)); 27.21 (C(15´)); 28.03 (C(23´));
29.44 (C(16´)); 29.93 (C(21´)); 30.74 (C(3)); 34.08 (C(22´));
34.32 (C(7´)); 37.04 (C(10´)); 37.37 (C(13´)); 37.81 (C(1´));
38.16 (C(4´)); 40.92 (C(8´)); 42.68 (C(14´)); 48.06 (C(17´),
(6ꢀHydroxyꢀ2,5,7,8ꢀtetramethylꢀ3,4ꢀdihydroꢀ2Hꢀ1ꢀbenzoꢀ
pyranꢀ2ꢀyl)methyl 3ꢀoxolupanꢀ28ꢀoate (16b). Yield 75%, white
20
crystals, m.p. 110—112 °C, [α]D +5.6° (c 0.55, CH2Cl2).
Found (%): C, 78.21; H, 9.90. C44H66O5. Calculated (%):
C, 78.29; H, 9.86. IR, ν/cm–1: 1723 (C=O). UV (CHCl3),
λ
max/nm (ε): 295 (3244). 1H NMR, δ: 0.78, 0.88 (both d, 3 H each,
Me in Pri, J = 7 Hz); 0.93, 0.95, 0.99, 1.04, 1.09 (all s, 3 H each,
H(23´), H(24´), H(25´), H(26´), H(27´)); 1.25—2.55 (m, 31 H,
CH2, CH in the betulonic acid residue + Me—C(2), H(3) in the
chromanol residue); 2.12, 2.14, 2.20 (all s, 3 H each, Me—Ar);
2.68 (t, 2 H, H(4), J = 6.4 Hz); 4.02, 4.04, 4.13, 4.15 (all d,
0.5 H each, CH2—O, J = 11.2 Hz); 4.35 (s, 1 H, OH). 13C NMR,
δ: 11.30, 11.82, 12.22 (Me—Ar); 14.58 (C(27´)); 14.69 (C(25´),
Me in Pri); 15.85, 15.91 (C(26´)); 19.65 (C(6´)); 20.29 (C(4));
21.06 (C(24´)); 21.48 (C(11´)); 22.15 (Me—C(2)); 22.70 (Me in
Pri); 26.63 (C(12´)); 26.96 (C(23´)); 28.94, 29.07 (C(3)); 29.70

2228
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 12, December, 2010
Spivak et al.
C(18´)); 48.39 (C(19´)); 50.26 (C(9´)); 55.31 (C(5´)); 60.47
(C(28´)); 73.50 (C(2)); 81.79 (C(3´)); 109.40 (C(29´)); 116.80,
116.90 (C(5)); 118.32, 118.46 (C(7)); 121.08, 121.20 (C(8));
122.48 (C(4a)); 145.13, 145.18 (C(8a)); 145.85, 146.03 (C(6));
150.91 (C(20´)); 173.69, 173.87 (COO). MS, m/z: 789.819 [M]+,
812.638 [M + Na]+.
collected cells were placed in flatꢀbottomed 96ꢀwell cell culture
plates (2.0•105 cells per well) and cultured with LPS (Sigma)
and the test compounds for 48 h (37 °C, 5% CO2). Then a part of
wells were used to estimate the cytotoxicity in an MTT assay.32
Another part of wells was used to determine the nitrite content in
their supernatants with the Griess reagent on a Titertek spectroꢀ
photometer (λ = 540 nm); the arginase activity in cells was estiꢀ
mated according to a modified procedure.33 To do this, 0.1%
Triton Xꢀ100 (0.1 mL) was added to the wells with macrophages
and cells were cultured with constant agitation at ~20 °C for
30 min. Then 0.025 M TrisꢀHCl (0.1 mL) and 0.01 M MnCl2
(0.035 mL) were added to test tubes for activation of arginase
and cells were incubated at 56 °C for 10 min, whereupon 0.5 M
Lꢀarginine (pH 9.7, 0.05 mL) (Sigma) was added to the lysate
(0.05 mL) and cells were cultured at 37 °C for 60 min. The
reaction was terminated by adding a mixture of conc. H2SO4 and
H3PO4 with water (1 : 3 : 7 v/v, 0.8 mL). The urea concentraꢀ
tion in the resulting solution was determined on a spectrophotoꢀ
meter (540 nm) using a Mochevinaꢀ450 test system ("BioꢀLAꢀ
Test") according to the enclosed protocol. The amount of arginꢀ
ase catalyzing the formation of 1 mmol of urea per minute was
taken to be a unit of enzyme activity. To estimate cytotoxicity,
3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide
(MTT, Serva) was added to the wells four hours before the terꢀ
mination of the cultivation; the MTT concentration in the wells
was 200 μg mL–1. Then the supernatants were removed from the
wells and the precipitate was dissolved in DMSO (dimexid, "Tatꢀ
khimfarmpreparaty"). Absorption of the resulting solutions was
measured on a multichannel spectrophotometer at λ = 550 nm.
The data obtained were processed using a tꢀtest procedure.
The difference between experimental values were believed to be
reliable at p < 0.05.
3βꢀ[(6ꢀHydroxyꢀ2,5,7,8ꢀtetramethylꢀ3,4ꢀdihydroꢀ2Hꢀ1ꢀ
benzopyranꢀ2ꢀyl)carbonyloxy]lupꢀ20(29)ꢀenꢀ28ꢀol (18b). A soluꢀ
tion of TBAF (0.04 g, 0.15 mmol) in THF (0.15 mL) was added
dropwise under argon at 0 °C to a solution of compound 18a
(0.06 g, 0.08 mmol) in dry THF (3 mL). The reaction mixture
was stirred at ~20 °C for 24 h (monitoring by TLC in system B)
and diluted with water. The product was extracted with AcOEt
and the extract was washed with brine, dried over MgSO4, and
concentrated. The residue was chromatographed on SiO2 with
CHCl3 as an eluent. The yield of compound 18b was 57%, amorꢀ
20
phous powder, [α]D +17.4° (c 0.69, CHCl3). Found (%):
C, 78.21; H, 9.89. C44H66O5. Calculated (%): C, 78.29; H, 9.86.
IR, ν/cm–1: 1726 (O—C=O). UV (CHCl3), λmax/nm (ε): 295
(2153). 1H NMR, δ: 0.84, 0.88, 0.90, 0.96, 1.02 (all s, 3 H each,
H(23´), H(24´), H(25´), H(26´), H(27´)); 1.20—1.70 (m, 24 H,
CH2, CH in the betulin residue + 5 H, Me—C(2), H(3) in the
Trolox acid residue); 1.70 (s, 3 H, H(30´)); 2.06, 2.16, 2.19 (all s,
3 H each, Me—Ar); 2.37—2.68 (m, 3 H, H(4), H(19´)); 3.34,
3.82 (both d, 1 H each, CH2—O, J = 10.5 Hz); 4.27, 8.13 (s, 1 H,
OH); 4.39 (m, 1 H, H(3´)); 4.60, 4.70 (both s, 1 H each, H(29´)).
13C NMR, δ: 11.21, 11.88, 12.18 (Me—Ar); 14.76 (C(27´)); 15.91
(C(24´)); 16.09 (C(26´)); 16.40 (C(25´)); 18.11 (C(6´)); 19.05
(C(30´)); 20.82 (C(11´)); 21.17 (C(4)); 25.39 (Me—C(2)); 25.14
(C(12´)); 27.03 (C(2´)); 27.21 (C(15´)); 28.02 (C(23´)); 29.73
(C(16´)); 30.73 (C(3), C(21´)); 33.96 (C(22´)); 34.10 (C(7´));
37.03 (C(10´)); 37.28 (C(13´)); 37.81 (C(1´)); 38.17 (C(4´));
40.91 (C(8´)); 42.71 (C(14´)); 47.77 (C(17´)); 47.80 (C(18´));
48.73 (C(19´)); 50.23 (C(9´)); 55.30 (C(5´)); 60.56 (C(28´));
77.34 (C(2)); 81.78 (C(3´)); 109.40 (C(29´)); 116.91 (C(5));
118.44 (C(7)); 121.05 (C(8)); 122.50 (C(4a)); 145.17, 145.18
(C(8a)); 145.85 (C(6)); 150.90 (C(20´)); 173.70 (COO). MS,
m/z: 674.548 [M]+, 697.560 [M + Na]+.
This work was financially supported by the Russian
Foundation for Basic Research (Project No. 10ꢀ03ꢀ00105)
and the Division of Chemistry and Materials Science of
the Russian Academy of Sciences (Program "Medicinal
and Biomolecular Chemistry").
The influence of conjugates 6, 7, 19, and 20 and acids 1b and 4
on the functional state of macrophages was studied in both male
and female mice (line C57BL/6) aged eight through twelve weeks
(firstꢀquality category), which had been provided by the Departꢀ
ment of Experimental Biological Models of the Research Instiꢀ
tute for Pharmacology, Siberian Branch of the Russian Acadeꢀ
my of Medical Sciences. The animals were housed in an incomꢀ
plete barrier system (lightꢀdark cycle 12/12) with free access to
water and granulated food.
References
1. S. V. Vasil´eva, in Khimicheskaya i biologicheskaya kinetika.
Novye gorizonty. Biologicheskaya kinetika: Sb. obzornykh statei
[Chemical and Biological Kinetics. New Horizons. Biological
Kinetics: A Collection of Reviews], Khimiya, Moscow, 2005,
2, p. 579 (in Russian).
Macrophages were isolated from the mouse peritoneal fluid.
To do this, the abdominal cavities of mice were washed with
cold physiological fluid (an isotonic 0.9% solution of NaCl, OOO
"Zavod Medsintez"). Cells were precipitated, resuspended in
a nutrient medium consisting of RPMI 1640 (GNTs VB "Vektor"),
10 vol.% fetal calf serum (FCS) ("HyClone"), 2ꢀ[4ꢀ(2ꢀhydroxyꢀ
2. S. Goerdt, C. E. Orfanos, Immunity, 1999, 10, 137.
3. S. Ehrt, D. Schnappinger, S. Bekiranov, J. Exp. Med., 2001,
194, 1123.
4. D. M. Mosser, J. Leukoc. Biol., 2003, 73, 209.
5. A. Mantovani, Blood, 2006, 108, 408.
6. M. Munder, K. Eichmann, M. Modolell, J. Immunol., 1998,
160, 5347.
ethyl)piperazinꢀ1ꢀyl]ethanesulfonic acid (HEPES) (20 mmol L–1
,
Sigma), 2ꢀmercaptoethanol (0.05 mmol L–1, Sigma), gentamiꢀ
cin (50 μg mL–1, Sigma), and Lꢀglutamine (2 mmol L–1, Sigma),
transferred to plastic Petri dishes (c = (1.5—2.0)•106 cells per
1 mL, and cultured for 2 h (37 °C, 5% CO2). Only the cells
adhered to the plastic were collected.
7. T. Kreider, R. M. Anthony, J. F. Urban, Jr., W. C. Gause,
Curr. Opin. Immunol., 2007, 19, 448.
8. H. Maeda, T. Akaike, Biochemistry (Moscow), 1998, 63, 854.
9. T. Akaike, M. Suga, H. Maeda, Pros. Soc. Exp. Biol. Med.,
1998, 217, 64.
To study the influence of hybrid compounds on the funcꢀ
tional state of macrophages and estimate their cytotoxicity, the
10. J. Wang, G. Mazza, J. Agric. Food Chem., 2002, 50, 850.
11. T. Motai, S. Kitanaka, J. Nat. Prod., 2005, 68, 1732.

Conjugates of lupane and tocopherol
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 12, December, 2010
2229
12. LiꢀYue Nie, J.ꢀJ. Qin, Y. Huang, L. Yan, YuꢀBo Liu, Yueꢀ
Xing Pan, HuiꢀZi Jin, WeiꢀDong Zhang, J. Nat. Prod., 2010,
73, 1117.
23. A. Yu. Spivak, R. R. Mufazzalova, E. R. Shakurova, V. N.
Odinokov, Zh. Org. Khim., 2010, 46, 1355 [Russ. J. Org. Chem.
(Engl. Transl.), 2010, 46].
13. T. Honda, B.ꢀA. V. Rounds, L. Bore, H. J. Finlay, F. G.
Favaloro, N. Suh, Y. Wang, M. B. Sporn, G. W. Gribble,
J. Med. Chem., 2000, 43, 4233.
24. G. A. Tolstikov, M. I. Goryaev, Kh. O. Kim, R. A. Khegai,
Zh. Prikl. Khim., 1967, 40, 920 [J. Appl. Chem. USSR
(Engl. Transl.), 1967, 40].
14. T. Honda, G. W. Gribble, N. Suh, H. J. Finlay, B.ꢀA. V.
Rounds, L. Bore, F. G. Favaloro, Jr., Y. Wang, M. B. Sporn,
J. Med. Chem., 2000, 43, 1866.
15. T. Honda, Y. Honda, F. G. Favaloro, G. W. Gribble, N. Suh,
A. E. Place, M. H. Rendi, M. B. Sporn, Bioorg. Med. Chem.
Lett., 2002, 12, 1027.
16. T. Honda, K. T. Liby, X. Su, C. Sundararajan, Y. Honda,
N. Suh, R. Risingsong, C. R. Williams, D. B. Royce, M. B.
Sporn, G. W. Gribble, Bioorg. Med. Chem. Lett., 2006,
15, 6306.
25. O. B. Flekhter, E. I. Boreko, L. R. Nigmatullina, E. V.
Tret´yakova, N. I. Pavlova, L. A. Baltina, S. N. Nikolaeva,
O. V. Savinova, V. F. Eremin, F. Z. Galin, G. A. Tolstiꢀ
kov, Bioorg. Khim., 2004, 30, 89 [Russ. J. Bioorg. Chem.
(Engl. Transl.), 2004, 30].
26. R. M. Kondratenko, L. A. Baltina, E. V. Vasil´eva, L. A.
Baltina, Jr., A. F. Ismagilova, Kh. M. Nasyrov, N. Zh.
Baschenko, R. M. Kireeva, S. M. Fridman, G. A. Tolstiꢀ
kov, Bioorg. Khim., 2004, 30, 61 [Russ. J. Bioorg. Chem.
(Engl. Transl.), 2004, 30].
17. K. Liby, T. Honda, C. R. Williams, R. Risingsong, D. B.
Royce, N. Suh, A. T. DinkovaꢀKostova, K. K. Stephenson,
P. Talalay, C. Sundararajan, G. W. Gribble, M. B. Sporn,
Mol. Cancer Ther., 2007, 6, 2123.
18. V. N. Odinokov, A. Yu. Spivak, O. V. Knyshenko, Bioorg.
Khim., 2007, 33, 387 [Russ. J. Bioorg. Chem. (Engl. Transl.),
2007, 33].
27. K. Hiroya, T. Takahashi, N. Miura, A. Naganuma, T. Sakaꢀ
moto, Bioorg. Med. Chem., 2002, 10, 3229.
28. S. A. S. Pope, G. E. Burtin, P. T. Clayton, D. J. Madge,
D. P. R. Muller, Free Radical Biol. Med., 2002, 33, 807.
29. H. Lei, J. Atkinson, J. Org. Chem., 2000, 65, 2560.
30. K. Hiroya, T. Takahashi, N. Miura, A. Naganuma, T. Sakaꢀ
moto, Bioorg. Med. Chem., 2002, 10, 3229.
19. A. Detsi, D. Bouloumbasi, K. C. Prousis, M. Koufaki,
G. Athanasellis, G. Melagraki, A. Afantitis, O. Igglessiꢀ
Markopoulou, Ch. Kontogiorgis, D. J. HadjipavlouꢀLitina,
J. Med. Chem., 2007, 50, 2450.
20. A. Di Stefano, P. Sozio, A. Cocco, A. Iannitelli, E. Santussi,
M. Costa, L. Pessi, C. Nasuti, F. Cantalamessa, F. Pinnen,
J. Med. Chem., 2006, 49, 1486.
21. M. Nozawa, K. Takhashi, K. Kato, H. Akita, Chem. Pharm.
Bull., 2000, 48, 272.
22. S. Genet, A. Strehle, C. Schmidt, G. Boudjelal, A. Lobstein,
K. Schoonjans, M. Souchet, J. Auwerx, R. Saladin, A. Wagꢀ
ner, J. Med. Chem., 2010, 53, 178.
31. K. A. Lawson, K. Anderson, M. Menchaca, J. Atkinson,
L. Sun, V. Knight, B. E. Gilbert, C. Conti, B. G. Sanders,
K. Kline, Mol. Cancer Therapeutics, 2002, 2, 437.
32. ISO 10993ꢀ5. Biological Evaluation of Medical Devices, Pt 5:
Tests for Cytotoxicity, in vitro Methods, International Stanꢀ
dardization Organisation, Geneva, 1992.
33. M. G. Danilets, Yu. P. Bel´skii, N. V. Bel´skaya, E. S. Trofiꢀ
mova, E. G. Uchasova, V. I. Agafonov, Byull. Eksp. Biol.
Med. [Bulletin of Experimental Biological Medicine], 2007,
App. 1, 97 (in Russian).
Received October 17, 2010