Synthesis of novel 2-cyano substituted glycyrrhetinic acid derivatives as inhibitors of cancer cells growth and NO production in LPS-activated J-774 cells

Article abstract of DOI:10.1016/j.bmc.2013.10.049

Here we report the synthesis and biological activity of new semi-synthetic derivatives of naturally occurring glycyrrhetinic acid bearing a 2-cyano-3-oxo-1-en moiety in the A-ring and double bonds and carbonyl groups in the C, D and E rings. Bioassays using murine macrophage-like and tumor cells show that compound 4, which differs from Soloxolone methyl by the absence of a 9(11)-double bond in the C-ring, displays anti-inflammatory and inhibitory activities with respect to tumor cells with a high selectivity index value.

Full text of DOI:10.1016/j.bmc.2013.10.049

Bioorganic & Medicinal Chemistry xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of novel 2-cyano substituted glycyrrhetinic acid
derivatives as inhibitors of cancer cells growth and NO production
in LPS-activated J-774 cells
Oksana V. Salomatina , Andrey V. Markov b, , Evgeniya B. Logashenko , Dina V. Korchagina ,
a,
b,
⇑
a
b
a
b
a
Marina A. Zenkova , Nariman F. Salakhutdinov , Valentin V. Vlassov , Genrikh A. Tolstikov
a
N.N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch Russian Academy of Sciences, 9, Lavrentiev ave., Novosibirsk 630090, Russian Federation
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch Russian Academy of Sciences, 8, Lavrentiev ave., Novosibirsk 630090, Russian Federation
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Here we report the synthesis and biological activity of new semi-synthetic derivatives of naturally occur-
ring glycyrrhetinic acid bearing a 2-cyano-3-oxo-1-en moiety in the A-ring and double bonds and car-
bonyl groups in the C, D and E rings. Bioassays using murine macrophage-like and tumor cells show
that compound 4, which differs from Soloxolone methyl by the absence of a 9(11)-double bond in the
C-ring, displays anti-inflammatory and inhibitory activities with respect to tumor cells with a high selec-
tivity index value.
Received 25 July 2013
Revised 22 October 2013
Accepted 29 October 2013
Available online xxxx
Keywords:
Glycyrrhetinic acid derivatives
Cytotoxicity
Ó 2013 Elsevier Ltd. All rights reserved.
Nitric oxide
Biological activity
Selectivity index
8
1
. Introduction
system into A-rings of different triterpenes, such as betulin, betu-
9
,10
linic acid,
arjuonic acid, boswellic acid and glycyrrhetinic acid
1
1,12
The inorganic free radical NO, synthesized by a family of en-
(GA).
zymes termed NO-synthases (NOS), acts as a host defense mecha-
18bH-Glycyrrhetinic acid is the aglycone of glycyrrhizin, a ma-
jor component of licorice root, which has a long history of use in
herbal medicine in both eastern and western countries. In addition,
the level of glycyrrhizin can reach 25–30% in Glycyrrhiza glabra
roots, and 90% in triterpene extracts of licorice root. Therefore,
compared to other triterpenoic acids, GA can be obtained in larger
amounts for a reasonable price. Recent reviews have described a
wide spectrum of natural bioactivities exhibited by GA, including
anti-inflammatory, antiviral, hepatoprotective and immunomodu-
1
nism, damaging pathogenic DNA, and as a regulatory molecule
with homeostatic activities.2 However, excessive production of
NO, due to the reaction with superoxide in biological systems,
gives rise to various diseases. NO is a multifactorial molecule in
tumorogenesis, known to participate in carcinogenesis by inducing
DNA damage, supporting tumor progression and suppressing the
3
immune response. NO also plays an important role in angiogene-
sis, invasion and metastasis progression.4 Therefore, inhibition of
,5
1
3,14
NO production could facilitate anticancer therapy.
latory activities.
The ability of GA to inhibit cancer cell growth
1
5
The discovery that the CDDO (2-cyano-3,12-dioxoolean-
and induce tumor cell apoptosis has stimulated extensive use of
6
16
1
,9(11)-diene-28-oic acid) series of oleanolic acid, synthesized
GA as a scaffold for the synthesis of new anticancer agents. From
from naturally abundant oleanolic acid, display diverse biological
activities including suppression of NO production and proliferation
of malignant cells as well as induction of differentiation in malig-
nant cells (Suh et al., 1999) has renewed interest in the pentacy-
clic triterpenoids and natural products. This success has sparked
much interest in the introduction of the 2-cyano-1-en-3-one
a chemical standpoint, 18bH-glycyrrhetinic acid contains an
11-oxo function, which is rather rare among naturally occurring
1
7,18
triterpenoids.
7
The fact that minor changes in triterpenoid structure can cause
extensive changes in biological activity has long intrigued medici-
nal chemists. The most actively explored ‘simple’ derivatives of GA,
obtained by modification of 3-hydroxy (or 3-oxo) with different
1
9–23
aminoacids or/and carboxylic groups (C-30)
by esterification
⇑
Corresponding author. Tel.: +7 383 363 5161; fax: +7 383 363 5153.
E-mail address: evg_log@niboch.nsc.ru (E.B. Logashenko).
These authors contributed equally to this work.
exhibited a higher activity than GA and a better selectivity towards
0
968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.10.049
Please cite this article in press as: Salomatina, O. V.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.10.049

2
O. V. Salomatina et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
tumour cells.19 The cytotoxicity of GA bearing different substitu-
tions in the A ring was also higher than that of GA.2
due to the ability of Michael electrophiles to target specific nucle-
ophiles and exert selective biological functions. In the structure of
GA only one Michael acceptor site is presented. Soloxolone methyl
contains an additional Michael acceptor site in the C-9 position
and, earlier, we demonstrated that the anti-proliferative activity
of Soloxolone methyl is determined by the active Michael accep-
0,23
Modification of the C-3 hydroxy group with diamines of differ-
ent length leads to GA derivatives with significantly enhanced bio-
logical activity, able to induce apoptosis in tumor cells.21 Similar
2
0
levels of cytotoxicity were observed for 3-amino-GA derivatives
and for conjugates of the GA carboxylic group with dehydrozinger-
2
6–28
tor.
Compound 2 is characterized by the presence of an addi-
22
one containing different substitutors in an aromatic ring.
tional Michael acceptor site in the C-13 position. To achieve a
higher reactivity of derivatives in the Michael reaction, we intro-
duced 18,19 double bond in the E ring (compound 1). This modifi-
cation leads to more favorable steric arrangements of the Michael
center as a result of vinylogy at the C-19 position.
Synthesis of GA derivatives with major changes to the A ring-
oxidation of 3-hydroxy to 3-oxo group, conversion of this to a
methylene group, changing a 6-member ring to a 5-member ring,
different location of substituents or/and double bonds in the cyclo-
pentane ring as well as reduction of the carboxylic group to a hy-
droxyl group was also reported.24
12(13)-en (compound 3) and 12-oxo moities (compound 4)
6
were introduced into ring C since it was shown earlier that the
Earlier, we reported that a semisynthetic derivative Soloxolone
methyl (methyl 2-cyano-3,12-dioxo-18bH-olean-9(11),1(2)-dien-
presence of such functional groups in the framework of oleanolic
acid leads to compounds with enhanced antiproliferative
properties.
3
0-oate), obtained by direct modification of the A- and C-rings of
GA, possesses high antiproliferative activity with respect to cancer
cells. It was shown that human epidermoid cancer cells are sensi-
tive to this compound, as well as other tested tumor cell lines,
The sequence of reactions to introduce different functions
including double bonds and carbonyl groups in the C, D and E rings
and a 2-cyano-1-en-3-one moiety in the A-ring of 18bH-glycyrrh-
etinic acid is shown in Scheme 1. The methyl ester of 18bH-glyc-
2
5
including cells exhibiting a multidrug resistant phenotype.
2
5
This work was aimed at revealing the structure–activity rela-
tionships (SAR) of new semi-synthetic derivatives of GA. A series
of compounds based on GA was synthesized using the following
strategies: (a) building up a 2-cyano-1-en-3-one moiety in the A
ring of GA; (b) preserving the native 11-oxo-12(13)-en moiety of
GA in ring C, (c) lengthening the native system of conjugated bonds
to 11-oxo-12(13),18(19)-dien, converting the 11-oxo-12-en of GA
in ring C to 12-oxo-9(11)-en, 12-en and 12 oxo moieties (Fig. 1).
These derivatives and intermediates were then screened in vitro
for their ability to inhibit NO production and for their cytotoxic
activities.
yrrhetinic acid 5 was used as a starting material.
Methyl 2-cyano-3,11-dioxo-18bH-olean-1(2),9(11)-dien-30-
oate 2 and intermediate compounds 14–18 were synthesized
1
1
according to the described method. Compound 2 was used for
comparison since data on its biological activity were available.11
Methyl
2-cyano-3,11-dioxoolean-1(2),9(11),18(19)-trien-30-
oate 1 was obtained by the combined modification of rings A and
E of the starting triterpenoid. The formation of the 18,19-double
bond was carried out by the bromination–dehydrobromination of
compound 6 with bromine in acetic acid at 80 °C. Deprotection of
the acetate group by KOH in methanol freed the 3-hydroxy group
(
compound 9), followed by Jones oxidation to give the carbonyl
group at C-3 10. Subsequent formylation at C-2 was performed
by condensation with HCO Et/NaOMe in benzene and the resulting
2
2
. Results and discussion
2
hydroxymethylene derivative 11 was cyclized into its isoxazole
derivative 12 by reaction with hydroxylamine hydrochloride in
aqueous ethanol (reflux). The isoxazole ring opening between the
N–O bond was prompted uneventfully by NaOMe to deliver the
.1. Chemical synthesis
To improve the anti-inflammatory and anti-cancer activity of
GA, novel derivatives of GA were synthesized by chemical modifi-
cations in rings A, C and E (Fig. 1). The modification of ring A was
similar to that described by Honda et al.,6 and was aimed at
improving the native anti-inflammatory and cytotoxic properties
of triterpenoid.
2
-cyano group 13. The new 1,2-double bond was formed by dehy-
drogenation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
DDQ) in benzene (reflux) to complete construction of the 2-cya-
(
no-1-en-3-one fragment in the A ring 1.
The starting material for the synthesis of compound 3, the
methyl ester of 11-deoxoglycyrrhetinic acid acetate 7, was ob-
tained by reduction of the carbonyl group at C-11 by Zn/HCl in
It is known that the presence of Michael acceptor groups at crit-
ical positions is essential for inhibition of proliferation, promotion
of differentiation, and induction of apoptosis in various cell lines
2
9
9
COOCH3
C3O0 OH
COOCH
3
2
0
1
21
22
O
1
2
E
O
O
13 18
2
5
11
9
26
14
17
C
8
D
28
16
NC
1
15
NC
O
2
10
B
7
27
3
A
5
4
6
O
HO
1
2
24
23
1
8βH-Glycyrrhetinic acid
Soloxolone methyl
COOCH3
COOCH3
COOCH
3
O
O
NC
NC
NC
O
O
O
3
4
Figure 1. Semisynthetic derivatives of 18bH-glycyrrhetinic acid (1–4).
Please cite this article in press as: Salomatina, O. V.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.10.049

O. V. Salomatina et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
3
COOCH3
COOCH3
COOCH3
COOCH3
O
O
O
b)
a)
c)
AcO
6
AcO
AcO
8
5
AcO
7
d)
d)
d)
d)
HO
HO
HO
HO
9
14
15
24
25
e)
f)
e)
e)
1
9
e)
O
O
O
O
10
f)
H
2
0
H
O
f)
HO
H
HO
O
HO
16
O
1
1
g)
2
1
g)
g)
N
N
O
O
N
1
7
O
12
2
2
h)
h)
h)
NC
HO
NC
O
NC
HO
NC
NC
HO
NC
O
1
8
O
i)
13
i)
23
i)
j)
1
3
4
2
Scheme 1. Combined modification of 18bH-glycyrrhetinic acid rings (A) and (C–E). Reagent and conditions: (a) Br
2
, AcOH (80 °C); (b) Zn (powder), HCl (concd), dioxane (5–
, AcOH (80 °C); (d) KOH, MeOH (rt); (e) Jones reagent, acetone (rt); (f) HCOOEt, NaOMe, benzene (rt); (g) NH OHꢁHCl, EtOH–H O (reflux); (h) NaOMe, MeOH–
2
O (0 °C to rt); (i) 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), benzene (reflux); (j) AcOOH, chloroform (rt).
1
0 °C); (c) H
2
O
2
2
2
Et
dioxane at 5–10 °C. A further six-step modification of the A ring in
compound 7, similar to compound 1, led to compound 3. The car-
bonyl group at C-12 in derivative 8 was formed by interaction of
the 11,12-double bond of compound 4 with hydrogen peroxide
in acetic acid at 80 °C. Deprotection of the acetate group by KOH
in methanol freed the 3-hydroxy group (compound 24), followed
by Jones oxidation to give diketone 25. Compound 3 was converted
into 12-oxoderivative 4 by treating with peracetic acid. The car-
bonyl group at C-12 was formed as a result of the formation and
subsequent opening of the epoxide ring in acid media.
(Soloxolone methyl) exhibited antiproliferative activity via induc-
tion caspase-dependent intrinsic apoptotic pathway.25 Here we
screened compounds 1–4 and all intermediates leading to them
(see Scheme 1) for their cytotoxicity with respect to cancer cells.
Among the tested compounds, compound 3 was found to be insol-
uble in dimethylsulfoxide, so we could not perform any biological
assays with this derivative. As also mentioned above, compound 2,
containing the 11-oxo-12(13)-en fragment in its structure (boxed
1
1
in Fig. 1), has been described earlier and was used in the present
study for comparison. We also compared the cytotoxicity of the
new compounds with that of Soloxolone methyl.
All target compounds and intermediates were characterized by
H and C NMR and high-resolution mass-spectroscopy (HRMS).
1
13
The cytotoxic activity of the novel derivatives of GA against hu-
man epidermoid cancer cells KB-3-1 was determined using the
MTT assay, a colorimetric test of cell viability during treatment
So, as a result of the direct modification of the GA framework, 3
new target compounds (compounds 1, 3 and 4) were synthesized.
KB
with each compound. The IC50 values (concentration of a com-
2
.2. Biological activity
pound allowing survival of 50% of the cells in a population) of
the tested compounds in KB-3-1 cells are listed in Table 1.
In the previous study, we reported that the GA derivative
methyl 2-cyano-3,12-dioxo-18bH-olean-9(11),1(2)-dien-30-oate
As can be seen from the data presented in Table 1, intermedi-
KB
ates 9–25, except for 24 ( IC50 ¼ 6:8 ꢀ 3:5lM ), exhibit low toxicity
Please cite this article in press as: Salomatina, O. V.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.10.049

4
O. V. Salomatina et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
(25, 21, 23) or are not toxic for tumor cells, while compounds 1 and
Comparison of the inhibitory activity of the compounds with
4
exhibited similar cytotoxicity (4 and 5.5
l
M, respectively). How-
their cytotoxicity with respect to J-774 cells highlighted com-
pounds 2, 4 and Soloxolone methyl, which all displayed high inhib-
itory activity. Altogether, the data on cytotoxicity with respect to
tumor cells and inhibition of NO production position compound
4 and Soloxolone methyl as the leading molecules since they are
cytotoxic for tumor cells, efficiently inhibit NO production in LPS
stimulated macrophage-like cells and are characterized by high
SI values. This set of properties mark compound 4 and Soloxolone
methyl as leading molecules and puts them on a par with other
semisynthetic derivatives of pentacyclic triterpenes that are under
extensive investigation today to treat cancer by different modes of
action, including inflammation. It must also be emphasized that
compound 4, unlike Soloxolone methyl, was not toxic for normal
cells.
Comparison of the structures of the synthesized compounds
and their biological properties makes it possible to identify some
structure/biological activity relationships for synthetic analogs of
GA:
ever, their cytotoxicity is much less than that of Soloxolone methyl
0.3 M). Among compounds 1, 2 and 4, tested in parallel under
identical conditions, compound 2 had the least activity with a
(
l
KB
0
IC5 value of 20 lM. As our compounds have a structure which
resembles the one of Soloxolone methyl we assume that they exhi-
bit antiproliferative activity via the similar mechanism.
We used a Selectivity Index (SI) as the criterion to determine
the effectiveness of the compounds. The SI was defined as the ratio
of the cytotoxicity of a compound with respect to normal cells
J774
KB
(
IC5 ) versus cancer cells (IC50 ). For normal cells we used macro-
0
J774
32
phage-like J-774 cells; IC50 values for J-774 cells are listed in
Table 1. Taking into account that a higher SI corresponds to greater
overall anticancer activity, it can be seen that among the com-
pound tested only compounds 4 and Soloxolone methyl displayed
remarkable selectivity (>18.2 and 11, respectively).
It is known that in biological systems excessive production of
NO, due to reaction with superoxide, gives rise to various diseases
such as inflammation, carcinogenesis, and atherosclerosis.
2
9,30
Therefore, inhibition of NO production can be of therapeutic value.
The inhibitory activities (IC50 value) of semi-synthetic triterpe-
(i) lengthening of the conjugating system (11-oxo-
12(13),18(19)-dien (compound 1) versus 11-oxo-12(13)-en
(compound 2) led to a strong increase in the cytotoxicity
of the compound with respect to macrophage-like and
tumor cells (Table 1);
(ii) the location of the second Michael acceptor site significantly
affected the SI of the compound (SI values for Soloxolone
methyl, compound 1 and 2 are 11, 1.8 and >5, respectively);
(iii) the 9(11) double bond significantly increased the cytotoxic-
ity of the compounds; compare Soloxolone methyl (9(11)-en)
with compound 4: 4, lacking the 9(11) double bond, dis-
NO
noids and indomethacine (used as a positive control) on NO pro-
duction induced by LPS in J-774 macrophage-like cells are shown
in Table 1. Also, we analyzed the anti-inflammatory potential of ac-
tion of Soloxolone methyl for the first time.
The preliminary screening showed that most of the new deriv-
atives of GA exhibit inhibitory activities against LPS-induced NO
production in J-774 cells. The inhibitory activity of the compounds
decreased
in
the
order:
Soloxolone
methyl ꢂ 2 ꢃ 4 >
1
> 23 P 21 P 22 P 11 > 10 > 25 > indomethacine > 24.
Com-
pounds 9 and 14 were inactive in inhibiting LPS stimulated NO pro-
played high inhibitory activity and extremely low cytotoxic-
ity (IC50 >100 lM) with respect to macrophage-like cells;
(iv) replacement of the 11-oxo-12(13)-en structural element in
GA (compound 2) by 12-oxo-9(11)-en (Soloxolone methyl)
significantly increased the biological activity of the com-
pound in terms of cytotoxicity with respect to tumor cells
J774
duction. Ten compounds displayed inhibition rates greater than
31
that of the positive control indomethacine. Only compounds 1,
, 4, Soloxolone methyl and intermediate compound 23 were able
to inhibit NO production within a concentration range below
M. As in the case of cytotoxicity with respect to cancer cells,
the inhibitory activity of Soloxolone methyl was the highest
0.11 M).
2
10 l
(0.3 and 20
M) and cytotoxicity with respect to macrophage-like cells
3.3 and >100 M, for Soloxolone methyl and 2,
respectively).
lM), inhibition of NO production (0.11 and
(
l
2 l
(
l
Table 1
In vitro biological evaluation of the intermediates and target compounds
SIc
NO
50
d
Similar behavior was observed in the study of synthetic olean-
ane and ursane triterpenoids.
KB
IC5
a
J774
50
b
Compound
(lM)
IC
(lM)
IC
(lM)
0
6
1
2
4
4.0 ± 0.2
20 ± 0.6
5.5 ± 0.7
0.3 ± 0.1
ND
ND
7.0 ± 0.2
>100
>100
3.32 ± 0.1
>100
20.1 ± 1.3
13.4 ± 0.2
>100
>100
>100
>100
56.1 ± 2.7
>100
1.8
>5
>18.2
11
—
—
5.6 ± 1.4
2.0 ± 0.3
2.8 ± 0.3
0.11 ± 0.02
>100
16.3 ± 1.4
14.5 ± 0.8
>100
10.5 ± 1.9
12.4 ± 5.0
8.0 ± 1.0
59.4 ± 13.1
23.4 ± 1.0
29.6 ± 1.4
Unfortunately, we could not evaluate the influence of the
pharmacon group in the A ring on the biological activity of the
compounds since 3, 19 and 20 were insoluble in DMSO. Most likely
this effect is determined by a decrease in the polarity of the C ring
due to the absence of the carbonyl group at C-11. This fact is sup-
ported by the improved solubility of compounds 21 (2-hydroxy-
metylene group) and 22 (isoxazole ring) with increased polarity
of ring A. Moreover, 21 and 22 are characterized by overt biological
activity (Table 1). It is interesting that similar derivatives of olean-
Soloxolone methyl
9
1
1
1
2
2
2
2
2
0
1
4
1
2
3
4
5
ND
>100
—
—
40.0 ± 0.7
>100
80.0 ± 0.9
6.8 ± 3.5
25.2 ± 0.6
N/D
>2.5
—
>1.3
8.3
>3.9
N/D
6
olic and ursolic acid that differ from compound 3 by the location
of an ester group (C-20 vs C-17) were not only soluble but demon-
strated high biological activity. In our case the location of the ester
group significantly affected the solubility of the compounds.
Indomethacine
>100
The results are presented as the mean ± SEM of three independent experiments.
a
KB
The IC50 was defined as the compound concentration providing 50% tumor KB-
-1 cell survival measured by MTT assay as described in the section 4; incubation
time: 24 h.
The IC50 was defined as the compound concentration providing 50% J-774 cell
survival measured by MTT assay as described in the section 4; incubation time:
4 h.
Selectivity index for cytotoxicity against human carcinoma cells. The selectivity
index (SI) was the ratio of IC50 (cytotoxicity on normal J-774 cells) to IC50 (cyto-
toxicity on human cancer cells).
3
b
J774
3. Conclusion
2
We have synthesized GA derivatives with inhibitory effects on
overproduction of NO and with in vitro antiproliferative activity
with respect to tumor cells. Based on the obtained data we can
conclude that for the design of GA derivatives one needs to consider
that lengthening of the pairing system (11-oxo-12(13),18(19)-dien
c
J774
KB
d
NO
The IC50 was defined as the compound concentration providing a 50% decrease
in NO production by LPS-stimulated J-774 cells compared with control.
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O. V. Salomatina et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
5
0
(
compound 1), while giving the compounds the ability to inhibit
1H, H-22 ), 1.49 (ddd, 1H, J7e,7a 12.5, J7e,6a 3.8, J7e,6e 2.5 Hz, H-7e),
1.73 (m, 4H: H-5, 2H-6, H-7a), 1.58 (dd, 1H, J19a,19e 13.5, J19a,18a
13.5 Hz, H-19a), 1.83 (ddd, 1H, J15a,15e 13.6, J15a,16a 13.6, J15a,16e
4.5 Hz, H-15a) 1.90 (ddd, 1H, J19e,19a 13.5, J19e,18a 4.2, J19e,21e
2.5 Hz, H-19e), 1.98 (br d, 1H, J21e,21a 9.7 Hz, H-21e), 2.03 (ddd,
1H, J16a,16e 13.6, J16a,15a 13.6, J16a,15e 4.5 Hz, H-16a), 2.15 (dd, 1H,
excessive NO production and cancer cell growth, is accompanied
by a substantial increase in cytotoxicity with respect to normal
(
nonmalignant) cells. The presence in the structure of the com-
pound of a 11-oxo-12(13)-en structural element in GA (compound
2
4
) and the absence of a 9(11) double bond in the C ring (compound
) led to high biological activity of the resulting molecule but low
J
18a,19a 13.5, J18a,19e 4.2 Hz, H-18a), 2.68 (s, 1H, H-9a), 3.68 (s, 3H,
1
3
cytotoxicity with respect to normal cells.
3 3
OCH -31), 5.77 (s, 1H, H-12), 8.51 (s, 1H, H-1). C NMR (CDCl ):
One of the important criteria for a therapeutic drug for cancer is
to have minimal or no side effects on normal body cells of patients
undergoing chemotherapy. One way to achieve this is by employ-
ing lower doses of drugs. This invariably implies that the drug
should not only have high potent activity at lower concentrations
but should also exhibit a high degree of selectivity. The present
in vitro studies demonstrate the ability of synthetic compound 4
for highly selective toxicity at lower concentrations (SI = >18). In
addition, this compound also showed potent anti-inflammatory
activity.
d = 172.4 (d, C-1), 113.2 (s, C-2), 197.4 (s, C-3), 45.0 (s, C-4), 51.8
(d, C-5), 17.9 (t, C-6), 31.5 (t, C-7), 43.5 (s, C-8), 54.3 (d, C-9),
39.6 (s, C-10), 197.9 (s, C-11), 127.7 (d, C-12), 171.6 (s, C-13),
45.5 (s, C-14), 26.4 (t, C-15), 26.2 (t, C-16), 31.7 (s, C-17), 48.4 (d,
C-18), 41.0 (t, C-19), 43.9 (s, C-20), 31.0 (t, C-21), 37.6 (t, C-22),
27.4 (q, C-23), 21.4 (q, C-24), 19.4 (q, C-25), 18.9 (q, C-26), 23.3
(q, C-27), 28.5 (q, C-28), 28.1 (q, C-29), 176.6 (s, C-30), 51.7
2
5
(q, C-31), 114.8 (s, C-32). ½
a
ꢅ
D
3
+257° (c 0.12, CHCl ).
4.1.3. Methyl 3b-acetoxy-11-oxoolean-12(13),18(19)-dien-30-
oate (6)
Compound 6 was synthesized according to a known method.33
4
4
4
. Experimental
.1. Synthesis
Briefly, a solution of Br
(
(
2
(10.0 g, 21.8 mmol) in glacial AcOH
70 mL) was added dropwise at 80 °C to a stirred solution of 5
10.0 g, 19.0 mmol) in glacial AcOH (400 mL) for 1 h. The reaction
mixture was stirred for a further 1 h at 80 °C, cooled to room tem-
perature and diluted with H O (1.5 L). The solid was filtered,
washed with H O and dried. The crude product was purified by
.1.1. General experimental procedures
2
Melting points were determined on a Hoover melting point
2
apparatus and are uncorrected. The element composition of the
products was determined from high-resolution mass spectra re-
corded on a DFS (Double Focusing Sector) Thermo Electron Corpo-
ration instrument. Optical rotations were measured with a PolAAr
flash column chromatography (silica gel, chloroform) to yield com-
pound 6 (6.5 g, 65%) as a white solid. This material was used for the
next reaction without further purification. An analytically pure
sample was obtained by recrystallization from CH
3
OH. Mp
: 524.3490; found:
): d = 0.74 (dd, 1H, J5a,6a 12, J5a,6e 1.5 Hz,
-23), 0.83 (s, 3H, CH -24), 0.89 (s, 3H,
-28), 0.98 (ddd, 1H, J1a,1e 13.5, J1a,2a 13.5, J1a,2e 4 Hz, H-1a),
.11 (s, 6H,CH -26, CH -27), 1.17 (s, 3H, CH -25), 1.23 (ddd, 1H,
15e,15a 12.5, J15e,16a 4, J15e,16e 3 Hz, H-15e), 1.25 (s, 3H, CH -29),
.39 (dddd, 1H, J6a,6e 13, J6a,7a 13, J6a,5a 12, J6a,7e 3 Hz, H-6a), 1.44–
.60 (m: 9H, [1.48]—2H-22; [1.48] and [1.52]—2H-16; [1.49] and
1
13
3
005 polarimeter. H and C NMR spectra were measured on Bru-
2
5
48 5
44–247 °C. HRMS: m/z calcd for C33H O
ker spectrometers: AM-400 (operating frequency 400.13 MHz for
1
24.3496. H NMR (CDCl
3
1
13
1
H and 100.61 MHz for C) and DRX-500 (500.13 MHz for
and 125.76 MHz for 13C) for CDCl
solutions of the substances.
The signal from chloroform was used as an internal standard (d
.24 ppm, d 76.90 ppm). The structure of the compounds was
determined by NMR from proton spin–spin coupling constants in
H
H-5a), 0.81 (s, 3H, CH
CH
3
3
3
3
H
1
3
3
3
7
C
J
1
1
3
1
1
13
H– H double resonance spectra, and by analyzing C NMR spec-
tra using proton selective and off-resonance saturation spectra, 2D
[
1.55]—2H-7; [1.55]—H-21e; [1.57]—H-2e; [1.51]—H-6e), 1.66
1
3
1
1
C– H correlated spectroscopy on C–H constants (COSY,
J
C,H
(
dddd, 1H, J2a,2e 13.5, J2a,1a 13.5, J2a,3a 12, J2a,1e 4 Hz, H-2a), 1.94–
2
,3
13
1
1
35 Hz and COLOC,
JC,H 10 Hz, correspondingly), and 1D C– H
2
2
.06 (m, 2H, [1.99]—H-15a; [2.01]—H-21a), 1.99 (s, 3H, CH
.23 (s, 1H, H-9a), 2.65 (ddd, 1H, J1e,1a 13.5, J1e,2a 4, J1e,2e 3.5 Hz,
-31), 4.45 (dd, 1H, J3a,2a 12, J3a,2e 5 Hz, H-
3
-33),
long-range J modulation difference (LRJMD, JC,H 10 Hz). Purity of
the final compounds and intermediates for biological testing was
confirmed to be P95% as determined by HPLC analysis (for data,
see Supplementary data). HPLC analyses were carried out on a Mil-
ichrom A-02, using a ProntoSIL 120-5-C18 AQ column (BISCHOFF,
H-1e), 3.62 (s, 3H, OCH
a), 5.72 (s, 1H, H-12), 5.76 (d, 1H, J19,21e 1.2 Hz, H-19). C NMR
CDCl ): d = 38.6 (t, C-1), 23.3 (t, C-2), 80.4 (d, C-3), 37.8 (s, C-4),
5.0 (d, C-5), 17.3 (t, C-6), 33.7 (t, C-7), 43.3 (s, C-8), 60.6 (d,
3
13
3
(
5
3
2
.0 ꢄ 75 mm column, grain size 5.0
lm). Mobile phase: Millipore
C-9), 36.6 (s, C-10), 199.9 (s, C-11), 124.0 (d, C-12), 162.6 (s,
C-13), 45.0 (s, C-14), 25.8 (t, C-15), 35.9 (t, C-16), 34.7 (s, C-17),
purified water with 0.1% trifluoroacetic acid at a flow rate of
1
2
0
50 lL/min at 35 °C and UV detection at 210, 220, 240, 260 and
80 nm. A typical run time was 25 min with a linear gradient of
–100% methanol. Flash column chromatography was done with
1
42.6 (s, C-18), 129.5 (d, C-19), 44.2 (s, C-20), 27.7 (t, C-21), 34.6
(
t, C-22), 27.9 (q, C-23), 16.6 (q, C-24), 16.58 (q, C-25), 18.2 (q,
C-26), 19.5 (q, C-27), 24.2 (q, C-28), 24.9 (q, C-29), 176.6 (s,
C-30), 52.0 (q, C-31), 170.7 (s, C-32), 21.1 (q, C-33).
silica gel (Merck, 60–200 mesh) and neutral alumina (Chemapol,
4
0–250 mesh).
4
.1.4. Methyl 3b-hydroxy-11-oxoolean-12(13),18(19)-dien-30-
oate (9)
A mixture of 6 (6.5 g, 12.4 mmol) and KOH (45 g, 775 mmol) in
4
.1.2. Reagents
The synthesis and complete physicochemical description of
compounds 5, 7, 8 and Soloxolone methyl were presented in the
MeOH (650 mL) was stirred at room temperature for 3 h (the reac-
tion course was monitored by TLC). The resulting solution was con-
centrated under reduced pressure, and aq HCl (10%) was added
until the pH reached 4–5. The mixture was extracted with chloro-
form/ethyl acetate (1:4) (3 ꢄ 100 mL). The combined organic lay-
2
5
work.
Compound
2
was synthesized according to
a described
1
1
method with a yield of 20%; herein we present a complete de-
1
13
tailed description of the NMR H and C data:
1
H NMR (CDCl
.13 (s, 3H, CH -29), 1.16 (s, 3H, CH
.36 (s, 3H, CH -27), 1.42 (s, 3H, CH
3
): d = 0.80 (s, 3H, CH
3
-28), 1.12 (s, 3H, CH
3
-24),
-23),
ers were washed with satd aq NaHCO
3
, brine (3 ꢄ 50 mL) and
1
1
1
1
3
3
-26), 1.19 (s, 3H, CH
3
dried over anhydrous MgSO
4
. The solvent was removed to give a
3
3
-25), 1.04 (ddd, 1H, J16e,16a
solid 9 (5.9 g, 99%). This material was used for the next reaction
without further purification. An analytically pure sample was ob-
tained by recrystallization from methanol. Mp 208–210 °C. HRMS:
3.6, J16e,15a 4.5 J16e,15e 2.3 Hz, H-16e), 1.21 (dm, 1H, J15e,15a
3.6 Hz, H-15e), 1.27–1.32 (m, 2H, H-21a, H-22), 1.36–1.42 (m,
Please cite this article in press as: Salomatina, O. V.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.10.049

6
O. V. Salomatina et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
m/z calcd for C31
H
46
O
4
: 482.3396; found: 482.3385. 1H NMR
purification. An analytically pure sample was obtained by flash col-
umn chromatography (silica gel, n-hexane/AcOEt (9:1, v/v) fol-
lowed by n-hexane/AcOEt (3:1, v/v)) as an amorphous white
(
CDCl
CH -24), 0.90 (s, 3H, CH
1a,2e 4 Hz, H-1a), 0.95 (s, 3H, CH
3
): d = 0.65 (dd, 1H, J5a,6a 12, J5a,6e 1.5 Hz, H-5a), 0.81 (s, 3H,
-28), 0.91 (ddd, 1H, J1a,1e 13, J1a,2a 13,
-23), 1.13 (s, 6H, CH -26, CH
-25), 1.25 (ddd, 1H, J15e,15a 13, J15e,16a 4,
-29), 1.42 (m, 1H, H-6a),
.45–1.62 (m, 9H, H-6e, [1.49]—2H-22; [1.50] and [1.53]—2H-16,
1.50] and [1.57]—2H-7, [1.57]—H-21e, [1.58]—H-2e), 1.63 (m,
H, H-2a), 1.96–2.07 (m, 2H, [2.01]—H-15a; [2.03]—H-21a), 2.23
3
3
J
2
3
3
3
-
solid. HRMS: m/z calcd for C32
H
43
O
5
: 508.3177; found: 508.3101.
): d = 0.92 (s, 3H, CH -28), 1.09 (s, 3H, CH -24),
-27), 1.15 (s, 6H, CH -23 and CH -25), 1.17 (s, 3H,
-26), 1.26 (s, 3H, CH -29), 1.29 (s, 3H, CH -25), [1.10] (m, 1H,
1
7), 1.16 (s, 3H, CH
3
H NMR (CDCl
1.13 (s, 3H, CH
CH
3
3
3
J15e,16e 3 Hz, H-15e), 1.26 (s, 3H, CH
3
3
3
3
1
[
1
3
3
3
H-5a), 1.29 (ddd, 1H, J15e,15a 13, J15e,16a 4, J15e,16e 3 Hz, H-15e),
1.40–1.62 (m, 9H: [1.45]—2H-6; [1.50]—2H-22; [1.53]—2H-16,
[1.58]—2H-7, [1.58]—H-21e), 1.88 (d, 1H, J1a,1e 15 Hz, H-1a), 2.02
(m, 2H, H-15a, H-21a), 2.32 (s, 1H, H-9a), 3.32 (d, 1H, J1e,1a 15 Hz,
1
e
(
s, 1H, H-9a), 2.66 (ddd, 1H, J1e,1a 13, J1e,2a 3.5, J1e,2e 3.5 Hz, H ),
3
5
.18 (dd, 1H, J3a,2a 11, J3a,2e 5 Hz, H-3a), 3.64 (s, 3H, OCH
.74 (s, 1H, H-12), 5.76 (d, 1H, J19,21e 1.2 Hz, H-19). 13C NMR
): d = 38.9 (t, C-1), 27.1 (t, C-2), 78.6 (d, C-3), 38.9 (s, C-4),
5.0 (d, C-5), 17.4 (t, C-6), 33.8 (t, C-7), 43.4 (s, C-8), 60.7 (d,
3
-31),
3
H-1e), 3.64 (s, 3H, OCH -31), 5.78 (d, 1H, J19,21e 1.2 Hz, H-19),
(
CDCl
3
5.79 (s, 1H, H-12), 8.57 (d, J32,OH 3.2 Hz, CHOH-32), 14.84 (d, 1H,
1
3
5
3
JOH,32 3.2 Uw, CHOH-32). C NMR (CDCl ): d = 39.5 (t, C-1): 39.54
C-9), 36.8 (s, C-10), 200.1 (s, C-11), 124.0 (d, C-12), 162.6 (s,
C-13), 45.1 (s, C-14), 25.8 (t, C-15), 36.0 (t, C-16), 34.8 (s, C-17),
(t, C-1), 105.58 (s, C-2), 189.52 (s, C-3), 39.82 (s, C-4), 52.22 (d,
C-5), 18.65 (t, C-6), 32.79 (t, C-7), 42.96 (s, C-8), 58.57 (d, C-9),
35.97 (s, C-10), 199.42 (s, C-11), 123.93 (d, C-12), 163.15 (s,
C-13), 45.10 (s, C-14), 25.73 (t, C-15), 35.91 (t, C-16), 34.77 (s,
C-17), 142.55 (s, C-18), 129.78 (d, C-19), 44.27 (s, C-20), 27.73 (t,
C-21), 34.31 (t, C-22), 28.29 (q, C-23), 20.79 (q, C-24), 14.92 (q,
C-25), 18.02 (q, C-26), 19.51 (q, C-27), 24.24 (q, C-28), 24.88 (q,
C-29), 176.57 (s, C-30), 52.04 (q, C-31), 188.64 (d, C-32).
1
42.7 (s, C-18), 129.4 (d, C-19), 44.2 (s, C-20), 27.8 (t, C-21), 34.6
(
t, C-22), 28.0 (q, C-23), 15.5 (q, C-24), 16.6 (q, C-25), 18.3 (q,
C-26), 19.6 (q, C-27), 24.2 (q, C-28), 24.9 (q, C-29), 176.7 (s,
C-30), 52.0 (q, C-31).
4
.1.5. Methyl 3,11-dioxoolean-12(13),18(19)-dien-30-oate (10)
Jones reagent (6 mL), prepared from Na Cr *2H O in aq
was added dropwise to a solution of compound 9 (5.8 g,
2
2
O
7
2
3
4
H
2
SO
4
,
4.1.7. Methyl 11-Oxoisoxazolo[4,5-b]-olean-9(11),18(19)-dien-
30-oate (12)
1
2.0 mmol) in acetone (500 mL)) at 0 °C over a period of 30 min till
the brown color persisted. The mixture was stirred for a further
.5 h at room temperature (the reaction course was monitored
by TLC) and EtOH (10 mL) was added. The resulting mixture was
concentrated under reduced pressure (
ꢆ 100 mL) and diluted
with H O (1 L). The solid was filtered and dried. The ketone 10
5.2 g; 90%) was purified by flash column chromatography (neutral
NH
(4.2 g, 8.3 mmol) in EtOH (120 mL) and H
under reflux for 2 h, the solution was cooled to room temperature
and concentrated under reduced pressure (
ꢆ 10 mL). The mixture
was then diluted with AcOEt (150 mL) and washed with H O, satd
aq NaHCO , brine. The organic phase was dried over anhydrous
MgSO and evaporated to dryness. The crude product was purified
2
OH*HCl (5.8 g, 83.5 mmol) was added to a solution of 11
2
2
O (12 mL). After heating
v
v
2
2
(
3
alumina, chloroform) as a white solid. This material was used for
the next reaction without further purification. An analytically pure
sample was obtained by recrystallization from a chloroform/meth-
4
by flash column chromatography (silica gel, n-hexane/ethyl acetate
3:1, v/v) to yield the isoxazole derivative 12 (4.0 g, 95%). HRMS: m/
1
anol mixture. Mp 200 °C. HRMS: m/z calcd for C31
H
44
O
4
: 480.3240;
-28), 1.02 (s,
-27), 1.16 (s, 3H,
-25), 1.23–1.33 (m,
z calcd for C32
(CDCl ): d = 0.92 (s, 3H, CH
-27), 1.17 (s, 3H, CH -26), 1.19 (s, 3H, CH
-29), 1.26 (s, 3H, CH -23), [1.26] (m, 1H, H-5a), 1.29 (ddd, 1H,
4
H43NO : 505.3181; found: 508.3164. H NMR
1
found: 480.3235. H NMR (CDCl
H, CH -24), 1.05 (s, 3H, CH -23), 1.14 (s, 3H, CH
CH -26), 1.26 (s, 3H, CH -29), 1.29 (s, 3H, CH
3
): d = 0.91 (s, 3H, CH
3
3
3
-28), 1.11 (s, 3H, CH
3
-25), 1.14 (s, 3H,
3
3
3
3
CH
CH
3
3
3
-24), 1.25 (s, 3H,
3
3
3
3
3
2
1
H, [1.26] – H-5a; [1.28]—H-15e), 1.36 (ddd, 1H, J1a,1e 13.5, J1a,2a
1, J1a,2e 7 Hz, H-1a), 1.40–1.64 (m, 9H, [1.48] and [1.55]—2H-6;
J15e,15a 13, J15e,16a 3.5, J15e,16e 3.5 Hz, H-15e), 1.43–1.65 (m, 9H:
[1.49]—2H-22; [1.51] and [1.57]—2H-6; [1.53]—2H-16, [1.58]—H-
21e; [1.61]—2H-7), 1.95 (d, 1H, J1a,1e 15.5 Hz, H-1a), 1.96–2.07
(m, 2H, H-15a and H-21a), 2.41 (s, 1H, H-9a), 3.51 (d, 1H, J1e,1a
[
[
1.50]—2H-22; [1.51] and [1.55]—2H-16, [1.54] and [1.58]—2H-7,
1.59]—H-21e), 2.03 (m, 2H, H-15a, H-21a), 2.32 (s, 1H, H-9a),
2
J
7
(
.32 (ddd, 1H, J2e,2a 16, J2e,1a 7, J2e,1e 4 Hz, H-2e), 2.59 (ddd, 1H,
2a,2e 16, J2a,1a 11, J2a,1e 7 Hz, H-2a), 2.84 (ddd, 1H, J1e,1a 13.5, J1e,2a
, J1e,2e 4 Hz, H-1e), 3.64 (s, 3H, OCH -31), 5.77 (s, 1H, H-12), 5.78
d, 1H, J19,21e 1.2 Hz, H-19). 13C NMR (CDCl
): d = 39.5 (t, C-1),
4.0 (t, C-2), 217.0 (s, C-3), 47.5 (s, C-4), 55.3 (d, C-5), 18.7 (t,
C-6), 33.2 (t, C-7), 43.2 (s, C-8), 60.0 (d, C-9), 36.4 (s, C-10), 199.3
s, C-11), 124.0 (d, C-12), 163.0 (s, C-13), 45.1 (s, C-14), 25.8 (t,
15.5 Hz, H-1e), 3.64 (s, 3H, OCH
H-19), 5.80 (s, 1H, H-12), 7.94 (d, 1H, H-32). H NMR (CDCl ):
3
-31), 5.78 (d, 1H, J19,21e 1.2 Hz,
1
3
3
d = 39.77 (t, C-1), 108.82 (s, C-2), 172.12 (s, C-3), 38.21 (s, C-4),
53.32 (d, C-5), 18.01 (t, C-6), 32.89 (t, C-7), 43.21 (s, C-8), 59.05
(d, C-9), 34.79 (s, C-10), 199.27 (s, C-11), 123.95 (d, C-12), 163.11
(s, C-13), 45.13 (s, C-14), 25.81 (t, C-15), 35.92 (t, C-16), 34.96 (s,
C-17)⁄, 142.53 (s, C-18), 129.83 (d, C-19), 44.24 (s, C-20), 27.75
(t, C-21), 34.63 (t, C-22), 28.74 (q, C-23), 21.43 (q, C-24), 15.89
(q, C-25), 18.01 (q, C-26), 19.57 (q, C-27), 24.26 (q, C-28), 24.90
(q, C-29), 176.57 (s, C-30), 52.08 (q, C-31), 150.17 (d, C-32).
3
3
(
C-15), 36.0 (t, C-16), 34.8 (s, C-17), 142.6 (s, C-18), 129.7 (d,
C-19), 44.3 (s, C-20), 27.7 (t, C-21), 34.6 (t, C-22), 26.4 (q, C-23),
2
1.3 (q, C-24), 15.9 (q, C-25), 18.2 (q, C-26), 19.5 (q, C-27), 24.2
(
q, C-28), 24.9 (q, C-29), 176.6 (s, C-30), 52.0 (q, C-31).
4
.1.8. The mixture of tautomers (13)
4
1
.1.6. Methyl 2-hydroxymethylene-3,11-dioxoolean-
2(13),18(19)-dien-30-oate (11)
NaOMe (15 g) was added to a stirring solution of isoxazole
derivative 10 (3.9 g, 7.7 mmol), methanol (85 mL) and diethyl
NaOMe (3.0 g, 55.5 mmol) was added to a stirring solution of
ether (170 mL) at 0 °C. The stirring was continued at room temper-
ketone 10 (4.2 g, 8.8 mmol) and HCOOEt (4.0 mL, 50.5 mmol) in
dry benzene (50 mL). After stirring for 2 h at room temperature,
ature for 1 h. Then the resulting mixture was diluted with CHCl
Et O (100 mL, 1:3, v/v) and aq HCl (5%) was added until the pH
reached 4–5. The organic layer was separated; the aqueous layer
was extracted with CHCl /Et O (50 mL, 1:3, v/v). The combined or-
ganic layers were washed with satd aq NaHCO , brine and dried
over anhydrous MgSO . The solvent was removed to give a mixture
3
/
2
the mixture was diluted with CHCl
aq HCl (5%) was added until pH reached 4–5. The organic layer
was separated; the aqueous layer was extracted with CHCl /Et
3 2
/Et O (100 mL, 1:3, v/v), and
3
2
3
2
O
3
(
3 ꢄ 50 mL, 1:3, v/v). The combined organic layers were washed
4
with satd aq NaHCO
3
, brine and dried over anhydrous MgSO
4
.
of tautomers 13 (yield = 3.0 g, 100%) as a solid. This material was
The solvent was removed to give an amorphous solid 11 (4.3 g,
7%). This material was used for the next reaction without further
used for the next reaction without further purification. HRMS:
m/z calcd for C32H43NO : 505.3181; found: 507.3187.
4
9
Please cite this article in press as: Salomatina, O. V.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.10.049

O. V. Salomatina et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
7
4
3
.1.9. Methyl 2-cyano-3,11-dioxoolean-1(2),9(11),18(19)-trien-
0-oate (1)
C
31
H
48
O
3
: 468.7108; found: 468.3603. 1H NMR (CDCl
(s, 3H, CH -28), 0.99 (s, 3H, CH -26), 1.03 (s, 3H, CH
3H, CH -25), 1.07 (s, 3H, CH -23), 1.10 (s, 3H, CH
3H, CH -27), 0.86 (dm, 1H, J16e16a 13.2 Hz, H-16e), 0.96–1.01 (m,
3
): d = 0.76
-24), 1.05 (s,
3
-29), 1.12 (s,
3
3
3
A mixture of tautomers 13 (3.7 g, 7.3 mmol) and 2,3-dichloro-
,6-dicyano-1,4-benzoquinone (DDQ) (1.9 g, 8.4 mmol) in dry ben-
3
3
5
3
zene (200 mL) was heated under reflux for 4 h (the reaction course
was monitored by TLC). Then the insoluble matter was removed by
filtration and the filtrate was evaporated to dryness. The solid was
subjected to flash column chromatography (silica gel, benzene fol-
lowed by benzene/acetone (10:1, v:v) to give a crude 12. The crude
1H, H-15a), 1.59 (dd, 1H, J19a,19e 13.1, J19a,18a 13.1 Hz, H-19a), 1.63
(dd, 1H, J9a,11a 11.4, J9a,11e 6.4 Hz, H-9a), 1.77 (ddd, 1H, J15a,15e
13.6, J15a,16a 13.6, J15a,16e 4.5 Hz, H-15a), 2.35 (ddd, 1H, J2e,2a 16.0,
J2e,1a 6.8, J2e,1e 3.7 Hz, H-2e), 2.52 (ddd, 1H, J2a,2e 16.0, J2a,1a 11.2,
2e,1e 7.3 Hz, H-2a), 3.66 (s, 3H, OCH -31), 5.27 (t, 1H, J12,11 3.6 Hz,
J
3
product was purified by recrystallization from MeOH/CHCl
3
to give
H-12), the signals of the other proton 1.21–1.55 (H-1, H-5, 2H-6,
25
0
crystals 12 (yield 3.0 g; 84%). Mp 214–217 °C. ½
aꢅ
+440° (c 0.21,
2H-7, H-21, 2H-22) and 1.82–2.00 (H-1 , 2H-11, H-16a, H-18, H-
D
ꢇ1
0
13
3 3
CHCl ). IR (CHCl , cm
)
mmax: 3021; 2237 (C„N); 1726, 1687,
3
19e, H-21 ). C NMR (CDCl ): d = 39.2 (t, C-1), 34.0 (t, C-2), 217.5
1
5
CH
CH
656 (C@O). HRMS: m/z calcd for C32
H
41NO
4
: 503.3181; found:
): d = 0.92 (s, 3H, CH -28), 1.11 (s, 3H,
-27), 1.16 (s, 3H, CH -23), 1.19 (s, 3H,
-29), 1.47 (s, 3H, CH -25), 1.30 (ddd, 1H,
(s, C-3), 47.3 (s, C-4), 55.2 (d, C-5), 19.5 (t, C-6), 32.1 (t, C-7), 39.7
(s, C-8), 46.8 (d, C-9), 36.6 (s, C-10), 23.5 (t, C-11), 122.3 (d, C-
12), 144.3 (s, C-13), 41.6 (s, C-14), 26.0 (t, C-15), 26.9 (t, C-16),
31.9 (s, C-17), 48.2 (d, C-18), 42.7 (t, C-19), 44.2 (s, C-20), 31.2 (t,
C-21), 38.3 (t, C-22), 26.4 (q, C-23), 21.4 (q, C-24), 15.1 (q, C-25),
16.6 (q, C-26), 25.7 (q, C-27), 28.1 (q, C-28), 28.4 (q, C-29), 177.5
(s, C-30), 51.4 (q, C-31).
1
08.3025. H NMR (CDCl
3
3
3
-24), 1.15 (s, 3H, CH
-26), 1.27 (s, 3H, CH
3
3
3
3
3
J
15e,15a 13, J15e,16a 3.5, J15e,16e 3.5 Hz, H-15e), 1.48–1.66 (m, 10H:
[
[
1.51]—2H-22; [1.55]—H-5a; [1.56]—2H-16, [1.59]—H-21e;
1.60]—2H-6; [1.62]—2H-7), 2.02 (m, 1H, H-21a), 2.04 (m, 1H, H-
1
1
3
5a), 2.60 (s, 1H, H-9a), 3.66 (s, 3H, OCH -31), 5.82 (d, 1H, J19,21e
19
13
.2 Hz, H ), 5.86 (s, 1H, H-12), 8.39 (s, 1H, H-1). C NMR (CDCl
3
):
4.1.12. Methyl 2-hydroxymethylene-3-oxo-18bH-olean-12(13)-
en-30-oate (21)
d = 172.53 (d, C-1), 114.73 (s, C-2), 197.48 (s, C-3), 44.87 (s, C-4),
1.85 (d, C-5), 17.99 (t, C-6), 32.61 (t, C-7), 43.62 (s, C-8), 53.39
5
3-Oxoderivative 20 (0.8 g, 1.7 mmol) and HCOOEt (0.6 mL,
7.3 mmol) in dry benzene (8 mL) were reacted in the presence of
NaOMe (0.7 g, 13.0 mmol) in a similar way as described in the syn-
thesis of 11 to give 21 (0.75 g, 89.0%) as a amorphous solid. This
material was used for the next reaction without further purifica-
tion. An analytically pure sample was obtained by flash column
chromatography (silica gel, n-hexane/AcOEt (9:1, v/v) followed
(
(
d, C-9), 39.42 (s, C-10), 197.97 (s, C-11), 123.20 (d, C-12), 164.78
s, C-13), 45.43 (s, C-14), 25.76 (t, C-15), 35.73 (t, C-16), 34.77 (s,
C-17), 142.23 (s, C-18), 130.62 (d, C-19), 44.29 (s, C-20), 27.69 (t,
C-21), 34.54 (t, C-22), 27.42 (q, C-23), 21.39 (q, C-24), 19.90 (q,
C-25), 18.51 (q, C-26), 19.56 (q, C-27), 24.28 (q, C-28), 24.82 (q,
C-29), 176.41 (s, C-30), 52.09 (q, C-31), 113.10 (s, C-32).
2
5
by n-hexane/AcOEt (8:1, v/v)). ½
m/z calcd for C32 : 496.7211; found: 496.3550. H NMR
(CDCl ): d = 0.76 (s, 3H, CH -28), 0.909 (s, 3H, CH -25), 0.99 (s,
3H, CH -26), 1.09 (s, 3H, CH -24), 1.10 (s, 3H, CH -29), 1.12 (s,
3H, CH -27), 1.17 (s, 3H, CH -23), 0.86 (dm, 1H, J16e,16a 13.2 Hz,
aꢅ
D
3
+185° (c 0.08, CHCl ). HRMS:
1
4
.1.10. Methyl 3b-hydroxy-18bH-olean-12(13)-en-30-oate (19)
Methyl 3b-acetoxy-18bH-olean-12(13)-en-30-oate (2.0 g,
.9 mmol) was deprotected with ROH (13 g, 232 mmol) in CH OH
48 4
H O
7
3
3
3
3
3
3
3
3
(
130 mL) in a similar way as described in the synthesis of 9 to fin-
3
3
ish 19 (1.8 g, 98%). This material was used for the next reaction
H-16e), 0.96–1.03 (m, 1H, H-15a), 1.11-1.15 (m, 1H, H-5), 1.59
without further purification. An analytically pure sample was ob-
(dd, 1H, J19a,19e 13.1, J19a,18a 13.1 Hz, H-19a), 1.64 (dd, 1H, J9a,11a
tained by recrystallization from CH
HRMS: m/z calcd for C31 : 470.7269; found: 470.3760.
NMR (CDCl ): d = 0.74 (s, 3H, CH -28), 0.81 (dd, 1H, J5a,6a 12.0 Hz,
5a,6e 1.6 Hz; H-5a), 0.82–0.86 (m, 1H, H-16e), 0.83 (s, 3H,
CH -24), 0.84 (s, 3H, CH -23), 0.93 (s, 6H, CH -25 and CH -26),
.94 (dm, J15e,15a 13.5 Hz, H-15e), 1.02 (m, 1H, H-1), 1.09 (s, 3H,
3
OH/CHCl
3
. Mp 242–214 °C.
11.5, J9a,11e 6.2 Hz, H-9a), 1.76 (ddd, 1H, J15a,15e 13.7, J15a,16a 13.7,
1
0
H
50
O
3
H
J
15a,16e 4.4 Hz, H-15a), 1.90 (d, 1H, J1,1 14.3 Hz, H-1), 2.27 (d, 1H,
0
0
3
3
3
J1 ,1 14.3 Hz, H-1 ), 3.66 (s, 3H, OCH -31), 5.30 (t, 1H, J12,11 3.6 Hz,
J
H-12),), 8.86 (d, 1H, J32,OH 3.2 Hz, H-32), 14.91 (d, 1H, JOH,32
3.2 Hz, OH), the signals of the other proton 1.82–2.03 (2H-11, H-
3
3
3
3
0
0
0
16a, H-18, H-19e, H-21 ) and 1.21–1.56 (H-1 , 2H-11, H-16a, H-
0
13
CH
3
-29), 1.10 (s, 3H, CH
3
-27), 1.17–1.35 (m, 4H, H-7, H-21,
3
18, H-19e, H-21 ). C NMR (CDCl ): d = 39.2 (t, C-1), 105.7 (s, C-
0
2
H-22), 1.39 (m, 1H, H-6a), 1.44–1.64 (m, 7H, H-1 , 2H-2, H-6e,
2), 190.6 (s, C-3), 40.0 (s, C-4), 51.9 (d, C-5), 19.4 (t, C-6), 31.7 (t,
C-7), 39.5 (s, C-8), 45.5 (d, C-9), 36.1 (s, C-10), 23.3 (t, C-11),
122.2 (d, C-12), 144.2 (s, C-13), 41.5 (s, C-14), 26.0 (t, C-15), 26.8
(t, C-16), 31.8 (s, C-17), 48.1 (d, C-18), 42.7 (t, C-19), 44.1 (s, C-
20), 31.1 (t, C-21), 38.2 (t, C-22), 28.3 (q, C-23), 20.8 (q, C-24),
14.5 (q, C-25), 16.4 (q, C-26), 25.6 (q, C-27), 28.1 (q, C-28), 28.4
(q, C-29), 177.5 (s, C-30), 51.5 (q, C-31), 188.3 (d, C-32).
0
H-7 , H-9a, H-19), 1.73 (ddd, 1H, J15a,15e 13.5 Hz, J15a,16a 13.5 Hz,
0
J
2
15a,16e 4.6 Hz; H-15a), 1.79–1.93 (m, 5H; 2H-11, H-18, H-19 , H-
1 ), 1.92 (m; 1H, H-16a), 3.23 (dd, 1H, J3a,2a 10.0, 3
0
J
3a,2e 6.0 Hz;
1
3
H-3a), 3.64 (s, 3H; OCH
NMR (CDCl ): d = 38.1 (t, C-1), 23.4 (t, C-2), 78.6 (d, C-3), 37.56
s, C-4), 55.1 (d, C-5), 18.1 (t, C-6), 32.5 (t, C-7), 39.7 (s, C-8), 47.4
d, C-9), 36.7 (s, C-10), 23.3 (t, C-11), 122.3 (d, C-12), 144.2 (s,
3
-31), 5.23 (t, J12,11) 3.6 Hz, H-12).
C
3
(
(
C-13), 41.4 (s, C-14), 26.0 (t; C-15), 26.8 (t, C-16), 31.8 (s, C-17),
8.1 (d, C-18), 42.7 (t, C-19), 44.1 (s, C-20), 31.15 (t, C-21), 38.3
t, C-22), 27.9 (q, C-23), 16.54 (q, C-24), 15.4 (q, C-25), 16.6 (q,
4.1.13. Methyl isoxazolo[4,5-b]-18bH-olean-12(13)-en-30-oate
(22)
4
(
The isoxazole derivative 22 was obtained by condensation of
C-26), 25.8 (q, C-27), 28.0 (q, C-28), 28.4 (q, C-29), 177.0 (s,
C-30), 51.4 (q, C-31).
compound 21 (0.75 g, 1.5 mmol) and NH
2
OH*HCl (1.0 g,
14.4 mmol) in EtOH/H 0 (40 mL, 10:1, v/v) in a similar way as
2
described in the synthesis of 12. Flash column chromatography
purification (silica gel, n-hexane/AcOEt (9:1, v/v)) followed by
4
.1.11. Methyl 3-oxo-18bH-olean-12(13)-en-30-oate (20)
Methyl 3b-hydroxy-18bH-olean-12(13)-en-30-oate 19 (1.9 g,
.8 mmol) was oxidized by Jones reagent (2 mL) in acetone
2
5
n-hexane/AcOEt (8:1, v/v)) yielded 22 (0.7 g, 95%). ½
0.10, CHCl ). HRMS: m/z calcd for C32 : 493.7205; found:
): d = 0.78 (s, 3H, CH -28), 0.89 (s, 3H,
-26), 1.11 (s, 3H, CH -29), 1.14 (s, 3H,
-24), 1.30 (s, 3H, CH -23), 0.84–0.90 (m,
aꢅ
+234° (c
D
3
3
H47NO
3
1
(
200 mL) in a similar way as described in the synthesis of 10. Flash
493.3556. H NMR (CDCl
CH -25), 1.00 (s, 3H, CH
CH -27), 1.20 (s, 3H, CH
3
3
column chromatography purification (neutral alumina, chloro-
form) afforded 20 (1.4 g, 78%) as a white solid. This material was
used for the next reaction without further purification. An analyt-
ically pure sample was obtained by recrystallization from a chloro-
form/methanol mixture. Mp 188–190 °C. HRMS: m/z calcd for
3
3
3
3
3
3
H-16e), 1.02 (ddd, 1H, J15e,15a 13.5, J15e,16a 4.2, J15e,16e 2.3 Hz, H-
15e), 1.61 (dd, 1H, J19a,19e 13.1, J19a,18a 13.1 Hz, H-19a), 1.74 (dd,
1H, J9a,11a 11.5, J9a,11e 6.2 Hz, H-9a), 1.79 (ddd, 1H, J15a,15e 13.5,
Please cite this article in press as: Salomatina, O. V.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.10.049

8
O. V. Salomatina et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
0
J
2
J
15a,16a 13.5, J15a,16e 4.5 Hz, H-15a), 1.99 (d, 1H, J1,1 15.0 Hz, H-1),
C-1), 23.3 (t, C-2), 80.4 (d, C-3), 37.8 (s, C-4), 55.0 (d, C-5), 17.3
0
0
.42 (d, 1H, J1 ,1 15.0 Hz, H-1 ), 3.67 (s, 3H, OCH
3
-31), 5.31 (t, 1H,
12,11 3.6 Hz, H-12), 7.96 (s, 1H, H-32), the signals of the other pro-
(t, C-6), 33.7 (t, C-7), 43.3 (s, C-8), 60.6 (d, C-9), 36.6 (s, C-10),
199.9 (s, C-11), 124.0 (d, C-12), 162.6 (s, C-13), 45.0 (s, C-14),
25.8 (t, C-15), 35.9 (t, C-16), 34.7 (s, C-17), 142.6 (s, C-18), 129.5
(d, C-19), 44.2 (s, C-20), 27.7 (t, C-21), 34.6 (t, C-22), 27.9 (q, C-
23), 16.6 (q, C-24), 16.58 (q, C-25), 18.2 (q, C-26), 19.5 (q, C-27),
24.2 (q, C-28), 24.9 (q, C-29), 176.6 (s, C-30), 52.0 (q, C-31), 170.7
(s, C-32), 21.1 (q, C-33).
13
ton 1.83–2.06 and 1.22–1.62. C NMR (CDCl
08.7 (s, C-2), 172.9 (s, C-3), 34.6 (s, C-4), 53.4 (d, C-5), 18.8 (t,
C-6), 31.8 (t, C-7), 39.7 (s, C-8), 46.0 (d, C-9), 38.5 (s, C-10), 23.3
t, C-11), 122.2 (d, C-12), 144.3 (s, C-13), 41.6 (s, C-14), 26.1 (t, C-
3
): d = 35.4 (t, C-1),
1
(
1
4
5), 26.9 (t, C-16), 31.9 (s, C-17), 48.1 (d, C-18), 42.8 (t, C-19),
4.2 (s, C-20), 31.2 (t, C-21), 38.3 (t, C-22), 28.7 (q, C-23), 21.3 (q,
C-24), 15.3 (q, C-25), 16.4 (q, C-26), 25.6 (q, C-27), 28.1 (q, C-28),
4
.1.17. Methyl 3,12-dioxo-18bH-olean-30-oate (25)
2
8.4 (q, C-29), 177.5 (s, C-30), 51.4 (q, C-31), 150.1 (d C-32).
Methyl 3b-hydroxy-12-oxo-18bH-olean-30-oate 24 (2.5 g,
.1 mmol) was oxidized by Jones reagent (2 mL) in acetone
5
4
.1.14. Cleavage of the isoxazole ring in compound 22
(
200 mL) in a similar way as described in the synthesis of 10. Flash
The isoxazole ring in compound 22 (0.6 g, 1.2 mmol) was
column chromatography purification (neutral alumina, chloro-
form) afforded 25 (1.9 g, 76%) as a white solid. An analytically pure
sample was obtained by recrystallization from a mixture of chloro-
cleaved by NaOMe (2.5 g) in a mixture of Et
2
2
2
O/MeOH (60 mL,
:1, v/v) in a similar way as described in the synthesis of 13 to give
3 (0.6 g, quantitative yield) as an amorphous solid. This material
25
D
form/methanol. ½
aꢅ
3
+47° (c 0.28, CHCl ). HRMS: m/z calcd for
was used for the next reaction without further purification. HRMS:
m/z calcd for C32 : 493.7205; found: 493.3401.
1
C
(
H
31 48
O
4
: 484.7104; found: 484.3553. H NMR (CDCl
s, 3H, CH -28), 0.88 (s, 3H, CH -27), 0.94 (s, 3H, CH -25), 0.99 (s,
-24), 1.03 (s, 3H, CH -23), 1.06 (s, 3H, CH -29), 1.13 (s,
-26), 0.83 (dm, 1H, J16e,16a 13.2 Hz, H-16e), 0.93–1.00 (m,
H, H-15a), 1.16 (dd, 1H, J19a,19e 13.3, J19a,18a 13.3 Hz, H-19a), 2.16
3
): d = 0.80
H47NO
3
3
3
3
3
3
1
H, CH
H, CH
3
3
3
4
.1.15. Methyl 2-cyano-3,11-dioxo-18bH-olean-1(2),12(13)-
3
dien-30-oate (3)
Compound 3 was obtained by oxidation of a mixture of tautom-
ers 23 (0.6 g, 1.0 mmol) DDQ (0.25 g, 1.0 mmol) in benzene (40 mL)
in a similar way as described in the synthesis of 1. Flash column
chromatography purification (silica gel, benzene followed by ben-
zene/acetone (10:1, v:v) yielded 3. The crude product was recrys-
(
dd, 1H, J11a,11e 16.6, J11a,9a 12.8 Hz, H-11a), 2.22 (dd, 1H, J11e,11a
1
3
2
2
6.6, J11e,9a 5.3 Hz, H-11e), 2.31 (ddd, 1H, J2e,2a 15.9, J2e,1a 7.0, J2e,1e
.7 Hz, H-2e), 2.46 (ddd, 1H, J2a,2e 15.9, J2a,1a 10.5, J2a,1e 7.0 Hz, H-
a), 2.53 (ddd, 1H, J19e,19a 13.3, J19e,18a 3.7, J19e,21e 2.1 Hz, H-19e),
3
.73 (d, 1H, J13,18 4.4 Hz, H-13), 3.64 (s, 3H, OCH -31), the signals
tallizated from MeOH/CHCl
3
to give crystals 3 (yield 0.25 g; 50%).
13
3
of the other protons 1.15–1.60 and 1.65–1.92. C NMR (CDCl ):
25
ꢇ1
½
a
ꢅ
+294° (c 0.21, CHCl
3
). Mp 303–305 °C. IR (CHCl
3
, cm
)
m
max
:
D
d = 38.4 (t, C-1), 33.6 (t, C-2), 216.4 (s, C-3), 47.1 (s, C-4), 54.7 (d,
C-5), 19.4 (t, C-6), 31.0 (t, C-7), 41.3 (s, C-8), 48.8 (d, C-9), 36.4 (s,
C-10), 38.3 (t, C-11), 211.2 (s, C-12), 50.0 (d, C-13), 42.0 (s, C-14),
2
952; 2236 (C„N); 1723, 1686 (C@O). HRMS: m/z calcd for
1
C
32
H
45NO
3
: 491.3181; found: 491.3025. H NMR (CDCl
-28), 1.04 (s, 3H, CH -26), 1.11 (s, 3H, CH -29), 1.12 (s,
-24), 1.13 (s, 3H, CH -27), 1.19 (s, 3H, CH -23), 1.21 (s,
-25), 0.88 (dm, 1H, J16e,16a 13.3 Hz, H-16e), 1.30 (ddd, 1H,
3
): d = 0.76
(
s, 3H, CH
3
3
3
2
5.8 (t, C-15), 26.2 (t, C-16), 31.9 (s, C-17), 38.3 (d, C-18), 34.0 (t,
C-19), 43.9 (s, C-20), 31.1 (t, C-21), 38.2 (t, C-22), 26.1 (q, C-23),
1.0 (q, C-24), 14.7 (q, C-25), 15.6 (q, C-26), 20.6 (q, C-27), 26.8
q, C-28), 28.5 (q, C-29), 177.3 (s, C-30), 51.3 (q, C-31).
3
3
H, CH
H, CH
3
3
3
3
2
(
J15e,15a 13.6, J15e,16a 4.2, J15e,16e 2.3 Hz, H-15e), 1.57 (dd, 1H, J19a,19e
1
1
3.2, J19a,18a 13.2 Hz, H-19a), 1.76 (ddd, 1H, J15a,15e 13.6, J15a,16a
3.6, J15a,16e 4.5 Hz, H-15a), 1.84 (ddd, 1H, J19e,19a 13.2, J19e,18a 4.3,
J
3
1
19e,21e 2.5 Hz, H-19e), 3.66 (s, 3H, OCH
3
-31), 5.32 (t, 1H, J12,11
.6 Hz, H-12), 7.72 (s, 1H, H-1). 13C NMR (CDCl
): d = 169.9(d, C-
), 113.7 (s, C-2), 198.0 (s, C-3), 44.8 (s, C-4), 52.4 (d, C-5), 18.7
4.1.18. Methyl 2-cyano-3,12-dioxo-18bH-olean-30-oate (4)
A solution of AcOOH (0.24 mmol) in CH Cl (0.2 mL) was added
dropwise to compound 3 (0.1 g, 0.2 mmol) in CH Cl (5 mL). The
3
2
2
2
2
(
2
(
1
2
t, C-6), 32.0 (t, C-7), 40.7 (s, C-8), 41.0 (d, C-9), 40.4 (s, C-10),
3.2 (t, C-11), 121.0 (d, C-12), 145.1 (s, C-13), 41.9 (s, C-14), 25.9
t, C-15), 26.7 (t, C-16), 31.9 (s, C-17), 48.1 (d, C-18), 42.4 (t, C-
9), 44.1 (s, C-20), 31.1 (t, C-21), 38.1 (t, C-22), 27.6 (q, C-23),
1.5 (q, C-24), 17.9 (q, C-25), 17.2 (q, C-26), 25.6 (q, C-27), 28.0
mixture was stirred at room temperature (the reaction course
was monitored by TLC). The resulting mixture was diluted with
H
2
O (10 mL). The mixture was extracted with ethyl acetate
(3 ꢄ 50 mL). The combined organic layers were washed with satd
aq NaHCO , brine and dried over anhydrous MgSO . Compound 4
3
4
(
q, C-28), 28.4 (q, C-29), 177.4 (s, C-30), 51.5 (q, C-31), 114.8 (s,
(0.6 g; 60%) was purified by flash column chromatography (silica
gel, chloroform) as a white solid. An analytically pure sample
was obtained by recrystallization from a mixture of chloroform/
C-32).
2
5
-1
4
.1.16. Methyl 3b-hydroxy-12-oxo-18bH-olean-30-oate (24)
Methyl 3b-acetoxy-18bH-olean-12(13)-en-30-oate (3.0 g,
.7 mmol) was deprotected with ROH (20 g, 356 mmol) in CH OH
methanol. ½
a
ꢅ
D
3 3
+52° (c 0.15, CHCl ). IR (CHCl , cm ) mmax: 2954;
8
2236 (C„N); 1726, 1696, 1690 (C@O). HRMS: m/z calcd for
1
5
3
C
32
H
45NO
4
: 507.7040; found: 507.3416. H NMR (CDCl
-28), 0.94 (s, 3H, CH -27), 1.12 (s, 3H, CH -29), 1.13 (s,
-24), 1.16 (s, 3H, CH -25), 1.20 (s, 3H, CH -23), 1.23 (s,
-26), 0.90 (dm, 1H, J16e,16a 13.3 Hz, H-16e), 1.03 (ddd, 1H,
J15e,15a 13.5, J15e,16a 3.8, J15e,16e 2.1 Hz, H-15e), 1.99 (dd, 1H, J9a,11a
3
): d = 0.85
(
200 mL) in a similar way as described in the synthesis of 9 to fin-
(s, 3H, CH
3H, CH
3H, CH
3
3
3
ish 24 (2.5 g, 92%). This material was used for the next reaction
without further purification. An analytically pure sample was ob-
3
3
3
3
tained by recrystallization from CH
HRMS: m/z calcd for C31 : 486.7263; found: 486.3709.
NMR (CDCl ): d = 0.68 (dd, 1H, J5a,6a 11.5, J5a,6e 2.1 Hz, H-5a), 0.74
s, 3H, CH -24), 0.80 (s, 3H, CH -28), 0.82 (s, 3H, CH -25), 0.82–
.89 (m, 1H, H-16e), 0.88 (s, 3H, CH -27), 0.92–0.98 (m, 1H,
H-15e), 0.94 (s, 3H, CH -23), 1.08 (s, 3H, CH -29), 1.09 (s, 3H,
CH -26), 1.17 (dd, 1H, J19a,19e 13.4, J19a,18a 13.4 Hz, H-19a), 2.10
dd, 1H, J11a,11e 16.8, J11a,9a 13.1 Hz, H-11a), 2.22 (dd, 1H, J11e,11a
6.8, J11e,9a 5.1 Hz, H-11e), 2.53 (ddd, 1H, J19e,19a 13.4, J19e,18a 3.3,
19e,21e 2.3 Hz, H-19e), 2.71 (d, 1H, J13,18a 4.4 Hz, H-13), 3.14 (dd,
H, J3a,3e 11.3, J3a,2e 4.8 Hz, H-19e), 3.66 (s, 3H, OCH -31), the sig-
): d = 38.6 (t,
3
OH/CHCl
3
. Mp 258–260 °C.
1
50
H O
4
H
13.1, J9a,11e 4.6 Hz, H-9a), 2.34 (dd, 1H, J11a,11e 16.2, J11a,9a 13.1 Hz,
H-11a), 2.45 (dd, 1H, J11e,11a 16.2, J11e,9a 4.6 Hz, H-11e), 2.58 (ddd,
1H, J19e,19a 13.2, J19e,18a 3.8, J19e,21e 2.1 Hz, H-19e), 2.81 (d, 1H,
3
(
0
3
3
3
J
13,18 4.4 Hz, H-13), 3.70 (s, 3H, OCH
nals of the other proton 1.15–1.30, 1.40–1.55 and 1.59–1.97.
NMR (CDCl ): d = 167.7 (d, C-1), 114.5 (s, C-2), 197.4 (s, C-3), 44.9
3
-31), 7.63 (s, 1H, H-1), the sig-
3
13
C
3
3
3
3
(
1
J
1
(s, C-4), 52.2 (d, C-5), 18.7 (t, C-6), 31.0 (t, C-7), 42.5 (s, C-8), 43.0
(d, C-9), 40.3 (s, C-10), 38.1 (t, C-11), 209.0 (s, C-12), 50.3 (d, C-
13), 42.2 (s, C-14), 25.9 (t, C-15), 26.2 (t, C-16), 32.0 (s, C-17),
38.4 (d, C-18), 34.0 (t, C-19), 44.0 (s, C-20), 31.2 (t, C-21), 38.3 (t,
C-22), 27.5 (q, C-23), 21.3 (q, C-24), 17.5 (q, C-25), 16.3 (q, C-26),
3
13
nals of the other protons 1.16–1.93. C NMR (CDCl
3
Please cite this article in press as: Salomatina, O. V.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.10.049

O. V. Salomatina et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
9
2
0.8 (q, C-27), 26.9 (q, C-28), 28.6 (q, C-29), 177.3 (s, C-30), 51.5 (q,
and Scientific School No. 2972.2012.4, by the Ministry of education
and science of Russian Federation, project N. 8277.
C-31), 114.5 (q, C-32).
We thank Mrs. Albina V. Vladimirova (Institute of Chemical
Biology and Fundamental Medicine SB RAS) for cell maintenance.
4
4
.2. Biological assay
.2.1. Cell culture and GA derivatives
The murine J-774 macrophage cell line and human epidermoid
Supplementary data
cancer cell line KB-3-1 (Russian Cell Culture Collection, St. Peters-
burg) were cultured in Dulbecco’s modified Eagle’s medium
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.10.049.
(
DMEM) (Sigma–Aldrich Inc., USA) supplemented with 10% (v/v)
heat-inactivated fetal bovine serum (FBS) (BioloT, Russia), 100 U/
mL penicillin (ICN Biomedicals Inc., USA), 100 g/mL streptomycin
and 0.25 g/mL amphotericin and incubated at 37 °C in a humid-
ified atmosphere containing 5% CO and 95% air.
l
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v
This work was supported by RAS programs ‘Molecular and
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Please cite this article in press as: Salomatina, O. V.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.10.049