Synthesis, Modification, and Antimicrobial Activity of the N-Methylpiperazinyl Amides of Triterpenic Acids

Article abstract of DOI:10.1134/S1068162010030155

N-methylpyperazinyl amides of betulinic, platanic, glycyrrhetic, oleanolic, ursolic, and moronic acids were synthesized and modified. Betulin and betulonic acid showed antimicrobial activity against Staphylococcus aureus at a concentration of 90 mg/ml, and betulin manifested a bacteriostatic effect against Klebsiella pneumoniae at a concentration of 60 mg/ml. Among the studied N-methylpyperazinyl amides, the highest activity against S. aureus was observed for a betulonic acid derivative. Pleiades Publishing, Ltd., 2010.

Full text of DOI:10.1134/S1068162010030155

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2010, Vol. 36, No. 3, pp. 383–389. © Pleiades Publishing, Ltd., 2010.
Original Russian Text © O.B. Kazakova, G.V. Giniyatullina, G.A. Tolstikov, N.I. Medvedeva, T.M. Utkina, O.L. Kartashova, 2010, published in Bioorganicheskaya Khimiya, 2010,
Vol. 36, No. 3, pp. 416–422.
Synthesis, Modification, and Antimicrobial Activity
of the NꢀMethylpiperazinyl Amides of Triterpenic Acids
1
O. B. Kazakovaa, , G. V. Giniyatullinaa, G. A. Tolstikova, N. I. Medvedevaa,
T. M. Utkinab, and O. L. Kartashovab
a Institute of Organic Chemistry, Ufa Scientific Center, Russian Academy of Sciences, pr. Oktyabrya 71, Ufa, 450054 Russia
b Institute of Cellular and Intracellular Symbiosis, Ural Division, Russian Academy of Sciences, Orenburg, Russia
Received October 16, 2009; in final form, November 30, 2009
Abstract—Nꢀmethylpyperazinyl amides of betulinic, platanic, glycyrrhetic, oleanolic, ursolic, and moronic
acids were synthesized and modified. Betulin and betulonic acid showed antimicrobial activity against Staꢀ
phylococcus aureus at a concentration of 90 mg/ml, and betulin manifested a bacteriostatic effect against
Klebsiella pneumoniae at a concentration of 60 mg/ml. Among the studied
Nꢀmethylpyperazinyl amides, the
highest activity against S. aureus was observed for a betulonic acid derivative.
Key words: antimicrobial activity, Nꢀmethylpyperazinyl amides, synthesis, triterpenoids
DOI: 10.1134/S1068162010030155
1
INTRODUCTION
concentrations (EC50) of 0.001–0.30
μM [12]. The
interaction of the ꢀBocꢀbisaminopropylpiperazinyl
N
Triterpenoid derivatives of the lupine line proved to
amide of ursolic acid with aldehydes resulted in a
series of imines with antimalarial activity against Plasꢀ
modium falciparum resochinꢀsensitive and resistant
be promising blocks for the design of new drugs with
various activities [1, 2]. Dimethylsuccinate of betuꢀ
linic acid served as the basic structure for the antiviral
(HIV, enteroviruses) product Bevirimat [3]. A series of
valuable compounds was found among the amides of
betulonic acid. Antioxidant [4], anticancer, and antiꢀ
metastatic effects, as well as the capacity to decrease
the toxic properties of cytostatics used in antitumor
chemotherapy [6], were found for amides of betulonic
strains (IC50 0.08–46.04
that the ꢀmethylpiperazinyl amide of betulonic acid
) displayed expressed antitumor activity, a decreased
μM) [13]. Recently, we found
N
(
I
severity of tissue pathological changes, and exhibited
noticeable antioxidant and cytoprotector effects in
normal cells [14]. Therefore, it is a new promising
agent for both chemical modifications and studies of
the spectrum of biological activity.
acid and
α
ꢀ and ꢀalanines. Amides containing fragꢀ
β
ments of caprylic, pelargonic, and undecanoic acid
inhibited the growth of MTꢀ4, MOLTꢀ4, CEM, and
Hep G2 cells [7]. The dipeptide of betulonic acid (
N'ꢀ
{
N
ꢀ[3ꢀoxolupꢀ20(29)eneꢀ28ꢀoyl]–9ꢀaminononanoyl}ꢀ3ꢀ
RESULTS AND DISCUSSION
aminoꢀ3ꢀphenylpropionic acid) is patented as an antiꢀ
viral (antiꢀHIV, antiherpetic) and immunostimulating
agent [8]. In addition, this dipeptide effectively
induces in vitro apoptosis in leucosis and hepatocarciꢀ
noma cells and inhibits the growth of tumor MTꢀ4,
MOLTꢀ4, CEM, and Hep G2 cells [9]. Amides of betꢀ
ulonic acid and
influenza virus A [10]. The amide of betulonic acid
and ꢀBocꢀlysine was reported to possess specific
activity in vitro and in vivo towards prostate cancer
[11].
The potential of the introduction of a piperazine
fragment into triterpene acid structures was described
In this work, we describe the chemical transformaꢀ
tions of the
Nꢀmethylpiperazinyl amide of betulonic
acid ( ); the synthesis of similar amides of platanic,
glycyrrhetic, oleanolic, ursolic, and moronic acids;
and the evaluation of their antimicrobial activities.
I
Lꢀamino acids are effective against
(4ꢀMethylpiperazinꢀ1ꢀyl)amide of betulonic acid
N
(
I
) was obtained by the treatment of the corresponding
acyl chloride with ꢀmethylpiperazine [14]. Comꢀ
) was modified using the known approaches
ꢀmethylpiperazinyl amides of 3 ꢀbetulinic
ꢀnicotinoyl betulinic (III), and 2ꢀfurfuꢀ
rylidenebetulonic acids (IV) (Scheme 1). The
ꢀmethylpiperazinyl amide of 3ꢀketoplatanic acid (
was quantitatively formed in the reaction of the ozoꢀ
nolysis of amide ( ).
N
pound (
to give
I
N
β
(
II), 3 ꢀO
β
in [12, 13]. For example, piperazinyl amides of 3 ꢀ
glutaryl betulinic acid manifested antiꢀHIV activity at
β
N
V)
1
Corresponding author; phone/fax: +7 (347) 2356ꢀ066; eꢀmail:
obf@anrb.ru.
I
383

384
KAZAKOVA et al.
R
20 29
2' 3'
17
CON N CH3
CON N CH3
CON N CH3
6' 5'
O
i
2
3
3
O
O
R1O
iii
(
I) R = CH
2
iv
(IV)
(V
) R = O
O
1
(
II) R = H
ii
1
(III) R
=
N
i
NaBH /
isopropanol; ii nicotinoyl chloride/pyridineꢀDMAP;
Reaction conditions:
4
iii 2ꢀfuryl aldehyde, 40% KOH/EtOH; iv O3/CH2Cl2.
Scheme 1.
For the preparation of triterpeneꢀbased
ylpiperazinyl amides, we also used oleanolic (VI), tion of amide bonds in compounds (
ursolic (VII), and moronic ( ) acids. The latter was
IX), (XI), and (XIII)–(XV). In the 1H NMR spectra,
obtained using an earlier developed procedure [15]. the resonances at 2.06–2.56 ppm (H'3 and H'5) and
Nꢀmethꢀ the region of
δ
173.1–175.4 ppm supported the formaꢀ
I
)–( ), (VIII),
V
X
(
δ
Amides (VIII), (IX), and (XI) were synthesized from
the corresponding acyl chlorides in 78–86% yields
δ
2.84–3.69 ppm (H2', H6') were characteristic of
ꢀmethylpiperazine, including an intense singlet of
N
(Scheme 2). In the case of the
amide of glycyrrhetic acid (XIII), the performed modꢀ the chemical shifts of these atoms and the appearance
ification led to 3ꢀketoꢀ and 3 ꢀnicotinoyl derivaꢀ of the corresponding resonances of furfurylidene and
N
ꢀmethylpiperazinyl the NꢀCH3 group at 2.11–2.29 ppm. The changes of
δ
β
ꢀO
tives (XIV) and (XV) (Scheme 3). The structures of the nicotinoyl fragments and keto groups in the NMR
prepared compounds were confirmed by NMR specꢀ spectra unambiguously confirmed the amide modifiꢀ
13
troscopy. The resonances in the C NMR spectra in cations at positions C2, C3, and C29.
R1
R1
R2
R2
20
19
2' 3'
6' 5'
i, ii
17
COOH
CON
N CH3
3
O
O
(
VI), (VII
VI), (VIII) R1 = CH3, R2 = H,
VII), (IX) R1 = H, R2 = CH3
)
(VIII), (IX)
(
(
19
18
2' 3'
i, ii
17
CON
N CH3
COOH
6' 5'
O
O
(X
)
(XI)
i
(COCl)2/benzene; ii Nꢀ /chloroform.
methylpiperazine
Reaction conditions:
Scheme
2.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 36
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2010

SYNTHESIS, MODIFICATION, AND ANTIMICROBIAL ACTIVITY
385
Table 1. Comparative antimicrobial activity of betulin, betulonic acid, and amides (I), (IV), (IX), and (XV) towards S. aureus*
Concentration, mg/ml
80
Control**
Compound
40
60
90
100
1
2
(I)
1
×
105
5
×
104
1
×
103
single
colonies
no growth
1
×
106
1
×
106
(
(
(
IV
IX
XV
)
1
5
1
1
×
×
×
×
105
104
106
105
1
1
×
104
104
not tested
not tested
not tested
not tested
not tested
not tested
no growth
not tested
not tested
not tested
no growth
1
1
1
1
×
×
×
×
106
1
1
1
1
×
×
×
×
106
106
106
106
)
×
106
106
106
)
not tested
Betulin
1
×
103
single
colonies
Betulonic acid
1
×
103
1
×
103
<1
×
103
no growth
no growth
1
×
106
1
×
106
* The number of grown colonies is given.
** Control 1, physiological solution; control 2, DMSO.
2' 3'
COOH
30
CON N CH3
CON N CH3
6' 5'
20
12
O
O
O
11
iii
3
(
XII)
(XIV)
HO
R1O
O
i, ii
1
(
XIII) R = H
O
iv
1
(XV) R
=
N
Reaction conditions
:
i
(COCl)2/benzene; ii Nꢀmethylpiperazinyl/chloroform; iii Jones reagent/acetone;
iv nicotinoyl chloride/pyridineꢀDMAP.
Scheme 3.
It was interesting to study the effects of triterpeneꢀ at a concentration of 90 mg/ml it manifested a bacteꢀ
based ꢀmethylpiperazinyl amides on the growth of riostatic effect, and at a concentration of 100 mg/ml
Staphylococcus aureus and Klebsiella pneumoniae
demonstrated bactericidal activity. Betulin and betuꢀ
N
.
According to published data, natural triterpenoids lonic acid were most effective against S. aureus (the
possess antimicrobial activity towards S. aureus, presence of betulin at a concentration of 80 mg/ml
Escherichia coli, Bacillus subtilis, Micrococcus luteus, induced the growth of single staphylococcus coloꢀ
Staphylococcus epidermidis, and Enterococcus faecalis nies), and at a concentration of 90 mg/ml they disꢀ
at concentrations of 0.006–200 mg/ml [16–20].
played bactericidal effects.
Betulin was most effective against K. pneumoniae
:
The study of the effect of triterpenoid ꢀmethꢀ
N
as its concentration was increased to 60 mg/ml, it
gradually reduced the colony number down to single
colonies (Table 2). Betulonic acid showed bacterioꢀ
static properties in the concentration range of 50–
ylpiperazinyl amides on S. aureus showed that comꢀ
pound (XV) was indifferent at a concentration of
40 mg/ml, whereas compounds (I), (IV), and (VIII)
insignificantly inhibited microorganism growth. At
the same time, betulonic acid at this concentration
decreased the number of grown colonies by three
orders of magnitude (Table 1). Compounds (IV) and
IX) at a concentration of 60 mg/ml reduced the numꢀ
ber of grown microorganisms by two orders of magniꢀ
tude as compared to the control, whereas betulin and
60 mg/ml. The studied
at concentrations of 60 mg/ml inhibited the growth of
K. pneumoniae to 1
105 CFU/ml (compounds (III
105 CFU/ml (compound IX) as
Nꢀmethylpiperazinyl amides
×
)
and (XV)) and 5
×
(
compared to the control.
To summarize, we synthesized and carried out
betulonic acid, by three orders of magnitude. Comꢀ some modifications of the
pound ( ) at a concentration 80 mg/ml inhibited the amides of betulonic, platanic, glycyrrhetic, oleanolic,
growth of 50% of S. aureus clones and decreased the ursolic, and moronic acids. Betulin and betulonic acid
number of microorganisms down to 1
103 CFU/ml; displayed antimicrobial activities against S. aureus at a
Nꢀmethylpiperazinyl
I
×
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 36
No. 3
2010

386
Table 2. Comparative antimicrobial activity of betulin, betulonic acid, and amides (III), (IX), and (XV) towards K. pneumoniae*
Concentration, mg/ml Control
40 50
KAZAKOVA et al.
Compound
20
60
1
2
(
(
(
III
IX
XV
)
5
5
1
1
×
106
106
105
105
1
1
1
1
×
106
106
105
104
5
5
1
1
×
105
105
105
103
1
5
1
×
105
105
105
5
5
5
5
×
×
×
×
105
5
5
5
5
×
×
×
×
105
)
×
×
×
×
×
×
×
×
×
×
105
105
105
105
105
105
)
×
Betulin
single
colonies
Betulonic acid
1
×
104
1
×
103
<1
×
103
<1
×
103
5
×
105
5
×
105
* See the footnotes to Table 1.
concentration of 90 mg/ml, and betulin showed bacteꢀ the routine procedure [10]. Nicotinoyl chloride was
riostatic properties towards K. pneumoniae at a conꢀ obtained according to [24].
centration of 60 mg/ml. The
Nꢀmethylpiperazinyl
Preparation of compounds (VIII), (IX), (XI), and
(XIII). ꢀmethylpiperazine (1 mmol, 0.1 ml) was
added to a solution of acyl chloride (1 mmol) in dry
CHCl3 (30 ml), and Et3N (3 ml) was added dropwise.
The mixture was refluxed for 3 h, washed with 5% HCl
amides of betulonic acid demonstrated an antimicroꢀ
bial affect against S. aureus at a concentration of
100 mg/ml. None of the studied amides showed bacꢀ
tericidal activity towards K. pneumoniae. It is noteꢀ
worthy that in spite of the weak antibacterial activity,
triterpenoids, including those described in this work,
can change the bacterial biological characteristics,
first of all, the capacity of microorganisms to persisꢀ
tence [21, 22].
N
(2
× 50 ml) and water (50 ml), dried with CaCl2, and
evaporated in a vacuum. Compound (XIII) was used
without further purification; compounds (VIII), (IX),
and (XI) were purified by column chromatography on
Al2O3 eluted with chloroform.
3ꢀOxoꢀ17ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)carbonyloleanꢀ
12(13)ꢀene (VIII). Yield 0.42 g (78%). Rf 0.29; mp.
EXPERIMENTAL
20
159–161°C. [
%: C 77.92; H 10.14; N 4.96. C35H56N2O2 (
Calc., %: C 78.31;
α
]
+69
°
(
c
0.82, CH3ОH). Found,
536.839)
H 10.51; N 5.22. H NMR: 0.78,
D
The 1H and 13C NMR spectra were registered on a
Bruker AMꢀ300 spectrometer (Germany, 300 and
75.5 MHz, respectively) in CDCl3 with tetramethylsiꢀ
lane as an internal standard. The melting points were
measured on a Boetius microtable. The optical
absorption was measured on a PerkinꢀElmer 241 MC
(Germany) polarimeter in a tube 1 dm in length. TLC
analysis was carried out on Silufol plates (Chemapol,
Czech Republic) eluted with a 25 : 1 chloroform :
methanol mixture. Compounds were developed with
5% phosphotungstic acid in ethanol followed by heatꢀ
ing at 100–120°C for 2–3 min. For ozonation, an
Ozonꢀ2K (Russia) ozonator was used. Column chroꢀ
matography was performed on neutral Al2O3 (Reaꢀ
khim). Moronic acid was obtained as described in
M
.
1
0.89, 0.93,1.01, 1.03, 1.11, 1.14 (21 H, 7 s, 7 CH3),
1.22–2.02 (22 H, m, CH2, CH), 2.26 (3 H, s, 4'ꢀCH3),
2.38–2.51 (4 H, m, H3', H5'), 2.83–3.97 (1
H, m,
H11), 3.36–3.52 (4 H, m, H2', H6'), 5.02–5.12 (1 H,
13
s, H12). C NMR: 14.9,16.7, 17.8, 19.5, 21.3, 22.6,
23.4, 24.0, 25.7, 26.3, 27.3, 27.9, 29.8, 30.2, 32.2,
32.3, 33.0, 33.9, 34.0, 36.7, 38.1, 41.9, 43.6, 45.1,
45.8, 46.3, 46.9, 47.3, 55.1, 55.3, 121.2 (C12), 144.8
(C14), 174.9 (CON), 217.6 (C3).
3ꢀOxoꢀ17ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)carbonyloursanꢀ
12(13)ꢀene (IX). Yield 0.45 g (84%).
R
f 0.27; mp 142–
20
145°C. [
α
]
+147
°
(
c
0.35, CHCl3). Found, %:
536.839). Calc.,
78.31; H 10.51; N 5.22. H NMR: 0.78, 0.87,
C
D
[15]. Oleanolic and ursolic acids and Nꢀmethylpiperꢀ
azine were purchased from Aldrich Chemical Co.
78.03; H 10.22; N 4.82. C35H56N2O2 (
M
1
%:
C
Glycyrrhetic acid (XII) was prepared as described in
0.93, 1.01, 1.03, 1.11, 1.14 ( (21 H, 7 s, 7CH3), 1.19–
2.18 (22 H, m, CH2, CH), 2.28 (3 H, s, 4'ꢀCH3),
[23]. The synthesis of the
Nꢀmethylpiperazinyl amide
of betulonic acid (I) is described in [14]. Acyl chloꢀ
rides were obtained by the interaction of the correꢀ 2.32–2.46 (4 H, m, H3', H5'), 2.74–3.02 (1 H, m,
sponding acids with oxalyl chloride in benzene using 11), 3.52–3.69 (4 H, m, 2', H6'), 5.22–5.31 (1 H,
H
H
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 36
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2010

SYNTHESIS, MODIFICATION, AND ANTIMICROBIAL ACTIVITY
387
13
4.62 (2 H, br s, H29), 7.40 (1 H, dd,
8.24 (1 H, ddd,
J
3.8, 4.7, H3''),
s, H12). C NMR: 15.0, 16.7, 17.3, 19.4, 21.1, 21.4,
J
4.7, 1.6, 1.7, H4''), 8.70 (1 H, t,
J
4.1,
1.8, 5.9, H1"). C NMR:
45.6, 46.7, 47.2, 48.4, 54.9, 55.2, 124.9 (C12), 138.6 14.5,15.8, 15.9, 19.3, 19.6, 20.9, 21.6, 22.9, 25.6, 26.5,
22.6, 23.3, 23.6, 24.8, 26.4, 28.1, 30.4, 32.5, 34.0,
13
34.1, 36.6, 37.5, 38.6, 39.1, 39.3, 39.4, 42.1, 44.9, H2"), 9.18 (1 H, dd,
J
(
C
14), 175.1 (CON), 217.5 (C3).
3ꢀOxoꢀ17ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)carbonyloleanꢀ
29.7, 31.3, 32.4, 33.6, 34.1, 35.9, 36.9, 38.1, 39.6,
40.5, 40.8, 41.8, 45.5, 45.9, 47.3, 50.2, 52.6, 54.3,
54.5, 55.0, 55.2, 82.0 (C3), 108.9(C29), 123.0, 126.5,
136.7, 150.6, 151.0 (C20), 152.9, 164.6, 173.1 (CON).
18(19)ꢀene (XI). Yield 0.46 g (86%).
R
f 0.26; mp 115–
20
D
117°С. [
H 10.18; N 5.06. С35Н56N2O2 (
C 78.31; H 10.51; N 5.22. 1H NMR: 0.76, 0.83, 0.83,
0.90, 0.95, 0.96, 1.04 (21 H, 7 s, 7 CH3), 1.17–1.92
(22 H, m, CH2, CH), 2.05–2.15 (1 H, m, H13), 2.17
(3 H, s, 4'ꢀCH3), 2.23–2.47 (4 H, m, H3', H5'), 3.48–
3.63 (4 H, m, H2', H6'), 5.05 (1 H, s, H19). 13C NMR:
14.4, 15.5, 16.0, 19.3, 20.7, 20.8, 21.2, 25.2, 26.3,
26.5, 29.0, 30.5, 31.3, 31.7, 32.9, 33.2, 33.6, 33.7,
36.6, 39.5, 40.6, 41.2, 41.5, 43.7, 45.6, 45.7, 46.9,
49.7, 49.9, 54.5, 54.7, 129.0 (C18), 140.9 (C19), 173.7
(CON), 217.5 (C3).
α
]
+70
°
(с
0.06, CHCl3). Found, %: C 78.14;
536.839). Calc., %:
3
β
ꢀ
O
ꢀNicotinoylꢀ20ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)carꢀ
M
bonylꢀ11ꢀoxooleanꢀ12(13)ꢀene (XV). Yield 0.59 g
20
D
(89%). Rf 0.18; mp 226–228°С. [
CHCl3). Found, %: C 74.52; H 8.76; N 6.01.
С41Н59N3O4 ( 657.933). Calc., %: C 74.85; H 9.04;
N 6.39. H NMR: 0.80, 0.82, 1.01, 1.13, 1.16, 1.35
(21 H, 7 s, 7CH3), 1.25–2.22 (19 H, m, CH2, CH),
2.26 (3 H, s, 4'ꢀCH3), 2.31 (1 H, s, H19), 2.35–2.49
(4 H, m, H3', H5'), 2.83 (1 H, m, H18), 2.84–3.04
(4 H, m, H2', H6'), 3.08–3.33 (1 H, m, H3), 5.66
α
]
+33.2
°
(с 0.37,
M
1
(1 H, s, H12), 7.40 (1 H, dd,
(1 H, ddd, 4.7, 1.6, H4''), 8.70 (1 H, t,
9.18 (1 H, dd,
1.8, 5.9, H1''). 13C NMR: 15.8, 15.9,
J
3.8, 4.7, H3''), 8.24
J
J
4.1, H2''),
3 ꢀHydroxyꢀ17ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)carboꢀ
β
J
nylolupaꢀ20(29)ꢀene (II). Sodium borohydride (50 mg,
1.3 mmol) was added under stirring for 10 min to a
solution of compound (I) (0.54 g, 1 mmol) in isoproꢀ
panol (20 ml) and kept for 2 h. The mixture was
diluted with 10% HCl (30 ml); the residue was filtered,
washed with water, dried, and recrystallized from
16.7, 19.3, 19.6, 20.9, 21.6, 22.9, 25.6, 26.5, 29.7,
31.3, 32.4, 33.6, 34.1, 35.9, 36.9, 38.1, 39.6, 40.5,
40.8, 41.8, 45.5, 45.9, 47.3, 50.2, 54.3, 54.5, 55.0, 61.5
(C9), 82.0 (C3), 123.4, 126.9, 128.3 (C12), 137.4,
150.6, 152.5, 164.6, 169.5 (C13), 175.4 (CON), 200.0
(C11).
EtOH. Yield 0.50 g (93%). Rf 0.13; mp 138–141°С
.
Found, %: C 77.86; H 10.63; N 4.92. С35Н58N2O2
2ꢀFurfurylideneꢀ3ꢀoxoꢀ17ꢀ(4ꢀmethylpiperazinꢀ1ꢀ
yl)carbonylolupaꢀ20(29)ꢀene (IV). Furfuryl aldehyde
(0.11 ml, 1.3 mmol) and 40% KOH in ethanol (2.5 ml)
(
M 538.855). Calc., %: C 78.01; H 10.85; N 5.20.
1H NMR: 0.86, 0.92, 0.96, 1.02, 1.12 (15 H, 5 s,
5 CH3), 1.22–2.02 (21 H, m, CH2, CH), 1.67 (3 H, s,
H30), 2.06–2.18 (4 H, m, H3', H5'), 2.29 (3 H, s,
4'ꢀCH3), 2.32–2.56 (3 H, m, H13, H16), 2.84–3.20
were added to a solution of compound (I) (0.54 g,
1 mmol) in ethanol (5 ml) under stirring and cooling
(below 10°C). The mixture was kept for 30 min at this
temperature and then for a day at room temperature,
pH was adjusted to neutral values with glacial AcOH,
and the mixture was poured into cold water (50 ml).
The residue was filtered, washed with water, and dried.
Yield 0.55 g (90%). Rf 0.13; mp 147–149°С.
(7 H, m, H19, H3, H2', H6',
ОH), 4.62 (2 H, br s,
13
H29). C NMR: 14.5, 15.8, 15.9, 19.3, 19.6, 20.9,
21.6, 22.9, 25.6, 26.5, 29.7, 31.3, 32.4, 33.6, 34.1,
35.9, 36.9, 38.1, 39.6, 40.5, 40.8, 41.8, 45.5, 45.9,
47.3, 50.2, 52.6, 54.3, 54.5, 55.0, 55.2, 77.3 (C3),
109.1 (C29), 151.2 (C20), 173.4 (CON).
[
α D
]
20 +54.5
°
(
с
0.5, CHCl3). Found, %: C 77.84;
614.909). Calc., %:
H 9.22; N 4.28. С40Н58N2O3 (
M
Preparation of compounds (III) and (XV). Freshly
prepared nicotinoyl chloride (1.5 mmol) and dimethꢀ
ylaminopyridine (0.1 mmol) were added to a solution
of compound (II) or (XIII) (1 mmol) in anhydrous
pyridine and refluxed for 4 h. The reaction mixture
was poured into 5% HCl (200 ml); the residue was
washed with water, dried, and purified by column
chromatography on Al2O3 eluted with chloroform.
1
C 78.13; H 9.51; N 4.56. H NMR: 0.86, 0.92, 0.96,
1.02, 1.12 (15 H, 5 s, 5CH3), 1.22–2.12 (19 H, m,
CH2, CH), 1.67 (3 H, s, H30), 2.15–2.23 (4 H, m,
H3', H5'), 2.26 (3 H, s, 4'ꢀCH3), 2.32–2.56 (3 H, m,
H13, H16), 2.84–3.04 (5 H, m, H19, H2', H6'), 4.64
(1 H, br s, H29), 6.64–6.72 (2 H, d, J 3.7, H3'', H4''),
7.21–7.33 (1 H, m, H1''), 7.34–7.48 (1 H, m, H5'').
13C NMR: 14.5,15.8, 15.9, 19.3, 19.6, 20.9, 21.6, 22.9,
25.6, 26.5, 29.7, 31.3, 32.4, 33.6, 35.9, 36.9, 38.1,
39.6, 40.5, 40.8, 41.8, 45.5, 45.9, 47.3, 50.2, 52.6,
54.3, 54.5, 55.0, 55.2, 108.9 (C29), 110.2, 116.2,
122.1, 138.2 (C2), 148.7, 150.1 (C20), 151.2, 173.2
(CON), 207.2 (C3).
3 ꢀOꢀNicotinoylꢀ17ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)carꢀ
β
bonylolupaꢀ20(29)ꢀene (III). Yield 0.55 g (85%). Rf
20
D
0.15; mp 224–225°С. [
α
]
+ 32.5° (с 0.65, CHCl3).
Found, %: C 76.21; H 9.34; N 6.27. С41Н61N3O3
643.950). Calc., %: C 76.47; H 9.55; N 6.53.
1Н NMR: 0.86, 0.92, 0.96, 1.02, 1.12 (15 H, 5 s,
5 CH3), 1.22 2.12 (22 H, m, CH2, CH), 1.67 (3 H, s,
H30), 2.29 (3 H, s, 4'ꢀCH3), 2.32–2.56 (7 H, m, H13,
(M
3,20ꢀDioxoꢀ17ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)carbonylꢀ29ꢀ
–
norlupane (V). Ozone was passed through a solution of
compound (I) (1 mmol) in CH2Cl2 (20 ml) at –60°C
H16, H3', H5'), 2.84–3.04 (5 H, m, H19, H2', H6'), until the starting compound disappeared (TLC conꢀ
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 36
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2010

388
KAZAKOVA et al.
3. Qian, K., MorrisꢀNatschke, S.L., and Lee, K.H., Med.
Res. Rev, 2009, vol. 29, pp. 369–393.
trol). The mixture was heated to room temperature,
kept for a day, the solvent was evaporated in a vacuum,
and the residue was crystallized from ethanol. Yield
0.39 g (74%). Rf 0.14; mp 124–126°С. Found, %: C
4. Sorokina, I.V., Tolstikova, T.G., Zhukova, N.A.,
Petrenko, N.I., Shul’ts, E.E., Uzenkova, N.V.,
Grek, O.R., Pozdnyakova, S.V., and Tolstikov, G.A.,
Dokl. Akad. Nauk, 2004, vol. 399, pp. 274–277 [Dokl.
(Engl. Transl.), vol. 399, pp. 94–97].
75.46; H 9.75; N 4.97. С34Н54N2O3 (
M 538.811). Calc.,
13
%: C 75.79; H 10.10; N 5.20. C NMR: 0.86, 0.92,
0.96, 1.02, 1.12 (15 H, 5 s, 5CH3), 1.22–2.12 (21 H,
m, CH2, CH), 2.06–2.18 (4 H, m, H3', H5'), 2.11 (3
H, s, 4'ꢀCH3), 2.29 (3 H, s, CH3), 2.32–2.56 (3 H, m,
H13, H16), 2.84–3.04 (5 H, m, H19,H2',H6').
5. Sorokina, I.V., Tolstikova, T.G., Zhukova, N.A.,
Petrenko, N.I., Uzenkova, N.V., Shul’ts, E.E., and
Popova, N.A., Byull. Eksp. Biol. Med, 2006, vol. 15,
pp. 69–72.
3,11ꢀDioxoꢀ20ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)carbonyꢀ
loleanꢀ12(13)ꢀene (XIV). The Jones reagent (1.56 ml)
was added to a solution of compound (XIII) (0.60 g,
1 mmol) in acetone (30 ml) at 0°C and the mixture
was stirred at 0°C for 4 h. Methanol (1 ml) was added
and the mixture was poured out onto ice (10 g). The
precipitate was filtered off, washed with water, dried,
and the residue was purified by column chromatograꢀ
phy on Al2O3 eluted with chloroform. Yield 0.41 g
20
6. Zhukova, N.A., Semenov, D.E., Sorokina, I.V., Tolꢀ
stikova, T.G., Pozdnyakova, S.V., and Grek, O.R.,
Byull. Eksp. Biol. Med, 2005, vol. 14, pp. 348–351.
7. Pokrovskii, A.G., Shintyapina, A.B., Pronkina, N.V.,
Kozhevnikov, V.S., Plyasunova, O.A., Shul’ts, E.E.,
and Tolstikov, G.A., Dokl. Akad. Nauk, 2006, vol. 407,
pp. 698ꢀ701 [Dokl. (Engl. Transl.), vol. 407].
8. RF Patent No. 2211843 (2002).
9. Shintyapina, A.V., Shults, E.E., Petrenko, N.I., Uzenꢀ
kova, N.V., Tolstikov, G.A., Pronkina, N.V., Kozhevniꢀ
kov, V.S., and Pokrovsky, A.G., Bioorg. Khim. 2007.
vol. 33. pp. 620–626 [Russ. J. Bioorg. Chem. (Engl.
Transl.), 2007, vol. 33, pp. 579–583].
(75%). Rf 0.15; mp 272–274°С. [
CHCl3). Found, %: C 76.03; H 9.61; N 4.78.
С35Н54N2O3 ( 550.822). Calc., %: C 76.32; H 9.88;
α
]
+18.4
°
(с 0.05,
D
M
1
N 5.09. H NMR: 0.80, 0.82, 1.01, 1.13, 1.16, 1.35
(21 H, 7 s, 7CH3), 1.25–2.21 (18 H, m, CH2, CH),
2.24 (3 H, C, 4'ꢀCH3), 2.31 (1 H, s, H19), 2.83 (1 H,
m, H18), 2.35–2.48 (4 H, m, H3', H5'), 2.84–3.20
(4 H, m, H2', H6'), 3.08–3.33 (1 H, m, H3), 5.66
(1 H, s, H12). 13C NMR: 15.8, 15.9, 16.8, 19.3, 19.6,
20.9, 21.6, 22.9, 25.6, 26.5, 29.7, 31.3, 32.4, 33.6,
34.1, 35.9, 36.9, 38.1, 39.6, 40.5, 40.8, 41.8, 45.5,
45.9, 47.3, 50.2, 54.3, 54.5, 55.0, 61.5 (C9), 128.3
(C12), 169.5 (C13), 175.4 (CON), 200.0 (C11), 215.8
(C3).
10. Flekhter, O.B., Boreko, E.I., Nigmatullina, L.R.,
Tret’yakova, E.V., Pavlova, N.I., Baltina, L.A.,
Nikolaeva, S.N., Savinova, O.V., Eremin, V.F.,
Galin, F.Z., Tolstikov, G.A., Bioorg. Khim., 2004 [Russ. J.
Bioorg. Chem. (Engl. Transl.), 2004, vol. 30, pp. 80–88].
11. Saxena, B.B., Zhu, L., Hao, M., Kisilis, E.,
Katdare, M., Oktem, O., Bomshteyn, A., and Rathꢀ
nam, P., Bioorg. Med. Chem. Lett., 2006, vol. 14,
pp. 6349–6358.
12. Robinson, G.N., Wild, C.T., Ashton, M., Thomas, R.,
Montalbetty, C., Coulter, T.S., Magaraci, F.,
Townsend, R.J., Nitz, T.J., and Covert, J.M., Patent
WO No. 2006/053255, 2006.
Antimicrobial activity was determined by the
method of serial dilutions according to [25] towards
S. aureus and K. pneumoniae [26]. A suspension of the
tested microorganisms (0.1 ml, 109 CFU) was added to
each dilution and the mixtures were incubated at 37°C
for 1 h and cultivated on a thick nutrient medium
(NPO Microgen, Ministry of Health of the Russian
Federation). The grown colonies were calculated.
Sterile isotonic 10% sodium chloride and DMSO
(Barcelona, Spain) were added to the controls.
13. Gnoatto, S.C.B., Susplugas, S., Vechia, L.D.,
Ferreira, T.B., DassonvilleꢀKlimpt, A., Zimmer, K.R.,
Demailly, C., Nascimento, S.D., Guillon, J.,
Grellier, P., Verli, H., Gosmann, G., and Sonnet, P.,
Bioorg. Med. Chem., 2008, vol. 16, pp. 771–882.
14. Giniyatullina, G.V., Kazakova, O.B., Tolstikova, T.G.,
Sorokina, I.V., and Tolstikov, G.A., in Tez. dokl.
XI shkolyꢀkonferentsii po organicheskoi khimii (Abstr.
XI School Conference on Organic Chemistry), Yekatꢀ
erinburg: GU VPO Ural. Gos. Tekhn. Univ. UPIU im.
pervogo Prezidenta Rossii B.N. El’tsina, 2008,
pp. 287–289.
ACKNOWLEDGMENTS
15. Flekhter, O.B., Medvedeva, N.I., Tolstikov, G.A.,
Galin, F.Z., Yunusov, M.S., Huong Nguen Thi Mai, Le
Viet Tien, Savinova, O.V., Boreko, E.I., Titov L.P., and
Glukhov, I.V., Bioorg. Khim., 2009, vol. 35, pp. 253ꢀ259
The work was supported by the Russian Foundaꢀ
tion for Basic Research, project no. 08ꢀ03ꢀ00868.
[
Russ. J. Bioorg. Chem. (Eng. Transl.), 2009, vol. 35,
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