Antitumor activity of amides of dihydrobetulonic acid in vitro and in vivo

Article abstract of DOI:10.1134/S106816201302012X

Amides containing homopiperidine and piperazine cycles were synthesized from dihydrobetulonic acid obtained by the oxidation of dihydrobetulin. All substances were shown to have a high antitumor activity (CCID₅₀ = 3.5-36.2 μM) in vitro in lymphoid (CEM-13, U-937) and monocytic (MT-4) human cell lines. Amides containing the methyl- and ethylpiperazine residues do not influence the viability of Lung Lewis Carcinoma cells in culture and do not have a significant effect on its transplants in C57BL/6 mice. However, these amides efficiently inhibit the development of metastases in lungs of these mice. The antimetastatic activity of the studied amides increases with changing the methyl by ethyl aliphatic residue in the piperazine cycle.

Full text of DOI:10.1134/S106816201302012X

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2013, Vol. 39, No. 2, pp. 194–201. © Pleiades Publishing, Ltd., 2013.
Original Russian Text © I.V. Sorokina, I.Ya. Mainagashev, N.A. Zhukova, D.V. Korchagina, T.G. Tolstikova, V.P. Nikolin, N.A. Popova, M.A. Pokrovskii, A.G. Pokrovskii, N.F.
Salakhutdinov, 2013, published in Bioorganicheskaya Khimiya, 2013, Vol. 39, No. 2, pp. 221–229.
Antitumor Activity of Amides of Dihydrobetulonic Acid
in vitro and in vivo
, 1
I. V. Sorokinaa , I. Ya. Mainagasheva, N. A. Zhukovaa, D. V. Korchaginaa, T. G. Tolstikovaa,
V. P. Nikolinb, N. A. Popovab, M. A. Pokrovskiic, A. G. Pokrovskiic , and N. F. Salakhutdinova
a Vorozhtsov Institute of Organic Chemistry, Siberian Branch, Russian Academy of Sciences,
pr. Lavrent’eva 9, Novosibirsk, 630090, Russia
b Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences, Novosibirsk, Russia
c Novosibirsk State University, Novosibirsk, Russia
Received August 10, 2012; in final form, October 8, 2012
Abstract—Amides containing homopiperidine and piperazine cycles were synthesized from dihydrobetuꢀ
lonic acid obtained by the oxidation of dihydrobetulin. All substances were shown to have a high antitumor
activity (CCID50 = 3.5–36.2 M) in vitro in lymphoid (CEMꢀ13, Uꢀ937) and monocytic (MTꢀ4) human
µ
cell lines. Amides containing the methylꢀ and ethylpiperazine residues do not influence the viability of Lung
Lewis Carcinoma cells in culture and do not have a significant effect on its transplants in C57BL/6 mice.
However, these amides efficiently inhibit the development of metastases in lungs of these mice. The antimeꢀ
tastatic activity of the studied amides increases with changing the methyl by ethyl aliphatic residue in the pipꢀ
erazine cycle.
Keywords: dihydrobetulonic acid, amides, piperazine derivatives, antitumor activity, antimetastatic effect
DOI: 10.1134/S106816201302012X
1
INTRODUCTION
Dihydrobetulonic acid (I) can be considered as a
promising compound in the series of biologically active
lupans. Hydrogenation of betulonic acid derivatives in
the C20 position increases their antitumor activity as
compared to nonhydrogenated analogues [9]. It was
Over the past decades, pentacyclic triterpenoids of
the lupane series have been in the focus of medical
chemistry as lowꢀtoxicity compounds with a high
activity, which stimulate apoptosis of tumor cells [1].
At the same time, they are searching among agentꢀ
modifiers, which can be used in traditional chemoꢀ
therapy to improve the efficiency, and reduce the toxꢀ
icity, of cytostatic drugs. To obtain libraries of derivaꢀ
tives from starting triterpene compounds, the derivaꢀ
tives of the available plant metabolite, betulin, are
often used. We have previously found that the derivaꢀ
tives of betulonic acid containing the fragments of
shown with an example of 3ꢀOꢀglutaryldihydrobetulin
that the use of the hydrogenated analogue of betulin,
instead of its common derivative, leads to an increase in
the antiꢀHIV activity by at least three orders of magniꢀ
tude [10]. The pronounced antitumor activity in colon
cancer xenografts was found for the dihydrobetulonic
acid derivative containing the 4ꢀnitrobenzyloxyimine
substituent in the C3 position [11].
ω
ꢀamino acids are active inductors of apoptosis in leuꢀ
kemic and hepatocellular carcinoma cells in vitro [2].
Amides of betulonic acid were shown to be more effiꢀ
cient inductors of apoptosis than the corresponding
acids [3]. Betulonic acid and its alanylamide derivaꢀ
tives enhance the antitumor effect of cytostatic polyꢀ
chemotherapy in mice with transplantable tumors [4,
5] and reduce the severity of necrobiotic disorders in
the liver, kidney, and myocardium caused by cytostatic
drugs [6–8].
RESULTS AND DISCUSSION
The goal of the work was the synthesis of amides of
dihydrobetulonic acid and the study of its antitumor
properties in tumor cell cultures and in animals with
transplantable tumors. Moreover, we evaluated the
antitumor and antimetastatic efficiency of individual
lupan derivatives along with cytotoxic polychemotherꢀ
apy in tumorꢀbearing mice.
Betulin was obtained by the extraction of birch bark
by benzene and used as the starting compound to synꢀ
thesize amides (III)–(VII). Hydrogenation of betulin
with hydrogen in an autoclave led to dihydrobetulin,
which was oxidized with Fieser’s solution. At the next
Abbreviations: CCID , concentration leading to the death of
50
50% of cells; TGI, index of tumor growth inhibition; MII,
metastasis inhibition index; MF, metastasis frequency.
Corresponding author: phone: 7(383)330ꢀ07ꢀ31; eꢀmail:
sorokina@nioch.nsc.ru.
1
194

ANTITUMOR ACTIVITY OF AMIDES
195
stage, chloroanhydride of dihydrobetulonic acid was excess of the corresponding amine resulting in target
obtained, which was then interacted with double compounds (III)–(VII) (Scheme).
Conditions: а. (COCl)2, CH2Cl2, 20°C, 4 h; b. HNR1R2, CH2Cl2, 20°C, 18–20 h.
29
30
20
21
22
19
18
12
28
OH
Cl
11
26
9
17
16
25
13
14
15
a
b
1
4
O
O
2
3
10
8
7
5
27
6
O
O
24
23
(I
)
(II)
NR1R2
O
O
(
III)–(VII
)
9'
8'
O
7'
7'
7'
5'
2'
5'
2'
5'
8'
N
3'
4'N
3'
O
6'
4'
3'
6'
1'
N
6'
1'
N
4'
N1'
2'
(
(
III) NR1R2 =
(
IV) NR1R2 =
(V
) NR1R2 =
11'
10'
9'
8'
7'
6'
5'
5'
7'
N1'
4'N
3'
8'
6'
4'
3'
N1'
9' 11'
10'
2'
2'
VI) NR1R2 =
(
VII) NR1R2 =
Scheme.
The structure of the prepared compounds was conꢀ
The data allow one to consider betulonic acid (I)
firmed by 1H and 13C NMR and IR spectra. The speꢀ and its amides (III)–(VII) as potential promising antiꢀ
cific rotation and melting point values were deterꢀ tumor drugs with a high activity against carcinoma
mined for all synthesized compounds. Brutto formulas tumor lines CEMꢀ13, Uꢀ937, and MTꢀ4.
of the synthesized compounds were determined from
the element analysis or mass spectra of high resoluꢀ
tion.
Two piperazine derivatives (III) and (IV), which
showed high activity in the MTT test, were chosen for
the following in vivo experiments. These experiments
The in vitro screening by the MTT test was used to were carried out with female mice C57BL/6 inocuꢀ
study the effect of hydrogenated betulonic acid ( ) and lated intramuscularly with Lewis lung carcinoma.
I
its amides (III)–(VII) on the viability of human canꢀ Compounds (III) and (IV) were administered intraꢀ
cer cells: leukemic (CEMꢀ13 and Uꢀ937) and monoꢀ gastrically in a dose of 20 mg/kg daily from the third to
cytic (MTꢀ4) cell lines. Compounds (
VII) were found to exhibit high antitumor activity in sen according to the data on the antitumor activity of
all tumor cultures used. All agents have similar pentacyclic triterpenoids in the dose range of 5 to
CCID50 values in the range of 3.5–36.2 M (Table 1). 50 mg/kg [12]. The measurement of tumor nodules
I) and (III)– the tenth day after transplantation. The dose was choꢀ
(
μ
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 39
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196
SOROKINA et al.
Table 1. CCID50 values of hydrogenated betulonic acid (
and its amides (III)–(VII) for different human carcinoma
cell lines
I)
Thus, the eightꢀday intragastric introduction of
methylꢀ and ethylpiperazine of dihydrobetulonic acid
in a dose of 20 mg/kg at the early stage of the neoplasꢀ
tic process had no antitumor effect due, probably, both
to an insufficient dose of the agents and the low sensiꢀ
tivity of this tumor to the piperazine derivative of dihyꢀ
drobetulonic acid. To confirm these assumptions, we
performed an additional study of the activity of the
compounds in a Lewis carcinoma cell culture, which
was obtained from the corresponding solid tumor
transplants. After excision of the tumor tissue, cells
were washed by saline, crushed and placed in Versen
solution. The cells were then washed and placed in a
nutrient medium. The compounds were found to have
no cytotoxic effects even at the maximum concentraꢀ
CCID , μM
50
Compound
CEMꢀ13
Uꢀ937
MTꢀ4
(
(
(
(
(
(
I
)
4.4
13.0
6.9
3.8
7.8
3.5
33.4
7.2
III
)
IV
)
6.0
V
)
12.4
26.0
10.6
23.5
36.2
11.9
7.7
VI
)
10.9
11.9
VII
)
tion used (100 M). Thus, in the in vitro and in vivo
μ
experiments, no significant cytotoxic effect against
Lewis lung carcinoma was found for hydrogenated
betulonic acid and its amides.
Table 2. Indices of tumor growth inhibition and average life
span of mice after transplantation of Lewis lung carcinoma
and 8ꢀday administration of compounds (III) and (IV
)
We evaluated the efficiency of amides (III) and (IV
)
Indexes of tumor
growth inhibition, %
Comꢀ
pound
Life span
(days)
when administered at the stage of tumor progression
and dissemination, in particular, along with cytotoxic
polychemotherapy (PCT). In the latter case, the criteꢀ
rion for efficiency was the increase in the antitumor
and antimetastatic activity of the cytostatic preparaꢀ
tions. Lewis lung carcinoma was transplanted into
11th day 13th day 15th day
Control
–
–
–
19.6 1.7
22.8 2.4
22.4 1.1
(
III)
1.9
4.7*
8.7
7.6
19.9
20.3
(
IV)
female mice C57BL/6. Compounds (III) and (IV
)
were intragastrically administered as the waterꢀtween
suspension in a dose of 20 mg/kg from day 11 to day 18
after the transplantation. The day before the adminisꢀ
tration of agents, half of the mice were parenterally
administered a oneꢀtime cytostatic complex simulatꢀ
ing the standard chemotherapy scheme (cyclophosꢀ
phamide, doxorubicin, vincristine, and prednisolone)
[6]. During the administration of the agents, the volꢀ
ume of tumor nodules and the TGI indexes were evalꢀ
uated. The size of metastatic foci in the lungs was
counted morphometrically on 19th day after transꢀ
plantation.
*
P < 0.05, significant difference from the control.
Table 3. Indices of tumor growth inhibition and lethality in
mice with Lewis lung carcinoma during isolated and comꢀ
bined with polychemotherapy 8ꢀday administration of
compounds (III) and (IV
)
Indexes of tumor growth
inhibition, %
Lethality,
%
Compound
Control
14th day 16th day 18th day
–
–
22
5
–
30
40
40
20
20
0
It was shown that the methylꢀ and ethylpiperazine
derivatives in a standꢀalone mode of administration
exhibit low antitumor activity and a slightly lower surꢀ
vival rate of the mice compared to the control group.
During the course administration along with PCT,
methylpiperazine derivative (III) insignificantly
reduces the cytotoxic effect of chemotherapy and does
not affect the mortality percentage of mice. Ethylpipꢀ
erazine derivative (IV) more markedly reduces the
efficiency of chemotherapy and improves the survival
of animals (Table 3).
(
III)
0
12
12
26
17
12
(
IV)
0
11
11
13
PCT
25
21
9
PCT + (III
)
PCT + (IV
)
started after visualization and continued until the
death of animals (from 11 to 15 days after transplantaꢀ
tion).
Compounds (III) and (IV) showed a stable tenꢀ
The compounds studied were found to exhibit the
dency to the tumor growth delay, but this effect was not antimetastatic effect in the standꢀalone mode of
statistically significant. The index of tumor growth administration into animals. The more pronounced
inhibition (TGI) for both compounds was 20% on the activity was registered for ethylpiperazine derivative
15th day of the experiment. Also noted was a slight (the 8ꢀfold reduction in the bulk density of metastases)
increase in the average life expectancy of mice, which compared with methylpiperazine (reduction by a facꢀ
may indicate the stimulation of antitumor resistance tor of 2.5) (Table 4). It should be noted that the antiꢀ
in the animals (Table 2).
metastataic effect of ethylpiperazine derivative (IV)
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ANTITUMOR ACTIVITY OF AMIDES
197
Table 4. Indicators of metastasis of Lewis lung carcinoma in the lungs of mice after isolated and combined with polychemoꢀ
therapy 8ꢀday administration of compounds (III) and (IV
)
Compound
Control
Metastasis volume density,
μM
Metastasis frequency, %
MII, %
319.78 51.36
123.93 49.22
39.08 2.44*
50.66 9.15*
78.05 14.91*
40.06 4.14*
100
100
100
88
–
(III
)
61.2
87.8
86.0
75.6
87.5
(IV)
PCT
PCT + (III
)
100
100
PCT+ (IV
)
*
Р < 0.001 with respect to control.
was not inferior to that of cytostatic PCT, which 30 A/G (Germany). The elemental composition of the
reduced the area of metastatic foci by a factor of more substances was determined by elemental analysis and
than six. Nevertheless, the administration of ethylpipꢀ mass spectrometry of a high resolution recorded on a
erazine derivative along with the standard chemotherꢀ DFS (double Focusing Sector) spectrometer (Thermo
1
13
apy did not increase its antimetastatic effect. Methꢀ Electron Corporation, Germany). H and C NMR
ylpiperazine derivative (III) slightly reduced the corꢀ spectra were recorded in CDCl3 on a DRXꢀ500 specꢀ
1
responding effect of PCT (MII was 75.6 vs 86.0 for trometer (500.13 MHz and 125.76 MHz for H and
PCT).
13C, respectively; Bruker, Germany). Chemical shifts
(
δ
) are in ppm, CSSI are in Hz. The residual signals of
Thus, ethylpiperazine derivative (IV) at the standꢀ
the solvent were used as the internal standard (δН
=
alone mode administration exhibits pronounced antiꢀ
metastatic activity. When used in combination with
cytostatic polychemotherapy, this compound’s antiꢀ
metastatic effect is not reduced. The methylpiperazine
derivative (III) itself exhibits moderate antimetastatic
action and when administered along with chemotherꢀ
apy, slightly decreases its effect.
It can be concluded that methylꢀ and ethylpiperaꢀ
zine derivatives of dihydrobetulonic acid have a cytoꢀ
static effect in vitro on CEMꢀ13, Uꢀ937, and MTꢀ4
cells. Lewis lung carcinoma in the form of either cell
culture or solid tumor in mice is not very sensitive to
the action of the compounds. Administration of
amides of dihydrobetulonic acid (III) and (IV) in mice
at the stage of tumor progression causes a significant
delay in its dissemination, and has little effect on the
growth of transplants. The replacement of the methyl
substituent by the ethyl group in the piperazine fragꢀ
ment leads to the enhancement of the antimetastatic
effect, which, probably, provides the reduction in morꢀ
tality of animals during the administration of agent
7.24 ppm; δС = 76.9 ppm). The structure of the comꢀ
pounds was established from the 1H–1H NMR spectra
involving the doubleꢀresonance spectra, 13C NMR
spectra using standard recording techniques in the
J
ꢀmodulation (JMOD) regime with nonresonant and
selective suppression of protons, twoꢀdimensional
spectra of the 13Сꢀ1Н heteronuclear correlation of the
1
direct (CꢀH COSY. JC,H 135 Hz) and longꢀrange
spinꢀspin coupling constants (COLOC, 2,3JC,H 10 Hz).
Betulin was isolated from the outer bark of birch
[13]; dihydrobetulin was synthesized by hydrogenaꢀ
tion of betulin over Raney nickel according to the
known method [14].
3ꢀOxolupanꢀ28ꢀic acid (dihydrobetulonic acid (I))
.
A suspension of dihydrobetuline (10 g, 22.5 mol) in
glacial acetic acid (165 mL) and acetone (110 mL) was
cooled to 0°С, and the freshly prepared Fieser’s soluꢀ
tion (11 g, 0.11 mol of chromic anhydride in 12 mL of
glacial acetic acid and 15 mL of Н2О) was added under
stirring for 1 h. The reaction mixture was kept at room
temperature for 3 h, followed by the addition of methꢀ
anol (50 mL), benzene (300 mL), and 10% NaCl soluꢀ
tion (300 mL). The benzene layer was removed, and the
(
IV). The use of the piperazine derivatives of dihydroꢀ
betulonic acid in combination with PCT does not
result in the enhancement of the antitumor and antiꢀ
metastatic efficiency of the latter.
aqueous layer was extracted with benzene (
2 150 mL).
×
The combined extracts were washed with a 10% soluꢀ
tion of NaCl and dried with MgSO4. The mixture was
filtered and the benzene evaporated to ~100 mL and
poured into a 5% solution of KOH (200 mL). The resꢀ
idue of the potassium salt of dihydrobetulonic acid was
filtered and dried. The dry salt was dissolved in ethanol
(50 mL), the undissolved portion was filtered, and the filꢀ
trate was poured into a 5% solution of HCl (250 mL).
The precipitate was filtered, washed with H2O and
EXPERIMENTAL
IR spectra (
22 spectrometer (Germany) in KBr. The special rotaꢀ
tion ( D) was evaluated using a polAAr 3005 specꢀ
trometer (Great Britain). Concentrations of solutions
) in СНСl3 are in g/100 mL. Melting points were
ν
, cm–1) were recorded on a VECTOR
[α]
(
c
determined using a Termosystem FP 900 instrument
(Mettler Toledo, Switzerland) and on a Kofler table S
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 39
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198
SOROKINA et al.
dried. We obtained 5.5 g (54%) of the product, Tm
=
C25), 15.60 (q, C26), 14.43 (q, C27), 177.15 (s, C28),
14.50 (q, C29), 22.68 (q, C30).
27
253–254оС (lit. Tm
0.41). Found:
=
253–254°С [15]).
M
+10.7
α
[ ]D
]+. C30H48O3. Calꢀ
culated: 456.3603. IR spectrum: 1705 (C=O). H
General method of the synthesis of amides of dihyꢀ
drobetulonic acid (III)–(VII). The synthesis was carꢀ
ried out similarly to method [15], except that an equivꢀ
alent excess of the reacting amine was used instead of
triethylamine. The corresponding amine (2.2 mol)
was added to the solution of chloroanhydride of dihyꢀ
drobetulonic acid (II) (0.5 g, 1.05 mmol) in anhydrous
СН2Сl2 (40 mL). The reaction mixture was kept at room
temperature for 18–20 h, followed by the dilution with
(c
m
/
z
456.3608 [
1
NMR spectrum: 0.73 (3 H, d,
d, 7.0, H30); 0.90 (3 H, s, H25); 0.93 (6 H, s, H26,
H27); 0.98 (3 H, s, H24); 1.04 (3 H, s, H23); 1.11–
1.70 (18 H, m, СН2,СН); 1.76 (1 H, septet d, 7.0,
or
1e,2e 4.3,
J 7.0, H29); 0.83 (3 H,
J
J
J
20,19 2.5, H20); 1.86 (1 H, dd, 2
H22 ); 1.88 (1 H, ddd, 1e,1a 13.0,
J
12.4,
J
7.3, H22
α
β
J
J1e,2a 7.5, J
H1e); 2.15–2.27 (3 H, m, СН2,СН); 2.38 (1 H, ddd,
J2е,2а 15.8, J2е,1а 7.5, J2е,1е 4.3, H2e); 2.47 (1 H, ddd,
J2а,2е 15.8, J2а,1а 9.7, J2а,1е 7.5, H2a); 9.48 (1 H, br.s,
СН2Сl2 to 20 mL, washing with H2O (
3 × 20 mL), and
drying with MgSO4. The desiccant was filtered off, and
the solvent was removed. The residue was separated by
OH). 13C NMR spectrum: 39.43 (t, C1), 33.97 (t, C2),
218.10 (s, C3), 47.19 (s, C4), 54.75 (d, C5), 19.48 (t,
C6), 33.52 (t, C7), 40.49 (s, C8), 49.45 (d, C9), 36.74
(s, C10), 21.25 (t, C11), 26.72 (t, C12), 38.22 (d,
C13), 42.48 (s, C14), 29.54 (t, C15), 31.86 (t, C16),
56.72 (s, C17), 48.54 (d, C18), 44.01 (d, C19), 29.59
(d, C20), 22.60 (t, C21), 37.26 (t, C22), 26.50 (q,
C23), 20.86 (q, C24), 15.73 and 15.76 (qq, C25 and
C26), 14.40 (q, C27), 182.79 (s, C28), 14.53 (q, C29),
22.84 (q, C30).
column chromatography on silica gel (60–200 m,
μ
Merck, Germany). Different eluents were used for
13
each product (see below). C NMR data for comꢀ
pounds (III) and (IV) are presented in Table 5.
(4'ꢀMethylpiperazineꢀ1'ꢀyl)amid of 3ꢀoxolupanꢀ
28ꢀoic acid (III). Eluent: СН2Сl2ꢀMeOH (80 : 20).
Isolated: 0.41 g (72%) of the product with Tm
=
241–
22
242°С.
–25.7 (c 0.20). Found: m/z 538.4486
α
[ ]D
[M
]+. C35H58N2O2. Calculated: 538.4493. IR specꢀ
3ꢀOxolupanꢀ28ꢀoyl chloride (chloroanhydride of
dihydrobetulonic acid (II)). Oxalyl chloride (2.3 mL,
26.36 mmol) was added to the solution of dihydrobetꢀ
ulonic acid (I) (5.7 g, 12.48 mol) in anhydrous СН2Сl2
α
β
(70 mL) and the mixture was kept at room temperaꢀ
ture for 4 h. After evaporation of the solvent, the same
volume of СН2Сl2 was added to the residue, and the
mixture was reꢀevaporated. The residue was treated
with anhydrous ether, the precipitate was filtered and
washed with ether. We obtained 4.6 g (91%) of chloroꢀ
anhydride of dihydrobetulonic acid with Tm
=
206–
207°С. 27 +3.9 (
c
0.41). Found: 474.3258 [
m
/
z
M
]+.
α
[ ]D
C30H47ClO2. Calculated: 474.3264. IR spectrum: 1811
(COCl), 1705 (C=O). 1H NMR spectrum: 0.71 (3 H,
d, J 7.0, H29); 0.82 (3 H, d, J 7.0, H30); 0.91 (3 H, s,
H25); 0.93 (3 H, s, H27); 0.95 (3 H, s, H26); 0.99 (3
H, s, H24); 1.04 (3 H, s, H23); 1.07–1.56 (17 H, m,
СН2,СН); 1.63 (1 H, dm, 2J 12.6, H12e); 1.76 (1 H,
septet d,
J 7.0, J20,19 2.5, H20); 1.89 (1 H, ddd, J1е,1а
13.0,
11.0,
12.4,
J
J
J
10.5,
7.2,
J
J
4.1,
1.3, Н22
13a,18а 10.8, J
J
19,20 2.5, H19); 2.10 (1 H, ddd, 2J
or Н22 ); 2.19 (1 H, ddd,
13a,12е 3.8, H13a); 2.37 (1 H,
α
β
J13a,12а 13.0,
J
ddd, J2е,2а 15.8, J2е,1а 7.5, J2е,1е 4.3, H2e); 2.48 (1 H,
13
ddd, J2а,2е 15.8, J2а,1а 9.8, J2а,1е 7.5, H2a). C NMR:
39.47 (t, C1), 33.96 (t, C2), 217.86 (s, C3), 47.20 (s,
C4), 54.83 (d, C5), 19.46 (t, C6), 33.51 (t, C7), 40.53
(s, C8), 49.52 (d, C9), 36.74 (s, C10), 21.20 (t, C11),
26.60 (t, C12), 37.46 (d, C13), 42.52 (s, C14), 29.42 (t,
C15), 31.98 (t, C16), 68.15 (s, C17), 49.02 (d, C18),
43.07 (d, C19), 29.47 (d, C20), 22.00 (t, C21), 36.22
(t, C22), 26.44 (q, C23), 20.91 (q, C24), 15.78 (q,
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 39
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ANTITUMOR ACTIVITY OF AMIDES
199
Table 5. Data of 13C NMR spectra for amides of dihydrobetulonic acid (III)–(VII) in CDCl3)
, ppm)
δ
a
a
a
b
b
C atom
(III
)
(
IV)
(
V)
(VI)
(VII)
C1
39.48 t
33.98 t
217.91 s
47.15 s
54.90 d
19.49 t
33.62 t
40.47 s
49.80 d
36.75 s
21.54 t
27.01 t
36.54 d
41.88 s
29.65 t
32.15 t
54.79 s
52.06 d
42.60 d
29.63 d
23.35 t
35.97 t
26.44 q
20.86 q
15.78 q
15.77 q
14.26 q
173.50 s
14.51 q
22.77 q
55.06 t
45.76 q
39.46 t
33.95 t
217.85 s
47.12 s
54.88 d
19.47 t
33.60 t
40.45 s
49.79 d
36.73 s
21.53 t
26.99 t
36.51 d
41.86 s
29.61 t
32.13 t
54.75 s
52.04 d
42.59 d
29.61 d
23.33 t
35.95 t
26.43 q
20.84 q
15.76 q
15.76 q
14.24 q
173.41 s
14.49 q
22.75 q
52.90 t
52.08 q
11.73 q
39.45 t
33.94 t
217.82 s
47.12 s
54.86 d
19.45 t
33.60 t
40.44 s
49.76 d
36.72 s
21.51 t
26.95 t
36.55 d
41.88 s
29.63 t
32.20 t
54.87 s
52.06 d
42.58 d
29.60 d
23.32 t
35.99 t
26.41 q
20.84 q
15.75 q
15.74 q
14.25 q
173.85 s
14.47 q
22.73 q
43.64 t
155.34 s
61.38 t
14.45 q
39.50 t
34.00 t
217.97 s
47.18 s
39.50 t
33.98 t
217.96 s
47.15 s
54.90 d
19.51 t
33.65 t
40.52 s
49.88 d
36.76 s
21.59 t
27.04 t
36.51 d
41.98 s
29.80 t
31.92 t
55.21 s
52.50 d
42.76 d
29.73 d
23.43 t
36.23 t
26.46 q
20.84 q
15.80 q
15.74 q
14.34 q
173.88 s
14.56 q
22.82 q
C2
C3
C4
C5
54.93 d
19.52 t
33.67 t
40.49 s
49.83 d
36.77 s
21.56 t
27.03 t
36.56 d
41.90 s
29.65 t
32.13 t
54.80 s
52.07 d
42.66 d
29.65 d
23.37 t
35.96 t
26.47 q
20.90 q
15.80 q
15.80 q
14.28 q
173.47 s
14.52 q
22.79 q
52.16 t
76.12 d
142.26 s
126.66d
C6
C7
C8
C9
C10
C11
C12
C13
C14
C15
C16
C17
C18
C19
C20
C21
C22
C23
C24
C25
C26
C27
C28
C29
C30
C3', C5'
C7'
C8'
C9'
a)
b)
Very broad signals (~45 ppm) are observed for C2' and C6' atoms of compounds (III)–(V);
Signals of C2' and C6' of compound (VI) and C2' and C7' of compound (VII) are not observed; chemical shifts of signals of aromatic
part in compound (VI) are 128.43 (d, C10') and 126.94 (d, C11') ppm.
22
J
J
16е,15е 3.0, H16e); 2.13 (1 H, dddd,
J19,18а 10.7, J 10.0,
g (63%) of the product with Tm
=
185–186°С.
–
α
[ ]D
3.8, 19,20 2.7, H19); 2.25–2.38 (5 H, m, H2e, 2H3',
J
82.0 ( 0.20). Found: m/z 596.4551 [
с
M
]+. C37H60N2O4
.
2H5'); 2.34 (2 H, q, J7',8' 7.2, 2H7'); 2.43 (1 H, ddd,
J2а,2е 15.7, J2а,1а 9.6, J2а,1е 7.6, H2a); 2.91 (1 H, ddd,
Calculated: 596.4548. IR spectrum: 1632 (CON);
1707 (C=O). 1H NMR spectrum: 0.68 (3 H, d, J 6.8,
H29); 0.78 (3 H, d, J 6.8, H30); 0.88 (3 H, s, H25);
0.89 (3 H, s, H27); 0.91 (3 H, s, H26); 0.96 (3 H, s,
H24); 1.01 (3 H, s, H23); 1.03–1.16 (3 H, m, H12a,
J13а,12а 12.8,
J13а,18а 10.7, J13а,12е 3.8, H13a); 3.56 (4 H,
br.s, 2Н2', 2Н6').
(4'ꢀEthoxycarbonylpiperazineꢀ1'ꢀyl)amid of 3ꢀoxoꢀ
lupanꢀ28ꢀoic acid (V). Eluent: СН2Сl2. Isolated: 0.39
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 39
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200
SOROKINA et al.
H15e, H22); 1.21 (3 H, t, J 7.1, 3H9'); 1.25 (1 H, dd,
J18a,13а 10.6, J18a,19 10.6, H18a); 1.26 (1 H, dd, J5a,6а vitro. The Tꢀcell leukemia cell lines CEMꢀ13 and
Antitumor activity of compounds (I), (III)–(VII) in
Uꢀ937 and the tumor TMꢀ4 cell line of human monoꢀ
cytes were cultured in RPMI 1640 containing 10%
fetal calf serum and antibiotics (100 U/mL of penicilꢀ
lin and 0.1 mg/mL of streptomycin) in 5% СО2 at
11.5,
1.63 (1 H, dm,
H20,
6.8, J20,19 2.6); 1.81 (1 H, dd, 2J 11.7,
H22 or H22 ); 1.86 (1 H, ddd, 1е,1а 13.2, 1е,2а 7.6,
1е,2е 4.4, H1e); 2.02 (1 H, ddd, 16е,16а 13.2, 16е,15а 3.5,
16е,15е 3.0, H16e,); 2.13 (1 H, dddd, 19,18а 10.6, 10.2,
3.6, 19,20 2.6, H19,); 2.34 (1 H, ddd, 2е,2а 15.8, J2е,1а
2е,1е 4.4, H2e); 2.44 (1 H, ddd, 2а,2е 15.8, 2а,1а 9.7,
J
5a,6е 2.5, H5a); 1.28–1.51 (12 H, m, СH2,СH);
12е,12а 11.0, H12e); 1.73 (1 H, septet d,
6.3,
J
J
α
J
β
J
J
37°С.
J
J
J
J
J
The viability of cells after the incubation with comꢀ
pounds ( ), (III)–(VII) was evaluated by the MTT
J
J
I
J
J
test, which allows for the spectrophotometric determiꢀ
nation of the amount of living cells in a sample. For
7.5,
J
J
J
J2а,1е 7.6, H2a); 2.88 (1 H, ddd, J13а,12а 13.0, J13а,18а
10.6, J13а,12е 3.7, H13a,); 3.38 (4 H, m, 2H3', 2H5');
3.52 (4 H, br.s, 2H2', 2H6'); 4.09 (2 H, q, J 7.1, 2H8').
this, the cells were planted in 96ꢀwell plates (100
cells at a concentration of 500000 cells/mL), followed
by the addition of the solution of compounds ( ),
μ
L of
I
(
III)–(VII) in DMSO to the final concentration in the
(4'ꢀDiphenylmethylpiperazineꢀ1'ꢀyl)amid of 3ꢀoxoꢀ
lupanꢀ28ꢀoic acid (VI). Eluent: СНСl3. Isolated: 0.68 g
medium of 0.1–100 g/mL, and incubated with these
μ
compounds for three days under the same conditions.
The MTT solution (5 mg/mL) in phosphateꢀsaline
buffer was then added to the cells without changing the
medium to a concentration of 0.5 mg/mL, and the
incubation continued for 3 h under the same condiꢀ
22
(93%) of the product with Tm
=
146–147°С.
M
–
.
α
[ ]D
20.7 ( 0.21). Found: 690.5098 [
с
m/z
]+. C47H66N2O2
Calculated: 690.5119. IR spectrum: 1630 (CON);
1704 (C=O). 1H NMR spectrum: 0.70 (3 H, d, J 6.8,
H29); 0.81 (3 H, d, J 6.8, H30); 0.909 (3 H, s, H25);
0.911 (3 H, s, H27); 0.95 (3 H, s, H26); 1.00 (3 H, s,
H24); 1.05 (3 H, s, H23); 1.02–1.14 (3 H, m, H12a,
tions. The medium was removed, DMSO (100 L) was
μ
added, and the optical absorption was measured on a
multichannel spectrophotometer at 570 nm and 630 nm,
where А570 is the absorption of formazan and А630 is the
background of cells. The data are presented as the
amount of living cells vs. the control. The number of
cells in the control was 100% when cells were incuꢀ
bated only in the presence of the solvent (DMSO).
H15e, H22); 1.25 (1 H, dd, J18a,13а 10.7, J18a,19 10.7
,
H18a); 1.30 (1 H, dd, J5a,6а 11.5, J5a,6е 2.6, H5a,);
1.30–1.51 (12 H, m, СH2, CH); 1.66 (1 H, dm, J12е,12а
11.0, H12e); 1.76 (1 H, septet d,
1.85 (1 H, br.dd, 2J 11.7,
6.1, H22
(1 H, ddd, 1е,1а 13.2, 1е,2а 7.6, 1е,2е 4.4, H1e); 2.05 (1
H, dm, 16е,16а 12.8, H16e); 2.17 (1 H, m, H19); 2.31
(4 H, br.s, 2H3', 2H5'); 2.38 (1 H, ddd, 2е,2а 15.6, J2е,1а
7.3, J2е,1е 4.4, H2e); 2.47 (1 H, ddd, J2а,2е 15.6, J2а,1а
9.7, 2а,1е 7.6, H2a); 2.94 (1 H, ddd, 13а,12а 12.8, J13а,18а
J
6.8,
J20,19 2.4, H20);
J
α
or H22 ,); 1.89
β
Antitumor and antimetastatic activity of compounds
(III) and (IV) in vivo. The study was performed on
mice with transplanted Lewis lung carcinoma in two
series of experiments: the agents were introduced in
the early or late stages of cancer.
J
J
J
J
J
J
J
We used female mice of the C57BL/6 line weighing
20–25 g, which were kept under normal vivarium conꢀ
ditions with natural light regime and fed with a stanꢀ
dard feed and water. All manipulations with animals
were carried out in accordance with the European
Convention on humane treatment of laboratory aniꢀ
mals. Transplantation of Lewis lung carcinoma cells
10.7, J13а,12е 3.5, H13a); 3.57 (4 H, br.s, 2H2', 2H6');
4.17 (1 H, s, H7'); 7.15 (2 H, t, J 7.5, 2H11'); 7.25 (4
H, t, J 7.5, 4H10'); 7.39 (4 H, d, J 7.5, 4H9').
(Homopiperidinꢀ1'ꢀyl)amid of 3ꢀoxolupanꢀ28ꢀoic
acid (VII). Eluent: СHСl3. Isolated: 0.37 g (65%) of
22
the product with Tm
0.20). Found: 537.4544 [
culated: 537.4540. IR spectrum: 1622 (CON); 1709
(C=O). 1H NMR spectrum: 0.69 (3 H, d,
6.8, H29);
0.78 (3 H, d, 6.8, H30); 0.88 (3 H, s, H25); 0.90 (3
H, s, H27); 0.94 (3 H, s, H26); 0.96 (3 H, s, H24); 1.01
(3 H, s, H23); 1.00–1.13 (3 H, m, H12a, H15e, H22
or H22 ); 1.22 (1 H, dd, J18a,13а 10.8, J18a,19 10.8
H18a); 1.27 (1 H, dd,
=
242–243°С.
–38.0
α
[ ]D
was performed intramuscularly (
2
106 cells in 0.1 mL
×
(с
m/z
M
]+. C36H59NO2. Calꢀ
of physiological solution). Measurement of each
tumor nodule was performed with a caliper in three
mutually perpendicular directions. The antitumor
activity was expressed as an index of tumor growth
inhibition (TGI), which was defined as the ratio of the
difference of the tumor mass in the control and experꢀ
imental groups to the tumor mass in the control. Each
group contained at least 10 mice.
J
J
α
β
,
J
5a,6а 11.2,
J
5a,6е 2.5, H5a); 1.28–
1.49 (12 H, m, СH2, CH); 1.71 (1 H, septet d,
20,19 2.5, H20); 1.54–1.76 (4 H, m, СH2, CH); 1.86 (1
H, m, H1); 1.88 (1 H, dd, 2J 11.7,
6.1, Н22 or
Н22 ); 2.14 (1 H, dm, 16е,16а 12.6, H16e); 2.17 (1 H,
m, H19); 2.35 (1 H, ddd,
H2e); 2.44 (1 H, ddd, J2а,2е 15.7, J2а,1а 9.6, J2а,1е 7.6
H2a); 3.02 (1 H, ddd, 13а,12а 12.8,
3.6, H13a); 3.10–3.8 (4 H, m, 2H2', 2H7').
J 6.8,
In the first experimental series, amides of dihydroꢀ
betulonic acid (III) and (IV) were administered intraꢀ
gastrically as the waterꢀtween suspension in a dose of
20 mg/kg for 8 days, starting from the third day after
the tumor transplantation (the total dose was
160 mg/kg). The control group of animals with the
tumor intragastrically received the waterꢀtween mixꢀ
ture. The dynamics of the growth of tumor transplants
J
J
α
β
J
J2е,2а 15.7,
J2е,1а 7.5,
J
2е,1е 4.3
,
,
J
J13а,18а 10.8, J13а,12е
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 39
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ANTITUMOR ACTIVITY OF AMIDES
201
was evaluated from the day after withdrawal of the
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ACKNOWLEDGMENTS
The work was supported by the Interdisciplinary
integration project no. 41 of the Siberian Branch of the
Russian Academy of Sciences.
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RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 39
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2013