Synthesis of oxalic acid derivatives and their antitumor activity in experiments in vivo

Article abstract of DOI:10.1007/s11172-018-2124-5

A series of amino acid derivatives of oxalic acid were obtained. In combination therapy with conventional cytostatics used in lower doses, the new compounds substantially increase the efficacy of the drugs in the treatment of experimental P388 murine leukemia and some its drug-resistant strains.

Full text of DOI:10.1007/s11172-018-2124-5

694
Russian Chemical Bulletin, International Edition, Vol. 67, No. 4, pp. 694—699, April, 2018
Synthesis of oxalic acid derivatives
and their antitumor activity in experiments in vivo
†
B. S. Fedorov, S. A. Goncharova, T. A. Rajevskaya, A. B. Eremeev,
†
A. N. Utenyshev, M. A. Fadeev, G. N. Bogdanov, N. P. Konovalova, and S. M. Aldoshin
Institute of Problems of Chemical Physics, Russian Academy of Sciences,
prosp. Akad. Semenova, 142432 Chernogolovka, Moscow Region, Russian Federation.
Fax: +7 (496) 522 3507. E-mail: tara@icp.ac.ru
1
A series of amino acid derivatives of oxalic acid were obtained. In combination therapy with
conventional cytostatics used in lower doses, the new compounds substantially increase the
efficacy of the drugs in the treatment of experimental P388 murine leukemia and some its drug-
resistant strains.
Key words: hydroxamic acids, anticancer drugs, adjuvant effect, combination therapy, murine
leukemia, drug-resistance, in vivo.
Anticancer drugs are known to have severe side effects on
antimetabolites and is associated with the mechanisms of
their action. Multidrug resistance arises simultaneously
against numerous drugs, mainly of natural origin (anthra-
cycline antibiotics, vinca alkaloids, epipodophyllotoxins,
taxol, actinomycin D, colchicine, etc.), in addition to
normal tissues and organs due to their high toxicity, resulting
in that the patients, as a rule, cannot undergo the complete
course of treatment. Therefore, of particular interest are
attempts to reduce the toxic effect of conventional cytostat-
ics by using low-toxic adjuvant compounds based on
metabolically active substances, thereby significantly re-
ducing therapeutic doses of cytostatics (and, consequently,
decreasing their toxicity) with retention of efficacy.
6
a single drug initially administered. The development of
MDR is not related to the mechanisms of action of drugs
but is attributed to amplification (or overexpression) of
closely related genes (mdr) encoding transmembrane
glycoprotein P-gp that mediates energy-dependent efflux
from the cells particularly of those agents, against which
resistance is developed.
Over the years, our research interest has focused on the
synthesis of dicarboxylic acid derivatives and evaluation of
their antitumor activity.1 We have prepared3 oxalic,
tartaric, aspartic, and maleic acid hydroxamides. These
compounds, when used as chemosensitizers in combination
therapy of transplanted tumors with cisplatin or cyclophos-
phamide, completely inhibit B-16 melanoma metastasis
and metastatic Lewis lung carcinoma, resulting in 100%
survival of experimental leukemic animals. We believe that
these compounds can also be effective sensitizers of con-
ventional anticancer drugs in drug-resistant tumors.
Drug resistance is a serious impediment to cancer
chemotherapy because it is developed in response to the
treatment with drugs, to which cancer cells were highly
,2
Many details of the mechanisms of DR and MDR have
been elucidated. Although these data are helpful in finding
7
ways to overcome these types of drug resistance, there
are no strategies for reversing DR and MDR. Therefore,
the development of new treatments of cancer cells with
acquired DR or MDR is a challenging task that has re-
ceived great attention in cancer research.
In this study, we examined the possibility of using
oxalic acid derivatives to increase the efficacy of conven-
tional cytostatics applied in lower doses in experimental
P388 leukemia and its drug-resistant strains in vivo.
4
sensitive at the beginning of the treatment. In the clinics,
the diversity of tumors relapsing after the treatment with
different drugs is due particularly to outgrowth of drug-
resistant cell clones. Currently, there are no drugs, includ-
ing the latest and targeted pharmaceuticals, to which no
resistance is developed.5
Results and Discussion
The goal of this study was to synthesize new oxalic acid
derivatives of the general formula RC(O)—C(O)R, where
R = —NHCH C(O)OMe (1) or —NHCH(Me)C(O)OMe
2
There are two main types of drug resistance — drug
resistance (DR) and multidrug resistance (MDR). Drug
resistance is acquired in response to alkylating agents and
(2) and also (2,2,6,6-tetramethylpiperidin-4-yl)amino (3).
The method for the synthesis of compounds 1—3 is based
on the treatment of oxalyl dichloride with amino acid
methyl ester followed by the separation of oxalylbis(amino
acid) dimethyl ester and its subsequent treatment with an
†
Deceased.
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 4, pp. 0694—0699, April, 2018.
066-5285/18/6704-0694 © 2018 Springer Science+Business Media, Inc.
1

Oxalamide derivatives
Russ. Chem. Bull., Int. Ed., Vol. 67, No. 4, April, 2018
695
C(8A)
C(7A)
C(5)
O(0A)
C(6A)
C(9A)
C(3)
C(4)
C(10A) C(2A)
C(1A)
N(1)
N(2)
C(9)
C(1)
O
N(2A)
N(1A)
C(2) C(10)
C(4A)
C(7)
C(3A)
C(6)
C(8)
C(5A)
OW
Cl
Fig. 1. Overall view of the molecular structure of compound 3 (hydrogen atoms are omitted).
aqueous sodium hydroxide solution in methanol. The
target product was isolated by filtration. It should be noted
that the use of sodium salts ensures good solubility of the
compounds in water, which is necessary for their admin-
istration to animals as aqueous solutions.
tial in order to reduce their high toxicity, which often re-
quires treatment interruption much earlier than necessary.
The results of evaluation of the efficacy of compound 2
in the initial P388 tumor (drug-sensitive) and in a number
of its drug-resistant strains are summarized in Table 1.
As can be seen in Table 1, the use of compound 2 in the
initial P388 strain led to a 109% and 110% increase in me-
dian life span (ILS), when used in combination with cispla-
tin and cyclophosphan, respectively; with etoposide, the
increase in median life span was 137%. In combination with
etoposide, the survival rate of animals increased from 17%
to 50%. In the P388/cPt strain, compound 2 caused a 85%
increase in the efficacy of etoposide; in the P388/CP strain,
the efficacy of doxorubicin (DOX) was enhanced by 61%.
The data on the effect of compound 3 on the efficacy
of therapy with conventional cytostatics of drug-resistant
strains of P388 leukemia are given in Table 2.
As can be seen in Table 2, compound 3 did not
have intrinsic antitumor activity against DR and MDR
strains. However, compound 3 can, in some cases, in-
crease the efficacy of conventional cytostatics. For in-
stance, in the P388/rub strain compound 3 enhanced
the efficacy of mitomycin C by 64%. In the P388/CP
strain, the use of compound 3 in combination with
etoposide resulted in a 43% increase in ILS. In the
P388/cPt strain, compound 3 enhanced the efficacy of
etoposide by 48%.
Compound 3 was prepared as a dihydrochloride salt.
The nitrogen atoms N(2) and N(2A) are protonated
(
Fig. 1). The bond lengths between the N atom and carbons
are 1.528 Å (N(2)—C(4)) and 1.525 Å (N(2)—C(7)). The
nitrogen-containing heterocycle adopts a chair conforma-
tion. The angles between the plane of the central part of
the ring formed by the C(3)C(10)C(4)C(6) atoms and the
planes through the C(3)C(10)C(2) and C(4)C(7)N(2)
atoms are 48.1 and 49.1, respectively. The bond lengths in
the central moiety of molecule 3 are as follows: C(1)—C(1A),
1
.541 Å; C(1)—N(1), 1.337 Å; C(1)—O, 1.229 Å;
N(1)—C(2), 1.456 Å. The crystal structure of compound 3
contains a water molecule of crystallization, the oxygen
atom of which is involved in an intermolecular N—H…O
hydrogen bond having the following parameters:
HN…O (A), 2.261 Å; N(1)…O (A), 2.997 Å; N(1)
W
W
HNO (A), 143.7. The hydrogen atoms of the water
W
molecule of crystallization form contacts with chloride
anions: H (1A)…Cl(A) = 2.536 Å and H (2A)…Cl(B) =
W
W
=
2.448 Å (Fig. 2).
We examined the possibility of using the synthesized
oxalic acid derivatives to increase the efficacy of conven-
tional cytostatics, when applied in lower doses, in experi-
mental P388 leukemia and its drug-resistant strains in vivo.
The evaluation of acute toxicity of the new compounds
in mice demonstrated that compounds 1 and 2 have low
toxicity. The administration in increasing doses up to
O
C(1)
–
1
1
250 mg kg did not lead to death of animals. In this case,
–
1
the therapeutic dose was taken as 400 mg kg . For com-
N(1)
O (A)
HN
–
1
pound 3, LD50 was 138 mg kg and the therapeutic dose
was 46 mg kg 1.
–
W
In combination therapy experiments, the synthesized
compounds were combined with conventional cytostatics, the
doses of the latter being substantially decreased compared
to their therapeutic doses: fivefold for cyclophosphan (CP),
H
W(1A)
HW(2A)
Cl(A)
Cl(B)
6
.8-fold for cisplatin (cPt), 1.5-fold for mitomycin C
(
(
MitC), sixfold for etoposide, and fourfold for doxorubicin
DOX). The use of lower doses of anticancer drugs is essen-
Fig. 2. System of hydrogen bonds formed by a water molecule of
crystallization in the crystal of compound 3.

6
96
Russ. Chem. Bull., Int. Ed., Vol. 67, No. 4, April, 2018
Fedorov et al.
Table 1. Efficacy of compound 2 in P388 leukemia and its drug-resistant strains in vivo in combination
therapy with conventional cytostatics used in lower doses
Agent
Dose
Dosage regimen
(days after transplantation)
Number of
survivals (%)
ILS
(%)
–
1
/
mg kg
Р388
2
cPt
CP
400
0.6
20
1.0
2.5
1.0
1—7
1—7
0
0
28
169
163
145
273
126
278
273
96
1, 6
0
DOX
Etoposide
MitC
2
2
2
2
2
1, 7
0
17
0
0
17
0
50
0
0
1, 5, 9
1, 6
+ cPt
400 + 0.6
400 + 20
400 + 1.0
400 +2.5
400 + 1.0
—
(1—7) + (1—7)
(1—7) + (1, 6)
(1—7) + (1, 7)
(1—7) + (1, 5, 9)
(1—7) + (1, 6)
—
+ CP
+ DOX
+ etoposide
+ MitC
410
108
—
Control
Р388/CP
2
cPt
400
0.6
1—7
1—7
0
0
7
32
89
98
62
32
150
128
75
DOX
Etoposide
MitC
2
2
2
2
1.0
1, 7
0
2.5
1.0
400 + 0.6
400 + 1.0
400 + 2.5
400 + 1.0
—
1, 5, 9
1, 6
(1—7) + (1—7)
(1—7) + (1, 7)
(1—7) + (1, 5, 9)
(1—7) + (1, 6)
—
0
0
+ cPt
0
+ DOX
+ etoposide
+ MitC
17
0
0
0
Control
—
Р38 8/cPt
2
CP
400
20
1—7
1, 6
0
0
0
3
88
200
19
5
90
285
14
—
DOX
Etoposide
MitC
2
2
2
2
1,0
1, 7
0
2.5
1.0
400 + 20
400 + 1.0
400 + 2.5
400 + 1.0
—
1, 5, 9
1, 6
(1—7) + (1, 6)
(1—7) + (1, 7)
(1—7)+(1, 5, 9)
(1—7) + (1, 6)
—
17
0
0
50
0
0
0
+ CP
+ DOX
+ etoposide
+ MitC
Control
Р388/rub
2
400
0.6
1—7
1—7
0
0
0
cPt
CP
MitC
2
2
2
143
230
174
101
126
100
—
20
1, 6
17
0
1.0
1, 6
+ cPt
400 + 0.6
400 + 20
400 + 1
—
(1—7)+(1—7)
(1—7) + (1, 6)
(1—7) + (1, 6)
—
0
+ CP
0
+ MitC
0
Control
0
Compound 1 was tested in four drug-resistant strains
therapy with known cytostatics in very low doses, had, in
some cases, significant adjuvant effect. For instance,
compound 2 in the initial P388 strain increased the efficacy
of cPt, CP, and etoposide by more than 100%. Besides,
in combination with etoposide, the number of surviving
animals increased from 17% to 50%. In two drug-resistant
strains, the adjuvant effect was observed with the use of
compound 2. Thus, in the P388/cPt strain, compound 2
enhanced the efficacy of etoposide by 85%, and it caused
of P388 leukemia in vivo (Table 3).
As can be seen in Table 3, the monotherapy with com-
pound 1 was in general inefficient, but in some cases
compound 1 somewhat enhanced the efficacy of conven-
tional cytostatics.
Therefore, no one of the tested compounds had intrin-
sic antitumor activity in all the tumors used. However
the synthesized compounds, when used in combination

Oxalamide derivatives
Russ. Chem. Bull., Int. Ed., Vol. 67, No. 4, April, 2018
697
Table 2. Efficacy of compound 3 in drug-resistant strains of P388 leukemia in vivo in combination
therapy with conventional cytostatics used in lower doses
Agent
Dose
Dosage regimen
(days after transplantation)
Number of
survivals (%)
ILS
(%)
–
1
/
mg kg
Р388/vcr
3
46
20
0.6
1
46 + 20
46 + 0.6
46 + 1
—
1—7
1, 5
0
0
0
0
0
0
0
0
0
87
90
82
102
91
CP
cPt
MitC
3
3
3
1—7
1, 6
+ CP
(1—7) + (1, 5)
(1—7) + (1—7)
(1—7) + (1, 6)
—
+ cPt
+ MitC
88
—
Control
Р388/rub
3
46
20
1—7
1, 5
0
0
0
0
0
0
0
0
4
119
176
117
121
179
181
—
CP
cPt
Mit.C
3
3
3
0.6
1—7
1
1, 6
+ CP
46 + 20
46 + 0.6
46 + 1
—
(1—7) + (1, 5)
(1—7) + (1—7)
(1—7) + (1, 6)
—
+ cPt
+ MitC
Control
Р388/CP
3
cPt
46
0.6
1—7
1—7
0
0
50
0
0
0
17
0
1
36
DOX
Etoposide
MitC
3
3
3
3
1.0
1, 7
203
112
64
2.5
1
46 + 0.6
46 + 1
46 + 2.5
46 + 1
—
1, 5, 9
1, 6
(1—7) + (1—7)
(1—7) + (1, 7)
(1—7) + (1, 5, 9)
(1—7) + (1, 6)
—
+ cPt
69
+ DOX
+ etoposide
+ MitC
108
155
62
0
0
Control
—
Р388/cPt
3
CP
46
20
1—7
1, 5
0
0
0
0
0
0
0
0
0
0
1
10
DOX
Etoposide
MitC
3
3
3
3
1.0
1, 7
106
127
62
2.5
1
46 + 20
46 + 1
46 + 2.5
46 + 1
—
1, 5, 9
1, 6
(1—7) + (1, 5)
(1—7) + (1, 7)
(1—7) + (1, 5, 9)
(1—7) + (1, 6)
—
+ CP
26
+ DOX
+ etoposide
+ MitC
116
175
59
Control
—
a 61% increase in the efficacy of doxorubicin in the P388/
CP strain. In the P388/rub strain, compound 1 enhanced
the efficacy of CP by 44%; in the P388/cPt strain, it caused
a 40% increase in the activity of DOX. Compound 3 had
adjuvant effect in three drug-resistant tumors: it enhanced
the efficacy of mitomycin C in the P388/rub strain by 64%
and caused a 43% and 48% increase in the efficacy of eto-
poside in the P388/CP and P388/cPt strains, respectively.
Once again, it should be emphasized that in all com-
bination therapy experiments, the known anticancer drugs
were used in doses substantially lower than therapeutic
one. These drugs are commonly known to be highly toxic.
Therefore, their use in lower doses (and consequently, with
lower toxicity) and with high efficacy will make it possible
to increase the duration of treatment and achieve better
therapeutic outcome.
The results of the present study demonstrate that this
line of research holds promise.
Experimental
Elemental analysis was performed on an Euro EA-3000 ana-
1
lyzer. The H NMR spectra were measured on an Avance III
spectrometer (Bruker, 500 MHz).

6
98
Russ. Chem. Bull., Int. Ed., Vol. 67, No. 4, April, 2018
Fedorov et al.
Table 3. Efficacy of compound 1 in drug-resistant strains of P388 leukemia in vivo in combination
therapy with conventional cytostatics used in lower doses
Agent
Dose
Dosage regimen
(days after transplantation)
Number of
survivals (%)
ILS
(%)
–
1
/
mg kg
Р388/vcr
1
CP
cPt
1
1
400
20
1—7
1, 6
0
0
0
0
0
0
0
112
137
87
0.6
1—7
+ CP
+ cPt
400 + 20
400 + 0.6
—
(1—7) + (1, 6)
(1—7) + (1—7)
—
79
Control
—
Р388/rub
1
CP
cPt
1
1
400
20
1—7
1, 6
0
0
0
0
0
0
0
98
210
142
135
—
0.6
1—7
+ CP
+ cPt
400 + 20
400 + 0.6
—
(1—7) + (1, 6)
(1—7) + (1—7)
—
Control
Р388/CP
1
cPt
400
0.6
1—7
1— 7
0
0
0
47
122
128
50
7
84
165
64
—
DOX
Etoposide
MitC
1
1
1
1
1.0
1, 7
0
2.5
1.0
400 + 0.6
400 + 1.0
400 + 2.5
400 + 1.0
—
1, 5, 9
1, 6
(1—7) + (1—7)
(1—7) + (1, 7)
(1—7) + (1, 5, 9)
(1—7) + (1, 6)
—
0
0
+ cPt
0
+ DOX
+ etoposide
+ MitC
0
17
0
Control
0
Р388/cPt
1
CP
400
20
1—7
1, 6
0
0
0
0
0
0
0
0
0
0
3
12
DOX
Etoposide
MitC
1
1
1
1
1.0
1, 7
77
2.5
1.0
400 + 20
400 + 1.0
400 +2.5
400 + 1.0
—
1, 5, 9
1, 6
(1—7) + (1, 6)
(1—7) + (1, 7)
(1—7) + (1, 5, 9)
(1—7) + (1, 6)
—
190
58
12
+ CP
+ DOX
+ etoposide
+ MitC
117
185
47
Control
—
N,N´-Bis(2-methoxy-2-oxoethyl)oxalamide (1). Triethylamine
6.43 mL, 0.19 mol) was added with vigorous stirring to a suspen-
ester hydrochloride (8.35 g) in chloroform (150 mL). The reaction
mixture was cooled to 0 C, and oxalyl dichloride (2.6 mL) was
added. Then the reaction mixture was warmed to ~20 C, stirred
for 1.5 h, transferred into a separatory funnel, and washed with
2
sion of glycine methyl ester hydrochloride (11.92 g, 0.095 mol)
in chloroform (150 mL) at ~20 C. The reaction mixture was
cooled to 0 C, and oxalyl dichloride (6.02 g, 0.048 mol) was
added. Then the reaction mixture was warmed to ~20 C, stirred
for 1.5 h, transferred into a separatory funnel, and washed with
a 2% aqueous solution of NaHCO (75 mL). The aqueous solu-
3
tion was extracted with chloroform (450 mL). The extracts were
combined and dried over MgSO . The chloroform was removed
4
a 2% aqueous solution of NaHCO (75 mL). The aqueous solu-
under reduced pressure. Colorless crystalline compound 2 was
3
tion was extracted with chloroform (450 mL). The chloroform
obtained in a yield of 4.54 g (58%). M.p. 125—126 C. Found (%):
extracts were combined and dried over MgSO . The chloroform
C, 45,95; H, 6.02; N, 11.09. C10H16N O . Calculated (%):
2 6
4
1
was removed under reduced pressure, and colorless crystals
were isolated. The crystals were washed with diethyl ether and
dried in air. The yield of compound 1 was 8.7 g (78%),
m.p. 155—157 C. Found (%): C, 41.34; H, 4.96; N, 12.26.
C, 46.15; H, 6.15; N, 10.76. H NMR (DMSO-d ), : 9.02 (d, 2 H,
6
NH, J = 6.2 Hz); 4.38 (m, 2 H, CH); 3.64 (s, 6 H, OCH ); 1.35
3
(d, 6 H, CH , J = 7.3 Hz).
3
N,N´-Bis(2,2,6,6-tetramethylpiperidin-4-yl)oxalamide di-
hydrochloride (3). Oxalyl dichloride (2.03 g) was added to a solu-
tion of 4-amino-2,2,6,6-tetramethylpiperidine (5 g) in dichlo-
romethane (50 mL). The reaction mixture was stirred for 2 h at
10 C. The colorless precipitate that formed was filtered off,
washed with diethyl ether (20 mL), and dried at ~20 C. Crystals
C H N O . Calculated (%): C, 41.44; H, 5.17; N, 12.07.
8
12
2
6
1
H NMR (DMSO-d ),
d, 4 H, CH , J = 6.2 Hz); 3.65 (s, 6 H, OCH ).

: 9.12 (2 H, NH, J = 6.2 Hz); 3.92
6
(
2
3
N,N´-Bis(1-methoxy-1-oxoprop-2-yl)oxalamide (2). Triethyl-
amine (16.72 g) was added to a suspension of DL-alanine methyl

Oxalamide derivatives
Russ. Chem. Bull., Int. Ed., Vol. 67, No. 4, April, 2018
699
of compound 3 were obtained in a yield of 7.24 g, m.p. 296 C.
According to the X-ray diffraction data, the crystals are com-
pound 3 dichloride containing water of crystallization.
X-ray diffraction study. The unit cell parameters for com-
pound 3 were measured and the three-dimensional X-ray dif-
fraction intensity set was collected on a Kuma-4 automatic dif-
fractometer (Mo-K radiation, graphite monochromator) at
the plot of mortality of animals versus the dose. The therapeutic
dose corresponds to 1/3 of LD50
Dilution of preparations. The synthesized compounds were
dissolved in sterile distilled water. All drugs and compounds were
administered intraperitoneally in a volume of 0.2 mL per 20 g of
body weight.
.
2
93 K. Transparent single crystals of compound 3 (C20H40N O •
4 2
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2
3
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1
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–1
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a single crystal of dimensions 0.220.140.11 mm. The empirical
absorption correction was applied using the Multiscan method.
After rejection of systematic absences and merging of equivalent
3
2
2
reflections, the working set of measured F (hkl) and (F ) con-
sisted of 3749 unique reflections, of which 3299 reflections were
with F2 > 2(F ). The structure was solved by direct methods
4
5
6
2
2
and refined by the full-matrix least-squares method based on F
with anisotropic displacement parameters for nonhydrogen atoms
using the SHELXTL program package. In the crystal structure
8
. J. P. Gillet, M. M. Gottesman, Methods Mol. Biol., 2010,
of the complex, most H atoms were located in difference Fourier
maps. The coordinates and isotropic displacement parameters
of all H atoms were refined by the least-squares method using
5
96, 47—76.
7
. M. Saraswathy, S. Gong, Biotechnol. Adv., 2013, 31, 1397—1407.
8
. G. M. Sheldrik, SHELX-86, Program for the Crystal Struc-
turе Determination, University of Cambridge (England), 1986.
. G. M. Sheldrick, SHELXTL v. 6.14, Structure Determination
Software Suite, Bruker AXS, Madison (Wisconsin, USA), 2000.
9
a riding model. In the final cycle of the full-matrix refinement,
the absolute shifts of all 202 variable parameters were less than
9
0
.001. The final parameters of the refinement were R = 0.037,
1
Rw = 0.13; GOOF 0.613 based on the observed reflections. After
10. Rukovodstvo po provedeniyu doklinicheskikh issledovanii lek-
arstvennykh sredstv [Manual on Preclinical Studies of Drugs],
Part 1, Eds A. N. Mironov, N. D. Bunatyan, Moscow, 2012,
the refinement, the maximum and minimum difference electron
density residuals were 0.531 and –0.267 e А– , respectively.
All structural data were deposited with the Cambridge
Crystallographic Data Centre (CCDC 1564933).
3
9
39 pp. (in Russian).
1
1. E. M. Treshchalina, O. S. Zhukova, G. K. Gerasimova, N. V.
Biological assays. Tests in animals were performed in ac-
cordance with the European Convention for the protection of
vertebrate animals used for experimental and other scientific
Andronova, A. M. Garin, Metodicheskie ukazaniya po izuche-
niyu protivoopukholevoi aktivnosti farmakologicheskikh veshchestv
[
Methodological Guidelines for Study of Antitumor Activity of Phar-
macological Agents] in Rukovodstvo po eksperimental´nomu (do-
purposes (1997) and the guidelines for implementation of pre-
clinical studies of pharmaceutical agents.1
0—13
The P388 murine
klinicheskomu) izucheniyu novykh farmakologicheskikh veshchestv
leukemia was maintained in the DBA/2 mouse line; its
drug-resistant variants — the strains resistant to rubomycine
[
Manual on Experimental (Preclinical) Study of New Pharmaco-
logical Substances], Meditsina, Moscow, 2005, 106 (in Russian).
(
P388/rub), vincristine (P388/vcr), cyclophosphan (P388/CP),
1
2. Rukovodstvo po eksperimental´nomu (doklinicheskomu) izuche-
niyu novykh farmakologicheskikh veshchestv [Manual on
Experimental (Preclinical) Study of New Pharmacological
Substances], Ed. R. U. Habriev, Meditsina, Moscow, 2005,
and cisplatin (P388/cPt) — were maintained in BDF mice by
1
6
intraperitoneal transplantation of 10 cells per animal. Besides,
P388 leukemia was transplanted into BDF mice. The increase
1
in median life span (ILS) served as the activity criterion
8
32 pp. (in Russian).
1
3. M. L. Belen´kii, Elementy kolichestvennoi otsenki farmako-
ILS (%) = [(MLSt – MLSc)/MLSc]•100,
logicheskogo effekta [Elements of Quantitative Assessment of
Pharmacological Effect], Gos. izd-vo med. lit-ry, Leningrad,
where MLSt and MLSc is the median life span (in days) of
treated and control animals, respectively. Each experimental
group contained six—eight animals.
1
963, 146 pp. (in Russian).
1
4. S. A. Goncharova, N. S. Demidova, O. A. Shiryaeva, V. N.
Shevtsova, N. P. Konovalova, Eksperim. Onkologiya [Exp.
Oncology], 1987, 9, 42—47 (in Russian).
Drug-resistant strains of P388 leukemia were produced and
characterized in our previous studies.1
4—16
The P388/rub and
1
5. N. S. Demidova, S. A. Goncharova, O. B. Chernova, B. P.
P388/vcr strains carried MDR phenotype and genotype. Cells
of resistant strains were stored in glass vials in liquid nitrogen.
The vials were thawed when required, and the cells were admin-
Kopnin, A. V. Gudkov, Genetika [Soviet Genetics], 1987, 23,
1797—1806 (in Russian).
1
6. N. S. Demidova, O. B. Chernova, E. Y. Siyanova, S. A.
istered intraperitoneally to BDF mice. The tumors in the third
1
Goncharova, B. P. Kopnin, Somatic Cell and Molecular
Genetics, 1991, 17, 581—590.
transplant generation were used in experiments.
The general toxicity of the tested compounds was evaluated
in BDF hybrid mice after a single intraperitoneal administration
Received May 18, 2017;
in revised form July 28, 2017;
accepted December 20, 2017
1
at different doses. The doses that cause death in 50% and 100%
of animals (LD50 and LD100, respectively) were determined from