Antitumor activity of actinocin-based lipid intercalators

Full text of DOI:10.1023/A:1005266521565

Pharmaceutical Chemistry Journal
Vol. 34, No. 7, 2000
SEARCH FOR NEW DRUGS
ANTITUMOR ACTIVITY OF ACTINOCIN-BASED
LIPID INTERCALATORS
I. S. Golubeva,1 I. Yu. Kubasova,1 N. P. Yavorskaya,1 E. N. Glibin,2 and E. B. Popova1
Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 34, No. 7, pp. 3 – 5, July, 2000.
Original article submitted November 30, 1999.
In the previous communication [1] we demonstrated that
the class of actinocin derivatives includes compounds pos-
sessing antitumor activity, the effect of which in tests on ani-
mals bearing model tumors was comparable with that of
actinomycin D. The results of that investigation showed that
the antitumor activity in the series of compounds studied de-
pends both on the ability to complex formation with DNA
and on the presence of lipophilic-hydrophilic mobility in the
molecules.
and octadecyl (IV)] in the amide group of the quinoid ring.
Differing in length, the substituents account for different
lipophilicities of the synthesized compounds [4].
(CH2)3N(CH3)
2 · HCl
Alk
NH
CO
NH
CO
NH2
N
O
O
Earlier, Denny et al. [2] also concluded that the balance
of lipophilic-hydrophilic properties is one of the factors de-
termining the antitumor properties of acridine derivatives.
Behr [3] synthesized an acridine-based compound, contain-
ing a cationoid center and an aliphatic radical. He demon-
strated that the presence of these fragments in the molecule
makes it capable of intercalating into DNA and modifying
the DNA properties as a result of the aliphatic fragment
“sticking” in the drug – DNA complex, for which reason the
compound was classified as a lipid intercalator. Unfortu-
nately, the biological properties of such compounds were
never reported.
The presence of two nonequivalent carboxy groups in the
actinomycin D molecule implies the possibility to introduce
substituents of various types into amides of actinocin and its
derivatives. For example, by introducing an alkyl radical into
one amide group and a substituent with cationoid center into
another amide group we may synthesize compounds with
properties analogous to those of the lipid intercalator de-
signed in [3]. We aimed at studying the effects of lipophilic
fragments and cationoid groups (imparting hydrophilic prop-
erties to a molecule) in DNA complexes. For this purpose,
we have synthesized a series of 6-demethylactinocin diami-
des (with 4-methylcinnabarinic acid) containing various
alkyl substituents [butyl (I), cyclohexyl (II), dodecyl (III),
CH3
I – IV
Alk = n-C4H9 (I); cyclo-C9H11 (II); n-C12H25 (III); n-C18H37 (IV).
In order to study the dependence of the antitumor proper-
ties on the positions of the alkyl substituent and the radical
with cationoid center, we have also synthesized compound V,
in which the alkyl radical is situated (in contrast to com-
pound II) in the quinoid ring. This compound, as well as
compounds I – IV [4], was synthesized by mixed oxidative
condensation of a 3-hydroxyanthranilic acid derivative (VI)
and a 3-hydroxy-4-methylanthranylic acid derivative (VII).
The latter compound was obtained by the catalytic hydroge-
nation of N-cyclohexyl-2-nitro-3-hydroxy-4-methylbenza-
mide hydrochloride [6].
The accompanying “symmetric” derivatives of cin-
nabarinic acid (VIII, cf. [5]) and actinocin (IX) were sepa-
rated from the target compound V using the difference in
their solubilities in water and organic solvents. The proposed
structure of compound V was confirmed by the absence of a
signal characteristic of protons of the 6-CH3 group in the 1H
NMR spectrum (see data reported in [5] and in the experi-
mental chemical part below) and by the presence of absorp-
tion characteristic of cinnabarinic acid and actinocin [5] in the
electronic absorption spectrum. The corresponding character-
istics were also obtained for the “symmetric” derivative IX.
1
Blokhin Oncological Research Center, Russian Academy of Medical Sci-
ences, Moscow, Russia.
2
St. Petersburg State Technological Institute (Technical University), St. Pe-
tersburg, Russia.
339
0091-150X/00/3407-0339$25.00 © 2000 Kluwer Academic/Plenum Publishers

340
I. S. Golubeva et al.
HCl · (CH3)2N
(CH2)3
drogen at room temperature and atmospheric pressure in the
presence of 0.2 g of 10% Pd on charcoal. The formation of
aminophenols VI and VII was monitored by TLC in systems
A and B. Upon completion of the reduction process, the cata-
lyst was separated by filtration and the filtrate was mixed by
stirring at room temperature with 0.972 g of p-benzoquinone
in 10 ml of methanol. The mixture was stirred at room tem-
perature for 2 h and allowed to stand without stirring for
15 h. The precipitate (0.38 g), separated by filtration after the
mixture standing and identified by TLC in systems A and B
as compound IX, was washed with ethanol and ether. The fil-
trate was evaporated to half volume, after which an addi-
tional amout of precipitated compound IX was obtained by
filtration. The residual filtrate was diluted with 150 ml of ab-
solute ether and allowed to stand for 16 h without stirring.
The precipitate was separated by filtration and dissolved in
50 ml of water. The aqueous solution was filtered through a
thin charcoal layer, saturated with NaCl to a concentration of
100 – 110 g/liter, and extracted with chloroform to obtain
compound V (the aqueous layer analyzed by chromatogra-
phy showed the presence of compound VIII). The chloro-
form extract containing compound V was doubly washed
with a 100 – 110 g/liter aqueous NaCl solution, evaporated
in vacuum to a residual volume of 10 – 15 ml, diluted with
100 – 150 ml of anhydrous ether, and filtered to obtain 0.389 g
of compound V (chromatographically homogeneous in sol-
vent systems A and B) in the form of finely crystalline pow-
NH
CO
NH
CO
NH2
OH
NH2
OH
+
CH3
VI
HCl · (CH3)2N
(CH2)3
VII
NH
CO
NH
CO
NH2
O
N
O
CH3
V
N(CH3)
(CH2)3
2 · HCl
HCl · (CH3)2N
(CH2)3
NH
CO
NH
CO
NH
CO
NH
CO
NH2
O
N
N
O
NH2
O
O
1
CH3
CH3
VIII
der; m.p., > 230°C; H NMR spectrum (d, ppm): 7.56 (m,
IX
3H, Harom), 2.85 (s, 6H, N+(CH3)2), 2.12 (s, 3H, 4-CH3), plus
several multiplets in the region of 1 – 3 ppm attributed to
methylene and methine protons of the propyl and cyclohexyl
radicals; UV spectrum in EtOH, lmax (log e): 239 (4.53), 445
(4.42); C26H33N5O4 × HCl.
Below we report on a comparative study of the antitumor
properties of compounds I – V, the previously reported
N,N¢-di(3-dimethylaminopropyl)actinocyldiamide (compo-
und X), and actinomycin D.
1,9-Di(cyclohexylcarbamoyl)-2-amino-4,6-dimethyl-3H-
3-oxophenoxazine (IX). N-cyclohexyl-2-nitro-3-hydroxy-
4-methylbenzamide (0.28 g) [6] in 10 ml of methanol was
hydrogenated at room temperature and atmospheric pressure
in the presence of palladium black. The reduction process
completion was monitored by hydrogen absorption and by
TLC in solvent systems A and B. Upon completion of the re-
duction process, the catalyst was separated by filtration and
the filtrate was mixed by stirring at room temperature with
0.162 g of p-benzoquinone in 3 ml of methanol. The mixture
was allowed to stand at room temperature for 18 h and fil-
tered to separate the precipitate of compound IX (0.23 g);
EXPERIMENTAL CHEMICAL PART
The UV spectra were recorded with a Specord UV-VIS
1
spectrophotometer (Germany). The H NMR spectra were
measured on a Tesla BS-497 100-MHz spectrometer (Czech
Republic) using freshly prepared solutions in trifluoroacetic
acid with TMS internal standard. The melting temperatures
were determined using an HMK heating table of the Koefler
type. TLC analyses were conducted on Silufol UV-254
plates eluted in the systems chloroform – methanol – 25%
aqueous ammonia, 8 : 2 : 0.2 (A), or 25%-aqueous-ammo-
nia-saturated chloroform – methanol, 2 : 1 (B).
The data of elemental analyses (C, H, N) for compounds
V and IX coincide with the results of analytical calculations.
The physicochemical parameters of the synthesized com-
pounds I – IV agree with the published data [4].
1
m.p., > 230°C (chloroform – benzene); H NMR spectrum
(d, ppm): 7.38 (m, 1H, 8-H), 7.18 (1H, J 8 Hz, 7-H), 3.86 (m,
2H, CH), 2.46 (s, 3H, 6-CH3), 2.17 (s, 3H, 4-CH3), 1.93 m,
1.69 m, 1.25 m (20H, CH2); UV spectrum in EtOH, lmax
(log e), nm: 245 (4.58), 445 (4.45); C28H34N4O4.
1-(N-Cyclohexylcarbamoyl)-9-(3-dimethylaminopro-
pyl)carbamoyl-2-amino-4-methyl-3H-3-oxophenoxazine-
hydrochloride (V). A mixture of 0.84 g of N-cyclohexyl-2-
nitro-3-hydroxy-4-methylbenzamide [6] and 1.18 g of
N-(3-dimethylaminopropyl)-2-nitro-3-hydroxybenzamide
hydrochloride [5] in 30 ml of methanol was reduced by hy-
EXPERIMENTAL BIOLOGICAL PART
The antitumor activity was studied using a method devel-
oped at the Blokhin Oncological Research Center [7]. The
experiments were performed on first-generation BDF1 hy-

Antitumor Activity of Actinocin-Based Lipid Intercalators
341
TABLE 1. Antitumor Activity of Lipid Intercalators I – V,
Actinomycin D, and Compound X in Mice with Ca-755 Model
Adenocarcinoma
TABLE 2. Antitumor Activity of Actinocin Derivatives
Single
dose,**
mg/kg
TGI, % (24 h after last drug injection)
SLI, %
Com-
pound
Ca-755
LLC
B-16
RShM-5
P-388
TGI, %
Single
I
120
120
53 – 64
84*
20
48 – 53
–
83*
n/a***
n/a
Compound
dose,**
days after tumor inoculation:
SLI, %
mg/kg
II
34 – 41
79 – 93*
7
10 – 11 13 – 14 17 – 18
III
IV
V
X
30 96 – 98* 76 – 84* 66*
82*
–
n/a
I
120
120
30
64*
84*
98*
46
49
26
25
24
42
9
–
38
39
–
80
46
–
–
21
–
n/a
II
88*
99*
14
30
120
83*
–
n/a
III
IV
V
X
85*
0
30 68 – 89* 40 – 54 65 – 75* 64 – 75* 13 – 20
80
Actinomy- 0.04 81 – 98* 63 – 64* 68 – 75* 75 – 77* 38 – 81
cin D
120
30
83*
89*
96*
93*
75*
93*
74*
56*
81*
65*
23
52*
–
21
36
*
Actinomycin D 0.04
Reliable difference from control ( p < 0.05).
Treatment schedule: single daily injection over a period of
**
*
Reliable difference from control ( p < 0.05).
Treatment schedule: single daily injection over a period of
5 days.
***
n/a = no antitumor activity.
**
5 days.
In the tests on solid tumors, the maximum effect was also
produced by compound III (Table 2), the TGI values of
which for Ca-755, LLC, RShM-5, and B-16 were compara-
ble with that of actinomycin D. Compound II (with
cyclohexyl radical in position 9 of the amide group) pro-
duced the most pronounced effect with respect to the model
cervical carcinoma RShM-5.
None of the compounds tested, as well as the previously
studied actinocin derivatives [1], was effective in the treat-
ment of lymphocyte leukemia P-388; compounds I and II
produced no protective effect in mice inoculated with Lewis
lung carcinoma (Table 2).
brid mice inoculated with mammary adenocarcinoma
Ca-755, which is the most sensitive model for both
actinomycin D (XI) and actinocin derivatives [1].
The spectrum of antitumor action was determined on a
series of transferred tumors, including lymphocyte leukemia
P-388, Lewis lung carcinoma (LLC), melanoma B-16, and
cervical carcinoma RShM-5. The lymphocyte leukemia
P-388 was inoculated by intraperitoneally injecting 106 tu-
mor cells with a 0.5 ml volume of a type 199 nutrient me-
dium. The solid tumors were subcutaneously inoculated in
the right axillary region (0.5 ml of a 10% tumor cell suspen-
sion in the same medium). The treatment was started 24 after
intraperitoneal injections and 48 h after subcutaneous inocu-
lation of the solid tumors. The drug solutions were prepared
ex tempore and intraperitoneally injected in a range of doses.
The drug efficacy was evaluated by calculating the per-
centage tumor growth inhibition (TGI) for 7, 10 – 11,
13 – 14, and 16 – 17 days after the tumor transfer and by de-
termining the survival lifetime increase (SLI) for the test
mice with leukemia relative to the untreated control. Com-
pounds inhibiting tumor growth by not less than 50% and/or
increasing the lifetime by more than 25% were classified as
possessing antitumor activity.
Thus, the antitumor activity of the drugs studied is re-
lated to a certain extent to the hydrophilic-lipophilic proper-
ties of these compounds.
ACKNOWLEDGMENTS
This work was supported by the Russian Foundation for
Basic Research (project No. 99–03–33–044) and by the
INTAS Foundation (grant No. 97–31753).
The toxicity of the tested compounds was judged by the
number of lost animals, the macroscopic morphology pattern
of the internal organs, and the gain in total body and spleen
weights.
REFERENCES
1. N. P. Yavorskaya, I. Yu. Kubasova,
E. N. Glibin, Khim.-Farm. Zh., 30(12), 22 – 26 (1996).
I. S. Golubeva,
and
2. W. A. Denny, B. F. Cain, G. J. Atwell, et al., J. Med. Chem.,
25(3), 276 – 315 (1982).
RESULTS AND DISCUSSION
3. J. P. Behr, Tetrahedron Lett., 48, 5861 – 5864 (1986).
4. E. N. Glibin and E. B. Popova, Zh. Org. Khim., 35(3), 408 – 411
(1999).
As seen from the data presented in Table 1, all the stud-
ied compounds (except for derivative IV) injected in tolera-
ble doses suppressed the growth of adenocarcinoma Ca-755.
The maximum antitumor action (in some experiments, com-
parable with or even exceeding that of actinomycin D) was
observed for derivative III (with dodecyl radical in position 9
of the amide group).
5. Z. I. Korshunova,
O. F. Ginzburg, Zh. Org. Khim., 27(2), 369 – 376 (1991).
6. E. N. Glibin, B. V. Tsukerman, S. S. Tsymbalova,
O. F. Ginzburg, Zh. Org. Khim., 13(2), 462 – 463 (1977).
E. B. Popova,
E. N. Glibin,
and
and
7. Z. P. Sof’ina, Chemotherapy of Tumor Disorders [in Russian],
Meditsina, Moscow (1984), pp. 11 – 42.