Synthesis, cytotoxicity, antiviral activity and interferon inducing ability of 6-(2-aminoethyl)-6H-indolo[2,3-b]quinoxalines

Article abstract of DOI:10.1016/j.ejmech.2009.12.014

New 6-(2-aminoethyl)-6H-indolo[2,3-b]quinoxalines were synthesized with high yields using bromoethylisatin and 6-(2-bromoethyl)-6H-indolo[2,3-b]quinoxaline as intermediates. These compounds were screened for the cytotoxicity, antiviral activity and interferon inducing ability. It was shown, that tested 6-(2-aminoethyl)-6H-indolo[2,3-b]quinoxalines are low toxic potent interferon inducers and antivirals. Morpholine and 4-methyl-piperidine derivatives appeared as the most active antivirals and the least cytotoxic in the investigated series.

Full text of DOI:10.1016/j.ejmech.2009.12.014

European Journal of Medicinal Chemistry 45 (2010) 1237–1243
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis, cytotoxicity, antiviral activity and interferon inducing ability
of 6-(2-aminoethyl)-6H-indolo[2,3-b]quinoxalines
,
b
,
a
a,b
Marina O. Shibinskaya a, Sergey A. Lyakhov a , Alexander V. Mazepa , Sergey A. Andronati
,
*
Alexander V. Turov c, Nadezhda M. Zholobak d, Nikolay Ya. Spivak d
a A.V. Bogatsky Physico-Chemical Institute of NAS of Ukraine, Lyustdorfskaya doroga, 86, Odessa 65080, Ukraine
b Odessa National I.I. Mechnikov University, Dvoryanskaya str., 2, Odessa 65026, Ukraine
c National Taras Shevchenko University of Kyiv, Volodymyrska str., 64, Kyiv 01033, Ukraine
d D.K. Zabolotny Institute of microbiology and virology of the NAS of Ukraine, Zabolotnogo str., 154, Kiev 03143, Ukraine
a r t i c l e i n f o
a b s t r a c t
Article history:
New 6-(2-aminoethyl)-6H-indolo[2,3-b]quinoxalines were synthesized with high yields using bromoe-
thylisatin and 6-(2-bromoethyl)-6H-indolo[2,3-b]quinoxaline as intermediates. These compounds were
screened for the cytotoxicity, antiviral activity and interferon inducing ability. It was shown, that tested
6-(2-aminoethyl)-6H-indolo[2,3-b]quinoxalines are low toxic potent interferon inducers and antivirals.
Morpholine and 4-methyl-piperidine derivatives appeared as the most active antivirals and the least
cytotoxic in the investigated series.
Received 23 June 2009
Received in revised form
30 November 2009
Accepted 3 December 2009
Available online 28 December 2009
Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords:
Indolo[2,3-b]quinoxaline
Synthesis
Antiviral activity
Interferon
1. Introduction
H3C
CH3
Interferons (IFN) are ones among the most broadly used drugs
for the human viral diseases treatment [1–4]. Though, side effects
and sometimes low effectiveness are occurred, especially while
long-term IFN introduction [2,5,6]. Inductors of the endogenous IFN
are seemed as a good alternative for the exogenous IFN using [7].
Some of such inductors are widely used in the clinical practice.
Thus, Imiquimod (1) as 5% cream is used for the viral and cancer
diseases treatment [8]. Tilorone (2) [9,10] as Amixine [11] and
carboxymethylacridone derivatives (3a and 3b) as Cycloferon
and Neovir [7] were proved in Ukraine and Russia as a highly active
and tolerant human drugs for the prophylactics and treatment of
different acute respiratory viral diseases, hepatitis (A and B), HSV-
diseases, etc.
N
N
NH+
H3C
HN+
CH3
O
O
H3C
CH3
Cl
O
Cl
N
NH2
2
1
O
N+
H2
O
O
X+
H3C
OH
N
3a
3b
HO
OH
, where X =
OH
Na+
O
3
Tilorone’s ability to the DNA intercalation attributed to the
presence of planar polycyclic system [12–14] is seemed as a main
cause of it’s IFN-inducing ability and antiviral action [15,16].
Furthermore, we speculated [16] that these properties are general
* Corresponding author. A.V. Bogatsky Physico-Chemical Institute of NAS of
Ukraine, Lyustdorfskaya doroga, 86, Odessa 65080, Ukraine. Tel.: þ38 48 765 51 26;
fax: þ38 48 765 96 02.
E-mail address: Sergey_A_Lyakhov@ukr.net (S.A. Lyakhov).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.12.014

1238
M.O. Shibinskaya et al. / European Journal of Medicinal Chemistry 45 (2010) 1237–1243
ones of the DNA intercalators. Parallelism of the intercalating ability
on the one hand and IFN induction and antiviral activity on the
other one demonstrated for fluorenes [15,17], dibenzofurans
[18,19], anthraquinones [20], 9-aminoacridines [16,21], and many
other compounds argue for this speculation validity.
Purity of all synthesized compounds was controlled by thin-
layer chromatography on pre-coated silica gel F254 plates using
eluents of different composition.
The structure of the synthesized compounds was proved by
mass-spectrometry, IR spectroscopy and NMR-spectroscopy.
Molecular ions peaks are present in electron impact mass-spectra
and in mass-spectra with ionization method of fast atom
bombardment (FAB). Fragment ions set correspond to suggested
structures. Vibrations of aromatic C–H bonds are observed in IR
spectrums of compounds 8–19 at 3064–3055 cmꢀ1, aliphatic –
2953–2773 cmꢀ1. Double bonds vibrations of heterocyclic frag-
ments exhibit a band set at 1631–1440 cmꢀ1. The bands at 1240–
10600 cmꢀ1 are corresponds to CH2–N vibrations.
Two ‘‘parts’’ corresponded to aromatic and aliphatic parts of
molecule are observed in 1H NMR-spectra of synthesized indolo-
quinoxalines derivatives. The ‘‘aliphatic parts’’’ spectrums are
different according terminal amino-group structures and corre-
spond to them. On the contrary, differences in the ‘‘aromatic parts’’’
of spectra are much less manifest. The complex multiplet with
integral intensity 3H and five separate signals with integral inten-
sity 1H each are present in ‘‘aromatic’’ fragments of spectrum. All
detailed information is present in supplement. Details on mass-
spectra and NMR signal assigning one can find in the supplement.
Indoloquinoxalines are ones among DNA intercalators possess-
ing highly defined anti-herpesvirus activity [22]. In respect to the
mentioned above, we should wait that these compounds can
demonstrate activity not only against herpes simplex, but against
other viruses also, as well as interferon inducing properties. Coin-
cidence in almost of the tilorone and 2,3-dimetyl-6(2-dimethyla-
minoethlyl)-6H-indolo[2,3-b]quinoxalin (B-220, 4) activity types
pays attention to itself: inhibition of Herpesviridae species’ repro-
duction [9,22], inhibition of the spontaneous peroxide oxidation of
lipids (POL) [23,24], antimutagenic action [25–27], antitumoral
activity [28–30]. It is unlike, that such coincidence is occasional.
Therefore we have supposed that indoloquinoxaline derivatives
have also other properties like to tilorone, namely, the anticarci-
nogenic activity [26], IFN induction [31–33] and inhibition of
cytochrome P-450 [34].
According above we have attempted the aminoethyl-6H-
indolo[2,3-b]quinoxalines’ (9–20) synthesis, and studying of their
titled properties and SAR.
2.2. Biological activity
CH3
In vitro cytotoxicity, antiviral and IFN-inducing activities of all
synthesized compounds 8–19 were tested using both murine
fibroblasts (L929) and transferable piglet testicular (EPT) cells.
Antiviral and IFN-inducing activities were determined against
vesicular stomatitis virus (VSV). The obtained results are summa-
rized in Tables 1–3.
N
CH3
N
N
As shown in Table 1 most of the synthesized 6H-indolo[2,3-
b]quinoxaline derivatives showed low cytotoxicity in comparison
with tilorone, their cytotoxicity for both cell lines differ insig-
nificantly P > 0.05, using Van der Waerden’s nonparametric
(distribution-free) test X criteria [36,37]. The compounds with
morpholine (19) and 4-methyl-piperidine (16) as terminal
amino-group appeared as nontoxic (or slightly toxic) up to
N
CH3
4
B220
H3C
2. Results and discussion
2.1. Chemistry
maximum test concentration of 1000
mM using both cell lines. On
the contrary, pyrrolidine derivative (13) appears as the most
Targeted compounds were synthesized via 3-step procedure
(Scheme 1) starting from isatin (5) through it’s 1-bromoethyl-
derivative (6) and 6-(2-bromoethyl)indoloquinoxaline (7) as
intermediates. Final amination using of corresponding amines
access leads us to aminoethyl-indoloquinoxalines (8–19).
cytotoxic. Cytotoxic concentration (CC50) of this compound was
found as 76 mM against L929 cells.
All synthesized compounds 8–19 demonstrate significant anti-
viral activity (Table 2) against vesicular stomatitis virus (VSV) using
both cell lines. Their activity using L929 is higher (P < 0.01) than the
same using EPT cell line for both preventive (n ¼ 24, X ¼ 6.31 vs
Xt ¼ 5.55) and therapeutic (n ¼ 23, X ¼ 6.55 vs Xt ¼ 5.40) regimen of
injection.
It should be noted that antiviral activity of the 6H-indolo[2,3-
b]quinoxaline derivatives is higher (P < 0.01) when the tested
compounds were added to the cell monolayer 24 h before virus
infection (preventive regimen) than they were added immediately
after virus infection (therapeutic regimen) for both L929 (n ¼ 24,
X ¼ 8.11 vs Xt ¼ 5.55) and EPT (n ¼ 23, X ¼ 7.19 vs Xt ¼ 5.40). This is
in accordance with an interferon-mediated mechanism of viral
inhibition. Compound (12) contained ethyl-butyl-amine as
a terminal amino-group, becomes as exception – its antiviral action
was identical in both cases.
1-(2-Bromoethyl)-indole-2,3-dion (6) was synthesized via isatin
alkylation by excess of dibromoethane in DMF at room temperature
in the presence of potassium carbonate. Potassium carbonate using
instead of sodium hydride as described previously [35] leads both
to the procedure simplifying and yield increasing up to 80%. The
separation of the solution from the inorganic precipitate, the
evaporation of the solution to dryness and the re-crystallization of
the residue from the ethanol results in chromatographically pure 6
with 80% yield. Further condensation of 6 with 1,2-diaminobenzene
under the boiling in acetic acid lead to the 6-bromoethyl-6H-
indolo-[2,3-b]quinoxaline. Chromatographically homogenous
product (with respect to the admixtures of the aromatic nature)
falls out directly from the reacting mixture under the cooling down
to room temperature. In some cases, when precipitated product
contained uncertain admixtures, it was recrystallized from the
glacial acetic acid or ethanol yielding 80%.
Compounds 9–18 when injected 24 h before virus to the L929
monolayer (preventive regimen) demonstrate activity similar to
tilorone – no significant difference between their ꢀlg IC50 values
and the same one of tilorone is occurred. While EPT culture used,
compounds 9–15 and 17 appeared less active then tilorone while
compounds 8, 16, 18, 19 demonstrated activity similar to tilorone.
The compounds 9–19 were obtained by aminodebromination of
7 by excess secondary amines in boiling benzene. In contrast to
them, compound 8 was obtained at room temperature in DMF.

M.O. Shibinskaya et al. / European Journal of Medicinal Chemistry 45 (2010) 1237–1243
1239
o-phenylene-
diamine
O
O
N
Br
Br
K2CO3, DMF
CH3COOH,
boiling, 4 h
80 %
N
O
O
N
H
N
N
r.t., 2 h
80 %
5
6
Br
Br
7
RH
N
benzene, boiling, 6 - 8 h;
DMF, r.t.
N
N
80 - 90 %
R
8 - 19
CH3
CH3
CH3
CH3
CH3
CH3
(8)
;
(10)
;
N
N
CH3
CH3
N
N
R =
(9);
(11); N
(16); N
CH3 (12);
N
CH3
CH3
H3C
N
N
(13) N
;
(14)
;
(15);
(17);
N
CH3
O
(19)
(18);
N
N
Scheme 1.
Compound 12 appears as the least active one among investi-
gated in both cases.
prophylactic and therapeutic regimens) appear as the most active
IFN inducers exceeding tilorone by this property in the L929 cell
line.
The selectively index (SI) value as the integral parameter of the
antiviral effectiveness was determined as the ratio of the CC50 to
the IC50 (SI ¼ CC50/EC50). Compounds 16 and 19 appeared as potent
antivirals and low cytotoxic. Thus, the SI (Table 4) for compounds 16
and 19 was 1783 and 1258, respectively, which is 20 and 15 times
larger than the same for tilorone. Generally, SI of compounds for
L929 and EPT cell lines don’t differ significantly (P > 0.05) neither
Difference in activity of the tested compounds is seemed as
more prominent when injected in therapeutic regimen. In general,
in this case the most of indoloquinoxalines appeared more active
then tilorone using both cell cultures. Compounds 13 and 15
demonstrate the maximal antiviral activity among others.
All synthesized 6H-indolo[2,3-b]quinoxaline derivatives
exhibited interferon inducing activity (Table 3), which different
insignificantly (P > 0.05) using the EPT cells vs L929. The
compounds 9, 13, 14 and 17 induced highest titers of interferon. It’s
have been noted, that active therapeutic agents induce as high
interferon titers as tilorone or larger. On the other hand,
compounds 14 and 17 (not the most active antivirals in both
Table 2
Antiviral activity of synthesized 6H-indolo[2,3-b]quinoxaline derivatives.
Compound L929
TEa
EPT
PEb
TE
PE
Table 1
ed ꢀlg IC50
ꢁ
e
ꢀlg IC50
ꢁ
e
ꢀlg IC50
ꢁ
e
c
Cytotoxicity of synthesized 6H-indolo[2,3-b]quinoxaline derivatives.
ꢀlg IC50
4.95
ꢁ
8
0.08 5.84
0.05 5.89
0.12 5.89
0.07 5.84
0.05 5.50
0.04 5.88
0.06 5.87
0.03 5.90
0.11 5.79
0.09 5.89
0.09 5.86
0.10 5.79
0.06 5.84
0.12 5.02
0.05 5.21
0.06 5.18
0.09 5.32
0.10 4.88
0.05 5.39
0.03 5.25
0.07 5.39
0.09 4.70
0.06 5.10
0.06 5.84
0.04 5.47
0.10 5.57
0.13 5.52
0.04 4.88
0.02 5.57
0.09 5.54
0.05 5.58
0.11 5.80
0.12 5.57
0.04 5.84
0.12
0.04
0.06
0.11
0.10
0.06
0.03
0.07
0.10
0.05
0.10
0.13
0.08
Compound
L929
EPT
9
10
11
12
13
14
15
16
17
18
19
Tilorone
5.66
5.45
5.63
5.47
5.65
5.59
5.72
5.64
5.42
5.32
5.35
4.86
eb
ꢀlg CC50
ꢁ
e
a
ꢀlg CC50
ꢁ
8
9
10
11
12
13
14
15
16
17
18
19
4.04
3.19
3.66
3.25
3.76
4.12
3.75
3.63
3.24
3.84
4.01
2.97c
3.95
0.05
0.03
0.05
0.06
0.04
0.06
0.03
0.04
0.05
0.09
0.02
–
4.09
3.34
3.83
3.69
4.08
3.79
3.03
3.4
0.02
0.02
0.02
0.07
0.09
0.06
0.02
0.03
–
0.10 5.33
e
2.55c
3.88
4.09
2.69c
3.97
0.13
–
–
5.79
0.06 5.88
0.05
0.03
–
0.08 4.87
a
The tested compounds were added to cell monolayer immediately after virus
infection (TE – "therapeutic effect").
Tilorone
0.03
0.04
b
The tested compounds were added to cell monolayer 24 h before virus infection
a
ꢀlg CC50 – negative common logarithm of tested compound concentration (M)
(PE – "preventive effect").
c
which leads to 50% cell monolayer destruction.
ꢀlg IC50 – negative common logarithm of the tested compound concentration
b
Confidence interval value (ꢁ
e
) was calculated using P < 0.05.
(M) which lead to 50% cytopathic action of VVS inhibition.
c
d
ꢀlg CC50 value determination was failed (the compound in all investigated
Confidence interval values (ꢁ
e
) were calculated using P < 0.05.
ꢀlg IC50 value was failed to determine (the compound in all investigated
concentrations prevents less than 50% of VSV cytopathic action).
e
concentration range lead to cell monolayer destruction less than 50% cell);
extrapolated value is given.

1240
M.O. Shibinskaya et al. / European Journal of Medicinal Chemistry 45 (2010) 1237–1243
Table 3
4. Experimental
IFN-inducing activity of synthesized 6H-indolo[2,3-b]quinoxaline derivatives.
4.1. Chemistry
Compound
L929
EPT
T IFN at C ¼ 1.1 ꢃ 10ꢀ6 Ma
16
64
8
16
8
32
64
32
8
T IFN at C ¼ 1.5 ꢃ 10ꢀ6 M
Melting points are uncorrected. The 1H NMR-spectra (300 MHz)
of all compounds were recorded on a ‘‘Varian VXR-300’’ spec-
trometer in CDCl3 solution using TMS as an internal standard. COSY
and NOESY spectra were recorded using a ‘‘Varian Mercury-400’’
spectrometer. The mass-spectra of electron impact were recorded
using MX-1321 spectrometer with straight sample introduction.
The ionization energy of electrons was 70 eV, the source temper-
ature was 220 ꢂC. The mass-spectra with the fast atom bombard-
ment (FAB) ionization were recorded using VG 70-70 EQ
spectrometer. Ionization was realized using beam of argon-atoms
with energy 10 kV (the compounds were dissolved in 3-nitrobenzyl
alcohol). Thin-layer chromatography was performed on pre-coated
silica gel F254 plates (Merck).
8
9
10
11
12
13
14
15
16
17
18
19
32
32
32
32
8
64
32
32
32
32
32
16
32
64
4
8
Tilorone
32
a
T IFN – titer of the induced IFN – maximal IFN-containing cultural medium
dilution, under which it’s ability to the cytopathic action of VSV inhibition remains.
4.1.1. 1-(2-Bromoethyl)-1H-indole-2,3-dione (6)
Table 4
Potassium carbonate (18.9 g, 0.102 mol) was added to a stirred
solution of 1H-indole-2,3-dione (10 g, 0.068 mol) in 40 mL DMF,
then 1,2-dibromo-ethane (255.5 g, 1.36 mol, 117.2 mL) was added.
After stirring at room temperature for 2 h, the inorganic precipi-
tate was filtered and washed with DMF 3 ꢃ 5 mL. Filtrate was
evaporated under reduced pressure; the residue was washed with
water and filtered. The product was purified by crystallization
from ethanol. Yield: 80% (13.8 g); m.p. 132.8–133.4 ꢂC (lit. [35]
m.p. 131.0–132.0 ꢂC from ethanol]). MW. 254.08. Mass-spectrum
(electron impact) – m/z (I, %): 255 (21); 253 (20); 146 (100); 132
Selectivity index value of synthesized 6H-indolo[2,3-b]quinoxaline.
Compound
L929
EPT
SI (TE)a
SI (PE)
SI (TE)
SI (PE)
8
9
10
11
58
79
158
41
63
499
169
388
54
9
56
139
55
68
6
76
23
43
6
12
27
13
14
15
16
17
18
19
Tilorone
468
224
74
78
62
46
17
10
59
40
164
98
142
17
17
–
60
132
185
358
110
71
323
149
1783
49
57
1258
80
(55); 90 (7); 77 (10). 1H NMR (CDCl3)
d: 3.601 (t, 2H, BrCH2CH2N,
J ¼ 6.9 Hz); 4.136 (t, 2H, BrCH2CH2N, J ¼ 6.9 Hz); 7.007 (d, 1H,
arom, J ¼ 8.4 Hz); 7.134 (t, 1H, arom, J ¼ 7.5 Hz); 7.577–7.627 (m,
2H, arom). Rf 0.43 (benzene–triethylamine 10:1); Rf 0.63 (chloro-
form–acetone 10:1).
659
80
10
a
SI – selectivity index calculated as the ratio of CC50 to IC50
.
4.1.2. 6-(2-Bromoethyl)-6H-indolo[2,3-b]quinoxaline (7)
A
mixture of 1-(2-bromoethyl)-1H-indole-2,3-dione (10 g,
for therapeutic using (n ¼ 23, X ¼ 3.26 vs Xt ¼ 4.18) nor for
preventive one (n ¼ 24, X ¼ 2.26 vs Xt ¼ 4.29). SI for the tested
compounds was significantly (n ¼ 24, X ¼ 4.42 vs Xt ¼ 4.29) higher
under preventive regimen than under therapeutic one using L929
cell culture. Difference in the compounds SI under these regimens
appeared as insignificant (n ¼ 23, X ¼ 3.95 vs Xt ¼ 4.18) using EPT
cell line.
Obtained results are seemed as promised regarding further
investigations of indoloquinoxalines as perspective antivirals and
interferon inducers.
0.04 mol), o-phenylenediamine (4.32 g, 0.04 mol) and acetic acid
(60 mL) was boiled under reflux for 4 h. Precipitate formed after
reaction mixture cooling was collected, washed with acetic acid
(3 ꢃ 5 mL), and recrystallized from acetic acid. Yield: 80% (10.4 g);
m.p. 169–170 ꢂC. MW. 326.20. Mass-spectrum (electron impact) –
m/z (I, %): 329 (14); 327 (21); 232 (4); 220 (5); 219 (96); 102 (8); 90
(15); 69 (6); 60 (32); 45 (100); 43 (58). 1H NMR (CDCl3)
d: 3.863 (t,
2H, BrCH2CH2N, J ¼ 7.2 Hz); 4.843 (t, 2H, BrCH2CH2N, J ¼ 7.2 Hz);
7.386 (t, 1H, arom, J ¼ 7.5 Hz); 7.523 (d, 1H, arom, J ¼ 8.1 Hz); 7.662–
7.791 (m, 3H, arom); 8.132 (dd, 1H, J ¼ 8.4 Hz, J ¼ 1.5 Hz); 8.333 (dd,
1H, J ¼ 8.1 Hz, J ¼ 1.5 Hz); 8.503 (d, 1H, arom, J ¼ 7.5 Hz). Rf 0.60
(benzene–triethylamine 10:1); Rf 0.84 (chloroform–acetone 10:1).
3. Conclusions
New 6-(2-aminoethyl)-6H-indolo[2,3-b]quinoxalines where
synthesized with high yields via developed scheme using bro-
moethylisatin and 6-(2-bromoethyl)-6H-indolo[2,3-b]quinoxaline
as intermediates. These compounds were identified as a low toxic
potent interferon inducers an antivirals through L929 and EPT cells
based assays. Regarding compounds were more active as antivirals
when they were added to the cell monolayer before virus infection
than they were added immediately after virus infection we
suppose, their antiviral action is mediated by interferon first. On the
other hand, mode of their interferon inducing activity is still
unassigned, but may be speculated as a DNA intercalation medi-
ated. Thus, aminoethylquinoxalines 8–19 really demonstrate high
interferon inducing activity according to the initial speculation.
Additional studies to identify the mechanism of action of these
compounds are in progress.
4.1.3. (2-Indolo[2,3-b]quinoxalin-6-yl-ethyl)-dimethylamine (8)
A 33% water solution of dimethylamine (0.225 mL, 0.005 mol)
was added to a solution of 6-(2-bromoethyl)-6H-indolo[2,3-b]qui-
noxaline (0.81 g, 0.0025 mol) in 40 mL DMF. After stirring at room
temperature for 6 h, a mixture was evaporated under reduced
pressure. The residue was dissolved in 25 mL benzene and extracted
with 10% acetic acid (3 ꢃ 20 mL). This acetous extract was neutral-
ized with saturated solution of sodium carbonate to pH ¼ 8–9. The
obtained precipitate was collected, washed with water (3 ꢃ 5 mL)
and dried. Yield: 78% (0.57 g); m.p. 95–95.5 ꢂC (lit. [38] m.p. 88–
90 ꢂC from methyl acetate). MW. 290.37. Mass-spectrum (electron
impact) – m/z (I, %): 290 (2); 232 (5); 219 (7); 71 (30); 58 (55). Mass-
spectrum (FAB) – m/z (I, %): 291 (100) [M þ H]þ; 246 (16); 232 (8);
219 (12); 72 (20); 57 (55). 1H NMR (CDCl3)
2.878 (t, 2H, (al)CH2CH2N(ar)
J ¼ 6.9 Hz); 4.613 (t, 2H,
d: 2.408 (s, 6H, (CH3)2N);
N
,

M.O. Shibinskaya et al. / European Journal of Medicinal Chemistry 45 (2010) 1237–1243
1241
N(al)CH2CH2N(ar), J ¼ 7.2 Hz); 7.355 (t, 1H, arom, J ¼ 7.5 Hz); 7.520 (d,
CH3(CH2)3N(al)CH2CH3,
J ¼ 7.2 Hz);
1.243–1.404
(m,
5H,
1H, arom, J ¼ 7.8 Hz); 7.625–7.752 (m, 3H, arom); 8.124 (dd, 1H,
J ¼ 8.1 Hz, J ¼ 1.2 Hz); 8.289 (dd, 1H, J ¼ 8.1 Hz, J ¼ 1.5 Hz); 8.460 (d,
1H, arom, J ¼ 7.5 Hz). Rf 0.46 (benzene–triethylamine 10:1); Rf 0.08
(chloroform–acetone 10:1).
CH3CH2(CH2)2N(al)CH2CH3); 1.758–1.915 (m, 2H, CH3CH2CH2
CH2N(al)CH2CH3); 3.047–3.126 (m, 2H, CH3(CH2)2CH2N(al)CH2CH3);
3.184–3.315 (m, 2H, CH3(CH2)3N(al)CH2CH3); 3.460 (t, 2H,
N
(al)CH2CH2N(ar)
,
J ¼ 7.8 Hz); 5.075 (t, 2H, N(al)CH2CH2N(ar)
,
J ¼ 7.5 Hz); 7.414 (t, 1H, arom, J ¼ 7.8 Hz); 7.677–7.799 (m, 3H,
arom); 7.943 (d, 1H, arom, J ¼ 7.8 Hz); 8.095 (dd, 1H, J ¼ 7.8 Hz,
J ¼ 1.5 Hz); 8.324 (dd, 1H, J ¼ 8.1 Hz, J ¼ 1.5 Hz); 8.467 (d, 1H, arom,
J ¼ 8.1 Hz). Rf 0.68 (benzene–triethylamine 10:1); Rf 0.02 (chloro-
form–acetone 10:1).
4.1.4. Diethyl-(2-indolo[2,3-b]quinoxalin-6-yl-ethyl)-amine (9)
Diethylamine (0.36 g, 0.005 mol) was added to a solution of
6-(2-bromoethyl)-6H-indolo[2,3-b]quinoxaline (0.81 g, 0.0025 mol)
in 40 mL benzene. A mixture was boiled under reflux for 6 h.
After cooling the precipitate was filtered and washed with
benzene 3 ꢃ 5 mL. Filtrate was evaporated under reduced pres-
sure, the residue was dissolved in 25 mL benzene and extracted
with 10% acetic acid (3 ꢃ 20 mL). This acetous extract was
neutralized with saturated solution of sodium carbonate to
pH ¼ 8–9. The obtained precipitate was collected, washed with
water (3 ꢃ 5 mL) and dried. The product was purified by crys-
tallization from heptane. Yield: 83% (0.66 g); m.p. 107–107.5 ꢂC
(lit. [39] m.p. 108 ꢂC from petroleum-ether). MW. 318.43. Mass-
spectrum (electron impact) – m/z (I, %): 319 (1); 232 (4); 100
(12); 87 (100); 59(6). Mass-spectrum (FAB) – m/z (I, %): 319
(100) [M þ H]þ; 246 (10); 232 (8); 100 (6); 86 (12); 69 (6); 53
4.1.8. 6-(2-Pyrrolidin-1-yl-ethyl)-6H-indolo[2,3-b]quinoxaline (13)
Yield: 79% (0.63 g); m.p. 93–95 ꢂC. MW. 316.41. Mass-spectrum
(FAB) – m/z (I, %): 317 (100) [M þ H]þ; 247 (18); 233 (8); 221 (10); 84
(45); 54 (5). 1H NMR (CDCl3)
d: 1.698–1.875 (m, 4H, (CH2CH2)2N(al));
2.616–2.831 (m, 4H, (CH2CH2)2N(al)); 3.049 (t, 2H, N(al)CH2CH2N(ar)
,
J ¼ 7.8 Hz); 4.657 (t, 2H, N(al)CH2CH2N(ar), J ¼ 7.8 Hz); 7.346 (t, 1H,
arom, J ¼ 7.5 Hz); 7.553 (d,1H, arom, J ¼ 8.1 Hz); 7.619–7.751 (m, 3H,
arom); 8.120 (dd, 1H, J ¼ 8.4 Hz, J ¼ 1.8 Hz); 8.284 (dd,1H, J ¼ 8.1 Hz,
J ¼ 1.5 Hz); 8.451 (d, 1H, arom, J ¼ 7.8 Hz). Rf 0.46 (benzene–trie-
thylamine 10:1); Rf 0.08 (chloroform–acetone 10:1).
(8). 1H NMR (CDCl3)
d
: 1.029 (t, 6H, (CH3CH2)2N, J ¼ 5.4 Hz);
4.1.9. 6-(2-Piperidin-1-yl-ethyl)-6H-indolo[2,3-b]quinoxaline (14)
Yield: 87% (0.72 g); m.p. 130–131 ꢂC (lit. [39] m.p. 124–125 ꢂC
from petroleum-ether). MW. 330.44. Mass-spectrum (electron
impact) – m/z (I, %): 330 (2); 232 (5); 112 (25); 98 (100); 55(5).
Mass-spectrum (FAB) – m/z (I, %): 331 (100) [M þ H]þ; 246 (17); 232
2.683 (q, 4H, (CH3CH2)2N, J ¼ 3.9 Hz); 2.980 (t, 2H, N(al)CH2CH2
-
N(ar), J ¼ 6.9 Hz); 4.51 (t, 2H, N(al)CH2CH2N(ar), J ¼ 7.5 Hz); 7.360 (t,
1H, arom, J ¼ 7.5 Hz); 7.360–7.760 (m, 4H, arom); 8.05 (d, 1H,
J ¼ 8.1 Hz); 8.21 (d, 1H, J ¼ 8.1 Hz); 8.36 (d, 1H, arom, J ¼ 7.2 Hz).
Rf 0.62 (benzene–triethylamine 10:1); Rf 0.04 (chloroform–
acetone 10:1).
(7); 220 (9); 112 (20); 98 (60); 53(7). 1H NMR (CDCl3)
d: 1.432–1.480
(m, 2H, CH2(CH2CH2)2N(al)); 1.580–1.615 (m, 4H, CH2(CH2
CH2)2N(al)); 2.451–2.603 (m, 4H, CH2(CH2CH2)2N(al)); 2.865 (t, 2H,
The compounds 10–19 were obtained in a similar manner.
N
(al)CH2CH2N(ar)
,
J ¼ 7.2 Hz); 4.647 (t, 2H, N(al)CH2CH2N(ar)
,
4.1.5. (2-Indolo[2,3-b]quinoxalin-6-yl-ethyl)-dipropylamine (10)
Yield: 82% (0.7 g); m.p. 75–76 ꢂC. MW. 346.48. Mass-spectrum
(electron impact) – m/z (I, %): 346 (1); 232 (2); 127 (13); 114 (100);
72 (6); 43 (8). Mass-spectrum (FAB) – m/z (I, %): 347 (100) [M þ H]þ;
246 (20); 232 (6); 220 (9); 128 (17); 114 (83); 72 (5). 1H NMR
J ¼ 7.2 Hz); 7.354 (t, 1H, arom, J ¼ 7.2 Hz); 7.561 (d, 1H, arom,
J ¼ 8.1 Hz); 7.629–7.760 (m, 3H, arom); 8.127 (dd, 1H, J ¼ 8.4 Hz,
J ¼ 1.5 Hz); 8.298 (dd, 1H, J ¼ 8.1 Hz, J ¼ 1.2 Hz); 8.463 (d, 1H, arom,
J ¼ 7.8 Hz). Rf 0.59 (benzene–triethylamine 10:1); Rf 0.09 (chloro-
form–acetone 10:1).
(CDCl3)
4H, (CH3CH2CH2)2N); 2.504 (t, 4H, (CH3CH2CH2)2N, J ¼ 7.5 Hz);
2.945 (t, 2H, (al)CH2CH2N(ar)
N(al)CH2CH2N(ar), J ¼ 7.5 Hz); 7.364 (t, 1H, arom, J ¼ 6.9 Hz); 7.518 (d,
1H, arom, J ¼ 8.4 Hz); 7.639–7.775 (m, 3H, arom); 8.136 (dd, 1H,
J ¼ 8.4 Hz, J ¼ 1.2 Hz); 8.306 (dd, 1H, J ¼ 8.1 Hz, J ¼ 1.2 Hz); 8.476 (d,
1H, arom, J ¼ 7.8 Hz). Rf 0.71 (benzene–triethylamine 10:1); Rf 0.09
(chloroform–acetone 10:1).
d
: 0.802 (t, 6H, (CH3CH2CH2)2N, J ¼ 7.2 Hz); 1.339–1.463 (m,
4.1.10. 6-[2-(2-Methylpiperidin-1-yl)-ethyl]-6H-indolo
[2,3-b]quinoxaline (15)
N
,
J ¼ 7.5 Hz); 4.569 (t, 2H,
Yield: 85% (0.75 g); m.p. 113–115 ꢂC. MW. 344.46. Mass-spec-
trum (FAB) – m/z (I, %): 345 (100) [M þ H]þ; 247 (17); 233 (5); 221
(7); 126 (15); 113 (63); 54 (8). 1H NMR (CDCl3)
d: 1.120 (d, 3H,
CH(CH3)N(al), J ¼ 6.3 Hz); 1.307–1.434 (m, 2H, CH2(CH2)2CH2CH);
1.634–1.813 (m, 4H, CH2(CH2)2CH2CH); 2.486–2.548 (m, 2H,
CH2(CH2)2CH2CH(CH3)N(al)); 2.834–2.925 (m,1H, CH2CH(CH3)N(al));
4.1.6. Butyl-(2-indolo[2,3-b]quinoxalin-6-yl-ethyl)-
methylamine (11)
3.098–3.270 (m, 2H, N(al)CH2CH2N(ar)); 4.607–4.688 (m, 2H,
N(al)CH2CH2N(ar)); 7.350 (t, 1H, arom, J ¼ 7.5 Hz); 7.577 (d, 1H, arom,
J ¼ 7.8 Hz); 7.647–7.756 (m, 3H, arom); 8.114 (dd, 1H, J ¼ 8.4 Hz,
J ¼ 1.5 Hz); 8.291 (dd, 1H, J ¼ 8.4 Hz, J ¼ 1.8 Hz); 8.454 (d, 1H, arom,
J ¼ 7.8 Hz). Rf 0.19 (benzene–triethylamine 10:1); Rf 0.02 (chloro-
form–acetone 10:1).
Yield: 80% (0.66 g); m.p.of hydrochloride 214.5–215 ꢂC. MW.
332.45. Mass-spectrum (FAB) – m/z (I, %): 333 (100) [M þ H]þ; 246
(8); 232 (6), 100 (12); 53 (5). 1H NMR (CDCl3)
d: 0.926 (t, 3H,
CH3(CH2)3N(al)CH3, J ¼ 7.5 Hz); 1.281 (s, 1H, CH3(CH2)3N(al)CH3);
1.302–1.491 (m, 2H, CH3CH2(CH2)2N(al)CH3); 1.694–1.977 (m, 2H,
CH3CH2CH2CH2N(al)CH3); 2.921 (t, 2H, CH3(CH2)2CH2N(al)CH3,
J ¼ 6.6 Hz); 3.619 (t, 2H, N(al)CH2CH2N(ar), J ¼ 8.1 Hz); 5.220 (t, 2H,
N(al)CH2CH2N(ar), J ¼ 7.8 Hz); 7.446 (t, 1H, arom, J ¼ 8.2 Hz); 7.718–
7.830 (m, 3H, arom); 8.126 (d, 1H, arom, J ¼ 7.8 Hz); 8.193 (d, 1H,
J ¼ 7.8 Hz); 8.374 (dd, 1H, J ¼ 8.1 Hz, J ¼ 1.2 Hz); 8.512 (d, 1H, arom,
J ¼ 8.1 Hz). Rf 0.53 (benzene–triethylamine 10:1); Rf 0.01 (chloro-
form–acetone 10:1).
4.1.11. 6-[2-(4-Methylpiperidin-1-yl)-ethyl]-6H-indolo[2,3-
b]quinoxaline (16)
Yield: 80% (0.70 g); m.p. 141–142 ꢂC. MW. 344.46. Mass-spec-
trum (electron impact) – m/z (I, %): 344 (1); 232 (4); 125 (27); 112
(100); 69 (5); 44 (11). Mass-spectrum (FAB) – m/z (I, %): 345 (100)
[M þ H]þ; 246 (15); 232 (8); 126 (17); 112 (75); 69 (5); 53(8).
1H NMR (CDCl3)
d
: 0.916 (d, 3H, CH3CH, J ¼ 5.7 Hz); 1.234–1.346
(m, 3H, CH3CH(CH2CH2A)2N(al)); 1.619–1.662 (m, 2H, CH3
CH(CH2ACH2)2N(al)); 2.156–2.227 (m, 2H, CH3CH(CH2CH2B)2N(al));
2.910 (t, 2H, N(al)CH2CH2N(ar), J ¼ 7.2 Hz); 3.094–3.131 (m, 2H,
CH3CH(CH2BCH2)2N(al)); 4.651 (t, 2H, N(al)CH2CH2N(ar), J ¼ 7.2 Hz);
7.353 (t, 1H, arom, J ¼ 6.9 Hz); 7.585 (d, 1H, arom, J ¼ 8.1 Hz);
7.624–7.757 (m, 3H, arom); 8.116 (dd, 1H, J ¼ 8.4 Hz, J ¼ 1.2 Hz);
4.1.7. Butylethyl-(2-indolo[2,3-b]quinoxalin-6-yl-ethyl)-amine (12)
Yield: 84% (0.73 g); m.p. 53–54 ꢂC. MW. 346.48. Mass-spectrum
(electron impact) – m/z (I, %): 346 (1); 232 (2); 127 (13); 114 (100);
72 (10); 58 (9). Mass-spectrum (FAB) – m/z (I, %): 347 (100)
[M þ H]þ; 232 (6), 246 (8); 114 (10). 1H NMR (CDCl3)
d: 0.917 (t, 3H,

1242
M.O. Shibinskaya et al. / European Journal of Medicinal Chemistry 45 (2010) 1237–1243
8.289 (dd, 1H, J ¼ 8.4 Hz, J ¼ 1.2 Hz); 8.454 (d, 1H, arom, J ¼ 7.5 Hz).
Rf 0.62 (benzene–triethylamine 10:1); Rf 0.08 (chloroform–acetone
10:1).
4.2.2. Antiviral activity assay
Vesicular stomatitis virus (VSV) was grown in EPT and L929 cells
to titers of about 106 ID50/0.1 mL. Assay of antiviral effects of
compounds was based on evaluation of virus cell pathogen effects
for VSV. The tested compounds properly diluted in appropriate
media were added to cell monolayer grown in 96-well microtiter
plates (FaCCon) at 37 ꢂC in 5% CO2 (0.2 mL per well) either 24 h
before ("preventive effect") or immediately after virus infection
4.1.12. 6-(2-Azepan-1-yl-ethyl)-6H-indolo[2,3-b]quinoxaline (17)
Yield: 84% (0.73 g); m.p. 75–76 ꢂC. MW. 344.46. Mass-spectrum
(FAB) – m/z (I, %): 345 (100) [M þ H]þ; 246 (16); 232 (7); 219 (8);
126 (17); 112 (52); 69 (17); 53 (35). 1H NMR (CDCl3)
d: 0.822–0.888
(m, 4H, (CH2CH2CH2)2N(al)); 1.563–1.592 (m, 4H, (CH2CH2
CH2)2N(al)); 2.940–3.055 (m, 4H, (CH2CH2CH2)2N(al)); 3.160 (t, 2H,
("therapeutic effect"). Concentrations range 0.1–100
compounds was used. Triplicate wells were employed as a rule. The
multiplicity of infection was 0.1 ID50/cell for VSV. Virus yields (virus
m
M
of
N
(al)CH2CH2N(ar)
,
J ¼ 7.2 Hz); 4.710 (t, 2H, N(al)CH2CH2N(ar)
,
J ¼ 7.2 Hz); 7.381 (t, 1H, arom, J ¼ 7.2 Hz); 7.624 (d, 1H, arom,
J ¼ 8.1 Hz); 7.649–7.781 (m, 3H, arom); 8.136 (dd, 1H, J ¼ 8.1 Hz,
J ¼ 1.8 Hz); 8.311 (dd, 1H, J ¼ 8.1 Hz, J ¼ 1.2 Hz); 8.477 (d, 1H, arom,
J ¼ 7.8 Hz). Rf 0.56 (benzene–triethylamine 10:1); Rf 0.01 (chloro-
form–acetone 10:1).
cytodestructive action) were estimated 24 h post infection. EC50,
concentration (M) which lead to 50% cytopathic action of VVS
inhibition, was determined using regression analysis method
(Microsoft Excel).
Statistical evaluation of results was made by Student’s t-test
under P < 0.05.
4.1.13. 6-[2-(4-Methylpiperazin-1-yl)-ethyl]-6H-indolo
[2,3-b]quinoxaline (18)
4.2.3. IFN-inducing activity assay
Yield: 88% (0.76 g); m.p. 143–144 ꢂC. MW. 345.45. Mass-spec-
trum (FAB) – m/z (I, %): 346 (100) [M þ H]þ; 247 (13); 233 (8); 221
IFN-inducing activity was tested in vitro on murine fibroblast
(L929) cells and piglet testicular (EPT) cells line. The supernatant
was took after 24 and 48 h cultivation at 37 ꢂC in 5% CO2 and
activity of IFN was determined in it. Titers of the IFN induced by
compounds were determined on L929 and EPT cell cultures. The
tested compound samples (0.1 mL) in serial double dilution were
added to cell monolayer. The culture medium (0.1 mL) was added in
control well. VSV suspension (0.05 mL) was added in each well after
24 h incubation 37 ꢂC in 5% CO2. Plates were cultivated in the same
condition for 24 h, result estimate was determined by microscop-
ical method.
(10); 114 (26); 70 (22). 1H NMR (CDCl3)
d: 2.270 (s, 3H, CH3N);
2.347–2.443 (m, 4H, CH3N(CH2CH2)2N(al)); 2.598–2.696 (m, 4H,
CH3N(CH2CH2)2N(al)); 2.874 (t, 2H, N(al)CH2CH2N(ar), J ¼ 7.2 Hz);
4.583 (t, 2H, N(al)CH2CH2N(ar), J ¼ 6.9 Hz); 7.350 (t, 1H, arom,
J ¼ 7.8 Hz); 7.473 (d, 1H, arom, J ¼ 8.1 Hz); 7.621–7.753 (m, 3H,
arom); 8.094 (dd, 1H, J ¼ 8.4 Hz, J ¼ 1.5 Hz); e.e. 8.288 (dd, 1H,
J ¼ 8.4 Hz, J ¼ 1.5 Hz); 8.460 (d, 1H, arom, J ¼ 7.8 Hz). Rf 0.19
(benzene–triethylamine 10:1); Rf 0.02 (chloroform–acetone 10:1).
4.1.14. 6-(2-Morpholin-4-yl-ethyl)-6H-indolo[2,3-b]quinoxaline
(19)
Yield: 93% (0.78 g); m.p. 125–126 ꢂC (lit. [40] m.p. 135 ꢂC from
ethyl acetate). MW. 332.41. Mass-spectrum (FAB) – m/z (I, %): 333
(100) [M þ H]þ; 247 (22); 233 (10); 220 (14); 100 (43); 77 (6) 1H
Maximal dilution of the interferon contained media allowed the
cell culture protection against cytopathic action of the VSV 100
CPA50 was taken as interferon titer.
Appendix. Supplementary data
NMR (CDCl3)
d
: 2.600 (t, 4H, O(CH2CH2)2N(al), J ¼ 4.5 Hz); 2.864 (t,
2H, N(al)CH2CH2N(ar), J ¼ 6.9 Hz); 3.613 (t, 4H, O(CH2CH2)2N(al)
,
The supplementary data associated with this article can be
found in the on-line version at doi:10.1016/j.ejmech.2009.12.014.
J ¼ 4.8 Hz); 4.613 (t, 2H, N(al)CH2 CH2N(ar), J ¼ 6.9 Hz); 7.377 (t, 1H,
arom, J ¼ 7.2 Hz); 7.502 (d, 1H, arom, J ¼ 8.1 Hz); 7.643–7.774 (m,
3H, arom); 8.113 (dd, 1H, J ¼ 7.8 Hz, J ¼ 1.2 Hz); 8.292 (dd, 1H,
J ¼ 7.8 Hz, J ¼ 1.5 Hz); 8.487 (d, 1H, arom, J ¼ 7.8 Hz). Rf 0.53
(benzene–triethylamine 10:1); Rf 0.29 (chloroform–acetone 10:1).
References
[1] E. De Clercq, J. Infect. Dis. 194 (Suppl. 1) (2006) S19–S26.
[2] E. De Clercq, in: , Advances in Antiviral Drug Design, vol. 5, Elsevier, New York,
2007.
4.2. Biological assays
[3] A. Meager (Ed.), The Interferons: Characterization and Application, Elsevier,
New York, 2006.
[4] M.H. Woo, T.G. Burnakis, Ann. Pharmacother. 31 (1997) 330–337.
[5] E.J. Bini, E.H. Weinshel, Mayo Clin. Proc. 74 (1999) 367–370.
[6] R.J. Wills, S. Dennis, H.E. Spiegel, D.M. Gibson, P.I. Nadler, Clin. Pharmacol. Ther.
35 (1984) 722–727.
[7] E.B. Tazulakhova, O.V. Parshina, T.S. Guseva, F.I. Ershov, J. Interferon Cytokine
Res. 21 (2001) 65–73.
[8] B. Berman, U. Hengge, S. Barton, Acta Derm. Venereol. Suppl. (Stockh) 214
(2003) 12–17.
[9] R.F. Krueger, G.D. Mayer, Science 169 (1970) 1213–1214.
[10] G.D. Mayer, R.F. Krueger, Science 169 (1970) 1214–1215.
[11] S.A. Andronati, L.A. Litvinova, N.Ya. Golovenko, J. Med. Sci. Acad. Ukraine 5
(1999) 53–66.
[12] P. Chandra, F. Zunino, V.P. Gaur, A. Zaccara, M. Woltersdorf, G. Luoni, A. Gotz,
FEBS Lett. 28 (1972) 5–9.
[13] J. Sturm, L. Schreiber, M. Daune, Biopolymers 20 (1981) 765–785.
[14] T. Nishimura, T. Okobira, A.M. Kelly, N. Shimada, Y. Takeda, K. Sakurai,
Biochemistry 46 (2007) 8156–8163.
[15] S. Alcaro, A. Arena, S. Neri, R. Ottana, F. Ortuso, B. Pavone, M.G. Vigorita, Bioorg.
Med. Chem. 12 (2004) 1781–1791.
4.2.1. Cytotoxicity assay
Lines of murine fibroblast L929 and established piglet testicular
EPT cells were obtained from the Research Institute of Veterinary
Medicine, Ukrainian Academy of Agriculture, Kiev, Ukraine. EPT
cells were cultivated in Medium 199 supplemented with 10% of calf
embryo serum (Sigma) and 50 mg/mL kanamycin (Virion, Russian
Federation) while L929 cells were cultivated in Eagle’s Minimum
Essential medium supplemented with the same components as
above plus 300 mg/mL glutamine by standard techniques [41].
The in vitro cytotoxicity of synthesized compounds was deter-
mined against monolayer cell cultures EPT and L929 using 96-well
microtiter plates (FaCCon) at 37 ꢂC in 5% CO2. The culture medium
was substituted for cell maintenance medium (supplemented
with 5% of newborn calf serum) with compounds in concentration
1–1000
registered in 24–48 h using device Labsystem Myltiscan MS
¼ 540/620) past stained with 0.5% crystal violet in 70% ethanol.
LC50 concentration (M) which lead to 50% cell monolayer
mM. Toxic action of different compound concentrations was
[16] S.A. Lyakhov, L.A. Litvinova, Y. Suveyzdis, S.A. Andronati, S.L. Rybalko,
S.T. Dyadyun, Pharm. Chem. J. 34 (2000) 474–475.
[17] E.R. Andrews, R.W. Fleming, J.M. Grisar, J.C. Kihm, D.L. Wenstrup, G.D. Mayer, J.
Med. Chem. 17 (1974) 882–886.
[18] W.L. Albrecht, R.W. Fleming, S.W. Horgan, G.D. Mayer, J. Med. Chem. 20 (1977)
364–371.
(l
,
destruction, was determined using regression analysis method
(Microsoft Excel).

M.O. Shibinskaya et al. / European Journal of Medicinal Chemistry 45 (2010) 1237–1243
1243
[19] A. Guiotto, P. Rodighiero, M.S. Marciani, O. Gia, G. Pastorini, P. Manzini,
M. Zucca, I. Viano, Farmaco Sci. 34 (1979) 774–788.
[30] K. Hirata, J. Araya, S. Nakaike, K. Kitamura, T. Ishida, Chem. Pharm. Bull. 49
(2001) 44–48.
[20] A.D. Sill, E.R. Andrews, F.W. Sweet, J.W. Hoffman, P.L. Tiernan, J.M. Grisar,
R.W. Fleming, G.D. Mayer, J. Med. Chem. 17 (1974) 965–968.
[21] I. Mucsi, J. Molnar, M. Tanaka, C. Santelli-Rouvier, A.M. Patelis, J.P. Galy, J. Barbe,
Anticancer Res. 18 (1998) 3011–3015.
[22] J. Harmenberg, B. Wahren, J. Bergman, S. Akerfeldt, L. Lundblad, Antimicrob.
Agents Chemother. 32 (1988) 1720–1724.
[31] D.A. Stringfellow, L.A. Glasgow, Antimicrob. Agents Chemother.
73–78.
[32] E. De Clercq, Top. Curr. Chem. 52 (1974) 173–208.
[33] M. Degre, E.T. Glaz, Acta Pathol. Microbiol. Scand. [B] 85 (1977) 189–194.
[34] G.J. Mannering, K.W. Renton, R. el Azhary, L.B. Deloria, Ann. N.Y. Acad. Sci. 350
(1980) 314–331.
2 (1972)
[23] S.A. Lyakhov, L.A. Litvinova, S.A. Andronati, L.K. Berezina, B.N. Galkin,
V.E. Osetrov, T.O. Filippova, N.Ya. Golovenko, Ukr. Biokhim. Zh. 4 (2001)
108–113.
[24] O. Harbecke, C. Dahlgren, J. Bergman, L. Mo¨ller, J. Leukocyte Biol. 65 (1999)
771–777.
[35] G.I. Zhungietu, M.A. Rekhter, Isatin and its derivatives. Kishinev, Shtinitsa
(1977).
[36] B.L. Van Der Waerden, Mathematische Statistic. SpringerꢀVerlag, Berlin ꢀ
Gottingen – Heidelberg, 1957.
[37] B.L. Van der Waerden, E. Nievergelt, Tafeln zum Vergleich zweier Stichprobcn
mittels X-Test und Zeichentest. Springer – Verlag, Berlin ꢀ Gottingen – Hei-
delberg, 1956.
[25] N.S. Loginova, A.S. Kinzirskii, O.V. Parshina, E.E. Mirskaia, F.I. Ershov, Vestn.
Ross. Akad. Med. Nauk 3 (1996) 28–33.
[26] G.N. Zolotareva, A.S. Kinzirski, N.S. Loginova, Z.I. Galochkina, F.I. Ershov, Dokl.
Akad. Nauk SSSR 317 (1991) 992–996.
[38] I. Zegar, A. Graslund, J. Bergman, M. Eriksson, B. Norden, Chem.-Biol. Interact.
72 (1989) 277–293.
[27] J. Bergman, L. Mo¨ller, H. Vallberg, US Patent 6,465,466 B1; 2002.
[28] J.A. Kellen, A. Mirakian, Res. Commun. Chem. Pathol. Pharmacol. 32 (1981)
185–188.
[39] F. Knotz, W. Wendelin, Sci. Pharm. 43 (1975) 249–260.
[40] F.-A. Alphonse, S. Routier, G. Coudert, J.-Y. Me´rour, Heterocycles 55 (2001)
925–940.
[29] W.A. Basley, R.C. Rees, J.A. Greager, R.W. Baldwin, Chemotherapy 27 (1981)
44–52.
[41] R.I. Freshney, Animal Cell Culture. A Practical Approach. IRL Press, Oxford –
Washington, 1992.