Evaluation of antioxidant activity and cytotoxicity of polyfluorinated diarylacetylenes and indoles toward human cancer cells

Article abstract of DOI:10.1016/j.jfluchem.2019.109353

A large series of polyfluorinated diarylacetylenes and indoles was evaluated for antitumor activity in MCF-7, RPMI 8226, T98 G, HCT 116 human cancer cells and for cytotoxicity effect on HEK-293, WI-38, LMTK, AG 17 normal mammalian cell lines. It was found that with the increased number of fluorine atoms on the benzene ring of the compounds, their cytotoxicity increases, reaching a maximum in case of the perfluorinated structures. Polyfluorinated diarylacetylenes containing three fluorine atoms and trifluoromethyl and amino groups in one aryl ring, inhibit the proliferation of human myeloma RPMI 8226 and human breast cancer MCF-7 cells. Indoles showed more pronounced cytotoxicity as compared with their arylacetylene precursors. Compounds I₉, I₁₁, and I₁₃ were found to have a high growth-inhibitory activity toward the human cancer cell lines tested, with half-inhibitory concentrations (IC₅₀) of 1–10 μM. The structures of these compounds are characterized by complete fluorination of the benzene part of the indole core and the presence of an aromatic moiety at the 2nd position of the five-membered ring. Further, DNA fragmentation and Annexin V-FITC/PI staining assays confirm that I₉ – I₁₁ induce apoptosis. Mutagenic and antioxidant properties of the polyfluorinated indoles and of selected diarylacetylenes were also studied. On a Salmonella tester strain, it was demonstrated that the compounds are good antioxidants.

Full text of DOI:10.1016/j.jfluchem.2019.109353

Accepted Manuscript
Title: Evaluation of antioxidant activity and cytotoxicity of
polyfluorinated diarylacetylenes and indoles toward human
cancer cells
Authors: Olga Zakharova, Georgy Nevinsky, Larisa
Politanskaya, Dmitry Baev, Ludmila Ovchinnikova, Evgeny
Tretyakov
PII:
DOI:
Article Number:
S0022-1139(19)30157-5
https://doi.org/10.1016/j.jfluchem.2019.109353
109353
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22 July 2019
Please cite this article as: Zakharova O, Nevinsky G, Politanskaya L, Baev D,
Ovchinnikova L, Tretyakov E, Evaluation of antioxidant activity and cytotoxicity of
polyfluorinated diarylacetylenes and indoles toward human cancer cells, Journal of
Fluorine Chemistry (2019), https://doi.org/10.1016/j.jfluchem.2019.109353
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Evaluation of antioxidant activity and cytotoxicity of polyfluorinated
diarylacetylenes and indoles toward human cancer cells
Olga Zakharovaa, Georgy Nevinskya,b*, Larisa Politanskayab,c* Dmitry Baevb,c, Ludmila
Ovchinnikovad, Evgeny Tretyakovb,c
[a]
[b]
[c]
[d]
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences
Ac. Lavrentiev Ave. 8, Novosibirsk 630090, Russian Federation
Novosibirsk State University
Pirogova Str. 2, Novosibirsk 630090, Russian Federation
N.N. Vorozhtsov Institute of Organic Chemistry, Siberian Branch of Russian Academy of Sciences
Ac. Lavrentiev Ave. 9, Novosibirsk 630090, Russian Federation
Institute of Cytology and Genetics, Siberian Division of Russian Academy of Sciences
Ac. Lavrentiev Ave. 10, Novosibirsk 630090, Russian Federation
 Corresponding author. e-mail: nevinsky@niboch.nsc.ru (G. Nevinsky)
 Corresponding author. e-mail: plv@nioch.nsc.ru (L. Politanskaya)
Graphical abstract
Highlights
 Three polyfluorinated indoles exhibited good potency against human cancer cells.
 DNA fragmentation and Annexin V-FITC/PI staining assays revealed the apoptotic cell
death.
 Structure-activity relationship of compounds was illustrated by molecule docking studies
 Compounds good suppress spontaneous and H2O2-induced mutagenesis of bacterial cells.
Abstract
A large series of polyfluorinated diarylacetylenes and indoles was evaluated for antitumor
activity in MCF-7, RPMI 8226, T98G, HCT 116 human cancer cells and for cytotoxicity effect
on HEK-293, WI-38, LMTK, AG 17 normal mammalian cell lines. It was found that with the
increased number of fluorine atoms on the benzene ring of the compounds, their cytotoxicity
increases, reaching a maximum in case of the perfluorinated structures. Polyfluorinated
diarylacetylenes containing three fluorine atoms and trifluoromethyl and amino groups in one
aryl ring, inhibit the proliferation of human myeloma RPMI 8226 and human breast cancer
MCF-7 cells. Indoles showed more pronounced cytotoxicity as compared with their
arylacetylene precursors. Compounds I9, I11, and I13 were found to have a high growth-inhibitory
activity toward the human cancer cell lines tested, with half-inhibitory concentrations (IC50) of
1–10 μM. The structures of these compounds are characterized by complete fluorination of the
1

benzene part of the indole core and the presence of an aromatic moiety at the 2nd position of the
five-membered ring. Further, DNA fragmentation and Annexin V-FITC/PI staining assays
confirm that I9 – I11 induce apoptosis. Mutagenic and antioxidant properties of the
polyfluorinated indoles and of selected diarylacetylenes were also studied. On a Salmonella
tester strain, it was demonstrated that the compounds are good antioxidants.
Keywords: Polyfluorinated 2-arylindoles; Polyfluorinated diarylacetylenes; Cytotoxicity;
Structure-activity relationship; Mutagenic and antioxidant properties
1. Introduction
Privileged structures, such as the indole class, represent numerous types of molecules that
are known to have high binding affinity for various receptors. Because of its presence in a
variety of pharmacologically active molecules, the indole moiety is believed to represent the key
structural class in the field of drug design and discovery [1]. For instance, the indole nucleus is
found in various natural products such as Catharanthus alkaloids, well-known mitosis inhibitors
[2].
It is general knowledge that substituted 2-arylindoles suppress polymerization of tubulin by
selectively binding to the colchicine-binding site in tubulin and thereby stall the cell cycle at the
G2/M transition and cause apoptosis [1–4]. In an in vitro assay involving breast cancer cell lines,
these derivatives showed potency similar to that of vincristine and combretastatin A-4 [3].
Furthermore, 2-arylindoles are inhibitors of polymerization of tubulin [5–7].
These data have aroused substantial interest in the discovery of indole ring-carrying
compounds that could be more effective or more specific to tumors or tissues of interest.
Introduction of a fluorine atom into biologically active polynuclear aromatic hydrocarbons is
known to be effective at modifying their therapeutic efficacy [8]. Because of the specific
characteristics of fluorine, such as a small atomic radius, strong electronegativity, and weak
polarizability of the C–F bond, substitution of the hydrogen atom by a fluorine atom in
functional molecules tends to alter the parent molecule in terms of physicochemical properties
(e.g., lipophilicity and electronegativity) and physiological characteristics. The higher activity of
the fluorine derivative may be explained by improved pharmacokinetic properties, i.e., greater
resistance to metabolic degradation owing to a halogen bond.
In comparison with less fluorinated analogs, benzo-perfluorinated derivatives of 2,2-
dimethyl-2,3-dihydro-1H-quinolin-4-ones have a stronger cytotoxic effect on RPMI 8226
(human myeloma) cells [9]. A promising antiproliferation activity toward human cancer cells has
been detected in polyfluorinated analogs of quinolone based on the skeleton of 3-benzylidene-2-
aryl-2,3-dihydroquinolin-4-ones [10].
Indole fluorine derivatives are prominent members of the class of biologically active
benzoheterocycles [11]. These observations led to the present study on the cytotoxic and
anticancer activities of numerous polyfluorinated 2-arylindoles and their synthetic precursors
(polyfluorinated diarylacetylenes), which can also suppress cancer cell proliferation [12,13].
2. Results and discussion
2.1. Synthesis
2

In Scheme 1 outlined the synthesis of diarylacetylenes A and phenylindoles I obtained in the
present work starting from their corresponding iodoanilines and acetylene derivative. Other
investigated compounds were synthesized as described early [14, 15].
2.2. Evaluation of cytotoxicity of polyfluorinated derivatives
All target compounds were tested as growth inhibitors of four human tumorous and four
normal mammalian cell lines. Tables 1 and 2 summarize the data on antiproliferative activities of
polyfluorinated diarylacetylenes A and indoles I toward human breast cancer MCF-7 cells,
human myeloma RPMI 8226 cells, human glioblastoma T98G cells, human colon carcinoma
HCT 116 cells, human embryonic kidney HEK-293 cells, diploid human cell line WI-38, normal
mouse fibroblast line LMTK, and Chinese hamster AG 17 cells.
Tables 1 and 2 shows that arylacetylenes have lower cytotoxic activity toward the tested cell
lines as compared with the corresponding 2-arylindoles. One also pay attention that
nonfluorinated 2-(phenylethynyl)aniline (А18) has lower cytotoxicity compared to that
manifested by fluorinated diarylacetylenes А10–А16. For a number of compounds having a
phenyl moiety as one of the substituents at the triple bond (А18–А13), inhibitory action on the
growth of human cancer cells increases with the accumulation of fluorine atoms in the second
aromatic moiety, reaching its maximum at the complete substitution of all hydrogen atoms with
fluorine atoms and the trifluoromethyl group (Table 1, entry 13). For this compound (А13) the
values of IC50 are comparable to those observed for polyfluorinated indoles, most of them far
exceed diarylethylene in cytotoxicity.
Indoles I9, I11, and I13 manifested the strongest antiproliferative activity; it was found that
their IC50 values were lower than 10 М toward all the human tumor cell lines tested (Table 2,
entries 9, 11 and 13). The structures of these compounds are characterized by complete
fluorination of the benzene part of the indole core and the presence of an aromatic moiety at the
2nd position of five-membered ring. At the same time, the degree of fluorination of the
peripheral aromatic fragment and the presence of the p-amino group only slightly effect on the
cytotoxic activity of indoles. It follows that the degree of fluorination of the indole benzene
moiety has a primary influence on the relative cytotoxicity of the indoles.
The cytotoxic properties of indole I19 having the benzo-perfluorinated core and a butyl
group at position 2, are slightly inferior to those manifested by one of the most active indoles,
namely compound I9 (Table 2, entries 19 and 9). It is important to note that compounds I20 and
I21 containing a primary or tertiary alcohol function (CH2OH or C(CH3)2OH) at position 2 of the
indole nuclear have relatively low activity despite they are structural analogs of the most active
benzo-perfluorinated 2-arylindoles (Table 2, entries 20, 21).
Comparison of the IC50 values related to different cell lines indicates nonselectivity of the
action of all compounds on cancerous and normal cells, similar to the effect of colchicine (Table
2, entry 22). However, a fundamental difference between studied compounds and colchicine is
how they induce cell cycle perturbations. The flow cytometric analyses of propidium iodide
stained nuclei cells showed that the treating of A-549 cells with indole I11 or diarylacetylene A9
caused a significant accumulation of cells in the G1 phase and a decrease of cells in the G2/M
and S phases (see SI). As compared with indole I11 and diarylacetylene A9, colchicine arrest cells
at G2/M phase, which mean that it interferes with the mitotic division, that is more promising.
3

Nevertheless, a malfunctioning of cell cycle control in G1 phase is considered as one of the
most critical molecular bases for suppressing of tumor progression, because many of the
important processes in the G1 phase play a crucial role in proliferation, differentiation,
transformation, and programmed cell death (apoptosis).
2.3. Morphological analysis of apoptosis
Apoptosis is one of the major pathways that lead to the process of cell death. Chromatin
condensation, nuclear shrinking and fragmented nuclei are known as classic characteristics of
apoptosis. To quantify the apoptotic effect of one of the most active compound I9, Annexin V-
FITC/Propidium Iodide staining assay was carried out. This study facilitates the detection of
necrotic cells (Q1-FITC-/PI+); late apoptotic cells (Q2-FITC+/PI+); live cells (Q3-FITC-/PI-)
and early apoptotic cells (Q4-FITC+/PI-). In I9-treated cells, the annexin V/PI subpopulation
increased substantially compared with that in cells treated with DMSO (Fig. 1). According to the
data presented in Table 3, the apoptotic population (Q4) increased after incubation of the cells
with I9 compared to the control group treated with DMSO. This assay reveals that compound I9
shows apoptosis with an increment in early apoptotic cells [control (11.6%), I9 (35.3%)].
Apoptosis was also assessed by electrophoresis of extracted genomic DNA from cells.
Endonuclease mediated cleavage of nuclear DNA results in the formation of oligonucleosomal
DNA fragments a biochemical hallmark of apoptosis in many cell types [16]. DNA laddering
assay was performed with RPMI 8226 cells by treatment of indoles I9–I11 at 7.5 μM
concentration for 72 h, then the genomic DNA was isolated and electrophoresis in a 1.8%
agarose gel. The tested compounds induce DNA fragmentation which is observed as
characteristic ladder pattern in RPMI 8226 cells, while no laddering was observed in control
cells (DMSO) indicating that I9–I11 induce apoptotic cell death (Fig. 2).
2.4. Docking analysis
As it was mentioned in the introduction, substituted 2-arylindoles are known to suppress
polymerization of tubulin. Based on this assumption, we tried to link the observed cytotoxic
properties of indoles and acetylenes with their structure when it is placed in the binding site of
tubulin by molecular docking. Despite the fact that this study is purely speculative, it, in our
opinion, could help to understand the cause of changes in the cytotoxic properties of 2-
arylindoles when fluorine atoms are introduced into their structure. Compounds A13 and I9, I11,
and I13, which manifested the highest cytotoxicity, as well as nonfluorinated compounds A18 and
I18 (for comparison) were chosen for molecular docking study.
Tubulin can bind with various cytostatic drugs, leading to the stabilization of the
microtubules of the cytoskeleton of tumor cells, which leads to disruption of the cell cycle and
mitosis. There are three types of cytostatics with different binding sites on the surface of tubulin:
similar to colchicine, vinblastine and paclitaxel (taxol). As a result of execution of the docking
scoring function, the following values of the docking scores of the tested compounds with
tubulin in binding sites were obtained (Table 4).
4

Indole derivatives that are most active in terms of cytostatic activity show the lowest
docking score in the binding site of colchicine, although conformations that can be incorporated
into other binding sites are possible. However, the interaction of these compounds with the
binding sites of vinblastine and taxol seems less likely, due to the significantly larger sizes of
vinblastine and taxol. For a more detailed study of the possible binding of new derivatives, we
performed a pharmacophore analysis, presented in SI.
All these new molecules have potent affinity for tubulin, and this affinity is comparable
to that of colchicine in the model in question. The colchicine-binding site is formed by the B
chain of tubulin at the border with the A chain. The colchicine molecule is prone to the
formation of a hydrogen bond between the oxygen of the keto-group of its seven-membered ring
and the hydrogen of the amino group of chain A Val181 of tubulin. Within the hydrophobic
cavity of the binding site, the colchicine molecule is stabilized by hydrophobic interactions of the
-system of the trimethoxyphenyl moiety. It is possible that the high theoretical affinity of the
new derivatives determines hydrophobic interactions of the -systems of their polyfluorinated
rings, which can be got inserted into the hydrophobic pocket of the binding site just as the
trimethoxybenzyl cycle of colchicine can. It is also possible that C–F groups play an important
role in the formation of very weak interactions both with polar protons and the highly
electronegative atoms of amino acid residues that are capable of stabilizing the molecules of
these new compounds in the binding site (Fig. 3).
An interesting feature of the new compounds is the possible deeper placement of their
polyfluorinated aromatic rings into the hydrophobic pocket of the binding site as compared to
colchicine. The compound with the highest theoretical affinity, I9, possibly engages in a weak
interaction (via its C–F group of the indole ring) with the chain A amino acid residue Val181,
which is key for colchicine binding. The amino group of the aniline ring of this molecule can
serve as a proton donor for the oxygen of the chain B amino acid residue Val238, forming a
hydrogen bond. The trifluoromethyl group in compound A13 is capable of stabilizing the
molecule deep in the pocket of the binding site, forming weak interactions via fluorine with the
already mentioned oxygen atom of the chain B amino acid residue: Val238. It is also possible for
this compound to form a hydrogen bond between the proton of the amino group and the oxygen
atom of the chain B amino acid residue Asp251, which can form a weak interaction with one of
the C–F groups of the polyfluorinated aromatic ring.
The effect of polyfluorination of aromatic cycles in these compounds on their ability to bind
to tubulin is noteworthy. If we compare the calculated spatial configurations of compounds I18
and A18 in the binding site of tubulin with those of their polyfluorinated analogs I9 and A13, it
becomes apparent that numerous C–F groups allow I9 and A13 molecules to be positioned deep in
the hydrophobic binding site. Compounds I18 and A18 can occupy only the central region of the
binding site by means of one of their aromatic rings; this arrangement appears to be insufficient
for effective binding to tubulin (Fig. 4).
2.5. Antioxidant properties of of polyfluorinated derivatives
Some biologically active compounds interacting with many cell targets (which is true of
indoles) can be polyfunctional and possess cytoprotective properties, or conversely, can be
mutagenic or carcinogenic. Evidently, drugs are more successful when they are not mutagenic at
5

least at the therapeutic concentrations. The Salmonella Typhimurium TA102 strain is widely
used both for evaluation of mutagenicity of different compounds, including fluorinated arenes,
and for detection of antioxidant properties, judging by suppression of spontaneous mutagenesis
in this strain and by a decrease in the mutagenicity of oxidants (Н2O2) [9,17–19]. The mutagenic
activity of compounds A and I was estimated by the Ames test [20] based on S. Typhimurium
TA102 [21]. Mutagenicity in the Ames test is assessed by computing the frequency of reversion
from histidine auxotrophy to prototrophy under the influence of the substance being tested. The
mutation induction in the Ames test is estimated by calculating the frequency of reversion from
histidine auxotrophy to prototrophy in response to the substance under study [20,21].
The data on the four analyzed polyfluorinated diarylacetylenes and 13 indoles are
summarized in Table 5.
It is noteworthy that 15 of 17 compounds had comparable potency (IC50 values 0.43–1.34
M) in suppressing the spontaneous appearance of mutants, whereas two indoles (I12 and I20)
turned out to have lower values (Table 5). Fig. 5 shows the representative data on compounds I12
and I15. These compounds differ primarily in a set of substituents on the peripheral aromatic ring.
Perfluorinated structure I12, containing a para-amino group, had a more pronounced antioxidant
effect as compared to that of compound I15. Along with compound I12, indole I20 (containing a
tertiary alcohol function in its structure) also has low IC50 (µM): 0.22 (I12) and 0.34 (I20).
Meanwhile, perfluorinated structure A12 was found to have the lowest IC50 (0.72 µM) among the
diarylacetylenes under study. Thus, perfluorinated compounds possess more pronounced
cytotoxic and antioxidant activities in comparison with their less fluorinated analogs.
Some antioxidant compounds can efficiently decrease the mutagenic effect of H2O2. [9,17–
20]. In the Ames test, H2O2 was added to TA102 cells at the optimal concentration, 3 mM, and the
concentrations of test compound were varied (Fig. 5). At relatively low concentrations (0.028–
0.079 M), all 16 fluorinated derivatives effectively suppressed the H2O2-dependent formation
of mutants: from 138% to 100% of revertants (the number of revertants observed in the control
group without H2O2 was set to 100%). Compound I15 was found to have remarkably higher IC50:
0.25 M (Table 5). The concentrations corresponding to a decrease in the emergence of mutants
from 100% to 50% in the presence of H2O2 varied from 0.13 to 1.1 M, and the lowest IC50 was
observed for I20 (0.13 M) and I12 (0.16 M; Table 5). These data indicate that all 17 compounds
analyzed are not mutagenic themselves and can effectively decrease the magnitude of
spontaneous mutagenesis and the mutagenic effect of H2O2.
Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) is a water-soluble analog
of vitamin E, it is often used in biological or biochemical applications to reduce oxidative stress
or damage, as well as a standard in determination of the antioxidant properties of new
compounds. Previously, we found that at low concentrations (0.3 µM) control trolox suppressed
the t-BuO2H-dependent formation of mutants only for 8.8 % (decrease from 100 to 91.2%) of
revertants in the Ames test [21]. Herewith the most active indole I12 at the same concentration
(0.3 µM) suppressed the H2O2-dependent formation of mutants from 100 to 40% (Fig. 5, A).
Thus, it can be estimated that at concentration 0.3 µM compound I12 protects cells ~7-fold better
than trolox (Table 5). On the average the active indole compounds can be compared or more
active then -tocopherol succinate (Fig. 6).
6

Comparison of the results on biological testing of polyfluorinated diarylacetylenes and
indoles leads to the following observations.
Among 18 compounds of group A (diarylacetylenes) studied regarding suppression of the
growth of cancerous and normal cells, only 4 compounds were analyzed in experiments on
antioxidant activity (A2, A5, A10, and A12). In terms of growth suppression of tumorous and
normal cells, A10 and A12 have to some extent lower IC50 values (6–42 M) in comparison with
compounds A2 and A5 (35 to >75 M, Table 1). At the same time, their abilities to suppress the
appearance of spontaneous and H2O2-dependent mutants were very similar (Table 5).
Among the 21 compounds of group I (indoles), only 13 compounds were tested in the
experiments on antioxidant activity. The maximum capacity for suppressing the growth of
cancerous and normal cells was revealed for compounds I9 (1–10 M), I11 (1–5 M), and I13 (5–
35 M, Table 2). Antimutagenic activity of these three compounds was detected at their lower
(comparable) concentrations: 0.71 M (I9), 0.76 M (I11), and 0.94 M (I13). Of note, the
compound I12 and I20 (Table 5) having the maximum antioxidant activity suppressed the growth
of various cancerous and normal cells remarkably worse than did compounds I9, I11, and I13: I12
(6 to >75 M), and I20 (29–57 M, Table 2).
Because none of the compounds enhanced the spontaneous mutagenesis, and all effectively
suppressed the mutagenic effect of H2O2, they can be considered effective antioxidants. In
addition, the 50% decrease in spontaneous and H2O2-dependent mutagenesis of bacterial cells
was observed at significantly lower concentrations (Table 5) than those exerting a detectable
effect on tumor cells (Tables 1 and 2).
In the case of compounds subjected to the analysis of cell protection from spontaneous and
peroxide-dependent mutagenesis, the average IC50 values were calculated for suppression of the
growth of all tumorous and noncancer cells. Then, correlation coefficients between these IC50
values of protection from revertant formation and IC50 values in experiments with spontaneous
and H2O2-dependent mutagenesis were calculated (0.34 and 0.32, respectively). The correlation
coefficients (0.32–0.34) turned out to be quite low. This means that there is no strict dependence
between the antioxidant properties of these compounds and their ability to suppress the growth
of cancer cells. The relatively low correlation coefficients may be explained as follows: in
addition to interacting with various oxidants in cells, these compounds can in different ways
interact with cells and with their various components, including proteins and enzymes, important
for the growth of cells.
3. Conclusions
In conclusion, this study revealed that polyfluorinated 2-arylindoles are an interesting class
of compounds with high antiproliferative activity toward four cancer cell lines: MCF-7, RPMI
8226, T98G, and HCT 116. The series of in vitro experiments showed that benzo-perfluorinated
indoles I9, I11, and I13 have prominent G1-targeting anticancer effects. Furthermore, DNA
fragmentation and Annexin V-FITC/PI staining assays revealed that indoles I9–I11 induced the
apoptotic cell death. Additionally, indoles exert strong antioxidant action on bacterial cells in the
presence or absence of H2O2. Generally, it was shown that that fluorinated 2-arylindoles have
more pronounced cytotoxic, antioxidant, and antimutagenic properties as compared with their
non-fluorinated analogs.
7

4. Experimental section
4.1. General experimental procedures
All solvents were purified by standard procedures. The starting polyfluorinated iodoanilines
were synthesized according to previously described methods [22,23]. Other chemicals were
obtained from commercial sources and were used without further purification. Preparative TLC
was performed on Merck precoated silica gel 60 PF254 containing gypsum. Visualization of the
developed chromatograms was performed by means of UV light. NMR spectra were recorded on
Bruker Avance-300 (300.13 MHz for 1H and 282.37 MHz for 19F), Bruker Avance-400 (400.13
MHz for 1H) and DRX-500 (500.13 MHz for 1H and 125.76 MHz for 13C) spectrometers. CDCl3
and acetone-d6 served as solvents, with residual CHCl3 (δH = 7.26) or CDCl3 (δC = 77.0) and
acetone (δH = 2.15) or acetone-d6 (δC = 28.6 and 205.0) acting as internal standards. C6F6 (δF =
163.0) was used as an external reference for recording 19F NMR spectra. 13C NMR spectra were
recorded with C–H spin decoupling. Masses of molecular ions were determined by HRMS on a
DFS Thermo scientific instrument (EI, 70 eV). Melting points were recorded on a Mettler-
Toledo FP81 Thermosystem apparatus. Elemental analyses were performed on а Euro EA-3000
CHNS analyzer, or on Carlo Erba 1106 CHN elemental analyzer. The IR spectra were recorded
on a Bruker Vector 22 spectrometer. The Raman spectrum was recorded on Ramanscope
Senterra (25 mV, 785 nm).
4.1.1 Synthesis of phenylethynylanilines A; General Procedure
To a solution of 2,3,4-trifluoro-6-iodoaniline [or o-iodoaniline] (1 mmol) and
ethynylbenzene (150 mg, 1.5 mmol) in dry MeCN (10 mL) in a Schlenk flask under argon
Pd(PPh3)2Cl2 (28 mg, 0.04 mmol), CuI (17 mg, 0.09 mmol) and Et3N (3 mL) were added. The
reaction mixture was stirred at 60 °C for 3 h. Then, the mixture was allowed to cool down to r.t.,
and CH2Cl2 (10 mL) was added. The mixture was poured into H2O (20 mL) and extracted with
CH2Cl2 (3 × 50 mL). The combined organic layers were washed with H2O (20 mL) and dried
(MgSO4). After evaporation of the solvent in vacuo, the crude product was purified by
preparative TLC to obtain the target product.
4.1.1.1 2,3,4-Trifluoro-6-(phenylethynyl)aniline (A16)
Yellowish solid; yield: 210 mg (84%); Rf = 0.58 (EtOAc/hexane, 1:10); mp 61.8–62.7 °C. IR
(KBr): 3504, 3399, 3070, 2926, 2854, 1642, 1589, 1523, 1477, 1442, 1387, 1318, 1283, 1241,
1203, 1155, 1070, 1009, 913, 868, 760, 690, 570, 524 cm–1. Raman: 2209 (vs), 1596, 1387,
1094, 998 cm–1. 1H NMR (300 MHz, CDCl3): 7.54–7.51 (m, 2 H, Hm), 7.38–7.36 (m, 3 H, Hp +
Ho), 6.98 (ddd, J (H5,F4) = 10.3 Hz, J (H5,F3) = 7.7 Hz, J (H5,F2) = 2.2 Hz, 1 H, H5), 4.25 (s, 2 H,
NH2). 13C NMR (125.8 MHz, CDCl3):  = 142.9 (ddd, 1J (C4,F4) = 239.3 Hz, 2J (C4,F3) = 10.8
Hz, C4), 140.9 (ddd, 1J (C3,F3) = 251.8 Hz, 2J (C3,F2) = 16.5 Hz, 2J (C3,F4) = 13.4 Hz, C3), 140.1
(ddd, 1J (C2,F2) = 242.2 Hz, 2J (C2,F3) = 12.4 Hz, C2), 134.5 (d, 2J (C1,F2) = 10.6 Hz, C1), 131.6
(s, C10), 129.0 (s, C12), 128.6 (s, C11), 122.4 (s, C9), 114.0 (dd, 2J (C5,F4) = 19.2 Hz, 3J (C5,F3) =
3.2 Hz, C5), 103.4 (m, C6), 96.1 (m, C7), 82.9 (m, C8). 19F NMR (282 MHz, Acetone-d6): δ = –
150.9 (dd, J (F4,F3) = 21.6 Hz, J (F4,H5) = 10.3 Hz, 1 F, F4), –156.3 (dm, J (F2,F3) = 20 Hz, J
(F2,H5) = 2.2 Hz, 1 F, F2), –159.2 (m, J (F3,F4) = 21.5 Hz, J (F3,F2) = 20 Hz, J (F3,H5) = 7.7 Hz,
8

1 F, F3). HRMS (EI): m/z [M]+ calcd for C14H8F3N: 247.0603; found: 247.0602. Anal. Calcd for
C14H8F3N: C, 68.02; H, 3.26; N, 5.67. Found: C, 67.89; H, 3.38; N, 5.53.
4.1.1.2. 2-(Phenylethynyl)aniline (A18)
Yellowish solid; yield: 150 mg (89%); Rf = 0.58 (EtOAc/hexane, 1:5); mp 85.8 °C (decomp.)
(86–87 °C [24]). NMR spectra are close to that published in literature [24]. 1H NMR (400 MHz,
Acetone-d6): 7.57–7.55 (m, 2 H, Hm), 7.39–7.35 (m, 3 H, Hp + Ho), 7.28 (dd, J (H3,H4) = 7.7 Hz,
1 H, H3), 7.09 (tm, J (H5,H4) ≈ J (H5,H6) = 8 Hz, 1 H, H5), 6.79 (dd, J (H6,H5) = 8.2 Hz, 1 H, H6),
6.60 (tm, J (H4,H5) ≈ J (H4,H3) = 7.7 Hz, 1 H, H4), 5.13 (s, 2 H, NH2). 13C NMR (125.8 MHz,
Acetone-d6):  = 150.9 (s, C1), 133.3 (s, C3), 132.6 (s, C10), 131.1 (s, C5), 129.9 (s, C12), 129.5 (s,
C11), 125.0 (s, C9), 117.8 (s, C4), 115.4 (s, C6), 108.0 (s, C2), 95.4 (s, C7), 87.8 (m, C8). Anal.
Calcd for C14H1N: C, 87.01; H, 5.74; N, 7.25. Found: C, 87.24; H, 5.66; N, 7.22.
4.1.2 Synthesis of 2-phenylindoles I; General Procedure
To a solution of A (0.5 mmol) in MeCN (3 mL), PdCl2 (24 mg, 0.1 mmol) was added, and
the mixture was heated at reflux with stirring for 3 h. The desired product was isolated by TLC.
4.1.2.1 5,6,7-Trifluoro-2-phenyl-1H-indole (I16)
Yellowish solid; yield: 118 mg (96%); Rf = 0.50 (EtOAc/hexane, 1:10); mp 160.2–160.4 °C. IR
(KBr): 3437, 3089, 1603, 1552, 1523, 1471, 1452, 1400, 1377, 1352, 1313, 1255, 1228, 1184,
1063, 962, 931, 852, 767, 727, 692, 673, 588, 553, 497, 432 cm–1. 1H NMR (300 MHz, Acetone-
d6): 11.15 (s, 1 H, NH), 7.89–7.86 (m, 2 H, Hm), 7.45 (tm, J (Har,Har) = 7.2 Hz, 2 H, Ho), 7.34 (tt,
J (Har,Har) = 7.2 Hz, 1 H, Hp), 7.25 (m, J (H4,F5) = 10.5 Hz, J (H4,F6) = 6.7 Hz, J (H4,H3) = 2 Hz,
1 H, H4), 6.91 (dd, J (H3,F7) = 3 Hz, J (H3,H4) = 2 Hz, 1 H, H3). 13C NMR (125.8 MHz, Acetone-
d6):  = 147.8 (dd, 1J (C5,F5) = 237.0 Hz, 2J (C5,F6) = 12.0 Hz, C5), 142.5 (m, C2), 139.1 (ddd, 1J
(C7,F7) = 247.5 Hz, 2J (C7,F6) = 13.4 Hz, 3J (C7,F5) = 2.4 Hz, C7), 137.4 (ddd, 1J (C6,F6) = 239.3
Hz, 2J (C6,F5) = 18.6 Hz, 2J (C6,F7) = 12.4 Hz, C6), 132.9 (s, C8), 130.3 (s, C9), 129.5 (s, C11),
126.9 (s, C10), 126.6 (m, C3a), 123.1 (dm, 2J (C7a,F7) = 10.0 Hz, C7a), 102.9 (dd, 2J (C4,F5) = 19.4
Hz, 3J (C4,F6) = 3.7 Hz, C4), 101.4 (m, C3). 19F NMR (282 MHz, Acetone-d6): δ = –145.8 (dd, J
(F5,F6) = 19.9 Hz, J (F5,H4) = 10.5 Hz, 1 F, F5), –155.7 (dm, J (F7,F6) = 19.1 Hz, J (F7,H3) = 3
Hz, J (F7,Ho) = 2 Hz, 1 F, F7), –170.0 (td, J (F6,F5) ≈ J (F6,F7) = 19.5 Hz, J (F6,H4) = 6.7 Hz, 1 F,
F6). HRMS (EI): m/z [M]+ calcd for C14H8F3N: 247.0603; found: 247.0602. Anal. Calcd for
C14H8F3N: C, 68.02; H, 3.26; N, 5.67. Found: C, 68.21; H, 3.28; N, 5.60.
4.1.2.2 2-Phenyl-1H-indole (I18)
Yellowish solid; yield: 67 mg (77%); Rf = 0.61 (EtOAc/hexane, 1:5); mp 189.1–189.6 °C (189–
191 °C [25]). NMR spectra are close to that published in literature [25]. 1H NMR (500 MHz,
Acetone-d6): 10.67 (s, 1 H, NH), 7.87–7.85 (m, 2 H, Hm), 7.57 (d, J (H4,H5) = 7.9 Hz, 1 H, H4),
7.45–7.41 (m, 3 H, Hp + Ho), 7.30 (t, J (H7,H6) ≈ J (H7,Ho) = 7.4 Hz, 1 H, H7), 7.11 (t, J (H6,H7)
≈ J (H6,H5) = 7.2 Hz, 1 H, H6), 7.03 (t, J (H5,H6) ≈ J (H5,H4) = 7.3 Hz, 1 H, H5), 6.90 (m, 1 H,
H3). 13C NMR (125.8 MHz, Acetone-d6):  = 138.7 (s, C2), 138.3 (s, C7a), 133.5 (s, C8), 130.1 (s,
C3a), 129.7 (s, C9), 128.2 (s, C11), 125.8 (s, C10), 122.6 (s, C6), 121.1 (s, C4), 120.4 (s, C5), 112.0
(s, C7), 99.9 (s, C3). Anal. Calcd for C14H1N: C, 87.01; H, 5.74; N, 7.25. Found: C, 86.98; H,
5.95; N, 7.27.
9

The other polyfluorinated compounds studied in this work were obtained according to the
methods published earlier: A1 – A12 [15], A13 – A15, A17 [22]; I1 – I12 [15], I13 – I15, I17, I19 – I21.
[14].
4.2. Cell culture
MCF-7, human myeloma RPMI 8226, human glioblastoma cells T98G, human colon
carcinoma cells HCT116 and human embryonic kidney cells HEK-293, embryonic lung
fibroblasts WI-38 (SV40 transformed), normal mouse fibroblasts LMTK, Chinese hamster AG
17 cells were used.
Cell lines RPMI 8226, MCF-7, T98G, HCT116, HEK-293, WI-38, LMTK, AG-17 were
obtained from the Russian cell culture collection (Russian Branch of the ETCS, Russia, St.
Petersburg). Cells were cultivated in Iscove’s modified Dulbecco’s media (Sigma) with 10%
FBS (GibcoBRL Co., Gaithersburg, MD), 2 mM L-glutamine (Sigma), 250 mg/mL amphotericin
B and 100 U/mL penicillin/streptomycin (GIBCO BRL Co., Gaithersburg, MD). RPMI 8226
cells were cultivated in RPMI 1640 media (Sigma) supplemented with 10% FBS, 2 mM L-
glutamine, 250 mg/ml amphotericin B and 100 U/mL penicillin/streptomycin. Cells were grown
in a humidified atmosphere of 5% CO2 in air at 37 oC and were passaged with 0.05% trypsin-
EDTA every 3–4 days.
4.3. In vitro cytotoxicity assay
To evaluate antiproliferative properties of compounds A and I, the MTT assay (by using 3-
(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) [26] was performed. Briefly,
cells were seeded at 2×103 cells/well in a 96-well plates in a total volume of 100 μL in IMDM
medium supplemented 2 mM L-glutamine (Sigma), 250 mg/mL amphotericin B and 100 U/mL
penicillin/streptomycin and 10% FBS. Cells (except for suspension cell line RPMI 8226) were
incubated overnight and 1 μL of DMSO solutions of substances with different concentrations
were added to each well. After 72-hours incubation at 37 °C, MTT was added to final
concentration of 0.5 mg/mL. Three hours later culture medium was removed, MTT formazan
crystals were solubilized in 100 μL isopropanol and absorbance was measured at 570 nm in plate
reader Multiscan-FS (Thermo Fisher Scientific). The experiments were performed in triplicate
and repeated 3÷5 times for each compound per cell line. IC50 values reflecting the relative
number of living cells normalized to the number of cells treated with 1% DMSO were calculated
from dose-response curve using Origin 8.0 software. Topothecan was used as a cell death
inducing positive control.
4.4. Annexin V binding assay
In order to determine the apoptosis induced cell death annexin V FITC and propidium
iodide staining was performed by using the Annexin V FITC apoptosis detection kit according to
manufacturer’s protocol (Sigma Aldrich). The cells were treated with concentrations of 7.5 μM
compound I9 along with the control (DMSO). After 72 h of incubation, the cells were harvested,
washed with PBS and stained with Annexin V FITC and propidium iodide. Early and late
10

apoptotic cells were quantified using Accuri C6 flow cytometer based software (BD
Biosciences).
4.5. DNA fragmentation analysis
RPMI 8226 cancer cells were seeded in well plates and incubated for 24 h. After
incubation, cells were treated with tested fluorinated compounds at a 7.5 μM concentration for
72 h. Subsequently, cells were collected and centrifuged for 5 min at 4 °C. Pellet was collected
and washed with Phosphate buffered saline (PBS), added 200 μL of Lysis buffer (6M GuSCN), 4
μL of mercapthoethanol and incubated for 20 min at 56 °C. Later 280 μL of GuSCN-containing
adsorption buffer was added. The binding solution was mixed thoroughly and transferred to the
silica spin column. The column was put in a centrifuge, centrifuged for 1 min (13000 rpm),
washed twice with buffer (4.5 M GuSCN) and centrifuged again (1 min, 13000 rpm). GuSCN
was removed by washing the column with ethanol, containing buffer (500 mkL×2). The column
was centrifuged (1 min, 13000 rpm). DNA was eluted with 30 μL of milli-Q water by
centrifugation (1 min, 13000 rpm). DNA laddering was determined by 1.8% agarose gel
electrophoresis.
4.6. Docking studies
Molecular modeling was carried out in the Schrodinger Maestro visualization environment
using applications from the Schrodinger Small Molecule Drug Discovery Suite 2017-1 package
[Schrödinger, LLC, New York, NY, 2017]. Three-dimensional structures of the derivatives were
obtained empirically in the LigPrep application using the OPLS3 force field [27]. All possible
tautomeric forms of compounds, as well as various states of polar protons of molecules in the pH
range of 7.0±2.0 were taken into account. X-ray crystallographic models of interaction of
colchicine and tubulin with PDB ID 4O2B (resolution of 2.3 Å) [28], vinblastine and tubulin
with PDB ID 5J2T (resolution 2.2 Å) [29], dictyostatin and tubulin with PDB ID 5MF4
(resolution 2.3 Å) [30] were used for the calculations. To simulate the possible mechanism of
tubulin binding, molecular docking of new compounds to the binding sites of cytostaticshas been
performed in the Glide application [31]. The search area for docking was selected automatically,
based on the size and physico-chemical properties ofcytostatics. The extra precision (XP)
algorithm of docking was applied. Docking was performed in comparison withcytostatics. The
three-dimensional structures of cytostatics were obtained in the PubChem database and prepared
in the LigPrep application. Non-covalent interactions of compounds in the binding site were
visualized using Biovia Discovery Studio Visualizer [Dassault Systèmes BIOVIA, San Diego,
2016] and UCSF Chimera 1.11.2 [32].
4.7. Determination of mutagenicity
In the Ames test, the histidine-dependent strain of S. typhimurium TA102 was used, which
carries a mutation at the histidine operon [20]. The mutagenic activity of the samples was
analyzed by the standard method without metabolic activation [20]. A liquid culture of TA102
was obtained by 16-h growth of cells from a frozen stock at 37 °C in LB medium with penicillin.
Then cells were plated on minimal glucose agar, antibiotics and histidine at the density sufficient
11

to obtain isolated colonies. A separate bacterial colony was inoculated into LB medium (5 mL)
containing ampicillin (50 g/mL) and tetracycline (2 g/mL), and grown with shaking (130 rpm)
for 15 h at 37 °C.
The Ames test was carried out using the described double-layer method [21]. The overnight
culture of bacteria (100 L) containing one of the tested compounds in different concentrations
and, if required, 3 mM H2O2, were mixed at 42 °C with 2 ml of liquid 0.6% top agar. The
mixture was poured onto plates with a minimal medium containing 0.2% glucose and 3% agar,
taking care to distribute the mixture uniformly on the surface of the solid agar. The plates were
incubated for 48 h at 37 °C, and the revertants were counted. The cells incubated with H2O2 in
the absence of compounds analyzed were used as positive controls, and the cells grown in the
absence of H2O2 and antioxidants served as negative controls for the mutation induction. The
results are expressed as mean  standard deviation of at least 3 independent experiments.
Acknowledgments
The work was supported by the Russian Foundation for Fundamental Studies under Grant № 19-
53-53003\19. The authors thank the Multi-Access Chemical Service Center SB RAS and Olga
Koval for spectral and analytical measurements.
12

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15

Reagents and conditions: (a) HCCPh, Pd(PPh3)2Cl2, CuI, Et3N, MeCN, Ar, 60 °C, 3 h; (b) PdCl2, MeCN, Ar,
reflux, 3 h.
Scheme 1. Synthesis of polyfluorinated diarylacetylenes and indoles
DMSO treated cells (control)
I9 treated cells
Fig. 1. Annexin V-FITC/propidium iodide dual staining assay. RPMI 8226 cells were treated with compound I9
(7.5 μM) for 72 h, labelled with Annexin V-FITC/PI and examined for apoptosis using flow-cytometer. Cells in the
upper left quadrant (Q1): necrotic cells; upper right quadrant (Q2): late apoptotic cells; lower left quadrant (Q3): live
cells; lower right quadrant (Q4): early apoptotic cells.
16

Fig. 2. DNA fragmentations of conjugate DMSO and I9–I11 in RPMI 8226 cell line, M is the DNA Marker 50
Kb.
Fig. 3. A: binding site for colchicine on the tubulin surface; secondary structure of chain A is orange, chain B is
blue, and the molecule of colchicine is yellow. Noncovalent interactions of molecules in the binding site: B,
colchicine; C, I9; D, A13. Hydrophobic interactions are omitted, the surface of the binding site is colored in
accordance with the degree of hydrophobicity, and noncovalent interactions are indicated by dashed lines: green,
hydrogen bonds; blue, possible weak interactions of fluorine atoms.
17

Fig. 4. Superposition of structures in the colchicine-binding site (the surface of the binding site is colored in
accordance with the degree of hydrophobicity) on the surface of tubulin. The structures are colored as follows: gray,
colchicine; green, A13 (A) and I9 (B); orange, A18 (A) and I18 (B).
A
B
140
120
100
80
140
120
100
80
- without H2O2
- with H2O2
- without H2O2
- with H2O2
60
60
40
40
20
20
0
0
0.0
0.5
1.0
1.5
2.0
2.5
0.0
0.5
1.0
1.5
2.0
[I15], M
[I12], M
Fig. 5. Analysis of the mutagenic and antioxidant activities of compounds I12 (A) and I15 (B) by a standard Ames
test on S. Typhimurium strain TA102 in the absence or presence of 3 mM H2O2. The number of revertants in the
absence of H2O2 was set to 100%. The average error in three experiments for any compound concentration did not
exceed 5–10%.
18

Inhibition of t-BuO2H-dependent mutants formation by trolox, trolox
succinate, a-tocopherol and a-tocopherl succinate.
140
120
100
80
60
40
20
0
Trolox
Trolox succinate
a-tocopherol
a-tocopheryl succinate
-0.5
0
0.5
1
1.5
2
2.5
3
3.5
mM
Fig. 6. Analysis of the mutagenic and antioxidant activities of trolox, trolox succinate, tocopherol, tocopheryl
succinate by a standard Ames test on S. Typhimurium strain TA102 in the absence or presence of 3 mM H2O2. The
number of revertants in the absence of H2O2 was set to 100%. The average error in three experiments for any
compound concentration did not exceed 5–10%.
19

Table 1. In vitro cytotoxic activities (IC50, M) of polyfluorinated diarylacetylenes A1–A22
toward cancerous and normal cell lines.
Cell viabilitya IC50 [M]
Entry Compound
RPMI
8226
HCT
116
HEK
293
WI38 LMTK
AG 17
MCF-7
T98G
1
>75
>75
>75
>75
>75
472
503
726
743
715
2
3
4
>75
>75
>75
507
725 743
>75
>75
>75
365
327
377
283
688
509
208
324
4010 397
272 313
634
5
>75
>75
>75
>75
>75
>75
353
725
732
742
6
7
8
>75
>75
>75
>75
>75
>75
634
282
603
722
>75
>75
439
736
665
736
566 373
6010
443 526
605
20

9
>75
>75
>75
>75
>75
646
724
716
243
10
196
62
245
202
145 216
254
11
629
404
666
492
382 365
726
363
12
13
215
154
143
232
342
517
365 342
425
406
155
4010 487
347 237
4010
14
15
16
>75
287
228
322
173
263
284
538
686
543
517 384
474 393
394 295
358
615
365
295
295
185
525
464
21

17
18
>75
>75
>75
>75
>75
478
555
624
443
723 595
714 492
645
465
636
[a] Compound concentration that caused 50% cell growth inhibition (IC50) was determined after 72 h treatment with
the indicated compounds. The percentage of viable cells relative to DMSO control (mean ± SD from three
independent experiments, each conducted in triplicate) was determined by the MTT assay.
Table 2. In vitro cytotoxic activities (IC50, M) of polyfluorinated indoles I1–I21 toward
cancerous and normal cell lines.
Cell viabilitya IC50 [M]
Entry Compound
RPMI
8226
HCT
116
HEK
293
AG
17
WI38 LMTK
MCF-7
>75
T98G
1
>75
>75
>75
>75
>75
>75
562
503
494
685
554
2
3
>75
>75
>75
>75
>75
ND
ND
NDb
607
385
563
437
4
5
6
7
8
345
325
484
275
364
82
196
339
373
134
710
273
2010
324 3010 272
>75
>75
243
282
106
73
446
414
182
62
499
355
204
23
325
391
256
333
192
255
244 468
211 13
22

9
102
215
33
33
11
92
44
72
12
65
10
11
12
13
14
92
123
55
194
51
199
12
247 284
193
23
31
31
52
>75
174
54
144
66
253
114
205
349
74
429
113
183
263
63
107 218
184 315
358
194
436
77
213
142
15
16
228
324
147
334
256
455
252
414
214
364
234 495
385 504
204
>75
17
18
>75
>75
ND
ND
306
502
3710
394
727
13
225
665
283
523
605
19
20
21
1510
574
547
186
5010
507
285
503
524
182
294
494
284
272
402
174 142
474 537
494 635
242
417
516
23

22
NDc
2.20.5 0.40.5
0.10.5




Colchicine (control)
[a] Compound concentration that caused 50% cell growth inhibition (IC50) was determined after 72 h treatment with
the indicated compounds. The percentage of viable cells relative to DMSO control (mean ± SD from three
independent experiments, each conducted in triplicate) was determined by the MTT assay.
[b] ND means not effective. Cell viability was equal to or greater than 50% of the DMSO control at 100 M
compound concentration; therefore, accurate IC50 values for these compounds were not determined.
[c] See SI.
24

Table 3. I9-induces phosphatidylserine exposure. RPMI 8226 cells were treated with 7.5 μM I9
for 72 h and analyzed by flow cytometry after annexin V/ PI staining.
Cell population
Q1 FITC-/PI+
Q2 FITC+/PI+
Q3 FITC-/PI-
Q4 FITC+/PI-
Cells treated with DMSO
Cells treated with I9
0.1%
0.1%
9.4%
7.0%
78.8%
11.6 %
57.6%
35.3%
Table 4. Docking scores of the tested compounds with tubulin in the colchicine-, dictyostatin-
and vinblastine-binding sites.
Compound
Control
Predicted docking score, kcal/mol
Colchicine
-7.49
Dictyostatin
Vinblastin
-6.16
-4.60
-4.98
-5.15
-4.87
-3.90
-5.03
-4.66
-6.21
-5.12
-4.34
-5.00
-3.88
-4.97
-4.38
-3.84
-6.63
-6.45
I9
I11
I13
I18
A12
A13
A18
-5.91
-5.80
-5.65
-6.24
-5.88
Table 5. IC50 values characterizing suppression of spontaneous and H2O2-induced mutagenesis
by polyfluorinated compounds.
IC50, µM*
Suppression of H2O2-induced and
spontaneous mutagenesis
Suppression of spontaneous
mutagenesis
(from 100% to 50%)
Compound
From 138% to
100%
From 100% to
50%
A2
A5
A10
A12
I2
I4
I7
I9
I10
I11
I12
I14
I15
I17
I19
I20
I21
1.21±0.01
1.31±0.01
0.72±0.05
0.72±0.04
1.00±0.07
0.43±0.02
0.60±0.04
0.71±0.05
0.63±0.03
0.76±0.05
0.22±0.01
0.94±0.06
0.94±0.05
1.18±0.07
1.34±0.08
0.34±0.02
0.71±0.05
0.050±0.003
0.060±0.004
0.043±0.002
0.037±0.002
0.053±0.003
0.057±0.003
0.074±0.004
0.037±0.002
0.037±0.002
0.030±0.001
0.028±0.001
0.049±0.002
0.25±0.001
0.079±0.004
0.073±0.003
0.040±0.002
0.056±0.004
0.93±0.04
1.0±0.06
0.52±0.03
0.72±0.03
0.78±0.04
0.75±0.03
0.77±0.04
0.43±0.02
0.37±0.02
0.57±0.03
0.16±0.01
1.10±0.05
1.0±0.06
1.1±0.05
0.86±0.04
0.13±0.01
0.78±0.03
*Mean ± S.D. from three independent experiments.
25