Fluorinated analogs of malachite green: Synthesis and toxicity

Article abstract of DOI:10.3390/molecules13040986

A series of fluorinated analogs of malachite green (MG) have been synthesized and their toxicity to Saccharomyces cerevisiae and a human ovarian epithelial cell line examined. The toxicity profiles were found to be different for these two species. Two ana

Full text of DOI:10.3390/molecules13040986

Molecules 2008, 13, 986-994
molecules
ISSN 1420-3049
© 2008 by MDPI
www.mdpi.org/molecules
Full Paper
Fluorinated Analogs of Malachite Green: Synthesis and Toxicity
George A. Kraus 1,*, Insik Jeon 1, Marit Nilsen-Hamilton 2, Ahmed M. Awad 2,
Jayeeta Banerjee 2 and Bahram Parvin 3
1
Iowa State University Department of Chemistry, Ames, IA 50011, USA; Tel. +1 515-294-7794;
Fax: +1 515-294-0105
2
Iowa State University Department of Biochemistry, Biophysics and Molecular Biology, Ames, IA
50011, USA; Tel. +1 515-9996; Fax: +1 515-294-0453
3 Lawrence Berkeley Laboratory Department of Cancer Biology, Berkeley, CA 94720, USA; Tel. +1
510-486-6203; Fax: +1 510-486-6363
* Author to whom correspondence should be addressed; E-mail: gakraus@iastate.edu
Received: 21 March 2008; in revised form: 22 April 2008 / Accepted: 22 April 2008 / Published: 27
April 2008
Abstract: A series of fluorinated analogs of malachite green (MG) have been synthesized
and their toxicity to Saccharomyces cerevisiae and a human ovarian epithelial cell line
examined. The toxicity profiles were found to be different for these two species. Two
analogs, one with 2,4-difluoro substitution and the other with 2-fluoro substitution seem to
be the most promising analogs because they showed the lowest toxicity to the human cells.
Keywords: Malachite Green; Malachite Green analogs; Bathochromic shift; Toxicity;
Saccharomyces cerevisiae; human ovarian epithelial cells.
Introduction
We are developing a new procedure for real-time imaging of gene expression. It involves a nucleic
acid probe that can be used in conjunction with a 18F-labeled target molecule to image gene expression
in vivo [1]. The probe has the basic feature that its terminal segments hybridize to form a stem in the
absence of the target mRNA. One of the terminal segments is an aptamer and its activity is inhibited

Molecules 2008, 13
987
by hybridization with the other terminal segment (the attenuator). The presence of the target mRNA
will result in hybridization of the probe via the intervening sequence (located between the aptamer and
attenuator) with the target. Hybridization will release of the constraint on the aptamer with the result
that the aptamer will bind its target and localize the 18F-labeled malachite green (MG) to the tissue site
of gene expression.
In considering the aptamer to use for developing this imaging approach, we chose the malachite
green one for our initial studies. This aptamer has a high affinity for its ligand and has been shown to
function inside yeast cells. The use of MG in an imaging context would also constitute an entirely new
18
application for this compound, which could easily be linked to F for imaging purposes. In addition,
the fluorescent output of MG is increased over 2000 fold when it binds to the aptamer, which would
allow imaging in cell culture without the use of a radiolabeled MG [2].
Fluorinated derivatives of MG have the potential for being labeled with 18F for in vivo tracking and
are likely to substitute for MG in binding the MG aptamer. However, a critical question is whether
these MG derivatives are toxic to human cells. In this study we have tested this question and compared
the toxicity levels for human cells with those for yeast cells in which the MG aptamer has been
previously shown to function [3].
The general usefulness of this study is: 1) the intrinsic value of the defined toxicity range of MG
and some new derivatives for cells from humans and yeast, 2) specific information for groups working
with the MG aptamer to provide information regarding potential intracellular ligands for probe
development, and 3) the report of new chemical syntheses and the properties of new MG analogs.
Only two isolated reports of malachite green analogs bearing fluorines directly attached to the
aromatic rings have been published [4,5]. Compared with MG (1), substitution of a fluorinated moiety
reduced electron-donating capability resulting in a hypsochromic shift [6]. Here we present the
chemical syntheses of a series of analogs with fluorine substituents located at various positions on the
aromatic rings of MG. Their spectral properties and toxicities in yeast and humans are described.
Figure 1
Cl-
N
N
1: R1 = R2 = R3 = H
2: R1 = H, R2 = F, R3 = H
3: R1 = F, R2 = R3 = H
4: R1 = R2 = H, R3 = F
5: R1 = H, R2 = R3 = F
6: R1 = H, R2 = Cl, R3 = H
R1
R1
R2
R3
Results and Discussion
Preparation and Characteristics of MG analogs
Analogs 2, 4, and 5 were synthesized from the reaction of dimethyl aniline and the requisite
benzaldehyde in the presence of para-toluenesulfonic acid, as shown in Scheme 1. Oxidation to the
MG analog was achieved using lead dioxide. This oxidant provided the MG analogs in almost
quantitative yields. The oxidation of leucomalachite green using manganese dioxide, potassium

Molecules 2008, 13
988
permanganate and potassium persulfate has been reported [7]. Our oxidation procedure is rapid and
convenient. Analog 3 was prepared from 3-fluoro dimethylaniline and benzaldehyde.
Scheme 1.
O
N
X
H
N
N
X
N
N
X
R
PTSA
PbO2
+
X
R
R
X
R'
R'
R'
X = H, F R, R' = F, H
The absorption spectra in the visible range of each analog dissolved in water were determined in
the range of 300 to 700 nm. (Figure 2). All analogs were measured at 20 μM except analog 3, which
was measured at 500 μM. The figure inset shows the wavelengths of maximal absorption (λmax) in nm
and the molar extinction coefficient in units of M-1cm-1. All analogs showed a strong hypochromatic
shift compared with MG as demonstrated by the extinction coefficients at the wavelength of maximum
absorbance.
Figure 2. Visible absorption spectra.
The extinction coefficients for MG (1) are comparable to two literature values of 186,100 M-1 cm-1
determined in 20 mM sodium acetate buffer pH 4.0 [8], and 148,900 M-1 cm-1 determined in water [9].
The λmax of 617 nm for MG is the same as has been previously published [8, 9]. The three ortho-
substituted rings as well as the 3',3'' analog showed a red-shift in λmax of over 10 nm (9 nm for the 3',3''

Molecules 2008, 13
989
analog). It has been reported recently that MG also showed a red-shift in the maximum absorption
frequency by 14 nm after binding to the RNA MG aptamer [10]. In association with the aptamer, the
MG rings adopt a more coplanar relationship, compared with their orientation in untethered MG. The
more planar conformation leads to a more extended π-system in the aptamer-bound MG and a red-shift
in the MG λmax. A substitution in the 2-position of the 4',4''-di-(N-methyl-N-2,2,2-trifluoroethyl-
amino)-triphenylmethyl cation was also reported to result in bathochromic shifts of λmax [6]. Charge
delocalization by the fluorine substituents would result in rotation of the phenyl rings to produce a
more planar overall conformation of the analogs compared with MG. The observed red-shift in λmax of
analogs 2, 3, and 5, can be similarly explained by charge delocalization that require the rings to adopt a
coplanar orientation.
By contrast with the other analogs, the para-substituted ring in the analog 4 shows a small
hypsochromic shift (from 617 to 614 nm). This small shift can be attributed to reduction of the
negative charge on the phenyl ring produced by introduction of the electron withdrawing fluorine atom
and resulting in less intense π→π* transitions.
Toxicity of MG analogs
The synthesized analogs were tested for their toxicity to yeast as a representative fungus and to
cultured A2780 human ovarian epithelial cells as representative mammalian cells (Figure 3).
Figure 3. Comparison of MG analog toxicities for mammalian cells.
A2780 human ovarian epithelial cells were treated with the indicated analogs at concentrations
from 6.3 x 10-12 M to 1 x 10-4 M. The results represent the average of results from nine independently
performed experiments, which provided compiled data such that the effect of each analog was
measured in triplicate in each of two or three independent experiments. Shown are the data from 5 x

Molecules 2008, 13
990
10-8 M to 1 x 10-5 M. Within each experiment, the fraction of dead cells from the DMSO vehicle
controls were subtracted from each analog treatment. On average over all experiments, the fraction of
dead cells (with the vehicle addition alone) was 0.12 ± 0.04 and the average coefficient of variation for
all values was 33%. The limiting toxic concentrations determined from these experiments are shown in
Table 1. The toxicity of each of the analogs to A2780 cells was determined as described in
Experimental Procedures. Thirteen concentrations of each analog were tested including the highest
(10-4 M) and lowest (6.25 x 10-12 M). The limiting toxic concentration is the lowest concentration at
which toxicity of 10% or more above the control (vehicle) was detected. The differences of 5-10-fold
in limiting toxic concentration between analogs are well outside the estimated average range of error
(33%) of these estimates.
Table 1. Limiting toxic concentration for malachite green analogs.
Limiting toxic
Analog
concentration
1
2
3
4
5
6
1 x 10-7
1 x 10-6
1 x 10-7
1 x 10-7
1 x 10-6
5 x 10-7
First, the limiting toxic concentration of the analogs for A2780 cells was determined using a YO-
PRO®-1-based assay with automated image analysis. The limiting toxic concentration was defined as
the lowest concentration at which the percentage of dead cells was increased above the basal level. The
basal level was established by a control series to which the vehicle, DMSO, alone was added at the
same concentrations as included with the analogs. The limiting toxic concentrations determined from
these studies showed that 1 and analogs 3 and 4 were equally toxic as MG for human cells, whereas
analogs 2, 5 and 6 were less toxic (Table 1).
The toxicity profile for these analogs was different for yeast and humans (Figure 4). Saccharo-
myces cerevisiae, strain Y2158 and A2780 human ovarian epithelial cells were treated with the
indicated analogs at concentrations of 0, 0.1, 0.5, 1 and 5 μM. In particular, analog 3, one of the most
toxic for animal cells, was not toxic to yeast in the concentration range tested. On the other hand,
human cells were less sensitive to analog 5 than yeast cells. These studies have revealed one analog of
malachite green that can be used in yeast up to at least 5 μM and two analogs that can be used with
mammalian cells at concentrations below 1 μM without toxicity.

Molecules 2008, 13
991
Figure 4. Comparison of toxicity of MG analogs for yeast and human cells. All cell numbers are
expressed as fractions of the DMSO control. Bars: 0.1 μM (parallel slash), 0.5 μM (white), 1 μM (45°
slash), 5 μM (black). The error bars show standard deviations. Each yeast point is the average of 3
independent sets of experiments. The mammalian data is the average of two to three experiments with
triplicate independent estimates in each experiment. Error bars are only shown where the values for
more than one experiment have been averaged.
Experimental
General
YO-PRO®-1 iodide was from Invitrogen and DMSO was from Fisher. Hoechst 33342, YPD broth
and malachite green oxalate were from Sigma. 2Cl-MG was purchased from TCI. Spectra were
measured using a UV/VIS-S2100 Diode Array Spectrophotometer (Biochrom Ltd). Toxicity for yeast

Molecules 2008, 13
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was measured by growing the yeast in a New Brunswick Scientific C24 shaker incubator set for 220
1
rpm. Toxicity for human cells was measured using a Cellomics Arrayscan II HCS system (Zeiss). H-
13
and C-NMR experiments were performed with a Varian 300 MHz instrument operating at 300 and
75 MHz, respectively. All chemical shifts are reported relative to the solvent (CDCl3) peak (7.27 ppm
1
for H and 77.23 ppm for 13C). Coupling constants (J) are reported in Hz with peak multiplicities
indicated by the following abbreviaions: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet.
High resolution mass spectra were recorded on a Kratos model MS-50 spectrometer. Standard grade
silica gel (60 A, 32-63 μm) was used for flash column chromatography.
General procedure for the synthesis of fluorinated malachite green analogs
Aldehyde (10 mmol), N,N-dimethylaniline (40 mmol), p-toluenesulfonic acid (10 mmol), and
benzene (20 mL) were placed in a flask fitted with a Dean-Stark trap and boiled for 9 h. The reaction
mixture was diluted with benzene (40 mL) and washed twice with 10% sodium bicarbonate solution
and brine. After solvent evaporation, the residue was purified by column chromatography on silica gel
(10:1 hexane-ethyl acetate) to afford 95 – 100 % yield of the fluorinated leucomalachite green analog.
This compound (0.5 mmol) in H2O (3 mL) was then treated with PbO2 (0.65 mmol) and 15 drops of
concentrated HCl. The reaction was stirred at room temperature overnight. The mixture was poured
into a separatory funnel containing methanol (30 mL) and HCl and methylene chloride. After
extraction, drying and concentration in vacuo, the residue was purified by chromatography on silica
gel (CH2Cl2 -methanol = 9:1) to afford pure MG analogs (100% yield).
1
(2) 2-F-MG: H-NMR: δ 7.65-7.58 (m, 1H), 7.36 (d, J = 9.0 Hz, 4H), 7.32-7.13 (m, 3H), 6.95 (d, J =
9.3 Hz, 4H), 3.37 (s, 12H); 13C-NMR: δ 169.02, 162.92, 159.54, 157.14, 140.13, 135.11, 134.59,
134.47, 127.41, 127.39, 127.12, 126.96, 124.65, 124.60, 116.72, 116.43, 114.34, 41.43; EI-HRMS
calcd for C23H24FN2+ m/z [M+] 347.19235, found 347.19278.
(3) 3′, 3′′ DiF-MG: 1H-NMR: δ 7.69 (t, J = 7.5 Hz, 1H), 7.52 (t, J = 7.8 Hz, 2H), 7.31 (d, J = 7.2 Hz,
2H), 7.02 (t, J = 9.0 Hz, 2H), 6.77 (dd, J = 9.3, 2.4 Hz, 2H), 6.54 (dd, J = 14.7, 2.4 Hz, 2H), 3.40 (s,
13
12H); C-NMR: δ 169.01, 167.64, 167.62, 164.13, 164.10, 159.31, 159.13, 140.29, 140.27, 140.24,
139.97, 134.54, 133.98, 129.01, 118.55, 118.37, 110.83, 100.17, 99.80, 41.74; EI-HRMS calcd for
C23H23F2N2+ m/z [M+] 365.18293, found 365.18340.
1
(4) 4F-MG: H-NMR: δ 7.36-7.20 (m, 8H), 6.97 (d, J = 9.3 Hz, 4H), 3.38 (s, 12H); 13C-NMR: δ
175.86, 167.72, 164.31, 157.14, 140.88, 137.15, 137.03, 135.70, 135.66, 127.31, 116.42, 116.13,
114.13, 41.38; EI-HRMS calcd for C23H24FN2+ m/z [M+] 347.19235, found 347.19278.
1
(5) 2,4-DiF-MG: H-NMR: δ 7.40 (d, J = 9.3 Hz, 4H), 7.24-7.19 (m, 1H), 7.12-7.01 (m, 6H), 3.42 (s,
13
12H); C-NMR: δ 167.45, 163.96, 163.80, 163.46, 163.29, 160.10, 160.04, 159.88, 159.29, 157.03,
139.86, 136.57, 136.53, 136.44, 136.40, 127.10, 123.28, 123.24, 123.12, 123.07, 114.29, 112.50,
112.46, 112.22, 112.17, 105.38, 105.04, 104.70, 41.27; EI-HRMS calcd for C23H23F2N2+ m/z [M+]
365.18293, found 365.18340.

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Procedure for Measuring Toxicity of MG Analogs to Yeast
Saccharomyces cerevisiae, strain Y2158 were grown at 30°C in YPD culture medium (Sigma-
Aldrich, St. Louis MO) for 18 h and then diluted to OD600=0.4 into YPD medium with 0.032% DMSO
and the indicated concentration of the MG analog. The cells were incubated with shaking (18
mL/flask) at 30°C for 24h with samples being removed at 4, 6, 8, 10, and 24 h. The samples were
analyzed for OD600 and growth curves plotted. For most cases, all cell cultures had reached a growth
plateau by 24 h. The 10 h time points were chosen for comparison of toxicity because the cultures
were still in the log phase of growth at that time.
Procedure for Measuring Toxicity of MG Analogs to Human Cells
Human A2780 ovarian epithelial cancer cells were plated in RPMI (GIBCO) containing 10% fetal
bovine serum at 2,000 cells per well in a 96-well plate (100 μL/well) and grown for 24 h at 37°C in a
5% CO2 atmosphere. The MG analogs were added at the indicated concentrations and the cells
incubated for another 48 h. The medium was removed and fresh medium containing 1 μM YO-PRO®-
1 iodide (Invitrogen) and 10 μg/mL Hoechst 33342 (Sigma) stain was added to the cells. The cells
were incubated for 30 min at 37°C in a 5% CO2 atmosphere in the dark before counting the YO-
PRO®-1 and Hoechst stained nuclei using the optimal wavelengths YO-PRO®-1of λem=485 nm and
λex=530 nm. Ten fields were counted in each well at a magnification of 10X which resulted in 2,000-
6,000 nuclei being counted in control wells. The number of live cells per well were determined by
subtracting the number of nuclei labeled with YO-PRO®-1 from the number of nuclei labeled with
Hoechst in each well. Treatments were done in triplicate in each experiment. Cells were considered to
be dead if they stained with YO-PRO®-1 or were not attached to the substratum. Because higher
concentrations of drugs resulted in loss of significant numbers of cells from the substratum, the
averages of triplicates (Hoechst minus YO-PRO®-1) for each treatment were normalized to the total
number of cells in the control wells (no addition) to determine the fraction of live cells. These values
were subtracted from 1.0 to determine the fraction of dead cells. Before averaging the results from
different experiments, the average % dead cells in the vehicle control was subtracted from each value.
Acknowledgements
The authors extend their thanks to Dr. Nicolas Wang for running Cellomic assays for the
mammalian cell toxicity tests. This work is supported by the Director, Office of Energy Science
Research, Office Medical Sciences, Life Sciences of the U. S. Department of Energy under Contracts
W-7405-Eng-82 with the Ames National Laboratories and DE-AC03-76SF00098 with the University
of California. This is publication number LBNL-59259.
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