2′,6′-Dihalostyrylanilines, pyridines, and pyrimidines for the inhibition of the catalytic subunit of methionine s-adenosyltransferase-2

Article abstract of DOI:10.1021/jm5004864

Inhibition of the catalytic subunit of the heterodimeric methionine S-adenosyl transferase-2 (MAT2A) with fluorinated N,N-dialkylaminostilbenes (FIDAS agents) offers a potential avenue for the treatment of liver and colorectal cancers where upregulation of this enzyme occurs. A study of structure-activity relationships led to the identification of the most active compounds as those with (1) either a 2,6-difluorostyryl or 2-chloro-6- fluorostyryl subunit, (2) either an N-methylamino or N,N-dimethylamino group attached in a para orientation relative to the 2,6-dihalostyryl subunit, and (3) either an N-methylaniline or a 2-(N,N-dimethylamino)pyridine ring. These modifications led to FIDAS agents that were active in the low nanomolar range, that formed water-soluble hydrochloride salts, and that possessed the desired property of not inhibiting the human hERG potassium ion channel at concentrations at which the FIDAS agents inhibit MAT2A. The active FIDAS agents may inhibit cancer cells through alterations of methylation reactions essential for cancer cell survival and growth.

Full text of DOI:10.1021/jm5004864

Article
pubs.acs.org/jmc
Open Access on 06/20/2015
2′,6′-Dihalostyrylanilines, Pyridines, and Pyrimidines for the
Inhibition of the Catalytic Subunit of Methionine
S‑Adenosyltransferase‑2
Vitaliy M. Sviripa,†,§ Wen Zhang,†,∥ Andrii G. Balia,†,§ Oleg V. Tsodikov,‡ Justin R. Nickell,‡
Florence Gizard,‡ Tianxin Yu,†,∥ Eun Y. Lee,∥ Linda P. Dwoskin,‡ Chunming Liu,*,†,∥
and David S. Watt*,†,§,∥
‡
§
†Department of Molecular and Cellular Biochemistry, Department of Pharmaceutical Sciences, College of Pharmacy, Center for
∥
Pharmaceutical Research and Innovation, and Markey Cancer Center, University of Kentucky, Lexington, Kentucky 40506-0509,
United States
S
* Supporting Information
ABSTRACT: Inhibition of the catalytic subunit of the heterodimeric
methionine S-adenosyl transferase-2 (MAT2A) with fluorinated N,N-
dialkylaminostilbenes (FIDAS agents) offers a potential avenue for the
treatment of liver and colorectal cancers where upregulation of this
enzyme occurs. A study of structure−activity relationships led to the
identification of the most active compounds as those with (1) either a 2,6-difluorostyryl or 2-chloro-6-fluorostyryl subunit, (2)
either an N-methylamino or N,N-dimethylamino group attached in a para orientation relative to the 2,6-dihalostyryl subunit, and
(3) either an N-methylaniline or a 2-(N,N-dimethylamino)pyridine ring. These modifications led to FIDAS agents that were
active in the low nanomolar range, that formed water-soluble hydrochloride salts, and that possessed the desired property of not
inhibiting the human hERG potassium ion channel at concentrations at which the FIDAS agents inhibit MAT2A. The active
FIDAS agents may inhibit cancer cells through alterations of methylation reactions essential for cancer cell survival and growth.
improved (nanomolar) potency, water solubility, and the
desired property of not interacting with the human ether-a-
INTRODUCTION
■
̀
The development of antineoplastic agents with novel molecular
targets opens the door to new, potentially valuable treatment
strategies. We previously reported the effect of difluorinated
N,N′-dialkylaminostilbenes (FIDAS agents) on the prolifer-
ation of colon and liver cancer cells. We identified (E)-4-(2′,6′-
difluorostyryl)-N,N-dimethylaniline (FIDAS 1a, Figure 1) as a
lead structure with in vitro activity in LS174T cells in the low
micromolar range1−3 and utilized a biotinylated analogue of this
FIDAS agent to identify the catalytic subunit of the
heterodimeric enzyme, methionine S-adenosyl transferase-2
(MAT2A), as the sole binding partner.2 We validated MAT2A
as the target by suppression using shRNA, which demonstrated
in vitro depression in S-adenosylmethionine (SAM) and S-
adenosylhomocysteine (SAH) levels in cells exposed to FIDAS
agents. Also, we demonstrated in vivo activity of these FIDAS
agents on human colon cancer using mouse xenograft studies1
and in a three-dimensional culture model of primary CRC
organoids that mimics the microenvironment of tumors
(Supporting Information Figure S2). Other reports describe
hydroxy- or methoxy-substituted stilbenes as potential antineo-
plastic agents,4−10 but experience directed our study of
structure−activity relationships (SAR) away from oxygenated
stilbenes because of their potential for redox reactions and off-
target biological effects.
go-go-related (hERG) potassium channel. In general, drug
candidates must avoid hERG activation to progress toward
investigational new drug (IND) status, and our commitment to
moving FIDAS agents down this pathway led us to incorporate
hERG testing in our evaluation of candidates produced in this
SAR study. Binding to the hERG channel results in QT interval
prolongation in the electrocardiogram and adverse cardiac
events.15 Drug-induced ventricular fibrillation, in these cases,
may lead to sudden death and hence the need to identify drug
candidates that do not bind hERG at concentrations of the
FIDAS agents that inhibit MAT2A.
RESULTS
■
Synthesis of FIDAS Agents. A Wadsworth−Emmons
condensation of 2,6-dihalobenzyl diethyl phosphonates with
the appropriate 4-(N,N-dimethylamino)benzaldehydes secured
the (E)-4-(2′,6′-dihalostyryl)-N,N-dimethylanilines (1a−3a)
(Figure 2).17 Demethylation of 1a−3a using cyanogen
bromide18 secured the (E)-4-(2′,6′-dihalostyryl)-N-methylani-
lines (1b−3b). Synthesis of the corresponding pyridine and
pyrimidine analogues in the N,N-dimethylamino series also
employed the Wadsworth−Emmons condensation of the 2,6-
dihalobenzyl diethyl phosphonates with the appropriate
Since FIDAS agents offer a potentially new avenue for the
treatment of liver and colorectal cancers where MAT2 levels are
upregulated,11−14 we sought analogues of FIDAS 1a with
Received: March 27, 2014
Published: June 20, 2014
© 2014 American Chemical Society
6083
dx.doi.org/10.1021/jm5004864 | J. Med. Chem. 2014, 57, 6083−6091

Journal of Medicinal Chemistry
Article
Figure 1. FIDAS agents: Top row contains FIDAS agents in the aniline family; second row contains FIDAS agents in the 5-aminopyridine family;
third row contains FIDAS agents in the 2-aminopyridine family; and bottom row contains FIDAS agents in the 2-aminopyrimidine family.
pyridine-2-carboxaldehyde, pyridine-3-carboxaldehyde, and pyr-
imidine-5-carboxaldehyde and secured (E)-2-(2′,6′-dihalostyr-
yl)-5-(N,N-dimethylamino)pyridines (4a−6a), (E)-5-(2′,6′-di-
halostyryl)-2-(N,N-dimethylamino)pyridines (7a−9a), and
(E)-5-(2′,6′-dihalostyryl)-2-(N,N-dimethylamino)pyrimidines
(10a−12a), respectively (Figure 2). Synthesis of the corre-
sponding pyridine and pyrimidine analogues in the N-
methylamino series employed the Wadsworth−Emmons
condensation of the 2,6-dihalobenzyl diethyl phosphonates
with the appropriate pyridine-3-carboxaldehyde and pyrimi-
dine-5-carboxaldehyde and secured (E)-5-(2′,6′-dihalostyryl)-2-
(N-methylamino)pyridines (7b−9b) and (E)-5-(2′,6′-dihalos-
tyryl)-2-(N-methylamino)pyrimidines (10b−12b), respectively
(Figure 2). Synthesis of 1c involved the N-monoethylation of
(E)-4-(2′,6′-difluorostyryl)aniline3 (Figure 3). Synthesis of 7c
involved the initial Wadsworth−Emmons condensation of 2,6-
difluorobenzyl diethyl phosphonate with 2-(tert-
butyloxycarbonyl)aminopyridine-5-carboxaldehyde, N-ethyla-
tion, and deprotection of the tert-butoxycarbonyl group with
trifluoracetic acid (Figure 3). Synthesis of 10c involved the
Wadsworth−Emmons condensation of 2,6-difluorobenzyl
diethyl phosphonate with 2-(N-ethylamino)pyridine-5-carbox-
aldehyde (Figure 3). No E/Z isomerization of these FIDAS
agents, as determined by 1H NMR of freshly prepared
solutions, occurred when samples were stored as solids in the
dark at low temperatures. For biological experiments, freshly
prepared DMSO stock solutions were prepared, diluted with
buffer, and used immediately as described below.
MAT2A Inhibition Assays. Inherent fluorescence of the
2′,6′-difluorostyryl- and 2′-chloro-6′-fluorostyryl-substituted
FIDAS agents at 454 nm facilitated the development of a
fluorescence anisotropy assay to evaluate the binding between
MAT2A and FIDAS agents.19 Restriction of this assay to only
those FIDAS agents that displayed inherent fluorescence led to
the application of another, previously reported, MAT2A
inhibition assay to evaluate the activity of FIDAS agents.2
Recombinant MAT2A holoenzyme was purified and used to
synthesize SAM from methionine and ATP.16 For this assay,
each FIDAS analogue was preincubated with MAT2A
holoenzyme and then mixed with methionine and ATP.
Phosphate Pi concentrations were analyzed to determine the
level of MAT2A activity.20 Final 30 μM concentrations of
FIDAS agents were used for these experiments, which were
repeated in triplicate. The ratio of MAT2A inhibition for
6084
dx.doi.org/10.1021/jm5004864 | J. Med. Chem. 2014, 57, 6083−6091

Journal of Medicinal Chemistry
Article
Figure 2. Synthetic route to FIDAS agents with N-methylamino or N,N-dimethylamino groups. Reagents: (a) NaH, DMF followed by 4-
(CH3)2N(C6H4)CHO to give 1a−3a; (b) CNBr, acetone, 16 h, 56 °C followed by conc. HCl, 3 h, reflux; (c) NaH, DMF followed by 5-
(CH3)2N(C6H3N)-2-CHO to give 4a−6a; (d) NaH, DMF followed by 2-(CH3)2N(C6H3N)-5-CHO to give 7a−9a; (e) NaH, DMF followed by 2-
CH3NH(C6H3N)-5-CHO to give 7b−9b; (f) NaH, DMF followed by 2-(CH3)2N(C6H2N2)-5-CHO to give 10a−12a; (g) NaH, DMF followed by
2-CH3NH(C6H2N2)-5-CHO to give 10b−12b.
Figure 3. Synthetic route to FIDAS agents with N-ethylamino groups. Reagents: (a) NaH, DMF followed by 4-O2N(C6H4)CHO; (b) SnCl2, HOAc,
conc. HCl; (c) K2CO3, CH3CH2I, acetone; (d) NaH, DMF followed by 4-tBuOCONH(C6H4)CHO; (e) NaH, C2H5I; (f) CF3CO2H; (g) NaH,
DMF followed by 2-CH3CH2NH(C6H2N2)-5-CHO.
selected FIDAS agents relative to FIDAS 1a is summarized in
Figure 4.
(Table 1 and Supporting Information Figure S1). The IC50
values were calculated using Prism 5 analysis of the data from
the concentration−response curves for each compound.
Concentrations of FIDAS agents ranging from 1 to 300 nM
were used for these experiments, which were repeated in
triplicate at each concentration. Among these four families, only
compound 5a in the 5-aminopyridine family showed an IC50
value comparable to that of FIDAS 1a. Because this outcome
In Vitro Testing of FIDAS Agents with LS174T
Colorectal Cancer Cells. We treated LS174T colon cancer
cells with FIDAS agents 1−3 in the aniline family, 4−6 in the 5-
aminopyridine family, 7−9 in the 2-aminopyridine family, and
10−12 in the 2-aminopyrimidine family, and we determined
the IC50 value of each compound on LS174T cell proliferation
6085
dx.doi.org/10.1021/jm5004864 | J. Med. Chem. 2014, 57, 6083−6091

Journal of Medicinal Chemistry
Article
≈ IC50 value for 2a; IC50 value for 5a < IC50 value for 4a; and
IC50 value for 8a ≈ IC50 value for 7a. Replacing the 2′,6′-
difluoro substituents in FIDAS 1a with 2′,6′-dichloro
substituents led, in general, to relatively inactive compounds
in comparison with that of their difluorinated counterparts:
IC50 value for 3a > IC50 value for 1a; IC50 value for 6a ≈ IC50
value for 4a; IC50 value for 9a > IC50 value for 7a; and IC50
value for 12a > IC50 value for 10a.
Replacing the N,N-(dimethylamino)-substituent in FIDAS
1a with an N-(methylamino) substituent led, generally, to
compounds with increased inhibitory activity: IC50 value for 1b
< IC50 value for FIDAS 1a; IC50 value for 2b ≈ IC50 value for
2a; and IC50 value for 3b < IC50 value for 3a. Replacing the N-
methylamino substituent in 1b with the sterically larger N-
ethylamino-substituent in 1c led to inactive compounds in
some cases (e.g., IC50 for 1c > IC50 for 1b) or compounds with
comparable activity (e.g., IC50 for 7b ≈ IC50 for 7c; IC50 for
10b ≈ IC50 for 10c). In summary, the introduction of either the
N-methylamino substituent in 1b and 2b or the introduction of
the 2′-chloro-6′-fluorostyryl moiety in 8a and 8b led to the
most potent compounds with IC50 values less than 10 nM
(Table 1).
Figure 4. Ratio of MAT2A inhibition for selected FIDAS agents
relative to FIDAS 1a. Recombinant MAT2A holoenzyme was purified
and used to synthesize SAM from methionine and ATP. For inhibition
assay, each FIDAS analogue was preincubated with MAT2A
holoenzyme and then mixed together with methionine and ATP.
The reaction products are SAM and Pi. Pi concentration was analyzed
to determine the MAT2A activity. Inhibition activity of each
compound was compared with that of FIDAS 1a.
[3H]-Dofetilide Binding to Plasma Membranes Over-
expressing the hERG Channel. [3H]-Dofetilide competition
binding assays using HEK-293 cell membranes stably
expressing the hERG channel (hERG-HEK) correlate well
with results from voltage-clamp assays and provide useful
predictive screening assays for QT prolongation.21 We utilized
a [3H]-dofetilide binding assay to evaluate FIDAS agent
interaction with hERG. Amitriptyline (final concentration, 1
mM) was used as the positive control and exhibited an IC50
value (10.7 2.25 μM) that was in agreement with published
values.22 We selected a subset of the FIDAS agents in Figure 1
that possessed potent in vitro inhibition with LS174T
colorectal cancer cells and that represented the structural
subtypes in this SAR study (i.e., (E)-4-(2′,6′-difluorostyryl)-
N,N-(dimethyl)aniline (FIDAS 1a); (E)-2-(2′,6′-difluorostyr-
yl)-5-N,N-(dimethylamino)pyridine (4a); (E)-5-(2′,6′-difluor-
ostyryl)-2-N,N-(dimethylamino)pyridine (7a); and (E)-5-
(2′,6′-difluorostyryl)-2-N,N-(dimethylamino)pyrimidine (10a)
as well as variants with different halogenation and methylation
patterns). Concentrations of FIDAS agents ranging from 10−9
to 10−4 M were assayed in duplicate for these experiments (n =
3 experiments/analogue). IC50 values for hERG inhibition of
[3H]-dofetilide binding ranged from 20 to 60 μM. Ratios of the
IC50 values for the hERG inhibition and IC50 values for the
inhibition of LS174T cell proliferation (Table 1) ranged from 2
to 4 orders of magnitude for the subset of FIDAS agents that
were studied.
Table 1. Comparison of IC50 Values for the Inhibition of
LS174T Cancer Cells, IC50 for hERG Inhibition Values, and
the Ratio of IC50 for hERG Inhibition to the IC50 Values for
the Inhibition of LS174T Cancer Cells
inhibition of
LS174T cell
proliferation IC50
(nM)
ratio of IC50 for hERG
inhibition to IC50 for inhibition
of LS174T cell proliferation
(× 103)
hERG
inhibition
IC50 (μM)
FIDAS
1a
26.9 6.5
7.6 1.2
199.7 3.3
19.2 6.4
7.6 0.5
213.8 7.3
50.1 6.7
57.9 11.6
20.4 4.1
59.5 10.2
13.3 7.8
30.1 5.8
31.3 5.5
4.7 0.6
4.0 0.2
30.1 3.1
19.6 8.5
>200
32.1.1 4.4
49.1 11.1
1.2
6.5
1b
1c
2a
2b
3a
49.8 5.1
6.6
3b
4a
21.1 1.1
20.7 1.8
0.4
1.0
5a
6a
7a
22.0 3.6
1.7
7b
7c
8a
59.5 17.8
19.9 4.1
12.7
5.0
8b
9a
9b
10a
10b
10c
11a
11b
12a
12b
40.5 9.7
0.5
>200
>200
DISCUSSION
■
>200
We reported that (E)-4-(2′,6′-difluorostyryl)-N,N-dimethylani-
line (FIDAS 1a) selectively bound to the catalytic subunit of
methionine S-adenosyltranferase-2 (MAT2A), an enzyme
upregulated in various liver and colorectal cancers.1−3 FIDAS
1a possessed in vitro and in vivo activity against various liver
and colorectal cancer cell lines, possessed good oral
bioavailability and a reasonable half-life in vivo, and displayed
minimal gross toxicity in terms of body weight loss/death at
doses exceeding those that produced in vivo tumor reduction in
a xenograft model.1,2 In seeking analogues with increased
potency and improved water solubility relative to that of
FIDAS 1a, we undertook a SAR study that included pyridine
>200
>200
>200
did not represent a significant advance, further work with the 5-
aminopyridine family was not pursued. Similarly, the 2-
aminopyrimidines 10−12 displayed IC50 values in the 200−
900 nM range and also were not pursued in detail.
Replacing the 2′,6′-difluoro-substituents in FIDAS 1a with
2′-chloro-6′-fluoro substituents led to compounds with
comparable or increased inhibitory activity: IC50 value for 1a
6086
dx.doi.org/10.1021/jm5004864 | J. Med. Chem. 2014, 57, 6083−6091

Journal of Medicinal Chemistry
Article
and pyrimidine rings into the FIDAS platform (Figure 1). We
also focused on the modification of substituents, such as the
halogen and the N,N-dimethylamino substituents in FIDAS 1a,
as a means of improving potency. Finally, potency alone is no
longer sufficient to guide translational drug development. In
developing these FIDAS agents as potential drug candidates, we
sought FIDAS agents that avoided binding to the hERG
potassium channel associated with drug-induced, adverse
cardiac events.15 This study describes our success in meeting
these requirements (i.e., nanomolar potency, water solubility,
and absence of hERG activation) into the FIDAS platform.
We synthesized and evaluated four families of FIDAS agents,
as shown in Figure 1: (E)-4-(2′,6′-dihalostyryl)-N-alkyl- and
(E)-4-(2′,6′-dihalostyryl)-N,N-(dialkyl)anilines (1−3), which
we refer to as the aniline family; (E)-2-(2′,6′-dihalostyryl)-5-
(N-alkylamino)- and (E)-2-(2′,6′-dihalostyryl)-5-(N,N-
dialkylamino)pyridines (4−6), which we refer to as the 5-
aminopyridine family; (E)-5-(2′,6′-dihalostyryl)-2-(N-alkylami-
no)- and (E)-5-(2′,6′-dihalostyryl)-2-(N,N-dialkylamino)-
pyridines (7−9), which we refer to as the 2-aminopyridine
family; and (E)-5-(2′,6′-dihalostyryl)-2-(N-alkylamino)- and
(E)-5-(2′,6′-dihalostyryl)-2-(N,N-dialkylamino)pyrimidines
(10−12), which we refer to as the 2-aminopyrimidine family.
Syntheses relied principally on the Wadsworth−Emmons
coupling of arylphosphonates with aryl aldehydes (Figures 2
and 3). FIDAS agents were characterized fully, and their purity
was established by a combination of 13C nuclear magnetic
resonance (NMR) and combustion analyses.
We explored variations in the location, number, and nature of
the two halogen substituents within FIDAS 1a. As reported
previously, alterations in the location of the two halogens, other
than the 2,6-dihalostyryl arrangement, invariably led to
diminished activity in a cell proliferation in vitro assay using
LS174T cells.1−3 The inclusion of halogen substituents larger
than chlorine or the introduction of more than two halogen
substituents diminished or produced no appreciable increase in
activity (data not shown). We found, however, that the
substitution of one, but not both, of the fluorines in FIDAS 1a
with a chlorine substituent resulted in slightly improved levels
of in vitro activity. For example, the 2′-chloro-6′-fluoro
analogue, 2a, was comparable in activity to that of FIDAS 1a
and was 10-fold better than that of 3a (Table 1).
Modification of the alkyl groups in the N,N-(dialkylamino)-
phenyl subunit of FIDAS 1a produced an even more dramatic
improvement in activity than alteration in just the halogen
substituents. Prior studies1−3 had established that only the para
orientation of the N,N-dimethylamino group and the 2,6-
dihalostyryl group possessed good biological activity; con-
sequently, we did not explore ortho or meta orientations of
these groups in this study. Within the para series, the removal
of one of the methyl groups, as in the monomethyl analogues,
1b, 2b, and 3b, produced an active series with in vitro potencies
in the 7−50 nM range (Table 1). Additional modifications
involving substitution of a larger N-alkyl group than N-methyl,
such as N-ethyl in FIDAS 1c, led to diminished in vitro activity.
Inclusion of either N-alkylamino- or N,N-(dialkylamino)-
pyridine or pyrimidine rings in place of the N,N-
(dimethylamino)phenyl ring in FIDAS 1a offered an attractive
option for addressing the water-solubility issue and improving
potency. There are some inconsistencies between MAT2A
inhibition and cell proliferation inhibition, particularly in the
case of 8b, suggesting that other characteristics, such as stability
and solubility, may also contribute to the efficacy of FIDAS
agents in cell or in vivo assays. Although the hydrochloride salt
of FIDAS 1a was readily absorbed in a mouse bioavailability
study, the in vitro IC50 was only 27 nM in LS174T cells. We
found experimentally that the N-methyl analogues, 1b and 2b,
had IC50 values for the inhibition of LS174T cells that were 4-
fold greater than that of 1a. We also performed a proof-of-
concept experiment using primary CRC cells. We found that 2b
significantly inhibited the growth of CRC organoids (Support-
ing Information Figure 2), which further suggests that these
agents are potential candidates for CRC treatment.
With the objective of developing antineoplastic drugs with
limited affinity for human hERG, we utilized a [3H]-dofetilide
binding assay to evaluate the interaction of a subset of these
FIDAS agents with hERG. Ratios of the IC50 values for the
inhibition of LS174T cell proliferation and the IC50 values for
hERG inhibition (Table 1) were, as desired, larger than 2−4
orders of magnitude, with the exceptions of 4a and 10a.
Because these latter two FIDAS agents were also in families that
were the least active of the four families studied, namely, the 5-
aminopyridines (4−6) and the 2-aminopyrimidines (10−12),
they were set aside for further SAR development. The most
active compounds (i.e., 1b, 2b, 8a, and 8b) did not interact
appreciably with hERG and possess ratios of IC50 values for
hERG inhibition relative to IC50 values for inhibition of
LS174T cell proliferation (Table 1) that were greater than that
of FIDAS 1a and that are well within the selectivity range for
drugs entering preclinical development.23
In summary, four key findings emerged from these studies:
(1) FIDAS agents in the aniline family (1−3) and in the 2-
aminopyridine family (7−9) possess lower IC50 values in the
inhibition of LS174T cell proliferation than that of the 5-
aminopyridines (4−6) and the 2-aminopyrimidines (10−12),
(2) the most active FIDAS agents possessed either 2,6-
difluorostyryl or 2-chloro-6-fluorostyryl subunits, (3) the most
active FIDAS agents possessed small N-alkyl groups, specifically
the N-methylamino or the N,N-dimethylamino groups, in a
para orientation relative to the 2,6-dihalostyryl subunit, and (4)
2-aminopyridines 8a and 8b not only displayed IC50 values less
than 10 nM but also formed water-soluble hydrochloride salts.
In summary, we developed potent analogues of FIDAS 1a that
exhibited significantly increased ratios of IC50 for hERG
inhibition to IC50 for inhibition of LS174T cell proliferation,
thereby opening the door to further development of these
compounds as potential antineoplastic drugs.
EXPERIMENTAL PROCEDURES
■
Chemistry. Chemicals were purchased from Sigma-Aldrich
(Milwaukee, WI) or Fisher Scientific (Pittsburgh, PA) or were
synthesized according to literature procedures. Solvents were used
from commercial vendors without further purification unless otherwise
noted. Nuclear magnetic resonance spectra were determined on a
Varian instrument (1H, 400 MHz; 13C, 100Mz). High-resolution
electrospray ionization (ESI) mass spectra were recorded on a LTQ-
Orbitrap Velos mass spectrometer (Thermo Fisher Scientific,
Waltham, MA, USA). Resolution was set at 100 000 (at 400 m/z).
Samples were introduced through direct infusion using a syringe pump
with a flow rate of 5 μL/min. Purity of compounds was greater than
95%, as established using combustion analyses determined by Atlantic
Microlabs, Inc., Norcross, GA. Compounds were chromatographed on
preparative layer Merck silica gel F254 unless otherwise indicated.
General Procedure for the Synthesis of FIDAS Agents. To a
solution of 1.65 mmol (1.1 equiv) of diethyl phosphonate in 5 mL of
anhydrous DMF at 0 °C was added 2.25 mmol (1.5 equiv) of sodium
hydride (washed with anhydrous hexanes to remove oil). The mixture
was stirred for 15 min, and 1.5 mmol (1 equiv) of appropriate
6087
dx.doi.org/10.1021/jm5004864 | J. Med. Chem. 2014, 57, 6083−6091

Journal of Medicinal Chemistry
Article
aldehyde dissolved in 1 mL of anhydrous DMF was added dropwise at
0 °C. The mixture was stirred 12 h at 25 °C and quenched by pouring
into 30 mL of water with stirring. A precipitate was collected by
filtration and purified by recrystallization and/or chromatography as
noted for individual compounds described below. Several compounds
in this study were reported previously: FIDAS 1a,1−3 1b,2,3 2a,2,3 2b,2,3
3a,3 and 3b.3
(m, 2H), 6.87−6.81 (m, 2H), 6.49 (d, 1H, J = 9.2 Hz), 2.80 (d, 3H, J =
4.8 Hz). 13C NMR (DMSO-d6): δ 159.95 (dd, J1 = 247.4 Hz, J = 8.4
Hz, two C), 159.37, 148.44, 133.03, 132.9 (t, J = 8.0 Hz), 127.97 (t, J =
10.6 Hz), 120.60, 114.61 (t, J = 15.9 Hz), 111.96 (dd, J1 = 19.4 Hz, J2
= 6.4 Hz, two C), 109.55, 108.31, 27.89. HRMS (ESI) calcd for
C14H13F2N2 [MH+], 247.10413; found, 247.10318. Anal. Calcd for
C14H12F2N2: C, 68.28; H, 4.91. Found: C, 68.12; H, 5.04.
(E)-5-(2′,6′-Difluorostyryl)-2-(N-ethylamino)pyridine (7c).
The general procedure was repeated using 2-(tert-butyloxycarbonyl)-
aminopyridine-5-carboxaldehyde and 2,6-difluorobenzyl diethyl phos-
phonate to afford (E)-2-(tert-butoxycarbonylamino)-5-(2′,6′-
difluorostyryl)pyridine: yield 86%, mp 190−191 °C (from dichloro-
(E)-4-(2′,6′-Difluorostyryl)-N-ethylaniline (1c). To a mixture of
150 mg (0.65 mmol) of (E)-4-(2′,6′-difluorostyryl)aniline3 and 100
mg (0.72 mmol, 1.1 equiv) of K2CO3 in 3 mL of acetone was added
101 mg (0.65 mmol, 1 equiv) of iodoethane. The mixture was refluxed
for 12 h, poured into water, and extracted with CH2Cl2. The combined
organic phases were dried over anhydrous MgSO4 and evaporated to
give a product that was purified by chromatography using 1:7 ethyl
acetate−hexane (Rf = 0.43) to afford 103 mg (61%) of 1c as a white
1
methane). H NMR (CDCl3): δ 9.16 (br s, 1H), 8.45 (d, 1H, J = 2.4
Hz), 8.04 (d, 1H, J = 8.8 Hz), 7.89 (dd, 1H, J1 = 8.8 Hz, J2 = 2.4), 7.36
(d, 1H, J = 16.8 Hz), 7.12−7.20 (m, 1H), 7.08 (d, 1H, J = 16.8 Hz),
6.88−6.95 (m, 2H), 1.58 (s, 9H). 13C NMR (CDCl3): δ 160.95 (dd, J1
= 250.4 Hz, J = 7.6 Hz, two C), 152.61, 152.15, 146.88, 135.12, 131.10
(t, J = 8.6 Hz), 128.04 (t, J = 10.6 Hz), 115.02, 114.50 (t, J = 15.1 Hz),
112.28, 111.61 (dd, J1 = 19.4 Hz, J2 = 6.4 Hz, two C), 81.10, 28.38
(three C). HRMS (ESI) calcd for C18H19F2N2O2 [MH+], 333.14091;
found, 333.13949. Anal. Calcd for C18H18F2N2O2: C, 65.05; H, 5.46.
Found: C, 65.12; H, 5.59. To 615 mg of (E)-2-(tert-butoxycarbony-
lamino)-5-(2′,6′-difluorostyryl)pyridine (1.85 mmol) in 18 mL of
anhydrous N,N-dimethylformamide at 0 °C was added 163 mg of 60%
sodium hydride (4.07 mmol) in portions. The suspension was stirred
for 20 min while maintaining the temperature below 5 °C, and 0.16
mL of ethyl iodide (2.04 mmol) was added dropwise. The mixture was
stirred at 5 °C for 30 min and allowed to stir at 25 °C for 12 h. The
reaction was quenched with water, extracted with dichloromethane,
and washed successively with water, 0.1 M hydrochloric acid solution,
saturated aqueous NaHCO3 solution, and brine, and dried over
anhydrous MgSO4 to afford 633 mg (95%) of (E)-2-(N-(tert-
butoxycarbonyl)-N-ethylamino)-5-(2′,6′-difluorostyryl)pyridine as a
clear, colorless oil that was used in the next step without further
purification. To 613 mg (1.7 mmol) of (E)-2-(N-(tert-butoxycarbon-
yl)-N-ethylamino)-5-(2′,6′-difluorostyryl)pyridine in 17 mL of di-
chloromethane was added 4.4 mL of trifluoroacetic acid (57.1 mmol).
The mixture was stirred for 12 h at 25 °C. A precipitate was collected
and dissolved in water, cooled to 0 °C, and neutralized with aqueous
solution of Na2CO3. The product was dissolved in dichloromethane,
washed with water and brine, and dried over anhydrous MgSO4. The
product was purified by chromatography using 1:3 ethyl acetate−
hexane (Rf = 0.23) to afford 398 mg (90%) of 7c as a white solid. mp
96−97 °C. 1H NMR (CDCl3): δ 8.16 (br s, 1H), 7.70 (d, 1H, J = 7.6
Hz), 7.31 (d, 1H, J = 16.8 Hz), 7.13−7.06 (m, 1H), 6.91−6.86 (m,
3H), 6.39 (d, 1H, J = 8.8 Hz), 4.73 (br s, 1H), 3.33 (m, 2H, J = 7.2
Hz), 1.26 (t, 3H, J = 7.2 Hz). 13C NMR (CDCl3): δ 160.79 (dd, J1 =
249.7 Hz, J = 7.6 Hz, two C), 158.48, 148.32, 134.06, 132.12 (t, J = 8.3
Hz), 127.10 (t, J = 10.6 Hz), 122.74, 115.11 (t, J = 15.1 Hz), 111.53,
111.47 (dd, J1 = 19.7 Hz, J2 = 6.8 Hz, two C), 106.56, 36.92, 14.82.
HRMS (ESI) calcd for C15H15F2N2 [MH+], 261.11978; found,
261.11883. Anal. Calcd for C15H14F2N2: C, 69.22; H, 5.42. Found: C,
69.27; H, 5.43.
1
solid: mp 50−51 °C. H NMR (CDCl3): δ 7.38 (d, 2H, J = 8.4 Hz),
7.36 (d, 1H, J = 16.4 Hz), 7.12−7.03 (m, 1H), 6.94−6.84 (m, 3H),
6.59 (d, 2H, J = 8.8 Hz), 3.71 (br s, 1 H), 3.19 (q, 2H, J = 7.2 Hz),
1.27 (t, 3H, J = 7.2 Hz). 13C NMR (CDCl3): δ 160.80 (dd, J1 = 248.9
Hz, J2 = 8.4 Hz, two C), 148.55, 135.33 (t, J = 8.4 Hz), 128.02 (two
C), 127.71, 126.61 (t, J = 10.0 Hz), 115.58 (t, J = 15.6 Hz), 112.63
(two C), 111.41 (dd, J1 = 19.8 Hz, J2 = 6.8 Hz, two C), 110.61, 38.30,
14.80. HRMS (ESI) calcd for C16H16F2N [MH+], 260.12453; found,
260.12384. Anal. Calcd for C16H15F2N: C, 74.11; H, 5.83. Found: C,
74.20; H, 5.90.
(E)-2-(2′,6′-Difluorostyryl)-5-(N,N-dimethylamino)pyridine
(4a). Yield 81%, mp 90−92 °C (from hexane). 1H NMR (DMSO-d6):
δ 8.17 (d, 1H, J = 3.2 Hz), 7.42−7.28 (m, 4H), 7.18−7.11 (m, 2H),
7.06 (dd, 1H, J1 = 8.4 Hz, J2 = 3.2 Hz), 2.98 (s, 6H). 13C NMR
(DMSO-d6): δ 160.24 (dd, J1 = 248.2 Hz, J = 8.4 Hz, two C), 145.47,
142.08, 134.78, 134.42 (t, J = 8.0 Hz), 128.49 (t, J = 11.0 Hz), 123.50,
118.19, 114.20 (t, J = 15.2 Hz), 112.62, 112.00 (dd, J1 = 19.4 Hz, J2 =
6.4 Hz, two C), 39.56 (two C). HRMS (ESI) calcd for C15H15F2N2
[MH+], 261.11978; found, 261.11884. Anal. Calcd for C15H14F2N2: C,
69.22; H, 5.42. Found: C, 69.33; H, 5.59.
(E)-2-(2′-Chloro-6′-fluorostyryl)-5-(N,N-dimethylamino)-
pyridine (5a). Yield 80%, Rf = 0.44 (ethyl acetate−hexane 1:5), mp
78−80 °C. 1H NMR (DMSO-d6): δ 8.20 (d, 1H, J = 2.4 Hz), 7.53 (d,
1H, J = 16.4 Hz), 7.39−7.25 (m, 5H), 7.06 (dd, 1H, J1 = 8.4 Hz, J2 =
3.2 Hz), 2.98 (s, 6H). 13C NMR (DMSO-d6): δ 160.61 (d, J = 249.7
Hz), 145.52, 141.91, 135.26 (d, J = 12.9 Hz), 134.88, 133.39 (d, J = 6.1
Hz), 128.64 (d, J = 9.9 Hz), 125.96 (d, J = 3.8 Hz), 123.72, 123.55 (d,
J = 14.4 Hz), 118.12, 116.88 (d, J = 2.3 Hz), 115.18 (d, J = 23.5 Hz),
39.54 (two C). HRMS (ESI) calcd for C15H15ClFN2 [MH+],
277.09023; found, 277.08939. Anal. Calcd for C15H14ClFN2: C,
65.10; H, 5.10. Found: C, 65.04; H, 5.20.
(E)-2-(2′,6′-Dichlorostyryl)-5-(N,N-dimethylamino)pyridine
(6a). Yield 86%, mp 85−86 °C (from hexane). 1H NMR (DMSO-d6):
δ 8.19 (d, 1H, J = 3.2 Hz), 7.52 (d, 2H, J = 7.6 Hz), 7.42−7.36 (m,
2H), 7.30−7.26 (m, 1H), 7.15 (d, 1H, J = 16.0 Hz), 7.07 (dd, 1H, J1 =
8.8 Hz, J2 = 3.2 Hz), 2.98 (s, 6H). 13C NMR (DMSO-d6): δ 145.56,
141.62, 136.25, 134.78, 134.03, 133.53 (two C), 129.00 (two C),
128.78, 123.39, 120.27, 118.20, 39.58 (two C). HRMS (ESI) calcd for
C15H15Cl2N2 [MH+], 293.06068; found, 293.05986. Anal. Calcd for
C15H14Cl2N2: C, 61.45; H, 4.81. Found: C, 61.55; H, 4.75.
(E)-5-(2′-Chloro-6′-fluorostyryl)-2-(N,N-dimethylamino)-
1
pyridine (8a). Yield 76%, mp 63−65 °C (from hexane). H NMR
(DMSO-d6): δ 8.23 (d, 1H, J = 2.4 Hz), 7.87 (dd, 1H, J1 = 9.0 Hz, J2 =
2.6 Hz), 7.38−7.35 (m, 1H), 7.29−7.26 (m, 2H), 7.20 (d, 1H, J = 16.8
Hz), 6.97 (d, 1H, J = 16.8 Hz), 6.70 (d, 1H, J = 9.2 Hz), 3.07 (s, 6H).
13C NMR (DMSO-d6): δ 160.30 (d, J = 248.9 Hz), 158.75, 148.02,
133.81, 133.50 (d, J = 12.1 Hz), 132.97 (d, J = 6.0 Hz), 128.40 (d, J =
10.6 Hz), 125.87 (d, J = 3.1 Hz), 123.95 (d, J = 15.1 Hz), 120.30,
115.14 (d, J = 23.5 Hz), 114.25 (d, J = 2.3 Hz), 106.05, 37.64 (two C).
HRMS (ESI) calcd for C15H15ClFN2 [MH+], 277.09023; found,
277.08944. Anal. Calcd for C15H14ClFN2: C, 65.10; H, 5.10. Found: C,
65.01; H, 5.21.
(E)-5-(2′,6′-Difluorostyryl)-2-(N,N-dimethylamino)pyridine
1
(7a). Yield 87%, mp 103−104 °C (from hexane). H NMR (DMSO-
d6): δ 8.23 (d, 1H, J = 2.4 Hz), 7.89 (dd, 1H, J1 = 9.0 Hz, J2 = 2.4 Hz),
7.33−7.24 (m, 2H), 7.16−7.09 (m, 2H), 6.89 (d, 1H, J = 16.8 Hz),
6.68 (d, 1H, J = 8.8 Hz) 3.06 (s, 6H). 13C NMR (DMSO-d6): δ 159.97
(dd, J1 = 247.0 Hz, J = 7.2 Hz, two C), 158.70, 148.03, 133.79, 132.60
(t, J = 8.0 Hz), 128.13 (t, J = 10.6 Hz), 120.48, 114.54 (t, J = 15.6 Hz),
111.97 (dd, J1 = 19.4 Hz, J2 = 6.4 Hz, two C), 110.14, 106,04, 37.65
(two C). HRMS (ESI) calcd for C15H15F2N2 [MH+], 261.11978;
found, 261.11885. Anal. Calcd for C15H14F2N2: C, 69.22; H, 5.42.
Found: C, 69.32; H, 5.44.
(E)-5-(2′-Chloro-6′-fluorostyryl)-2-(N-methylamino)pyridine
1
(E)-5-(2′,6′-Difluorostyryl)-2-(N-methylamino)pyridine (7b).
Yield 83%, mp 130−132 °C (from ethanol−water). 1H NMR
(DMSO-d6): δ 8.12 (d, 1H, J = 2.4 Hz), 7.78 (dd, 1H, J1 = 8.6 Hz,
J2 = 2.6 Hz), 7.32−7.24 (m, 1H), 7.23 (d, 1H, J = 16.8 Hz), 7.16−7.08
(8b). Yield 86%, mp 106−108 °C (from hexane). H NMR (DMSO-
d6): δ 8.12 (d, 1H, J = 2.4 Hz), 7.76 (dd, 1H, J1 = 9.0 Hz, J2 = 2.6 Hz),
7.37−7.33 (m, 1H), 7.29−7.24 (m, 2H), 7.17 (d, 1H, J = 16.4 Hz),
6.91 (d, 1H, J = 16.4 Hz), 6.87 (q, 1H, J = 4.6 Hz), 6.51 (d, 1H, J = 8.8
6088
dx.doi.org/10.1021/jm5004864 | J. Med. Chem. 2014, 57, 6083−6091

Journal of Medicinal Chemistry
Article
Hz), 2.80 (d, 3H, J = 4.8 Hz). 13C NMR (DMSO-d6): δ 160.29 (d, J =
248.1 Hz), 159.44, 148.46, 133.80 (d, J = 11.4 Hz), 133.06, 132.93 (d,
J = 5.3 Hz), 128.28 (d, J = 8.3 Hz), 125.86 (d, J = 3.0 Hz), 124.02 (d, J
= 15.2 Hz), 120.43, 114.14 (d, J = 22.7 Hz), 113.68, 108.33, 27.90.
HRMS (ESI) calcd for C14H13ClFN2 [MH+], 263.07458; found,
263.07375. Anal. Calcd for C14H12ClFN2: C, 64.01; H, 4.60. Found: C,
64.13; H, 4.63.
(E)-5-(2′,6′-Dichlorostyryl)-2-(N,N-dimethylamino)pyridine
(9a). Yield 78%, mp 54−56 °C (from hexane). 1H NMR (DMSO-d6):
δ 8.22 (d, 1H, J = 2.8 Hz), 7.87 (dd, 1H, J1 = 8.8 Hz, J2 = 2.8 Hz), 7.51
(d, 2H, J = 8.0 Hz), 7.29−7.25 (m, 1H), 7.00 (d, 1H, J = 16.8 Hz),
6.92 (d, 1H, J = 16.8 Hz), 6.70 (d, 1H, J = 8.8 Hz), 3.07 (s, 6H). 13C
NMR (DMSO-d6): δ 158.78, 147.82, 134.47, 134.19, 133.98, 133.44
(two C), 128.90 (two C), 128.69, 119.86, 117.80, 106.01, 37.66 (two
C). HRMS (ESI) calcd for C15H15Cl2N2 [MH+], 293.06068; found,
293.05978. Anal. Calcd for C15H14Cl2N2: C, 61.45; H, 4.81. Found: C,
61.51; H, 4.96.
(m, 1H), 7.33−7.25 (m, 2H), 7.12 (d, 1H, J = 16.8 Hz), 7.04 (d, 1H, J
= 16.8 Hz), 2.83 (d, 3H, J = 4.8 Hz). 13C NMR (DMSO-d6): δ 162.30,
160.38 (d, J = 248.9 Hz), 156.45 (two C), 133.18 (d, J = 5.3 Hz),
130.97 (d, J = 11.4 Hz), 128.86 (d, J = 9.9 Hz), 125.97 (d, J = 3.1 Hz),
123.79 (d, J = 14.4 Hz), 118.98, 115.36, 115.13, 27.99. HRMS (ESI)
calcd for C13H12ClFN3 [MH+], 264.06983; found, 264.06918. Anal.
Calcd for C13H11ClFN3: C, 59.21; H, 4.20. Found: C, 59.19; H, 4.34.
(E)-5-(2′,6′-Dichlorostyryl)-2-(N,N-dimethylamino)-
1
pyrimidine (12a). Yield 82%, mp 103−104 °C (from hexane). H
NMR (DMSO-d6): δ 8.64 (s, 2H), 7.52 (d, 2H, J = 8.0 Hz), 7.31−7.27
(m, 1H), 7.04 (d, 1H, J = 16.8 Hz), 6.94 (d, 1H, J = 16.8 Hz), 3.16 (s,
6H). 13C NMR (DMSO-d6): δ 161.28, 156.09 (two C), 134.20, 133.49
(two C), 131.42, 129.03, 128.90 (two C), 118.92, 117.84, 36.76 (two
C). HRMS (ESI) calcd for C14H14Cl2N3 [MH+], 294.05593; found,
294.05516. Anal. Calcd for C14H13Cl2N3: C, 57.16; H, 4.45. Found: C,
57.26; H, 4.59.
(E)-5-(2′,6′-Dichlorostyryl)-2-(N-methylamino)pyrimidine
(12b). Yield 91%, mp 191−193 °C (from ethanol). 1H NMR
(DMSO-d6): δ 8.58 (s, 2H), 7.52 (d, 2H, J = 8.0 Hz), 7.42 (q, 1H, J =
4.8 Hz), 7.31−7.27 (m, 1H), 7.02 (d, 1H, J = 16.8 Hz), 6.91 (d, 1H, J
= 16.8 Hz), 2.83 (d, 3H, J = 5.2 Hz). 13C NMR (DMSO-d6): δ 162.29,
156.34 (two C), 134.26, 133.50 (two C), 131.57, 128.99, 128.88 (two
C), 118.62, 118.46, 27.93. HRMS (ESI) calcd for C13H12Cl2N3 [MH
+], 280.04028; found, 280.03979. Anal. Calcd for C13H11Cl2N3: C,
55.73; H, 3.96. Found: C, 55.77; H, 4.07.
Biological Studies. MAT2A Inhibition Assay. L-Methionine (50
μM) and ATP (50 μM) were incubated with purified His-tagged
MAT2A holoenzyme (3 μg) in 0.3 mL of reaction buffer (50 mM Tris
pH8.0, 50 mM KCl, 10 mL MgCl2) at room temperature for 25 min.
The Pi released from the reaction was measured with SensoLyte
Malachite Green (MG) phosphate assay kit (AnaSpec, 71103). The
absorbance was measured at 635 nm on a microplate reader (Spectra
MR, DYNEX Technologies). For the inhibition assay, MAT2A
holoenzyme was incubated with FIDAS agents at room temperature
for 10 min and then mixed with L-methionine and ATP in 0.3 mL of
reaction buffer.
Cell Proliferation Studies. LS174T colon cancer cells were plated
into 12-well plates (3 × 104 cells/mL). On the next day, the cells were
treated with FIDAS agents. Effects of FIDAS analogues on cell
proliferation were analyzed using Cell Viability Analyzer (Beckman
Coulter, Vi-Cell XR).
hERG Binding Studies. The HEK-293 cell line stably expressing
the hERG potassium channel (accession no. U04270), referred to as
hERG-HEK cells, were received at passage 11 (P11) from Millipore
(CYL3006, lot 2, Billerica, MA). [3H]-Dofetilide (specific activity of 80
Ci/mmol; labeled on the N-methyl group) was obtained from
American Radiolabeled Chemicals (St. Louis, MO). Other chemicals
and solvents were obtained from Sigma-Aldrich (Milwaukee, WI) with
exceptions of polyethylenimine (PEI), which was obtained from
Fluka/Sigma-Aldrich (St. Louis, MO), and minimium essential
medium (MEM) with GlutaMAX and phenol red, MEM nonessential
amino acids solution (NEAA, 100×), G418 disulfate salt solution, fetal
bovine serum (FBS), 0.05% Trypsin-EDTA 1× with phenol red, and
Hank’s balanced salt solution (HBSS), which were obtained from Life
Technologies (Carlsbad, CA).
hERG-HEK Cell Culture. hERG-HEK cells were cultured
according to the protocol provided by Millipore. Cells were
maintained in MEM (with glutamax and phenol red) supplemented
with 10% FBS, 1% NEAA. and 400 μg/mL Geneticin, and incubated at
37 °C in a humidified atmosphere with 5% CO2. Frozen aliquots of
cells were transferred into T-75 cm2 flasks and allowed to adhere for
4−8 h. The medium was replaced every 2 days. Passages were carried
out at least three times at 6 day intervals after thawing. Cells were
dissociated with trypsin/EDTA and seeded into new 150 × 25 mm
dishes at (2−3) × 106 cells per dish and placed at 30 °C, 5% CO2, for
40−48 h prior to membrane preparation. Membrane preparation
occurred 6 days after the last passage (passage 20).
(E)-5-(2′,6′-Dichlorostyryl)-2-(N-methylamino)pyridine (9b).
1
Yield 88%, mp 129−131 °C (from hexane). H NMR (DMSO-d6):
δ 8.11 (d, 1H, J = 2.0 Hz), 7.77 (dd, 1H, J1 = 9.0 Hz, J2 = 2.6 Hz), 7.50
(d, 2H, J = 8.0 Hz), 7.28−7.24 (m, 1H), 6.97 (d, 1H, J = 16.4 Hz),
6.88−6.83 (m, 2H), 6.50 (d, 1H, J = 8.4 Hz), 2.80 (d, 3H, J = 4.8 Hz).
13C NMR (DMSO-d6): δ 159.44 148.23, 134.52, 134.47, 133.43 (two
C), 133.24, 128.89 (two C), 128.60, 119.97, 117.21, 108.22, 27.89.
HRMS (ESI) calcd for C14H13Cl2N2 [MH+], 279.04503; found,
279.04430. Anal. Calcd for C14H12Cl2N2: C, 60.23; H, 4.33. Found: C,
60.35; H, 4.51.
(E)-5-(2′,6′-Difluorostyryl)-2-(N,N-dimethylamino)pyrimidine
(10a). Yield 85%, mp 134−136 °C (from hexane). 1H NMR (DMSO-
d6): δ 8.65 (s, 2H), 7.35−7.28 (m, 1H), 7.20 (d, 1H, J = 17.2 Hz),
7.17−7.10 (m, 2H), 7.00 (d, 1H, J = 17.2 Hz), 3.16 (s, 6H). 13C NMR
(DMSO-d6): δ 161.61, 160.43 (dd, J1 = 248.2 Hz, J = 7.6 Hz, two C),
156.55 (two C), 130.23 (t, J = 8.0 Hz), 128.99 (t, J = 10.6 Hz), 118.88,
114.65 (t, J = 15.2 Hz), 112.43 (dd, J1 = 19.4 Hz, J2 = 6.4 Hz, two C),
111.75, 37.17 (two C). HRMS (ESI) calcd for C14H14F2N3 [MH+],
262.11503; found, 262.11417. Anal. Calcd for C14H13F2N3: C, 64.36;
H, 5.02. Found: C, 64.45; H, 5.03.
(E)-5-(2′,6′-Difluorostyryl)-2-(N-methylamino)pyrimidine
(10b). Yield 90%, mp 143−144 °C (from ethanol). 1H NMR
(DMSO-d6): δ 8.60 (s, 2H), 7.42 (q, 1H, J = 4.6 Hz), 7.36−7.28 (m,
1H), 7.21−7.11 (m, 3H), 6.99 (d, 1H, J = 16.8 Hz), 2.83 (d, 3H, J =
4.8 Hz). 13C NMR (DMSO-d6): δ 162.19, 160.00 (dd, J1 = 248.2 Hz, J
= 7.6 Hz, two C), 156.38 (two C), 129.97 (t, J = 7.6 Hz), 128.50 (t, J =
10.6 Hz), 119.08, 114.26 (t, J = 15.2 Hz), 112.02 (dd, J1 = 19.4 Hz, J2
= 6.4 Hz, two C), 111.02, 27.93. HRMS (ESI) calcd for C13H12F2N3
[MH+], 248.09938; found, 248.09838. Anal. Calcd for C13H11F2N3: C,
63.15; H, 4.48. Found: C, 63.14; H, 4.55.
(E)-5-(2′,6′-Difluorostyryl)-2-(N-ethylamino)pyrimidine
(10c). Yield 89%, mp 147−149 °C (from ethanol). 1H NMR (DMSO-
d6): δ 8.58 (s, 2H), 7.48 (t, 1H, J = 5.6 Hz), 7.35−7.28 (m, 1H), 7.20−
7.10 (m, 3H), 6.98 (d, 1H, J = 16.8 Hz), 3.56−3.29 (m, 2H), 1.12 (t,
3H, J = 7.4 Hz). 13C NMR (DMSO-d6): δ 161.62, 160.00 (dd, J1 =
248.2 Hz, J = 7.6 Hz, two C), 156.40 (two C), 129.97 (t, J = 7.6 Hz),
128.51 (t, J = 10.6 Hz), 119.08, 114.27 (t, J = 15.6 Hz), 112.00 (dd, J1
= 19.4 Hz, J2 = 6.1 Hz, two C), 110.94, 35.50, 14.65. HRMS (ESI)
calcd for C14H14F2N3 [MH+], 262.11503; found, 262.11424. Anal.
Calcd for C14H13F2N3: C, 64.36; H, 5.02. Found: C, 64.54; H, 4.96.
(E)-5-(2′-Chloro-6′-fluorostyryl)-2-(N,N-dimethylamino)-
1
pyrimidine (11a). Yield 74%, mp 105−106 °C (from hexane). H
NMR (DMSO-d6): δ 8.64 (s, 2H), 7.38−7.25 (m, 3H), 7.14 (d, 1H, J
= 16.8 Hz), 7.42 (d, 1H, J = 16.8 Hz), 3.16 (s, 6H). 13C NMR
(DMSO-d6): δ 161.22, 160.32 (d, J = 248.9 Hz), 156.13 (two C),
133.12 (d, J = 5.3 Hz), 130.73 (d, J = 11.3 Hz), 128.83 (d, J = 8.4 Hz),
125.91 (d, J = 1.5 Hz), 123.67 (d, J = 14.5 Hz), 118.29, 115.36, 115.17
(d, J = 22.7 Hz), 36.73 (two C). HRMS (ESI) calcd for C14H14ClFN3
[MH+], 278.08548; found, 278.08458. Anal. Calcd for C14H13ClFN3:
C, 60.55; H, 4.72. Found: C, 60.66; H, 4.79.
Membrane Preparation. Cell membrane preparation was based
on previous methods.19−22 Cells were rinsed twice with HBSS at 37
°C and collected by scraping the dishes in ∼20 mL of ice-cold 0.32 M
(E)-5-(2′-Chloro-6′-fluorostyryl)-2-(N-methylamino)-
1
pyrimidine (11b). Yield 78%, mp 163−165 °C (from ethanol). H
NMR (DMSO-d6): δ 8.58 (s, 2H), 7.43 (q, 1H, J = 4.6 Hz), 7.38−7.36
6089
dx.doi.org/10.1021/jm5004864 | J. Med. Chem. 2014, 57, 6083−6091

Journal of Medicinal Chemistry
Article
sucrose and homogenized on ice with a Teflon pestle using a Maximal
Digital homogenizer (Fisher Scientific, Pittsburgh, PA) at ∼280 rpm
for 30 s. Homogenates were centrifuged at 300g and 800g for 4 min
each at 4 °C. Pellets were resuspended in 9 mL of ice-cold Milli-Q
water, and osmolarity was restored by addition of 1 mL of 500 mM
Tris buffer (pH 7.4) followed by suspension and centrifugation at 20
000g for 30 min at 4 °C. Pellets were homogenized in 2 mL assay
buffer (50 mM Tris, 10 mM KCl, and 1 mM MgCl2, 4 °C), and
aliquots of cell membrane suspensions were stored at −80 °C and
thawed the day of the [3H]-dofetilide binding assay. Protein content
was determined prior to the assay using a Bradford protein assay with
bovine albumin as the standard.
Craig Vander Kooi for helpful discussions regarding the
fluorescence assays.
ABBREVIATIONS USED
■
ATP, adenosine triphosphate; MAT2A, catalytic subunit of
methionine S-adenosyltransferase-2; CRC, colorectal cancer;
FIDAS, difluorinated N,N′-dialkylaminostilbenes; DMSO,
dimethyl sulfoxide; ESI, electrospray ionization; FBS, fetal
bovine serum; HBSS, Hank’s balanced salt solution; hERG-
HEK, HEK-293 cell membranes stably expressing the hERG
[3H]-Dofetilide Binding Assay. [3H]-Dofetilide binding assays
using hERG-HEK293 cell membranes were based on previous
methods.19 Assays determining concentration−response were con-
ducted in duplicate, and three independent assays were performed for
each analogue evaluated. Cell membrane suspension (5 μg) was added
to duplicate tubes containing assay buffer, 25 μL of a single
concentration of FIDAS agent (concentration range of 10 nM to
100 μM for each experiment), and 25 μL of [3H]-dofetilide (5 nM,
final concentration) for an assay volume of 250 μL. Binding occurred
for 60 min at 25 °C and was terminated by rapid filtration through
Whatman GF/B filters, which were presoaked in 0.25% PEI overnight,
using a Brandel cell/membrane harvester (M-48; Brandel Inc.,
Gaithersburg, MD). Filters were washed three times with ∼1 mL of
ice-cold assay buffer. Radioactivity was determined by liquid
scintillation spectrometry using the Tri-Carb 2100-TR liquid
scintillation analyzer (PerkinElmer Life and Analytical Sciences).
Data Analysis. Compound concentrations that produced 50%
inhibition (IC50) in the biological studies were determined from the
concentration−response curves via the nonlinear regression one-site
competition-fitting program (Prism 5.04; GraphPad Software Inc., San
Diego, CA).
̀
channel; hERG, human ether-a-go-go-related protein; IND,
investigational new drug; LC, liver cancer; MEM, minimum
essential medium; NMR, nuclear magnetic resonance; NEAA,
nonessential amino acids; PEI, polyethylenimine; SAH, S-
adenosylhomocysteine; SAM, S-adenosylmethionine; SAR,
structure−activity relationship
REFERENCES
■
(1) Zhang, W.; Sviripa, V.; Kril, L. M.; Chen, X.; Yu, T.; Shi, J.;
Rychahou, P.; Evers, B. M.; Watt, D. S.; Liu, C. Fluorinated N,N-
dialkylaminostilbenes for Wnt pathway inhibition and colon cancer
repression. J. Med. Chem. 2011, 54, 1288−1297.
(2) Zhang, W.; Sviripa, V.; Chen, X.; Shi, J.; Yu, T.; Hamza, A.; Ward,
N. S.; Kril, L. M.; Vander Kooi, C. W.; Zhan, C.-G.; Evers, B. M.; Watt,
D. S.; Liu, C. Fluorinated N,N-dialkylaminostilbenes repress colon
cancer by targeting methionine S-adenosyltransferase 2A. ACS Chem.
Biol. 2013, 8, 796−803.
(3) Watt, D. S.; Liu, C.; Sviripa, V.; Zhang, W. Stilbene analogs and
methods of treating cancer. Patent US2012/0196874, August 2, 2012.
(4) Paul, S.; Mizuno, C. S.; Lee, H. J.; Zheng, X.; Chajkowisk, S.;
Rimoldi, J. M.; Conney, A.; Suh, N.; Rimando, A. M. In vitro and in
vivo studies on stilbene analogs as potential treatment agents for colon
cancer. Eur. J. Med. Chem. 2010, 45, 3702−3708.
(5) Orsini, F.; Sello, G. Natural stilbenes and analogues as
antineoplastic agents. Stud. Nat. Prod. Chem. 2008, 34, 77−127.
(6) Maccario, C.; Savio, M.; Ferraro, D.; Bianchi, L.; Pizzala, R.;
Pretali, L.; Forti, L.; Stivala, L. A. The resveratrol analog 4,4′-
dihydroxy-trans-stilbene suppresses transformation in normal mouse
fibroblasts and inhibits proliferation and invasion of human breast
cancer cells. Carcinogenesis 2012, 33, 2172−2180.
ASSOCIATED CONTENT
* Supporting Information
■
S
Examples of IC50 determination for the cell proliferation assay
and effects of FIDAS agent on colon cancer organoids. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*(C.L.) Phone: 859-323-4558; Fax: 859-257-6030; E-mail:
chunming.liu@uky.edu.
■
(7) Mikuła-Pietrasik, J.; Sosin
ska, P.; Wierzchowski, M.; Piwocka, K.;
Ksiązek, K. Synthetic resveratrol analogue, 3,3′,4,4′,5,5′-hexahydroxy-
̇
trans-stilbene, accelerates senescence in peritoneal mesothelium and
promotes senescence-dependent growth of gastrointestinal cancers.
Int. J. Mol. Sci. 2013, 14, 22483−22498.
*(D.S.W.) Phone: 859-218-1026; Fax: 859-257-6030; E-mail:
dwatt@uky.edu.
(8) Chae, Y. S.; Kim, J. G.; Jung, H. J.; Yang, J. D.; Jung, J. H.; Aiyar,
S. E.; Kim, S.; Park, H. Anticancer effect of (E)-2-hydroxy-3′,4′,5′-
trimethoxystilbene on breast cancer cells by mitochondrial depolariza-
tion. Cancer Chemother. Pharmacol. 2011, 68, 349−358.
(9) Kelkel, M.; Jacob, C.; Dicato, M.; Diederich, M. Potential of the
dietary antioxidants resveratrol and curcumin in prevention and
treatment of hematologic malignancies. Molecules 2010, 15, 7035−
7074.
Notes
The authors declare the following competing financial
interest(s): In accord with University policy, Wen Zhang,
Chunming Liu, David S. Watt, and Vitaliy M. Sviripa have
disclosed this work to the University’s Intellectual Property
Committee that has sought patent protection and that has
given a private firm, Liu-Watt, LLC, in which the four principals
have partial ownership, an option to license this technology.
(10) Fulda, S. Resveratrol and derivatives for the prevention and
treatment of cancer. Drug Discovery Today 2010, 15, 757−765.
(11) Cai, J.; Sun, W. M.; Hwang, J. J.; Stain, S. C.; Lu, S. C. Changes
in S-adenosylmethionine synthetase in human liver cancer: molecular
characterization and significance. Hepatology 1996, 24, 1090−1097.
(12) Chen, H.; Xia, M.; Lin, M.; Yang, H.; Kuhlenkamp, J.; Li, T.;
Sodir, N. M.; Chen, Y. H.; Josef-Lenz, H.; Laird, P. W.; Clarke, S.;
Mato, J. M.; Lu, S. C. Role of methionine adenosyltransferase 2A and
S-adenosylmethionine in mitogen-induced growth of human colon
cancer cells. Gastroenterology 2007, 133, 207−218.
ACKNOWLEDGMENTS
■
C.L. and D.S.W. were supported by grant nos. R21 CA139359
and CA172379 from the NIH. L.P.D. and J.R.N. were
supported by grant nos. U01 DA013519, UL1TR000117, and
T32 DA016176 from the NIH. D.S.W. was supported by the
Office of the Dean of the College of Medicine and by NIH
grant no. P20 RR020171 from the National Institute of General
Medical Sciences to L. Hersh, PI. Its contents are solely the
responsibility of the authors and do not necessarily represent
the official views of the NIH or the NIGMS. The authors thank
(13) Ito, K.; Ikeda, S.; Kojima, N.; Miura, M.; Shimizu-Saito, K.;
Yamaguchi, I.; Katsuyama, I.; Sanada, K.; Iwai, T.; Senoo, H.;
Horikawa, S. Correlation between the expression of methionine
6090
dx.doi.org/10.1021/jm5004864 | J. Med. Chem. 2014, 57, 6083−6091

Journal of Medicinal Chemistry
Article
adenosyltransferase and the stages of human colorectal carcinoma.
Surg. Today 2000, 30, 706−710.
(14) Liu, Q.; Chen, J.; Liu, L.; Zhang, J.; Wang, D.; Ma, L.; He, Y.;
Liu, Y.; Liu, Z.; Wu, J. The X protein of hepatitis B virus inhibits
apoptosis in hepatoma cells through enhancing the methionine
adenosyltransferase 2A gene expression and reducing S-adenosylme-
thionine production. J. Biol. Chem. 2011, 286, 17168−17180.
(15) Kumar, D.; Elliott, P. Principles and Practice of Clinical
Cardiovascular Genetics; Oxford University Press: New York, 2010.
(16) Shafqat, N.; Muniz, J. R. C.; Pilka, E. S.; Papagrigoriou, E.; von
Delft, F.; Oppermann, U.; Yue, W. W. Insight into S-adenosylme-
thionine biosynthesis from the crystal structures of the human
methionine adenosyltransferase catalytic and regulatory subunits.
Biochem. J. 2013, 452, 27−36.
(17) Maryanoff, B. E.; Reitz, A. B. The Wittig olefination reaction
and modifications involving phosphoryl-stabilized carbanions. Stereo-
chemistry, mechanism, and selected synthetic aspects. Chem. Rev.
1989, 89, 863−927.
(18) Hageman, H. A. The von Braun cyanogen bromide reaction.
Org. React. 1953, 7, 198−262.
(19) A Practical Guide to Assay Development and High-Throughput
Screening in Drug Discovery; Chen, T., Ed.; CRC Press: Boca Raton, FL,
2010.
́
(20) Biswas, T.; Resto-Roldan, E.; Sawyer, S. K.; Artsimovitch, I.;
Tsodikov, O. V. A novel non-radioactive primase-pyrophosphatase
activity assay and its application to the discovery of inhibitors of
Mycobacterium tuberculosis primase DnaG. Nucleic Acids Res. 2013,
41, e56.
(21) Greengrass, P. M.; Stewart, M.; Wood, C. M. Affinity-assay for
the human ERG potassium channel. Patent WO2003021271 A2,
March 13, 2003.
(22) Jo, S.-H.; Youm, J. B.; Lee, C. O.; Earm, Y. E.; Ho, W.-K.
Blockade of the HERG human cardiac K+ channel by the
antidepressant drug amitryptyline. Br. J. Pharmacol. 2000, 129,
1474−1480.
(23) Redfern, W. S.; Carlsson, L.; Davis, A. S.; Lynch, W. G.;
MacKenzie, I.; Palethorpe, S.; Siegl, P. K.; Strang, I.; Sullivan, A. T.;
Wallis, R.; Camm, A. J.; Hammond, T. G. Relationships between
preclinical cardiac electrophysiology, clinical QT interval prolongation
and torsade de pointes for a broad range of drugs: evidence for a
provisional safety margin in drug development. Cardiovasc. Res. 2003,
58, 32−45.
6091
dx.doi.org/10.1021/jm5004864 | J. Med. Chem. 2014, 57, 6083−6091