Halogenated diarylacetylenes repress c-myc expression in cancer cells

Article abstract of DOI:10.1016/j.bmcl.2014.04.113

Halogenated diarylacetylenes that possess fluorine or chlorine substituents in one aryl ring and N-methylamino or N,N-dimethylamino in the other aryl ring inhibit the proliferation of LS174T colon cancer cells through the repression of c-myc expression an

Full text of DOI:10.1016/j.bmcl.2014.04.113

Accepted Manuscript
Halogenated Diarylacetylenes Repress c-Myc Expression in Cancer Cells
Vitaliy M. Sviripa, Wen Zhang, Liliia M. Kril, Alice X. Liu, Yaxia Yuan, Chang-
Guo Zhan, Chunming Liu, David S. Watt
PII:
S0960-894X(14)00480-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.04.113
BMCL 21606
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 March 2014
25 April 2014
28 April 2014
Please cite this article as: Sviripa, V.M., Zhang, W., Kril, L.M., Liu, A.X., Yuan, Y., Zhan, C-G., Liu, C., Watt,
D.S., Halogenated Diarylacetylenes Repress c-Myc Expression in Cancer Cells, Bioorganic & Medicinal Chemistry
Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.04.113
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Halogenated Diarylacetylenes Repress c-Myc
Expression in Cancer Cells
Vitaliy M. Sviripaa,b, Wen Zhanga,d, Liliia M. Krila,b, Alice X. Liua,b, Yaxia Yuanc,e,
Chang-Guo Zhanb,c,e, Chunming Liua,d,*, David S. Watta,b,d,e,*
aDepartment of Molecular and Cellular Biochemistry, College of Medicine, University of
Kentucky, Lexington, KY 40536-0509
bCenter for Pharmaceutical Research and Innovation, College of Pharmacy, University of
Kentucky, Lexington, KY 40536-0596
cCenter for Computational Drug Design, College of Pharmacy, University of Kentucky,
Lexington, KY 40536-0596
dLucille Parker Markey Cancer Center, University of Kentucky, Lexington, KY 40536-
0093
eDepartment of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky,
Lexington, KY 40536-0596
*Correspondence to: chunming.liu@uky.edu; dwatt@uky.edu

Abstract
Halogenated diarylacetylenes that possess fluorine or chlorine substituents in one aryl
ring and N-methylamino or N,N-dimethylamino in the other aryl ring inhibit the
proliferation of LS174T colon cancer cells through the repression of c-myc expression
and induction of the cyclin-dependent kinase inhibitor-1 (i.e., p21(Wif1/Cip1)) and
represent potentially useful antineoplastic agents.

In the course of developing new agents for the treatment of colorectal cancer, we
identified a family of fluorinated N,N- dialkylaminostilbene analogs (FIDAS agents) that
inhibit the expression of Wnt target genes, such as c-myc1, and repress colon cancer cell
growth in vitro and in vivo2-4. Recently, we found that (E)-4-(2’,6’-difluorostyryl)-N,N-
dimethylaniline (1) (Fig. 1) targeted exclusively the catalytic subunit3 of methionine S-
adenosyltransferase-2 (MAT-2) that serves as a source of S-adenosylmethionine (SAM)
in colorectal and liver cancers where MAT-2 is upregulated5-8. Presumably, neoplastic
tissues make effective use of SAM from this isoform of MAT to manage crucial
epigenetic modifications of histone proteins and thereby regulate gene expression.
Interference with this process represented a new approach for developing potential
antineoplastic agents, and consequently, we explored related compounds that might alter
c-myc expression. In addition, we sought compounds that would avoid the facile E/Z-
isomerizations that afflict the stilbenes and that complicate pharmacodynamic and
pharmacokinetic studies.
Among the possible analogs that would avoid this isomerization problem, the
diarylacetylenes 2 (Fig. 1) were an obvious choice. The only prior reports of acetylenic
compounds as antineoplastic agents include monoalkylacetylenes from aquatic
organisms9 and diarylacetylenic analogs of combrestatin10. The latter compounds
showed cytotoxic activity against a murine leukemia cell line and one showed activity as
an inhibitor of tubulin polymerization. The Sonogashira coupling11,12 of 4-(N,N-
dimethylamino)phenylacetylene with various aryl iodides provided access to the desired
diarylacetylenes 2 (Table 1). Prior work from our laboratories established that stilbenes

with N-methylamino and N,N-dimethylamino groups in a para-orientation relative to the
central double bond as well as 2,6-difluoro, 2-chloro-6-fluoro or 2,6-dichloro
halogenation patterns in the other aromatic ring were the most potent analogs in the
repression of c-myc expression and colon cancer cell LS174T proliferation2.
As a consequence, we limited SAR studies to those diarylacetylenes 2 that
possessed fluorine or chlorine substituents in one aryl ring and N-methylamino or N,N-
dimethylamino in the other aryl ring. We reported previously that stilbenes repressed
colon cancer cell proliferation by inhibiting c-myc expression and inducing the cell cycle
inhibitor, p21(Wif1/Cip1)2. The similarity of the diarylacetylenes to the stilbenes
prompted an in silico modeling study of the binding of (E)-4-(2’,6’-difluorostyryl)-N,N-
dimethylaniline (1) and 4-((2,6-difluorophenyl)ethynyl)-N,N-dimethylaniline (2m).
Using a computationally constructed open-state model of the homodimer of MAT2A (see
Supplementary Data for details), we observed that 1 and 2m bound to the same active site
(Fig. 2) and that the diarylacetylene 2m inhibited MAT2 at concentrations comparable to
that of the stilbene 1 (data not shown). Hydrophobic residues I139, F272, I274, and I344
define the boundaries of the binding pocket, and van der Waals contact between F272
and the halogen substituents accounted for the increased activity of ortho-halogens in 1
or 2m over the corresponding analogs with meta- or para-halogens. In addition, a cleft
formed by residues K203, S269, I274, and D280 accommodated the shape of these
particular ligands (Fig. 2).

Variability in the MAT2A inhibition assay made the measurement of the levels of
c-myc expression a preferred analytical tool for assessing the potency of diarylacetylenes.
We tested the effect of these diarylacetylenes 2 on the proliferation of LS174T colon
cancer cells. The expression of c-myc and p21(Wif1/Cip1) was analyzed by western
blotting (Fig. 3). The most active diarylacetylenes 2 inhibited c-myc expression at 1 µM
concentrations and as expected, induced p21(wif1/Cip1) at the same time. Consistent
with prior results in the stilbene family, the diarylacetylenes 2 lacking halogen
substituents (e.g., 2c) or possessing only one fluorine substituent at a meta-position
relative to the acetylenic linkage (e.g., 2f) had very low potency (Table 1).
Diarylacetylenes with one or two halogen substituents at ortho-positions relative to the
acetylenic linkage (e.g., 2b, 2d, 2e, 2m and 2n) possessed potencies as inhibitors of
LS174T cell proliferation that exceeded that of the related stilbene 1 with IC50 values less
than 50 nM (Table 1). Isomers of these diarylacetylenes (e.g., 2f, 2g, and 2j) with
halogens in meta- or para-positions were significantly less active than the
diarylacetylenes with ortho-halogens. Once again, these results are in consistent with the
SAR findings in the stilbene family that also repress c-myc expression2. Finally, the N-
methylation pattern in the diarylacetylenes suggested that N-methyl and N,N-
dimethylaniline subunits led to equipotent repression of c-myc expression (i.e., IC50 of 2b
≈ IC50 of 2m) but the desmethyl analog was considerably less active (IC50 of 2a = 55 7.8
nm).
In summary, diarylacetylenes 2 have a dramatic effect on the proliferation of
LS174T colon cancer cells by altering the expression of c-myc and thereby inducing

p21(Wif1/Cip1). These results are consistent with similar findings using halogenated
stilbenes2 and suggest that diarylacetylenes and stilbenes repress colon cancer
proliferation through similar mechanisms. Examining this question in detail is the
subject of on-going investigations.
Acknowledgments
CL and DSW were supported by R21 CA139359 and CA172379 from the NIH. CGZ
was supported by CHE-1111761 from the NSF. DSW was supported by the Office of the
Dean of the College of Medicine and by NIH Grant Number 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.
References and notes
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M.; Watt, D. S.; Liu, C. J. Med. Chem. 2011, 54, 1288.
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Vander Kooi, C. W.; Zhan, C.-G.; Evers, B. M.; Watt, D. S.; Liu, C. ACS Chem. Biol.
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4. Watt, D.; Liu, C.; Sviripa, V. M.; Zhang, W. U.S. Patent 8,664,276 March 4, 2014.
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N(CH3)2
F
F
Y
1
X1
X1
C
C
X2
X3
X2
X3
I
a or b
X5
X5
X4
X4
2
Figure 1. Synthesis of halogenated diarylacetylenes 2. Reagents: a, HC≡CHC6H4Y;
0.5% Pd(PPh3)4, 1% CuI, H2O, 75oC, 1-2 h; b, a, HC≡CHC6H4NH2; 0.5% Pd(PPh3)4, 1%
CuI, H2O, 75oC, 1-2 h followed by CH3I, K2CO3, acetone, 5 h, 56oC.

Figure 2. Docked structures of MAT2A binding with stilbene 1 (Panel A) and
diarylacetylene 2m (Panel B). The letters A and B following the amino acid
designations refer to the individual oligomers in the MAT2A homodimer.

Figure 3. Repression of c-myc expression and induction of p21(Wif1/Cip1) by
diarylacetylenes 2 in colon cancer cells. LS174T cells were treated with 1 µM of each
diarylacetylenes 2 for 36 h. DMSO and 1 were used as control. Cell lysates were
analyzed by western blotting with β-tubulin as a loading control.

Inhibition of
LS174T Cell
Proliferation
IC50 (nM)
Compound
1
X1
X2
X3
X4
X5
Y
F
F
F
F
N(CH3)2
NH2
59 7.5
55 7.8
23 10.3
>3000
39 6.0
31 3.1
>3000
>3000
119 4.6
56 8.1
>3000
>3000
55 6.0
23 6.0
19 5.0
52 7.1
2a
2b
2c
2d
2e
2f
F
F
NHCH3
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
F
Cl
F
F
F
2g
2h
2i
F
F
F
F
F
F
2j
F
F
2k
2l
F
F
F
2m
2n
2o
F
Cl
Cl
Cl
Table 1. Halogenated N,N-diarylacetylenes 2 and their IC50 values in the inhibition
of LS174T cell proliferation.

Graphical Abstract
Halogenated diarylacetylenes 2 (X1-5 = H, F and/or Cl and Y = 4-NHCH3 or 4-N(CH3)2)
inhibit that inhibit the expression of Wnt target genes, such as c-myc, at 1-3 µM
concentrations and induce the cyclin-dependent kinase inhibitor-1 (i.e., p21(Wif1/Cip1)).
X1
X2
X3
Y
C C
X5
X4
2