C.J.A. Ribeiro et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Table 2
However, all modifications led to a decrease in potency.
TDP2
inhibitory
activities
of
derivatives
17p–ai,
Lastly, we synthesized compounds in which the carboxamide moiety
of 17 is replaced by an amine (19) and or a sulfonamide (20). Both
substitutions led to inactive derivatives, highlighting the importance of
the amide group to activity.
19–20.
Since the goal of developing TDP2 inhibitors is to sensitize cancer
cells toward TOP2 poisons to ultimately overcome drug resistance and
potentially also allow TOP2 therapeutic efficiency at lower and less
toxic concentrations, it is imperative that these inhibitors are cell
permeable and non-cytotoxic. Toward these ends, selected active
compounds (7a, 17a, 17e, and 17z) were first tested in the PAMPA
permeability assay along with the most potent deazaflavin inhibitor 1
(Table 3). From this assay, excellent permeability was observed only
with our compound 17z. Another analogue 17e also exhibited bor-
derline Pe value which is about 10 times higher than that of 1. These
findings are consistent with the report that deazaflavin compounds
lacked cellular permeability. We then assessed the two cell-permeable
compounds 17e and 17z for cytotoxicity in two different cell lines
(HepG2 and HeLa), from which no cytotoxicity was observed at con-
centrations up to 100 µM (Table 3). These results indicate that despite
potential to be developed into cell permeable and noncytotoxic che-
mical probes for studying cellular functions.
Compds
R1
17p
4-ClPh
80.8
7.5
17q
4-OHPh
> 100
> 100
59.0
17r
4-CF3Ph
17s
4-OCH3Ph
4-FPh
3.7
4.6
17t
85.2
17u
17v
3-ClPh
> 100
> 100
> 100
> 100
> 100
21.0
3-OCH3Ph
3-CF3Ph
17w
17x
2-OCH3Ph
2-ClPh
17y
17z
pyridin-3-yl
furan-2-yl
thiophen-2-yl
furan-3-yl
3-SO2CH3Ph
3-COCH3Ph
3-COOHPh
pyridin-4-yl
quinolin-3-yl
pyrimidin-5-yl
2.1
6.8
17aa
17ab
17ac
17ad
17ae
17af
17ag
17ah
17ai
94.5
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
From a previous screening campaign against TDP2, compound
P10A10, reported as 7-phenyl triazolopyrimidine 6a, was identified as a
hit. The present study on hit validation via resynthesis and structure
elucidation revealed that the correct structure for P10A10 (Chembridge
ID 7236827) should be 5-phenyl isomer 7a. To probe TDP2 inhibition
potentialities of this scaffold we synthesized a total of 47 compounds:
11 derivatives inhibited TDP2 (IC50 < 100 µM), with four derivatives
(7a, 17a, 17e, and 17z) reaching potencies below 50 µM. Although we
were unable to increase significantly the potency of the original hit,
compounds 17e and 17z, particularly the latter, demonstrated good
cellular permeability and no cytotoxicity and can be further developed
into useful chemical probes for studying cellular functions of TDP2 in
host DNA repair as well as virus genome repair.
Compds
X
IC50 (µM)
19
20
CH2
SO2
> 100
> 100
a
IC50 values are the mean of three independent experiments performed in
triplicate.
Table 3
TDP2 inhibition, permeability and cytotoxicity of the four best derivatives.
Compds TDP2 IC50 (µM)a Pe (10−6 cm/s)b HepG2 CC50
HeLa CC50
7a
22.0
16.6
43.8
21.0
0.040
2.5
0.4
3.9
2.1
0.16
0.15
0.9
–
–
–
Acknowledgments
17a
17e
17z
1
–
> 100
> 100
> 100
–
This research was supported by the Academic Health Center Faculty
Research Development Grant Program (FRD #14.23), University of
Minnesota, and partially by the Center for Drug Design, University of
Minnesota, and NIH grant GM118047 to HA. We acknowledge
Professor Bert Semler at University of California, Irvine and Professor
Haitao Guo at Indiana University School of Medicine, for providing
HeLa and HepG2 cells, respectively. We also acknowledge Victor G.
Young, Jr. and the X-ray Crystallographic Laboratory for the X-ray
crystallography studies. The Bruker-AXS D8 Venture diffractometer was
6.3
> 100
0.095
–
a
IC50 values are the mean of three independent experiments performed in
triplicate.
b
c
Permeability is considered high when Pe > 1.5.
Compounds were tested at 50 µM and 100 µM (two independent experi-
ments were performed in triplicate).
50 µM. Moreover, changing chloro to para and ortho positions (17l–m)
or adding a second chloro (17o) further reduced activity.
purchased through
a grant from NSF/MRI (#1224900) and the
University of Minnesota.
Another SAR trend was that the phenyl group at different positions
in the main core favored position 7 (17a, IC50 = 16.6 µM vs. 18,
IC50 = 77.3 µM vs. 6a, IC50 > 100 µM). Although the potency of
compound 17e was found to be 2-fold lower than the hit compound
(IC50 = 43.8 µM vs 22.0 µM), its permeability in PAMPA assay was 6-
fold higher (Table 3), and therefore we decided to maintain a 3-
pyridine ring (17p–ac, Table 2). Only 17s with 4-methoxyphenyl group
was able to retain almost the same activity (IC50 = 59.0 µM), with
pyridine-3-yl substitution leading to a two-fold improve of potency
(17z, IC50 = 21.0 µM vs. 17e, 43.8 µM) and 7-fold increase in perme-
ability (Table 3). The increase in potency observed for derivative 17z
led us to further test substitution at meta position with potential H bond
acceptor properties (3-methylsulfonyl, 17ad; 3-acetyl, 17ae; 3-carbox-
ylate 17af) and also additional derivatives with aromatic nitrogens
(pyridine-4-yl, 17ag; quinoline-3-yl, 17ah; pyrimidin-5-yl, 17ai).
Appendix A. Supplementary data
Supplementary data (Experimental procedures, NMR characteriza-
tion, as well as crystal data and structure refinement for compound 11)
References
4