Semisynthetic aurones inhibit tubulin polymerization at the colchicine-binding site and repress PC-3 tumor xenografts in nude mice and myc-induced T-ALL in zebrafish

Abstract

Structure-activity relationships (SAR) in the aurone pharmacophore identified heterocyclic variants of the (Z)-2-benzylidene-6-hydroxybenzofuran-3(2H)-one scaffold that possessed low nanomolar in vitro potency in cell proliferation assays using various cancer cell lines, in vivo potency in prostate cancer PC-3 xenograft and zebrafish models, selectivity for the colchicine-binding site on tubulin, and absence of appreciable toxicity. Among the leading, biologically active analogs were (Z)-2-((2-((1-ethyl-5-methoxy-1H-indol-3-yl)methylene)-3-oxo-2,3-dihydrobenzofuran-6-yl)oxy)acetonitrile (5a) and (Z)-6-((2,6-dichlorobenzyl)oxy)-2-(pyridin-4-ylmethylene)benzofuran-3(2H)-one (5b) that inhibited in vitro PC-3 prostate cancer cell proliferation with IC50 values below 100 nM. A xenograft study in nude mice using 10 mg/kg of 5a had no effect on mice weight, and aurone 5a did not inhibit, as desired, the human ether-à-go-go-related (hERG) potassium channel. Cell cycle arrest data, comparisons of the inhibition of cancer cell proliferation by aurones and known antineoplastic agents, and in vitro inhibition of tubulin polymerization indicated that aurone 5a disrupted tubulin dynamics. Based on molecular docking and confirmed by liquid chromatography-electrospray ionization-tandem mass spectrometry studies, aurone 5a targets the colchicine-binding site on tubulin. In addition to solid tumors, aurones 5a and 5b strongly inhibited in vitro a panel of human leukemia cancer cell lines and the in vivo myc-induced T cell acute lymphoblastic leukemia (T-ALL) in a zebrafish model.

Full text

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Semisynthetic aurones inhibit
tubulin polymerization at the
colchicine-binding site and repress
PC-3 tumor xenografts in nude mice
and myc-induced T-ALL in zebrash
Yanqi Xie1,2, Liliia M. Kril1,2, Tianxin Yu1,3, Wen Zhang1,3, Mykhaylo S. Frasinyuk
1,2,4,
Svitlana P. Bondarenko5, Kostyantyn M. Kondratyuk4, Elizabeth Hausman1,
Zachary M. Martin1,2, Przemyslaw P. Wyrebek1,2, Xifu Liu6, Agripina Deaciuc7,
Linda P. Dwoskin7, Jing Chen1, Haining Zhu1, Chang-Guo Zhan2,7,8, Vitaliy M. Sviripa2,3,7,
Jessica Blackburn1, David S. Watt1,2,3,7 & Chunming Liu1,3
Structure-activity relationships (SAR) in the aurone pharmacophore identied heterocyclic variants
of the (Z)-2-benzylidene-6-hydroxybenzofuran-3(2H)-one scaold that possessed low nanomolar
in vitro potency in cell proliferation assays using various cancer cell lines, in vivo potency in prostate
cancer PC-3 xenograft and zebrash models, selectivity for the colchicine-binding site on tubulin, and
absence of appreciable toxicity. Among the leading, biologically active analogs were (Z)-2-((2-((1-ethyl-
5-methoxy-1H-indol-3-yl)methylene)-3-oxo-2,3-dihydrobenzofuran-6-yl)oxy)acetonitrile (5a) and (Z)-
6-((2,6-dichlorobenzyl)oxy)-2-(pyridin-4-ylmethylene)benzofuran-3(2H)-one (5b) that inhibited in vitro
PC-3 prostate cancer cell proliferation with IC50 values below 100 nM. A xenograft study in nude mice
using 10 mg/kg of 5a had no eect on mice weight, and aurone 5a did not inhibit, as desired, the human
ether--go-go-related (hERG) potassium channel. Cell cycle arrest data, comparisons of the inhibition of
cancer cell proliferation by aurones and known antineoplastic agents, and in vitro inhibition of tubulin
polymerization indicated that aurone 5a disrupted tubulin dynamics. Based on molecular docking and
conrmed by liquid chromatography-electrospray ionization-tandem mass spectrometry studies,
aurone 5a targets the colchicine-binding site on tubulin. In addition to solid tumors, aurones 5a and 5b
strongly inhibited in vitro a panel of human leukemia cancer cell lines and the in vivo myc-induced T cell
acute lymphoblastic leukemia (T-ALL) in a zebrash model.
e aurones comprise a family of plant-derived avonoids that arise out of a mixed polyketide-shikimate path-
way, contribute to the yellow coloration of certain owers1 and possess a range of biological properties2–4 aect-
ing organisms ranging from protazoans to mammals. e antineoplastic activity5 of several naturally occurring
aurones led to studies of natural and semisynthetic aurones as inhibitors of in vitro cancer cell proliferation6–8,
typically at low micromolar concentrations. Additional studies identified a panoply of roles at a molecular
1Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, Lexington, KY,
40536-0509, USA. 2Center for Pharmaceutical Research and Innovation, College of Pharmacy, University of Kentucky,
Lexington, KY, 40536-0596, USA. 3Lucille Parker Markey Cancer Center, University of Kentucky, Lexington, KY,
40536-0093, USA. 4Institute of Bioorganic Chemistry and Petrochemistry, National Academy of Science of Ukraine,
Kyiv, 02094, Ukraine. 5National University of Food Technologies, Kyiv, 01601, Ukraine. 6Center for Drug Innovation
and Discovery, Hebei Normal University, Shijiazhuang, Hebei, 050024, People’s Republic of China. 7Department of
Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY, 40536-0596, USA. 8Molecular
Modeling and Pharmaceutical Center, College of Pharmacy, University of Kentucky, Lexington, KY, 40536-0596,
USA. Correspondence and requests for materials should be addressed to D.S.W. (email: dwatt@uky.edu) or C.L.
(email: chunming.liu@uky.edu)
Received: 22 May 2018
Accepted: 17 December 2018
Published: xx xx xxxx
OPEN
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level: drug eux modulators2,9–15 of P-glycoprotein (P-gp) or ATP-binding cassette sub-family G member 2
(ABCG2), modifiers of adenosine-receptor interactions16,17, DNA sission-promoters18, teleomerase inhibi-
tors19, sphingosine-kinase inhibitors20, phosphatidylinositol-3-kinases (PI3−α) inhibitors21, cyclin-dependent
kinase inhibitors22, inducers of cytoprotective NAD(P)H:quinone oxidoreductase-123 (NQO1), and scavengers
of reactive-oxygen-species24 (ROS). Although these ndings suggested that aurones would disrupt biological
systems non-specically, our studies of the aurone pharmacophore identied heterocyclic variants of the (Z)-
2-benzylidene-6-hydroxybenzofuran-3(2H)-one scaold that possessed the low nanomolar in vitro potency,
encouraging in vivo potency in mouse xenogra and zebrash models, selectivity for the colchicine-binding site
in tubulin25–31, and the absence of appreciable toxicity.
Prior SAR studies of aurones as antineoplastic agents replaced the C-2 benzylidene subunit found in naturally
occurring aurones, such as sulfuretin (1a) and aureusidin (1b) (Fig.1A), with a C-2 heteroarylmethylene group.
Aurones with 2-(coumarin-4-yl)methylene groups32 or 2-(furan-2-yl)methylene groups33 displayed in vitro activ-
ity against human leukemia K562 cells; aurones with 2-(piperazin-1-yl)methylene groups possessed IC50 values in
the low micromolar range against various solid tumor cell lines34; and benzofuran-3(2H)-ones with 2-(indol-3-yl)
methylene groups inhibited cell proliferation in breast cancer MCF-7 and MDA-MB-231 cell lines35. e relative
potencies among these heterocyclic- and heteroarylmethylene-substituted aurones, the in vivo activity of these
aurones, and the specic biological target or targets in these cases was unclear.
We determined that semisynthetic aurones with either 3-indolylmethylene or 4-pyridylmethylene groups at
C-2 in place of the naturally occurring C-2 benzylidene group and with selected alkoxy groups at C-6 possessed in
vitro potencies in the mid- to low nanomolar range using in vitro PC-3 cancer cell proliferation assays. e most
potent of these aurones in these in vitro assays also displayed good activity in an in vivo PC-3 xenogra study.
Although our studies focused on developing agents for the treatment of prostate cancers, the prior report that
aurones with 2-(coumarin-4-yl)methylene groups32 or 2-(furan-2-yl)methylene groups33 displayed in vitro activ-
ity against human leukemia K562 cells prompted a study of myc-induced T-cell acute lymphoblastic leukemia
(T-ALL) in a zebrash model where these aurones also exhibited minimal toxicity. In summary, the aurones
reported in this paper showed activity in two dierent animal models, displayed no apparent toxicity in two dif-
ferent species, and, like the literature reports cited above, showed activity against not only against prostate cancer
PC-3 cells but also against leukemia cells. Finally, using a competition assay with mass spectrometry as an analyt-
ical tool, we established that these aurones functioned at a molecular level as tubulin polymerization inhibitors by
binding to the colchicine-binding site.
Results
Synthesis of semisynthetic aurones. e condensation of 6-hydroxybenzofuran-3(2H)-one (2) with a
spectrum of heteroaryl carboxaldehydes 3 under basic conditions led to aurones 4 (Fig.1B). A mixture of 50%
aqueous potassium hydroxide (2 eq) in 1:1 ethanol-N,N-dimethylformamide (DMF) was preferred over other
conditions23,36–38 reported for similar condensations. e assignment of (Z)-stereochemistry in 4 was in accord
with prior acid- or base-catalyzed condensations of benzofuran-3(2H)-ones with aromatic aldehydes39,40. e
subsequent alkylation of the C-6 hydroxyl group in aurones 4 using various alkyl bromides and anhydrous potas-
sium carbonate in DMF led to the 6-alkoxyaurones 5 (Fig.1B).
Figure 1. (A) Representative naturally occurring aurones, sulfuretin (1a) and aureusidin (1b). (B) Synthesis of
aurones 4 and 5. Legend: a, heterocyclic-substituted benzaldehydes or heteroaryl carboxaldehydes 3, 50% aq.
KOH, 1:1 EtOH:DMF, b, BrCH2CN, K2CO3, DMF; c, ClCH2C6H3-2,6-Cl2, K2CO3, DMF. (C) Biologically active
aurones 5a and 5b.
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Structure-activity relationships (SAR). A reiterative process of synthesis and screening using in vitro
prostate cancer PC-3 cell proliferation assays identied an intersection of modications at the C-2 and C-6
positions in semisynthetic aurones that were the most promising for further study (Table1). Initial screening
identied heteroarylmethylene-substituted aurones 4a–4d with 1-isoquinolylmethylene, 2-quinolylmethylene,
8-methoxy-2-quinolylmethylene, and 5-methoxy-N-ethyl-3-indolylmethylene groups at C-2 and hydroxyl
groups at C-6 as the most potent analogs at 10 μM concentrations but with only minimal activity at 1 μM con-
centrations (Table1). Modications at the C-4 and C-7 positions in the benzofuran ring in aurones 4 proved
unrewarding in terms of increased potency (data not shown). Eorts to identify benzylidene-substituted aurones
4 with saturated, heterocyclic groups attached to the phenyl ring were equally unrewarding with the exception of
(2Z)-6-hydroxy-2-(4-pyrrolidin-1-ylbenzylidene)-1-benzofuran-3(2H)-one (4e) (Table1).
Additional eorts to improve potency in aurones 4a–4e led to the alkylation of the C-6 hydroxyl group with
a range of alkylating agents to obtain 6-alkoxyaurones 5 (Fig.1B). An SAR study involving dual modications of
the C-6 alkoxy group and the C-2 heteroarylmethyelene group identied two aurones with 90%+ inhibition of in
vitro prostate cancer PC-3 cell proliferation at 300 nM concentration: (Z)-2-((2-((1-ethyl-5-methoxy-1H-indol-
3-yl)methylene)-3-oxo-2,3-dihydrobenzofuran-6-yl)oxy)acetonitrile (5a) and (Z)-6-((2,6-dichlorobenzyl)
oxy)-2-(pyridin-4-ylmethylene)benzofuran-3(2H)-one (5b) (Fig.1C). In a dose-response study, aurone 5a and
5b displayed IC50 values of 58.7 ± 1.1 nM and 66 ± 1.1 nM (Fig.2A), respectively. Aurone 5a displayed an IC50
value of 1.3 ± 0.2 μM using normal human embryo lung HEL299 cells that indicated that aurone 5a was selec-
tively more toxic to a cancer cell line than a normal cell line.
e pairing of the cyanomethoxy group at C-6 with the (N-ethyl-5-methoxy-1H-indol-3-yl)methylene at C-2
in aurone 5a and the pairing of the 2,6-dichlorobenzyloxy group at C-6 with the (pyridin-4-yl)methylene at
C-2 in aurone 5b (Fig.1C) were essential to potency. Alternate pairings, modication in the halogenation type
and pattern in the 2,6-dichlorobenzyloxy group, changes in the N-ethyl-5-methoxy-1H-indol-3-yl group (e.g.,
replacement of the N-ethyl with an N-methyl group; replacement of the 5-methoxy with a 5-hydroxy group), and
modications at still other positions in the benzofuran (e.g., methyl groups at C-7) led to diminished activity in
the prostate cancer PC-3 cell proliferation assay relative to aurones 5a and 5b. Finally, we performed additional
cell proliferation inhibition studies using other cancer cell lines, and aurones 5a and 5b showed potent low nano-
molar activities against these cell lines (Table2).
Prostate cancer PC-3 xenograft study in mice using aurone 5a. We evaluated the in vivo tumor
inhibitory eect of aurone 5a using prostate cancer PC-3 xenogras in immune-defective nude mice. PC-3 cells
were subcutaneously injected into both anks of nude mice. Two weeks aer the inoculation, the mice were ran-
domized to two groups (n = 5), treated with aurone 5a or control vehicle by intraperitoneal administration for
18 days and then sacriced. Compared to vehicle, the administration of 5a at 10 mg/kg/day showed signicant,
tumor-growth suppression (Fig.2B). Importantly, aurone 5a achieved tumor regression with no apparent gross
toxicity as reected by minimal changes in mice weights (Fig.2C). To understand the mechanisms of aurone
5a-induced tumor repression, we performed another PC-3 xenogra study by treating the tumors with vehi-
cle and aurone 5a for one week. Tumor sections were analyzed by H&E (Fig.2D) and immunohistochemistry
(IHC) stainings (Fig.2E,F). We observed increased apoptosis (Fig.2E) and decreased angiogenesis marker, VEGF
(Fig.2F), in aurone 5a-treated tumors.
Eect of aurone 5a on tubulin polymerization. An analysis of the screening data (Table3) from the
NCI-60 human tumor cell lines available through the developmental therapeutics program of NCI showed excel-
lent response to aurone 5a with IC50 values in the range of 200–500 nM. ese values were consistent with the
IC50 values determined by Vi-CELL XR 2.03 (Fig.2A and Table2). An analysis of the NCI-60 data from aurone
5a using the COMPARE algorithm41 matched the response of cell lines to aurone 5a with the response of other
tubulin-polymerization inhibitors. An analysis of the eects of aurone 5a on cell cycle progression using PC-3
cells indicated signicant cell cycle arrest at G2/M phases (Fig.3A,B), again consistent with the inhibition of tubu-
lin microtubule assembly. We then investigated the level of tubulin polymerization in PC-3 cells treated by aurone
5a at indicated concentrations. Aer cell treatment and lysis, we separated the cell lysates by centrifugation into
supernatants and pellets, that were individually subjected to western blotting using antibodies against β-tubulin.
Aer treatment with aurone 5a for 6 hours, the amount of tubulin in pellets was signicantly less than that in cell
lysates from dimethyl sulfoxide (DMSO)-treatment alone, even at a concentration as low as 300 nM (Fig.3C). We
Aurone C-6 C-2 Aryl or Heteroaryl Group
% Inhibition of PC-3 Cells
10 μM 1 µM300 nM
4a OH 1-isoquinolyl 88 ± 5.5
4b OH 2-quinolyl 99 ± 0.2 26 ± 8.6
4c OH 8-methoxy-2-quinolyl 97 ± 0.8 40 ± 8.4
4d OH N-ethyl-5-methoxy-3indolyl 69 ± 16 13 ± 9.5
4e OH 4-(pyrrolidin-1-yl)phenyl 95 ± 4.7 2.1 ± 7.1
5a OCH2CN N-ethyl-5-methoxy-3-indolyl 95 ± 2.5 93 ± 2.8
5b OCH2C6H3-2,6-Cl24-pyridyl 92 ± 0.4 95 ± 1.1
Table 1. Abbreviated SAR study involving modications aurone at the C-2 and C-6 positions using prostate
cancer PC-3 cell proliferation assays.
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also performed an in vitro tubulin polymerization assay in the presence and absence of aurone 5a. In the presence
of glycerol and guanosine triphosphate, either aurone 5a at 5 µM or colchicine at 5 µM decreased the formation
of microtubules in a similar fashion whereas a DMSO-treated control group showed, as expected, substantial
tubulin polymerization (Fig.3D).
Competition study of colchicine and aurone 5a for the colchicine-binding site on tubulin. A
competitive, tubulin-binding assay42 conrmed that aurone 5a bound to the colchicine-binding site. Aurone
5a was added at various concentrations to a solution of α/β-tubulins (1.3 mg/mL) and colchicine (1.25 μM).
Unbound colchicine was separated from either tubulin-colchicine or tubulin-aurone 5a complex by Amicon
Ultra-0.5 mL Centrifugal Filters (30 kDa Cut-o). e level of unbound colchicine was measured by liquid
chromatography-electrospray ionization-tandem mass spectrometry (LC-MS/MS). Aurone 5a released colchicine
from tubulin in a dose-dependent manner (Fig.3E) that indicated that aurone 5a bound to the colchicine-binding
site on tubulin.
Figure 2. (A) Dose responses of aurones 5a and 5b in PC-3 cell proliferation inhibition assay. (B) Eect of
aurone 5a on PC-3 tumor xenogras in nude mice (n = 5) at 10 mg/kg/day. (C) Eect on aurone 5a on body
weights of the treated mice: *P < 0.05, t-test. (D) H&E analysis of tumor sections. (E). Apoptosis analysis by
TUNEL assay. (F). IHC analysis of angiogenesis marker, VEGF-A.
Cell lines
IC50 (nM)
Aurone 5a Aurone 5b
PC-3 58.7 ± 1.1 66.0 ± 1.1
LS174T 155.2 ± 1.1 158.3 ± 1.0
A549 173.6 ± 1.0 113.0 ± 1.0
MCF-7 244.3 ± 1.2 185.6 ± 1.1
NCI/ADR-res 85.9 ± 1.0 190.3 ± 1.1
OVCAR-8 181.9 ± 1.0 257.7 ± 1.1
Table 2. IC50 values of aurones 5a and 5b in cancer cell line proliferation inhibition assays.
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Eects of aurone 5a on microtubule networks. We analyzed microtubule networks in PC-3 cells by
immunouorescence staining using an anti-α-tubulin Ab (Fig.3F–H). e control, DMSO-treated cells retained
their normal microtubule network and an overall, shuttle-like morphology (Fig.3F) whereas aurone 5a-treated
cells demonstrated signicant microtubule depolymerization and adopted a round morphology (Fig.3G,H).
Molecular docking analysis. We performed molecular docking using AutoDock Vina43 to explore the pos-
sible binding of aurone 5a to the colchicine-binding site (CBS) on αβ-tubulin heterodimers because this site was
well known to host a plethora of chemically unrelated compounds44. A less active aurone 4d (Fig.4A) than aurone
5a and colchicine were also docked into the CBS for comparison. We observed that aurone 5a, 4d and colchicine
occupied the CBS at the interface of the α-tubulin and α-tubulin heterodimer (Fig.4B). A hydrophobic pocket
formed by Ala, Ile and Leu residues from β-tubulin accommodated the hydrophobic indole moiety of aurone
5a (Fig.4C). e benzofuran-3(2H)-one and cyanomethoxy groups in aurone 5a participated in hydrophobic
contacts with the loop T7 and helix H8 of β-tubulin and with the loops T3, T4 and T5 from α-tubulin (Fig.4C)45.
In addition, hydrogen-bonding interactions between the carbonyl oxygen of the benzofuran-3(2H)-one and
βAsn258 and hydrogen-bonding interactions between the nitrogen of the cyanomethoxy group and αTyr224 and
αGln11 provided additional binding stabilization (Fig.4D).
e indole moiety and a portion of the benzofuran-3(2H)-one in aurone 5a superimposed well with the col-
chicine A and B rings (Fig.4E); however, aurone 5a did not occupy the hydrophobic pocket within β-tubulin in
which the colchicine C ring resided. Instead, aurone 5a formed contacts with loops T3, T4, and T5 of α-tubulin
Panel/Cell Line
GI50 (nM)
Panel/Cell Line
GI50 (nM)Leukemia Melanoma
CCRG-CEM 289 LOX IMVI 696
HL-60(TB) 236 MALME-3M >100 µM
K-562 212 M14 319
MOLT-4 523 MDA-MB-435 174
RPMI-8226 352 SK-MEL-2 836
SR 275 SK-MEL-28 10.2 µM
Non-Small Cell
Lung Cancer GI50 (nM) SK-MEL-5 405
UACC-257 67.1 µM
A549(ATCC) 5.1 µM UACC-62 499
EKVX 2.73 µMOvarian Cancer GI50 (nM)
HOP-62 542
HOP-92 NA IGROV1 774
NCI-H226 57.4 µM OVCAR-3 377
NCI-H23 812 OVCAR-4 19 µM
NCI-H322M 1.43 µM OVCAR-5 2.52 µM
NCI-H460 337 OVCAR-8 483
NCI-H522 3.13 µM NCI/ADR-RES 406
Colon Cancer GI50 (nM) SK-OV-3 669
Renal Cancer GI50 (nM)
COLO 205 446
HCC-2998 3.44 µM 786-0 470
HCT-116 386 A498 10.3 µM
HCT-15 399 ACHN 794
HT29 356 RXF 393 182
KM12 546 SN 12C 763
SW-620 345 TK-10 56.9 µM
CNS Cancer GI50 (nM) UO-31 864
Breast Cancer GI50 (nM)
SF-268 848
SF-295 307 MCF7 311
SF-539 269 MDA-MB-231 2.66 µM
SNB-19 468 HS 578T 360
SNB-75 5.65 µM BT-549 571
U251 453 T-47D NA
Prostate Cancer GI50 (nM) MDA-MB-468 2.16 µM
PC-3 367
DU-145 643
Table 3. IC50 values of aurone 5a in NCI-60 cell line proliferation inhibition assays (Data were produced by the
Nation Cancer Institute (Maryland, USA).
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using the benzofuran-3(2H)-one and cyanomethoxy groups. Additionally, a comparison of the binding poses of
5a and 4d revealed why 5a possessed better potency than 4d. Aurone 4d had major interactions with β-tubulin
but lacked the bifurcated, hydrogen-bonding between the nitrogen of the cyanomethoxy group and αTyr224 and
αGln11 of α-tubulin. is deciency weakened the binding anity of aurone 4d relative to the potent aurone 5a
(Fig.4D).
Leukemia cell study in zebrash using aurone 5a. In addition to the in vivo PC-3 xenogra study
in mice, we sought to test these aurones in a second species. Two prior reports indicated that aurones with
2-(coumarin-4-yl)methylene groups or 2-(furan-2-yl)methylene groups displayed in vitro activity against a leu-
kemia cell line. Consequently, we tested various leukemia cell lines and found that the IC50 values for aurone
5a were in the mid-nanomolar range (Table4). e IC50 values of two normal B-lymphoblast cells were much
higher than the leukemia cell lines and suggested a preferential toxicity of aurone 5a toward leukemia cells.
Because these leukemia cell lines had various mutations, we tested the activity of aurone 5a in vivo using a genet-
ically well-dened, zebrash myc-induced T-ALL leukemia model46,47 (Fig.5). e zebrash (Danio rerio) is a
vertebrate system that develops tumors similar to those in humans and that provides a plaform that is easy to
manipulate for in vivo assays even in large-scale screens. According to previous studies46,47, the zebrash Rag2
promotor controlling the myc-GFP transgene specically targets gene expression to lymphoid cells. e Rag2:
myc-GFP transgene was micro-injected into wild-type zebrash embryos at the one-cell development stage, and
a small fraction of injected embryos developed c-myc induced leukemia. We treated GFP-labeled leukemia cells
in zebrash with either DMSO (Fig.5A at day 0 and 5D at day 5); aurone 5a in DMSO (Fig.5B at day 0 and 5E
at day 5); or aurone 5b in DMSO (Fig.5C at day 0 and 5 F at day 5). Since aurone 5a had auto-uorescence that
interfered with visualizing the loss of the GFP-labeled leukemia cells (Fig.5E), we selected aurone 5b that lacked
this auto-uorescence and clearly displayed the loss of the GFP-labeled leukemia cells (Fig.5F). Aurone 5a and 5b
signicantly blocked the progression of T-ALL in zebrash (Fig.5D versus 5F, Fig.5G).
Figure 3. (A,B) Aurone 5a induced cell cycle arrest. (C) Aurone 5a decreased tubulin polymerization. (D)
Aurone 5a (5 µM) and colchicine (5 µM) inhibited tubulin polymerization in vitro in a similar fashion. (E)
Competitive tubulin binding assay with colchicine in the presence of increasing concentrations of aurones 5a.
(F–H) Aurone 5a treatment (6 h) inhibited microtubule structures and caused cell morphology change in PC-3
cells as shown in panels F, DMSO; G, 5a (1 µM); H, 5a (300 nM). Red immunouorescence: α-tubulin; blue:
DAPI.
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Interaction of aurone 5a with potassium channel derived from human ether-a-go-go related
gene (hERG). Inhibition of the hERG potassium channel derived from hERG oen leads to drug failure
in preclinical studies or even in clinical trials. We utilized a well-established [3H]-dofetilide binding assay48 to
evaluate the interaction of aurones with hERG. [3H]-Dofetilide competition binding assays using HEK-293 cell
membranes stably expressing the hERG channel (hERG-HEK) correlated well with results from voltage-clamp
assays and provided useful predictive screening assays for QT prolongation49. Amitriptyline (nal concentration,
1 mM) was used as the positive control and exhibited an IC50 value (10.7 ± 2.25 μM) in agreement with published
values50. Concentrations of aurones 5a and 5b ranging from 10−9 to 10−4 M were assayed in duplicate for these
experiments (n = 3 experiments/analog). As desired, aurones 5a and 5b displayed no hERG inhibition (IC50 val-
ues > 100 μM).
Discussion
Two types of inhibitors target tubulin microtubule dynamics: stabilizing agents, such as paclitaxel, and destabiliz-
ing agents, such as the Vinca alkaloids and colchicine. ese agents bind tubulin subunits at well-characterized,
binding sites, some of which nd broad application in cancer therapeutics, including prostate cancer. Until
recently, few agents were known that targeted the colchicine-binding site, but various pharmacophores25–31
now appear to exhibit excellent potency and selective binding to the colchicine-tubulin site. e impetus for
Figure 4. (A) Structures of aurone 5a, a less active aurone 4d, and colchicine. (B) Aurone 5a bound to the
colchicine-binding site (CBS) in the interface of αβ-tubulin dimers (cyan for β, green for α). (C) Close-up view
of the interaction environment of 5a (gray sticks) and tubulin (carton). (D) Superimposition of 5a (gray sticks)
and 4d (magenta sticks) in the colchicine-binding site. Hydrogen bonding is represented by yellow, dashed lines.
(E) Superimposition of 5a (gray sticks) and colchicine (purple sticks) in the colchicine-binding site.
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developing these agents derives in part from the continuing need for new tubulin-targeting drugs to meet
the needs of patients experiencing resistance or developing mutations crippling the use of traditional taxol or
Vinca-based therapies. e semisynthetic aurones reported here provide a new pharmacophore for the develop-
ment of colchicine-targeting microtubule inhibitors for cancer treatment.
Prior reports that naturally occurring aurones, such as sulfuretin (1a) and aureusidin (1b) (Fig.1A), and several
semisynthetic aurones possessed in vitro antineoplastic activity encouraged our interest in exploring SAR relation-
ships within the aurone pharmacophore. A straightforward condensation of 6-hydroxybenzofuran-3(2H)-one (2)
with various aryl or heteroaryl carboxaldehydes 3 furnished aurones 4 in which the C-2 benzylidine groups were
either substituted with or replaced by heterocycles (Fig.1B). Using a PC-3 cell proliferation assay as a readout, we
found that the aurones 4a-4e bearing nitrogen-containing heterocycles at C-2 were marginally active in a 1–10
μM concentration range (Table1). Alkylation of the C-6 hydroxyl group in concert with alterations in the C-2
heteroarylmethylene subunit led ultimately to two aurones 5a and 5b (Fig.1C) with IC50 values of 58.7 ± 1.1 nM
and 66 ± 1.1 nM, respectively (Fig.2A). e pairing of the unusual cyanomethoxy group at C-6 with the (N-ethyl-
5-methoxy-1H-indol-3-yl)methylene at C-2 in aurone 5a and the pairing of the 2,6-dichlorobenzyloxy group at C-6
with the (pyridin-4-yl)methylene at C-2 in aurone 5b were essential to achieve nanomolar potency.
Cell Line C el l Typ e IC50 (nM) 95% Condence
Interval (nM)
CCRF-CEM T-ALL 244 197–301
DND41 T-ALL 210 116–379
Jurkat T-ALL 273 226–344
HBP-ALL T-ALL 94 51–173
Loucy T-ALL 334 285–391
Molt-4 T-ALL 241 114–402
Molt-16 T-ALL 234 218–250
RPMI8402 T-ALL 301 248–364
Nalm-16 B-ALL 272 248–291
REH B-ALL 287 252–326
NCI-BL2009a Normal B-Lymphoblast 1,253 429–3,658
HCC1007-BL Normal B-Lymphoblast 1,379 372–2,490
Table 4. IC50 values of aurone 5a in leukemia cell line proliferation inhibition assays.
Figure 5. Aurones 5a and 5b inhibited myc-induce T-ALL in a zebrash model. (A,D) Treatment of GFP-
labeled thymic lymphoma cells with DMSO alone at day 0 and day 5, respectively. (B,E) Treatment of GFP-
labeled thymic lymphoma cells with aurone 5a in DMSO at day 0 and day 5, respectively. (C,F) Treatment of
GFP-labeled thymic lymphoma cells with aurone 5b at day 0 and day 5, respectively. (G) Percent change in
uorescence (i.e., number of GFP-labeled thymic lymphoma cells) as a function of time from administration of
DMSO alone to the administration of aurone 5b in each zebrash (n = 8).
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In addition to these in vitro studies, we evaluated the in vivo tumor inhibitory eect of aurone 5a using pros-
tate cancer PC-3 xenogras in immune-defective nude mice. Compared to vehicle, the administration of aurone
5a at 10 mg/kg/day showed signicant, tumor-growth suppression (Fig.2B). Importantly, aurone 5a achieved
tumor regression with no apparent gross toxicity as reected by minimal changes in mice weights (Fig.2C). IHC
staining suggested that aurone 5a treatment induced apoptosis and decreased angiogenesis in the xenograed
tumors (Fig.2E,F), which is consistent with the function other microtubule inhibitors51. In summary, SAR stud-
ies identied aurone 5a that possessed good in vitro activity in cancer cell proliferation studies in the nanomolar
range, good reduction in tumor volume in an in vivo prostate PC-3 xenogra study, and minimal gross toxicity
based on minimal weight loss during the in vivo studies. Preliminary indications involving the minimal eects
on normal cell proliferation, the minimal changes in mice weights during xenogra studies, the absence of hERG
inhibition and absence of toxic eects on zebrash in studies, as described below, suggested that aurone 5a had an
acceptable “toxicity window” that was sucient to warrant further study.
Knowledge about the binding site between a ligand and its biological target is pivotal for structure-guided,
rational design of compounds with improved properties including potency and solubility. Molecular docking
studies showed that aurone 5a binds to the colchicine-binding site between the α-tubulin and β-tubulin. e
indole moiety and part of the benzofuran-3-one of aurone 5a as well as the A and B rings of colchicine occupied a
hydrophobic pocket in β-tubulin (Fig.4C,E). However, aurone 5a did not occupy another hydrophobic pocket in
which the colchicine C ring normally resided. Instead, aurone 5a interacted more with α-tubulin than β-tubulin
and participated in bifurcated hydrogen-bonding between the nitrogen of the cyanomethoxy group and αTyr224
and αGln11 of α-tubulin (Fig.4D). e relatively inactive aurone 4d failed to form this same interaction because
it lacked a cyanomethyl group.
To conrm that aurone 5a bound to the colchicine-binding site, we performed a tubulin polymerization assay
and a competitive tubulin-binding assay42. Aurone 5a inhibited tubulin polymerization in vitro (Fig.3D). In
addition, aurone 5a bound to the CBS, resulting in an increased amount of unbound colchicine (Fig.3E). ese
data were consistent with molecular docking results, and echoed the fact that the CBS would accommodate chem-
ically diverse compounds. Mechanistically, previous crystallography studies show that free tubulin dimers are in
a “straight” state and polymerized tubulin dimers in microtubules are in a “curved” conformation45,52–54. During
tubulin polymerization, tubulin dimers structurally transitioned from a straight state to a curved state, during
which the T7 loop of β-tubulin ipped inwards into the CBS. As a mechanism of action, colchicine bound to the
CBS, prevented the T7 loop ipping towards the CBS, and thus inhibited tubulin polymerization53,55. Importantly,
our leading compounds showed strong interaction with T7 loop (Fig.4C,E) and reected a similar mechanism
of action seen with colchicine. As a result, aurone 5a strongly inhibited cell cycle progression at G2/M phases
(Fig.3A,B) and disrupted microtubule networks in PC-3 cells (Fig.3F–H).
By testing the efficacy of aurone 5a in the NCI-60 and other cell lines, we found that 5a demonstrated
broad-spectrum, anticancer activity (Fig.2A, Tables2–4). e NCI/ADR-RES cell line that was normally resist-
ant to adriamycin and many other cancer chemotherapeutics due to the expression of P-glycoprotein exhibited
inhibition by aurone 5a, and hence, aurone 5a was not a likely substrate of P-glycoprotein. As previously noted,
aurone 5a showed no general toxicity in nude mice at doses that signicantly inhibited PC-3 tumor xenogras
(Fig.2C). We also tested aurones 5a and 5b in zebrash models where we again observed no gross toxicity on
zebrash but observed signicant inhibition of myc-induced T-ALL in vivo (Fig.5). e zebrash myc-induced
T-ALL model could be an important in vivo tool to screen and characterize future aurone analogs.
In summary, we identied two potent, semisynthetic aurones 5a and 5b that function as tubulin inhibitors
with IC50 values of 58.7 ± 1.1 nM and 66 ± 1.1 nM, respectively (Fig.2A). Importantly, aurone 5a displayed activ-
ity in an in vivo PC-3 prostate cancer xenogra model in nude mice at 10 mg/kg without aecting mice weight
(Fig.2B,C). Aurones 5a and 5b showed potent in vivo activity in a genetically well-dened, zebrash myc-induced
T-ALL leukemia model46,47 (Fig.5). Aurone 5a also displayed no appreciable anity for human hERG potassium
channel and was not a substrate of P-glycoprotein. An analysis of screening data from the NCI-60 human tumor
cell lines using the COMPARE algorithm41 matched the response to aurone 5a with other tubulin-polymerization
inhibitors. We used combination of experimental studies to examine this prediction: a competition study of col-
chicine and aurone 5a for the colchicine-binding site on tubulin (Fig.3E), a study of the comparative inhibition
of tubulin polymerization with aurone 5a and colchicine (Fig.3D), and detailed computational modeling of the
binding of these agents to tubulin (Fig.4). Liquid chromatography-electrospray ionization-tandem mass spec-
trometry studies further conrmed that aurone 5a targeted the colchicine-binding site on tubulin. Continued
studies will dene the pharmacokinetic and pharmacodynamics properties of aurones in this family.
Chemistry Materials and Methods. Chemicals were purchased from Sigma-Aldrich (St. Louis, MO)
or Fisher Scientic (Pittsburgh, PA) unless otherwise noted or were synthesized according to literature proce-
dures. Solvents were used from commercial vendors without further purication unless otherwise noted. Nuclear
magnetic resonance spectra were determined on Varian instruments (1H, 400 or 500 MHz; 13C, 100 or 126 Mz).
Low-resolution mass spectra were obtained using an Agilent 1100 (atmospheric pressure, chemical ionization)
instrument. High resolution mass data were obtained by direct infusion electrospray ionization mass spectrom-
etry (-MS) using a LTQ-Orbitrap mass spectrometer coupled with a Heated Electrospray Ionization (HESI-II)
Probe (ermo Fisher Scientic, Waltham, MA) and an FT analyzer at a resolution of 100,000. e reported
m/z mass was a mean of 20 scans. Melting points were determined in open capillarity tubes with a Buchi B-535
apparatus and are uncorrected. Compounds were puried by chromotography on preparative layer Merck silica
gel F254 unless otherwise noted.
General procedure for the synthesis of aurones 3a-3f and 4a-4o. To a suspension of 10 mmol of
6-hydroxybenzofuran-3(2H)-one (2) (Ark Pharm, Arlington Heights, IL USA) in 20 mL of a 1:1 mixture of DMF
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and absolute ethanol was added 2.3 mL of 50% aqueous potassium hydroxide. To this clear solution, obtained aer
stirring for ca. 30 min, was added 10 mmol of the appropriate carboxaldehyde. e mixture was stirred for 6–8 h
at 25 °C. e mixture was diluted with 100 mL of hot water, acidied with glacial acetic acid pH 5. e resulting
precipitate was collected by ltration, washed with water, dried and re-crystallized from DMF-methanol.
(2Z)-6-Hydroxy-2-(isoquinolin-1-ylmethylene)-1-benzofuran-3(2H)-one (4a). Yellow crystals
(78% yield); mp > 220 °C; 1H NMR (400 MHz, DMSO-d6) δ 6.71–6.76 (m, 2H), 7.43 (s, 1H), 7.69 (d, J = 8.3 Hz,
1H), 7.7–7.77 (m, 1H), 7.79–7.85 (m, 1H), 7.87 (d, J = 5.6 Hz, 1H), 8.03 (d, J = 8.1 Hz, 1H), 8.35 (d, J = 8.9 Hz,
1H), 8.69 (d, J = 5.6 Hz, 1H), 11.3 ppm (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ 98.78, 105.12, 112.39, 113.23,
120.99, 125, 126.42, 127.44, 128.25, 130.59, 135.8, 142.65, 149.81, 151.34, 167.15, 169.05, 182.01 ppm; MS (ACPI)
m/z 290.2 (MH+, 100); HRMS (ESI/HESI) m/z: [M + H]+ Calcd for C18H11NO3 290.0812; Found 290.0810.
(2Z)-6-Hydroxy-2-(quinolin-2-ylmethylene)-1-benzofuran-3(2H)-one (4b). Yellow crystals (72%
yield); mp 249–251 °C; 1H NMR (400 MHz, DMSO-d6) δ 6.75 (dd, J = 8.4, 2 Hz, 1H), 6.78–6.9 (m, 2H), 7.56–7.73
(m, 2H), 7.75–7.88 (m, 1H), 7.93–8.13 (m, 2H), 8.29 (d, J = 8.7 Hz, 1H), 8.48 (d, J = 8.7 Hz, 1H), 11.39 ppm (s,
1H); 13C NMR (126 MHz, DMSO-d6) δ 98.84, 110, 112.29, 113.41, 122.62, 126.29, 126.88, 127.47, 127.79, 129.08,
130.11, 136.72, 147.82, 149.55, 151.88, 167.13, 168.36, 181.53 ppm; MS (ACPI) m/z 290.0 (MH+, 100); HRMS
(ESI/HESI) m/z: [M + H]+ Calcd for C18H11NO3 290.0812; Found 290.0806.
(2Z)-6-Hydroxy-2-[(8-methoxyquinolin-2-yl)methylene]-1-benzofuran-3(2H)-one (4c). Ye ll ow
crystals (68% yield); mp 250–252 °C; 1H NMR (400 MHz, DMSO-d6) δ 4 (s, 3H), 6.75 (dd, J = 8.5, 2 Hz, 1H), 6.81
(s, 1H), 6.85 (d, J = 2 Hz, 1H), 7.09–7.30 (m, 1H), 7.45–7.62 (m, 2H), 7.67 (d, J = 8.5 Hz, 1H), 8.3 (d, J = 8.7 Hz,
1H), 8.42 (d, J = 8.7 Hz, 1H), 11.38 ppm (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ 55.75, 98.59, 109.12, 110.09,
112.2, 113.21, 119.05, 122.67, 125.9, 127.71, 127.77, 136.19, 139.81, 149.11, 150.13, 155.13, 166.87, 168.12, 181.22
ppm; MS (ACPI) m/z 320.0 (MH+, 100); HRMS (ESI/HESI) m/z: [M + H]+ Calcd for C19H13NO4 320.0917;
Found 320.0919.
(2Z)-2-[(1-Ethyl-5-methoxy-1H-indol-3-yl)methylene]-6-hydroxy-1-benzofuran-3(2H)-one
(4d). Yellow crystals (77% yield); mp 265–267 °C; 1H NMR (400 MHz, DMSO-d6); δ 1.39 (t, J = 7.2 Hz, 3H),
3.85 (s, 3H), 4.27 (q, J = 7.2 Hz, 2H), 6.72 (dd, J = 8.4, 2 Hz, 1H), 6.83 (d, J = 2 Hz, 1H), 6.87 (dd, J = 8.9, 2.4 Hz,
1H), 7.23 (s, 1H), 7.42 (d, J = 8.8 Hz, 1H), 7.56–7.64 (m, 2H), 8.18 (s, 1H), 10.98 ppm (s, 1H); 13C NMR (126 MHz,
DMSO-d6); δ 15.38, 41.3, 55.46, 98.49, 101.1, 105.37, 107.42, 111.41, 112.56, 112.76, 114.38, 125.36, 128.22,
130.74, 133.5, 144.76, 155.02, 165.47, 166.52, 180.12 ppm; MS (ACPI) m/z 336.0 (MH+, 100); HRMS (ESI/HESI)
m/z: [M + H]+ Calcd for C20H17NO4 336.1230; Found 336.1224.
(2Z)-6-Hydroxy-2-(4-pyrrolidin-1-ylbenzylidene)-1-benzofuran-3(2H)-one (4e). Yellow crys-
tals (83% yield); mp > 220 °C; 1H NMR (400 MHz, DMSO-d6) δ 1.83–2.07 (m, 4H), 3.26–3.32 (m, 4H), 6.61 (d,
J = 8.9 Hz, 2H), 6.66–6.72 (m, 2H), 6.77 (d, J = 1.9 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.77 (d, J = 8.9 Hz, 2H), 11
ppm (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ 24.95, 47.24, 98.42, 111.97, 112.58, 112.94, 113.69, 118.62, 125.39,
133.16, 144.73, 148.49, 165.58, 166.87, 180.64 ppm; MS (ACPI) m/z 308.1 (MH+, 100); HRMS (ESI/HESI) m/z:
[M + H]+ Calcd for C19H17NO3 308.1281; Found 308.1279.
(Z)-2-((2-((1-Ethyl-5-methoxy-1H-indol-3-yl)methylene)-3-oxo-2,3-dihydrobenzofuran-6-yl)
oxy)acetonitrile (5a). To a solution of 670 mg (2 mmol) of (2Z)-2-[(1-ethyl-5-methoxy-1H-indol-3-yl)
methylene]-6-hydroxy-1-benzofuran-3(2H)-one (4d) in 10 mL of DMF was added 830 mg (6 mmol, 3 eq) of
anhydrous potassium carbonate. e mixture was heated to 60 °C and 0.152 mL (2.4 mmol, 1.2 eq) of chloroace-
tonitrile was added. e mixture was stirred at 60 °C for an additional 8 h, cooled, and poured into 100 mL of 0.1 N
aqueous sulfuric acid. e precipitate was collected by ltration, washed with water, dried and re-crystallized
from DMF-methanol to afford 487 mg (65%) of 5a as yellow crystals: mp 230–232 °C; 1H NMR (400 MHz,
DMSO-d6) δ 1.44 (d, J = 7.2 Hz, 3H), 3.86 (s, 3H), 4.33 (q, J = 7.2 Hz, 2H), 5.39 (s, 2H), 6.9 (dd, J = 8.9, 2.4 Hz,
1H), 6.97 (dd, J = 8.6, 2.2 Hz, 1H), 7.29 (d, J = 2.2 Hz, 1H), 7.37 (s, 1H), 7.51 (d, J = 8.9 Hz, 1H), 7.63 (d, J = 2.4 Hz,
1H), 7.77 (d, J = 8.6 Hz, 1H), 8.23 ppm (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ 14.67, 40.97, 53.89, 55.34, 98.17,
101.45, 106.18, 107.17, 110.99, 111.68, 112.46, 115.45, 116.87, 124.84, 127.89, 130.8, 133.46, 144.2, 154.96, 162.6,
165.49, 179.58 ppm; MS (ACPI) m/z 375.2 (MH+, 100); HRMS (ESI/HESI) m/z: [M + H]+ Calcd for C22H19N2O4
375.1339; Found 375.1337.
(2Z)-6-[(2,6-Dichlorobenzyl)oxy]-2-(pyridin-4-ylmethylene)-1-benzofuran-3(2H)-one (5b). To
a solution of 1.5 g (10 mmol) of 6-hydroxybenzofuran-3(2H)-one (2) in 30 mL of DMF was added 4.14 g
(30 mmol, 3 eq) of anhydrous potassium carbonate followed by 2.35 g (12 mmol, 1.2 eq) of 2,6-dichlorobenzyl
chloride (ermosher Acros Organics, Geel, Belgium). e mixture was stirred at 25 °C for 8 h and diluted
with 200 mL of water. e precipitate was collected, washed with water, dried and puried by column chro-
matography using 1:100 dichloromethane-methanol to afford 1.79 g (58%) of 6-((2,6-dichlorobenzyl)oxy)
benzofuran-3(2H)-one as pale yellow crystals: mp 153–155 °C. 1H NMR (400 MHz, CDCl3) δ 4.64 (s, 2H), 5.34 (s,
2H), 6.67–6.77 (m, 2H), 7.29 (d, J = 7.2 Hz, 1H), 7.33–7.42 (m, 2H), 7.58 (d, J = 9 Hz, 1H); 13C NMR (100 MHz,
CDCl3) δ 65.57, 75.56, 97.32, 111.98, 114.76, 125.15, 128.56, 130.9, 130.96, 136.97, 167.18, 176.32, 197.49 ppm;
MS (ACPI) m/z 309.2 (MH+, 100). To 50 mL of a freshly prepared 0.2 M (5 eq) solution of sodium methoxide was
added a solution of 618 mg (2 mmol) of 6-((2,6-dichlorobenzyl)oxy)benzofuran-3(2H)-one and 214 mg (2 mmol,
1 eq) of 4-pyridinecarboxaldehyde in 5 mL of methanol. e mixture was stirred at 25 °C for 12 h. e solution
was concentrated and poured into 100 mL of water at 0 °C. e mixture was acidied with 1N aqueous hydrochlo-
ric acid solution to ca. pH 6. e precipitate was collected by ltration and recrystallized from 2:1 DMF-methanol
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to aord 445 mg (56%) of 5b: mp 219–222 °C; 1H NMR (400 MHz, CDCl3) δ 5.41 (s, 2H), 6.7 (s, 1H), 6.88 (dd,
J = 8.6, 2.2 Hz, 1H), 6.96 (d, J = 2.2 Hz, 1H), 7.28–7.36 (m, 1H), 7.36–7.45 (m, 2H), 7.68–7.78 (m, 3H), 8.7 ppm
(d, J = 5.2 Hz, 2H); 13C NMR (126 MHz, CDCl3) δ 66.04, 98, 108.3, 113.23, 114.8, 124.74, 126.45, 128.78, 130.92,
131.17, 137.19, 139.95, 150.3, 150.36, 167.19, 168.85, 182.73 ppm; MS (ACPI) m/z 398.0 (MH+, 100); HRMS (ESI/
HESI) m/z: [M + H]+ Calcd for C21H13Cl2NO3 398.0345; Found 398.0349.
Biological Studies. PC-3, MCF-7 and A549 cells were cultured in the medium recommended by American
Type Culture Collection at 37 °C with 5% CO2 atmosphere in a water jacketed incubator (NuAire). Ovcar-8
and NCI/ADR-RES cells were gis from Dr. Markos Leggas, University of Kentucky, Lexington, KY USA. e
beta-tubulin antibody was from Developmental Studies Hybridoma Bank. (Iowa city, IA USA).
Cell proliferation inhibition assay. Cancer cells were seeded into 24-well plates at a density of 20,000 cells
per well in 1 mL of culture medium and were cultured overnight at 37 °C. Compounds and the vehicle control
(DMSO) were added to the cells. Aer 6 days, the medium was removed, and 100 µL of trypsin was added. e
cells were re-suspended in phosphate-buered saline (PBS) and were counted by Vi-CELL XR 2.03 (Beckman
Coulter, Inc. USA). e ratio R of the number of viable cells in the compound treatment group to the number of
viable cells in DMSO treatment group was taken as relative growth, and the percentage growth inhibition was
calculated as (1 − R)*100. For initial testing, compounds were added to the cells at a nal concentration of 10 µM.
Active compounds at 10 µM were tested at lower concentrations than 10 µM.
In vitro tubulin polymerization assay. An in vitro tubulin polymerization assay was performed using
a protocol from Cytoskeleton, Inc. (Denver, CO USA). Tubulin powder (Cytoskeleton Inc. Denver, CO USA)
was dissolved in a buer prepared from 100 mM PIPES (pH 6.9), 2 mM MgCl2, 1 mM GTP, and 5% glycerol at
0 °C. Aliquots (80 µL, 3.75 µg/µL) of this tubulin solution were divided into the wells of a 96-well half-area plate
(Corning Inc., NY USA). Aer adding either DMSO or testing compounds, the plate was mounted on a Spectra
MRTM microplate spectrophotometer equipped with a thermal controller at 37 °C (Dynex Technologies, Inc.,
Chantilly, VA USA). Readings at 350 nm were recorded every 30 s for 1 h.
In vivo microtubule assembly assay. e amount of insoluble polymerized microtubules and soluble
tubulin dimers in cells aer exposure to aurones were detected using a reported method. Cells were seeded in
6-well plates at 50% conuency and cultured overnight. DMSO or aurones in DMSO solution were added, and
the cells were incubated for additional 6 h. e medium was removed, and cells were washed with PBS three times
followed by the addition of a lysis buer prepared from 20 mM Tris-HCl (pH 6.8), 1 mM MgCl2, 2 mM EGTA,
20 µg/mL aprotinin, 20 µg/mL leupeptin, 1 mM PMSF, 1 mM orthovanadate, and 0.5% NP40. e lysates were
centrifuged at 12,000 g for 10 min to obtain supernatants and pellets that were mixed with loading buer and
heated to 100 °C. Standard western blotting against α-tubulin was performed as described previously56.
Immunouorescence imaging. Tubulin networks were examined by confocal immunouorescence imag-
ing. Briey, PC3 cells were placed at a density of 80,000/mL to 24-well plates equipped with round microscope
glass cover slides. Aer culturing at 37 °C for 24 hours, DMSO or compounds were added to the cells and incu-
bated for additional 6 hours. en the medium was removed and the cells were washed with PBS three times.
Primary anti-α-tubulin antibody was added and incubated overnight at 4 °C. Aer additional washing, secondary
TRITC-conjugated anti-rabbit antibody was added for 40 min, followed by additional washing and staining with
DAPI. Final washing was performed and the cover slides were inverted onto glass slides. Images (40x) were taken
using a Nikon confocal microscope with excitation at 557 nm and emission at 576 nm.
Molecular docking studies. An X-ray crystal structure of αβ-tubulin binding with colchicine (pdb:
4O2B) was downloaded from RCSB Protein Data Bank and manipulated using AutoDockTools-1.5.6 (Molecular
Graphics Laboratory, e Scripps Research Institute, La Jolla, CA 92037 USA).e αβ-Tubulin dimer was sepa-
rated from 4O2B using PyMOL (Version 1.7.4.5 Edu). Water molecules were removed, and polar hydrogens and
Kollman charges were added. e docking pocket (colchicine-binding site) was dened as follows: Search space:
18 × 18 × 18 Å3; Center_x, y, z = 14.815, 9.422, −20.186. e aurones 4d, 5a, and colchicine were manipulated
by Openbabel. Molecular docking of 4d, 5a, and colchicine to the colchicine-binding site was executed using
AutoDock vina-1.1.2 using the iterated gradient-based local search method with a Broyden–Fletcher–Goldfarb–
Shanno (BFGS) method for local optimization43. Exhaustiveness was set at 14 and the number of modes was nine.
Other parameters were le at default values.
hERG binding studies. An HEK-293 cell line stably expressing the hERG potassium channel (accession
number U04270) referred to as hERG-HEK cells were received at passage 11 (P11) from Millipore (CYL3006,
lot 2, Billerica, MA USA). [3H]-Dofetilide (specic activity of 80 Ci/mmol; labeled on the N-methyl group)
was obtained from American Radiolabeled Chemicals, St. Louis, MO USA). Other chemicals and solvents
were obtained from Sigma-Aldrich (Milwaukee, WI USA) with exceptions of polyethylenimine (PEI), which
was obtained from Fluka/Sigma-Aldrich (St. Louis, MO USA), and Minimium Essential Medium (MEM) with
GlutaMAXTM and phenol red, MEM non-essential amino acids solution (NEAA, 100X), G418 disulfate salt solu-
tion, fetal bovine serum (FBS), 0.05% Trypsin-EDTA 1X with phenol red, and Hank’s balanced salt solution
(HBSS), which were obtained from Life Technologies (Carlsbad, CA USA).
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hERG-HEK Cell Culture. e hERG-HEK cells were cultured according to the protocol provided by Merck
Millipore (Burlington, MA USA). 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 humidied atmosphere with 5%
CO2. Frozen aliquots of cells were transferred into T-75 cm2 asks and allowed to adhere for 4–8 h. e medium
was replaced every 2 days. Passages were carried out at least 3 times aer thawing at 6 day intervals. 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 aer the last
passage (passage 20).
Membrane preparation. Cell membrane preparation was based on previous methods49,50,57. Cells were
rinsed twice with HBSS at 37 °C and collected by scraping the dishes in ca. 20 mL of ice-cold 0.32 M sucrose and
homogenized on ice with a Teon pestle using a Maximal Digital homogenizer (Fisher Scientic, Pittsburgh, PA
USA) at ~280 rpm for 30 sec. Homogenates were centrifuged at 300 g and 800 g for 4 min each at 4 °C. Pellets were
resuspended in 9 mL of ice-cold Milli-Q water and osmolarity restored by addition of 1 mL of 500 mM Tris buer
(pH 7.4) followed by suspension and centrifugation at 20,000 g for 30 min at 4 °C. Pellets were homogenized in
2 mL assay buer (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.
[3H]-Dofetilide binding assay. [3H]-Dofetilide binding assays using hERG-HEK293 cell membranes were
based on previous methods. Assays determining concentration-response were performed in duplicate, and three
independent assays were performed for each analog evaluated. Cell membrane suspension (5 μg) was added to
duplicate tubes containing assay buer, 25 μL of a single concentration of FIDAS agent (concentration range of 10
nM-100 µM for each experiment), and 25 μL of [3H]-dofetilide (5 nM, nal concentration) for an assay volume
of 250 μL. Binding occurred for 60 min at 25 °C and was terminated by rapid ltration through Whatman GF/B
lters, which were pre-soaked in 0.25% PEI overnight, using a Brandel cell/membrane harvester (M-48; Brandel
Inc., Gaithersburg, MD USA). Filters were washed three times with ca. 1 mL of ice-cold assay buer. Radioactivity
was determined by liquid scintillation spectrometry using the Tri-Carb 2100-TR Liquid Scintillation Analyzer
(Perkin-Elmer Life and Analytical Sciences).
In vivo evaluation of anti-leukemia activity in the zebrash model. Zebrash studies were car-
ried out with approval from the Institutional Animal Care and Use Committees of the University of Kentucky
(2015–2225). All methods were performed in accordance with the relevant guidelines and regulations according
to protocols. Rag2: myc-GFP zebrash (n = 8) at 21 days of age were treated with DMSO, either aurones 5a or 5b
in 1.5 mL of sh-system water in 12-well plates. Zebrash were treated with compound for 2 days, removed from
drug for 1 day, and treated for two more days with freshly prepared solutions of compound. Animals were imaged
at the start and end of treatment using a uorescence-equipped dissecting microscope at 350 ms exposure. e
GFP image was overlaid onto the bright-eld image of each animal in Photoshop, and the percent change in leu-
kemia burden was calculated by normalizing the GFP+ area to the total area of the animal in ImageJ (National
Institute of Health, USA).
In vivo evaluation of anti-cancer activity and gross toxicity in PC-3 xenografts. Mouse stud-
ies were carried out with approval from the Institutional Animal Care and Use Committees of the University
of Kentucky (2009–1064). All methods were performed in accordance with the relevant guidelines and regula-
tions according to protocols. PC-3 cells suspended in PBS were subcutaneously injected in the lower anks of
immune-decient nude mice (5 mice in each group, two tumors on each mouse) at a density of 2 × 106 cells in 200
μL of PBS. Aer tumors were established (in about two weeks), aurone 5a formulated in a mixture of Tween-80
(5%), DMSO (10%), PEG400 (25%) and PBS (60%) was intraperitoneally administered to mice at a daily dose of
10 mg of aurone 5a/kg (mouse). e rst day of treatment was set as day 1. At day 18 treatment was ceased and
mice were sacriced. Blank vehicle was used as a control. Tumors and mouse weights were measured, and tumor
volumes were calculated as Length × width2/2. For H&E and IHC studies, the tumors were treated with vehicle
and aurone 5a for 1 week. H&E and TUNEL staining was performed based on standard protocol by the Markey
Cancer Center Biospecimen Procurement & Translational Pathology Shared Resource Facility (BPTP SRF) at the
University of Kentucky. For IHC staining, the following antibody was used: anti- VEGF-A (Santa Cruz, sc-152,
1:100).
Competitive tubulin binding assay and Liquid Chromatography-Electrospray Ionization-
Tandem Mass Spectrometry (LC-ESI-MS/MS) method. Competitive tubulin binding assay was per-
formed as described to demonstrate that aurones bind to the colchicine-binding site of tubulins. e colchicine
quantication was performed at the University of Kentucky Proteomics Core using a protocol modied from a
previously published method. LC-MS/MS analysis was carried out using an TSQ Vantage mass spectrometer
(ermo Fisher Scientic, Waltham, MA USA) coupled with a Shimadzu high performance liquid chromatogra-
phy (HPLC) system (Shimadzu Scientic Instruments, Inc., Columbia, MD USA) through an electrospray ioni-
zation source. e colchicine-containing samples were separated with a Kinetex® reversed phase 2.6 μm XB-C18
100 Å LC column (100 × 4.6 mm) (Phenomenex Inc., Torrance, CA USA) at a ow rate of 300 μL/min. Mobile
phase A was water with 0.1% (v/v) formic acid while mobile phase B was acetonitrile with 0.1% (v/v) formic
acid. A 16 min gradient condition was applied: initial 60% mobile phase B was increased linearly to 100% in
3 min, remained 100% for 3 min, and quickly (0.01 min) decreased to 60% for re-equilibration. Multiple reaction
monitoring (MRM) mode was used to scan from m/z 400 to m/z 310 in the positive mode to obtain the most
sensitive signals for colchicine. e spraying voltage was set at 4000 V, vaporize temperature at 300 °C, capillary
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temperature at 350 °C and sheath gas pressure at 45 (arbitrary units). Collision energies (CE) were set at 25 volts.
Xcalibur soware (Ver. 2.1.0, ermo Fisher Scientic, Waltham, MA USA) was used for the data acquisition and
quantitative processing. A series of colchicine at concentrations of 20, 40, 80, 200, 400, 800 nM were prepared to
establish a linear calibration curve with a coecient of correlation R2 = 0.9944.
Statistics. Biological assays have been performed at least twice. Data were shown as mean ± SD or the 95%
condence intervals were provided. For the mice study, ve mice with two tumors on the lower anks of each
mouse were used in each treatment group. e data for the mouse study were analyzed by t-test. For the zebrash
study, eight sh were used in each treatment group.
Data Availability
e data related to this manuscript during the current study are available from the corresponding authors on
reasonable request.
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Acknowledgements
C.L. and D.S.W. were supported by NIH R01 CA172379. D.S.W. was also supported by the Oce of the Dean of
the College of Medicine, the Markey Cancer Center, and the Center for Pharmaceutical Research and Innovation
(CPRI) in the College of Pharmacy, NIH R21 CA205108 (to J. Mohler), Department of Defense Idea Development
Award PC150326P2, and NIH P20 RR020171 from the National Institute of General Medical Sciences (to L.
Hersh). e authors also thank the University of Kentucky Proteomics Core mass spectrometric measurements
using a LTQ-Orbitrap mass spectrometer that was acquired by NIH grant S10 RR029127 to Professor H. Zhu. is
research was also supported by the Flow Cytometry and Cell Sorting Shared Resource Facility of the University of
Kentucky Markey Cancer Center (P30CA177558). We thank Markey Biospecimen Procurement & Translational
Pathology Shared Resource Facility (BPTP SRF) for processing, H&E and TUNEL staining of the tumor tissues.
Author Contributions
Y.X., L.M.K., M.S.F., S.P.B., K.M.K., P.P.W. and V.M.S., performed chemical synthesis and characterization. Y.X.,
T.Y., W.Z., E.H., Z.M.M., A.D. and J.C., performed biological analysis. Y.X., T.Y., X.L., L.P.D., H.Z., C.G.Z., J.B.,
D.S.W. and C.L., analyzed the data. Y.X., D.S.W. and C.L., write the manuscript.
Additional Information
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-42917-0.
Competing Interests: C.L. and D.S.W. have partial ownership in a private venture, Epionc Inc., incorporated
to develop small-molecule inhibitors for cancer treatment. In accord with University of Kentucky policies,
C.L. and D.S.W. have disclosed this work to the University of Kentucky’s Intellectual Property Committee and
complied with stipulations of the University’s Conict of Interest Oversight Committee.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional aliations.
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