ArticlePDF Available

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

Authors:

Abstract and Figures

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.
This content is subject to copyright. Terms and conditions apply.
1
SCIENTIFIC REPORTS | (2019) 9:6439 | https://doi.org/10.1038/s41598-019-42917-0
www.nature.com/scientificreports
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 properties24 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 proliferation68,
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
Content courtesy of Springer Nature, terms of use apply. Rights reserved
2
SCIENTIFIC REPORTS | (2019) 9:6439 | https://doi.org/10.1038/s41598-019-42917-0
www.nature.com/scientificreports
www.nature.com/scientificreports/
level: drug eux modulators2,915 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 tubulin2531, 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
conditions23,3638 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.
Content courtesy of Springer Nature, terms of use apply. Rights reserved
3
SCIENTIFIC REPORTS | (2019) 9:6439 | https://doi.org/10.1038/s41598-019-42917-0
www.nature.com/scientificreports
www.nature.com/scientificreports/
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 4a4d 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 4a4e 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.
Content courtesy of Springer Nature, terms of use apply. Rights reserved
4
SCIENTIFIC REPORTS | (2019) 9:6439 | https://doi.org/10.1038/s41598-019-42917-0
www.nature.com/scientificreports
www.nature.com/scientificreports/
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.
Content courtesy of Springer Nature, terms of use apply. Rights reserved
5
SCIENTIFIC REPORTS | (2019) 9:6439 | https://doi.org/10.1038/s41598-019-42917-0
www.nature.com/scientificreports
www.nature.com/scientificreports/
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).
Content courtesy of Springer Nature, terms of use apply. Rights reserved
6
SCIENTIFIC REPORTS | (2019) 9:6439 | https://doi.org/10.1038/s41598-019-42917-0
www.nature.com/scientificreports
www.nature.com/scientificreports/
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.
(FH) 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.
Content courtesy of Springer Nature, terms of use apply. Rights reserved
7
SCIENTIFIC REPORTS | (2019) 9:6439 | https://doi.org/10.1038/s41598-019-42917-0
www.nature.com/scientificreports
www.nature.com/scientificreports/
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 109 to 104 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 pharmacophores2531
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.
Content courtesy of Springer Nature, terms of use apply. Rights reserved
8
SCIENTIFIC REPORTS | (2019) 9:6439 | https://doi.org/10.1038/s41598-019-42917-0
www.nature.com/scientificreports
www.nature.com/scientificreports/
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).
Content courtesy of Springer Nature, terms of use apply. Rights reserved
9
SCIENTIFIC REPORTS | (2019) 9:6439 | https://doi.org/10.1038/s41598-019-42917-0
www.nature.com/scientificreports
www.nature.com/scientificreports/
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” conformation45,5254. 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24). 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
Content courtesy of Springer Nature, terms of use apply. Rights reserved
10
SCIENTIFIC REPORTS | (2019) 9:6439 | https://doi.org/10.1038/s41598-019-42917-0
www.nature.com/scientificreports
www.nature.com/scientificreports/
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
Content courtesy of Springer Nature, terms of use apply. Rights reserved
11
SCIENTIFIC REPORTS | (2019) 9:6439 | https://doi.org/10.1038/s41598-019-42917-0
www.nature.com/scientificreports
www.nature.com/scientificreports/
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).
Content courtesy of Springer Nature, terms of use apply. Rights reserved
12
SCIENTIFIC REPORTS | (2019) 9:6439 | https://doi.org/10.1038/s41598-019-42917-0
www.nature.com/scientificreports
www.nature.com/scientificreports/
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
Content courtesy of Springer Nature, terms of use apply. Rights reserved
13
SCIENTIFIC REPORTS | (2019) 9:6439 | https://doi.org/10.1038/s41598-019-42917-0
www.nature.com/scientificreports
www.nature.com/scientificreports/
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.
References
1. Naayama, T. et al. Specicity analysis and mechanism of aurone synthesis catalyzed by aureusidin synthase, a polyphenol oxidase
homolog responsible for ower coloration. FEBS Lett 499, 107–111 (2001).
2. Boumendjel, A. Aurones: a subclass of avones with promising biological potential. Curr Med Chem 10, 2621–2630 (2003).
3. Zwergel, C. et al. Aurones: interesting natural and synthetic compounds with emerging biological potential. Nat Prod Commun 7,
389–394 (2012).
4. Haudecoeur, . & Boumendjel, A. ecent advances in the medicinal chemistry of aurones. Curr Med Chem 19, 2861–2875 (2012).
5. Liu, H. L., Jiang, W. B. & Xie, M. X. Flavonoids: recent advances as anticancer drugs. ecent Pat Anticancer Drug Discov 5, 152–164
(2010).
6. Cheng, H. et al. Design, synthesis and discovery of 5-hydroxyaurone derivatives as growth inhibitors against HUVEC and some
cancer cell lines. Eur J Med Chem 45, 5950–5957, https://doi.org/10.1016/j.ejmech.2010.09.061 (2010).
7. Zheng, X., Cao, J. G., Meng, W. D. & Qing, F. L. Synthesis and anticancer eect of B-ring triuoromethylated avonoids. Bioorg Med
Chem Lett 13, 3423–3427 (2003).
8. Lawrence, N. J., ennison, D., McGown, A. T. & Hadeld, J. A. e total synthesis of an aurone isolated from Uvaria hamiltonii:
aurones and avones as anticancer agents. Bioorg Med Chem Lett 13, 3759–3763 (2003).
9. Hadjeri, M. et al. Modulation of P-glycoprotein-mediated multidrug resistance by avonoid derivatives and analogues. J Med Chem
46, 2125–2131, https://doi.org/10.1021/jm021099i (2003).
10. Sim, H. M., Lee, C. Y., Ee, P. L. & Go, M. L. Dimethoxyaurones: Potent inhibitors of ABCG2 (breast cancer resistance protein). Eur J
Pharm Sci 35, 293–306, https://doi.org/10.1016/j.ejps.2008.07.008 (2008).
11. Sim, H. M., Loh, . Y., Yeo, W. ., Lee, C. Y. & Go, M. L. Aurones as modulators of ABCG2 and ABCB1: synthesis and structure-
activity relationships. Chem Med Chem 6, 713–724, https://doi.org/10.1002/cmdc.201000520 (2011).
12. Cherigo, L., Lopez, D. & Martinez-Luis, S. Marine natural products as breast cancer resistance protein inhibitors. Mar Drugs 13,
2010–2029, https://doi.org/10.3390/md13042010 (2015).
13. Boumendjel, A. et al. 4-Hydroxy-6-methoxyaurones with high-anity binding to cytosolic domain of P-glycoprotein. Chem Pharm
Bull (Toyo) 50, 854–856 (2002).
14. Boumendjel, A., Di Pietro, A., Dumontet, C. & Barron, D. ecent advances in the discovery of avonoids and analogs with high-
anity binding to P-glycoprotein responsible for cancer cell multidrug resistance. Med es ev 22, 512–529, https://doi.org/10.1002/
med.10015 (2002).
15. Vaclaviova, ., Boumendjel, A., Ehrlichova, M., ovar, J. & Gut, I. Modulation of paclitaxel transport by avonoid derivatives in
human breast cancer cells. Is there a correlation between binding anity to NBD of P-gp and modulation of transport? Bioorg Med
Chem 14, 4519–4525, https://doi.org/10.1016/j.bmc.2006.02.025 (2006).
16. Jacobson, . A., Moro, S., Manthey, J. A., West, P. L. & Ji, X. D. Interactions of avones and other phytochemicals with adenosine
receptors. Adv Exp Med Biol 505, 163–171 (2002).
17. Gao, Z. G. et al. Structural determinants of A(3) adenosine receptor activation: nucleoside ligands at the agonist/antagonist
boundary. J Med Chem 45, 4471–4484 (2002).
18. Huang, L. et al. New compounds with DNA strand-scission activity from the combined leaf and stem of Uvaria hamiltonii. J Nat
Prod 61, 446–450, https://doi.org/10.1021/np9703609 (1998).
19. Ballinari, D. B, Ermoli, A. G., Moll, M. J. & Vanotti, E. Aurones as telomerase inhibitors (2002).
20. Smith, C. D., French, . J. & Yun, J. . Sphingosine inase inhibitors (2003).
21. Bursavich, M. G. et al. Novel benzofuran-3-one indole inhibitors of PI3 inase-alpha and the mammalian target of rapamycin: hit to
lead studies. Bioorg Med Chem Lett 20, 2586–2590, https://doi.org/10.1016/j.bmcl.2010.02.082 (2010).
22. Schoepfer, J. et al. Structure-based design and synthesis of 2-benzylidene-benzofuran-3-ones as avopiridol mimics. J Med Chem
45, 1741–1747 (2002).
23. Lee, C. Y., Chew, E. H. & Go, M. L. Functionalized aurones as inducers of NAD(P)H:quinone oxidoreductase 1 that activate Ah/
XE and Nrf2/AE signaling pathways: synthesis, evaluation and SA. Eur J Med Chem 45, 2957–2971, https://doi.org/10.1016/j.
ejmech.2010.03.023 (2010).
24. Westenburg, H. E. et al. Activity-guided isolation of antioxidative constituents of Cotinus coggygria. J Nat Prod 63, 1696–1698
(2000).
25. Mirzaei, H. & Emami, S. ecent advances of cytotoxic chalconoids targeting tubulin polymerization: Synthesis and biological
activity. European journal of medicinal chemistry 121, 610–639, https://doi.org/10.1016/j.ejmech.2016.05.067 (2016).
26. Ji, Y. T., Liu, Y. N. & Liu, Z. P. Tubulin colchicine binding site inhibitors as vascular disrupting agents in clinical developments. Curr
Med Chem 22, 1348–1360 (2015).
27. Li, W., Sun, H., Xu, S., Zhu, Z. & Xu, J. Tubulin inhibitors targeting the colchicine binding site: a perspective of privileged structures.
Future Med Chem 9, 1765–1794, https://doi.org/10.4155/fmc-2017-0100 (2017).
28. Dong, M., Liu, F., Zhou, H., Zhai, S. & Yan, B. Novel Natural Product- and Privileged Scaold-Based Tubulin Inhibitors Targeting
the Colchicine Binding Site. Molecules 21, https://doi.org/10.3390/molecules21101375 (2016).
29. Dumontet, C. & Jordan, M. A. Microtubule-binding agents: a dynamic eld of cancer therapeutics. Nat ev Drug Discov 9, 790–803,
https://doi.org/10.1038/nrd3253 (2010).
30. Lu, Y., Chen, J., Xiao, M., Li, W. & Miller, D. D. An overview of tubulin inhibitors that interact with the colchicine binding site.
Pharm es 29, 2943–2971, https://doi.org/10.1007/s11095-012-0828-z (2012).
31. Bueno, O. et al . High-anity ligands of the colchicine domain in tubulin based on a structure-guided design. Sci ep 8, 4242, https://
doi.org/10.1038/s41598-018-22382-x (2018).
32. Zwergel, C. V. et al. benzofuran-chromone and -coumarin derivatives: synthesis and biological activity in 562 human leuemia
cells. Med. Chem. Commun. 4, 1571–1579 (2013).
Content courtesy of Springer Nature, terms of use apply. Rights reserved
14
SCIENTIFIC REPORTS | (2019) 9:6439 | https://doi.org/10.1038/s41598-019-42917-0
www.nature.com/scientificreports
www.nature.com/scientificreports/
33. Guo, Q. N., Zhou, L., Yu, Y. & Teng, Y. P. Design, synthesis and biological evaluation of the novel antitumor agent aurone derivatives.
Ad. Mat. es. 781-784, 1235–1239 (2013).
34. Huang, W., Liu, M. Z., Li, Y., Tan, Y. & Yang, G. F. Design, syntheses, and antitumor activity of novel chromone and aurone
derivatives. Bioorg Med Chem 15, 5191–5197, https://doi.org/10.1016/j.bmc.2007.05.022 (2007).
35. Patha, N. P. J. Design and synthesis of indole integrated aurones as potent anti breast cancer agents. Ind. J. App. es. 6, 800–802
(2016).
36. Oombi, S . et al. Discovery of benzylidenebenzofuran-3(2H)-one (aurones) as inhibitors of tyrosinase derived from human
melanocytes. J Med Chem 49, 329–333, https://doi.org/10.1021/jm050715i (2006).
37. Haudecoeur, . et al. Discovery of naturally occurring aurones that are potent allosteric inhibitors of hepatitis C virus NA-
dependent NA polymerase. J Med Chem 54, 5395–5402, https://doi.org/10.1021/jm200242p (2011).
38. Sheng, . et al. Design, synthesis and AChE inhibitory activity of indanone and aurone derivatives. Eur J Med Chem 44, 7–17,
https://doi.org/10.1016/j.ejmech.2008.03.003 (2009).
39. Hastings, J. H. H. e stereochemistry of aurones [2-substituted benzylidenebenzofuran-3-(2H)-ones]. J. Chem. Soc., Perin Trans.
1, 2128–2132 (1972).
40. ing, T. J. H., Heller, J. S. & X-ray, H. G. analysis of (Z)-2-p-methoxyphenylmethylenebenzofuran-3-(2H)-one. J. Chem. Soc., Perin
Trans. 1, 1455–1457 (1975).
41. Paull, . D. et al. Display and analysis of patterns of dierential activity of drugs against human tumor cell lines: development of
mean graph and COMPAE algorithm. J Natl Cancer Inst 81, 1088–1092 (1989).
42. Li, C. M. et al. Competitive mass spectrometry binding assay for characterization of three binding sites of tubulin. J Mass Spectrom
45, 1160–1166, https://doi.org/10.1002/jms.1804 (2010).
43. Trott, O. & Olson, A. J. AutoDoc Vina: improving the speed and accuracy of docing with a new scoring function, ecient
optimization, and multithreading. J Comput Chem 31, 455–461, https://doi.org/10.1002/jcc.21334 (2010).
44. Sanghai, N. et al. Combretastatin A-4 inspired novel 2-aryl-3-arylamino-imidazo-pyridines/pyrazines as tubulin polymerization
inhibitors, antimitotic and anticancer agents. MedChemComm 5, 766–782 (2014).
45. Lowe, J., Li, H., Downing, . H. & Nogales, E. ened structure of alpha beta-tubulin at 3.5 A resolution. J Mol Biol 313, 1045–1057,
https://doi.org/10.1006/jmbi.2001.5077 (2001).
46. Blacburn, J. S. et al. Notch signaling expands a pre-malignant pool of T-cell acute lymphoblastic leuemia clones without aecting
leuemia-propagating cell frequency. Leuemia 26, 2069–2078, https://doi.org/10.1038/leu.2012.116 (2012).
47. Langenau, D. M. et al. Myc-induced T cell leuemia in transgenic zebrafish. Science 299, 887–890, https://doi.org/10.1126/
science.1080280 (2003).
48. Sviripa, V. M. et al. 2,6-Dihalostyrylanilines, pyridines, and pyrimidines for the inhibition of the catalytic subunit of methionine
S-adenosyltransferase-2. J Med Chem 57, 6083–6091, https://doi.org/10.1021/jm5004864 (2014).
49. Greengrass, P. M. S. & Wood, M. C. M. Anity-assay for the human EG potassium channell (2003).
50. Jo, S. H., Youm, J. B., Lee, C. O., Earm, Y. E. & Ho, W. . Blocade of the HEG human cardiac (+) channel by the antidepressant
drug amitriptyline. Br J Pharmacol 129, 1474–1480, https://doi.org/10.1038/sj.bjp.0703222 (2000).
51. Pasquier, E., Andre, N. & Braguer, D. Targeting microtubules to inhibit angiogenesis and disrupt tumour vasculature: implications
for cancer treatment. Curr Cancer Drug Targets 7, 566–581 (2007).
52. Prota, A. E. et al. e novel microtubule-destabilizing drug BAL27862 binds to the colchicine site of tubulin with distinct eects on
microtubule organization. J Mol Biol 426, 1848–1860, https://doi.org/10.1016/j.jmb.2014.02.005 (2014).
53. avelli, . B. et al. Insight into tubulin regulation from a complex with colchicine and a stathmin-lie domain. Nature 428, 198–202,
https://doi.org/10.1038/nature02393 (2004).
54. Ayaz, P., Ye, X., Huddleston, P., Brautigam, C. A. & ice, L. M. A. TOG:alphabeta-tubulin complex structure reveals conformation-
based mechanisms for a microtubule polymerase. Science 337, 857–860, https://doi.org/10.1126/science.1221698 (2012).
55. Dorleans, A. et al. Variations in the colchicine-binding domain provide insight into the structural switch of tubulin. Proc Natl Acad
Sci USA 106, 13775–13779, https://doi.org/10.1073/pnas.0904223106 (2009).
56. Zhang, W. et al. Fluorinated N, N-dialylaminostilbenes repress colon cancer by targeting methionine S-adenosyltransferase 2A.
ACS Chem Biol 8, 796–803, https://doi.org/10.1021/cb3005353 (2013).
57. Chen, T. A Practical Guide to Assay Development and High-throughput Screening in Drug Discovery. (CC Press Taylor and Francis
Group, 2010).
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.
Content courtesy of Springer Nature, terms of use apply. Rights reserved
15
SCIENTIFIC REPORTS | (2019) 9:6439 | https://doi.org/10.1038/s41598-019-42917-0
www.nature.com/scientificreports
www.nature.com/scientificreports/
Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre-
ative Commons license, and indicate if changes were made. e images or other third party material in this
article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the
material. If material is not included in the article’s Creative Commons license and your intended use is not per-
mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the
copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
© e Author(s) 2019
Content courtesy of Springer Nature, terms of use apply. Rights reserved
1.
2.
3.
4.
5.
6.
Terms and Conditions
Springer Nature journal content, brought to you courtesy of Springer Nature Customer Service Center GmbH (“Springer Nature”).
Springer Nature supports a reasonable amount of sharing of research papers by authors, subscribers and authorised users (“Users”), for small-
scale personal, non-commercial use provided that all copyright, trade and service marks and other proprietary notices are maintained. By
accessing, sharing, receiving or otherwise using the Springer Nature journal content you agree to these terms of use (“Terms”). For these
purposes, Springer Nature considers academic use (by researchers and students) to be non-commercial.
These Terms are supplementary and will apply in addition to any applicable website terms and conditions, a relevant site licence or a personal
subscription. These Terms will prevail over any conflict or ambiguity with regards to the relevant terms, a site licence or a personal subscription
(to the extent of the conflict or ambiguity only). For Creative Commons-licensed articles, the terms of the Creative Commons license used will
apply.
We collect and use personal data to provide access to the Springer Nature journal content. We may also use these personal data internally within
ResearchGate and Springer Nature and as agreed share it, in an anonymised way, for purposes of tracking, analysis and reporting. We will not
otherwise disclose your personal data outside the ResearchGate or the Springer Nature group of companies unless we have your permission as
detailed in the Privacy Policy.
While Users may use the Springer Nature journal content for small scale, personal non-commercial use, it is important to note that Users may
not:
use such content for the purpose of providing other users with access on a regular or large scale basis or as a means to circumvent access
control;
use such content where to do so would be considered a criminal or statutory offence in any jurisdiction, or gives rise to civil liability, or is
otherwise unlawful;
falsely or misleadingly imply or suggest endorsement, approval , sponsorship, or association unless explicitly agreed to by Springer Nature in
writing;
use bots or other automated methods to access the content or redirect messages
override any security feature or exclusionary protocol; or
share the content in order to create substitute for Springer Nature products or services or a systematic database of Springer Nature journal
content.
In line with the restriction against commercial use, Springer Nature does not permit the creation of a product or service that creates revenue,
royalties, rent or income from our content or its inclusion as part of a paid for service or for other commercial gain. Springer Nature journal
content cannot be used for inter-library loans and librarians may not upload Springer Nature journal content on a large scale into their, or any
other, institutional repository.
These terms of use are reviewed regularly and may be amended at any time. Springer Nature is not obligated to publish any information or
content on this website and may remove it or features or functionality at our sole discretion, at any time with or without notice. Springer Nature
may revoke this licence to you at any time and remove access to any copies of the Springer Nature journal content which have been saved.
To the fullest extent permitted by law, Springer Nature makes no warranties, representations or guarantees to Users, either express or implied
with respect to the Springer nature journal content and all parties disclaim and waive any implied warranties or warranties imposed by law,
including merchantability or fitness for any particular purpose.
Please note that these rights do not automatically extend to content, data or other material published by Springer Nature that may be licensed
from third parties.
If you would like to use or distribute our Springer Nature journal content to a wider audience or on a regular basis or in any other manner not
expressly permitted by these Terms, please contact Springer Nature at
onlineservice@springernature.com

Supplementary resource (1)

... (Z)-2-((2-((1-Ethyl-5-methoxy-1H-indol-3-yl)methylene)-3-oxo-2,3-dihydrobenzofuran-6-yl)oxy) acetonitrile, also known as A14, is one kind of semisynthetic aurones. Previous studies have reported the inhibition effect of A14 on PC-3 tumor xenografts in nude mice [18], but little is known about the role of A14 on T-cell acute lymphoblastic leukemia mice. In this study, the effects of A14 on T-cell acute lymphoblastic leukemia were elevated in vitro and in vivo. ...
... Results showed that A14 inhibited Jurkat cell proliferation by 50% at 0.3 μM and THP-1 cells by 40% at 0.03 μM concentration (Fig. 1A). As we have demonstrated that A14 is a tubulin inhibitor in PC-3 cells [18], we wonder whether the proliferation inhibition is associated with the tubulin inhibitor. Results demonstrated A14 decreased tubulin polymerization especially in Jurkat cells, indicating that tubulin inhibition may be contributed the cell proliferation by the A14 (Fig. 1B). ...
... Though colchicine itself is not used as an agent to protect against cancer, several tubulin inhibitors are investigated in preclinical or clinical trials for the interaction with the colchicine binding site [21]. Previous studies demonstrated that semisynthetic aurones inhibit tubulin polymerization at the colchicine-binding site [18], indicating the possible mechanism of action of A14 on T-ALL. Indeed, we found A14 significantly inhibited proliferation. ...
Article
Full-text available
Background Acute lymphoblastic leukemia is an aggressive neoplasm and seriously threatens human health. A14 is one kind of semisynthetic aurone that exhibits the capability to inhibit prostate cancer, but little is known about the role of A14 on T-cell acute lymphoblastic leukemia. Methods Firstly, the effects of A14 on the ability of leukemia cells to proliferate were measured by Vi-cell counter. Then, we detected the cell cycle and apoptosis by flow cytometry and characterized the related protein expression using immunoblotting. In addition, we constructed stable luciferase expressing cell lines for use in a cell derived xenograft mouse model to measure the effect of A14 on T-cell acute lymphoblastic leukemia. Results Results exhibited that A14 markedly suppressed cell proliferation and induced G2/M phase arrest along with cell cycles regulating proteins changes. A14 led to apoptosis in leukemia cells, at least partly, through the cytochrome c signaling pathway. Experiments in cell derived xenograft mouse model also showed that A14 markedly ameliorated the survival rate. Conclusions The present study revealed that semisynthetic aurones A14 can effectively protect against T-cell acute lymphoblastic leukemia progression both in vitro and in vivo, indicating the capability of A14 as a promising drug for the treatment of T-cell acute lymphoblastic leukemia.
... Induction of microtubule depolymerization was also the mechanism of action of (Z)-2-((2-((1-ethyl-5-methoxy-1H-indol-3-yl)methylene)-3-oxo-2,3-dihydrobenzofuran-6yl)oxy)acetonitrile (5a) and (Z)-6-((2,6-dichlorobenzyl)oxy)-2-(pyridin-4ylmethylene)benzofuran-3(2H)-one (5b), which showed a selectivity for the colchicinebinding site on tubulin [123]. The molecules inhibited the growth of T-and B-ALL cell lines while being less effective against normal B-lymphoblast cells and blocked disease progression in a myc-induced T-ALL zebrafish model. ...
Article
Full-text available
Uncontrolled proliferative signals and cell cycle dysregulation due to genomic or functional alterations are important drivers of the expansion of undifferentiated blast cells in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) cells. Therefore, they are largely studied as potential therapeutic targets in the field. We here present the most recent advancements in the evaluation of novel compounds targeting cell cycle proteins or oncogenic mechanisms, including those showing an antiproliferative effect in acute leukemia, independently of the identification of a specific target. Several new kinase inhibitors have been synthesized that showed effectiveness in a nanomolar to micromolar concentration range as inhibitors of FLT3 and its mutant forms, a highly attractive therapeutic target due to its driver role in a significant fraction of AML cases. Moreover, we introduce novel molecules functioning as microtubule-depolymerizing or P53-restoring agents, G-quadruplex-stabilizing molecules and CDK2, CHK1, PI3Kd, STAT5, BRD4 and BRPF1 inhibitors. We here discuss their mechanisms of action, including the downstream intracellular changes induced by in vitro treatment, hematopoietic toxicity, in vivo bio-availability and efficacy in murine xenograft models. The promising activity profile demonstrated by some of these candidates deserves further development towards clinical investigation.
... Synthesis. The aldol condensation reaction 15 was finally selected to construct the tricyclic skeletons of the aurone and indanone derivatives ( Figure 3). For the synthesis of aurone derivatives, 2,4dihydroxyacetophenone was used as the starting material to generate intermediate hydroxybenzofuran- ...
... In 2019, Xie reported two potent, semisynthetic aurones 86 and 87 that function as tubulin polymerization inhibitors with IC 50 values of 58.7 nM and 66.0 nM, respectively (Fig. 18). 46 Importantly, aurone 86 displayed activity in an in vivo PC-3 prostate cancer xenograft model in nude mice at 10 mg/kg without affecting mice weight. 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 86 disrupted tubulin dynamics. ...
Article
Full-text available
Aurones are naturally occurring structural isomerides of flavones that have diverse bioactivities including antiviral, antibacterial, antifungal, anti-inflammatory, antitumor, antimalarial, antioxidant, neuropharmaco-logical activities and so on. They constitute an important class of pharmacologically active scaffolds that exhibit multiple biological activities via diverse mechanisms. This review article provides an update on the recent advances (2013-2020.4) in the synthesis and biological activities of these derivatives. In the cases where sufficient information is available, some important structure-activity relationships (SAR) of their biological activities were presented, and on the strength of our expertise in medicinal chemistry and careful analysis of the recent literature , for the potential of aurones as medicinal drugs is proposed.
... In 2019, Xie reported two potent, semisynthetic aurones 86 and 87 that function as tubulin polymerization inhibitors with IC 50 values of 58.7 nM and 66.0 nM, respectively (Fig. 18). 46 Importantly, aurone 86 displayed activity in an in vivo PC-3 prostate cancer xenograft model in nude mice at 10 mg/kg without affecting mice weight. 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 86 disrupted tubulin dynamics. ...
Preprint
Aurones are naturally occurring structural isomerides of flavones that have diverse bioactivities including antiviral, antibacterial, antifungal, anti-inflammatory, antitumor, antimalarial, antioxidant, neuropharmacological activities and so on. They constitute an important class of pharmacologically active scaffolds that exhibit multiple biological activities via diverse mechanisms. This review article provides an update on the recent advances (2013–2020.4) in the synthesis and biological activities of these derivatives. In the cases where sufficient information is available, some important structure–activity relationships (SAR) of their biological activities were presented, and on the strength of our expertise in medicinal chemistry and careful analysis of the recent literature, for the potential of aurones as medicinal drugs is proposed.
... We guided our research towards the thiazole ring, because previous studies reported in literature stated that the benzene ring bound to the benzofuranone through the vinylic carbon is not mandatory and it can be replaced with other cyclic compounds containing nitrogen, some of these compounds exhibiting remarkable activity [10,44]. Thiazole is already a common moiety found in the structure of certain molecules with anticancer activity [1,14,16,19,42], thus the research concerning the biological properties of the thiazole ring is ongoing and warrants further investigation. ...
Article
Cancer cells undergo significant "metabolic remodeling" to provide sufficient ATP to maintain cell survival and to promote rapid growth. In colorectal cancer (CRC) cells, ATP is produced by mitochondrial oxidative phosphorylation (OXPHOS) and by substantially elevated cytoplasmic glucose fermentation (i.e., the Warburg effect). Glucose transporter 1 (GLUT1) expression is significantly increased in CRC cells, and GLUT1 inhibitors block glucose uptake and hence glycolysis crucial for cancer cell growth. In addition to ATP, these metabolic pathways also provide macromolecule building blocks and signaling molecules required for tumor growth. In this study, we identify a diaminobutoxy-substituted isoflavonoid (DBI-1) that inhibits mitochondrial complex I and deprives rapidly growing cancer cells of energy needed for growth. DBI-1 and the GLUT1 inhibitor, BAY-876, synergistically inhibit CRC cell growth in vitro and in vivo. This study suggests that an electron transport chain (ETC) inhibitor (i.e. DBI-1) and a glucose transport inhibitor, (i.e. BAY-876) are potentially effective combination for CRC treatment.
Article
Developing effective treatments for colorectal cancers through combinations of small-molecule approaches and immunotherapies present intriguing possibilities for managing these otherwise intractable cancers. During a broad-based, screening effort against multiple colorectal cancer cell lines, we identified indole-substituted quinolines (ISQs), such as N7,N7-dimethyl-3-(1-methyl-1H-indol-3-yl)quinoline-2,7-diamine (ISQ-1), as potent in vitro inhibitors of several cancer cell lines. We found that ISQ-1 inhibited Wnt signaling, a main driver in the pathway governing colorectal cancer development, and ISQ-1 also activated adenosine monophosphate kinase (AMPK), a cellular energy-homeostasis master regulator. We explored the effect of ISQs on cell metabolism. Seahorse assays measuring oxygen consumption rate (OCR) indicated that ISQ-1 inhibited complex I (i.e., NADH ubiquinone oxidoreductase) in the mitochondrial, electron transport chain (ETC). In addition, ISQ-1 treatment showed remarkable synergistic depletion of oncogenic c-Myc protein level in vitro and induced strong tumor remission in vivo when administered together with BI2536, a polo-like kinase-1 (Plk1) inhibitor. These studies point toward the potential value of dual drug therapies targeting the ETC and Plk-1 for the treatment of c-Myc-driven cancers.
Article
Acute myeloid leukemias (AML) and acute lymphoid leukemias (ALL) are heterogenous diseases encompassing a wide array of genetic mutations with both loss and gain of function phenotypes. Ultimately, these both result in the clonal overgrowth of blast cells in the bone marrow, peripheral blood, and other tissues. As a consequence of this, normal hematopoietic stem cell function is severely hampered. Technologies allowing for the early detection of genetic alterations and understanding of these varied molecular pathologies have helped to advance our treatment regimens toward personalized targeted therapies. In spite of this, both AML and ALL continue to be a major cause of morbidity and mortality worldwide, in part because molecular therapies for the plethora of genetic abnormalities have not been developed. This underscores the current need for better model systems for therapy development. This article reviews the current zebrafish models of AML and ALL and discusses how novel gene editing tools can be implemented to generate better models of acute leukemias. This article is categorized under: • Adult Stem Cells, Tissue Renewal, and Regeneration > Stem Cells and Disease • Technologies > Perturbing Genes and Generating Modified Animals Abstract Zebrafish are useful models in research, and in particular make for good models of hematopoiesis due to their genetic conservation to humans and ease of genetic manipulation through microinjections. Xenografts of cells from other species, including human, may be tracked at various stages in the transluscent, developing fish and used for humanized research.
Article
Full-text available
Microtubule-targeting agents that bind at the colchicine-site of tubulin are of particular interest in antitumoral therapy due to their dual mechanism of action as antimitotics and vascular disrupting agents. Cyclohexanediones derivatives have been described as a new family of colchicine-domain binders with an association constant to tubulin similar to that of colchicine. Here, the high-resolution structures of tubulin in complex with cyclohexanediones TUB015 and TUB075 were solved by X-ray crystallography. A detailed analysis of the tubulin-TUB075 interaction by means of computational affinity maps allowed the identification of two additional regions at the binding site that were addressed with the design and synthesis of a new series of cyclohexanediones with a distal 2-substituted benzofurane. These new compounds showed potent antiproliferative activity with IC50values in the nM range, arrested cell cycle progression at the G2/M phase and induced apoptosis at sub μM concentrations. Moreover, they caused the destruction of a preformed vascular network in vitro and inhibited the migration of endothelial cells at non-toxic concentrations. Finally, these compounds displayed high affinity for tubulin as substantiated by a Kbvalue of 2.87 × 108 M-1which, to the best of our knowledge, represents the highest binding constant measured to date for a colchicine-domain ligand.
Article
Full-text available
Tubulin inhibitors are effective anticancer agents, however, there are many limitations to the use of available tubulin inhibitors in the clinic, such as multidrug resistance, severe side-effects, and generally poor bioavailability. Thus, there is a constant need to search for novel tubulin inhibitors that can overcome these limitations. Natural product and privileged structures targeting tubulin have promoted the discovery and optimization of tubulin inhibitors. This review will focus on novel tubulin inhibitors derived from natural products and privileged structures targeting the colchicine binding site on tubulin.
Article
Full-text available
The first review regarding the potential of aurones as promising drug candidates was reported in 2003. Since, considerable efforts have been made to explore the pharmacological and therapeutical activities of aurones. In this regard, many biological areas were concerned, including major pathological, such as cancer and neurodegenerative disorders. The aim of the present report is to highlight the progress made during the last ten years on the medicinal chemistry of aurones. A special focus will be made on the structure-activity relationship aspects among aurones and especially in case where aurones were found highly active than the corresponding flavones and chalcones.
Article
Full-text available
Breast cancer resistance protein (BCRP) is a protein belonging to the ATP-binding cassette (ABC) transporter superfamily that has clinical relevance due to its multi-drug resistance properties in cancer. BCRP can be associated with clinical cancer drug resistance, in particular acute myelogenous or acute lymphocytic leukemias. The overexpression of BCRP contributes to the resistance of several chemotherapeutic drugs, such as topotecan, methotrexate, mitoxantrone, doxorubicin and daunorubicin. The Food and Drugs Administration has already recognized that BCRP is clinically one of the most important drug transporters, mainly because it leads to a reduction of clinical efficacy of various anticancer drugs through its ATP-dependent drug efflux pump function as well as its apparent participation in drug resistance. This review article aims to summarize the different research findings on marine natural products with BCRP inhibiting activity. In this sense, the potential modulation of physiological targets of BCRP by natural or synthetic compounds offers a great possibility for the discovery of new drugs and valuable research tools to recognize the function of the complex ABC-transporters.
Article
Full-text available
Tumor vasculature is an important target in cancer treatment. Two distinct vascular-targeting therapeutic strategies are applied to attack cancer cells indirectly. The antiangiogenic approach intervenes in the neovascularization processes and blocks the formation of new blood vessels, while the antivascular approach targets the established tumor blood vessels, making vascular shutdown and resulting in rapid haemorrhagic necrosis and tumor cell death. A number of compounds with diverse structural scaffolds have been designed to target tumor vasculature and they are called vascular disrupting agents (VDAs). The biological or ligand-directed VDAs utilize antibodies, peptides or growth factors to deliver toxins or pro-coagulants or proapoptotic affectors to tumor-related blood vessels, while the small-molecule VDAs selectively target tumor blood vessels and have little effects on the normal endothelium. Among the small-molecule VDAs, the tubulin colchicine binding site inhibitors have been extensively studied and many of them entered the clinical trials, including CA-4P, CA-1P, AVE8062, OXi4503, CKD-516, BNC105P, ABT-751, CYT-997, ZD6126, NPI-2358, MN-029 and EPC2407. This review makes a summary of the small-molecule VDAs in clinical developments and highlights some potential VDA leads or candidates for the treatment of tumors.
Article
Full-text available
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)pyridin 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.
Article
The vital roles of microtubule in mitosis and cell division make it an attractive target for antitumor therapy. Colchicine binding site of tubulin is one of the most important pockets that have been focused on to design tubulin-destabilizing agents. Over the past few years, a large number of colchicine binding site inhibitors (CBSIs) have been developed inspired by natural products or synthetic origins, and many moieties frequently used in these CBSIs are structurally in common. In this review, we will classify the CBSIs into classical CBSIs and nonclassical CBSIs according to their spatial conformations and binding modes with tubulin, and highlight the privileged structures from these CBSIs in the development of tubulin inhibitors targeting the colchicine binding site.
Article
Since microtubules have an important role in mitosis and other vital cellular functions, tubulin-targeting chemotherapy has been received growing attention in anticancer drug design and development. It was found that a number of naturally occurring compounds including distinct chalcones exert their effect by inhibition of tubulin polymerization. After the identification of tubulin polymerization as potential target for chalcone-type compounds, extensive researches have been made to design and synthesis of new anti-tubulin chalconoids. Although diverse chalcones have found to be potent anticancer agents but in the present review, we focused on the recently reported tubulin polymerization inhibitors from chalcone origin and related synthetic compounds, and their detailed synthetic methods and biological activities.
Article
Aurones belong to a class of heterocyclic flavonoids which contains a benzofuran element associated with a benzylidene linked in position 2. Aurones possess a wide range of pharmacological activities and biological activities, such as antitumor, antifungal, phytoalexin and so on. A novel series of 2-ayl-yl (5-methacrylate) aurone analogues were synthesized in six steps with the overall yield of 11%-13% and characterized by 1H NMR. Among the key intermediates and target compounds, 2-(2-furan-ylmethylene)-5-methacrylate-benzofuran-3(2H)-one (7a) and 2-(2-thienyl-ylmethylene)-5-methacrylate-benzofuran-3(2H)-one (7b) have never been reported before. Primary biological activities evaluation showed that 7a exhibited good inhibitory activities against K562 with an IC50 of 2.18 μM and against HepG2 with an IC50 of 3.95μM.