Fluorescein hydrazones as novel nonintercalative topoisomerase catalytic inhibitors with low DNA toxicity

Article abstract of DOI:10.1021/jm501263m

Fluorescein hydrazones (3a-3l) were synthesized in three steps with 86-91% overall yields. Topo I- and IIα-mediated relaxation and cell viability assay were evaluated. 3d inhibited 47% Topo I (camptothecin, 34%) and 20% Topo II (etoposide 24%) at 20 μM. 3

Full text of DOI:10.1021/jm501263m

Article
pubs.acs.org/jmc
Fluorescein Hydrazones as Novel Nonintercalative Topoisomerase
Catalytic Inhibitors with Low DNA Toxicity
A. F. M. Motiur Rahman,*,† So-Eun Park,‡ Adnan A. Kadi,† and Youngjoo Kwon*,‡
†Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
‡College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul 120-750, Republic of Korea
S
* Supporting Information
ABSTRACT: Fluorescein hydrazones (3a−3l) were synthesized in three
steps with 86−91% overall yields. Topo I- and IIα-mediated relaxation and
cell viability assay were evaluated. 3d inhibited 47% Topo I (camptothecin,
34%) and 20% Topo II (etoposide 24%) at 20 μM. 3l inhibited 61% Topo
II (etoposide 24%) at 20 μM. 3d and 3l were further evaluated to
determine their mode of action with diverse methods of kDNA
decatenation, DNA−Topo cleavage complex, comet, DNA intercalating/
unwinding, and Topo IIα-mediated ATP hydrolysis assays. 3d functioned
as a nonintercalative dual inhibitor against the catalytic activities of Topo I
and Topo IIα. 3l acted as a Topo IIα specific nonintercalative catalytic
inhibitor. 3d activated apoptotic proteins as it increased the level of
cleaved capase-3 and cleaved PARP in a dose- and time-dependent
manner. The dose- and time-dependent increase of G1 phase population
was observed by treatment of 3d along with the increase of p27kip1 and the
decrease of cyclin D1 expression.
Topo inhibitors are grouped two types such as Topo poisons
and catalytic inhibitors based on their mode of action. Topo
poison is defined for compounds which stabilize the Topo−
DNA complex formed transiently and covalently, known as
“cleavable complex”, and prevent the cleaved DNA strand(s)
from religation, thus leading to an accumulation of undesired
truncated DNA. Topo poison is named due to severe DNA
damage and genetic toxicity induced by the Topo-mediated
undesired DNA truncation. The rest of the Topo inhibitors
besides Topo poisons can be grouped as a Topo catalytic
inhibitor.4,5,8,9
Fluorescein is a synthetic organic compound having open/
cyclic form (1, Figure 1), it is dark orange/red powder in color,
soluble in water as well as in alcohol. It was first synthesized
from phthalic anhydride and resorcinol in 1871 via Friedel−
Craft reaction in the presence of zinc chloride.10 Later on, a
INTRODUCTION
■
Cancer is not just one simple disease but a complex one. The
two main characteristics of cancer are uncontrolled cell growth
and cell migration from the original site to distant sites in
human body. Cancer is life threatening, and if the migration is
not controlled, cancer can lead to death.1 Cancerous cell
proliferation is mainly related with essential process of the cell
cycle (DNA synthesis, mitosis, and cytokinesis) which is
controlled by several cell-cycle controlling proteins.2,3 Among
these proteins, topoisomerases have been focused by many
researchers as an attractive drug target for therapeutic
intervention on cancerous cell proliferation.4,5 Two types of
isomers of topoisomerase exist in humans, type I of
topoisomerase (Topo I) and type II of topoisomerase (Topo
II). Both are nuclear enzymes essential to resolve topological
problems which unavoidably occur during DNA replication and
cell division.4,6 Topo I cuts one strand of a DNA double helix
to pass the other strand through the cut and then reseals the
Topo I-mediated cut, while Topo II cleaves both strands of one
DNA duplex to let the other noncleaved duplex pass through a
transiently formed Topo II-mediated break and then followed
by religating the cleaved strands. The differences of Topo II
from Topo I beside simultaneous cleavage of double strands are
(i) Topo II functions in a form of homodimer with requirement
of divalent metal ion, Mg2+, binding and ATP hydrolysis for
enzyme turn over and rapid kinetics; (ii) only Topo II enables
chromosomes to be disentangled and decatenated during DNA
replication by introducing transient double strand breaks.3−7
Figure 1. Structures of fluorescein.
Received: August 18, 2014
© XXXX American Chemical Society
A
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Figure 2. Structure of functionalized fluoresceins.
Scheme 1. Synthetic Scheme of Fluorescein Hydrazide (2)
synthetic method was developed for synthesizing fluorescein by
using methansulfonic acid as milder reagent and obtained high
yield.11 Fluorescein commonly used as a fluorescent tracer in
microscopy, a type of dye laser, and in forensics and serology to
detect latent blood stains.
hydrolysis studies,39 artificial photosynthesis and solar energy
conversion,40 and the conjugate addition of amino acid side
chains.41 In addition, fluorescein tetracysteine-tagged protein
conjugates linked with a diethylamine moiety was applied to
purify tetracysteine-tagged proteins42 and fluorescein hydra-
zide/hydrazone was utilized as a colorimetric logic chemo-
sensor for pH and Cu(II) ion detection.42−46
Fluorescein (1) has been derivatized and/or functionalized
particularly on the top ring with an isothiocyanate (−NC
S) group named fluorescein isothiocyanate (FITC, Figure 2).
FITC has been widely utilized as diverse forms to understand
various physicochemical, photophysical, and biological implica-
tions; FITC-labeled PEO−PCL−PEO triblock copolymer,12
FITC−deacetylated chitin conjugates,13 FITC-labeled human
plasma,14 FITC-labeled collagen,15 FITC−PEO conjugate,16
FITC−cyclodextrin conjugates,17 FITC−fullerol conjugates,18
insulin−fluorescein conjugates,19 bifunctional polyacrylamides
containing vancomycin and fluorescein,20 a fluorescein−
ruthenium−octa-arginine conjugate,21 lithocholyl−lysyl−fluo-
rescein conjugates,22 dimethylamionapthamide−fluorescein
conjugates,23 FITC-labeled lectin,24 FITC labeling at the 5′-
end of DNA with intercalating compound conjugates,25 and
FITC−dextran conjugates.26
Fluorescein also functionalized on the top ring with a
carboxylic acid group fluorescein carboxylic acid (FCA), which
has been used as precursor for the synthesis of oligonucleotides
(e.g., fluorescein phosphoramidites, FAM),27 fluorescein-tagged
peptoid conjugates,28,29 characterizing RecA−DNA interac-
tion,30 fluorescein-C-and O-glycosides,31 fluorescein labeled 7-
methylguanosinemonophosphate,32 and ifenprodil conju-
gates.33
Although fluorescein and fluorescein derivatives/conjugates
have diverse physicochemical, photophysical, and biological
properties, their physiological activity and relevance to drug
development still need to be clarified. A detailed search gave us
the information that fluorescein derivatives derived from cyclic
form of 1 (Figure 1, cyclic form) have been studied for the
detection of membrane-bound DNA,47 detection of damaged
nucleic acids,48 measurement of mammalian phosphoinositide-
specific phospholipase C activity,49 and proteolytic activities50
rather than the open form of 1 (Figure 1, open form). In
addition, fluorescein−naringenin conjugates shows 20% of the
activity of naringenin.51 It should be noted that, recently,
halogenated fluorescein analogues was reported with potent
microbial activity.52 As a part of our research finding
biologically active molecules for potential oncology therapeu-
tics, we have designed a number of derivatives of fluorescein
hydrazone (FH) containing no free carboxylic acid groups as
stated below. To the best of our knowledge, no report has been
made for the evaluation on cytotoxicity and topoisomerase
activity for the fluorescein hydrazones.
RESULTS AND DISCUSSION
■
The two hydroxyl groups on the bottom of 1 have been also
functionalized as hydroxyl group protected fluorescein
carboxylic acid (FCA-OR) and evaluated their various proper-
ties including the detection of the esterase activity of trace
copper(II),34 fluorescent derivatizing reagent,35 fluorescein
dye,36 fluorescent reagents in food samples,37 a tool for
determining the hydrolytic properties of fluorescein esters,38
Chemistry. Esterification of 1 was carried out in the
presence of catalytic amount of concentrate sulfuric acid in
ethanol to afford fluorescein ethyl ester (FLEt) in an excellent
yield (93%). The resulting ester was then refluxed with excess
amount of hydrazine hydrate in methanol to lead to the desired
fluorescein hydrazide (2) and obtained 99% yield. Compound
2 also can be prepared directly form 1 by refluxing in the
B
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Scheme 2. Synthetic Scheme Fluorescein Hydrazone Derivatives (3a−3l)
Chart 1. Structures of Synthesized Fluorescein Hydrazone Derivatives (3a−3l)
presence of excess amount of hydrazine hydrate in methanol
gave 80−90% yield (Scheme 1).
and Figure 3). Compounds 3d and 3i inhibited Topo I activity
at 100 μM by 66% and 45%, respectively. Moreover, compound
3d inhibited Topo I more strongly (47%) than camptothecin
(34%) at 20 μM concentration. Most of the compounds
displayed stronger Topo II inhibitory activity at 100 and 20
μM, except for compound 2. Compounds 3f (38%), 3k (79%),
and 3l (61%) exhibited stronger Topo II inhibitory activities
than etoposide (24%) at 20 μM concentration.
Structure−Activity Relationships (SARs) Study of 3a−
3l Based on Topoisomerases Activities. Synthesized
fluorescein hydrazone containing a 2-nitro group in a phenyl
ring attached with fluorescein by a hydrazone bond, compound
(3d) inhibited Topo I activity at 100 μM by 66% and at 20 μM
by 47%, whereas camptothecin inhibited 34% at 20 μM
concentration. While N,N-dimethyl group at the 4-position of
the phenyl ring attached with fluorescein by a hydrazone bond
(3i) decreased the inhibitory activity than 3d, it inhibited Topo
I activity at 100 μM by 45% and at 20 μM by 12%. On the
other hand, containing a 2,4-dichloro (3k) and a isatin moiety
Synthesis of 3a−3l was straightforward as shown in Scheme
2. Reactions of 2 with substituted benzaldehydes were carried
out in the presence of a catalytic amount of acetic acid in
ethanol under refluxing conditions to afford the corresponding
3a−3k in excellent yields (95−99%). To obtain 3l, hydrazide 2
was refluxed with isatin in similar fashion of which the yield was
98%. It should be noted that while condensing 2 with
substituted benzaldehydes in ethanol without acetic acid no
products and/or byproducts were obtained even in refluxing
condition for 2 days. After addition of a catalytic amount (1−3
drops) of glacial acetic acid, we obtained excellent yields (95−
99%). The yield and the purity of each compound were given
in Experimental Section. All the synthesized compounds are
listed in Chart 1.
Topo I and II Inhibitory Activities of Compounds 3a−
3l. Most tested compounds had no Topo I inhibitory activity at
100 μM treatment except for compounds 3d and 3i (Table 1
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Cell Proliferation Inhibitory Activities of Compounds
3a−3l. The cell proliferation inhibitory activities of compounds
are listed in Table 2 as values of IC50. Most of the tested
compounds, except for compound 2, displayed antiproliferation
activity against all tested cancer cell lines. Compound 3d
displayed the highest cytotoxicity against the HCT15 cells
Table 1. Topoisomerase I and II Inhibitory Activities of
Compounds 2 and 3a−3l
% inhibition against
Topo I
100 μM
Topo IIα
compd
20 μM
100 μM
20 μM
a
(1.59
0.05 μM). Compounds 3d and 3l were chosen for
camptothecin
52
NT
8
34
NT
82
7
NT
24
NT
17
17
15
20
19
38
15
22
21
19
79
61
further study because compound 3d was a Topo I and Topo II
dual inhibitor with the strongest antiproliferative activity among
tested compounds, and compound 3l inhibited Topo II
specifically.
etoposide
NT
NT
NT
NT
NT
47
2
3a
3b
3c
3d
3e
3f
0
80
76
74
72
75
72
74
75
78
83
92
2
Inhibitory Activity of Compounds 3d and 3l on Topo
IIα-Mediated Decatenation of Kinetoplast DNA. The
decatenation assay is suitable for screening Topo II specific
inhibitor because kinetoplast DNA (kDNA) can be decatenated
by Topo II but not by Topo I.53 Compound 3d inhibited the
decatenation activity of Topo II at 50 and 100 μM by 29% and
88%, respectively (Figure 4A,C). Treatment of compound 3l
with 50 μM did not inhibit the decatenation (data not shown),
therefore the concentration of compound 3l was increased to
100 and 200 μM, which inhibited the decatenation by 46% and
91%, respectively (Figure 4B,D). However, the inhibitory
activity of compound 3l on Topo II-mediated DNA relaxation
was more potent than 3d at both treated concentrations.
Ellipticine, used as a positive control, is a well-known DNA
intercalating and DNA binding Topo II inhibitor. The extent of
inhibition on Topo II-mediated decatenation of kDNA by
ellipticine is similar to that previously observed.54 Etoposide is a
well-known Topo II poison due to its function to stabilize the
transiently formed Topo II−DNA covalent complex.4,5 Less
inhibitory activity of etoposide against Topo II-mediated
decatenation than ellipticine attributed to its different mode
of action from that of ellipticine.55,56 On the basis of results of
relaxation and decatenation assays, both of compounds 3d and
3l were confirmed to inhibit potently Topo II.
4
66
0
NT
NT
NT
NT
16
0
3g
3h
3i
0
0
45
0
3j
NT
NT
NT
3k
3l
25
14
100
a
NT: nontested.
(3l) decrease very much inhibitory activity, its inhibited Topo I
activity at 100 μM by 25% and 14%, respectively. Except above
compounds (3d, 3i, 3k, and 3l), no compounds inhibited
expected Topo I activity. It should be noted that, having a 3-
hydroxy group (3e), 4-hydroxy group (3g), 4-methoxy group
(3h), and 3-hydroxy-4-methoxy groups (3j) did not inhibit
Topo I activity at all at 100 μM concentration. All the results
are summarized in Table 1.
All the compounds, 3a−3l inhibited strong Topo II activity
at 100 and 20 μM. However, having 3-hydroxy-4-methoxy
groups (3j, 83%), 2,4-dichloro groups (3k, 92%), and an isatin
moiety (3l, 100%) in the phenyl ring inhibited stronger Topo II
activity at 100 μM than the etoposide (82%, Table 1). Not only
that, but also at 20 μM concentration, 3k (79%) and 3l (61%)
show much more inhibition activity than etoposide (24%).
Compound 3d As a Nonintercalative Catalytic Topo I
and II Dual Inhibitor and Compound 3l As a Non-
intercalative Catalytic Topo II Specific Inhibitor. Com-
Figure 3. Topoisomerase I and IIα inhibitory activities of compounds 2 and 3a−3l. (A) Topo I: lane D, pBR322 DNA only; lane T, pBR322 DNA +
Topo I; lane C, pBR322 DNA + Topo I + camptothecin; lanes 2 and 3a−3l, pBR322 DNA + Topo I + compounds 2 and 3a−3l. (B) Topo IIα: lane
D, pBR322 DNA only; lane T, pBR322 DNA + Topo IIα; lane E, pBR322 DNA + Topo IIα + etoposide; lanes 2 and 3a−3l, pBR322 DNA + Topo
IIα + compounds 2, 3a−3l.
D
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a
Table 2. Cell Proliferation Inhibitory Activities of Compounds 2 and 3a−3l
IC50 (μM)
compd
T47D
HCT15
DU145
HEK293
adriamycin
0.57 0.18
3.26 1.54
1.21 0.76
>50
2.03 2.19
4.46 0.93
0.29 0.12
>50
0.88 0.16
9.29 0.71
1.05 0.69
>50
3.07 1.67
6.48 3.22
0.12 0.02
>50
etoposide
camptothecin
2
3a
3b
3c
3d
3e
3f
5.28 0.05
6.26 0.06
>50
5.48 0.06
6.06 0.06
>50
7.87 0.04
7.46 0.03
12.35 0.24
11.78 0.43
9.09 0.04
7.94 0.17
18.49 0.04
9.78 0.25
45.25 3.67
19.63 0.13
8.44 1.00
15.89 0.10
2.81 0.03
2.38 0.01
1.46 0.01
4.28 0.11
1.46 0.01
4.74 0.10
12.13 0.03
7.61 0.10
3.48 0.10
11.26 0.03
5.00 0.08
6.53 0.02
9.60 0.12
5.17 0.06
21.51 0.11
1.59 0.05
5.78 0.05
5.25 0.07
9.55 0.01
3.99 0.25
23.57 0.70
16.72 0.06
22.99 0.32
20.58 0.23
3g
3h
3i
15.42 0.19
13.79 0.27
18.65 0.17
11.15 0.16
4.12 0.01
37.78 0.79
3j
3k
3l
a
Each data represents mean SD obtained from three different experiments. Cell lines used as follows: HEK293, human embryonic kidney cell;
DU145, human prostate cancer cell; HCT15, human colon cancer cell; T47D, human breast cancer cell. Positive controls used are as follows:
etoposide, positive control for Topo IIα and cytotoxicity; camptothecin, positive control for Topo I and cytotoxicity; adriamycin, positive control for
cytotoxicity.
Figure 4. Compounds 3d (A) and 3l (B) inhibited Topo II-mediated kDNA decatenation: lane M, marker; lane D, kDNA only; lane T, kDNA +
Topo IIα; lanes Eto, Ellip, and Comp 3d and 3l, kDNA + Topo IIα in the presence of each of compound at the designated concentration. Eto,
etoposide; Ellip, ellipticine. (C,D) Quantification of decatenated products formed in (A) and (B), respectively.
pounds 3d and 3l were examined with cleavage complex assay
whether they functioned as a Topo II poisons or catalytic
inhibitor. Incubation of Topo IIα with etoposide resulted in a
linear DNA band, a marker of Topo poison, as depicted in
Figure 5A. However, this linear DNA band was not observed
with treatment of 100 μM compounds 3d and 3l, even at a
higher concentration of 300 μM. Therefore, we can conclude
that compounds 3d and 3l did not stabilize the enzyme−DNA
cleavage complex and did not act as classical poisons but
functioned as catalytic inhibitors. To gain more insight into the
fact that compounds 3d and 3l functioned as catalytic
inhibitors, we measured DNA damage by observing comet
tails using comet assay after treatment of etoposide or
compounds with HCT15 cells. The comet tails occur when
the stable enzyme−DNA cleavage complex is formed in cells,
therefore the formation of comet tail is an important property
of Topo II poisons.57 Etoposide, used as a positive control,
significantly induced DNA tails at 10 μM concentration. In
contrast, compounds 3d and 3l did not induce at 20 μM
concentration (Figure 5B,C), approving their mode of action as
catalytic inhibitors. The catalytic inhibitors of Topos can be
grouped into two types of intercalative and nonintercalative
catalytic inhibitors.4−6 The intercalative or nonintercalative
ability of compounds 3d and 3l were examined with DNA
intercalating/unwinding assay using a negatively supercoiled
pHOT1 DNA as a substrate because the formation of
supercoiled DNA in this assay is a practical characteristic of
intercalative drugs.58 Amsacrine (m-AMSA), a well-known
E
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Figure 5. Compounds 3d and 3l functioned as nonintercalative catalytic inhibitors against Topo I/II and Topo II specifically. (A) Cleavage complex
assay. In contrast to etoposide, compounds 3d and 3l did not induce a linear DNA band which was generated due to stabilization of a transient
cleavage complex of Topo II−DNA. (B) Comet assay. Images of control (nontreated), etoposide (Topo II poison), and compounds 3d or 3l treated
HCT15 cells. Compounds 3d and 3l did not induce DNA damage in HCT15 cells, but etoposide-treated HCT15 cells showed severe comet
formation. (C) Graphical representation of the selected comet lengths of untreated- and treated-HCT15 cells in pixels with corresponding to
concentration in (B). The percentage of tail DNA was obtained by analysis of images using Komet software. Columns and error bars indicate mean
SD (n = 50). ***P < 0.001 significantly different from the value of control. (D) DNA intercalating/unwinding assay. m-AMSA (intercalative Topo
II poison) blocked the unwinding of pHOT1 DNA, which was mediated by excess amount of Topo I and formed the supercoiled DNA in dose-
dependent manner, but compounds 3d and 3l did not up to 1000 μM treatment. (E) DNA retardation in migration during gel electrophoresis.
Ethidium bromide generated a noticeable retardation in migration of pBR322 helix DNA, but compounds 3d and 3l did not.
Figure 6. Compound 3d reduced migration and proliferation of HEK293 (A) and DLD1 (B) cells. Cells were cultured in 6-well plate until reached
to 90% confluence. A scratch wound in mono layer cells across the center of each well was created and followed by 4 h of serum starvation, then cells
were treated with compound 3d at designated concentrations for 24 or 48 h. The wound gap was assessed by a fluorescence inverted microscope
with 5× magnification at different time points (0, 12, 24, and 48 h).
intercalative Topo II poison,4,5 blocked unwinding of pHOT1
DNA and formed supercoiled DNA in the presence of excess
Topo I in a dose-dependent manner, which is consistent with
the result previously reported.59 While compounds 3d and 3l
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Figure 7. Compound 3d induced apoptosis through cleaving caspase-3 and PARP in HCT15 cells. Cleaved caspase-3 and PARP were increased in
HCT15 cells in a dose-dependent manner when treated with 2.5, 5, 10, and 15 μM of compound 3d for 24 h (A) and augmented in a time-
dependent manner when treated with 10 μM compound 3d for 4, 7, 9, and 12 h (B). (C,D) The levels of proteins in (A) and (B) were normalized
to β-actin and depicted as histograms. Values represent the mean SD obtained from three different experiments. *P < 0.05, **P < 0.01, ***P <
0.001 significantly different from the value of control (untreated).
did not block DNA unwinding at high concentration up to
1000 μM treatment (Figure 5D). In addition, the retardation of
pBR322 helix DNA migration during gel electrophoresis was
simply checked in the presence or absence of compounds.
Ethidium bromide, a well-known DNA intercalator, induced
noticeable retardation as shown in Figure 5E like the previous
observation,60 while compounds 3d and 3l did not. Taken
together, it can be inferred that compound 3d is a
nonintercalative catalytic Topo I and II dual inhibitor and
compound 3l is a nonintercalative catalytic Topo II specific
inhibitor.
Effect of Compound 3d on Migration and Prolifer-
ation in HEK293 and DLD1 Cells. As results of diverse assays
performed so far, compound 3d is inferred to be a more potent
Topo inhibitor than compound 3l. Compound 3d demon-
strated the most potent antiproliferative activity in HCT15 cells
(Table 1). Therefore, HCT15 cells were chosen as a model cell
line and compound 3d was selected for further investigating its
cellular mechanism. We first investigated the inhibitory activity
of compound 3d on migration and proliferation in HCT15
cells, but we failed in observing cell migration of untreated
HCT15 cells. Because both HCT15 and DLD1 are human
colorectal adenocarcinoma cells and derived from the same
individual which are described in the cell information on ATCC
and HEK293, a human embryonic kidney cell line is often used
for the test of cell migration.61,62 HEK293 and DLD1 were in
this study utilized to perform a scratch wound healing assay to
evaluate the effect of compound 3d on cell migration. After a
scratch wound was prepared with a sterilized microtip then the
wound was checked after the treatment of 5, 10, and 15 μM
compound 3d in HEK293 cells and 10 and 15 μM in DLD1
cells, respectively. The wound gap in untreated HEK 293 cells
was almost completely covered at 24 h, while cell migration was
inhibited in compound 3d-treated cells in a time and dose-
dependent manner (Figure 6A). Although the anticell
migratory activity of compound 3d in DLD1 cells was slower
than in HEK293 cells, DLD1 cell migration was also inhibited
by compound 3d in a time and dose-dependent manner, which
confirms that compound 3d possesses the inhibitory activity of
viability and migration of cancer cells.
Compound 3d Induced Apoptosis in HCT15 Colon
Cancer Cells. We observed the protein level of cleaved
caspase-3 and cleaved PARP in HCT15 cells by Western blot
analysis. Cleavage of caspase-3 (17 and 19 kDa), a marker of
apoptotic machinery activation, was increased in a dose- and
time-dependent manner when HCT15 cells were treated with
G
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Figure 8. Treatment of compound 3d induced G1 arrest along with an increase of p27kip1 and a decrease of cyclin D1 in a dose- and time-dependent
manner in HCT15 cells. (A,B) FACS analysis. HCT15 cells were treated with various concentrations (0, 2, 3, 4, and 5 μM) of compound 3d for 24 h
(A) and treated with 5 μM of compound 3d for various times (0, 4, 8, 12, and 24 h) (B). Contents of compound 3d-treated or -untreated cells
(control) were depicted as histograms. Values represent the mean SD obtained from three different experiments. (C,D) Western analysis. The
expression levels of cyclin D and p27kip1 were detected after HCT15 cells were treated with various concentrations (0, 2.5, 5, 7.5, and 10 μM) of
compound 3d for 24 h and treated with 10 μM of compound 3d for various times (0, 4, 8, 12, and 24 h). (E,F) The levels of proteins in (C) and (D)
were normalized to β-actin. The protein level ratios of cyclin D or p27kip1 to β-actin in compound 3d-treated or -untreated cells (control) were
depicted as histograms. Values represent the mean
significantly different from the value of control (untreated).
SD obtained from three different experiments. *P < 0.05, **P < 0.01, ***P < 0.001
2.5, 5, 10, and 15 μM compound 3d for 24 h (Figure 7A) and
treated with 10 μM compound 3d for 4, 7, 9, and 12 h (Figure
7B). The truncation of PARP from its native 116 kDa form to
89 kDa product is also characteristic of apoptosis.63 The
expression of cleaved PARP (89 kDa) was increased in a dose-
and time-dependent manner. The increment of cleaved
caspase-3 and cleaved PARP indicates that compound 3d
induced apoptosis in HCT15 cancer cells.
Compound 3d Induced G1 Arrest in HCT15 Colon
Cancer Cells. We examined the effect of compound 3d on the
cell cycle of HCT15 cells using a fluorescence activated cell
sorter (FACS) with staining of propidium iodide. As shown in
Figure 8, treatment of compound 3d at 2, 3, 4, and 5 μM for 24
h increased G1 phase population to 60.0%, 65.3%, 69.0% and
73.4%, respectively. Greater than 1.7-fold increases in the G1
phase population was observed at 5 μM treatment of
compound 3d compared to untreated cells (Figure 8A).
Furthermore, we treated HCT15 cells with 5 μM compound
3d for 4, 8, 12, and 24 h. A significant time-dependent increase
in G1 phase population was observed at treatment of 5 μM
compound 3d for 24 h (Figure 8B).
To confirm the G1 arrest effect of compound 3d, we
investigated changes in the expression of G1 checkpoint-related
proteins, including cyclin D1 and p27kip1 using Western blot
analysis. The level of p27kip1, a negative regulator of G1
progression, is increased concomitant with a decrease of cyclin
D1 expression when G1 arrest is induced in cell cycles.64,65 The
protein level of cyclin D1 was significantly decreased in a dose-
and time-dependent manner and reached the highest level at
treatment of 10 μM compound 3d for 24 h as shown in Figure
8C,D. The level of p27kip1 was substantially increased in a dose-
and time-dependent manner. The compound 3d-induced G1
phase arrest was confirmed by an increase of p27kip1
concomitant with a decrease of cyclin D1 in colon cancer cells.
CONCLUSION
■
Fluorescein and fluorescein derivatives or conjugates have been
reported to possess diverse physicochemical, photophysical,
and biological properties, however, their physiological activity
and relevance to drug development are not intensively studied
yet. Thirteen compounds of fluorescein hydrazones (3a−3l)
were simply synthesized with excellent yields (95−99%).
H
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spectra were taken in positive/negative mode and scan began at 50 m/
z and ended at 1000 m/z; the injection volume was 2 μL. Purity of all
fluorescein hydrazones was assessed using an Eclipse Plus C18 (2.1
mm × 150 mm, 3.5 μm) column (Agilent Technologies, Palo Alto,
CA, USA) in an Agilent high performance liquid chromatography
(HPLC) 1200 connected to an Agilent 6320 ion trap mass
spectrometer. The mobile phase was composed of two solvents,
methanol and water, eluting with a gradient of 5−95% methanol in
water for 10 min, flow rate of 0.3 mL/min, run time of 10 min.
According to these LC−MS analyses, final compounds showed a
purity of ≥98%.
Synthesis of FLEt. Fluorescein (1, 3.32 g, 0.01 mol) was dissolved
in 50 mL of absolute ethanol, a catalytic amount of concentrate
sulfuric acid was added, and the mixture was refluxed for overnight.
Solvent were evaporated under vacuum, ethyl acetate was added and
washed with water, 5% NaHCO3, and brine and dried over anhydrous
Na2SO4, filtered, and evaporated; the solvent obtained FLEt (3.35 g,
93%) as white powder; mp 241−242 °C.66 1H NMR (CD3OD, 500
MHz): δ, 8.30 (d, J = 7.0 Hz, 1H), 7.86 (t, J = 6.5 Hz 1H), 7.80 (t, J =
6.5 Hz 1H), 7.45 (d, J = 7.0 Hz 1H), 7.03 (d, J = 9.0 Hz, 1H), 6.77 (s,
2H), 7.03 (dd, J = 9.0, 2.0 Hz, 1H), 4.02 (q, J = 7.0 Hz, 1H), 0.92 (t, J
= 7.0 Hz, 1H) ppm. ESI mass (m/z) 358.9 [M − H]− (calculated
360.10).
Synthesis of 2. Fluorescein (3.32 g, 0.01 mol) was dissolved in 50
mL of absolute ethanol, followed by the addition of hydrazine
monohydrate (excess, 30 mL). The mixture was refluxed for 12 h until
the fluorescence of the solution disappeared. The reaction mixture was
dissolved in ethyl acetate (100 mL) then washed with water several
times. The organic phase was collected, dried over anhydrous Na2SO4,
filtered, and evaporated of the solvent. The crude product was
recrystallized from methanol to give 2 (3.35 g, 99%) as white powder;
Rf = 0.55 (CH2Cl2:methanol = 10:1); mp 272−273 °C.43 1H NMR
(CD3OD, 500 MHz): δ, 7.89 (d, J = 5 Hz, 1H), 7.58−7.52 (m, 2H),
7.05 (d, J = 8 Hz, 1H), 6.65 (s, 2H), 6.48 (s, 4H) ppm. 13C NMR
(CD3OD, 125 MHz): δ 168.6, 160.1, 154.7 153.3, 134.2, 130.5, 129.7,
129.1, 124.9, 123.8, 113.22, 110.3, 103.9, and 64.8 ppm. ESI mass (m/
z) 347.0796 [M + H]+, 368.9707 [M + Na]+ (calculated 346.0954).
General Procedure for the Synthesis of 3a−3l.43 A mixture of
fluorescein hydrazide (2, 0.3 mmol) and substituted aldehyde (0.3
mmol) in 10 mL of ethanol was added a drop of glacial acetic acid, and
the reaction mixture was refluxed in oil bath for 6 h. The precipitate
solid was filtered, washed with cold ethanol, and air-dried to obtain 3,
which was further purified by recrystallization using methanol/
dichloromethane.
Compounds 3a−3l were first evaluated by Topo I- and IIα-
mediated relaxation assay and cell viability assay in several
human cancer cell lines. On the basis of Topo inhibitory
activity and cytotoxicity of synthesized fluorescein hydrazones
(Figure 3 and Tables 1 and 2), compounds 3d and 3l were
further evaluated to determine their mode of action with
diverse methods of kDNA decatenation, DNA−Topo cleavage
complex, comet, DNA intercalating/unwinding, and Topo IIα-
mediated ATP hydrolysis assays (Figures 4 and 5); compound
3d functioned as a nonintercalative dual inhibitor against the
catalytic activities of Topo I and Topo IIα. Compound 3l acted
as a Topo IIα specific nonintercalative catalytic inhibitor. The
effect of compound 3d on migration and proliferation using
HEK293 and DLD1 cells was further examined due to its
strongest cytotoxicity and better Topo IIα-mediated kDNA
decatenation efficacy than compound 3l. Cell migration was
inhibited in compound 3d-treated cells in a dose- and time-
dependent manner (Figure 6). Compound 3d activated
apoptotic proteins as it increased the level of cleaved capase-
3 and cleaved PARP in a dose-and time-dependent manner
(Figure 7). The dose- and time-dependent increase of G1 phase
population was observed by treatment of compound 3d along
with the increase of p27kip1 and the decrease of cyclin D1
expression (Figure 8). It is the first time a fluorescein derivative
as a potent catalytic inhibitor against Topo I and IIα is reported
. Taken together, compound 3d and 3l can be a novel scaffold
for the development of antitumor agents targeting Topo I and
IIα catalytic activity with a further optimizing process.
Molecular docking study and in vivo experiment will be
performed by our group in the very near future and the results
will be explored in due course.
EXPERIMENTAL SECTION
■
Chemistry General. Chemicals and solvents were commercial
reagent grade and were used without further purifications. All reactions
were carried out under an atmosphere of nitrogen gas inside the fume
hood by using a flame-dried apparatus with magnetic stirrer, unless
otherwise indicated. TLC analysis was carried out on glass-backed
TLC silica plates (silica gel 60 F254, 0.25 mm) impregnated with
fluorescence indicator (Merck art. 1.05554). The detection of UV-
active substances was visualized using ultraviolet light (λmax = 254 nm),
and for non-UV active substances KMnO4/PMA/p-anisaldehyde stains
were used. Column chromatography was carried out on 60−120 mesh
Merck silica gel. Melting points were determined on a Barnstead
electrothermal digital melting point apparatus model 9100 and are
uncorrected. NMR spectra were obtained using a Bruker-500
(E)-2-(Benzylideneamino)-3′,6′-dihydroxyspiro[isoindoline-1,9′-
1
xanthen]-3-one (3a). White powder (96%); mp 268−267 °C. H
NMR (CD3OD, 500 MHz): δ 8.53 (s, 1H), 7.99 (d, J = 7 Hz, 1H),
7.64−7.57 (m, 2H), 7.49 (dd, J = 5.5, 2 Hz, 1H), 7.31 (s, 3H), 7.12 (d,
J = 7 Hz, 1H), 6.71 (d, J = 2 Hz 1H), 6.54 (d, J = 8.5 Hz, 1H), 6.47
(dd, J = 8.5, 2.5 Hz, 1H) ppm. 13C NMR (DMSO-d6, 125 MHz): δ
163.6, 158.6, 152.2, 150.3, 149.2, 134.4, 134.0, 130.3, 129.1, 128.8,
128.0, 126.7, 123.8, 123.2, 112.3, 110.2, 102.4, and 65.3 ppm. LC/MS/
MS: retention time: 4.9 min. ESI mass (m/z) 435.1486 [M + H]+,
457.0719 [M + Na]+ (calculated 434.1267).
1
spectrometer (500 MHz for H NMR and 125 MHz for 13C NMR)
and Jeol 400 MHz spectrometer and are reported as parts per million
(ppm) from the internal standard of tetramethylsilane. The coupling
constants J are reported in hertz (Hz). Peaks are described as broad
signals (br), singlet (s), doublets (d), doublets of doublets (dd),
doublets of doublets of doublets (ddd), triplets (t) and multiplets (m),
doublets of triplets (dt), and triplets of doublets (td). Electrospray
ionization (ESI) mass spectrometry (MS) experiments were
performed using an Agilent high performance liquid chromatography
(HPLC) 1200 connected to an Agilent 6320 ion trap mass
spectrometer fitted with an electrospray ionization (ESI) ion source
(Agilent Technologies, Palo Alto, CA, USA). Agilent 6300 series ion
trap LC/MS system software, version 6.2, was used for data acquisition
and analysis. An Eclipse Plus C18 (2.1 mm × 150 mm, 3.5 μm)
column (Agilent Technologies, Palo Alto, CA, USA) was used. The
mobile phase was composed of two solvents, methanol and water, with
a gradient of 1−95% methanol in water, 0.3 mL/min flow rate, run
time of 10 min. The ESI ion source capillary temperature was set at
325 °C; the dry gas flow rate was set to 10.0 L/min; nebulizer pressure
was set to 40.00 psi; maximum accumulation time was 300000 μs;
(E)-3′,6′-Dihydroxy-2-(2-hydroxybenzylideneamino)spiro-
[isoindoline-1,9′-xanthen]-3-one (3b). Yellow powder (99%); mp
325−326 °C. 1H NMR (DMSO-d6, 500 MHz): δ, 9.91 (brs, 2H), 9.18
(s, 1H), 7.90 (d, J = 8.2 Hz, 1H), 7.10 (pentet, J = 10.9 Hz, 2H), 7.43
(d, J = 9.2 Hz, 1H), 7.34 (t, J = 9.2 Hz, 1H), 7.11 (d, J = 9.2 Hz, 1H),
6.99 (d, J = 10.1 Hz 1H), 6.90 (t, J = 9.2 Hz, 1H), 6.64 (s, 2H), 6.46
(m, 4H) ppm. 13C NMR (DMSO-d6, 125 MHz): δ 163.4, 160.7, 158.7,
157.2, 152.2, 150.5, 150.4, 136.4, 134.1, 131.8, 129.3, 129.2, 129.0,
128.7, 128.1, 123.8, 123.2, 122.2, 119.4, 119.4, 118.8, 117.2, 116.4,
112.5, 109.4, and 62.2 ppm. LC/MS/MS: retention time: 5.1 min. ESI
mass (m/z) 451.1360 [M + H]+, 473.0663 [M + Na]+ (calculated
450.1216).
(E)-3′,6′-Dihydroxy-2-(2-methoxybenzylideneamino)spiro-
[isoindoline-1,9′-xanthen]-3-one (3c). White cream (99%); mp 297−
1
298 °C. H NMR (CD3OD, 500 MHz): δ, 8.56 (s, 1H), 7.87 (d, J =
10.5 Hz, 1H), 7.69 (d, J = 7.0 Hz, 1H), 7.52−7.45 (m, 2H), 7.17 (td, J
I
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= 7.5, 1.5 Hz, 1H), 6.97 (d, J = 8.5 Hz, 1H), 6.76 (t, J = 7.0 Hz, 1H),
6.58 (d, J = 2.0 Hz 1H), 6.40 (d, J = 8.5 Hz, 1H), 6.35 (dd, J = 8.5, 2.0
Hz, 1H), 3.61 (s, 3H) ppm. 13C NMR (DMSO-d6, 125 MHz): δ
163.5, 158.5, 157.8, 152.1, 150.6, 144.1, 133.9, 131.8, 129.0, 129.0,
128.0, 124.6, 123.8, 123.1, 122.5, 120.7, 112.3, 111.9, 110.0, 102.4,
65.1, and 55.7 ppm. LC/MS/MS: retention time: 3.6 min. ESI mass
(m/z) 465.1515 [M + H]+, 487.1260 [M + Na]+ (calculated
464.1372).
(overlapped, 4H), 2.91 (s, 6H) ppm. 13C NMR (DMSO-d6, 125
MHz): δ 163.5, 158.9, 152.8, 150.2, 151.8, 150.8, 133.9, 130.1, 129.4,
128.7, 128.5, 124.1, 123.4, 122.1, 112.7, 112.1, 111.0, 102.9, 65.7, and
19.0 ppm. LC/MS/MS: retention time: 4.0 min. ESI mass (m/z)
478.2045 [M + H]+ (calculated 477.1689).
(E)-3′,6′-Dihydroxy-2-(3-hydroxy-4-methoxybenzylideneamino)-
spiro[isoindoline-1,9′-xanthen]-3-one (3j). Yellowish-white cream
1
(95%); mp 301−303 °C. H NMR (DMSO-d6, 500 MHz): δ, 9.92
(s, 2H), 9.24 (s, 1H), 8.84 (s, 1H), 7.88 (d, J = 9.2 Hz, 1H), 7.59
(pentet, J = 9.2 Hz, 2H), 7.09 (d, J = 9.2 Hz, 1H), 6.90 (s, 1H), 6.88
(d, J = 11.0 Hz, 1H), 6.80 (d, J = 10.1 Hz, 1H), 6.64 (d, J = 1.9 Hz,
2H), 6.49 (d, J = 11.0 Hz, 2H), 6.43 (dd, J = 11.0, 1.8 Hz, 2H) 3.74 (s,
3H) ppm. 13C NMR (DMSO-d6, 125 MHz): δ 163.4, 158.5, 152.1,
150.6, 150.2, 150.1, 146.7, 133.7, 129.1, 129.0, 128.0, 127.2, 123.6,
123.0, 120.5, 112.3, 111.8, 110.2, 102.5, 65.2, and 56.0 ppm. LC/MS/
MS: retention time: 1.6 min. ESI mass (m/z) 481.1588 [M + H]+,
503.0988 [M + Na]+. LC/MS/MS: retention time: 2.4 min. ESI mass
(m/z) 481.1760 [M + H]+, 503.1167 [M + Na]+ (calculated
480.1321).
(E)-3′,6′-Dihydroxy-2-(2-nitrobenzylideneamino)spiro-
[isoindoline-1,9′-xanthen]-3-one (3d). Yellow needle (98%); mp
1
287−289 °C. H NMR (DMSO-d6, 500 MHz): δ, 9.95 (s, 2H), 9.23
(s, 1H), 7.95 (t, J = 10.1 Hz, 2H), 7.71 (d, J = 4.5 Hz, 2H), 7.69−7.57
(m, 3H), 7.12 (d, J = 9.2 Hz, 1H), 6.66 (s, 2H), 6.52 (d, J = 11.9 Hz,
2H), 6.45 (d, J = 11 Hz, 2H) ppm. 13C NMR (DMSO-d6, 125 MHz):
δ 164.2, 158.7, 152.0, 150.8, 148.0, 142.2, 134.4, 133.7, 130.8, 129.2,
128.6, 128.1, 127.8, 127.0, 124.6, 123.8, 123.4, 112.4, 109.5, 102.6, and
65.3 ppm. LC/MS/MS: retention time: 2.1 min. ESI mass (m/z)
434.1259 [(M − NO2) + H]+, 462.1417 [(M − OH) + H]+, 480.0977
[M + H]+, 502.0716 [M + Na]+ (calculated 479.1117). LC/MS/MS:
retention time: 3.7 min. ESI mass (m/z) 480.1333 [M + H]+,
502.0577 [M + Na]+ (calculated 479.1117).
(E)-2-(2,4-Dichlorobenzylideneamino)-3′,6′-dihydroxyspiro-
[isoindoline-1,9′-xanthen]-3-one (3k). Yellowish-white powder
1
(95%); mp 349−350 °C. H NMR (DMSO-d6, 500 MHz): δ, 9.97
(E)-3′,6′-Dihydroxy-2-(3-hydroxybenzylideneamino)spiro-
(s, 2H), 8.97 (s, 1H), 7.95 (d, J = 7.5 Hz, 1H), 7.69−7.60 (m, 4H),
7.44 (dd, J = 8.5, 1.5 Hz, 1H), 7.13 (d, J = 7.0 Hz, 1H), 6.67 (d, J = 2.0
Hz, 2H), 6.52 (d, J = 8.5 Hz, 2H), 6.47 (dd, J = 9.0, 2 Hz, 2H) ppm.
13C NMR (DMSO-d6, 125 MHz): δ 164.0, 158.7, 152.0, 150.9, 141.0,
[isoindoline-1,9′-xanthen]-3-one (3e). Yellowish-white cream (98%);
1
mp 289−291 °C. H NMR (DMSO-d6, 500 MHz): δ, 9.93 (s, 2H),
9.59 (s, 1H), 8.89 (s, 1H), 7.92 (d, J = 7.0 Hz, 1H), 7.65−7.58 (m,
2H), 7.15 (t, J = 7.5 Hz, 1H), 7.15 (d, J = 7.5 Hz, 1H), 6.85 (s, 1H),
6.85 (s, 1H), 6.83 (d, J = 7.5 Hz, 1H), 6.76 (dd, J = 7.5, 1.5 Hz, 1H),
6.66 (d, J = 2 Hz, 2H), 6.51 (d, J = 8.5 Hz, 2H), 6.46 (dd, J = 8.5, 2
Hz, 2H) ppm. 13C NMR (DMSO-d6, 125 MHz): δ 163.7, 158.5, 157.2,
152.1, 150.6, 149.3, 135.6, 133.9, 129.9, 129.0, 128.8, 128.0, 123.7,
123.2, 118.6, 117.8, 11.3, 112.2, 110.3, 102.5, and 65.2 ppm. LC/MS/
MS: retention time: 2.7 min. ESI mass (m/z) 451.1260 [M + H]+,
473.0589 [M + Na]+ (calculated 450.1216).
135.3, 134.4, 133.9, 130.5, 129.3, 129.2, 128.0, 128.0, 127.0, 123.8,
123.4, 112.6, 110.1, 102.5, and 65.0 ppm. LC/MS/MS: retention time:
8.1 min. ESI mass (m/z) 503.2564 [M(35Cl) + H]+, 502.8 [M(37Cl) +
H]+; (calculated 502.0487).
(E)-3′,6′-Dihydroxy-2-(2-oxoindolin-3-ylideneamino)spiro-
[isoindoline-1,9′-xanthen]-3-one (3l). Orange solid (98%); mp 261−
1
262 °C. H NMR (DMSO-d6, 500 MHz): δ, 10.87 (s, 1H), 9.94 (s,
2H), 7.98 (d, J = 9.1 Hz, 1H), 7.70−7.62 (m, 2H), 7.37 (t, J = 9.1 Hz,
1H), 7.18 (d, J = 9.2 Hz, 1H), 7.15 (d, J = 10.0 Hz, 1H), 6.99 (t, J =
9.2 Hz, 1H), 6.83 (d, J = 10.0 Hz, 1H), 6.59 (s, 2H), 6.56 (d, J = 11.0
Hz, 2H), 6.42 (dd, J = 11.0 Hz, 2H) ppm. 13C NMR (DMSO-d6, 125
MHz): δ 163.7, 159.9, 158.6, 152.1, 152.0, 151.3, 144.8, 134.3, 133.6,
129.3, 128.4, 128.2, 128.0, 124.0, 123.9, 122.4, 117.0, 112.4, 110.6,
109.8, 102.3, and 67.0 ppm. LC/MS/MS: retention time: 2.4 min. ESI
mass (m/z) 476.1468 [M + H]+ (calculated 475.1168).
(E)-2-(4-Chlorobenzylideneamino)-3′,6′-dihydroxyspiro-
[isoindoline-1,9′-xanthen]-3-one (3f). White powder (95%); mp
1
308−309 °C. H NMR (DMSO-d6, 500 MHz): δ, 9.93 (s, 2H), 9.10
(s, 1H), 7.93 (d, J = 7.0 Hz, 1H), 7.66−7.59 (m, 2H), 7.44 (d, J = 2.0
Hz, 4H), 7.15 (d, J = 7.5 Hz, 1H), 6.66 (d, J = 2.0 Hz, 2H), 6.50 (d, J
= 8.5 Hz, 2H), 6.46 (dd, J = 8.5, 2 Hz, 2H) ppm. 13C NMR (DMSO-
d6, 125 MHz): δ 163.7, 158.6, 152.2, 150.3, 147.9, 134.8, 134.1, 133.3,
129.1, 128.9, 128.3, 128.0, 123.8, 123.2, 112.3, 110.1, 102.5, and 65.4
ppm. LC/MS/MS: retention time: 6.2 min. ESI mass (m/z) 469.1834
[M(35Cl) + H]+, 470.9947 [M(37Cl) + H]+ (calculated 468.0877).
(E)-3′,6′-Dihydroxy-2-(4-hydroxybenzylideneamino)spiro-
[isoindoline-1,9′-xanthen]-3-one (3g). Yellowish-white cream (97%);
Human Topo I and Topo IIα Relaxation Assay. Top-
oisomerases I and II (Topo I and II) inhibition were measured by
assessing the relaxation of supercoiled pBR 322 plasmid DNA as
previously described.67 The mixture of 100 ng of supercoiled DNA
pBR322 (Thermo Scientific, USA) and 1 unit of human Topo I
(TopoGen, USA) or Topo IIα (USB Corp., USA) was incubated in
the absence or presence of the prepared compounds at 37 °C for 1 h
in the reaction buffer (Topo I, 10 mM Tris HCl (pH 7.9) containing 1
mM EDTA, 0.15 M NaCl, 0.1% BSA, 0.1 mM spermidine, and 5%
glycerol; Topo IIα, 10 mM tris-HCl (pH 7.9) containing 50 mM
NaCl, 50 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 15 μg/mL BSA, and
1 mM ATP). The 10 μL of the reaction mixture as a final volume was
terminated by adding 2.5 μL of the stop solution (Topo I, 0.5%
sarcosyl containing 0.00025% bromophenol blue and 2.5% glycerol;
Topo IIα, 0.7 mM EDTA). The reaction mixture was then
electrophoresed on a 0.8% agarose gel at 60 V for 1 h with TAE
running buffer. Gels were stained for 15 min in an aqueous solution of
ethidium bromide (0.5 μg/mL). DNA bands were visualized by trans-
illumination with UV light and were quantitated using an Alpha Tech
Imager (Alpha Innotech Corporation). Camptothecin (Sigma, USA)
and etoposide (Sigma, USA) were used as positive controls as Topo I
inhibitor and Topo II inhibitors, respectively.
1
mp 281−283 °C. H NMR (DMSO-d6, 500 MHz): δ, 9.93 (s, 2H),
8.93 (s, 1H), 7.89 (d, J = 6.5 Hz, 1H), 7.60 (pentet, J = 8.5 Hz, 2H),
7.26 (d, J = 8.5 Hz, 2H), 7.10 (d, J = 7.0 Hz, 1H), 6.73 (d, J = 8.5 Hz,
2H), 6.64 (d, J = 1.5 Hz, 2H), 6.49 (d, J = 8.5 Hz, 2H), 6.46 (dd, J =
8.5, 2 Hz, 2H) ppm. 13C NMR (DMSO-d6, 125 MHz): δ 163.3, 159.7,
158.4, 152.2, 150.4, 133.6, 129.2, 129.0, 128.6, 128.0, 125.3, 123.6,
123.0, 115.7, 112.2, 110.3, 102.4, and 65.2 ppm. LC/MS/MS:
retention time: 2.4 min. ESI mass (m/z) 451.1226 [M + H]+
(calculated 450.1216).
(E)-3′,6′-Dihydroxy-2-(4-methoxybenzylideneamino)spiro-
[isoindoline-1,9′-xanthen]-3-one (3h). White cream (99%); mp
274−275 °C. 1H NMR (DMSO-d6, 500 MHz): δ, 9.90 (s, 2H),
9.03 (s, 1H), 7.91 (d, J = 7.0 Hz, 1H), 7.65−7.58 (m, 2H), 7.38 (d, J =
8.5 Hz, 2H), 7.13 (d, J = 7.0 Hz, 1H), 6.93 (d, J = 8.5 Hz, 2H), 6.65
(d, J = 1.5 Hz, 2H), 6.49 (d, J = 8.5 Hz, 2H), 6.46 (dd, J = 8.5, 2 Hz,
2H), 3.75 (s, 3H) ppm. 13C NMR (DMSO-d6, 125 MHz): δ 163.4,
161.0, 160.8, 158.5, 152.2, 150.3, 149.7, 133.8, 129.3, 129.0, 128.6,
128.1, 127.0, 123.7, 123.1, 114.3, 112.2, 110.3,102.4, 65.3, and 55.2
ppm. LC/MS/MS: retention time: 3.8 min. ESI mass (m/z) 465.2472
[M + H]+, 487.1383 [M + Na]+ (calculated 464.1372).
Cytotoxicity Assay. Cytotoxicity were evaluated using diverse
cancer cell lines following the previous method.68 HCT15 (human
colon cancer cell lines), T47D (human breast cancer cell line), and
DU145 (human prostate cancer cell line) were grown in RPMI1640
(Hyclone, USA), and HEK293 (human kidney embryonic cell line)
was cultured in DMEM (Hyclone, USA). After seeding cells in 96-well
plates at a density of 2−4 × 104 cells per well with incubation
(E)-3′,6′-Dihydroxy-2-(4-(dimethylamino)benzylideneamino)-
spiro[isoindoline-1,9′-xanthen]-3-one (3i). Yellow powder (99%);
1
mp 316−317 °C. H NMR (DMSO-d6, 500 MHz): δ, 9.90 (s, 2H),
8.91 (s, 1H), 7.88 (d, J = 6.0 Hz, 1H), 7.59 (t, J = 7.5 Hz, 2H), 7.25 (d,
J = 8.0 Hz, 2H), 7.11 (d, J = 6.0 Hz, 1H), 6.64 (overlapped, 4H), 6.48
J
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overnight in 0.1 mL of media supplemented with 10% Fetal Bovine
Serum (FBS) (Hyclone, USA) in a 5% CO2 incubator at 37 °C,
culture medium in each well was exchanged with 0.1 mL aliquots of
medium containing graded concentrations of each compound and
incubated continuously for 72 h. Then 5 μL of the cell counting kit-8
solution (Dojindo, Japan) was added to each well and incubated for
additional 4 h. The absorbance of each well was measured using an
automatic ELISA reader system (Bio-Rad 3550, USA) at 450 nm. The
IC50 values were calculated with using TableCurve (Jandel Scientific
Software, USA).
kDNA Decatenation Assay. The assay was performed as
following the previous method67 in a total reaction volume of 10 μL
containing 50 ng of kDNA (TopoGEn, USA), compound, and 3 units
of human Topo IIα in the reaction buffer solution (10 mM tris-HCl
(pH 7.9) containing 50 mM NaCl, 50 mM KCl, 5 mM MgCl2, 0.1 mM
EDTA, 15 μg/mL BSA, and 1 mM ATP). The reaction mixtures were
incubated for 30 min at 37 °C and then terminated by the addition of
2.5 μL of stop solution containing 5% SDS, 25% ficoll, and 0.05%
bromphenol blue followed by treatment with 0.25 mg/mL proteinase
K (Roche) at 37 °C for 30 min to eliminate the protein. Reaction
mixtures were resolved by electrophoresis on a 1.2% (w/v) agarose gel
containing 0.5 μg/mL ethidium bromide in the TAE buffer. DNA
bands were visualized by UV and photographed and documented with
an Alpha Tech Imager.
cells was then created across the center of the well with a sterile
microtip. After starvation with low serum media (1% FBS in DMEM)
for 4 h, the cells were exposed to compound (5, 10, or 15 μM) for 24
or 48 h and allowed to migrate in the medium. The wound was
assessed by a fluorescence inverted microscope with 5× magnification
at different time points (0, 12, 24, and 48 h).
Western Blot Assay. HCT15 cells were grown on 60 mm tissue
culture dishes at 1 × 106 cells until reaching 80% confluency. The cells
were then treated with investigational compounds at various
concentrations for various incubation times as designated in figure
legend. Cells were lysed in lysis buffer solution containing 50 mM Tris
HCl, 300 mM NaCl, 1% Triton X-100, 10% glycerol, 1.5 mM MgCl2, 1
mM CaCl2, 1 mM PMSF, and 1% protease inhibitor cocktail. Then 70
μg of protein per sample was resolved by 8 or 10% SDS-PAGE and
transferred to a PVDF membrane (Millipore, USA). The membranes
were blocked with 5% skim milk in Tris buffered saline containing
0.1% Tween 20 (TBST) and probed with primary antibodies in a
dilution ratio of 1:1000 for 2−3 h. The blots were washed, exposed to
HRP-conjugated antirabbit IgG (Cell Signaling Technology Inc. USA)
in a dilution ratio of 1:2000 for 2 h, and detected with ECL Western
blotting detection reagent (Animal Genetics Inc., Korea). All primary
antibodies used were purchased from Cell Signaling Technology Inc.
(USA). Western blot images were taken by LAS-3000 (Fuji Photo
Film Co., Ltd., Japan) and analyzed using Multi-Gauge Software (Fuji
Photo Film Co. Ltd., Japan).
Cleavage Complex Stabilization Assay. Supercoiled DNA
pBR322 (100 ng) and 3 units of Topo IIα were mixed and incubated
for 5 min at 37 °C. Compound was then added and then incubated at
37 °C for 20 min and stopped with 10% SDS, followed by digestion
with proteinase K at 45 °C for 30 min.67 After the addition of the
loading buffer, the reaction mixture was heated for 2 min at 70 °C and
then electrophoresed on a 0.8% agarose gel in TAE buffer containing
0.5 μL/mL ethidium bromide at 20 V for 6 h. The gel was destained
with water for 10 min and visualized by an Alpha Tech Imager.
Comet Assay. Comet assay was performed using the Alkaline
Comet Assay kit (Trevigen, USA) according to the method previously
reported.69 HCT15 cells were seeded in a 6-well plate in a density of
105 cells per well with incubation for 24 h. Cells were then incubated
either without or with etoposide or compound for 24 h in serum free
media and harvested by trypsinization. The harvested cells were
resuspended in 1 mL of ice-cold PBS. Then 8 μL of resuspended cells
were mixed with 80 μL of low-melting agarose at 37 °C, followed in
the same way as the previous method.69 Comet images were captured
with Zeiss HBO100 microscope illumination system (Carl Zeiss,
Germany) equipped with epq100-isolated epifluorescence condenser.
A total of 50 HCT15 cells were randomly analyzed with an image
analysis system (Komet 5.5, Kinetic Imaging Ltd., UK). Komet 5.5
software calculated the lengths of the comet tails, and the mean values
represented the extent of the DNA damage.
FACS Analysis. HCT15 cells were seeded in 60 mm dishes at a
density of 5 × 105 cells per dish. After reached 80% confluency, cells
were treated with the test compounds at various concentrations for
various times as designated in figure legend. Cells were then washed
with ice-cold PBS (pH 7.4) and harvested by centrifugation at 2000
rpm for 5 min. The pellets were fixed with 70% ethanol, and fixed cells
were then washed with PBS before incubation with 50 mg/mL
propidium iodide (Sigma, USA) and 2.5 mg/mL RNase (Sigma,
USA). Fluorescence was measured with a fluorescence-activated cell
sorting (FACS)-Caliber flow cytometer (BD Biosciences, USA). At
least 10000 cells were measured for each sample.70
Statistical Analysis. Data are expressed as the means standard
deviation (SD), with each experiment performed in triplicate.
Comparison of the differences was conducted with an analysis of
variance (ANOVA) using the Prism statistical software package
(GraphPad Software, USA). The differences were considered
statistically significant when the p value was <0.05.
ASSOCIATED CONTENT
■
S
* Supporting Information
Proton (1H) and carbon (13C) NMR and mass spectra of
synthesized compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
DNA Unwinding/Intercalating Assay. The DNA-unwinding
capacity of compound was analyzed using a DNA unwinding kit
(TopoGEN, USA) according to the method previously reported.67,69
First, 100 ng of pHOT1 plasmid was treated with 4 units of Topo I in
reaction buffer (10 mM Tris HCl (pH 7.9) containing 1.5 M NaCl, 1%
BSA, 1 mM spermidine, and 50% glycerol) for 30 min at 37 °C. After
incubation, various concentrations of m-AMSA or compounds 3d or 3l
were added and incubated at 37 °C for an additional 30 min followed
by addition of 1 μL of Topo I stop buffer. The resulting aqueous phase
was then resolved on 1% agarose gels at 15 V/cm for 12−15 h. After
electrophoresis, gels were stained in TAE buffer with ethidium
bromide for 30 min and visualized using an Alpha Tech Imager. The
compound inhibitory activity of DNA unwinding was also evaluated by
monitoring retardation of DNA migration.67 Briefly, 125 ng of
negatively supercoiled DNA pBR322 and 100 or 300 μM of
investigational compounds in a total volume of 10 μL was incubated
at 37 °C for 20 min. Reaction mixtures were resolved on 1% agarose
gels at 20 V/cm for 12−16 h. After electrophoresis, gels were stained
in TAE buffer with ethidium bromide for 30 min and visualized using
an Alpha Tech Imager.
AUTHOR INFORMATION
Corresponding Authors
*For A.F.M.M.R.: phone, +966 11 46 70237; fax, +966 11 46
76220; E-mail, afmrahman@ksu.edu.sa.
*For Y.K.: phone, +82 23 277 4653; fax, +82 23 277 2851; E-
mail, ykwon@ewha.ac.kr.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work is funded by research grant from the The National
Plan for Science, Technology and Innovation, Kingdom of
Saudi Arabia (project no. 11-MED-1909-02).
ABBREVIATIONS USED
FACS, fluorescence activated cell sorter; FCA, fluorescein
carboxylic acid; FLEt, fluorescein ethyl ester; FH, fluorescein
■
Cell Migration Assay. HEK293 and DLD1 cells were cultured in a
6-well plate to 90% confluency. A clean wound in the cell monolayer
K
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Journal of Medicinal Chemistry
Article
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hydrazone; FITC, fluorescein isothiocyanate; FITC-PEO,
fluorescein isothiocyanate- poly(ethylene oxide); FAM, fluo-
rescein phosphoramidites; FCA-OR, hydroxyl group protected
fluorescein carboxylic acid; kDNA, kinetoplast DNA; PEO−
PCL−PEO, poly(ethylene oxide)-block-poly(3-caprolactone)-
block-poly(ethylene oxide); pHOT1 DNA, supercoiled plasmid
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