Fluorescein hydrazones: A series of novel non-intercalative topoisomerase IIα catalytic inhibitors induce G1 arrest and apoptosis in breast and colon cancer cells

Article abstract of DOI:10.1016/j.ejmech.2016.09.004

Fluorescein hydrazones (5 and 7) were synthesized in three/four steps with 82–92% yields. All synthesized compounds were evaluated by topoisomerase I (topo I) and topoisomerase IIα (topo IIα)-mediated relaxation and cell viability assays. Among them, most

Full text of DOI:10.1016/j.ejmech.2016.09.004

Accepted Manuscript
Fluorescein hydrazones: A series of novel non-intercalative topoisomerase IIα
catalytic inhibitors induce G1 arrest and apoptosis in breast and colon cancer cells
Mohammad Shahidul Islam, Seojeong Park, Chanju Song, Adnan A. Kadi, Youngjoo
Kwon, A.F.M. Motiur Rahman
PII:
S0223-5234(16)30732-2
10.1016/j.ejmech.2016.09.004
EJMECH 8870
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 July 2016
Revised Date: 29 August 2016
Accepted Date: 1 September 2016
Please cite this article as: M.S. Islam, S. Park, C. Song, A.A. Kadi, Y. Kwon, A.F.M.M. Rahman,
Fluorescein hydrazones: A series of novel non-intercalative topoisomerase IIα catalytic inhibitors induce
G1 arrest and apoptosis in breast and colon cancer cells, European Journal of Medicinal Chemistry
(2016), doi: 10.1016/j.ejmech.2016.09.004.
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ACCEPTED MANUSCRIPT
O
N
N
O N
2
EtO
O
OH
>
>
Inhibited 53% topo II (camptothecin, 29%) at 20 microM;
Showed excellent antiproliferative activity against
DU145 (1.43 ±0.04 microM), HCT15 (2.4 ± 0.03 microM)
and MCF7 (11.4 ± 0.5 microM) cell lines in comparison
with adriamycin, etoposide, and camptothecin.
Induce G1 arrest and apoptosis in breast and colon
cancer cells
>

ACCEPTED MANUSCRIPT
Fluorescein Hydrazones: A Series of Novel Non-intercalative Topoisomerase IIα
Catalytic Inhibitors Induce G1 Arrest and Apoptosis in Breast and Colon
Cancer Cells
a,#
b,#
b
c
Mohammad Shahidul Islam, Seojeong Park, Chanju Song, Adnan A. Kadi, Youngjoo
b,*
c,*
Kwon, A. F. M. Motiur Rahman
a
Department of Chemistry, College of Science, King Saud University, Riyadh 11451, Saudi Arabia.
College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Womans University,
b
Seoul 120-750, Republic of Korea.
c
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh
11451, Saudi Arabia.
#
*
These authors are equal contributors
Corresponding Authors:
Tel: +82232774653, Fax: +82232772851, Email: ykwon@ewha.ac.kr (Y. Kwon);
Tel: +966114670237, Fax: +966114676220, Email: afmrahman@ksu.edu.sa (A. F. M. M. Rahman)

ACCEPTED MANUSCRIPT
Abstract. Fluorescein hydrazones (5 and 7) were synthesized in three/four steps with 82-92%
yields. All synthesized compounds were evaluated by topoisomerase I (topo I) and topoisomerase
IIα (topo IIα)-mediated relaxation and cell viability assays. Among them, most of the compounds
showed topo I & IIα inhibitory activity and nineteen compounds showed strong anti-proliferative
activity against various cell lines. In brief, 5e inhibited 53% topo IIα (etoposide 29%) at 20 µM and
showed excellent antiproliferative activity against DU145 (1.43 ± 0.04 ꢀM), HCT15 (2.4 ± 0.03 ꢀM)
and MCF7 (11.4 ± 0.5 ꢀM) cell lines in comparison with adriamycin, etoposide, and camptothecin.
Compounds 5e, 5g and 5h were further evaluated to determine their mode of action. Compounds 5e,
5g and 5h functioned as non-intercalative topo IIα catalytic inhibitor with induction of G1 arrest
and activation of apoptotic proteins in dose-dependent manner.
Keywords: Fluorescein; Fluorescein hydrazones; topoisomerase IIα catalytic inhibitor; apoptosis.

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INTRODUCTION
Fluorescein (1) is a dark orange/red color synthetic organic molecule (Figure 1) and generally
utilized as a fluorescent tracer in microscopy, in a type of dye laser as gain medium, and in
forensics and serology to detect latent blood stains. Recently, applications of the fluorescein
derivatives are increasing rapidly, for details, please see our recently reported article [1] and
reference therein (Figure 2). Fluorescein derivatives have been widely utilized as diverse forms to
understand various physicochemical, photophysical and biological implications, for example,
fluorescein-conjugates [2-23], to purify tetracysteine-tagged proteins [24], to be precursor for the
synthesis of oligonucleotides [16], and to characterize RecA–DNA interaction [19]. Fluorescein
was also utilized for developing fluorescence-polarization assay [21], derivatizing fluorescent
reagent for aliphatic amines and food samples [25-27], and determining the hydrolytic properties of
fluorescein esters [28, 29], artificial photosynthesis and solar energy conversion [30], measurement
of mammalian phosphoinositide-specific phospholipase C. activity [31], proteolytic activities [32],
and the conjugate addition of amino acid side chains [33]. It should be noted that, recently,
halogenated fluorescein analogues was reported with potent microbial activity [34] and inhibition of
SecA ATPase [34].
Figure 1. Structures of fluorescein
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 developed (some of the fluorescein derivatives with divers application
are depicted in Figure 2). As we all know well that, cancer is life threatening and it can lead to death

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[
35] and the topoisomerases have been established as molecular targets of anticancer drugs [36, 37].
Two-type isomers of topoisomerase exist in human, 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 [38, 39]. 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 transiently and covalently formed topo-DNA complex,
known as “cleavable complex”, and prevent the cleaved DNA strand(s) from re-ligation 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
topo inhibitors besides topo poisons can be grouped as a topo catalytic inhibitor [38, 40-42].
As a part of our study, finding topo catalytic inhibitors for potential oncology therapeutics, recently,
we have reported a series of potential fluorescein hydrzone derivatives as novel non-intercalative
topo catalytic inhibitors with low DNA toxicity [1]. From the structure activity relationships based
on anti-proliferative activity and topo I & II inhibitory activity of the reported compounds, 2-nitro
group in phenyl ring attached with fluorescein by hydrazone bond displayed the highest anti-
proliferative activity against the HCT15 cells (1.59 ± 0.05 µM; etoposide showed 4.46 ± 0.93 µM)
and inhibited both topo I and II activity with better potency than camptothecin and comparable
efficacy with etoposide. It should be noted that, besides nitro group, we have reported many other
substituents which showed anti-proliferative activity and topo II inhibitory activity, but
interestingly, fluorescein hydrazide with free amino and hydroxyl groups did not shows neither anti-
proliferative activity nor topo inhibitory activity [1]. As continuing of this finding, here, we report
design and synthesis of a number of fluorescein hydrazone derivatives with either blocking one
and/or both hydroxyl groups, and evaluation of their anti-proliferative activity and topo inhibitory
activities.

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Figure 2. Structure of some reported fluorescein derivatives with diverse application
RESULTS AND DISCUSSION
Chemistry. Fluorescein (1) was reacted with ethyl iodide in the presence of potassium carbonate
(
K CO ) in DMF at room temperature for 24 hours to afford one pot hydroxyl group protection as
2 3
well as esterification and then fluorescein ethyl ester (2) was obtained in very good yield (82 %).
The resulting ester (2) was then refluxed with excess amount of hydrazine hydrate in methanol to
lead the desire fluorescein hydrazide 3 in quantitative yield (99 %) (Scheme 1).

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Scheme 1. Synthesis of fluorescein hydrazide (3)
Synthesis of the fluorescein hydrazone derivatives (7) was straightforward as shown in Scheme 2.
Scheme 2. Synthesis of fluorescein hydrazone derivatives
7
Reactions of fluorescein hydrazide (3) with substituted benzaldehydes were carried out in the
presence of catalytic amount of glacial acetic acid in ethanol under refluxing condition to afford the
corresponding fluorescein hydrazones (5) in excellent yields (95-99%) [1]. It should be noted that,
while condensing fluorescein hydrazide (3) with substituted benzaldehydes in ethanol without
acetic acid, no product and/or byproduct were obtained even in refluxing condition for 2 days, but,
addition of a catalytic amount of glacial acetic acid (1-3 drops) gave excellent yields (95–99%).
After successfully obtaining fluorescein hydrazones (5), it was then reacted with benzoyl chloride
derivative (6) in the presence of triethylamine in dichloromethane at room temperature to obtain
fluorescein hydrazone derivatives (7) and was yielded 82-92% (Scheme 2). A list of synthesized
fluorescein hydrazone derivatives 5 and 7 series are depicted in Figure 3.

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O
Cl
O
Cl
O
Cl
O
Cl
N
N
N
N
N
N
N
N
N
N
Cl
OH
Cl
O
O
O
EtO
EtO
EtO
O
OH
OH
EtO
O
O
R
EtO
O
5b
EtO
O
R
5
a
7aa-ad
7ba-bd
O
OCH3
O
OCH3
O
O
N
N
N
N
N
N
N
H CO
H CO
3
3
O
O
O
EtO
O
O
R
EtO
O
OH
OH
EtO
O
O
O
R
R
5
c
7ca-cd
5d
7da-dd
O
O
O
O
N
N
N
N
N
N
N
O
O N
O N
2
2
O
O
O
OH
EtO
O
O
R EtO
O
5f
OH EtO
O
O
R
O
R
O
5
e
7ea-ed
7fa-fd
O
O
O
O
O
R
O
N
N
N
N
N
N
N
N
OH
O
HO
O
O
EtO
O
OH
EtO
O
O
R
EtO
O
OH
Cl
EtO
O
R
5g
7ga-gd
5h
7ha-hd
7
7
H CO
3
aa, ba, ca,
ab, bb, cb,
db, eb, fb,
gb, hb, ib,
O
OCH3
O
OCH3
O
da, ea, fa, R =
R =
N
N
N
ga, ha, ia,
N
OH
O
O N
2
R
O
ac, bc, cc,
dc, ec, fc, R =
gc, hc, ic,
ad, bd, cd,
dd, ed, fd, R =
gd, hd, id,
EtO
O
OH
EtO
O
O
R
5
i
7ia-id
Figure 3. Structures of the synthesized fluorescein hydrazone derivatives (5 and 7)
Topo I and IIα inhibitory activities of the synthesized compounds (5 and 7). Most of the
synthesized compounds showed topo I and IIα inhibitory activity at 100 µM and the compounds
showing more than 30% inhibitory activity were selected to test their activity at 20 µM. The results
are listed in Table 1 and depicted in Figures 4 and 5. Among tested compounds, 7aa and 7bc
inhibited topo I at 20 ꢀM by 36.4% and 77.0%, respectively (camptothecin 35.6%); 7da, 7dc and
7dd inhibited by 30.0%, 34.4%, and 48.1%, respectively (camptothecin 36.0%); 7fa inhibited by
24.6% (camptothecin 36.7%); 5h and 7hd inhibited by 28.1% and 55.9%, respectively
(
camptothecin 39.5%); 7ib and 7ic inhibited by 13.9% and 15.8%, respectively (camptothecin
5.1%). On the other hand, only 5e (52.7%) exhibited stronger topo IIα inhibitory activity at 20 ꢀM
than etoposide (29.3%).
2

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Table 1. Topoisomerase I and IIα inhibitory activities of the synthesized compounds (5 and 7).
%
Inhibition against
Compounds
Topo I
Topo IIα
1
00 ꢀM
75.2
NT
20 ꢀM
35.6
NT
100 ꢀM
20 ꢀM
Camptothecin
Etoposide
NT
NT
80.9
81.6
83.7
6.5
29.3
1.3
5
a
22.7
NT
7
aa
ab
ac
ad
90.5
4.2
36.4
NT
NT
NT
NT
NT
10.0
77.0
2.5
1.5
7
NT
NT
NT
2.3
7
12.9
9.4
10.8
7.0
7
5
b
12.3
14.4
69.2
98.0
99.9
77.7
NT
80.0
8.5
7ba
NT
NT
2.9
7
bb
bc
bd
25.0
83.8
25.3
NT
7
7
NT
NT
29.3
3.6
Camptothecin
Etoposide
36
NT
NT
NT
17.3
3.5
70.7
65.0
5.6
5
c
0.8
7
ca
cb
cc
cd
3.3
NT
NT
NT
NT
3.3
7
35.6
54.6
44.6
28.1
55.2
74.0
79.3
81.3
85.1
NT
4.8
7
6.0
7
9.8
5.4
5
d
NT
30.0
6.8
58.3
7.8
7
da
db
dc
dd
NT
NT
NT
NT
NT
29.3
52.7
NT
7
8.1
7
34.4
48.1
36.7
NT
14.8
0
9.0
7
6.9
Camptothecin
Etoposide
NT
69.8
62.4
5.4
5
e
35.6
39.8
7ea

ACCEPTED MANUSCRIPT
7
eb
ec
ed
19.0
NT
NT
0
6.2
NT
NT
NT
3.3
NT
NT
NT
NT
NT
29.3
2.6
NT
NT
NT
NT
1.9
NT
NT
NT
NT
NT
29.3
2.7
NT
NT
NT
NT
7
16.9
55.4
36.5
30.1
0
7.5
6.1
7
7
5
f
0
73.8
13.1
11.4
13.2
7.9
fa
24.6
NT
NT
9.2
39.5
NT
NT
NT
NT
NT
NT
28.1
NT
NT
NT
55.9
25.1
NT
4.2
1.7
13.9
15.8
72
7
fb
fc
fd
7
22.7
75.3
69.9
NT
0
7
Camptothecin
Etoposide
NT
74.6
89.3
7.0
5
g
7
ga
gb
gc
gd
0
7
0
2.8
7
0
6.6
7
7
0
6.7
5
h
39.5
0
78.7
11.1
10.3
25.7
21.5
NT
ha
7
hb
hc
hd
0
7
0
7
73.2
83.6
NT
77.8
86.6
73.1
87.2
89.4
Camptothecin
Etoposide
74.4
37.9
4.8
5
i
7
ia
ib
ic
id
7
4.9
7
3.9
7
2.7
NT: non-tested

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Figure 4. Topoisomerase I inhibitory activities of compounds 5a-i, 7aa~ad - 7ia~id. (A) Lane D:
pBR322 DNA; Lane T: pBR322 DNA + Topo I; Lane C: pBR322 DNA + Topo I + Camptothecin;
Lane 5a / 7aa~7ad – 7ia-id: pBR322 DNA + Topo I + compound 5a / 7aa~7ad – 7ia-id.

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Figure 5. Topoisomerase IIα inhibitory activities of compounds 5a-i, 7aa~ad - 7ia~id. Lane D:
pBR322 DNA; Lane T: pBR322 DNA + Topo IIα; Lane E: pBR322 DNA + Topo IIα + Etoposide;
Lane 5a / 7aa~7ad – 7ia-id: pBR322 DNA + Topo IIα + compound 5a / 7aa~ad - 7ia~id.
Structure-Activity Relationships (SARs) study of fluorescein hydrazone derivatives (5 and 7)
based on topo inhibitory activities. As we have stated in the introduction part, synthesized
fluorescein hydrazones (FH-2N) containing 2-nitro group in phenyl ring attached with fluorescein
by hydrazone bond inhibited topo I activity at 100 ꢀM by 66% (camptothecin 52%) and at 20 ꢀM
by 47% (camptothecin 34%); as well as inhibited topo IIα activity at 100 ꢀM by 72% (etoposide
82%) and at 20 ꢀM by 20% (etoposide 24%) [1]. In this study, we blocked one hydroxyl group of
FH-2N by ethyl chain (5e) and interestingly it showed different result from that of FH-2N. Here,

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we found, topo I inhibitory activity decreased at both of 100 ꢀM (35.6%, camptothecin 85.1%) and
20 ꢀM (14.8%, camptothecin 36.7%) but topo IIα inhibitory activity increased dramatically at 20
ꢀ
M (52.7%, etoposide 29.3%) than the free hydroxyl compound (FH-2N). While we protected
another hydroxyl group by various aryl acid chlorides compounds 7ea-7ed we did not observe any
topo I or topo IIα inhibitory activity at 20 ꢀM but at 100 ꢀM only 7ea and 7ed showed 39.8% and
55.4% topo I inhibitory activity, respectively. Hence, we easily can understand that if we block one
free hydroxyl group of FH-2N, compound 5e increased topo IIα inhibitory activity whereas
decreased topo I inhibitory activity. On the other hand, blocking both hydroxyl groups of FH-2N,
decreased both topo I and topo IIα inhibitory activities. Instead of 2-nitro group, 4-chloro in phenyl
ring compound 5a and 2,4-dichloro compound 5b gave similar results that low topo I inhibitory
activity and high topo IIα inhibitory activity at 100 ꢀM but no or very low inhibitory activity at 20
ꢀ
M. Changing the substituents by methoxy group at 4-position of compound 5c and at 2-position of
compound 5d also showed similar results to those of 5a/5b showing high topo IIα inhibitory and
low topo I inhibitory activities. While we use hydroxyl substituents at 4-position (5f), 3-position
(5g), and at 2-position (5h) also showed similar results but very much increased topo IIα inhibitory
activity at 100 ꢀM and no or very low inhibitory activity at 20 ꢀM. Exceptionally, while we inserted
two substituents in phenyl ring at 3-position by hydroxy group and at 4-position by methoxy group,
compound 5i showed higher topo I inhibitory activity than topo IIα inhibitory activity at 100 ꢀM. It
is worthy to note that some of the compounds with protection on both hydroxyl groups, for
example, compound 5b was protected by ethyl and 2-notro benzoyl group (7bc), 5d with ethyl and
2
-notro benzoyl group (7dc), ethyl and benzoyl group (7dd), 5h with ethyl and benzoyl group
7hd), and three hydroxyl group of 5i with ethyl and benzoyl group (7hd) showed higher topo I
inhibitory activity at both concentrations of 100ꢀM and 20ꢀM than camptothecin.
(

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Cell proliferation inhibitory activities of the synthesized compounds (5 and 7). The cell
proliferation inhibitory activities of compounds 5 and 7 series are listed in Table 2 as values of IC .
5
0
A total of 19 compounds showed anti-proliferation activity out of 45 tested compounds. Among
them, compounds 5e, 5f, 5g, 5h, and 5i displayed excellent anti-proliferative; 5e against DU145
(
1.43 ± 0.04 ꢀM), HCT15 (2.4 ± 0.03 ꢀM) and MCF7 (11.4 ± 0.5 ꢀM) cell lines; 5f against T47D
0.78 ± 0.16 ꢀM) DU145 (1.06 ± 0.01 ꢀM), HCT15 (0.03 ± 0.0001 ꢀM) and MCF7 (4.66 ± 0.02
(
ꢀ
M) cell lines; 5g against T47D (0.35 ± 0.02 ꢀM) DU145 (0.04 ± 0.0001 ꢀM), HCT15 (0.83 ±
0.0001 ꢀM) and MCF7 (2.23 ± 0.02 ꢀM) cell lines; 5h against T47D (0.43 ± 0.04 ꢀM) DU145
(
0.55 ± 0.01 ꢀM), HCT15 (1.84 ± 0.02 ꢀM) and MCF7 (4.74 ± 0.03 ꢀM) cell lines; 5i against T47D
1.61± 0.16 ꢀM) DU145 (1.93 ± 0.02 ꢀM), HCT15 (0.29 ± 0.01 ꢀM) and MCF7 (3.64 ± 0.12 ꢀM)
(
cell lines. Compounds 5g and 5h showed much stronger anti-proliferative activity against T47D
cells than all tested positive controls such as adriamycin, etoposide, and camptothecin. In addition,
compound 5g inhibited topo IIα specifically, while 5h inhibited both of topo I and IIα but
compounds 5g and 5h have a subtle difference in their structures i.e. different position of hydroxy
group (Figure 3). Compound 5e showed the strongest topo IIα inhibitory activity at 20 µM among
all the tested compounds. Compound 5e has the dual inhibitory activity against topo I and IIα like
compound 5h. Both compounds 5e and 5h have the nitro group and hydroxy group in ortho position
in the phenyl ring, respectively. Therefore, compounds 5e, 5g and 5h were chosen for further study.

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Table 2. Cell proliferation inhibitory activities of synthesized compounds (5 and 7)
IC (µM)
5
0
Compounds
T47D
1.5 ± 0.063
4.85 ± 0.017
0.59 ± 0.008
5.74 ± 0.01
> 50
DU145
1.59 ± 0.06
2.19 ± 0.14
0.38 ± 0.07
2.3 ± 0.06
> 50
HCT15
0.74 ± 0.007
1.27 ± 0.007
0.35 ± 0.018
2.19 ± 0.14
28.73 ± 0.22
3.17 ± 0.01
6.25 ± 0.03
0.67 ± 0.03
> 50
MCF7
5.31 ± 0.099
35.55 ± 1.311
4.54 ± 0.038
9.77 ± 0.16
> 50
Adriamycin
Etoposide
Camptothecin
5a
7
aa
5
b
> 50
> 50
23.25 ± 0.03
> 50
7
bc
> 50
6.74±0.06
> 50
5
d
> 50
> 50
7
da
> 50
6.3 ± 0.17
> 50
> 50
7
7
7
db
21.39 ± 0.36
>50
1.92 ± 0.06
2.40 ± 0.03
47.02 ± 0.87
0.03 ± 0.0001
> 50
> 50
5e
1.43 ± 0.04
> 50
11.4 ± 0.5
> 50
ea
7.75 ± 0.21
0.78 ± 0.16
44.78 ± 1.21
0.35 ± 0.02
> 50
5
f
1.06 ± 0.01
> 50
4.66 ± 0.02
> 50
fb
5g
0.04 ± 0.0001
> 50
0.83 ± 0.0001
28.85 ± 0.65
1.84 ± 0.02
1.56 ± 0.01
> 50
2.23 ± 0.02
> 50
7
gd
5
h
0.43 ± 0.04
6.22 ± 0.03
5.45 ± 0.02
> 50
0.55 ± 0.01
> 50
4.74 ± 0.03
> 50
7
hb
hc
hd
7
> 50
> 50
7
> 50
8.29 ± 0.19
0.29 ± 0.01
> 50
>5 0
5
i
1.61 ± 0.16
23.56 ± 0.24
1.93 ± 0.02
> 50
3.64 ± 0.12
> 50
7
ib
Each data represents mean ± S.D. obtained from three different experiments. Cell lines used as
follows, T47D: human breast cancer cell, MCF-7: human breast cancer cell, DU145: human
prostate cancer cell, HCT15: human colon cancer cell. Adriamycin, etoposide, and camptothecin
were used as positive controls. Compounds labeled with > 50 inhibited the cell growth less than
50% at 50 µM.

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Compounds 5e, 5g, and 5h are non-intercalative topo IIα catalytic inhibitors. To evaluate
catalytic inhibition properties of compounds 5e, 5g, and 5h, we measured DNA damage by
observing comet tails using comet assay after treatment of 5e with HCT15 cells and 5g and 5h with
T4D7 cells. Etoposide, used as a positive control was treated with both HCT15 and T47D cells.
The comet tails occur when the stable enzyme-DNA cleavable complex is formed in cells, therefore
the formation of comet tail is an important property of topo II poisons [43]. Etoposide, a well-
known topo II poison and used as a positive control, significantly induced DNA tails at 5 ꢀM and
10 ꢀM treatments. In contrast, the extent of comet tails when tested with compounds 5e, 5g and 5h
are almost same as that of untreated cells (Figure 6), approving their mode of action as catalytic
inhibitors.
Figure 6. Comet assay: (A) Images of control (untreated), etoposide (topo II poison), and
compound 5e-treated HCT15 cells, and compounds 5g- and 5h-treated T47D cells. Compounds 5e,
5g, and 5h did not induce DNA damage in HCT15 cells and T47D cells, respectively, but
etoposide-treated HCT15 cells showed severe comet formation with treatment for 24 hours at
designated concentrations. (B) Graphical representation of the selected comet lengths of untreated-
and treated-HCT15 and treated-T47D cells in pixels with corresponding to concentration in (A).

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We further evaluated whether compounds 5e, 5g, and 5h could generate a linear truncated DNA
with the cleavable complex assay. As shown in Figure 7A, etoposide generated the linear DNA
because it stabilized the transiently formed enzyme-DNA complex leading to the blockage of
religation and induction of the linear DNA, while compounds 5e, 5g, and 5h did not generate the
linear truncated DNA even at 500 µM treatment. Co-treatment of 100 µM compounds 5e, 5g, and
5h with etoposide reduced the generation of linear DNA. This result suggests co-treatment of these
topo IIα catalytic inhibitors with topo II poisons may be beneficial to reduce the severe side effect
of gene toxicity of topo II poison and to enhance the efficacy of topo IIα inhibition. By a band
depletion assay, compounds 5e, 5g, and 5h were confirmed again as topo IIα catalytic inhibitors.
Topo II poison, etoposide induced covalently formed topo II-DNA complex, therefore free topo IIα
band was not detected by the topo IIα antibody as shown in Figure 7B. However free topo IIα
bands were detected in the compound 5e-treated HCT15 cells and 5h-treated T47D cells with
almost similar intensity to that of untreated cells. The compound 5g treatment showed also more
intense band of free topo IIα than etoposide treatment (Figure 7B).
Figure 7. (A) Cleavable complex assay of compounds 5e, 5g, and 5h: To determine whether topo II
poison or catalytic inhibitor, 250 ng of pBR322 DNA was treated with 3 units of topo IIα for 10
minutes prior to treatment of etoposide (100 ꢀM), each compound (100 ꢀM and 500 ꢀM) or co-

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treatment. After incubation for additional 30 minutes at 37 °C, the agarose gel electrophoresis were
performed. A linear band on the gel for compounds 5e, 5g, and 5h was not observed reflecting that
they are topo IIα catalytic inhibitors. (B) Band depletion assay of compounds 5e, 5g, and 5h:
Whether functioning as a topo poison or a catalytic inhibitor can be defined by looking at free topo
IIα band. The topo IIα covalently bound to DNA was depleted during DNA precipitation process
5
after treatment of topo II poison. The cells seeded in a density of 2 × 10 were treated with each of
etoposide, compounds 5e, 5g, and 5h for 2 hours at 37 °C and then were harvested. The harvested
cells were lysed by denaturing agent of 62.5 mM Tris-HCl (pH 6.8) containing 1mM EDTA and 2%
SDS. The prepared lysates were detected by Western blotting.
Compounds 5e, 5g, and 5h were examined whether to intercalate into DNA for their topo IIα
inhibitory activity with competitive ethidium bromide (EtBr) displacement assay. Since EtBr is a
well-known DNA intercalator and contains a fluorophore, EtBr displacement assay has been
utilized to identify the intercalation ability of compound [44]. The addition of m-AMSA
(amascrine), a well-known intercalative topo II poison quenched the fluorescence intensity of EtBr
intercalated into calf thymus DNA (ctDNA) through displacement of EtBr in dose-dependent
manner (Figure 8A). However, the addition of compounds 5e, 5g, and 5h, did not change the
fluorescence intensity (Figure 8B~8D). Base on the results of comet assay, cleavable complex assay,
band depletion assay, and competitive EtBr displacement assay, it can be agreeably concluded
compounds 5e, 5g, and 5h function as non-intercalative topo IIα catalytic inhibitors.

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Figure 8. Competitive EtBr displacement assay: The mixture of ctDNA (30 µM) and EtBr (20 µM)
in the 10 mM tris buffer solution (pH 7.2) was incubated for 30 minutes at room temperature
followed by addition of each compound at the designated concentrations with continuous incubation
for additional 30 minutes. The fluorescence intensity of each well was measured with excitation at
471 nm. The emission spectrum was collected at 500~700 nm.

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G1 arrest was induced in dose- and time-dependent manners after treatment of compound 5e
with HCT15 colon cancer cells and compounds 5g and 5h with T47D breast cancer cells. We
evaluated the effect of compounds 5e, 5g, and 5h on the cell cycle since these compounds inhibited
potently cancer cell growth through inhibition of topo. HCT15 colon cancer cells were selected for
cell cycle analysis for compound 5e because it was the most active in HCT15. T47D breast cancer
cells were used for compounds 5g and 5h by the same reason as compound 5e. Etoposide induced
G2/M-phase arrest in both HCT15 and T47D cancer cells in a dose-dependent manner as reported
previously [45]. Figures 9A and 9B demonstrate the cell cycle profile of HCT15 cells after exposure
to various concentrations of compound 5e (0, 1, 3, and 10 ꢀM) for 20 h and those of T47D cells
after treatment of each of compounds 5g and 5h (0, 1, 3, and 10 ꢀM) for 24 h. The G1-phase
fraction was substantially increased by compounds 5e, 5g, and 5h. The G1-phase arrest induced by
compounds 5e, 5g, and 5h was further confirmed by investigation of the changes in G1 checkpoint-
kip1
related proteins, including cyclin D1 and p27 . It is well known that the level of cyclin D1 is
kip1
decreased during G1 phases while p27
is increased when G1 arrest occurs [46]. The protein
kip1
levels of cyclin D1 was decreased and p27
was increased in dose-dependent manners after
treatment of compounds 5e, 5g, and 5h as shown in the bottom panel of Figures 9A and 9B. The
dependency on treatment time of compounds 5e, 5g, and 5h to induce the G1-phase arrest was also
evaluated with various incubation times (0, 5, 10, 15 and 20 h) at the concentration of 10 ꢀM for
compounds 5e, 5g, and 5h (Figures 9C and 9D). The G1-phase fraction was increased, the protein
kip1
level of cyclin D1 was decreased, and p27
was increased in according to increase of incubation
time, reflecting compounds 5e, 5g, and 5h induced the G1-phase arrest in time-dependent manners.

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Figure 9. The effect of compounds 5e, 5g, and 5h on the cell cycle: HCT15 cells were treated with
compound 5e for 20 h (A) and T47D cells were treated with compounds 5g and 5h for 24 h (B) in
various concentrations (0, 1, 3, and 10 µM), respectively. While etoposide was treated with HCT15
and T47D cells for 20 h and 24 h, respectively, in concentrations of 0.5 and 1 µM. HCT cells were
treated with 10 µM of compound 5e (C) and T47D cells were treated with 10 µM of compounds 5g
kip1
and 5h (D) for various times (0, 5, 10, 15, and 20 h). Cyclin D1 and p27 expression levels were
detected in compound 5e-treated HCT15 cells and compounds 5g and 5h-treated T47D cells in the
same condition of treatment as the cell cycle analysis.

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Apoptosis was induced in compound 5e-treated HCT15 and in compounds 5g and 5h-treated
T47D cells through bcl-2, bax, and cleaved PARP. The protein levels of apoptotic markers
including cleaved PARP, bcl-2, and bax were investigated by western blotting analysis after
treatment of compounds 5e, 5g, and 5h by western blot analysis. After HCT15 cells were treated
with 10 and 30 µM of compound 5e for 24 h and T47D cells with 10 and 30 µM of compounds 5g
and 5h for 24 h, the expression levels of cleaved PARP and bax were increased while that of bcl-2
was reduced (Figure 10). Based on this result, it can be concluded that compounds 5e, 5g, and 5h
induced apoptosis in HCT15 colon cancer cells and T47D breast cancer cells, respectively.
Figure 10. The effect of compounds 5e, 5g, and 5h on apoptotic proteins: After compound 5e was
treated with HCT15 cells and compounds 5g and 5h were treated with T47D cells in concentrations
of 10 and 30 ꢀM for 24 h, cells were harvested and lysed. The supernatants of each compound-
treated cell lysates after lysis were electrophoresed on 12% SDS-PAGE gels. Western blotting
analyses were then performed in according to the method described in the experimental. Cleaved
PARP, bcl-2, and bax were detected in 5e-treated HCT15 cells (A), 5g-treated T47D cells (B), and
5h-treated T47D cells (C).

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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. Forty five compounds of fluorescein
hydrazones with protection of free hydroxyl group (5 and 7 series) were synthesized with excellent
yields (82-92%). The series of compounds 5 and 7 were first evaluated by topo I- and IIα-mediated
relaxation assay and cell viability assay in several human cancer cell lines. Based on topo inhibitory
activity and anti-proliferative of synthesized fluorescein hydrazones, compounds 5e, 5g and 5h
were further evaluated to determine their mode of action with comet assay, stabilizing cleavable
complex assay, band depletion assay, and competitive EtBr displacement assay which found
compounds 5e, 5g and 5h functioned as a non-intercalative topo IIα catalytic inhibitor. Compounds
5e and 5h inhibited both topo I and IIα. Compound 5g is a non-intercalative topo IIα-specific
catalytic inhibitor. Compound 5e, 5g, and 5h induced the G1-pahse arrest through decrease of
kip1
cyclin D1 and increase of p27 in dose- and time-dependent manners. Compound 5e, 5g, and 5h
activated apoptotic proteins of cleaved PARP and bax and reduced anti-apoptotic protein of bacl-2
in the dose-dependent manner. We are reporting fluorescein derivatives as a potent catalytic
inhibitor against topo I and IIα. In our observation, compounds 5e, 5g and 5h can be a novel
scaffold for the development of antitumor agents targeting topo I and/or IIα catalytic activity with
further optimizing process and the results will be explored in due courses.

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EXPERIMENTAL
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 F , 0.25
2
54
mm) impregnated with fluorescence indicator (Merck art. 1.05554). The detection of UV-active
substances was visualized using Ultra-Violet light (λmax = 254 nm) and for non UV active
substances KMnO /PMA/p-anisaldehyde stains were used. Column chromatography was carried out
4
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 MHz, Agilent Technologies 400 MHz and Jeol 400 MHz spectrometers and are
reported as parts per million (ppm) from the internal standard of tetramethylsilane. The coupling
constants J are reported in Hertz (Hz). Peaks were described as broad singnals (br), singlet (s),
doublets (d), doublets of doublets (dd), doublets of doublets of doublets (ddd), triplets (t) and
multiplets (m), doublets of triplets (dt), 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 × 150 mm, 3.5 micron) 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 300,000 ꢀs; spectra were
taken in positive/negative mode and scan began at 50 m/z and ended at 1000 m/z; the injection

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volume was 2 ꢀL. Purity of all fluorescein hydrazones was assessed using an Eclipse Plus C18 (2.1
×
150 mm, 3.5 micron) 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 ethoxyfluorescein ethyl ester (2). Fluorescein (1, 30 g, 90 mmol) was suspended in
DMF (30 mL) in a 500 mL round bottom flask equipped with magnetic stirrer. K CO (2.2 equiv.)
2
3
was added to the reaction mixture followed by addition of ethyl iodide (2.2 equiv. g ~ 16 mL, d=
.94). The reaction mixture was stirred at room temperature until the starting materials were fully
1
consumed (approximately for 24 hours). The reaction was diluted with 500 mL water and the pale
yellow color solid precipitated was collected by filtration followed by washing with excess amount
of water (100 x 3) afforded analytical pure 2 (26 g, 74 %). R = 0.5 (Ethyl acetate: n-hexane = 3 : 7).
f
1
mp. 151-152 °C. H NMR (CDCl , 400 MHz): δ, 8.23 (d, J = 7.2 Hz, 1H), 7.71 (t, J = 6.6 Hz 1H),
3
7
6
6
.65 (t, J = 7.4 Hz 1H), 7.29 (d, J = 7.4 Hz 1H), 6.92 (d, J = 2.2 Hz, 1H), 6.87 (t, J = 9.5 Hz 2H),
.71 (dd, J = 8.8, 1.4 Hz, 1H), 6.53 (dd, J = 9.5, 1.4 Hz, 1H), 6.44 (d, J = 1.4 Hz, 1H), 4.12 (q, J =
1
3
.6 Hz, 2H), 4.01 (q, J = 6.6 Hz, 2H), 1.45 (t, J = 7.3 Hz, 3H), 0.94 (t, J = 7.2 Hz, 3H) ppm. C
NMR (CDCl , 100 MHz): δ 185.5, 165.4, 163.6, 159.0, 154.4, 134.8, 132.6, 131.8, 130.9, 130.5,
3
130.4, 129.8, 129.7, 129.1, 117.6, 114.9, 113.9, 105.8, 105.8, 100.7, 64.6, 61.4, 14.6 and 13.7 ppm.
+
ESI mass (m/z) 388.9 [M+H] (calculated 388.1).
Synthesis of ethoxyfluorescein hydrazide (3). Ethoxyfluorescein ethyl ester (2, 26 g, 66.94 mmol)
was suspended in methanol (260 mL) in a 500 mL round bottom flask equipped with magnetic
stirrer and condenser. Hydrazine hydrate (12.9 g, 258 mmol) was added to the solution and it was

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then heated to reflux for 10 hours. The solvent was then removed by reduced pressure and the
reaction mixture was diluted with water (100 mL), afforded analytical pure cream color compound
1
3
(23g, 92%). R = 0.4 (ethyl acetate: n-hexane = 3 : 7). mp. 196-197 °C. H NMR (DMSO-d , 400
f
6
MHz): δ 9.84 (s, 1H, -OH), 7.77 (dd, J = 4.4, 1.2 Hz, 1H), 7.48 (pentet, J = 4.0 Hz 2H), 6.97 (dd, J
3.5, 1.2 Hz 1H), 6.77 (d, J = 2.4 Hz 1H), 6.60 (d, J = 2.4 Hz, 1H), 6.57 (dd, J = 8.8, 2.4 Hz 1H),
.48 (d, J = 9.2 Hz, 1H), 6.46 (dd, J = 6.8, 2.0 Hz, 1H), 6.40 (d, J = 8.8 Hz, 1H), 4.39 (br, 2H, -
=
6
13
NH ), 4.02 (q, J = 6.8 Hz, 2H), 1.30 (t, J = 6.8 Hz, 3H) ppm. C NMR (DMSO-d , 100 MHz): δ
2
6
1
1
66.0, 159.6, 158.7, 152.9, 152.8, 151.8, 133.1, 129.8, 129.0, 128.5, 128.4, 123.9, 122.6, 111.9,
+
+
11.8, 110.3, 102.8, 101.7, 65.1, 63.9 and 15.0 ppm. ESI mass (m/z) 374.3 [M] , 375.2 [M+H] ,
(calculated 374.1).
General procedure for the synthesis of fluorescein hydrazone (5) [1]. A mixture of
ethoxyfluorescein hydrazide (3, 3 g, 8.01 mmol) and aryl aldehyde (4, 1 equiv.) in ethanol (30 mL)
was added a drop of glacial acetic acid and the reaction mixture was refluxed in oil bath for 6 h. The
solvent was then removed by reduced pressure and the reaction mixture was diluted with water (20
mL). The mixture was extracted with chloroform (20 x 3 mL). The combined organics layer were
washed with brine (40 mL) and concentrate under reduced pressure to obtain crude product 5,
which was then subjected to column chromatography using silica gel with 40% ethyl acetate in n-
hexane, afforded analytical pure 5.
(E)-2-((4-chlorobenzylidene)amino)-3'-ethoxy-6'-hydroxyspiro[isoindoline-1,9'-xanthen]-3-one
-1
(
5a). Pink powder (86 %); mp. 180-181 °C. IR (KBr, cm ): 3410, 2976, 2345, 1676, 1655, 1500,
-
1 1
1
7
2
8
559, 1421, 1313, 1263, 1180, 1108, 1042, 819 cm ; H NMR (CDCl , 400 MHz): δ 8.65 (s, 1H),
3
.97 (d, J = 7 Hz, 1H), 7.54-7.46 (m, 3H), 7.32 (dd, J = 8.8, 2.2 Hz, 2H), 7.13 (dd, J = 8.8, 1.5 Hz,
H), 7.07 (d, J = 8.0, 1.5 Hz, 1H), 6.78 (d, J = 2.2 Hz, 1H), 6.71 (d, J = 2.2 Hz, 1H), 6.60 (dd, J =
1
3
.6, 1.5 Hz, 1H), 6.45-6.54 (m, 3H) ppm. C NMR (CDCl , 100 MHz): δ 165.2, 160.1, 157.8,
3

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152.6, 147.3, 137.6, 134.0, 133.8, 133.2, 129.0, 128.6, 128.5, 128.0, 124.9, 124.8, 124.7, 66.0, 63.8
3
5
+
and 14.7 ppm. R (ethyl acetate / n-hexane 2:3, v/v): 0.55; ESI mass (m/z) 497.2 [M( Cl)+H] ,
f
37
+
35
+
37
+
35
+
4
99.2 [M( Cl)+H] , 519.1 [M( Cl)+Na] , 520.9 [M( Cl)+Na] , 535.1 [M( Cl)+K] , 537.1
37
+
[
M( Cl)+K] (calculated 496.1).
(E)-2-((2,4-dichlorobenzylidene)amino)-3'-ethoxy-6'-hydroxyspiro[isoindoline-1,9'-xanthen]-
-1
3
-one (5b). Brown powder (85 %); mp. 164-165 °C. IR (KBr, cm ): 3414, 2978, 1700, 1662, 1617,
-
1
1
1
8
6
3
1
1
500, 1465, 1319, 1271, 1176, 1111, 1030, 845 cm ; H NMR (CDCl , 400 MHz): δ, 8.35 (s, 1H),
3
.08 (s, 1H), 8.00 (d, J = 7.4 Hz, 1H), 7.74 (d, J = 8.8 Hz, 1H), 7.45 (pentet, J = 7.3 Hz, 2H), 7.04-
.99 (m, 3H), 6.78 (s, 1H), 6.70 (d, J = 1.5 Hz, 1H), 6.56 (d, J = 8.6 Hz, 1H), 6.50-6.42 (overleaped,
13
H), 3.98 (q, J = 6.6 Hz, 2H) and 1.36 (t, J = 6.6 Hz, 3H) ppm. C NMR (CDCl , 100 MHz): δ
3
65.8, 160.3, 158.2, 152.4, 152.3, 151.7, 141.8, 136.0, 135.0, 134.4, 130.2, 129.0, 128.1, 128.0,
27.9, 127.7, 127.2, 123.8, 112.6, 112.1, 109.9, 109.5, 103.4, 101.7, 65.7, 63.8 and 14.6 ppm. Rf
3
5
+
37
+
(
ethyl acetate / n-hexane 2:3, v/v): 0.50; ESI mass (m/z) 531.2 [M( Cl)+H] , 533.2 [M( Cl)+H] ,
3
5
+
37
+
35
+
37
+
5
5
53.2 [M( Cl)+Na] , 555.2 [M( Cl)+Na] , 569.2 [M( Cl)+K] , 571.1 [M( Cl)+K] (calculated
30.1).
(E)-3'-ethoxy-6'-hydroxy-2-((4-methoxybenzylidene)amino)spiro[isoindoline-1,9'-xanthen]-3-
-1
one (5c). Pink powder (86 %); mp. 152-153 °C. IR (KBr, cm ): 3411, 2970, 2345, 1700, 1686,
-
1
1
1
8
7
6
654, 1609, 1500, 1423, 1308, 1253, 1180, 1104, 1029, 830 cm ; H NMR (CDCl , 400 MHz): δ,
3
.62 (s, 1H), 7.95 (dd, J = 7.0, 1.5 Hz, 1H), 7.46 (pentet, J = 7.3 Hz, 2H), 7.34 (d, J = 8.8 Hz, 2H),
.03 (dd, J = 8.2, 2.2 Hz, 1H), 6.80 (s, 1H), 6.70-6.65 (overleaped, 3H), 6.59 (d, J = 8.8 Hz, 1H),
.52-6.46 (overleaped, 3H), 3.97 (q, J = 7.2 Hz, 2H), 3.70 (s, 3H) and 1.36 (t, J = 6.6 Hz, 3H) ppm.
1
3
C NMR (CDCl , 100 MHz): δ 165.0, 161.2, 160.0, 157.8, 152.6, 149.4, 133.5, 129.1, 128.8,
3
128.1, 123.6, 123.5, 114.4, 113.8, 112.4, 111.6, 111.4, 103.4, 101.7, 66.0, 63.7, 55.3 and 14.7 ppm.
+
+
R (ethyl acetate / n-hexane 2:3, v/v): 0.45; ESI mass (m/z) 493.3 [M+H] , 515.3 [M+Na] , 531.3
f
+
[
M+K] , (calculated 497.2).

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(E)-3'-ethoxy-6'-hydroxy-2-((2-methoxybenzylidene)amino)spiro[isoindoline-1,9'-xanthen]-3-
-1
one (5d). Yellow powder (89 %); mp. 268-269 °C. IR (KBr, cm ): 3412, 2928, 1700, 1685, 1636,
-1
1
1
560, 1500, 1421, 1313, 1263, 1180, 1108, 1042, 819 cm ; H NMR (CDCl , 400 MHz): 9.10 (s,
3
1H), 7.98 (dd, J = 7.0, 1.5 Hz, 1H), 7.65 (dd, J = 7.3, 1.5 Hz, 1H), 7.49 (m, 2H), 7.20 (m, 1H), 7.06
(dd, J = 8.2, 2.2 Hz, 1H), 6.84-6.70 (m, 4H), 6.63-6.42 (overleaped, 4H), 3.99 (q, J = 7.2 Hz, 2H),
1
3
3
1
1
1
.72 (s, 3H) and 1.36 (t, J = 6.6 Hz, 3H) ppm. C NMR (DMSO-d , 100 MHz): δ 164.0, 159.9,
6
59.1, 158.2, 152.7, 152.6, 150.9, 145.0, 144.9, 134.5, 134.4, 132.4, 132.3, 129.5, 128.5, 128.4,
25.1, 125.0, 124.3, 124.2, 123.7, 123.6, 122.9, 121.1, 112.9, 112.8, 112.4, 112.3, 112.2, 112.2,
12.1, 111.8, 110.3, 102.8, 102.7, 101.8, 65.5, 63.9, 56.2, 56.1, 15.0 and 14.9 ppm. R (ethyl
f
+
+
+
acetate / n-hexane 2:3, v/v): 0.50; ESI mass (m/z) 493.3 [M+H] , 515.3 [M+Na] , 531.3 [M+K] ,
(calculated 492.2).
(E)-3'-ethoxy-6'-hydroxy-2-((2-nitrobenzylidene)amino)spiro[isoindoline-1,9'-xanthen]-3-one
-1
(
5e). Yellow needle (88 %); mp. 244-245 °C. IR (KBr, cm ): 3412, 2972, 2345, 1675, 1656, 1500,
-
1 1
1
9
1
457, 1424, 1311, 1258, 1183, 1105, 1040, 816 cm ; H NMR (DMSO-d , 400 MHz): 9.94 (s, 1H),
6
.28 (s, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.69-7.50 (overlapped, 5H) 7.11 (d, J = 7.2 Hz, 1H), 6.85 (s,
H), 6.66 (d, J = 2.0 Hz, 1H), 6.59 (ab quartet, 2H), 6.53 (d, J = 8.1 Hz, 1H), 6.46 (dd, J = 8.1, 1.6
1
3
Hz, 1H), 4.01 (q, J = 7.2 Hz, 2H), and 1.28 (t, J = 6.8 Hz, 3H) ppm. C NMR (DMSO-d , 100
6
MHz): δ 164.6, 160.1, 159.2, 152.6, 152.4, 151.1, 148.5, 143.0, 135.0, 134.1, 131.3, 129.7, 129.1,
128.6, 128.4, 128.3, 127.4, 125.1, 124.3, 123.9, 113.1, 112.3, 111.4, 109.9, 103.0, 102.0, 65.7, 63.9
and 14.9 ppm. R (ethyl acetate / n-hexane 2:3, v/v): 0.50; R (ethyl acetate / n-hexane 2:3, v/v):
f
f
+
+
+
0
.45; ESI mass (m/z) 507.9 [M+H] , 529.8 [M+Na] , 546.8 [M+K] (calculated 507.1).
(E)-3'-ethoxy-6'-hydroxy-2-((4-hydroxybenzylidene)amino)spiro[isoindoline-1,9'-xanthen]-3-
-1
one (5f). Light pink powder (88 %); mp. 230-231 °C. IR (KBr, cm ): 3412, 2978, 2344, 1676,
-1
1
1
656, 1609, 1560, 1500, 1465, 1424, 1322, 1266, 1184, 1105, 1041, 831 cm ; H NMR (DMSO-
d6, 400 MHz): 9.92 (s, 1H), 8.97 (s, 1H), 7.88 (dd, J = 6.4, 2.0 Hz, 1H), 7.60-7.54 (pentet of