Anticancer activity and SAR studies of substituted 1,4-naphthoquinones

Article abstract of DOI:10.1016/j.bmc.2013.05.017

In this paper, we report the structure-activity relationship studies of substituted 1,4-naphthoquinones for its anticancer properties. 1,4-Naphthoquinone, Juglone, Menadione, Plumbagin and LLL12.1 were used as lead molecules to design PD compounds. Most o

Full text of DOI:10.1016/j.bmc.2013.05.017

Accepted Manuscript
Anticancer activity and SAR studies of Substituted 1, 4-Naphthoquinones
Deepak Bhasin, Somsundaram N. Chettiar, Jonathan P. Etter, May Mok, Pui-
Kai Li
PII:
S0968-0896(13)00442-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.05.017
BMC 10843
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
21 March 2013
30 April 2013
10 May 2013
Please cite this article as: Bhasin, D., Chettiar, S.N., Etter, J.P., Mok, M., Li, P-K., Anticancer activity and SAR
studies of Substituted 1, 4-Naphthoquinones, Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/
10.1016/j.bmc.2013.05.017
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Anticancer activity and SAR studies of Substituted 1, 4-Naphthoquinones
Deepak Bhasin,* Somsundaram N. Chettiar,* Jonathan P. Etter, May Mok, Pui-Kai Li#
* Equal contribution to the research work
Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University,
Columbus, Ohio 43210
# Correspondence to:
Pui-Kai Li, Ph.D.,
College of Pharmacy, The Ohio State University,
Rm 338 Parks Hall, 500 West 12th Avenue,
Columbus, OH 43210-1291
Tel: (614) 688-0153
Fax: (614) 292-2435
E-mail: li.27@osu.edu

Abstract
In this paper, we report the structure activity relationship studies of substituted 1,4-
naphthoquinones for its anticancer properties. 1,4-naphthoquinone, Juglone, Menadione, Plumbagin and
LLL12.1 were used as lead molecules to design PD compounds. Most of the PD compounds showed
improved antiproliferative activity in comparison to the lead molecule in prostate (DU-145), breast
(MDA-MB-231) and colon (HT-29) cancer cell lines. PD9, PD10, PD11, PD13, PD14 and PD15 were
found to be the most potent compound with an IC50 value of 1-3 µM in all cancer cell lines. Fluorescent
polarization assay was employed to study the inhibition of STAT3 dimerization by PD compounds. PD9
and PD18 were found to be potent STAT3 dimerization inhibitors.

1. Introduction
1,4-Naphthoquinones are one of the most important and widely distributed chemical class in the
quinone family. Chemically, naphthoquinones can generate reactive oxygen species such as superoxides
and hydroxyl radicals. In addition, the quinone moiety can also serve as an electrophile and can react with
different biological targets in various species including human. 1,4-naphthoquinones are reported to have
a variety of pharmacological properties, including antibacterial,1 antifungal,2,3 antiviral,4-6 anti-
inflammatory,7,8 antiartherosclerotic,9 and anticancer effects.10-12
Recently, 1,4-napthaquinones such as Menadione, Juglone and Plumbagin have gained importance
for their anticancer activity. Juglone, from its natural source, Juglans mandshuria, has been used for the
treatment of cancer in traditional Chinese medicine.13 Plumbagin, derived from the plant genus Plumbago,
has been reported to have antibacterial, antifungal as well as anticancer activities. It has been shown to
exhibit cytotoxic properties in various cancer cell lines as well as in vivo studies in animal models.14-17
Menadione (Vitamin K3) and its derivatives have been reported to have anticancer effect in hepatic,
breast, blood, bladder and oral cancer cell lines.18
Figure 1. Structure of substituted 1,4-napthaquinones
The goal of this study is to design more potent analogs with 1,4-naphthoquinone scaffold and study
the structure activity relationship (SAR) for anticancer activity. 1,4-naphthoquinone, Juglone, Menadione,
Plumbagin and LLL12.1 (Figure 1) were used as lead molecules.19 A total of 18 analogs were designed
and synthesized, and their antiproliferative activities in prostate (DU-145), breast (MDA-MB-231) and
colon (HT-29) cancer cell lines were determined (Table 1). The active analogs were further tested for
their ability to inhibit STAT3 dimerization using fluorescent polarization (FP) assay.

2. Results and Discussion
2.1 Chemistry
PD1 and PD2 were designed based on 1,4-naphthoquinone, while Juglone was used to design
compounds PD3-5, PD9 and PD10. PD6-8 were designed based on Menadione; PD16-18 were based on
Plumbagin and PD11-15 were designed using LLL12.1 as the lead compound. PD1-4, PD6, PD8 and
PD18 are known molecules reported in literature,20-26 while the other PD compounds are novel molecules.
PD1-5 synthesis was accomplished by reacting either naphthoquinone or Juglone with aniline or
anisidines in ethanol at room temperature (Scheme 1) according to reported procedure,27 but in absence of
CeCl3. PD13-15 was synthesized from LLL12.1 using similar reaction conditions.
Scheme 1. Synthesis of PD1-5, PD11, PD13-15
Synthesis of PD6, PD7, PD16 and PD17 were carried out by reacting appropriate naphthoquinone
with appropriate aniline in acetic acid using 10% copper acetate as catalyst (Scheme 2). It has been
shown that adding copper acetate in acetic acid readily promotes oxidative coupling of anilines to
naphthoquinones.28 Since plumbagin and menadione didn’t give sufficient yield with the earlier reaction
protocol in ethanol, we decided to utilize copper chemistry for the synthesis of the compounds. PD8-11,
PD12 and PD18 were synthesized by reacting the appropriate naphthoquinone with 2-thioethanol in a
solvent mixture of methanol and 2-propanol (Scheme 3).

Scheme 2. Synthesis of PD6, PD7, PD16, PD17
Scheme 3. Synthesis of PD8-12 and PD18
Juglone has been shown to predominantly yield one isomer on reaction with thioglycolic acid.29 A
few examples also exist, where Juglone have been shown to undergo Michael addition at position 2, such
as the addition of dimethylamine to Juglone and the reaction of various 2,3-dihalogen juglones with
aniline and p-toluidine.22,30,31 The structural assignment of the product obtained from the addition reaction
of 5-substituted naphthoquinones was achieved using two-dimensional NMR spectroscopy (2D NMR).
The reaction of various anilines with Juglone predominantly yielded the regioisomer with the
addition product at position 3. The other isomer (2-position) was obtained in trace amounts in this case.
The structural assignment of PD5 is used to illustrate this point and can be extrapolated as a
representation of other Juglone analogs. In the structural assignment of PD5, HMBC correlation is
observed between C-1 (δc 182.77 ppm) and H-8 (δH 7.45 ppm, doublet) which differentiates between C-1
(δc 182.77 ppm) and C-4 (δc 186.35 ppm). H-8 is also confirmed by COSY, which also confirms H-6 and
H-7 by showing correlation to each other. HMBC correlation observed between C-4 and the NH proton
(δH 9.2 ppm) further confirms the structure of PD5. HMBC correlation is also observed between the

hydrogen bonded carbonyl C-4 and H-2 (δH 6.0 ppm), which further confirms this regiochemistry. H-2
also shows HMBC correlation with C-8, C-8a and C-9, respectively (Figure 2).
Atoms
H-2
H-6
Atoms
C-1
δH (ppm)
6.08 (s, 1H)
7.25 (d, 1H)
7.73 (t, 1H)
7.45 (d, 1H)
δC (ppm)
182.77
186.35
C-4
H-7
H-8
Figure 2. 1H and 13C NMR assignments of PD5
Reaction of Juglone with thioethanol resulted in both isomers, PD9 & PD10, in almost equal
quantity indicating no regioselectivity. HMBC correlation is observed between C-1 (δc 182.00 ppm) and
H-8 (δH 7.56 ppm, doublet) which differentiates between C-1 and C-4 (δc 187.42 ppm). HMBC
correlation is also observed between C-1 and H-3 proton (δH 6.82 ppm) (Figure 3). In case of PD10,
which is the other regioisomer of PD9, the observed HMBC correlation between C-4 and H-8; and other
evidences confirms its structure.
Position
H-3
Position
C-1
δH (ppm)
6.82 (s, 1H)
δC (ppm)
182.00
H-6
7.36 (d, 1H)
C-4
187.42
H-7
7.76-7.68 (m, 1H)
7.56 (d, 1H)
H-8
Figure 3. 1H and 13C NMR assignments of PD9
In case of PD11, HMBC correlation is observed between C-1 (δc 181.75 ppm) and H-8 (δH 8.29
ppm, doublet). HMBC correlation is also seen between C-1 and H-3 (δH 6.90 ppm), which confirms the

structure of PD13. HMBC correlation of H-7 with C-8a (δC 143.5 ppm); and H-3 with C-4a (δC 129.96
ppm) further confirms this regiochemistry of PD11 (Figure 4).
Position
H-3
H-6
Position
C-1
δH (ppm)
6.90 (s, 1H)
8.44 (d, 1H)
8.05 (t, 1H)
8.29 (d, 1H)
δC (ppm)
181.75
181.45
C-4
H-7
C-4a
C-8a
129.96
H-8
143.49
Figure 4. 1H and 13C NMR assignments of PD11
In case of PD13, HMBC correlation is observed between C-1 (δC 181.86 ppm) and H-8 (δH 8.27 ppm,
doublet). H-8 is also confirmed by COSY, which also confirms H-6 (δH 8.38 ppm) and H-7 (δH 8.04 ppm)
by showing correlation with each other. HMBC correlation is also observed between C-4 (181.57 ppm)
and H-2 (δH 6.17 ppm). HMBC correlation observed between C-4 and the NH proton (δH 9.49 ppm)
further confirms the regiochemistry (Figure 5). COSY shows H-11 (δH 7.36 ppm, triplet) is coupled with
SO2NH2 and correlates with H-10 (δH 7.0 ppm, doublet) and H-12 (δH 6.8 ppm, doublet). As a result, H-14
is merged with H-10 in 1H NMR and appears at 6.96 ppm.
Position
Position
δH (ppm)
δC (ppm)
H-2
6.17 (s, 1H)
C-1
181.86
H-6
H-7
H-8
8.38 (d, 1H)
8.04 (t, 1H)
8.27 (d, 1H)
C-4
181.57
Figure 5. 1H and 13C NMR assignments of PD13

2.2 Biological activity
2.2.1 Antiproliferative activity
PD compounds were tested for their antiproliferative activity in prostate, breast and colon cancer
cell lines (Table 1). The 1,4-napthaquinone with R1 substitution as electron donating hydroxyl group
(Plumbagin or Juglone) had similar antiproliferative activity when compared to the unsubstituted 1,4-
napthaquinone, while R1 substitution with electron withdrawing sulfonamide group (LLL12.1) led to a
decrease in activity in all cell lines. The R3 substitution of unsubstituted 1,4-napthaquinone with aromatic
amine (PD1 & PD2) showed no major change in activity, except in breast cancer cells where decreased
activity was observed. The R3 substitution of Juglone with aromatic amine (PD3-5) increased its
antiproliferative activity in both prostate and breast cancer cells, whereas 2-thioethanol substitution at R2
or R3 position of Juglone showed an increase in activity across all cell lines. The R3 substitution of
Menadione (PD6-8) with aromatic amines or 2-thioethanol led to a decrease in activity with the exception
of 2-thioethanol derivative (PD8), increased activity was observed in breast cancer cells. Similarly, R3
substitution of Plumbagin (PD16-17) with various anilines led to a decrease in activity in prostate and
colon cancer cells. 2-thioethanol substituted Plumbagin derivative (PD18) showed increased activity in
breast and colon cancer cells. R3 substitution of LLL12.1 with aromatic amine (PD13-15) led to a 6-12
fold increase in antiproliferative activity in breast cancer cell lines; and 12-20 folds in colon cancer cells
in comparison to LLL12.1. The substitution of LLL12.1 with 2-thioethanol (PD11) at R2 position led to a
moderate increase in activity, while the di-substituted LLL12.1 analog (PD12) showed a decrease in
activity. These results indicate the general trend that substitution at either R2 or R3 of the 1,4-
napthoquinone ring increases antiproliferative activity, while di-substitution at both R2 and R3 decreases
activity.
LLL12.1 is the precursor of the potent STAT3 inhibitor, LLL12, which binds to the SH2 domain
of STAT3 protein and prevent its dimerization.19 Thus, the selected PD compounds were further
evaluated for their abilities to inhibit STAT3 dimerization using fluorescent polarization (FP) assay.

R1
R2
R3
DU-145
MDA-MB-231
HT-29
Compound
(IC50 µM)
(IC50 µM)
(IC50 µM)
Plumbagin
Juglone
1,4-Naphthoquinone
Menadione
LLL12.1
PD-1
OH
OH
CH3
H
5.23 ± 0.24
11.67 ± 0.05
10.89 ± 2.29
9.86 ± 0.22
14.39 ± 0.61
5.52 ± 1.25
6.02 ± 1.37
3.13 ± 0.7
21.24 ± 0.51
7.15 ± 1.43
4.48 ± 0.11
12.68 ± 0.50
16.13 ± 0.17
> 100
4.19 ± 0.06
7.71 ± 0.88
12.76 ± 1.12
9.69 ± 0.42
24.62 ± 1.40
31.25 ± 3.95
12.64 ± 0.14
10.02 ± 0.99
6.88 ± 0.30
5.51 ± 0.36
19.0 ± 6.65
> 100
H
H
H
H
H
H
CH3
H
SO2NH2
H
H
H
H
-NHPh
PD-2
H
H
-NHPh(4-OMe)
-NHPh
> 100
PD-3
OH
H
7.41 ± 0.06
2.67 ± 0.14
3.31 ± 0.03
>100
PD-4
OH
H
-NHPh(4-OMe)
-NHPh(3-OMe)
-NHPh(4-OMe)
-NHPh(3-OMe)
-SCH2CH2OH
H
2.85 ± 0.43
2.27 ± 0.38
8.12 ± 0.77
15.16 ± 1.78
22.3 ± 1.55
3.62 ± 0.05
3.37 ± 0.29
2.85 ± 0.17
19.5 ± 4.75
1.47 ± 0.08
2.44 ± 0.23
1.22 ± 0.01
14.22 ± 2.28
10.28 ± 1.64
8.16 ± 2.35
PD-5
OH
H
PD-6
H
CH3
PD-7
H
CH3
> 100
PD-8
H
CH3
3.27 ± 1.09
2.66 ± 0.29
1.69 ± 0.05
9.68 ± 1.50
22.9 ± 0.70
2.22 ± 0.03
3.39 ± 0.64
1.73 ± 0.26
21.40 ± 1.64
20.51 ± 0.86
5.09 ± 1.21
17.5 ± 2.85
2.17 ± 0.59
2.11 ± 0.78
9.68 ± 1.73
26.6 ± 2.50
1.89 ± 0.14
1.97 ± 0.05
1.05 ± 0.08
15.65 ± 11.3
> 100
PD-9
OH
-SCH2CH2OH
PD-10
OH
H
-SCH2CH2OH
H
PD-11
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
OH
-SCH2CH2OH
PD-12
-SCH2CH2OH
-SCH2CH2OH
-NHPh(3-OMe)
-NHPh
PD-13
H
H
PD-14
PD-15
H
-NHPh(4-OMe)
-NHPh(4-OMe)
-NHPh(3-OMe)
-SCH2CH2OH
PD-16
CH3
CH3
CH3
PD-17
OH
PD-18
OH
2.16 ± 0.50
Table 1. Antiproliferative activity of PD compounds
2.2.2 Validation of the fluorescence polarization assay
In order to obtain a full saturation curve, increasing concentrations of STAT3 (0-1200 nM) were
incubated in the presence of 10 nM 5-FAM-SpYLPQTV. The Kd value was determined to be 136 nM,
which is similar to the value of 150 nM obtained by Schust and Berg using the peptide 5-FAM-
GpYLPQTV-NH2.32 The slight increase in binding that was observed is expected due to the presence of
10% DMSO in the buffer, which has been shown to slightly decrease the Kd value. The unlabeled peptide
SpYLPQTV showed greater than 90% inhibition of the FP signal at concentrations as low as 2 µM, with a

calculated IC50 of 329 nM, which is similar to the value of 300 nM for the unlabeled peptide
GpYLPQTV-NH2 observed by Zhang and colleagues (Figure 6).33 At a concentration of 100 µM the
unlabeled, non-phosphorylated peptide SYLPQTV showed only 9 ± 4% inhibition. The small amount of
inhibition observed is presumably due to non-specific binding. These data demonstrates that only the
unlabeled, phosphorylated peptide inhibits the binding of the fluorescent probe to the STAT3 SH2
domain, and that the assay is functioning properly.
a)
b)
Figure 6. a) Saturation curve: Changes in specific binding that were observed when 10 nM 5-FAM-SpYLPQTV
was incubated with increasing concentrations of STAT3. Specific binding is equal to the contribution to the signal of
bound ligand. b) Percent inhibition of the fluorescence polarization signal when various concentrations of unlabeled
peptide (SpYLPQTV) were incubated with 150 nM STAT3 and 10 nM 5-FAM-SpYLPQTV
2.2.3 Inhibition of the fluorescence polarization signal in the presence of compounds
PD4 and PD5 were almost entirely insoluble in the fluorescent peptide buffer at a concentration
of 500 µM (which explains their lack of activity) while PD7, PD13 - PD17 were partially insoluble. Other
compounds had no solubility issues at the pre-mentioned concentration. PD9 and PD13 had slight issues
with internal fluorescence at the excitation/emission settings that were used.
PD compounds were first evaluated at 500 µM. At this concentration four of the compounds
(PD9, PD10, PD11 and PD18) inhibited the signal by greater than 50 percent and had comparable activity
as that of Plumbagin and Juglone (Figure 7). PD4, PD5, PD13, PD14 and PD15 had potent activity in the
cellular assays, but in contrast showed lower inhibitory activities in the FP assay. Their lack of solubility
in the buffer could explain the reduced potency in the assay. PD9 and PD18 were selected for further
evaluation due to their potent activity in the cellular assays and their suitability for the FP assay. The Ki

values of PD9 and PD18 were derived to be 13.3 ± 0.62 µM and 18.2 ± 4.78 µM, respectively, which is
similar to the activities of other potent compounds that have been reported through the use of this
assay.33-35 This data demonstrates the ability of these compounds to bind to the STAT3 SH2 domain and
inhibit dimerization.
Figure 7. Percent inhibition of the fluorescence polarization signal in the presence of the indicated
compounds (500 µM), SpYLPQTV (100 µM), or SYLPQTV (100 µM)
3. Conclusion
In summary, we have synthesized a series of 1,4-naphthoquinone analogues with improved
antiprofilerative activity. PD9, PD10, PD11, PD13, PD14 and PD15 were found to be the most potent
compound with an IC50 value of 1-3 µM in all cancer cell lines. The FP assay demonstrates STAT3
binding and inhibiton of STAT3 dimerization as a probable mechanism of action for their anticancer
activity. PD9 and PD18 were found to be potent STAT3 dimerization inhibitors. Several other substituted
anilines can be explored further in quest for more potent compounds.
4. Experimental
1
13
General Aspects: H, CNMR, COSY, HMBC, HSQC were obtained on Bruker 300 MHz FT-NMR.
Chemical shifts (δ) are reported in ppm from an internal TMS standard. Coupling constants are reported in
Hz. High resolution-Electron Spray Ionization (HR-ESI) mass spectra were obtained on a Micromass LCT
spectrometer. Melting points are uncorrected and were obtained on Hoover capillary melting point.

Chemicals and solvents were obtained from Sigma Aldrich. Unless specified otherwise, all reactions were
carried out under nitrogen atmosphere. Silica gel 60 (0.063-0.200mm) from Dynamic Adsorbents was
used for gravity column chromatography. Reagent grade solvents were used for column chromatography and were
obtained from VWR. Precoated aluminium backed TLC plates with silica gel 60 F254 from Dynamic
Adsorbents were used for TLC. General compound visualization for TLC was achieved by UV light and
I2 (adsorbed on silica gel in a container). Yields are unoptimized. Plumbagin, Juglone, Naphthoquinone
and Menadione were obtained commercially from Sigma Aldrich. LLL12.1 was synthesized using the
reported method in literature.19
4.1 General synthetic procedure for PD1-PD5, PD13-PD15: The appropriate naphthoquinone (1.58
mmol) was dissolved in 8 ml of EtOH followed by slow addition of aniline (1.18 mmol) at room
temperature. The reaction was stirred overnight. The reaction mixture was diluted with water and
extracted with ethyl acetate. The organic layer was collected, dried over sodium sulphate, concentrated
under reduced pressure and purified using silica gel column chromatography in 30-45% EtOAc/Hexane to
afford the pure compounds.
O
4.1.1. 2-(phenylamino)naphthalene-1,4-dione (PD1): Dark red solid, yield 32%; mp 189-190 C (Lit
O
190-191 C);20 1H NMR (400 MHz, CDCl3) δ 8.15 (t, J=7.9 Hz, 2H), 7.84-7.66 (m, 2H), 7.59-7.54 (m,
1H), 7.45 (t, J=8.4 Hz, 2H), 7.29-7.25 (m, 5H), 6.45 (s, 1H). MS (HR-ESI) M+Na calculated 272.0687;
Observed 272.0650.
4.1.2. 2-((4-methoxyphenyl)amino)naphthalene-1,4-dione (PD2): Dark red solid, yield 34%; mp 156-
158 OC (Lit 154-158 OC);21 1H NMR (400 MHz, CDCl3) δ 8.15-8.09 (m, 1H), 7.77 (t, J=7.6 Hz, 1H), 7.68
(t, J=8.2 Hz, 1H), 7.46 (s, 1H), 7.21 (d, J=8.9 Hz, 2H), 6.97 (d, J=8.9 Hz, 2H), 6.25 (s, 1H), 3.86 (s, 3H).
MS (HR-ESI) M+Na calculated 302.0792; Observed 302.0740.
4.1.3. 8-hydroxy-2-(phenylamino)naphthalene-1,4-dione (PD3): Dark red solid, yield 30%; mp 218-
O
O
220 C (Lit 228 C);22 1H NMR (400 MHz, CDCl3) δ 11.58 (s, 1H), 7.65-7.45 (m, 6H), 7.30-7.21 (m,
4H), 6.40 (s, 1H). MS (HR-ESI) M+Na calculated 288.0637; Observed 288.0570.
4.1.4. 8-hydroxy-2-((4-methoxyphenyl)amino)naphthalene-1,4-dione (PD4): Dark red solid, yield
O
1
28%; mp 206-208 C; H NMR (300 MHz, DMSO-d6) δ 11.55 (s, 1H), 9.21 (s, 1H), 7.73 (t, J=7.8 Hz,
1H), 7.45 (d, J=7.3 Hz, 1H), 7.32-7.23 (m, 3H), 7.02 (d, J=8.6 Hz, 2H), 5.85 (s, 1H), 3.78 (s, 3H). MS
(HR-ESI) M+Na calculated 318.0742; Observed 318.0694.

4.1.5. 8-hydroxy-2-((3-methoxyphenyl)amino)naphthalene-1,4-dione (PD5): Dark red solid, yield
27%; mp 174-176 OC; NMR (400 MHz, DMSO-d6) δ 11.53 (s, 1H), 9.21 (s, 1H), 7.73 (t, J=8.2 Hz, 1H),
7.45 (d, J=7.6 Hz, 1H), 7.35 (t, J=7.7 Hz, 1H), 7.25 (d, J=7.6 Hz, 1H), 6.97-6.95 (m, 2H), 6.83-6.80 (m,
13
1H), 6.08 (s, 1H), 3.77 (s, 3H). C NMR (400 MHz) δ 186.35, 182.77, 161.31, 160.75, 146.87, 140.00,
138.40, 133.72, 130.95, 123.13, 118.46, 116.53, 115.11, 111.71, 110.44, 103.55, 56.07. MS (HR-ESI)
M+Na calculated 318.0742; Observed 318.0703.
4.1.6. 7-((3-methoxyphenyl)amino)-5,8-dioxo-5,8-dihydronaphthalene-1-sulfonamide
(PD13):
O
Reddish brown solid, yield 20%; mp 202-204 C; 1H NMR (400 MHz, DMSO-d6) δ 9.49 (s, 1H), 8.38
(d, J=8.6 Hz, 1H), 8.27 (d, J=8.5 Hz, 1H), 8.04 (t, J=8.1 Hz, 1H), 7.37 (d, J=5.0 Hz, 3H), 6.99-6.97 (m,
2H), 6.82 (d, J=7.6 Hz, 1H), 6.17 (s, 1H), 3.77 (s, 3H). 13C NMR (400 MHz, DMSO-d6) δ 181.86,
181.57, 160.76, 147.97, 143.50, 139.94, 135.69, 135.65, 134.35, 133.08, 130.23, 128.47, 116.51, 111.85,
110.46, 102.20, 56.08. MS (HR-ESI) M+Na calculated 381.0521; Observed 381.0435.
4.1.7. 5,8-dioxo-7-(phenylamino)-5,8-dihydronaphthalene-1-sulfonamide (PD14): Reddish brown
solid, yield 17%; mp 237-239 OC; 1H NMR (300 MHz, DMSO-d6) δ 9.55 (s, 1H), 8.38 (d, J=8.5 Hz, 1H),
8.28 (d, J=8.0 Hz, 1H), 8.05 (t, J=8.2 Hz, 1H), 7.56-7.37 (m, 5H), 7.33-7.20 (m, 1H), 6.12 (s, 1H). MS
(HR-ESI) M+Na calculated 351.0415; Observed 351.0475.
4.1.8. 7-((4-methoxyphenyl)amino)-5,8-dioxo-5,8-dihydronaphthalene-1-sulfonamide (PD15): Dark
purple solid, yield 18%; mp >250 OC; 1H NMR (300 MHz, DMSO-d6) δ 9.72 (s, 1H), 8.41 (d, J=6.2 Hz,
1H), 8.12-8.00 (m, 2H), 7.34 (d, J=9.1 Hz, 2H), 7.05 (d, J=7.9 Hz, 2H), 5.98 (s, 1H), 3.79 (s, 3H). MS
(HR-ESI) M+Na calculated 381.0521; Observed 381.0518.
4.2. General synthetic procedure for PD6, PD7, PD16 and PD17- The appropriate naphthoquinone (1.26
mmol) was dissolved in 3 ml of glacial acetic acid followed by addition of copper acetate (0.126 mmol) at
room temperature. Then appropriate aniline (1.26 mmol) wad added to the reaction mixture and the
reaction was heated to 60 OC in air for 4 hours. The reaction mixture was diluted with water and extracted
with ethyl acetate. The organic layer was collected, dried over sodium sulphate, concentrated under
reduced pressure and purified using silica gel column chromatography in 15-25% EtOAc/Hexane to
afford the pure compounds.
4.2.1. 2-((4-methoxyphenyl)amino)-3-methylnaphthalene-1,4-dione (PD6): Red solid, yield 41%; mp
O
1
162-164 C; H NMR (300 MHz, CDCl3) δ 8.18-8.02 (m, 2H), 7.80-7.62 (m, 2H), 7.35 (s, 1H), 6.95 (d,

J=8.7 Hz, 2H), 6.8 (d, J=8.7 Hz, 1H), 3.85 (s, 3H), 1.85 (s, 3H). MS (HR-ESI) M+Na calculated
316.0950; Observed 316.0910.
4.2.2. 2-((3-methoxyphenyl)amino)-3-methylnaphthalene-1,4-dione (PD7): Red solid, yield 39%; mp
O
1
153-155 C; H NMR (300 MHz, DMSO-d6) δ 8.64 (s, 1H), 8.01 (t, J=7.0 Hz, 2H), 7.88-7.73 (m, 2H),
7.19 (t, J=7.6 Hz, 1H), 6.69-6.52 (m, 3H), 3.72 (s, 3H), 1.67 (s, 3H). MS (HR-ESI) M+Na calculated
316.0950; Observed 316.0900.
4.2.3. 5-hydroxy-3-((4-methoxyphenyl)amino)-2-methylnaphthalene-1,4-dione (PD16): Dark purple
O
1
solid, yield 30%; mp 145-147 C; H NMR (300 MHz, DMSO-d6) δ 11.57 (s, 1H), 8.62 (s, 1H), 7.70
(J=7.9 Hz, 1H), 7.52 (d, J=7.9 Hz, 1H), 7.21 (d, J=7.9 Hz, 1H), 7.00 (d, J=8.7 Hz, 2H), 6.89 (d, J=8.7 Hz,
2H), 3.73 (s, 3H), 1.54 (s, 3H). MS (HR-ESI) M+Na calculated 332.0899; Observed 332.0855.
4.2.4. 5-hydroxy-3-((3-methoxyphenyl)amino)-2-methylnaphthalene-1,4-dione (PD17): Dark red
O
color, yield 28%; mp 157-159 C; (300 MHz, CDCl3) δ 11.62 (s, 1H), 7.70-7.59 (m, 2H), 7.33 (s, 1H),
7.22 (dd, J = 12.4, 8.1 Hz, 2H), 6.69 (d, J = 8.3 Hz, 1H), 6.59 (d, J = 8.0 Hz, 1H), 6.53 (s, 1H), 3.82 (s,
3H), 1.80 (s, 3H).MS (HR-ESI) M+Na calculated 332.0899; Observed 332.0810.
4.3. General Synthetic procedure for PD8-PD12 and PD18- The appropriate naphthoquinone (2.32 mmol)
was dissolved in a mixture of 40 ml MeOH (0.058 M) and 32 ml of 2-propanol (0.072 M) at room
temperature. 2-Mercaptoethanol (0.154 ml, 2.20 mmol) was added to the reaction mixture and stirred for
24 hours. The reaction mixture was then poured in 0.019 M of water and extracted with ether. The ether
layer was washed with 10% Copper sulfate solution twice and then with water again. The organic layer
was collected, dried over sodium sulphate, concentrated under reduced pressure and purified using silica
gel column chromatography in 80-100% EtOAc/Hexane to afford the pure compounds.
4.3.1. 2-((2-hydroxyethyl)thio)-3-methylnaphthalene-1,4-dione (PD8): Yellow solid, yield 27%; mp
O
1
80-82 C; H NMR (300 MHz, CDCl3) δ 8.07-7.99 (m, 2H), 7.72-7.63 (m, 2H), 3.80 (t, J=5.7 Hz, 2H),
3.34 (t, J=5.7 Hz, 2H), 2.78 (bs, 1H), 2.36 (s, 3H). MS (HR-ESI) M+Na calculated 271.0405; Observed
271.0373.
4.3.2. 5-hydroxy-2-((2-hydroxyethyl)thio)naphthalene-1,4-dione (PD9): Yellowish red solid, yield
16%; mp 148-150 OC; NMR (400 MHz, DMSO-d6) δ 12.13 (s, 1H), 7.76-7.68 (m, 1H) 7.56 (d, J=7.4 Hz,
1H), 7.36 (d, J=8.7 Hz, 1H), 6.82 (s, 1H), 5.15 (t, J=5.6 Hz, 1H), 3.70 (d, J=6.0 Hz, 2H), 3.08 (t, J=6.0

Hz, 2H). 13C NMR (400 MHz, DMSO-d6) δ 187.42, 182.00, 161.14, 156.52, 136.88, 132.23, 127.58,
125.55, 120.06,115.16, 59.15, 33.95. MS (HR-ESI) M+Na calculated 273.0198; Observed 273.0164.
4.3.3. 8-hydroxy-2-((2-hydroxyethyl)thio)naphthalene-1,4-dione (PD10): Red solid, yield 15%; mp
135-137 OC; 1H NMR (400 MHz, DMSO-d6) δ 11.46 (s, 1H), 7.73 (d, J=7.8 Hz, 1H), 7.54 (d, J=5.3 Hz,
1H), 7.30 (d, J=6.8 Hz, 1H), 6.79 (s, 1H), 5.15 (s, 1H), 3.77-3.66 (m, 2H), 3.12-3.00 (m, 2H). MS (HR-
ESI) M+Na calculated 273.0198; Observed 273.0149.
4.3.4. 6-((2-hydroxyethyl)thio)-5,8-dioxo-5,8-dihydronaphthalene-1-sulfonamide (PD11): Yellow
O
1
solid, yield 28%; mp 203-205 C; H NMR (400 MHz, DMSO-d6) δ 8.44 (d, J=7.9 Hz, 1H), 8.29 (d,
J=8.8 Hz, 1H), 8.05 (t, J=7.9 Hz, 1H), 7.38 (s 2H), 6.90 (s, 1H), 3.71 (d, J=6.1 Hz, 2H), 3.10 (d, J=6.1
Hz, 2H). 13C NMR (400 MHz, DMSO-d6) δ 181.75, 181.45, 153.89, 143.49, 134.75, 134.74, 134.49,
131.27, 129.96, 129.13, 59.27, 33.66. MS (HR-ESI) M+Na calculated 335.9976; Observed 335.9949.
4.3.5. 6,7-bis((2-hydroxyethyl)thio)-5,8-dioxo-5,8-dihydronaphthalene-1-sulfonamide (PD12): 2 eq
O
1
of mercaptoethanol used. Yellow solid, yield 22%; mp 162-164 C; H NMR (300 MHz, DMSO-d6) δ
8.28 (d, J=8.3 Hz, 1H), 8.17 (d, J=8.2 Hz, 1H), 7.95 (s, 1H), 7.43 (s, 2H), 4.89 (dt, J=16.2, 5.1 Hz, 2H),
3.59 (d, J=5.6 Hz, 4H), 3.23 (dt, J =13.4, 6.8 Hz, 4H). MS (HR-ESI) M+Na calculated 411.9959;
Observed 411.9915.
4.3.6. 5-hydroxy-3-((2-hydroxyethyl)thio)-2-methylnaphthalene-1,4-dione (PD18): Yellow solid,
O
1
yield 18%; mp 87-90 C; H NMR (300 MHz, DMSO-d6) δ 11.74 (s, 1H), 7.70 (t, J=8.0 Hz, 1H), 7.51
(d, J=7.2 Hz, 1H), 7.30 (d, J=8.4 Hz, 1H), 4.90 (t, J=5.2 Hz, 1H), 3.59 (q, J=5.3 Hz, 2H), 3.22 (t, J=6.1
Hz, 2H), 2.26 (s, 3H). MS (HR-ESI) M+Na calculated 287.0354; Observed 287.0293.
4.4. Biological activity
4.4.1 Cell Lines and Culture
Prostate (DU-145), breast (MDA-MB-231) and colon (HT-29) cancer cell lines were obtained from
American Type Culture Collection. HT-29 and DU-145 cells were cultured in complete RPMI 1640
(Gibco, Invitrogen, Grand Island, NY) + 10% FBS (GIBCO), 2mM L-glutamine (GIBCO) and 1%
gentamicin (GIBCO). MDA-MB-231 cells were cultured in DMEM F-12 + 10% FBS, 2mM L-glutamine
and 1% gentamicin.

4.4.2 Anitproliferative assay
Cells were cultured at 37 OC (5% CO2). Cells were plated in 96-well plates at a cell density of 2,500
cells per well in 100 µL of media and were allowed to attach overnight. On the following day a step by
step serial dilution of the compounds was performed, and compounds were added to the cells (to bring the
total to 200 µl per well). The treated cells were then incubated for 72 hours. Cell viability was determined
by using the Promega CellTiter 96 Aqueous Non-Radioactive Cell Proliferation assay (MTS). IC50 values
were calculated with SoftMax Pro. Each compound was run at least twice in triplets.
4.4.3 Fluorescent polarization assay
4.4.3.1. Materials and Methods
STAT3 protein (>90% purity) was purchased from Abcam (Cambridge, MA). The fluorescent
peptide with the amino acid sequence 5-carboxyfluorescein-SpYLPQTV (5-FAM-SpYLPQTV) (95.1%
purity) was prepared by Genscript (Piscataway, NJ). The unlabeled peptide with the amino acid sequence
SpYLPQTV (98.7% purity) was prepared by Genscript (Piscataway, NJ). The unlabeled peptide with the
amino acid sequence SYLPQTV (98.4% purity) was prepared by Genscript (Piscataway, NJ). The
fluorescent peptide buffer conditions were 10 mM HEPES, 50 mM NaCl, 1 mM EDTA, 0.1% Nonidet P-
40, 10% DMSO, pH 7.6.
4.4.3.2. Fluorescence polarization assay development and optimization
The STAT3 FP assay was developed similarly to what has been previously reported.32,36 Buffer
conditions, similar to those of Schust and colleagues were applied in this assay.35 The fluorescent peptide
and unlabeled peptide used in this assay are identical to those used by Hao and colleagues. The assay was
performed in black 384-well microplates (Perkin Elmer, Waltham, MA) in total volumes of 25 μL per
well. Measurements were taken with a FlexStation 3 microplate reader (Molecular Devices, Sunnyvale,
CA). The FP values were recorded using an excitation filter at 480 nm and an emission filter at 530 nm.
The final concentration of fluorescent peptide used for binding experiments was 10 nM; which is identical
to what has been previously reported.32-35,37 To evaluate inhibitors, the compounds (diluted in buffer from
stock solutions) were incubated at room temperature at the indicated concentrations with STAT3 protein
(final concentration of 150 nM) in buffer for at least 60 minutes. The fluorescence probe was then added
and the mixtures were allowed to incubate for at least 30 minutes prior to readings being taken. This
method is similar to what has been previously reported.34-36 In evaluating compounds the experiments
were performed at least twice in triplicate. To determine the IC50 values, compounds were tested at
multiple concentrations (500 to 2 µM) using a 1:2 serial dilution. SoftMax Pro version 5.4.4 (Molecular

Devices, Sunnyvale, CA) was used to determine IC50 values via a standard 4-parameter logistic fit. Ki
values were determined using the following equation: Ki = IC50 / ( 1 + ( [STAT3] / Kd ). The unlabeled
peptide SpYLPQTV was used as a positive control while the peptide SYLPQTV was used as a negative
control.
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Graphical Abstract
Anticancer activity and SAR studies of Substituted
1,4-Naphthoquinones
Leave this area blank for abstract info.
Deepak Bhasin,* Somsundaram N. Chettiar,* Jonathan P. Etter, May Mok, Pui-Kai Li#