Synthesis and evaluation of (±)-dunnione and its ortho-quinone analogues as substrates for NAD(P)H:quinone oxidoreductase 1 (NQO1)

Article abstract of DOI:10.1016/j.bmcl.2015.01.057

Natural product (±)-dunnione (2) and its ortho-quinone analogues (3-8) were synthesized and found to be substrates for NQO1. The structure-activity relationship study revealed that the biological activity was favored by the presence of methyl group at the C ring and methoxy group at the A ring. The docking studies supported the rationalization of the metabolic studies. Deeper location in the active site of NQO1, interactions with hydrophobic pocket and C-H...π interactions with the adjacent Phe178 residue contributed to the better catalytic efficiency and specificity to NQO1. Cytotoxicity studies and determination of superoxide (O₂^-) production in the presence and absence of the NOQ1 inhibitor dicoumarol confirmed that the ortho-quinones exerted their antitumor activity through NQO1-mediated ROS production by redox cycling.

Full text of DOI:10.1016/j.bmcl.2015.01.057

Bioorganic & Medicinal Chemistry Letters 25 (2015) 1244–1248
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of ( )-dunnione and its ortho-quinone
analogues as substrates for NAD(P)H:quinone oxidoreductase 1
(NQO1)
Jinlei Bian a,c, Lili Xu a,c, Bang Deng a,c, Xue Qian a,c, Jun Fan c, Xiuwen Yang c, Fang Liu a,c, Xiaoli Xu a,c
,
Xiaoke Guo a,c, Xiang Li a,c, Haopeng Sun a,c, Qidong You a,b,c, , Xiaojin Zhang a,d,
⇑
⇑
a Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 210009, China
b State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China
c Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
d Department of Organic Chemistry, School of Science, China Pharmaceutical University, Nanjing 210009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Natural product ( )-dunnione (2) and its ortho-quinone analogues (3–8) were synthesized and found to be
substrates for NQO1. The structure–activity relationship study revealed that the biological activity was
favored by the presence of methyl group at the C ring and methoxy group at the A ring. The docking
studies supported the rationalization of the metabolic studies. Deeper location in the active site of
Received 7 October 2014
Revised 17 January 2015
Accepted 23 January 2015
Available online 31 January 2015
NQO1, interactions with hydrophobic pocket and C–H. . .p interactions with the adjacent Phe178 residue
contributed to the better catalytic efficiency and specificity to NQO1. Cytotoxicity studies and determina-
tion of superoxide (OÅ2À) production in the presence and absence of the NOQ1 inhibitor dicoumarol
confirmed that the ortho-quinones exerted their antitumor activity through NQO1-mediated ROS produc-
tion by redox cycling.
Keywords:
Natural product
Dunnione
NQO1
Antitumor
Ó 2015 Elsevier Ltd. All rights reserved.
Reactive oxygen species (ROS)
NAD(P)H:quinone oxidoreductase 1 (NQO1) is a ubiquitous
flavoenzyme that catalyzes the direct two-electron reduction of
quinones to hydroquinones, and it is highly expressed in many
solid tumors.1 This forms the development of efficient quinone
( )-Dunnione (2) is a natural dihydrofuran-fused ortho-
naphthoquinone with similar ortho-quinone moiety as compared
to 1 (Fig. 1). Recently, 2 has been reported to exhibit potent anti-
Trypanosoma cruzi activity5,6 as well as antitumor activity.7 How-
ever, the molecular target of 2 to elicit its biological effect remains
unrevealed. The structural similarity of the dihydrofuran-fused
ortho-naphthoquinone 2 to 1 prompted us to determine whether
NQO1 plays a pivotal role on initiating its antitumor effect. In
addition, few studies have investigated the structure–activity rela-
tionship (SAR) of the ortho-quinones such as 1 and 2 for their NQO1
mediated bioactivity. Thus we planned to explore the preliminary
SAR for this series of ortho-quinone compounds. Because the
quinone pharmacophore (B ring) is considered to be essential for
NQO1 reduction, we mainly focus our efforts on investigating com-
pounds with substitution at A ring and C ring in this study.
Compound 2 and its analogues 3–8 were synthesized in only
three steps (O-allylation, Claisen rearrangement and cyclization)
from lawsone8 (9a) or methoxy-substituted lawsone (9b–9d).
O-allylation of 9a (3,3-dimethyl allyl bromide, K2CO3, DMF) gave
3,3-dimethyl-allyl ether (10a) in 71% yield. Claisen rearrangement
of 10a (120 °C, toluene) gave 3,3-dimethyl allyl lawsone (11a)
(82%). 11a was cyclized to dunnione (2) mediated by Lewis acid
NbCl5 at room temperature in 85% yield (Scheme 1). Similarly,
substrates as potential NQO1-directed antitumor agents.2
A
number of quinone-based chemotherapeutic agents such as ortho-
naphthoquinone, para-naphthoquinone, para-indolequinone, para-
benzimidazolequinone, and para-quinolinequinone derivatives can
be bioactivated by NQO1.3 Compared to the structurally diverse
para-quinone substrates, the reported ortho-quinone substrates
for NQO1 are limited. Among them b-lapachone (b-lap, 1, Fig. 1),
a natural tetrahydropyran-fused ortho-naphanoquinone isolated
from the Bignoniaceae family, is the most prominent and represen-
tative example.3 1 is currently in multiple phase II clinical trials for
the treatment of pancreatic adenocarcinoma.4,3a It has been
reported to kill many human cancers selectively through rapid
reactive oxygen species (ROS) generation mediated by NQO1
bioreduction.4
⇑
Corresponding authors.
E-mail addresses: youqd@163.com (Q. You), zhangxiaojin@outlook.com
(X. Zhang).
http://dx.doi.org/10.1016/j.bmcl.2015.01.057
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

J. Bian et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1244–1248
1245
O
O
O
B
O
A
B
A
C
C
O
O
-lapachone ( -lap,1)
β
β
( ) −dunnione (2)
O
O
6
O
O
7
R4
R1
A
B
8
C3
∗
∗
R2
9
C
C2
O
O
R3
6, R4 = C7-OMe
7, R4 = C8-OMe
8, R4 = C9-OMe
3, R1 = H, R2 =H, R3 = Me
4, R1 = H, R2 = H, R3 = H
5, R1 = H, R2 = Me, R3 = H
Figure 1. Structures of b-lap, dunnione and analogues of dunnione.
compounds 3–8 were prepared by analogous routes in similar
yields. Most of these compounds were obtained with a mixture
of stereoisomers, and compound 4 was mainly in its trans-isomer
The steric configuration of structural formula was shown in the
Supporting information (Fig. S2).
After achieving the synthesis, the ability of NQO1 to process
these ortho-quinones (2–8) in vitro was assessed as previously
reported.3a From the results, Michaelis–Menten curves were gener-
ated (Fig. 2), and the NQO1 kinetic parameter (kcat/Km) was calcu-
lated (Table 1). The highest value of the NQO1 kinetic parameter
(kcat/Km) reflects the highest catalytic efficiency and specificity.
As shown in Table 1, it was observed that 4 (kcat/Km = 0.8 0.2 Â
106 MÀ1 sÀ1) showed worse catalytic efficiency and specificity than
the control b-lap (kcat/Km = 2.6 0.5 Â 106 MÀ1 sÀ1), most likely
because of the impact of C ring narrowing on binding of ortho-
Figure 2. Michaelis–Menten curves for the ortho-quinone substrates for NQO1.
(A) Michaelis–Menten curves for compounds 2–5. (B) Michaelis–Menten curves for
compounds 6–8 and the control b-lap.
Table 1
Kinetic and computation parameters for the dihydrofuran-fused ortho naphthoqui-
nones and b-lap
O
R3
O
Compd
kcat/Km (106 MÀ1 sÀ1
)
ChemScorea
C@O5. . .NH5a (Å)
6
O
R2
OH
7
8
2
3
4
5
6
7
8
1.9 0.4
1.0 0.2
0.8 0.2
1.4 0.5
2.0 0.4
3.1 0.5
2.4 0.6
2.6 0.5
70.52 (69.82)
66.98
3.52 (3.64)
3.59
R4
a
R4
A
B
A
B
R1
9
66.02 (65.50)
70.92 (68.98)
71.88 (70.23)
72.57 (70.29)
69.37 (67.01)
69.43
3.60 (3.91)
3.57 (3.72)
3.41 (3.97)
3.33 (3.88)
3.45 (3.66)
3.31
O
O
9a, R4 = H
1 = Me, R2 = Me, R3 = H, R4 = H
10a,
R
4
10b, R1 = H, R2 = H, R3 = Me, R4 = H
10c, R1 = H, R2 = H, R3 = H, R4 = H
9b,
R
= C7-OMe
9c, R4 = C8-OMe
9d, R4 = C9-OMe
10d,
R
1 = H, R2 = Me, R3 = H, R4 = H
b-Lap (1)
10e, R1 = Me, R2 = Me, R3 = H, R4 = C7-OMe
10f, R1 = Me, R2 = Me, R3 = H, R4 = C8-OMe
a
The computation parameters of the C2-R-isomer; the C2-S-isomer was shown
in parentheses.
10g,
R
1 = Me, R2 = Me, R3 = H, R4 = C9-OMe
b
quinone in the active site of NQO1. To further probe the effect,
we examined the ortho-quinone with two methyl groups (3 and
5) and three methyl groups (2) at the C ring. It was found that
compounds with more substituted methyl groups were more
specific toward enzyme. Especially for 2, which had three methyl
O
O
6
O
R1
R2
7
8
R3
OH
R4
c
A
B
R4
A
B
9
C
O
R2
O
R1
groups at
C ring, showed efficient catalyzed reduction rate
R3
(kcat/Km = 1.9 0.4 Â 106 MÀ1 sÀ1). Compounds (6–8) with meth-
oxy group at the A ring showed better enzyme catalytic efficiency
and specificity than 2, indicating that methoxy-substitution was
= Me, R2 = Me, R3 = H, R4 = H
1
1 = Me, R2 = Me, R3 = H, R4 = H
11a,
R
2,
R
3, R1 = H, R2 = H, R3 = Me, R4 = H
11b, R1 = H, R2 = H, R3 = Me, R4 = H
11c, R1 = H, R2 = H, R3 = H, R4 = H
4, R1 = H, R2 = H, R3 = H, R4 = H
= H, R2 = Me, R3 = H, R4 = H
1
1 = H, R2 = Me, R3 = H, R4 = H
11d,
R
5,
R
tolerable for NQO1 substrates. Among them, compound
7
6, R1 = Me, R2 = Me, R3 = H, R4 = C7-OMe
11e, R1 = Me, R2 = Me, R3 = H, R4 = C7-OMe
11f, R1 = Me, R2 = Me, R3 = H, R4 = C8-OMe
(kcat/Km = 3.1 0.5 Â 106 MÀ1 sÀ1) was the most efficient substrate,
which possessed slightly better kinetic parameters than the control
b-lap. It indicated that the introduction of methoxy group at the
A ring improved the catalytic efficiency and specificity of these
ortho-quinones.
7, R1 = Me, R2 = Me, R3 = H, R4 = C8-OMe
= Me, R2 = Me, R3 = H, R4 = C9-OMe
1
1 = Me, R2 = Me, R3 = H, R4 = C9-OMe
11g,
R
8,
R
Scheme 1. Reagents and conditions: (a) substituted allyl bromide, K2CO3, DMF,
45 °C, 57–72%; (b) toluene, 120 °C, 63–90%; (c) NbCl5, DCM, rt, 70–85%.

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J. Bian et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1244–1248
Figure 3. Docked conformation of compounds 2 C2-R-isomer (A), 2 C2-S-isomer (B), 7 C2-R-isomer (C) and 7 C2-S-isomer (D) into the active binding site of NQO1. The
interaction mode was obtained through molecular docking (PDB ID: 1DXO) and depicted using MOE 2013.08. The carbon atoms of the compounds and the key residues in the
active site of NQO1 were colored in yellow and green, respectively. The H-bonds and H–p-bonds were shown as black dot lines.
To elucidate the possible binding mode of NQO1 with the ortho-
quinones, a molecular docking study was performed based on the
crystal structure of NQO1 (PDB code 1DXO). All of the compounds
structures were further analysed relative to the stereoisomers of
these compounds (take compound 1 as examples), observed as
the likely products of chemical synthesis (Fig. 3). For both C2-
R-isomer and C2-S-isomer, the substituted methyl (C ring) can be
placed into the pocket formed by Tyr128, Gly149 and Gly150.
The Gold5.1 ChemScore scores for the R and S isomers are 70.57
and 69.82, respectively, favouring the R-isomer slightly. As for
the corresponding distances between the ortho-quinones and
FAD, C2-R-isomer (distance 3.52 Å, Table 1) was also fitted slightly
deeper into the active site than the C2-S-isomer (distance 3.64 Å,
Table 1), but the difference is not significant. The docking results
indicated that both isomers can be catalyzed by NQO1 which is
consistent with the result of the indistinguishable kinetic data of
these isomers from enzymatic assays. The docking poses of the R,
S-isomers of the representative compounds (1 and 7) were shown
in Figure 3.
were found to form p-stacking interactions with the isoalloxazine
ring of FAD which was very similar to the parent duroquinone.9
A correlation was previously observed between the substrate (qui-
none carbonyl) and the FAD cofactor (atom N5 which transfer the
hydride), with compounds that have a shorter predicted hydride
donor–acceptor distance being better NQO1 substrates.10 There-
fore, it was necessary to calculate and analyse the corresponding
distances between the ortho-quinones and FAD. As shown in
Table 1, all were within a reasonable distance (about 4 Å) for
hydride transfer, suggesting that they were potential substrates
for NQO1. Notably, compounds with methoxy substituent at the
C ring such as 7 (distance 3.33 Å, Table 1) fitted deeper into the
active site compared to 2 (distance 3.52 Å, Table 1), providing a
rationale for the better catalytic efficiency and specificity of the
compounds with methoxy substituent at the A ring on one hand.
The binding modes of these ortho-quinones with NQO1 were care-
fully analysed and the results for representative compounds 2 and
7 were shown in Figure 3 (Other compounds see Supplementary
data, the representative conformations were selected). As shown
in Figure 3, the methyl groups at the C ring can fit into the hydro-
phobic pocket formed by Tyr128, Gly149 and Gly150. It explained
the fact that 2 which contained three methyl substituents exhib-
ited higher NQO1 catalytic efficiency and specificity than other
compounds (3–5) with fewer methyl substituents at the C ring.
Particularly, as for compounds 7 and 8 with methoxy group at
Cytotoxicity studies and determination of ROS production were
also performed on all of the naphthoquinones. Cell survival was
measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
razolium bromide MTT assay. We utilized the human non-small
Table 2
Cytotoxicity of all of the naphthoquinones toward A549 cells in the presence and
absence of the NQO1 inhibitor (DIC, 25
l
mol LÀ1
mol LÀ1
)
Compd
Cytotoxicity IC50
A549
(l
)
Selectivity ratio IC50
(A549 + DIC)/IC50 (A549)
A549+DIC
2
3
4
5
6
7
8
6.1 1.2
10 6.5
7.7 0.8
7.0 1.3
9.2 2.2
6.8 1.1
16 5.0
7.0 0.5
24 5.9
20 6.0
30 3.5
17 2.1
34 5.9
41 4.9
79 4.3
25 2.3
3.9
2.0
3.8
2.4
3.7
6.0
4.9
3.6
the C-8 or C-9 position of the A ring, C–H. . .
the adjacent Phe178 residue was observed for enhancing
p
interaction with
-stack-
p
ing interaction with surrounding residues, supporting that they
would be excellent substrates for NQO1 from the other hand
(Fig. 3B). It should be noted that most of the synthesized
compounds (2, 4–8) are mixtures of stereoisomers. The docked
b-lap (1)

J. Bian et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1244–1248
1247
Figure 4. (A) Cell death of A549 Cells treated for 2 h with compound 7 in the presence and absence of the NQO1 inhibitor (DIC, 25
lM). (B) Rate of ROS production of all of the
naphthoquinones (2–8) in the absence (the blue bars) and presence (the green bars) of the NQO1 inhibitor (DIC, 25
lM) with NQO1. Rate of ROS production of b-lap and
menadione (the pink bars) in the absence of DIC were showed as control. The rates expressed mean SD, P <0.001 versus control (n = 3).
cell lung cancinoma A549 cell lines in the absence and presence of
the NQO1 inhibitor dicoumarol (DIC, 25
mol LÀ1) to compare the
interactions between the ortho-quinones and NQO1. Our docking
studies supported the rationalization of the metabolic studies.
Deeper location in the active site of NQO1, interactions with hydro-
l
cytotoxicity of the ortho-quinones (Table 2). DIC has been widely
used in studies of NQO1-mediated cell death, as incubation of cells
with the inhibitor was effective in blocking the enzymatic activity
of NQO1.11 As illustrated in Table 2, coincubation with DIC protects
A549 cells from the cell death mediated by these ortho-quinones,
shifting the IC50 from 2-fold to 6-fold. (The fold is the ratio of the
IC50 of cotreatment with ortho-quinones and DIC to the IC50 of
treatment with only ortho-quinones, and a higher ratio indicates
greater protection and greater NQO1 specificity). Compounds
(6–8) with methoxy group at the A ring of 2 even showed better
toxic selectivity to NQO1-rich A549 cell lines (selectivity ratio
P3.7) compared to the control b-lap (select ratio = 3.6). The results
suggested that the ortho-quinones exerted their antitumor activity
by an NQO1-dependent mechanism. As more clearly shown in
Figure 4A, coincubation with DIC greatly protected A549 cells from
the cell death mediated by the best compound 7, shifting the
IC50 = 6-fold.
phobic pocket and C–H. . .
p interaction with the adjacent Phe178
residue provided the rationale for the better catalytic efficiency
and specificity of compound 7 (named as DDO-7102). The evalua-
tion of the antitumor activity in human non-small cell lung carci-
noma A549 cell lines in the absence and presence of the NQO1
inhibitor DIC provided 7 as lead, with a better toxic selectivity than
the parent drug 2. Determination of superoxide (OÅ2À) production in
the presence and absence of the NQO1 inhibitor dicoumarol con-
firmed that these ortho-quinones exerted their antitumor activity
through NQO1-mediated ROS production by redox cycling. These
findings encourage further investigations of the novel dihydrofu-
ran-fused ortho-naphthoquinones as NQO1-directed antitumor
agents.
Acknowledgments
In addition, NQO1 substrates have been suggested to exert their
antitumor activity through rapid ROS generation.4 We thus further
investigated the ability of these compounds to produce ROS. As we
previously reported, menadione was used as positive control.3a The
production of the superoxide (OÅ2À), the main constitute of ROS, was
measured by a spectrophotometric assay with cytochrome c as the
We are thankful for the financial support of the National Natural
Science Foundation of China (No. 81302636), the Natural Science
Foundation of Jiangsu Province of China (No. BK20130656), the
Program of State Key Laboratory of Natural Medicines, China
Pharmaceutical University (No. SKLNMZZ201202), the National
Found for Fostering Talents of Basic Science (NFFTBS) of China
(No. J1030830), the Priority Academic Program Development of
Jiangsu Higher Education Institutions (PAPD).
terminal electron acceptor.3a The initial rates (
lmol cytochrome c
reduced/min/mg NQO1) were calculated from the liner portion
(0–30 s) of the reduction graphs. The highest value represented it
can produce maximum amount of ROS. As shown in Figure 4B,
compounds 2, 3, 4 and 5 showed similar rates of ROS generation
to b-lap. However, compounds 6, 7 and 8 showed high rates of
ROS generation, being much more efficient than b-lap. In fact, the
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.01.
057.
rate of ROS production for compound 8 (1380 120
c/min/mg NQO1) was even shown better than the positive control
menadione (1315 25 mol cyt c/min/mg NQO1). The results indi-
lmol cyt
l
References and notes
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