Synthesis of carbazoloquinone derivatives and their antileukemic activity via modulating cellular reactive oxygen species

Article abstract of DOI:10.1016/j.bmcl.2019.06.038

Carbazoloquinone alkaloids are of great interest as privileged structures for anticancer drug molecules. The purpose of this study was to investigate the structure-activity relationships of carbazoloquinone derivatives as anticancer agents. A series of ca

Full text of DOI:10.1016/j.bmcl.2019.06.038

Accepted Manuscript
Synthesis of carbazoloquinone derivatives and their antileukemic activity via
modulating cellular reactive oxygen species
Natsumi Suematsu, Masayuki Ninomiya, Hodaka Sugiyama, Taro Udagawa,
Kaori Tanaka, Mamoru Koketsu
PII:
S0960-894X(19)30418-4
https://doi.org/10.1016/j.bmcl.2019.06.038
BMCL 26516
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
22 April 2019
18 June 2019
19 June 2019
Please cite this article as: Suematsu, N., Ninomiya, M., Sugiyama, H., Udagawa, T., Tanaka, K., Koketsu, M.,
Synthesis of carbazoloquinone derivatives and their antileukemic activity via modulating cellular reactive oxygen
species, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.06.038
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Synthesis of carbazoloquinone derivatives and their antileukemic
activity via modulating cellular reactive oxygen species
Natsumi Suematsua, Masayuki Ninomiyaa, Hodaka Sugiyamaa, Taro Udagawaa, Kaori
Tanakab,c, and Mamoru Koketsua,*
aDepartment of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu
University, 1-1 Yanagido, Gifu 501-1193, Japan
bDivision of Anaerobe Research, Life Science Research Center, Gifu University, 1-1
Yanagido, Gifu 501-1194, Japan
cUnited Graduate School of Drug Discovery and Medicinal Information Sciences,
Gifu University, 1-1 Yanagido, Gifu 501-1194, Japan
*To whom correspondence should be addressed. Tel./Fax: +81-58-293-2619, E-mail:
koketsu@gifu-u.ac.jp (M.K.)
1

ABSTRACT
Carbazoloquinone alkaloids are of great interest as privileged structures for
anticancer drug molecules. The purpose of this study was to investigate the structure-
activity relationships of carbazoloquinone derivatives as anticancer agents. A series of
carbazoloquinones including murrayaquinone A, koeniginequinones A and B, and
related analogues were therefore prepared. Palladium-catalyzed intramolecular
cyclization reaction mechanism was well elucidated by DFT calculations. Treatment
of the synthesized derivatives showed cytotoxicity on human leukemia HL-60 cells in
a dose-dependent fashion. In addition, murrayaquinone A and -brazanquinone
elevated cellular levels of reactive oxygen species (ROS), thereby triggering apoptosis.
Our findings emphasize the excellent potential of carbazoloquinone derivatives as
ROS-inducing anticancer agents.
Keywords: Carbazoloquinone alkaloid; Murrayaquinone A; DFT calculation;
Reactive oxygen species; Apoptosis
2

Quinone molecules are widespread in nature, and some of them have been used to
treat cancer (e.g., doxorubicin, daunorubicin, and mitomycin C).1 Anticancer
quinones show remarkable potential and distinctive modes of actions, and because of
that, the exploration of natural quinone constituents for cancer chemotherapy attracts
a great deal of attention.2–4 There is continued raising interest in the development of
new chemotherapeutic agents containing the quinone motif.
Carbazoloquinones (carbazole-1,4-quinones) constitute a subclass of natural
carbazole alkaloids occurring in the Rutaceae (citrus) family of plants.5,6 The
Murraya genus of flowering shrubs is the greatest source of these constituents. Their
basic framework comprises fused 1,4-quinone and indole structures derived from
anthranilic acid and prenyl pyrophosphate.7 These quinones have been reported to
possess a broad range of biological properties such as anticancer, anti-inflammatory,
antibacterial, and anti-infective properties.8–11 Murrayaquinone A (3-methyl-1H-
carbazole-1,4(9H)-dione) was firstly isolated from the root bark of Murraya
euchrestifolia Hayata.12 This quinone displayed significant cytotoxicity against
human skin melanoma SK-MEL-5 and human colon adenocarcinoma Colo-205 cells.8
Additionally, murrayaquinone A was documented as a potential antibacterial agent
because of its pronounced inhibition against both of Escherichia coli and
Staphylococcus aureus.9 Murrayaquinone A also inhibited against Trichomonas
(Trichomonas gallinae) parasites.11 Methoxylated analogues, koeniginequinones A
and B were found primarily in the stem bark of curry tree, Murraya koenigii Spreng.13
Earlier observations prompted us to take up the carbazoloquinone unit as an
active pharmacophore for further diversification to exploit its medicinal potential. In
continuation of our work on naturally occurring quinones and related synthetic
analogues,14,15 the present study focuses on the structure-activity relationships of
3

carbazoloquinone derivatives as anticancer agents. Furthermore, the underlying
mechanisms associated with those effects were studied.
For construction of the carbazoloquinone core, the palladium-catalyzed
intramolecular cyclization of arylamino-p-toluquinones is a common approach.16
Initially, naturally occurring carbazoloquinones were prepared as follows: the addition
of anilines into p-toluquinone in acidic conditions furnished the required 2-anilino-5-
methyl-1,4-benzoquinones 1a–1c in 16–58% yields (Scheme 1). When aniline
(R1=R2=H) was used, 2-anilino-6-methyl-1,4-benzoquinone 2a was formed as a
byproduct in 30% yield. Its methyl position on quinone was determined by HMQC
and HMBC techniques. It is thought that this reaction proceeds through hydroquinone
intermediates,17 which are immediately converted into the quinones (aerial
oxidation).18,19 Tandon VK and Maurya HK reported aqueous conditions accelerate
this type addition.20 Refluxing 1a–1c with an equimolar amount of palladium(II)
acetate (Pd(OAc)2) in acetic acid allowed access to murrayaquinone A (3a, 49%
yield), koeniginequinones A (3b, 50% yield) and B (3c, 42% yield). Through the
same route, other derivatives bearing 6-methoxy (-OMe), 6-chloro (-Cl), and 6-
trifluoromethyl (-CF3) substituents on their benzene ring were synthesized. Detailed
synthetic conditions and spectroscopic data of compounds are given in the
Supplementary data.
R1
1
2
1
2
O
1a
R =R =H ( , 58%)
3a
R =R =H ( , 49%)
Pd(OAc)2
(1.0 equiv)
R1
1b
R =H, R =OMe ( , 26%)
O
1
2
1
2
3b
R =H, R =OMe ( , 50%)
R2
1
2
1
2
1c
R =R =OMe ( , 16%)
3c
R =R =OMe ( , 42%)
1
2
AcOH
reflux, 15 h
N
R2 R1=OMe, R2=H (1d, 28%)
3d
R =OMe, R =H ( , 14%)
O
N
H
H
1
2
1
2
1e
R =Cl, R =H ( , 15%)
O
O
3e
R =Cl, R =H ( , 34%)
1) H2O, 60oC, 30 min
1
2
O
O
1
2
1f
R =CF3, R =H ( , 9%)
3f
R =CF3, R =H ( , 39%)
+
+
2) Anilines (1.0 equiv)
H2O/AcOH, rt, 15 h
R1
Pd(OAc)2
(1.0 equiv)
O
R1
R2
R2
1
2
1
2
4a
R =R =H ( , 60%)
2a
R =R =H ( , 30%)
p-Toluquinone
1
2
1
2
4d
R =OMe, R =H ( , 27%)
2d
R =OMe, R =H ( , 18%)
AcOH
reflux, 15 h
N
N
H
H
O
O
Scheme 1. Synthesis of carbazoloquinone derivatives.
4

As a crucial step for the structure-activity relationships, we evaluated the
synthetic carbazoloquinone derivatives (3 and 4) for in vitro cytotoxicity against
human leukemia HL-60 cells using the CCK-8 assay method.21 The cells were treated
with increasing concentrations of each compound for 48ꢀh, with DMSO as control.
Even at a concentration of 10 M, most derivatives exhibited high cytotoxicity with
cell viability of less than 20% (% of control). Their IC50 values are as follows: 3a
(0.73 M), 3b (1.21 M), 3c (0.55 M), 3d (3.11 M), 3e (0.24 M), 3f (0.59 M),
4a (0.29 M), and 4d (5.06 M), respectively. The cytotoxic effects of derivatives 3a,
3c, 3e, 3f, and 4a are two orders of magnitude greater than that of 5-fluorouracil (5-
FU: anticancer chemotherapy drug,22 IC50: 42.8 M). There is not much difference
between the compounds tested. In other words, a functional group on the benzene ring
of carbazoloquinones has unexpectedly less impact on their cytotoxicity.
Next, we examined the effects of substructures located directly on the quinone
core on their efficacy. A methyl group was replaced by a benzene ring. Conversions
of the pyrrole to other five-membered heterocycles such as furan, thiophene, and
selenophene were carried out. Although the addition of aniline and benzenethiol into
p-benzoquinone gave the desired products 5a and 5c in 32% and 64% yields, the
reactions with phenol and benzeneselenol did not work at all under the same
conditions. As an alternative, 2-chloro-p-benzoquinone was employed for the two
cases. The nucleophilic substitution of phenol to 2-chloro-p-benzoquinone in the
presence of K2CO3 in DMF worked well, giving 5b in 78% yield. Since selenol (R-
SeH) easily condenses into the diselenide (R-Se-Se-R),23 phenylselenolate anion (Ph-
Se-) was generated in situ from diphenyl diselenide (Ph-Se-Se-Ph) by hydride
reduction using NaBH4. The anion successfully attacked to 2-chloro-p-benzoquinone,
affording 5d in 78% yield (Scheme 2). The Pd(OAc)2-catalyzed intramolecular
5

cyclization of 5a succeeded in 6a in 87% yield. Cyclized products 6b–6c were
obtained in yields of 24% for -brazanquinone (benzo[b]naphtho[2,3-d]furan-6,11-
dione) 6b, 3.7% for 6c, and 3.4% for 6d when AgOAc was used as an oxidant.24,25 It
is worth mentioning that the yields were affected by the heteroatom (X atom) in five-
membered rings (Scheme 3).
O
O
1) H2O, 60oC, 30 min
2) PhNH2 or PhSH
(1.0 equiv)
X
5a
X=NH ( , 32%)
5c
X=S ( , 64%)
O
O
H2O/AcOH, rt, 15 h
O
Phenol (1.5 equiv)
K2CO3 (3.0 equiv)
DMF, rt, 2 h
O
O
O
O
O
5b
5d
, 78%
, 78%
PhSeSePh (1.2 equiv)
NaBH4 (1.2 equiv)
Cl
EtOH (3.0 equiv)
DMF, 0oC to rt, 18 h
Se
O
Scheme 2. Addition of aniline, phenol, benzenethiol, and benzeneselenol.
O
O
Pd(OAc)2
(1.0 equiv)
6a
X=NH ( , 87%)
6b
X=O ( , 24%)*
6c
X=S ( , 3.7%)*
AcOH
reflux, 15 h
X
X
6d
X=Se ( , 3.4%)*
O
O
5a
X=NH ( ) X=S (
5c
)
5d
)
*AgOAc (2.0 equiv) was
used as an oxidant
5b
X=O (
)
X=Se (
Scheme 3. Pd(OAc)2-catalyzed intramolecular cyclization of 5.
6

O
O
O
O
OAc
Pd
O
O
O
O
Pd(OAc)2
H
Pd
X
X
X
Reactant
IM1
TS2a
5a
5c
)
X=NH (
X=O (
)
X=S (
or
O
5b
5d
)
)
X=Se (
OH
O
O
O
O
O
Pd
X
O
O
H
Pd
X
X
O
Product
IM2
TS2b
6a
6c
)
X=NH (
X=O (
)
X=S (
6b
6d
)
)
X=Se (
Scheme 4. Proposed mechanism for Pd(OAc)2-catalyzed intramolecular cyclization of
5.
To reveal the reaction mechanism of Pd(OAc)2-catalyzed intramolecular
cyclization of 5, we performed DFT calculations (see, SI for computational detail).
The proposed mechanism and relative Gibbs free energy profile for the reaction are
shown in Scheme 4 and Fig. 1, respectively. This reaction is stepwise reaction and has
three transition states corresponding to coordination of Pd(OAc) (TS1), ring closure
process to form six-membered ring (TS2), and elimination of Pd atom (TS3).
According to the previous study,16 we calculated two kinds of reaction pathways for
TS2 (TS2a and TS2b). In TS2b, AcOH participates to abstract H atom from phenyl
ring, whereas AcOH does not participate in TS2a. Our DFT calculation implies that
the ring closure reaction proceed without AcOH, because the relative Gibbs free
energy of TS2a is 5.4 ~ 8.8 kcal/mol lower than that of TS2b. Since the relative
Gibbs free energy of TS1 and TS2a are higher than TS3, the rate-determining step is
coordination of Pd(OAc) or ring closure reaction to form six-membered ring.
7

Fig. 1. Relative Gibbs free energy profile of Pd(OAc)2-catalyzed intramolecular
cyclization of 5.
The relative energies of TS1 and TS2a are the lowest in 5a, whereas the highest
values are found in 5d. Our DFT calculation implies that the reaction of 5a occurs
more easily than 5b–5d. This calculation result is in good agreement with the
experimental yields of 6a–6d (Scheme 3). To interpret the difference in reactivity
among 5a–5d, we focus on the atomic charge (natural charge) of carbon atom in
quinone ring in Reactant and in phenyl ring in IM1 (Fig. 2). These carbon atoms are
negatively-charged and their negative charges become smaller in the order of 5a > 5b
> 5c > 5d. The electron distribution around the carbon atom is, thus, clearly affected
by X atom. Since positively-charged Pd atom attacks the carbon atom to proceed the
reaction, it can be speculated that the negative charge on the carbon atom determines
the reactivity of compound.
8

Fig. 2. Calculated atomic charge of C (red-colored) atom in Reactant and IM1.
To gain insights into the structure-activity relationships, the synthetic derivatives
(6) were assessed their in vitro cytotoxicity on HL-60 cells. The furan derivative -
brazanquinone 6b exhibited strong cytotoxicity with a low IC50 value of 0.41 M. On
the other hand, the IC50 values of pyrrole, thiophene, and selenophene derivatives 6a,
6c, and 6d were over 50 M. Comparing 6a with murrayaquinone A (3a), the directly
attached benzene ring on the quinone moiety decreased the potential. Very
interestingly, the heteroatom in five-membered rings was most influential on
cytotoxicity of these compounds.
Reactive oxygen species (ROS) including hydroxyl radicals (•OH), superoxide
•-
anions (O2 ), singlet oxygen (1O2), and hydrogen peroxide (H2O2) are constantly
generated in organisms.26 Cells are particularly sensitive to ROS-resulted redox
imbalance, thereby causing damages of biological macromolecules and
genotoxicity.27 Therefore, in this study, it was hypothesized that the potent
carbazoloquinone derivatives exert inhibition of HL-60 cell proliferation via affecting
cellular redox environment. Intracellular and extracellular ROS levels were measured
following 3 h-treatment with murrayaquinone A (3a), 6a, and -brazanquinone (6b)
by the nitroblue tetrazolium (NBT) reduction method.28,29 Results are summarized in
Fig. 3. When exposed to 1.0 μM of compounds 3a and 6b, intracellular ROS levels
were increased approximately 2.3–2.4-fold compared to untreated controls, but the
9

increase in 6a-treated cells was minimal. Comparatively high extracellular ROS levels
were detected in 3a- and 6b-treated cell culture medium.
Fig. 3. Intracellular and extracellular ROS levels in compounds 3a-, 6a-, and 6b-
treated HL-60 cells. The cells were treated with 1.0 M of 3a, 6a, and 6b for 3 h.
NBT reduction assay was performed to determine cellular ROS levels and the results
were quantified relative to untreated controls (mean ± S.D., n = 3).
The nuclear morphology of HL-60 cells stained with Hoechst 33342 was
observed. The cells in the rapid phase of growth were exposed to the derivatives 3a
and 6b at a concentration of 1.0 μM for 24 h. The microscopic images are shown in
Fig. 4. In the vehicle control group, the cells had uniform round- or oval-shaped
nuclei with evenly distributed chromatin, while treatment with 3a and 6b resulted in
alterations in chromatin features including crenation, condensation, and fragmentation,
which are cues for the appearance of apoptosis.
10

Fig. 4. Morphological changes of HL-60 cells induced by compounds 3a and 6b at a
concentration of 1.0 M. The cells were treated with 3a and 6b for 24 h, and were
stained with Hoechst 33342. (A) Control; (B) Treatment with 3a; (C) Treatment with
6b.
In general, quinone molecules undergo redox cycling involved continuous
generation of ROS through successive reduction and oxidation reactions.30,31 There
are several evidences showing that ROS act as critical mediators in cell apoptosis in
the biological context.32,33 A potent naturally occurring quinone -lapachone isolated
from Tabebuia avellanedae (Bignoniaceae family), displayed promising cytotoxic
activity due to its ability to promote ROS-mediated apoptosis34,35 and to disrupt redox
homeostasis.15 Aziridinylquinone RH-1 (2,5-diaziridinyl-3-hydroxymethyl-6-methyl-
cyclohexa-2,5-diene-1,4-dione) induced apoptosis by the formation of ROS arising
from its redox cycling.36 It is believed that a drastic increase in cellular ROS
accumulation and redox imbalance caused by 3a and 6b profoundly inhibited cell
proliferation and induced apoptotic cell death. Mechanically, their quinone moieties
can be reductively metabolized to the corresponding hydroquinones, which easily
transfer electrons and protons to molecular oxygen under backformation of the
original quinones.37–39 It is presumed that ROS generated by their redox cycling
contribute largely to cytotoxic actions of these carbazoloquinone derivatives.
11

In conclusion, the present study reports on the synthesis of carbazoloquinone
derivatives and their inhibition on HL-60 cells via induction of ROS-mediated
apoptosis. The promising antileukemic properties of murrayaquinone A (3a) and -
brazanquinone (6b) would be helpful in the synthesis of a library of quinone-based
molecules for extensive anticancer studies, which would be used to develop more
appropriate drug candidates. Further research continues in our laboratory and will be
reported in due course.
Acknowledgments
This work was supported by a Grant-in-Aid for Science Research from the
Ministry of Education, Culture, Sports, Science and Technology of Japan (No.
18K05028) to which we are grateful.
Supplementary data
Supplementary data associated with this article can be found, in the online
version, at http://XXXXXXXX.
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14

 Palladium-catalyzed
intramolecular
cyclization
mechanism
of
carbazoloquinone derivatives was elucidated by DFT calculations.
 Murrayaquinone A and -brazanquinone elevated cellular levels of ROS in
HL-60 cells.
 Murrayaquinone A and -brazanquinone induced apoptotic cell death.
15

Synthesis of carbazoloquinone derivatives and their antileukemic
activity via modulating cellular reactive oxygen species
Natsumi Suematsu, Masayuki Ninomiya, Hodaka Sugiyama, Taro Udagawa,
Kaori Tanaka, and Mamoru Koketsu
Murrayaquinone A
-Brazanquinone
O
O
N
O
H
O
O
ROS
ROS
16