Quinonoid compounds via reactions of lawsone and 2-aminonaphthoquinone with α-bromonitroalkenes and nitroallylic acetates: Structural diversity by C-ring modification and cytotoxic evaluation against cancer cells

Article abstract of DOI:10.1016/j.ejmech.2018.03.079

Morita-Baylis-Hillman acetates and α-bromonitroalkenes have been employed in cascade reactions with lawsone and 2-aminonaphthoquinone for the one-pot synthesis of heterocycle fused quinonoid compounds. The reactions reported here utilized the 1,3-binucleo

Full text of DOI:10.1016/j.ejmech.2018.03.079

Accepted Manuscript
Quinonoid compounds via reactions of lawsone and 2-aminonaphthoquinone with α-
bromonitroalkenes and nitroallylic acetates: Structural diversity by C-ring modification
and cytotoxic evaluation against cancer cells
Thekke V. Baiju, Renata G. Almeida, Sudheesh T. Sivanandan, Carlos A. de Simone,
Lucas M. Brito, Bruno C. Cavalcanti, Claudia Pessoa, Irishi N.N. Namboothiri,
Eufrânio N. da Silva Júnior
PII:
S0223-5234(18)30322-2
10.1016/j.ejmech.2018.03.079
EJMECH 10344
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 January 2018
Revised Date: 27 March 2018
Accepted Date: 29 March 2018
Please cite this article as: T.V. Baiju, R.G. Almeida, S.T. Sivanandan, C.A. de Simone, L.M. Brito, B.C.
Cavalcanti, C. Pessoa, I.N.N. Namboothiri, Eufrâ.N. da Silva Júnior, Quinonoid compounds via reactions
of lawsone and 2-aminonaphthoquinone with α-bromonitroalkenes and nitroallylic acetates: Structural
diversity by C-ring modification and cytotoxic evaluation against cancer cells, European Journal of
Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.03.079.
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ACCEPTED MANUSCRIPT
Graphical abstract
Pyrrole and furan fused naphthoquinones were designed and synthesized from
corresponding 2-aminonaphthoquinones and 2-hydroxynaphthoquinone on reaction
with conjugated nitroalkenes and evaluated against several cancer cell lines showing, in
some cases, IC50 values below 1.5 µM.

ACCEPTED MANUSCRIPT
Quinonoid Compounds via Reactions of Lawsone and 2-Aminonaphthoquinone
with α-Bromonitroalkenes and Nitroallylic Acetates: Structural Diversity by
C-ring Modification and Cytotoxic Evaluation against Cancer Cells
Thekke V. Baiju,a Renata G. Almeida,b Sudheesh T. Sivanandan,a Carlos A. de Simone,c
Lucas M. Brito,d Bruno C. Cavalcanti,d Claudia Pessoa,d Irishi N. N. Namboothiria* and
Eufrânio N. da Silva Júniorb*
aDepartment of Chemistry, Indian Institute of Technology Bombay, Mumbai, 400 076,
India; bInstitute of Exact Sciences, Department of Chemistry, Federal University of
Minas Gerais, CEP 31270-901, Belo Horizonte, MG, Brazil; cDepartment of Physics
and Informatics, Institute of Physics, University of São Paulo, São Carlos, 13560-160,
SP, Brazil; dDepartment of Physiology and Pharmacology, Federal University of
Ceará, CEP 60180-900, Fortaleza, CE, Brazil.
Corresponding authors: I.N.N. Namboothiri, E-mail: irishi@chem.iitb.ac.in; E.N. da
Silva Júnior, E-mail: eufranio@ufmg.br
Abstract. Morita-Baylis-Hillman acetates and α-bromonitroalkenes have been
employed in cascade reactions with lawsone and 2-aminonaphthoquinone for the one-
pot synthesis of heterocycle fused quinonoid compounds. The reactions reported here
utilized the 1,3-binucleophilic potential of hydroxy- and aminonaphthoquinones and the
1,2/1,3-bielectrophilic potential of bromonitroalkenes and Morita-Baylis-Hillman
acetates for the synthesis of pyrrole and furan fused naphthoquinones. The synthesized
compounds were evaluated against HCT-116 (human colon carcinoma cells), PC3
(human prostate cancer cells), HL-60 (human promyelocytic leukemia cells), SF295
(human glioblastoma cells) and NCI-H460 (human lung cancer cells) and exhibited
antitumor activity with IC50 values as low as < 2 µM. Selected compounds were also
evaluated against OVCAR-8 (ovary), MX-1 (breast) and JURKAT (leukemia) cell lines.
The cytotoxic potential of the quinones evaluated was also assayed using non-tumor
cells, exemplified by peripheral blood mononuclear (PBMC) and L929 cells.
1

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Introduction
Quinonoids are potent bioactive compounds that are well-known for their
medicinal value, particularly, for their excellent cancer chemotherapeutic efficiency.
Naphthoquinones are important quinonoid compounds that are abundant in nature and
exhibit broad spectrum of biological activities such as anticancer [1], antifungal [2],
antibacterial [3], antiviral [4], anti-inflammatory [5], antimalarial [6], antiplatelet [7],
antithrombotic and antiallergic activities [7]. Because of these unique biological
activities and wide spread applications in pharmaceutical field, the design, synthesis and
biological screening of novel naphthoquinone derivatives are at the center stage of
research in the biomedical field [8]. Considering the increased mortality rate of tumor
patients, recent research has been immensely focused on developing novel antitumor
agents from naphthoquinones. Consequently, several reports appeared on the synthesis
as well as biological screening of naphthoquinone derivatives [9]. Also, there exists
tremendous interest in developing diverse heterocycle fused naphthoquinones owing to
their enhanced biological activity compared to the parent naphthoquinone moieties [10].
Heterocycle containing naphthoquinones, especially the pyrrole and furan fused
derivatives, are interesting candidates for medicinal chemistry purposes and are well
studied [11]. The impact of heterocycle on the naphthoquinone bioactivity was
established by structure-activity relationship studies [12]. Some of the bioactive
naphthoquinone motifs containing pyrrole and furan skeleton are illustrated in Scheme
1A [11,13].
Pyrrole fused naphthoquinones (p-indoloquinones) received great interest in
pharmaceutical field due to their applicability in drug release systems [14] and are
important precursors of bioactive molecules [15]. The lead role of these heterocycle
fused analogues in medicinal chemistry prompted synthetic chemists to develop novel
and efficacious synthetic strategies [16] and some of the recently practiced protocols in
this direction include ultrasound assisted reaction of 1,4-naphthoquinone and α-
aminoacetals [17], Diels-Alder reaction of indole-4,7-dione with conjugated dienes [18],
C,N-dialkylation of enaminones by 2,3-dichloronaphthoquinone [19] and various
transition metal (Pd, Cu, Au) catalyzed annulation reaction of naphthoquinone
derivatives [20]. Additionally, numerous other methods are also available for the
preparation of p-indoloquinone starting from 2-aminonaphthoquinone derivatives [21].
Even though many elegant protocols exist towards the synthesis of
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pyrrolonaphthoquinones, simple and milder methods are still highly desirable in view of
their pharmacological activities.
Diverse reactivity exhibited by conjugated nitroalkenes and their derivatives
were well-explored for the synthesis of various heterocycles [22]. In particular, the
nitroallylic acetate derived from Morita-Baylis-Hillman (MBH) adduct of nitroalkene
[23] and α-bromonitroalkene [24] serve as excellent substrates for the synthesis of
heterocycles. In 2014, our group reported an efficient and enantioselective method for
the synthesis of pyranonaphthoquinones from lawsone and nitroallylic acetate derived
MBH-adduct of nitrostyrene and formaldehyde in the presence of a chiral squaramide
catalyst [25]. In addition, α-bromonitroalkene was successfully employed to synthesize
chiral dihydrofuran and furan fused naphthoquinones (Scheme 1B) [26,27]. These two
types of compounds are important examples of C-ring modified lapachone-type
molecules with antitumor activities. Our group [28] and others [29] have utilized the
strategy of C-ring modification of quinones for preparing potential bioactive
compounds in a drug discovery program aiming new antitumor compounds (Scheme
1B).
Methodologies involving transition-metal catalysis [30], organocatalysis [31]
and acid catalysis [32] were developed for the synthesis of quinones with diverse
biological activities. However, Organic Chemists are often challenged to develop new
synthetic strategies for the preparation of new substances with potential bioactivities,
and still more, new methods that can be employed in the synthesis of natural bioactive
products of this class of compounds. Our continued interest in developing novel
synthetic strategies by utilizing nitroalkene derivatives together with synthesis and
antitumor evaluation of naphthoquinone derivatives prompted us to investigate the
reactivity of 2-aminonaphthoquinones and 2-hydroxynaphthoquinones with conjugated
nitroalkene derivatives (Scheme 1C). Thus, taking into account the reasons discussed
above, herein, we describe the synthesis of pyrrole and furan fused naphthoquinones
from corresponding 2-aminonaphthoquinones and 2-hydroxynaphthoquinone on
reaction with conjugated nitroalkenes and their evaluation against diverse cancer and
non-tumor cell lines.
3

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(A) Bioactive and natural products containing pyrrole and furan fused naphthoquinones
R1
O
O
OH
O
O
R2
O
OH
O
OH
O
O
Avicequinone B: R1 = R2 = H
Avicequinone D: R1 = OH, R2 = C(Me)2OH
O
O
Kigelinone
O
Maturone
O
O
CH2Ph
OH
O
H
CH3
N
N
CH3
CH3
N
O
FNQ3
CH3
OH
P(OEt)2
O
O
N
O
Utahmycin B
O
OH
(B) Bioactive quinones designed by ring modification of lapachone prototypes
O
O
O
Furonaphthoquinones
with antitumor activity
Ref. 28
C
O
C
-Lapachone derivatives
Asymmetric version
Ref. 25 and 26
N
N
O
N
SPh
O
O
Ph
R
O
O
C-ring
modification
NO2
C
O
H
C
O
O
O
O
O
R
C-ring
modification
C-ring
modification
H
O
O
C
O
NO2
C
O
Pterocarpanquinone with
antitumor activity
Ref. 29
n
n = 1 or 2
(C) This Work: New pyrrole and furan fused naphthoquinones and their antitumor evaluation
22 examples
23 examples
R1
O
NO2
E
O
R1
O
R
NO2
X
X
R
R1
X
AcO
Br
Base, THF
E
Base, THF
E = CO2Et
R
O
R
O
O
9 compounds with
IC50 < 10 µM
R = o-NO2C6H4: highly
active (IC50 < 2 µM)
X = -NH or O
5 compounds
with IC50 < 10 µM
*Most of the compounds assayed
against five or more cancer cell lines
Scheme 1. An Overview on Biological and Synthetic Aspects of Heterocycle Fused
Naphthoquinone Motifs.
Results and Discussion
Chemistry
Setting the goal to synthesize heterocycle fused naphthoquinones, we began our
investigation by reacting 2-aminonaphthoquinone derivative 1a and α-bromonitroalkene
2a in the presence 2 equiv CS2CO3 in THF at room temperature (Table 1). Gratifyingly,
as expected, pyrrole fused naphthoquinone derivative 3a was formed in 47% yield
(entry 1). The structure of the product 3a was established by various spectroscopic
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analyses and finally unambiguously confirmed by single crystal X-ray analysis (Table
1). After the preliminary experiment, optimization studies were conducted to improve
the yield by choosing 1a and 2a as model substrates. Initially, different bases were
screened to get the best reaction conditions. The organic bases such as DABCO and
TMG provided the product 3a in poor yield (entries 2 and 3). Inorganic bases such as
K2CO3 (entry 4), and KOH (entry 5), besides Cs2CO3 (entry 1) were screened in THF as
solvent and among these KOH afforded the product 3a in 55% yield. Further screening
of different solvents revealed that DMF and DMSO are not suitable for our reaction
(entries 6-7) and DCM was inferior to THF, especially in terms of reaction rate (entries
5 and 8). However, increasing the amount of α-bromonitroalkene 2a to 1.5 equiv
improved the yield of 3a to 73% (entry 9) which subsequently reached 86% when silica
gel was replaced by neutral alumina for column chromatography (entry 10).
Table 1. Optimization of Reaction Conditionsa
To explore the scope of the reaction, experiments were carried out with
differently
substituted
α-bromonitroalkenes
2
and
various
N-arylated
aminonaphthoquinones 1 under the optimized reaction conditions (Table 2). Irrespective
of the electronic nature, α-bromonitroalkenes bearing both electron rich and electron
5

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deficient aryl groups tolerated the reaction providing 1,3-diaryl pyrrolonaphthoquinones
3a-e in moderate to good yields (35-86%). Considering the relevance of heterocycles in
the naphthoquinone core, we examined the reactivity of furan containing α-
bromonitroalkene with 2-aminonaphthoquinone which also furnished 3-furyl
pyrrolonaphthoquinone 3f, though in moderate yield. Next, the substitution pattern on
the N-aryl group of 2-aminonaphthoquinone 1 was varied. Regardless of the electronic
properties of aryl substituents, various N-arylated aminonaphthoquinones 1 smoothly
reacted with α-bromonitroalkenes 2 and provided the products 3h-l in reasonable yields.
Furthermore, N-alkyl substituted 2-aminonaphthoquinone also participated in the
reaction and delivered the corresponding pyrrolonaphthoquinone derivative 3m in
moderate yield.
Table 2. Substrate Scope for the Synthesis of Pyrrolonaphthoquinonesa,b
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Encouraged by these results, we decided to examine the reactivity of Morita-
Baylis-Hillman (MBH) acetates of nitroalkenes (α-nitroallylic acetates) 4 with 2-
aminonaphthoquinones 1. Similar to α-bromonitroalkenes 2, nitroallylic acetates 4 also
behave as bielectrophiles. Several carbocycles and heterocycles were synthesized by
utilizing the 1,2- and 1,3-bielectrophilic nature of nitroallylic acetates 4 by several
research groups including ours [23]. Under the established reaction conditions, we
investigated the reactivity of various aryl substituted MBH-acetates 4 with 2-
aminonaphthouinones 1. As expected, diverse functionalized pyrrolonaphthoquinones 5
were formed in satisfactory yields and the results are summarized in Table 3. Various
N-aryl substituents were also tolerated for this reaction to provide 1,2,3-trisubstituted
pyrrolonaphthoquinones 5. Moreover, the reaction was also compatible for heterocylic
nitroallylic acetates and produced 3-furyl and 3-thiophene substituted
pyrrolonaphthoquinones 5e and 5f, respectively, in reasonable yields. The structure of
the formed pyrrolonaphthoquinones was characterized by various spectroscopic
analyses and the regiochemistry was further confirmed by single crystal X-ray analysis
of a representative product 5i (Table 3). It should be noted that the simple primary 2-
aminonaphthoquinone failed to yield the pyrrolonaphthoquinones on reaction with α-
bromonitroalkenes 2 and MBH acetates 4.
7

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Table 3. Synthesis of Pyrrolonaphthoquinones 5 from 2-Aminonaphthoquinones 1 and
MBH-Acetates 4a,b
After successful synthesis of pyrrole fused naphthoquinone derivatives, we
further proceeded to synthesize the furan fused naphthoquinones 7 by employing 2-
hydroxynaphthoquinone
6
(Table 4). The method for the synthesis of
furanonaphthoquiones 7 from 2-hydroxynaphthoquinone 6 and α-bromonitroalkenes 2
was previously reported [27]. Considering the biological potential of furan fused
naphthoquinones, we have synthesized these furanonaphthoquinone scaffolds 7 under
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conditions similar to those reported [27] and subjected them to antitumor evaluation
(vide infra). Various 3-aryl furanonaphthoquinones 7 were synthesized in good to
excellent yields (56-93%) and the results obtained are summarized in Table 4.
Table 4. Synthesis of Furanonaphthoquinone 7 from 2-Hydroxynaphthoquinone 6 and
α-Bromonitroalkenes 2a,b
O
O
OH
O
NaOAc, TBAB
H2O, 70 ºC, 7 h
NO2
R
+
Br
2
R
O
7
O
6
O
O
O
O
O
O
O
O
O
O
O
7a
87%
7b
73%
7c
56%
F
Br
SCH3
O
O
O
O
O
O
NO2
O
O
7g
75%
S
7f
R
7d, R = H
82%
7e, R = OCH3
80%
OCH3
67%
O
O
O
O
7i
74%
O
7h
93%
O
O
a Reaction scale: 2-Hydroxynaphthoquinone 6 (0.15 mmol), -bromonitroalkene 2
(0.3 mmol, 2 equiv), NaOAc (0.18 mmol, 1.2 equiv), TBAB (0.03 mmol, 20 mol%).
b Yields after recrystallization from EtOAc.
The reactivity of MBH-acetates 4 with 2-hydroxynaphthoquinone 6 was then
explored towards the synthesis of highly substituted furanonaphthoquinones 8 (Table 5).
The initial reaction was performed by reacting 2-hydroxynaphthoquinone 6 with MBH-
acetate 4a in the presence of 1 equiv of DABCO in THF at room temperature. As
observed in the case of 2-aminonaphthoquinone 1, the reaction afforded expected
furanonaphthoquinone derivative 8d in low (12%) yield (entry 1). Upon observation of
9

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this initial reactivity, efforts were made to optimize the reaction conditions to improve
the yield. Screening of other different organic bases such as DBU, TMG, DMAP
provided the product in inferior yield (entries 2-4). Further, we have screened few
inorganic bases such as KOH, Cs2CO3 and K2CO3 (entries 5-7) of which Cs2CO3
provided better yield of 51% (entry 6). Among various solvents screened, the initially
employed reaction medium, THF, turned out to be superior to others such as CH3CN
and toluene (entries 6, 8 and 9). Finally, the reaction was performed under microwave
irradiation conditions in the presence of Cs2CO3 in THF at 40 oC. In this case, the yield
slightly improved to 54% (entry 10). Changing the solvent from THF to CH3CN or base
from Cs2CO3 to KOH and DMAP under the microwave conditions led to unsatisfactory
results (entries 11-13). Subsequently, the effect of base loading on the product yield was
investigated. While increasing the base loading from 1.0 equiv to 1.5 and 2.0 equiv led
to no change and drop in the yield, respectively (entries 14-15), decreasing the base
loading from 1.0 equiv to 0.5 equiv caused enhancement in the yield to 65% (entry 16).
Further decreasing the base loading or increasing the reaction temperature did not
improve the yield (entries 17-18).
10

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Table 5. Optimization of Reaction Conditionsa,b
Under the above optimized conditions, the reaction was generalized by varying
the MBH acetates 4 and hydroxynaphthoquinones 6 (Table 6). MBH acetates 4
possessing electron donating and electron withdrawing substituents on the aryl ring,
heteroaryl and alkyl groups furnished the corresponding furanonaphthoquinones 8 in
moderate to good yield. When the reaction was performed by employing α-naphthyl
derived MBH-acetate, along with the major product, furanonaphthoquinone derivative
8k, which was formed in 30% yield, the pyran ring fused naphthoquinone 9 was
formed in low (10%) yield.
11

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Table 6. Synthesis of Furanonaphthoquinones 8 from 2-Hydroxynaphthoquinones 6 and
MBH-acetates 4a,b
Based on our previous reports [23], a possible mechanism for the formation of
pyrrole and furan fused naphthoquinones which is outlined in Scheme 2. The base
abstracts a proton from lawsone 6 or 2-aminonaphthoquinone 1 and generates
corresponding quinonoid anion. The anion reacts with α-bromonitroalkenes 2 in a
12

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Michael fashion to give intermediate IA (path A). Intramolecular nucleophilic
substitution of bromide in IA in a 5-exo-tet fashion provides the cyclized intermediate
IIA which then undergoes base assisted HNO2 elimination to afford pyrrole and furan
fused naphthoquinones 3 and 7. Similarly, the bielectrophilic Morita-Baylis-Hillman
acetate 4 reacts with quinone in an SN2′ fashion and generates intermediate IB.
Subsequently, intramolecular aza/oxa-Michael reaction in a 5-exo-trig manner results in
the cyclized intermediate IIB. Further, base facilitated elimination of HNO2 from
intermediate IIB affords the respective heterocycle fused naphthoquinones 5 and 8. The
pyranonaphthoquinone 9 (Table 6) was formed through a 6-endo-trig cyclization of
intermediate 1B (not shown).
Scheme 2. Possible Mechanism for the Formation of Pyrrole and Furan Fused
Naphthoquinones
13

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Table 7. Selected Single Crystal X-ray data for compounds 3a, 5i, 7i, 8e and 9.
Parameters
CCDC
Empirical formula
Formula weight
Temperature
Wavelength
3a
1570229
5i
1569289
C30 H24 Br N O6
574.41
7h
8e
1570230
C88 H60 Cl4 O20
1579.16
293(2) K
0.71073 Å
Trigonal
R -3
9
1569291
C22H12 O3
324.32
293(2) K
0.71073 Å
Monoclinic
P21/c
1569290
C26 H18 O5
410.40
293(2) K
1.54184 Å
Triclinic
P -1
C26 H19 N O3
393.42
293(2) K
0.71073 Å
Monoclinic
P 1 21/n 1
293(2) K
0.71073 Å
Triclinic
P -1
Crystal system
Space group
a = 15.2899(6) Å
b = 14.9166(6) Å
c = 17.4055(7) Å
α = 90°
a = 7.9504(6) Å
b = 10.8078(8) Å
c = 15.5154(12) Å
α = 81.431(6)°.
β = 83.263(6)°.
γ = 72.763(7)°.
a = 12.484(5) Å
b = 7.767(3) Å
c = 15.583(8) Å
α = 90°
a = 59.1248(14) Å
b = 59.1248(14) Å
c = 10.6783(3) Å
α = 90°.
a = 8.1391(6) Å
b = 10.1038(5) Å
c = 12.7566(9) Å
α = 106.719(6)°
β = 90.539(6)°
Unit cell dimensions
β = 102.107(4)°
β = 90.83(5)°
β = 90°.
γ = 120°.
γ = 102.947(5)°
γ = 90°
γ = 90°
Volume
Z
Density (calculated)
Absorption
coefficient
F(000)
3881.4(3) Å3
8
1255.30(17) Å3
2
1510.8(4) Å3
4
32327.5(15) Å3
18
976.09(11) Å3
2
1.346 Mg/m3
1.520 Mg/m3
1.426 Mg/m3
1.460 Mg/m3
1.396 Mg/m3
0.088 mm-1
1.685 mm-1
0.095 mm-1
0.246 mm-1
0.793 mm-1
1648
588
672
14688
428
0.086 x 0.126 x 0.174
mm3
0.144 x 0.204 x
0.057 x 0.162 x
0.30 x 0.24 x
.054 x 0.095 x
Crystal size
0.206 mm3
0.239 mm3
0.16 mm3
0.227 mm3
Theta range for data
collection
2.11 to 25.00°
1.99 to 25.00°
2.6 to 34.3°
1.82 to 25.00°
4.70 to 64.99°
-7<=h<=9,
-12<=k<=12,
-18<=l<=17
-18<=h<=18,
-17<=k<=16,
-20<=l<=20
-19≤h1≤18,
-11≤k≤11,
-24≤l≤23
-70<=h<=39,
-68<=k<=70,
-12<=l<=7
-9<=h<=9,
-11<=k<=7,
-14<=l<=14
Index ranges
Reflections collected
Independent
38195
6839 [R(int) =
0.0919]
8645
4398 [R(int) =
0.0416]
21067
6059 [R(int) =
0.06]
39518
12614 [R(int) =
0.0856]
6695
3245 [R(int) =
0.0637]
reflections
Completeness to
theta = 25.00° is
99.9%
Completeness to
theta = 25.00° is theta = 25.00° is
Completeness to
Completeness to
theta = 25.00° is
99.6 %
Completeness to theta
= 64.99° is 97.5 %
Completeness
99.3 %
98.9%
Full-matrix least-
squares on F2
Full-matrix
least-squares on
F2
Full-matrix least-
squares on F2
Full-matrix least-
squares on F2
Full-matrix least-
squares on F2
Refinement method
Data / restraints /
parameters
6839 / 0 / 545
1.052
4398 / 0 / 346
1.055
960/ 0 / 226
0.933
12614 / 0 / 1013
1.019
3245 / 0 / 280
1.049
Goodness-of-fit on
F2
Final R indices
[I>2sigma(I)]
R1 = 0.0594, wR2 =
0.1375
R1 = 0.0399, wR2
= 0.0842
R1 = 0.14, wR2
= 0.27
R1 = 0.0703, wR2
= 0.1616
R1 = 0.0598, wR2 =
0.1558
R1 = 0.0889, wR2 =
0.1636
R1 = 0.0504, wR2
= 0.0903
R1 = 0.29, wR2
= 0.35
R1 = 0.1325, wR2
= 0.2158
R1 = 0.0737, wR2 =
0.1729
R indices (all data)
Largest diff. peak
and hole
0.242 and -0.286
0.422 and -0.590
0.30 and -0.37
0.610 and -0.779
0.335 and -0.244
e.Å-3
e.Å-3
e.Å-3
e.Å-3
e.Å-3
14

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Biological Studies
The Cytotoxicity of compounds 3a-3m, 5a-5j, 7a-7i, 8a-8b, 8d-8g, 8i-8k and
8m described above (Tables 2-4 and 6), were evaluated in vitro using the MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colourimetric assay against five
cancer cell lines: HCT-116 (human colon carcinoma cells), PC3 (human prostate cancer
cells), HL-60 (human promyelocytic leukemia cells), SF295 (human glioblastoma cells)
and NCI-H460 (human lung cancer cells). Non-tumor cells, human peripheral blood
mononuclear (PBMC), and murine fibroblast immortalized cell lines (L929) were used
to evaluate the selectivity of the compounds (Table 8). As previously described [33], the
compounds were classified according to their activity as highly active (IC50 < 2 µM),
moderately active (2 µM < IC50 < 10 µM), or inactive (IC50 > 10 µM).
The first class of compounds, pyrrolonaphthoquinones 3a-3m, was considered
inactive (IC50 > 10 µM) with an exception of compound 3d. The activity exhibited by
compound 3d may be due to the presence of electron withdrawing chlorine atom. It is
important to highlight that 3d presented low cytotoxicity against the non-tumor cell
lines evaluated. For example, the selectivity index calculated for 3d (represented by the
ratio of cytotoxicities between non-tumor cells and different lines of cancer cells), when
we have considered NCI-H460 cancer cells was 3.1. In this same direction, the value for
doxorubicin, drug used in clinical, is 3.6 considering PBMC as non-tumor cells.
The second class of pyrrolonaphthoquinone derivatives 5a-5j was also evaluated
against cancer and healthy cells. Not surprisingly, since the first class of
pyrrolonaphthoquinones 3a-3m was inactive, quinones 5a-5j were also not efficient to
kill cancer cells. Against HL-60 cancer cells, two compounds 5a and 5f have presented
moderate activity and compound 5h was cytotoxic against SF-295 cells. Even with
these three compounds active against two different cancer cells, we considered this
series not remarkable related to their antitumor potential.
Finally, furanonaphthoquinones 7a-i, 8a-8b, 8d-8g, 8i-8k and 8m were also
studied. These compounds were active against all cancer cells used in this study.
Compounds from the family of type 8 have presented moderate activity with IC50 in the
range of 3.97 to 10 µM. Among all the compounds evaluated 7a-i were the most potent
with IC50 values < 6.33 µM. The quinones 7b, 7f and 7i were the best among the series
with IC50 in the range 2.23 to 5.39 µM corroborating that the presence of electron
withdrawing groups is important for the antitumor activity. As compounds 7a-i were the
15

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most active derivatives against the five cancer cell lines evaluated (Table 8), we decided
to expand the panel of cancer cells used in this study and we also assayed 7a-i against
OVCAR-8 (ovary), MX-1 (breast) and JURKAT (leukemia) cell lines (Table 9). All the
compounds in the 7a-i series were considered moderately active (IC50 < 6 µM) of which
furanonaphthoquinone 7f with IC50 values in the range of 1.47 to 2.85 µM found to be
most active. As these compounds exhibited low cytotoxicity against healthy cells L-929
with IC50 > 13 µM, definitely, we have herein identified important structures with
potential antitumor activity for further studies.
The mechanism of action of naphthoquinones is intrinsically related to formation
of reactive oxygen species (ROS), which cause oxidative stress by oxidizing lipids,
proteins and DNA [34]. Recently, our group has described the synthesis of fluorescent
quinone-based BODIPY hybrids with potent antitumor activity and some insights about
their mechanism of action. Studies involving lipid peroxidation and determination of
reduced (GSH) and oxidized (GSSG) glutathione, besides subcellular localization by
confocal microscopy were accomplished. Cell imaging experiments indicated that the
quinoidal derivatives might preferentially get localized in the lysosomes of cancer cells
[35]. These studies represent an important example of the ability of quinones to generate
reactive oxygen species and the participation of these species in the mechanism of
pharmacological action.
The complexity of biological systems associated with the capacity of quinoidal
derivatives to act as multitarget systems become the complete determination of the
mechanism of action of such compounds a difficult task which requires a thorough
investigation [36]. For instance, β-lapachone (β-lap) is a well-known quinoidal natural
product with antitumor activity, also named as ARQ 501. This quinone is currently in
advanced clinical trials for the treatment of some types of cancer, as for instance,
pancreatic cancer [37]. The mechanism of action is related to destruction of cancer cells
with elevated endogenous levels of NAD(P)H:quinone oxidoreductase 1 (NQO1) [38].
This enzyme is considered as an important target associated to the potential cytotoxicity
of β-lap. Although its mechanism of action is related to this enzyme, Ohayon and
coworkers have demonstrated the hypothesis that β-lap would be able to act
nonreversibly for inhibition of deubiquitinases, suggesting that the therapeutic effect
could be due to multiple pathways, with USP2 oxidation [39]. These results are
important examples of the complexity associated with the mode of action of
naphthoquinoidal compounds such as β-lapachone.
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Recently, several researchers have shown in independent studies novel
quinonoid derivatives as CDC25 inhibitors [40-42]. Known for its function as key
regulator of the cell cycle in human cells, the cell division cycle 25 phosphatases
(CDC25) are a valuable target for the development of small quinonoid inhibitors of
therapeutic importance [41]. This new approach represents another possibility within a
wide range of possibilities related to the mechanism of action of naphthoquinones
demonstrating the diversity of targets associated with the mode of action of this
important class of antitumor compounds.
Bearing in mind, the important aspects associated with the mechanism of action
of quinones discussed above, we have selected 7a-7i and accomplished experiments of
comet assay and evaluation of nitrate/nitrite levels in relation to oxidative stress
parameters to establish preliminary data about the mechanism in HCT-116. Our data
(not shown here) suggest that the cytotoxicity of the naphthoquinoidal derivatives may
be associated with late apoptosis. To understand important factors and details about the
complex mode of action of these compounds, further experiments are underway in our
laboratories and will be reported in due course for the compounds 7a-7i. As an
illustrative example, in order to determine the oxidative damage triggered by compound
7i, the production of nitrate/nitrite as a result of NO release after treatment of HCT-116
cells was studied. The treatment of HCT-116 cancer cells with 7i during 24 h has
resulted in a concentration-dependent increase in nitrate/nitrite at all concentrations
tested (p<0.05) after 24 h exposure. Increase in the levels of NO, oxidative metabolic
products, suggests that oxidative damage is involved in the cytotoxic effects of the
naphthoquinone studied. Thus, the measured levels (µM) were 5.33 ± 0.64 and 8.33 ±
0.88 after the treatment with 5 and 10 µM of 7i (Figure 1A). Considering the potential
cytotoxic and pro-oxidant properties of 7i, we have also investigated its ability to induce
DNA damage. The in vitro alkaline (pH > 13) comet assay, a well-known assay for
screening potential genotoxic agents, was used to evaluate DNA damage index (DI) in
HCT-116 cells, after 24 h exposure of 7i according to the comet assay (Figure 1B). Our
results clearly show a significant (p<0.05) increase in the means of DI values at both
concentrations in relation to the negative control group (DI = 8.3 ± 0.88), while for cell
cultures treated at 5 and 10 µM, DI values were 42.88 ± 1.97 and 82.77 ± 3.4,
respectively.
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(B)
(A)
Figure 1. (A) Nitrate/nitrite formation after 24 h treatment of HCT-116 cells with
compound 7i. Data are presented as mean values ± S.E.M. of three independent
experiments. *p<0.05 compared to the negative control by ANOVA followed by
Tuckey´s test. (B) Effects of 7i on DNA damage index after 24 h of exposure using
alkaline version of comet assay. Data are presented as mean values ± S.E.M. of three
independent experiments. *p<0.05 compared to the negative control by ANOVA
followed by Tuckey´s test. DOX = Doxorubicin.
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Table 8. Cytotoxic activity of furano- and pyrrolonaphthoquinones expressed by IC50 µM (95% CI) of compounds 3a-3m, 5a-5j, 7a-i, 8a-8b,
8d-8g, 8i-8k and 8m in cancer and non-tumor cell lines after 72 h exposure, obtained by nonlinear regression for all cell lines from three
independent experiments.
Compounds
HCT-116
>12.71
PC3
HL-60
>12.71
>13.18
SF-295
>12.71
>13.18
NCI-H460
>12.71
L-929
>12.71
>13.18
PBMC
>12.71
>13.18
>12.71
>13.18
3a
3b
>13.18
>13.18
>12.22
8.88
>12.22
8.66
>12.22
3.15
>12.22
4.28
>12.22
3.81
>12.22
>12.10
>12.22
>12.10
3c
3d
(8.30-9.24)
>12.59
(8.20-8.81)
>12.59
(3.09-3.25)
>12.59
(4.10-4.39)
>12.59
(3.70-3.92)
>12.59
>12.59
>13.54
>12.98
>13.18
>12.59
>12.10
>13.25
>11.33
>13.77
>12.59
>13.54
>12.98
>13.18
>12.59
>12.10
>13.25
>11.33
>13.77
3e
3f
>13.54
>12.98
>13.18
>12.59
>12.10
>13.25
>11.33
>13.77
>13.54
>12.98
>13.18
>12.59
>12.10
>13.25
>11.33
>13.77
>13.54
>12.98
>13.18
>12.59
>12.10
>13.25
>11.33
>13.77
>13.54
>12.98
>13.18
>12.59
>12.10
>13.25
>11.33
>13.77
>13.54
>12.98
>13.18
>12.59
>12.10
>13.25
>11.33
>13.77
3g
3h
3i
3j
3k
3l
3m
5a
3.36
(2.91-3.88)
>10.43
>10.43
>10.43
>10.43
>10.43
>10.43
5b
5c
5d
5e
5f
>10.02
>10.79
>11.13
>11.76
>10.02
>10.79
>11.13
>11.76
>10.02
>10.79
>11.13
>10.02
>10.79
>11.13
>11.76
>10.02
>10.79
>11.13
>11.76
>10.02
>10.79
>11.13
>11.76
>10.02
>10.79
>11.13
>11.76
>11.76
2.63
(2.29–3.01)
>11.34
>11.34
>11.34
>11.34
>11.34
>11.34
5g
5h
>9.45
>9.45
>9.45
>9.45
6.45
>9.45
>9.45
>9.45
>17.45
>17.45
>17.45
>17.45
>17.45
>17.45
19

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(5.23–7.96)
5i
5j
7a
>8.72
>8.29
6.02
(5.51-6.43)
5.39
>8.72
>8.29
3.97
(3.76-4.21)
2.61
>8.72
>8.29
3.28
(2.97-3.59)
2.64
>8.72
>8.29
6.33
(5.92-6.77)
3.49
>8.72
>8.29
4.34
(4.10-4.48)
4.88
>8.72
>8.72
>8.29
>17.12
>8.29
13.56
(13.11-14.38)
>14.20
>14.20
>15.62
>16.44
>14.96
>15.67
>17.85
>15.42
>15.72
7b
7c
7d
7e
7f
(5.25-5.59)
(2.41-2.95)
(2.44-2.81)
(3.32-3.72)
(4.65-5.25)
6.03
(5.84-6.12)
5.78
(5.19-6.38)
5.20
(4.84-5.56)
1.28
(1.03-1.53)
5.17
(4.92-5.53)
6.17
(5.89-6.38)
2.49
(2.28-2.74)
4.96
(4.66-5.19)
3.89
(3.62-4.31)
1.59
(1.50-1.66)
4.85
(4.64-5.17)
1.82
(1.57-1.97)
3.09
(2.87-3.24)
4.31
(4.14-4.54)
3.41
(3.05-3.74)
1.66
(1.50-1.78)
3.28
(2.92-3.61)
2.37
(2.19-2.62)
2.23
3.43
(3.21-3.59)
4.99
(4.83-5.22)
3.74
(3.59-4.01)
2.94
(2.75-3.32)
4.96
(4.46-5.28)
4.44
(4.19-4.62)
3.71
4.68
(4.43-4.84)
6.34
(6.12-6.81)
4.07
(3.86-4.37)
2.53
(2.31-2.75)
5.67
(5.24-6.17)
3.79
(3.67-4.01)
3.61
(3.33-3.99)
8.86
(8.12–9.67)
>13.36
>12.82
>15.62
>16.44
>14.96
>15.67
>17.85
7g
7h
7i
13.02
(12.74-13.42)
>15.72
2.76
(2.60-2.95)
2.54
(2.26-3.01)
(1.94-2.51)
8.38
(7.48–9.38)
(3.48-3.86)
12.50
(11.32–12.82)
>13.36
8a
>13.89
>13.89
>13.89
>13.89
8b
8d
8e
>13.36
>12.82
>13.36
>13.36
>12.82
>13.36
>12.82
>13.36
>12.82
>12.82
>12.82
10.23
(6.83–15.34)
3.97
(3.45–4.57)
>14.28
5.91
(5.04–6.91)
4.01
(3.52–4.57)
>12.69
>12.69
>12.69
>12.69
>12.69
>12.69
6.16
(5.57–6.80)
>14.28
13.67
7.53
(6.47–8.77)
>14.28
9.09
8.47
(7.59–9.45)
>14.28
8f
>11.90
>14.28
>27.32
>11.90
>14.28
>27.32
>11.90
>14.28
>27.32
8g
8i
>27.32
>11.11
(12.07–15.48)
(7.34–11.28)
8.61
(7.13–10.39)
10.34
(9.01–11.85)
8j
>11.11
>11.11
>11.11
20

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6.69
(5.43–8.24)
10.65
(9.06–12.51)
8k
8m
>12.19
>12.95
>12.19
>12.95
>12.19
>12.19
>12.95
>12.19)
>12.95
>12.19
>12.95
>12.95
0.21
0.76
0.02
0.41
0.15
1.72
0.55
Doxorubicin
(0.16–0.29)
(0.59–0.93)
(0.01–0.02)
(0.21–0.47)
(0.13–0.18)
(1.58–1.87)
(0.41–0.58)
Table 9. Cytotoxic activity of compounds 7a-i expressed by IC50 µM (95% CI) in cancer cell lines OVCAR-8, MX-1 and JURKAT and non-
tumor cells L-929 after 72 h exposure, obtained by nonlinear regression for all cell lines from three independent experiments.
Compounds
7a
OVCAR-8
MX-1
JURKAT
L-929
5.43
(4.75-4.86)
2.98
5.20
(5.03-5.47)
3.23
3.45
(3.01-3.76)
2.78
13.56
(13.11-14.38)
>14.20
7b
(2.58-3.32) (3.09-3.49)
(2.61-3.04)
2.18
(2.06-2.59)
4.09
(3.87-4.37)
6.01
4.74
(4.37-5.24)
5.59
>15.62
>16.44
>14.96
>15.67
>17.85
7c
3.81
7d
(5.32-6.67)
(5.45-5.75)
(3.55-3.97)
3.71
(3.56-3.89)
1.47
(1.31-1.66)
4.21
4.69
(4.51-4.84)
2.25
(1.91-2.60)
5.39
5.05
(4.72-5.29)
2.85
(2.53-3.25)
4.92
7e
7f
7g
(5.21-5.78)
3.88
(3.76-3.98)
3.08
(4.46-5.32) (3.96-4.53)
3.30
(2.65-3.51)
2.92
2.09
(1.88-2.28)
2.04
13.02
(12.74-13.42)
>15.72
7h
7i
(2.98-3.36)
0.45
(2.76-3.04)
0.42
(1.82-2.20)
0.02
1.72
Doxorubicin
(0.41-0.49)
(0.39-0.45)
(0.01-0.03)
(1.58–1.87)
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Conclusions
An efficient and convenient protocol towards the synthesis of heterocycle fused
naphthoquinones has been developed. Base mediated one-pot reaction of α-
bromonitroalkenes and nitroallylic acetates as bielectrophiles with 2-amino/hydroxy-
1,4-naphthoquinones as binucleophiles produced pyrrole and furan fused
naphthoquinones in moderate to good yields. The steps involved an SN2’ reaction
followed by an intramolecular nucleophilic substitution in a 5-exo-tet fashion or an
intramolecular oxa- or aza-Michael addition in a 5-exo-trig fashion. All the compounds
were evaluated against five cancer cell lines and two non-tumor cell lines. We have
expanded the panel of cancer cells used in this study for selected compounds which
exhibited potent cytotoxicity, thus evaluating a total of eight cancer cell lines. We
identified a very promising compound, a furanonaphthoquinone (7f) possessing an o-
nitrophenyl group at position 3, with IC50 in the range of 1.28 to 2.94 µM against cancer
cells and low cytotoxity against non-tumor cells. Consequently, this compound
presented high selectivity index, and we consider it as a promising candidate for
subsequent studies. This study represents the application of a simple and efficient
synthetic method for developing novel potential drugs against cancer.
Experimental Section
General Experimental Details: The melting points recorded are uncorrected. NMR
1
1
1
spectra (1H, H decoupled 13C, 13C-APT, H- H COSY and NOESY) were recorded
with TMS as the internal standard. The coupling constants (J values are given in Hz).
High resolution mass spectra were recorded under ESI Q-TOF conditions. X-ray data
were collected on a diffractometer equipped with graphite monochromated Mo Kα
radiation. The structures were solved by direct methods shelxs97 and refined by full-
matrix least squares against F2 using shelxl97 software. All H atoms were located by
geometric considerations placed (C-H = 0.93-0.96 Å; N-H = 0.86 Å) and refined as
riding with Uiso(H) = 1.5Ueq(C-methyl) or 1.2Ueq(other). Crystallographic data for the
structure was deposited in the Cambridge Crystallographic Data Centre, with numbers
CCDC 1570229, 1569289, 1569291, 1570230 and 1569290 for 3a, 5i, 7h, 8e and 9,
respectively. Starting materials α-bromonitroalkenes 2 [43], Morita-Baylis-Hillman
acetates 4 [44], and 2-aminonaphthoquinones 1 [45] were prepared by following
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literature methods. 2-Hydroxynaphthoquinone 6 was commercially available (Aldrich)
and 6-methoxy-2-hydroxynaphthoquinone was prepared by following the reported
literature procedure [46].
General Procedure for the Synthesis of Pyrrolonaphthoquinones 3a-3m. To a
stirred solution of 2-aminonaphthoquinone 1 (0.5 mmol, 1.0 equiv) in THF (4 mL),
KOH (56 mg, 1.0 mmol, 2 equiv) was added and the reaction mixture was stirred at rt
under N2 atmosphere. After 5 min, α-bromonitroalkene 2 (0.75 mmol, 1.5 equiv) in
THF (2 mL) was added dropwise to the reaction mixture and continued stirring at rt.
After completion of the reaction (as evidenced by TLC), the solvent was removed in
vacuo and water (4 mL) was added to the crude residue. The aqueous phase was
extracted with ethyl acetate (3 × 10 mL) and the combined organic layers were washed
with brine (10 mL), dried over anhydrous sodium sulfate and concentrated under
reduced pressure. The residue on purification by column chromatography (neutral
alumina) using petroleum ether-ethyl acetate (5:95 to 10:90) yielded
pyrrolonaphthoquinones 3.
1-(4-Methoxyphenyl)-3-(p-tolyl)-1H-benzo[f]indole-4,9-dione (3a). Red solid; Yield
169 mg, 86%; mp 142-146 °C; IR (film, cm−1) 1658 (s), 1592 (w), 1515 (m), 1483 (w),
1
1383 (w), 1266 (s), 1248 (s), 1170 (w), 1031 (w), 916 (m), 836 (w), 716 (w); H NMR
(500 MHz, CDCl3) δ 8.17 (dd, J = 7.0, 1.5 Hz, 1H), 8.06 (dd, J = 7.0, 1.5 Hz, 1H), 7.65
(td, J = 7.0, 1.5 Hz, 2H), 7.62 (d, J = 8.0 Hz, 2H), 7.39 (d, J = 9.0 Hz, 2H), 7.26 (d, J =
8.0 Hz, 2H), 7.09 (s, 1H), 7.03 (d, J = 9.0 Hz, 2H), 3.89 (s, 3H), 2.41 (s, 3H); 13C NMR
(125 MHz, CDCl3) δ 181.2, 175.6, 160.0, 137.8, 134.6, 133.8, 133.4, 133.1, 131.9,
131.8, 131.1, 129.6, 129.1, 129.0, 127.3, 127.2, 126.9, 126.4, 124.7, 114.3, 55.7, 21.5;
HRMS (ES+, Ar) calcd for C26H20NO3 (MH+) 394.1438, found 394.1433.
1-(4-Methoxyphenyl)-3-phenyl-1H-benzo[f]indole-4,9-dione (3b). Yellow solid;
Yield 128 mg, 67%; mp 164-166 °C; IR (film, cm−1) 3062 (w), 2931 (w), 2835 (w),
1659 (vs), 1592 (m), 1514 (s), 1437 (m), 1387 (m), 1265 (s), 1247 (s), 1169 (m), 1029
1
(m), 915 (s), 834 (m), 761 (m), 717 (m), 696 (m); H NMR (500 MHz, CDCl3) δ 8.18
(dd, J = 7.0, 1.5 Hz, 1H), 8.07 (dd, J = 7.0, 1.5 Hz, 1H), 7.71 (d, J = 8.0 Hz, 2H), 7.67
(td, J = 7.0, 1.5 Hz, 2H), 7.45 (t, J = 8.0 Hz, 2H), 7.40 (d, J = 9.0 Hz, 2H), 7.38 (t, J =
13
8.0 Hz, 1H), 7.11 (s, 1H), 7.03 (d, J = 9.0 Hz, 2H), 3.89 (s, 3H); C NMR (125 MHz,
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CDCl3) δ 181.3, 175.6, 160.0, 134.5, 133.8, 133.4, 133.2, 132.6, 131.8, 131.2, 129.8,
129.2, 128.3, 128.0, 127.3, 127.2, 126.9, 126.4, 124.6, 114.4, 55.7; HRMS (ES+, Ar)
calcd for C25H17NO3Na (MNa+) 402.1101, found 402.1100.
1,3-Bis(4-methoxyphenyl)-1H-benzo[f]indole-4,9-dione (3c). Orange Solid; Yield 96
mg, 47%; mp 178-180 °C; IR (film, cm−1) 2923 (s), 2852 (m), 1655 (s), 1475 (m), 1248
1
(vs), 1030 (m), 744 (w); H NMR (400 MHz, CDCl3) δ 8.18 (dd, J = 8.0, 1.8 Hz, 1H),
8.07 (dd, J = 7.6, 1.8 Hz, 1H), 7.67 (d, J = 8.8 Hz, 2H), 7.64 (td, J = 8.0, 1.8 Hz, 2H),
7.39 (d, J = 8.8 Hz, 2H), 7.08 (s, 1H), 7.03 (d, J = 8.8 Hz, 2H), 6.99 (d, J = 8.8 Hz, 2H),
13
3.90 (s, 3H), 3.87 (s, 3H); C NMR (100 MHz, CDCl3) δ 181.3, 175.6, 160.0, 159.6,
134.6, 133.9, 133.4, 133.1, 131.9, 131.7, 130.9, 130.4, 127.3, 127.0, 126.9, 126.4,
124.9, 124.5, 114.4, 113.8, 55.8, 55.5; HRMS (ES+, Ar) calcd for C26H19NO4Na
(MNa+) 432.1206, found 432.1205.
3-(4-Chlorophenyl)-1-(4-methoxyphenyl)-1H-benzo[f]indole-4,9-dione (3d). Yellow
solid; Yield 117 mg, 56%; mp 173-175 °C; IR (film, cm−1) 3065 (w), 2920 (w), 2838
(w), 1659 (vs), 1591 (m), 1513 (s), 1494 (m), 1266 (vs), 1248 (vs), 1169 (w), 1091 (w),
1031 (w), 915 (w), 834 (m), 736 (w), 715 (m); 1H NMR (500 MHz, CDCl3) δ 8.16 (dd,
J = 7.5, 1.5 Hz, 1H), 8.06 (dd, J = 7.5, 1.5 Hz, 1H), 7.66 (d, J = 8.5 Hz, 2H), 7.63-7.69
(m, 2H), 7.40 (d, J = 8.5 Hz, 2H), 7.38 (d, J = 9.0 Hz, 2H), 7.09 (s, 1H), 7.02 (d, J = 9.0
Hz, 2H), 3.89 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 181.3, 175.5, 160.1, 134.4, 133.9,
133.7, 133.5, 133.3, 132.0, 131.7, 131.1, 131.0, 130.5, 128.5, 127.3, 126.8, 126.4,
125.9, 124.5, 114.4, 55.7; HRMS (ES+, Ar) calcd for C25H16ClNNaO3 (MNa+)
436.0711, found 436.0710.
3-(4-Fluorophenyl)-1-(4-methoxyphenyl)-1H-benzo[f]indole-4,9-dione (3e). Yellow
solid; Yield 71 mg, 35%; mp 145-147 °C; IR (film, cm−1) 3069 (w), 2934 (w), 2839
(w), 1659 (vs), 1592 (m), 1549 (w), 1515 (s), 1506 (s), 1443 (m), 1416 (m), 1266 (vs),
1248 (vs), 1169 (m), 1160 (m), 1031 (m), 916 (m), 837 (m), 736 (m), 715 (m); 1H NMR
(500 MHz, CDCl3) δ 8.16 (dd, J = 7.0, 2.0 Hz, 1H), 8.06 (dd, J = 6.5, 2.0 Hz, 1H), 7.73-
7.64 (m, 4H), 7.39 (d, J = 8.5 Hz, 2H), 7.13 (t, J = 9.0 Hz, 2H), 7.08 (s, 1H), 7.03 (d, J
8.5 Hz, 2H), 3.90 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 181.3, 175.6, 162.7 (J = 246.0
Hz), 160.1, 134.5, 133.8, 133.5, 133.3, 131.9, 131.8, 131.0, 130.9 (d, J = 8.0 Hz), 128.6
24

ACCEPTED MANUSCRIPT
(d, J = 3.0 Hz), 127.3, 126.9, 126.4, 126.1, 124.5, 115.2 (d, J = 21.0 Hz), 114.4, 55.8;
HRMS (ES+, Ar) calcd for C25H16FNNaO3 (MNa+) 420.1006, found 420.1007.
3-(Furan-2-yl)-1-(4-methoxyphenyl)-1H-benzo[f]indole-4,9-dione (3f). Yellow solid;
Yield 64 mg, 35%; mp 190-192 °C; IR (film, cm−1) 2924 (m), 2850 (w), 1658 (vs),
1592 (m), 1513 (s), 1488 (s), 1390 (m), 1267 (s), 1169 (w), 1030 (w), 923 (w), 834 (w),
1
716 (w); H NMR (500 MHz, CDCl3) δ 8.24 (d, J = 7.8 Hz, 1H), 8.05 (d, J = 7.8 Hz,
1H), 7.76 (d, J = 3.5 Hz, 1H), 7.69 (t, J = 7.8 Hz, 1H), 7.64 (t, J = 7.8 Hz, 1H), 7.46 (s,
1H), 7.41-7.43 (unresolved m, 1H), 7.39 (d, J = 8.5 Hz, 2H), 7.03 (d, J = 8.5 Hz, 2H),
6.53 (t, J = 1.5 Hz, 1H), 3.90 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 180.8, 175.4,
160.1, 147.7, 141.8, 134.3, 133.8, 133.4, 133.2, 131.8, 131.4, 129.7, 127.3, 126.9,
126.4, 122.9, 117.1, 114.4, 112.0, 111.1, 55.8; HRMS (ES+, Ar) calcd for C23H15NO4Na
(M+) 392.0893, found 392.0900.
3-(4-Methoxyphenyl)-1-phenyl-1H-benzo[f]indole-4,9-dione (3g). Red solid; Yield
137 mg, 72%; mp 173-175 °C; IR (film, cm−1) 2923 (m), 2850 (w), 1658 (vs), 1594
(m), 1467 (m), 1383 (m), 1265 (vs), 1249 (vs), 1179 (m), 1029 (m), 915 (m), 716 (m);
1H NMR (500 MHz, CDCl3) δ 8.18 (dd, J = 7.5, 1.5 Hz, 1H), 8.06 (dd, J = 7.5, 1.5 Hz,
1H), 7.67 (d, J = 8.5 Hz, 2H), 7.69-7.63 (m, 2H), 7.56-7.52 (m, 3H), 7.49-7.47 (m, 2H),
7.11 (s, 1H), 6.99 (d, J = 8.5 Hz, 2H), 3.86 (s, 3H); 13C NMR (125 MHz, CDCl3) δ
181.3, 175.5, 159.6, 139.0, 134.5, 133.8, 133.4, 133.2, 131.6, 130.7, 130.4, 129.2 (× 2),
127.2, 126.9, 126.4, 126.2, 124.8, 124.7, 113.8, 55.5; HRMS (ES+, Ar) calcd for
C25H17NNaO3 (MNa+) 402.1101, found 402.1102.
1-(4-Fluorophenyl)-3-(4-methoxyphenyl)-1H-benzo[f]indole-4,9-dione (3h). Orange
solid; Yield 123 mg, 60%; mp 168-170 °C; IR (film, cm−1) 2919 (vs), 2850 (m), 1656
(s), 1591 (w), 1544 (w), 1505 (m), 1467 (w), 1381 (w), 1246 (s), 914 (m), 838 (m), 715
(m); 1H NMR (500 MHz, CDCl3) δ 8.18 (dd, J = 6.5, 1.5 Hz, 1H), 8.05 (dd, J = 6.5, 1.5
Hz, 1H), 7.65 (d, J = 9.0 Hz, 2H), 7.70-7.63 (m, 2H), 7.46 (dd, J = 8.5, 4.5 Hz, 2H),
13
7.22 (t, J = 8.5 Hz, 2H), 7.07 (s, 1H), 6.98 (d, J = 9.0 Hz, 2H), 3.86 (s, 3H); C NMR
(125 MHz, CDCl3) δ 181.2, 175.6, 163.7, 160.70 (d, J = 266.3 Hz), 135.0 (d, J = 3.8
Hz), 134.5, 133.7, 133.5, 133.2, 131.7, 130.6, 130.4, 128.1 (d, J = 8.8 Hz), 127.3, 126.9,
126.3, 124.7, 124.6, 116.2 (d, J = 23.8 Hz), 113.8, 55.5; HRMS (ES+, Ar) calcd for
C25H17FNO3 (MH+) 398.1187, found 398.1185.
25

ACCEPTED MANUSCRIPT
1-(4-Chlorophenyl)-3-(4-methoxyphenyl)-1H-benzo[f]indole-4,9-dione (3i). Orange
solid; Yield 61 mg, 29%; mp 193-195 °C; IR (film, cm−1) 2923 (w), 2854 (w), 1660
(vs), 1611 (w), 1591 (w), 1495 (m), 1419 (w), 1382 (w), 1278 (m), 1245 (s), 1182 (w),
1091 (w), 1031 (w), 915 (m), 833 (m), 714 (m); 1H NMR (400 MHz, CDCl3) δ 8.17 (dd,
J = 6.6, 2.0 Hz, 1H), 8.05 (dd, J = 6.6, 2.0 Hz, 1H), 7.70-7.62 (m, 2H), 7.65 (d, J = 8.8
Hz, 2H), 7.50, 7.42 (ABq, J = 8.4 Hz, 2H), 7.06 (s, 1H), 6.98 (d, J = 8.8 Hz, 2H), 3.86
(s, 3H); 13C NMR (100 MHz, CDCl3) δ 181.2, 175.6, 159.7, 137.5, 135.1, 134.5, 133.6,
133.5, 133.2, 131.6, 130.4, 129.4, 127.6, 127.5, 126.9, 126.4, 124.9, 124.6, 113.8, 55.5;
HRMS (ES+, Ar) calcd for C25H16ClNNaO3 (MNa+) 436.0711, found 436.0714.
1-(4-Bromophenyl)-3-(p-tolyl)-1H-benzo[f]indole-4,9-dione (3j). Yellow solid; Yield
76 mg, 34%; mp 179-181 °C; IR (film, cm−1) 2923 (s), 2851 (m), 1661 (vs), 1591 (w),
1
1493 (s), 1470 (m), 1278 (m), 1249 (m), 915 (m), 828 (w), 714 (w); H NMR (500
MHz, CDCl3) δ 8.17 (dd, J = 7.0, 2.0 Hz, 1H), 8.05 (dd, J = 7.0, 2.0 Hz, 1H), 7.69-7.63
(m, 2H), 7.66 (d, J = 9.0 Hz, 2H), 7.60 (d, J = 8.0 Hz, 2H), 7.36 (d, J = 9.0 Hz, 2H),
7.26 (d, J = 8.0 Hz, 2H), 7.08 (s, 1H), 2.41 (s, 3H); 13C NMR (125 MHz, CDCl3) δ
181.1, 175.6, 138.0, 137.9, 134.4, 133.6 (× 2), 133.2, 132.4, 131.6, 130.5, 129.3, 129.1
(× 2), 127.9, 127.7, 126.9, 126.4, 125.1, 123.1, 21.5; HRMS (ES+, Ar) calcd for
C25H16BrNO2Na (MNa+) 464.0257, found 464.0254.
1-(3-Bromophenyl)-3-(p-tolyl)-1H-benzo[f]indole-4,9-dione (3k). Yellow solid; Yield
103 mg, 46%; mp 153-155 °C; IR (film, cm−1) 3069 (w), 2923 (s), 2853 (m), 1661 (vs),
1593 (s), 1486 (vs), 1384 (m), 1273 (vs), 1171 (m), 1003 (m), 919 (s), 786 (m), 719 (s);
1H NMR (500 MHz, CDCl3) δ 8.20 (dd, J = 7.3, 1.8 Hz, 1H), 8.09 (dd, J = 7.3, 1.8 Hz,
1H), 7.72-7.66 (m, 4H), 7.63 (d, J = 8.0 Hz, 2H), 7.46 (dt, J = 8.0, 1.0 Hz, 1H), 7.42 (t,
13
J = 8.0 Hz, 1H), 7.28 (d, J = 8.0 Hz, 2H), 7.11 (s, 1H), 2.44 (s, 3H); C NMR (125
MHz, CDCl3) δ 181.0, 175.4, 140.1, 138.0, 134.4, 133.6, 133.2, 132.3, 131.7, 130.5,
130.4, 129.4, 129.2, 129.1, 129.0, 127.7, 126.9, 126.4, 125.2, 125.0, 122.5, 21.5; HRMS
(ES+, Ar) calcd for C25H16BrNNaO2 (MNa+) 464.0257, found 464.0252.
1,3-Di-p-tolyl-1H-benzo[f]indole-4,9-dione (3l). Orange solid; Yield 79 mg, 42%; mp
134-136 °C; IR (film, cm−1) 3028 (w), 2922 (m), 2862 (w), 1660 (vs), 1593 (m), 1517
1
(m), 1483 (s), 1266 (s), 1171 (w), 998 (w), 916 (s), 807 (m), 719 (m); H NMR (400
MHz, CDCl3) δ 8.19 (dd, J = 7.6, 1.8 Hz, 1H), 8.07 (dd, J = 7.6, 1.8 Hz, 1H), 7.69-7.62
26

ACCEPTED MANUSCRIPT
(m, 2H), 7.63 (d, J = 8.0 Hz, 2H), 7.37, 7.33 (ABq, J = 8.4 Hz, 4H), 7.26 (d, J = 8.0 Hz,
2H), 7.10 (s, 1H), 2.47 (s, 3H), 2.42 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 181.2,
175.5, 139.2, 137.8, 136.5, 134.5, 133.8, 133.4, 133.1, 131.7, 130.9, 129.8, 129.6,
129.1, 129.0, 127.3, 126.8, 126.3, 125.9, 124.7, 21.5, 21.4; HRMS (ES+, Ar) calcd for
C26H19NO2Na (MNa+) 400.1308, found 400.1307.
1-Butyl-3-(4-chlorophenyl)-1H-benzo[f]indole-4,9-dione (3m). Yellow solid; Yield
81 mg, 44%; mp 90-92 °C; IR (film, cm−1) 3068 (w), 2960 (s), 2931 (m), 2873 (m),
1654 (vs), 1592 (s), 1546 (m), 1488 (s), 1454 (s), 1411 (s), 1380 (s), 1275 (vs), 1214 (s),
1
1091 (s), 1013 (m), 930 (s), 836 (m), 791 (m), 718 (s); H NMR (400 MHz, CDCl3) δ
8.10-8.09 (m, 2H), 7.66-7.59 (m, 2H), 7.58 (d, J = 8.8 Hz, 2H), 7.35 (d, J = 8.8 Hz, 2H),
6.97 (s, 1H), 4.44 (t, J = 7.4 Hz, 2H), 1.85 (quintet, J = 7.4 Hz, 2H), 1.41 (sextet, J = 7.4
13
Hz, 2H), 0.98 (t, J = 7.2 Hz, 3H); C NMR (125 MHz, CDCl3) δ 180.8, 176.3, 134.4,
133.6, 133.5, 133.3, 133.0, 131.3, 131.1, 130.3, 129.9, 128.3, 126.7, 126.2, 125.2,
123.9, 49.7, 33.0, 20.0, 13.8; HRMS (ES+, Ar) calcd for C22H18ClNO2Na (MNa+)
386.0918, found 386.0918.
General Procedure for the Synthesis of Pyrrolonaphthoquinones 5a-j. To a stirred
solution of 2-aminonaphthoquinone 1 (0.18 mmol, 1.0 equiv) in THF (3 mL) under N2,
was added base KOH (20 mg, 0.36 mmol, 2 equiv) and the reaction mixture was stirred
at rt. After 5 min, MBH acetate 4 (0.36 mmol, 2.0 equiv) dissolved in THF (2 mL) was
added dropwise and continued stirring till the completion of the reaction (monitored by
TLC). The solvent was evaporated in vacuo and water (5 mL) was added to the crude
reaction mixture. The aqueous phase was extracted with ethyl acetate (3 × 10 mL) and
the combined organic layers were washed with brine (10 mL), dried over anhydrous
sodium sulfate and concentrated under reduced pressure. The crude residue was further
purified by neutral alumina column chromatography by eluting with 5-15% EtOAc-
petroleum ether (gradient elution) to afford 2,3-disubstituted pyrrolonaphthoquinones 5.
Ethyl 2-(1-(4-methoxyphenyl)-4,9-dioxo-3-(p-tolyl)-4,9-dihydro-1H-benzo[f]indol-
2-yl)acetate (5a). Yellow solid; Yield 38 mg, 44%; mp 136-138 °C; IR (film, cm−1)
2919 (m), 2849 (w), 1735 (s), 1659 (vs), 1593 (m), 1511 (vs), 1486 (m), 1472 (m), 1444
(m), 1250 (s), 1208 (m), 1184 (m), 1030 (m); 1H NMR (400 MHz, CDCl3) δ 8.11 (dd, J
= 7.2, 2.4 Hz, 1H), 8.02 (dd, J = 7.2, 2.4 Hz, 1H), 7.65-7.59 (m, 2H), 7.38 (d, J = 7.6
27

ACCEPTED MANUSCRIPT
Hz, 2H), 7.30 (d, J = 9.0 Hz, 2H), 7.26 (d, J = 7.6 Hz, 2H), 7.04 (d, J = 9.0 Hz, 2H),
4.04 (q, J = 7.2 Hz, 2H), 3.90 (s, 3H), 3.45 (s, 2H), 2.42 (s, 3H), 1.15 (t, J = 7.2 Hz,
3H); 13C NMR (100 MHz, CDCl3) δ 181.1, 175.3, 169.7, 160.3, 137.7, 135.0, 134.3,
133.9, 133.3, 133.1, 131.9, 130.1, 129.9, 129.6, 129.0, 128.7, 126.6, 126.3, 125.9,
124.8, 114.7, 61.5, 55.7, 31.0, 21.5, 14.2; HRMS (ES+, Ar) calcd for C30H26NO5 (MH+)
480.1805, found 480.1806.
Ethyl
2-(3-(4-chlorophenyl)-1-(4-methoxyphenyl)-4,9-dioxo-4,9-dihydro-1H-
benzo[f]indol-2-yl)acetate (5b). Yellow solid; Yield 20 mg, 22%; mp 147-149 °C; IR
(film, cm−1) 3065 (w), 2986 (w), 2936 (w), 1734 (s), 1660 (vs), 1592 (m), 1514 (s),
1250 (s), 1208 (s), 1095 (m), 1030 (m), 836 (m), 739 (m), 715 (m); 1H NMR (500 MHz,
CDCl3) δ 8.10 (dd, J = 7.0, 2.0 Hz, 1H), 8.02 (dd, J = 7.0, 2.0 Hz, 1H), 7.66-7.61 (m,
2H), 7.44-7.41 (unresolved m, 4H), 7.28 (d, J = 9.0 Hz, 2H), 7.04 (d, J = 9.0 Hz, 2H),
13
4.04 (q, J = 7.0 Hz, 2H), 3.90 (s, 3H), 3.41 (s, 2H), 1.15 (t, J = 7.0 Hz, 3H); C NMR
(125 MHz, CDCl3) δ 181.2, 175.3, 169.6, 160.4, 135.0, 134.1, 134.0, 133.8, 133.4,
133.3, 132.0, 131.6, 131.2, 129.7, 128.7, 128.6, 126.7, 126.4, 124.6, 124.5, 114.8, 61.6,
55.7, 31.0, 14.2; HRMS (ES+, Ar) calcd for C29H22ClNO5Na (MNa+) 522.1079, found
522.1078.
Ethyl 2-(4,9-dioxo-1,3-di-p-tolyl-4,9-dihydro-1H-benzo[f]indol-2-yl)acetate (5c).
Red viscous liquid; Yield 40 mg, 48%; IR (film, cm−1) 3065 (w), 2980 (w), 2924 (w),
1735 (s), 1659 (vs), 1594 (m), 1512 (m), 1486 (m), 1439 (m), 1326 (m), 1283 (m), 1208
1
(s), 1188 (s), 1158 (m), 1046 (m), 1012 (m), 998 (m), 824 (m), 740 (m), 715 (m); H
NMR (400 MHz, CDCl3) δ 8.12 (dd, J = 6.0, 2.4 Hz, 1H), 8.03 (dd, J = 6.0, 2.4 Hz,
1H), 7.65-7.58 (m, 2H), 7.40 (d, J = 8.0 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H), 7.28 (d, J =
8.0 Hz, 4H), 4.04 (q, J = 7.2 Hz, 2H), 3.46 (s, 2H), 2.49 (s, 3H), 2.43 (s, 3H), 1.15 (t, J
= 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 181.0, 175.1, 169.6, 139.6, 137.6, 134.7
(× 2), 134.2, 133.7, 133.1, 133.0, 131.7, 130.1, 130.0, 129.5, 128.9, 127.3, 126.5, 126.2,
125.9, 124.7, 61.4, 31.0, 21.5, 14.1; HRMS (ES+, Ar) calcd for C30H25NO4Na (MNa+)
486.1676, found 486.1671.
Ethyl 2-(4,9-dioxo-3-phenyl-1-(p-tolyl)-4,9-dihydro-1H-benzo[f]indol-2-yl)acetate
(5d). Red viscous liquid; Yield 21 mg, 26%; IR (film, cm−1) 2924 (s), 2851 (m), 1736
(s), 1661 (vs), 1594 (m), 1501 (m), 1440 (s), 1282 (m), 1211 (s), 1045 (m), 1024 (m),
28