Antiproliferative activity of naphthoquinones and indane carboxylic acids from lapachol against a panel of human cancer cell lines

Article abstract of DOI:10.1007/s00044-020-02545-0

Lapachol (1) is a well-studied natural product isolated from plants of the Bignoniaceae family and demonstrates diverse biological effects. Historically, chemical transformation of the lapachol scaffold has yielded new derivatives with impressive biological activity and rich chemical diversity. β-lapachone (2), α-lapachone (3), and 2-acetylfuronaphthoquinone (4) are examples of analogs derived from lapachol that show superior antitumor activity compared with the natural product. In the present study, novel indane carboxylic acid: 2,2-dimethyl-2,3-dihydroindeno[1,2-b]pyran-4,5-dione (9) and methyl 5-hydroxy-2,2-dimethyl-2,3,4,5-tetrahydroindeno[1,2-b]pyran-5-carboxylate (10) and naphthoquinone derivatives were synthesized from lapachol with structural similarities to the antitumor lapachol derivatives. The synthesized compounds were evaluated for antiproliferative activities against a panel of human cancer cell lines including in vitro models for neuroblastoma, melanoma, glioblastoma, and non-small cell lung cancer. As expected, the most potent derivatives were those incorporating β-naphthoquinone and α-naphthoquinono[2,3-b]furan skeletons. Many of these compounds possessed nanomolar to single digit micromolar antiproliferative potency. However, the most interesting analog evaluated was the dione 9 with an indeno[1,2-b]pyran skeleton, which demonstrated potent cytotoxic activity. The current investigation identified several new lead compounds that could be used as starting points for anticancer drug discovery.

Full text of DOI:10.1007/s00044-020-02545-0

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02545-0
ORIGINAL RESEARCH
Antiproliferative activity of naphthoquinones and indane carboxylic
acids from lapachol against a panel of human cancer cell lines
Kenneth O. Eyong1,2 Herve L. Ketsemen2 Zhenze Zhao3 Liqin Du3 Aude Ingels4 Veronique Mathieu4
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Alexander Kornienko3,5 Kenneth G. Hull1 Gabriel N. Folefoc2 Sundarababu Baskaran6 Daniel Romo1
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Received: 15 January 2020 / Accepted: 6 April 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
Lapachol (1) is a well-studied natural product isolated from plants of the Bignoniaceae family and demonstrates diverse
biological effects. Historically, chemical transformation of the lapachol scaffold has yielded new derivatives with impressive
biological activity and rich chemical diversity. β-lapachone (2), α-lapachone (3), and 2-acetylfuronaphthoquinone (4) are
examples of analogs derived from lapachol that show superior antitumor activity compared with the natural product. In the
present study, novel indane carboxylic acid: 2,2-dimethyl-2,3-dihydroindeno[1,2-b]pyran-4,5-dione (9) and methyl 5-
hydroxy-2,2-dimethyl-2,3,4,5-tetrahydroindeno[1,2-b]pyran-5-carboxylate (10) and naphthoquinone derivatives were
synthesized from lapachol with structural similarities to the antitumor lapachol derivatives. The synthesized compounds
were evaluated for antiproliferative activities against a panel of human cancer cell lines including in vitro models for
neuroblastoma, melanoma, glioblastoma, and non-small cell lung cancer. As expected, the most potent derivatives were
those incorporating β-naphthoquinone and α-naphthoquinono[2,3-b]furan skeletons. Many of these compounds possessed
nanomolar to single digit micromolar antiproliferative potency. However, the most interesting analog evaluated was the
dione 9 with an indeno[1,2-b]pyran skeleton, which demonstrated potent cytotoxic activity. The current investigation
identified several new lead compounds that could be used as starting points for anticancer drug discovery.
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Keywords Lapachol Naphthoquinone Indane carboxylic acid Cancer cell lines Benzilic acid rearrangement
Introduction
Natural products isolated from various traditional herbal
plants have had a huge impact on the development of var-
ious lead compounds used in the treatment of health issues
in societies throughout the world. The tremendous biodi-
versity of plants in tropical and subtropical regions has led
to the identification of equally diverse chemical structures
with a wide range of biological activity (Nepomuceno
2011). In 1882, the Italian scientist Emanuele Paterno
reported on the isolation of lapachol (1) (Fig. 1) from a
traditional herbal plant viz., Tabebuia impetiginosa (Mart.
ex DC) Standl (Castellanos et al. 2009). The first detailed
anticancer effects of lapachol (1) (Nepomuceno 2011) were
evaluated in 1968 and this compound was shown to possess
very potent effects toward cancerous tumors in rats
(Hussain et al. 2007). Unfortunately, in 1974, the National
Cancer Institute demonstrated in a phase I clinical trial that
lapachol (1) was not an effective treatment of cancer due to
lack of a therapeutic window and this essentially terminated
further clinical research at the time. However, in 1980, in a
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02545-0) contains supplementary
material, which is available to authorized users.
* Kenneth O. Eyong
eyongkk@yahoo.com
1
Department of Chemistry and Biochemistry and the CPRIT
Synthesis and Drug-Lead Discovery Laboratory, Baylor
University, Waco, TX 76798, USA
2
Department of Organic Chemistry, Faculty of Science, University
of Yaounde I, P.O. Box 812, Yaounde, Cameroon
3
Department of Chemistry and Biochemistry, Texas State
University, San Marcos, TX 78666, USA
4
Laboratoire de Cancérologie et de Toxicologie Expérimentale,
Faculté de Pharmacie, Université Libre de Bruxelles,
Brussels, Belgium
5
Department of Chemistry, North Caucasus Federal University, 1a
Pushkin St., Stavropol 355009, Russian Federation
6
Department of Chemistry, Indian Institute of Technology Madras,
Chennai 600 036, India

Medicinal Chemistry Research
O
O
O
O
O
Fig. 1 Structures of lapachol (1),
β-lapachone (2), α-lapachone
(3), 2-acetylfuronaphthoquinone
(4)
O
O
O
O
O
O
OH
O
1
2
3
4
small study with nine patients from Nice with various
cancers (liver, kidney, breast, prostate, and cervix), pure
lapachol (1) demonstrated the ability to shrink tumors,
reduce pain, and achieve complete remissions in three of the
patients without significant adverse effects (Hussain et al.
2007).
This discovery and the fact that additional antitumor
effects of lapachol (1) were found, led to it being considered
a good candidate for derivatization to identify new lead
compounds through structure–activity relationship (SAR)
studies. Moreover, various lapachol analogs have been
prepared and studied for the treatment of lung, breast,
prostate, melanoma, leukemic, ovarian, glioblastoma,
prostate, colon, and renal cancer (Hussain et al. 2007;
Fiorito et al. 2014). In addition to its cytotoxic activity,
lapachol (1) has also been shown to possess very broad
biological effects including antibacterial, antiviral, analge-
sic, anti-inflammatory, and fungicidal activity (Hussain
et al. 2007; Oliveira et al. 2002).
Derivatives of lapachol are also of interest due to their
diverse biological properties recently identified.
β-lapachone (2) and α-lapachone (3) isolated from T.
impetiginosa (Castellanos et al. 2009) and 2-
acetylfuronaphthoquinone (4) isolated from the Bignonia-
ceae Newbouldia laevis (Eyong et al. 2005) also possess
unique properties. For example, β-lapachone (2) displays a
broad spectrum of biological and pharmacological effects
including antifungal, anti-inflammatory, anticancer, anti-
bacterial, antiangiogenic, DNA damaging, and anti-
trypanocidal activity (Hussain et al. 2007; Queiroz et al.
2008; Rios-Luci et al. 2012; Li et al. 2000; Lim et al. 2015;
Ramos-Perez et al. 2014; Perez-Sacau et al. 2007; Sunassee
et al. 2013; da Silva et al. 2007). Interestingly, it has been
reported that β-lapachone (2) is able to selectively induce
cell death in various human cancer cells without killing
non-transformed cells and in combination therapy with
taxol it displays potent anticancer effects against many
cultured cancer cells including ovarian, prostate, lung,
breast, colon, melanoma, and pancreatic cell lines (Hussain
et al. 2007). In addition, α-lapachone (3) has been shown to
possess trypanocidal effects toward Trypanosoma cruzi and
its analogs display antileishmanial activity toward Leish-
mania amazonensis and L. braziliensis (Souza-Silva et al.
2014), while 2-acetylfuronaphthoquinone (4) is a potential
chemopreventive agent (Ueda 2005).
Results and discussion
Chemical transformation
The aim of this study was to prepare new derivatives of
lapachol that are structurally related to β-lapachone, α-
lapachone, and 2-acetylfuronaphtoquinone with potentially
enhanced biological activity. In addition, we sought to
probe how changes to the stereochemistry and electronics of
the core structures influence bioactivity. Finally, from pre-
vious studies, quinones represent a class of generally
cytotoxic moieties, which lead to a variety of generally
undesirable effects in vivo, including acute cytotoxicity,
immunotoxicity, and carcinogenesis. Quinones, often con-
sidered nuisance pharmacophores (Dahlin et al. 2015), are
Michael acceptors, and cellular damage can occur through
alkylation of crucial cellular proteins and/or DNA. Alter-
natively, quinones are highly redox active molecules, which
can engage in redox cycles with their semi-quinone radicals,
leading to formation of reactive oxygen species (ROS),
including superoxide, hydrogen peroxide, and ultimately
the hydroxyl radical. Production of ROS can cause severe
oxidative stress within cells through the formation of oxi-
dized cellular macromolecules, including lipids, proteins,
and DNA (Bolton et al. 2000; Monks et al. 1992). In a
pharmacomodulation attempt to remove these quinone
groups, orthoquinones were converted to indane carboxylic
acids via benzilic acid rearrangement and to phenazines
using a Schiff base reaction to avoid these potential pitfalls
associated with drugs that possess a quinone moiety.
To obtain new derivatives for anticancer screening,
lapachol (1) was treated with conc. H2SO4 at room tem-
perature (rt) to afforded β-lapachone (2), which undergoes a
benzilic acid rearrangement to yield compound 8 when
treated with 3 N sodium hydroxide as previously described
(Eyong et al. 2013). Treatment of 8 with diazomethane
generated in situ from nitrosomethylurea (NMU) afforded
an unexpected dione derivative 9 as the major compound
alongside the expected methyl ester 10 (Scheme 1).
The dione 9 was obtained as a red solid (mp 178–180 °C)
from silica gel purification by gradient elution (20 → 30%,
hexane/ethyl acetate). The molecular formula was deter-
mined to be C14H12O3 on the basis of LCMS (APCI+) m/z
Calcd for C14H13O3 [M+H]+ 229.08647, found 229.08606.
1
The H NMR spectrum (600 MHz, CDCl3) of compound 9

Medicinal Chemistry Research
13
12
O
O
OH
O
HO2C
OH
O
MeO2C
H2NCON(NO)CH3
50% KOH
Conc
H2SO4
O
5
3N NaOH
Lapachol (1)
+
rt, 1h, 99%
O
CH3CN, rt, 10H, 80%
0°C, 2h
O
O
1
(Benzilic Acid
2
8
9
10
10%
Rearrangement)
80%
Scheme 1 New indane derivatives from lapachol (1)
Table 1 1H and 13C NMR data (600 and 150 MHz) of compounds 9
and 10 (400 and 100 MHz) (δH, CDCl3, J in Hz)
C-10 (28.6), and C-11 (28.6) supporting H-3 connectivity in
2J or J and J. Decarboxylation followed by benzylic oxi-
dation occurs under the reaction conditions or during
workup to form dione 9. Further investigations into the
mechanism for the reaction resulting in compound 9 will be
pursued.
3
4
Compound 9
1H
Compound 10
1H
Position
13C
93.8
2
–
–
3
2.95 (s, 2H)
39.5
175.9
115.1
181.5
128.1
129.5
132.0
134.6
124.7
131.1
168.9
28.6
1.63 (t, J = 8.4, 2H)
Compound 10 was obtained as a white solid from silica
gel purification by gradient elution (2.5 → 5%, hexane/ethyl
acetate). The molecular formula was determined to be
C16H18O4 on the basis of HRMS (ESI-TOF) m/z Calcd for
C16H19O4 [M+H]+ 275.1283, found 275.1295. The 1H
NMR spectrum (400 MHz, CDCl3) of compound 10
exhibited signals indicative of four aromatic protons at δH
8.05 (2H, m) and 7.70 (2H, m), attributed to an AA′BB′
spin system. A singlet at 4.14 (3H, s) attributed to a
methoxy group. Two set of triplets at 2.69 (2H, t, J =
8.4 Hz) and at 1.63(2H, t, J = 8.4 Hz) corresponding to two
adjacent methylene groups. A singlet at δH 1.29 (6H, s) for
two methyl groups. Compound 10 is the expected ester
4
–
2.68 (t, J = 8.4, 2H)
4a
5
–
–
–
–
5a
6
–
–
8.15 (m, 1H)
8.05 (m, 1H)
7.69 (m, 1H)
7.69 (m, 1H)
8.05 (m, 1H)
–
7
7.60 (m, 1H)
8
7.65 (m, 1H)
9
7.65 (m, 1H)
9a
9b
10
11
12
13
–
–
–
1.61 (s, 3H)
1.29 (s, 3H)
1.29 (s, 3H)
–
1.61 (s, 3H)
28.6
1
from previously reported carboxylic acid 8. The H NMR
–
–
–
spectra of compound 10 is very similar to compound 8 with
just the appearance of the new methyl ester peak in com-
pound 10. See SI including a stacked spectra with com-
pounds 8 and 10.
–
4.14 (s, 3H)
exhibited signals indicative of four aromatic protons at δH
8.15 (1H, m), 7.65 (2H, m), and 7.60 (1H, m), a methylene
group at δH 2.95 (2H, s) and two methyl groups at δH 1.61
(6H, s). The 13C NMR spectrum (150 MHz, CDCl3)
exhibited 13 carbon signals that were assigned by a DEPT
from multiplicity edited HSQC experiment as four methine
sp2 at 134.6, 132.0, 129.5, and 124.7, one methylene at
39.5, two methyl at 28.6 appearing as a single peak, and
seven carbon signals including six sp2 carbon atoms at
181.5 (C=O), 175.9 (C=O), 168.9 (=C–O), 131.1 (C=C),
128.1 (C=C), 115.1 (C=C) and an sp3 carbon atom at 93.8
Previously synthesized compounds 5, 6, 7, 8, 11–23
(Eyong et al. 2013, 2015a; Tanis et al. 1988), which are
structurally related to α-lapachone, β-lapachone, and 2-
acetylfuronapthoquinone were re-synthesized to evaluate
their anticancer activities in a comprehensive panel of
human cancer cell line assays. Lapachol (1) under the
Hooker oxidation condition afforded nor-lapachol (5).
Treatment of compound 5 with conc. H2SO4 afforded nor-β
lapachone (6), which underwent benzilic acid rearrangement
using 3 N NaOH to give hydroxy acid 7 (Eyong et al. 2013).
Lapachol also afforded 3-bromo-β-lapachone (11) upon
bromination and the benzilic acid rearrangement of 11
provides compound 12 (Eyong et al. 2013). Finally,
hydrogen peroxide oxidation of lapachol afforded the
Hooker intermediate 13 which on esterification provided
methyl ester 14 (Eyong et al. 2013).
1
(C–O). The H and 13C NMR data (Table 1) were assigned
using 1H–1H COSY, HSQC, and HMBC spectra (see
1
Supporting Information). In the H–1H COSY, correlations
were observed between H-6 (δH 8.15, 1H, m) and H-7 (7.60,
1H, m), between H-7 (7.60, 1H, m) and H-8 (7.65, 1H, m),
and between H-8 (7.65, 1H, m) and H-9 (7.65, 1H, m)
indicating AA′BB′ spin system of four aromatic protons.
HMBC spectrum showed correlations of H-3 (δH 2.95, 2H,
s) with C-4 (175.9), C-9b (168.9), C-4a (115.1), C-2 (93.8),
In an attempt to obtain napthoquinones stereoselectively
for SAR studies, lapachol (1) was converted to lomatiol (15)
under allylic oxidation conditions (Tanis et al. 1988).
Subsequent treatment of lomatiol with m-CPBA afforded

Medicinal Chemistry Research
compounds 16 and 17 as a 1:1 mixture of diastereomers,
while under the Sharpless asymmetric epoxidation condi-
tions lomatiol afforded compounds 18 and 19 in 2:3 dia-
stereomeric ratio. On bromination, lomatiol afforded
monobrominated compounds 20, 21, 22, and 23 (Eyong
et al. 2015a).
U373 cell lines to determine their IC50 values (Table 3).
Most of these compounds possess the β-naphthoquinone
and the α-naphthoquinono[2,3-b]furan structural skeletons.
Indeed, a literature search reveals a significant number of
reports describing antiproliferative properties of compounds
in these classes (Rios-Luci et al. 2012; Yamashita et al.
2007). Our results show that submicromolar to single digit
micromolar IC50 values are associated with compounds in
which the β-naphthoquinone ring is fused with a saturated
pyran (2, 11, 20, 21) or a furan (6, 22, 23) moiety.
In the α-naphthoquinono[2,3-b]furan derivatives, the
furan ring can be aromatic (4, 24, 29) or saturated (16 and
28) and retain activity, exhibiting submicromolar to low
micromolar IC50 values. Within the α-naphthoquinono[2,3-
b]furan class, the acetyl furan 4 led to the highest cytotoxic
activity indicating that the carbonyl of the acetoxyl group
on the furan ring may play an important role in bioactivity
(cf. isopropylidene furan 29).
Knipholone anthrone (27) exhibited a mean IC50 of ca.
1 µM, a value that is consistent with the literature data
(Habtemariam 2010). It should be noted that this compound
was recently found to induce necrotic cell death in cancer
cells, identifying it as a promising anticancer agent to fight
cancers with intrinsic resistance to apoptosis (Kornienko
et al. 2013; Aksenov et al. 2015). Activity was found in the
indeno[1,2-b]pyran skeleton, as represented with the pro-
duct of the benzilic acid rearrangement, dione 9, showing
submicromolar to single digit micromolar potency
depending on the cell line tested.
These naphthoquinones together with some previously
isolated and synthesized quinone derivatives were screened
for
their
cytotoxicity,
including
7-methoxy-
2-acetylfuronapthoquinone (24), 2,3-dihydro-6-hydroxy-
2-(prop-1-en-2-yl)naphtha[2,3-b]furan-4,9-dione (25), 2-
acetylfuronapthoquinone (4), knipholone (26), knipholone
anthrone (27), 3-hydroxydehydroiso-α-lapachone (28), 2-
(prop-1-en-2-yl)naphtha[2,3-b]furan-4,9-dione (29), 3,4
dehydro-α-lapachone (30), tithoniamarin (31), new-
bouldiaquinone (32), acetyllapachol (33), 4-(1,4-dihydro-2-
hydroxy-1,4-dioxonaphthalen-3-yl)-2-methylbut-2-enal
(34), 2-hydroxy-3-(4,4-dimethoxy-3-methylbutyl)naphtha-
lene-1,4-dione (35), 1,4-dihydro-2-((3-(hydroxymethyl)-
3-methyloxiran-2-yl)methyl)
1,4-dioxonaphthalen-3
-yl acetate (36), 4-(1,4-dihydro-2-hydroxy-1,4-dioxo-
naphthalen-3-yl)-2-methylbutanal (37), acetyllomatiol (38),
methoxy lomatiol (39), 1,4-dihydro-2-((3-(hydroxymethyl)-
3-methyloxiran-2-yl)methyl)-1,4-dioxonaphthalen-3-yl
acetate (40), lapachol-OMe (41), 1a-methoxy-7a-(3-
methylbut-2-en-1-yl)-1a,7a-dihydronaphtho[2,3-b]oxirene-
2,7-dione (42), 6-hydroxy-6-(3-methylbut-2-enyl)benzo[a]
phenazin-5(6H)-one (43), β-lapachone-o-phenylene dia-
mine (44), and 3-bromo-5-hydroxy-2,2-dimethyl-3,4-dihy-
dro-2H-benzo[h]chromene-6 carbaldehyde (45) (Eyong
et al. 2005, 2006, 2008 2013, 2015a, 2015b; Kuete et al.
2007; Induli et al. 2013) (Fig. 2).
Conclusion
Cytotoxic activity
In this study, the cytotoxic activity of some previously iso-
lated quinones and derivatives of lapachol, which are struc-
turally related to known anticancer agents (i.e. β-lapachone, α-
lapachone, and 2-acetylfuronaphthoquinone), and two new
indeno[1,2-b]pyran derivatives were evaluated in a panel
of human cancer cell lines. Fourteen of the tested com-
pounds were most active with submicromolar to low
micromolar IC50 values. Among these compounds, seven
are based on the β-naphthoquinone and five on the α-
naphthoquinono[2,3-b]furan structural skeletons. These
results are comparable with values described in the lit-
erature for these classes of compounds (Rios-Luci et al.
2012; Yamashita et al. 2007). Among the two new indeno
[1,2-b]pyran derivatives, the further oxidized, benzilic
acid rearrangement product, dione 9 was the most inter-
esting finding given that anticancer activity with this
structural skeleton appears to be unprecedented. Given the
ease of synthesis and potent cytotoxic activity of dione 9
from commercially available lapachol (1), this tricyclic
pyranone-fused indenone represents a compelling starting
The compounds were evaluated in a panel (Dasari et al.
2015; Zhao et al. 2015) of human cell lines containing
in vitro models for cancers typically associated with dismal
prognosis, such as BE(2)-C, Kelly, SKNSH, CHLA-90 (all
neuroblastoma) (Esposito et al. 2017; Zhao et al. 2016),
A549 (non-small cell lung cancer) (Rossi et al. 2017),
SKMEL-28 (melanoma) (Amann et al. 2017), and U373
(glioblastoma) (Osuka and Van Meir 2017).
First, the compounds were tested at a single concentra-
tion of 10 µM and the percent residual growth of cancer
cells was recorded after a 72-h treatment (Table 2).
In most cases, the active compounds did not discriminate
between the cell lines used with the exception of a few, such
as β-lapachone derivative 21, which was essentially inactive
in three neuroblastoma cell lines, yet showed potent activity
against the other cell lines in the panel.
Fourteen of the most potent compounds were then
selected and evaluated against A549, SKMEL-28, and

Medicinal Chemistry Research
Fig. 2 Previously isolated and
synthesized quinone derivatives

Medicinal Chemistry Research
Table 2 Percent residual growth of cancer cell lines after a 72 h of treatment with 10 µM of each compound as determined by the colorimetric
MTT assay
Compound
Residual growth (%)
BE(2)-C
Kelly
SKNSH
CHLA-90
A549
SKMEL-28
U373
Mean
1
77
1
89
6
81
1
95
2
83
2
75
3
73
4
82
3
2
4
2
1
28
82
1
5
2
2
4
6
5
81
2
68
1
88
2
72
2
83
1
61
5
76
2
6
7
95
93
3
82
81
3
93
96
2
94
98
3
102
67
2
94
88
3
82
70
6
92
85
3
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
13
2
88
2
88
1
89
1
NTa
NT
1
NT
4
69
2
1
95
101
90
91
1
82
48
59
76
1
93
80
92
99
1
94
86
86
98
3
85
101
74
69
2
78
110
75
94
3
71
99
63
63
6
85
89
77
84
2
83
38
1
66
76
2
89
74
2
96
85
3
NT
78
NT
1
NT
87
NT
2
NT
74
NT
6
83
73
2
4
4
2
2
3
2
97
95
2
74
79
2
87
86
32
5
1
2
4
38
53
7
28
1
13
1
7
65
5
3
3
4
4
2
2
5
4
37
93
2
69
65
5
76
101
95
57
58
88
93
88
81
89
92
87
80
91
62
54
85
87
90
89
66
66
96
7
29
64
3
18
68
4
39
74
14
37
31
86
94
77
48
56
65
78
72
58
64
41
84
78
73
78
NT
47
80
19
34
25
81
82
72
68
73
78
78
67
68
53
24
83
76
80
81
78
6
69
44
79
65
79
84
61
73
89
99
84
77
35
91
88
64
75
90
65
34
96
85
95
97
93
94
98
91
97
74
29
94
101
89
88
85
2
3
1
1
2
68
82
76
24
87
85
78
36
9
88
78
40
66
56
67
51
17
58
59
3
63
76
48
76
67
66
64
77
80
20
3
16
2
58
4
93
78
67
77
NT
77
98
84
68
NT
92
94
72
aNot tested

Medicinal Chemistry Research
Table 3 In vitro growth
inhibition (IC50) after a 72 h of
treatment with each compound
as determined by the
Compound
Structural skeleton
IC50 (µM)
A549
SKMEL-28
U373
IC50 SEM
colorimetric MTT assay
2
β-naphthoquinone
β-naphthoquinone
β-naphthoquinone
β-naphthoquinone
β-naphthoquinone
β-naphthoquinone
β-naphthoquinone
α-naphthoquinono[2,3-b]furan
α-naphthoquinono[2,3-b]furan
α-naphthoquinono[2,3-b]furan
α-naphthoquinono[2,3-b]furan
α-naphthoquinono[2,3-b]furan
anthrone
0.03
3
0.05
2
0.24
2
0.1
2.2
2
0.07
11
20
21
6
0.2
0.4
0.5
0.1
1.3
0.6
0.07
0.7
0.4
0.2
0.0
0.5
0.5
3
3
2
2
1
1
1.4
2.5
3.5
1.0
0.09
0.9
2.4
2.6
3.0
1.1
1.1
3
2
3
22
23
4
5
2
NT
2.2
0.22
2
0.3
0.01
0.2
3
0.4
0.02
0.3
2
24
29
16
28
27
9
NT
3
2
3
3
3
3
0.1
0.8
2
2
indeno[1,2-b]pyran
0.6
2.0
point for further derivative synthesis and mode of action
studies.
which was purified by column chromatography over silica gel
using 10–20% EtOAc in hexane as solvent gradient afforded
pure compound 8 (43 mg, 80% yield) as a red solid (s).
Methylation of compound 8 with diazomethane. To a
stirred slurry of 50% aq. KOH (10 ml) in Et2O (5.0 ml) at
0 °C, N-nitroso-N-methylurea (22.5 mg, 0.16 mmol, 2
equiv) was added slowly. The resulting yellow colored
organic layer containing diazomethane was separated, dried
over KOH pellets, and added to compound 8 (20.8 mg,
0.08 mmol, 1 equiv) in Et2O (2 ml), the reaction mixture
was stirred for 2 h and the solvent was removed under
reduced pressure. Column chromatographic purification of
the crude products over silica gel using hexane-EtOAc
gradient systems afforded the unexpected dione 9 (18.4 mg,
Experimental
Synthetic procedures
Synthesis of 2,2-dimethyl-2,3-dihydroindeno[1,2-b]pyran-
4,5-dione (9) and methyl 5-hydroxy-2,2-dimethyl-2,3,4,5-
tetrahydroindeno[1,2-b]pyran-5-carboxylate (10)
To a stirred solution of lapachol (1), (242 mg, 1.0 mmol, 1
equiv) in CH2Cl2 (7 mL) was added conc. H2SO4 (0.8 ml,
1.5 mmol, 1.5 equiv), the resulting reaction mixture was
stirred for 3 h, at room temperature. The orange suspension
was filtered and washed with ice cold water (3 × 5 ml), the
crude product was dried under vacuum and column chro-
matographic purification of the crude compound over silica
gel using 20–30% EtOAc in hexane as solvent gradient
afforded pure β-lapachone (2) (218 mg, 90% yield), as an
orange solid (Eyong et al. 2013).
A solution of β-lapachone (2) (50 mg, 0.2 mmol) in
acetonitrile (2 ml) at 25 °C was treated with 3 N NaOH
solution (1 ml). The resultant mixture was stirred for 10 h at
25 °C, then the mixture was acidified to pH 5 using 5% HCl
(2 ml) and the solvent was removed under reduced pressure.
The residue was diluted with water (5 ml) and CH2Cl2
(5 ml). The layers were separated, the aqueous layer was
extracted with CH2Cl2 (2 × 5 ml) and the combined organic
layer was dried over anhydrous Na2SO4. The solvent was
removed under reduced pressure to give crude compound,
1
80% yield) and ester 10 (2.2 mg, 10% yield). dione 9: H
NMR (600 MHz, CDCl3) 8.15 (m, 1H), 7.60 (m, 1H), 7.65
(m, 1H), 7.65 (m, 1H), 2.95 (s, 2H), 1.61 (s, 6H); 13C NMR
(150 MHz, CDCl3) δ 181.5, 175.9, 168.9, 134.6, 132.0,
131.1, 129.5, 128.1, 124.7, 115.1, 93.8, 39.5, 28.6, 28.6.
LCMS (APCI+) m/z calcd for C14H13O3 [M+H]+:
229.08592; found: 229.08606. Ester 10: 1H NMR
(400 MHz, CDCl3) δ 8.05–8.04 (m, 2H), 7.70–7.68 (m,
2H), 4.14 (s, 3H), 2.70–2.66 (m, 2H), 1.75–1.65 (m, 2H)
1.29 (s, 6H). HRMS (ES+) m/z calcd for C16H19O4 [M+H]+:
275.1278; found: 275.1297.
Biological assays
Cell lines
BE(2)-C and SK-N-SH cells were purchased from the
American Type Culture Collection (ATCC). Kelly cells

Medicinal Chemistry Research
were obtained from the cell line repository at the Greehey
Children’s Cancer Research Institute, University of Texas
Health Science Center at San Antonio. CHLA-90 cells were
obtained from Children’s Oncology Group. The neuro-
blastoma cell lines were maintained in DMEM/
F12 supplemented with 10% fetal bovine serum (FBS) and
1% penicillin–streptomycin (P/S) under standard cell cul-
ture conditions. The A549 cells (DSMZ) were cultured in
RPMI culture medium supplemented with 10% heat-
inactivated FBS. SKMEL-28 cells (ATCC) and U373
GBM cells (ECACC) were cultured in RPMI culture med-
ium supplemented with 10% heat-inactivated FBS (Life
Technologies code 10270106). Cell culture media were
supplemented with 4 mM glutamine (Lonza code BE17-
605E), 100 µg/ml gentamicin (Lonza code 17-5182), and P/
S (200 units/ml and 200 µg/ml) (Lonza code 17-602E) at
37 °C with 5% CO2.
Compliance with ethical standards
Conflict of interest The authors declare no conflict of interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
References
Aksenov AV, Smirnov AN, Magedov IV, Reisenauer MR, Aksenov
NA, Aksenova IV, Pendleton AL, Nguyen G, Johnston RK,
Rubin M, De Carvalho A, Kiss R, Mathieu V, Lefranc F, Correa
J, Cavazos DA, Brenner AJ, Bryan BA, Rogelj S, Kornienko A,
Frolova LV (2015) Activity of 2-Aryl-2-(3-indolyl)acetohydrox-
amates against drug-resistant cancer cells. J Med Chem 58
(5):2206–2220
Amann VC, Ramelyte E, Thurneysen S, Pitocco R, Bentele-Jaberg N,
Goldinger SM, Dummer R, Mangana J (2017) Developments in
targeted therapy in melanoma. Eur J Surg Oncol 43(3):581–593
Bolton JL, Trush MA, Penning TM, Dryhurst G, Monks TJ (2000)
Role of quinones in toxicology. Chem Res Toxicol 13
(3):135–160
Cell proliferation assay
Castellanos JRG, Prieto JM, Heinrich M (2009) Red Lapacho (Tabe-
buia impetiginosa)—a global ethnopharmacological comomod-
ity? J Ethnopharmacol 121:1–13
The effect of the investigated compounds on cell pro-
liferation was determined by MTT (3-(4,5-Dimethylthia-
zol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole)
assay. The compounds were dissolved in DMSO or
MeOH/CH2Cl2 at a concentration of either 10 or 50 mM
prior to cell treatment. The cells were trypsinized and
seeded at various cell concentrations depending on the
cell type. The cells were grown for 24–72 h, treated with
test compounds at required concentrations and incubated
for 72 h in 100 or 200 µl media depending on the cell line
used. The number of experiments and replicates varied
depending on the cell line. Cells treated with 0.1%
DMSO, 1% CH2Cl2, or 1% MeOH were used as a nega-
tive control. The effect of compound treatment on cell
survival was evaluated by comparing compound-treated
cells to cells treated with control using two-tailed stu-
dent’s t test, with p < 0.05 considered as statistically sig-
nificant. The IC50 corresponds to the concentration of the
compound of interest that reduces by 50% the growth of
the cancer cell line of interest after having cultured it for
72 h in the presence of the compound in comparison to the
untreated control condition.
Dahlin JL, Nissink JWM, Strasser JM, Francis S, Higgins LA, Zhou
H, Zhang Z, Walters MA (2015) PAINS in the assay: chemical
mechanisms of assay interference and promiscuous enzymatic
inhibition observed during a sulfhydryl-scavenging HTS. J Med
Chem 58(5):2091–2113
Dasari R, De Carvalho A, Medellin DC, Middleton KN, Hague F,
Volmar MNM, Frolova LV, Rossato MF, De La Chapa JJ,
Dybdal-Hargreaves NF, Pillai A, Kälin RE, Mathieu V, Rogelj S,
Gonzales CB, Calixto JB, Evidente A, Gautier M, Munirathinam
G, Glass R, Burth P, Pelly SC, Van Otterlo WA, Kiss R, Kor-
nienko A (2015) Wittig derivatization of sesquiterpenoid poly-
godial leads to cytostatic agents with activity against drug
resistant cancer cells and capable of pyrrolylation of primary
amines. Eur J Med Chem 103:226–237
da Silva Jr EN, de Souza MCBV, Pinto AV, Pinto MCFR, Goulart
MOF, Barros FWA, Pessoa C, Costa-Lotufo LV, Montenegro
RC, de Moraes MO, Ferreira VF (2007) Synthesis and potent
antitumor activity of new arylamino derivatives of nor-beta-
lapachone and nor-alpha-lapachone. Bioorg Med Chem
15:7035–7041
Esposito MR, Aveic S, Seydel A, Tonini GP (2017) Neuroblastoma
treatment in the post-genomic era. J Biomed Sci 24(1):14
Eyong KO, Krohn K, Hussain H, Folefoc GN, Nkengfack AE, Schulz
B, Hu Q (2005) Newbouldiaquinone and newbouldiamide: a new
naphthoquinone– anthraquinone coupled pigment and a new
ceramide from Newbouldia laevis. Chem Pharm Bull 53
(6):616–619
Eyong KO, Folefoc GN, Kuete V, Beng VP, Hussain H, Krohn K,
Nkengfack AE, Saeftel M, Sarite SR, Achim H (2006) New-
bouldiaquinone A: a naphthoquinone-anthraquinone ether cou-
pled pigment, as a potential antimicrobial and antimalarial agent
from Newbouldia laevis. Phytochemistry 67:605–609
Eyong KO, Kumar SM, Folefoc GN, Nkengfack AE, Baskaran S
(2008) Semisynthesis and antitumoral activity of 2-
acetylfuranonaphthoquinone and other naphthoquinone deriva-
tives from lapachol. Bioorg Med Chem Lett 18:5387–5390
Eyong KO, Puppala M, Kumar PM, Lamshöft M, Folefoc GN, Spi-
teller M, Baskaran S (2013) A mechanistic study on the Hooker
Acknowledgements The authors would like to acknowledge the
council for International Exchange of Scholars (CIES) for the J.
William Fulbright Visiting Scholar program to Pr Kenneth Eyong. The
Molecular Bioscience Center, the CPRIT Synthesis and Drug-Lead
Discovery Laboratory and the Departments of Chemistry/Biochem-
istry and Biology at Baylor University, and Baylor University and
Texas State University, San Marcos for infrastructure and financial
support. The University of Yaounde I and the Government of
Cameroon for financial support through the Fonds d’Appuis a la
Recherche.

Medicinal Chemistry Research
oxidation: synthesis of novel indane carboxylic acid derivatives
from lapachol. Org Biomol Chem 11:459–468
Eyong KO, Chinthapally K, SenthilKumar S, Lamshöft M, Folefoc
GN, Baskaran S (2015a) Conversion of lapachol to lomatiol:
synthesis of novel naphthoquinone derivatives. New J Chem
39:9611–9616
Eyong KO, Ketsemen LH, Ghansenyuy YS, Folefoc NG (2015b)
Chemical constituents, the stereochemistry of 3-hydroxy fur-
onaphthoquinones from the root bark of Newbouldia laevis Seem
(Bignoniaceae), and screening against Onchocerca ochengi
parasites. Med Chem Res 24:965–969
Fiorito S, Epifano F, Bruyère C, Mathieu V, Kiss R, Genovese S
(2014) Growth inhibitory activity for cancer cell lines of lapachol
and its natural and semi-synthetic derivatives. Bioorg Med Chem
Lett 24:454–457
pharmacophore modeling of naphthoquinone derivatives with
cytotoxic activity in human promyelocytic leukemia HL-60 cell
line. J Med Chem 50:696–706
Queiroz MLS, Valadares MC, Torello CO, Ramos AL, Oliveira AB,
Rocha FD, Arruda VA, Accorei WR (2008) Comparative studies
of the effects of Tabebuia avellanedae bark extract and beta-
lapachone on the hematopoietic response of tumour-bearing mice.
J Ethnopharmacol 117:228–235
Ramos-Perez C, Lorenzo-Castrillejo I, Quevedo O, Garcia-Luis J,
Matos-Perdomo E, Medina-Coello C, Estevez-Braun A, Machin
F (2014) Yeast cytotoxic sensitivity to the antitumour agent
β-lapachone depends mainly on oxidative stress and is largely
independent of microtubule- or topoisomerase- mediated DNA
damage. Biochemical Pharmacol 92:206–219
Rios-Luci C, Bonifazi EL, Leon LG, Montero JC, Burton G, Pandiella
A, Misico RI, Padron JM (2012) β-Lapachone analogs with
Habtemariam S (2010) Knipholone anthrone from Kniphofia foliosa
induces a rapid onset of necrotic cell death in cancer cells.
Fitoterapia 81(8):1013–1019
enhanced antiproliferative activity. Eur
J Medicinal Chem
53:264–274
Hussain H, Krohn K, Ahmad VU, Miana GA, Green IR (2007)
Lapachol: an overview. Arkivoc 2:145–171
Induli M, Gebru M, Abdissa N, Akala H, Wekesa I, Byamukama R,
Heydenreic M, Murunga S, Dagne E, Yenesew A (2013) Anti-
plasmodial quinones from the rhizomes of Kniphofia foliosa. Nat
Prod Commun 8(9):1261–1264
Kuete V, Eyong KO, Folefoc NG, Beng VP, Hussain H, Krohn K,
Nkengfack AE (2007) Antimicrobial activity of the methanolic
extract and of the chemical constituents isolated from New-
bouldia laevis. Pharmazie 62:552–556
Kornienko A, Mathieu V, Rastogi SK, Lefranc F, Kiss R (2013)
Therapeutic agents triggering nonapoptotic cancer cell death. J
Med Chem 56(12):4823–4839
Rossi A, Sacco PC, Santabarbara G, Sgambato A, Casaluce F,
Palazzolo G, Maione P, Gridelli C (2017) Developments in
pharmacotherapy for treating metastatic non-small cell lung
cancer. Expert Opin Pharmacother 18(2):151–163
Souza-Silva F, Nascimento SB, Bourguignon SC, Pereira BA,
Carneiro PF, Da Silva WS, Alves CR, De Pinho RT (2014)
Evidences for leishmanicidal activity of the naphthoquinone
derivative epoxy-α-lapachone. Exp Parasitol 147:81–84
Sunassee SN, Veale CGL, Shunmoogam-Gounden N, Osoniyi O,
Hendricks DT, Caira MR, de la Mare J-A, Edkins AL, Pinto AV,
da Silva Jr EN, Davies-Coleman MT (2013) Cytotoxicity of
lapachol, β-lapachone and related synthetic 1,4-napthoquinones
against oesophageal cancer cells. Eur J Med Chem 62:98–110
Tanis SP, Chuang YH, Head DB (1988) Furans in synthesis. 8. Formal
total syntheses of (.+-.)- and (+)-aphidicolin. J Org Chem
53:4929–4938
Li Y, Li CJ, Yu D, Pardee AB (2000) Potent induction of apoptosis by
beta-lapachone in human multiple myeloma cell lines and patient
cells. Mol Med 6(12):1008–1015
Lim SM, Jeong Y, Lee S, Im H, Tae HS, Kim BG, Park HD, Park J,
Hong S (2015) Identification of β-Lapachone analogs as Novel
MALT1 inhibitors to treat an aggressive subtype of diffuse large
B-cell lymphoma. J Med Chem 58:8491–8502
Monks TJ, Hanzlik RP, Cohen GM, Ross D, Graham DG (1992) Qui-
none chemistry and toxicity. Toxicol Appl Pharmacol 112(1):2–16
Nepomuceno JC (2011) Lapachol and its derivatives as potential drugs
for cancer treatment. iConcept Press, Brazil
Oliveira MF, Lemos TLG, Mattos MCD, Segundo TA, Santiago GMP,
Braz-Filho R (2002) New enamine derivatives of lapachol and
biological activity. An da Academia Brasileira de Ciências
74:211–221
Osuka S, Van Meir EG (2017) Overcoming therapeutic resistance in
glioblastoma: the way forward. J Clin Invest 127(2):415–426
Perez-Sacau E, Dıaz-Penate RG, Estevez-Braun A, Ravelo AG, Gar-
cıa-Castellano JM, Pardo L, Campillo M (2007) Synthesis and
Ueda S (2005) The present invention relates to a novel compound
possessing antitumor activity, and an antitumor agent comprising
said compounds. US Patent 5663197
Yamashita M, Kaneko M, Iida A, Tokuda H, Nishimura K (2007)
Stereoselective synthesis and cytotoxicity of a cancer chemo-
preventive naphthoquinone from Tabebuia avellanedae. Bioorg
Med Chem Lett 17(23):6417–6420
Zhao Z, Ma X, Sung D, Li M, Kosti A, Lin G, Chen Y, Pertsemlidis A,
Th H, Du L (2015) microRNA-449a functions as a tumor sup-
pressor in neuroblastoma through inducing cell differentiation
and cell cycle arrest. RNA Biol 12(5):538–554
Zhao Z, Ma X, Shelton SD, Sung DC, Li M, Hernandez D, Zhang M,
Losiewicz MD, Chen Y, Pertsemlidis A, Yu X, Liu Y, Du L
(2016) A combined gene expression and functional study reveals
the crosstalk between N-Myc and differentiation-inducing
microRNAs in neuroblastoma cells. Oncotarget 7:79372–79387