Novel juglone and plumbagin 5-O derivatives and their in vitro growth inhibitory activity against apoptosis-resistant cancer cells

Article abstract of DOI:10.1016/j.bmcl.2015.12.017

Juglone 1 an plumbagin 2 are plant secondary metabolites nowadays well known for their anticancer properties. In this study we synthesized analogues of 1 and 2 deriving from the functionalization of the OH group in position 5 with different side chains in form of esters and ethers. Therefore the growth inhibitory activities of these adducts were evaluated in vitro on six cancer cell lines using the MTT colorimetric assays along with the two natural parent compounds. The data revealed that these latter displayed the strongest growth inhibitory activities in vitro. Quantitative videomicroscopy analyses were then carried out on human U373 glioblastoma cells, which are characterized by various level of resistance to pro-apoptotic stimuli. We compared the naturally occurring reference compounds 1 and 2 with the derivatives exerting the best activities in terms of IC₅₀ growth inhibitory values. These analyses showed that both juglone and plumbagin had a cytostatic effect on U373 cells and were able to overcome the intrinsic resistance of U373 cancer cells to pro-apoptotic stimuli.

Full text of DOI:10.1016/j.bmcl.2015.12.017

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel juglone and plumbagin 5-O derivatives and their in vitro
growth inhibitory activity against apoptosis-resistant cancer cells
Serena Fiorito a, Salvatore Genovese a, Vito Alessandro Taddeo a, Véronique Mathieu b, Robert Kiss b,
Francesco Epifano a,
⇑
a Dipartimento di Farmacia, Università ‘G. D’Annunzio’ Chieti-Pescara, Via dei Vestini 31, 66100 Chieti Scalo (CH), Italy
b Laboratoire de Cancérologie et de Toxicologie Expérimentale, Faculté de Pharmacie, Université Libre de Bruxelles, 1050 Brussels, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Juglone 1 an plumbagin 2 are plant secondary metabolites nowadays well known for their anticancer
properties. In this study we synthesized analogues of 1 and 2 deriving from the functionalization of
the OH group in position 5 with different side chains in form of esters and ethers. Therefore the growth
inhibitory activities of these adducts were evaluated in vitro on six cancer cell lines using the MTT
colorimetric assays along with the two natural parent compounds. The data revealed that these latter
displayed the strongest growth inhibitory activities in vitro. Quantitative videomicroscopy analyses were
then carried out on human U373 glioblastoma cells, which are characterized by various level of resistance
to pro-apoptotic stimuli. We compared the naturally occurring reference compounds 1 and 2 with the
derivatives exerting the best activities in terms of IC50 growth inhibitory values. These analyses showed
that both juglone and plumbagin had a cytostatic effect on U373 cells and were able to overcome the
intrinsic resistance of U373 cancer cells to pro-apoptotic stimuli.
Received 5 November 2015
Revised 3 December 2015
Accepted 7 December 2015
Available online xxxx
Keywords:
Juglone
Plumbagin
In vitro growth inhibition
Cancer
1,4-Naphthoquinones
Ó 2015 Elsevier Ltd. All rights reserved.
Cancer is among the main worldwide public health problem. In
this context the role of natural products has been well recognized
in terms of therapeutic or chemopreventive activities.1 A number
of important commercialized anticancer drugs have been obtained
from plants and these include vinblastine, vincristine, vinorelbine,
etoposide, teniposide, paclitaxel docetaxel, topotecan and irinote-
can. As emphasized by Newmann and Cragg natural products rep-
resent the most significant source of drugs, accounting for
approximately 74% of the anticancer ones nowadays at disposition
in therapy.2 During the last two decades we have gained experi-
ence in this context and have set-up and validated an original
screening approach based on the combination of the conventional
MTT colorimetric assay and quantitative videomicroscopy3–6 to
evaluate the efficiency and select anticancer drugs with putative
novel mechanisms of action. As reported below, we adopted this
strategy in the present investigation about the potentialities of nat-
ural and semi-synthetic 1,4-napthtoquinones structurally related
to juglone 1 and plumbagin 2. Naphthoquinones represent one of
the largest family of antitumor agents and the basic knowledge
on quinone studies has been used to design new anticancer drugs,
with the aim to improve their selectivity and providing a more
rational therapeutic application of these agents.7–9
1,4-Naphthoquinones represent a wide group of natural prod-
ucts and are found in several plant families, fungi, and bacteria.
The class of K vitamins represents a notable example of occurrence
of 1,4-naphthoquinones in nature. Such compounds have been
shown to exert valuable pharmacological effects, acting as cytotox-
ic, antibacterial, antifungal, antiviral, antiprotozoal, insecticidal,
anti-inflammatory, and antipyretic agents. Plants containing naph-
thoquinones are widely used in Southeast Asia and South America
to treat malignant and parasitic diseases.10 The mechanisms of
action underlying the observed effects mainly consists in the
capacity of 1,4-naphthoquinones to interact with topoisomerases
and to generate semiquinone radicals and reactive oxygen species
(ROS) in the endocellular environment.11
Juglone
1 (isolated from the black walnut, Juglans nigra
L. [Juglandaceae]), and plumbagin 2 (obtained from Plumbago,
Drosera, and Nepenthes spp.) represent well described
examples of research topics in natural product chemistry. Their
phytochemical and pharmacological properties have been recently
reviewed.12–15
In the current work, we synthesized and characterized the
in vitro growth inhibitory activity of 24 selected 1,4-naphtho-
quinones, including 1 and 2. The in vitro IC50 growth inhibitory
concentration was determined for each adduct in a panel of six
human cancer cell lines exhibiting different levels of resistance
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.bmcl.2015.12.017
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Fiorito, S.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.12.017

2
S. Fiorito et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
plumbagin acetate 19 is the pigment of tuberous roots and rhizome
of Drosera peltata Thunb. (Droseraceae);20 plumbagin methyl ether
20 has been extracted from the heartwood of Diospyros melanoxylon
Roxb. (Ebenaceae);21 and finally plumbagin 5-dimethylallyl ether
23 has been recently isolated from roots of Plumbago zeylanica L.22
Juglone 1 and plumbagin 2 are commercially available, while
the synthesis of all esters (laurate, myristate, palmitate, stearate,
and oleate) were performed by the reaction of juglone 1 and plum-
bagin 2 with the respective acyl chloride in Et2O in the presence of
Et3N at room temperature for 30 min. The yields of the desired
adducts were in the range 96–98%. Juglone acetate 8 and plumba-
gin acetate 19 were obtained using acetic anhydride for the same
time and using the same solvent and reagents at room temperature
(Scheme 1).
O-alkylations of 1 and 2 to obtain ethers 9–13 and 20–24,
respectively, were carried out in refluxing acetone for 1 h using
methyl iodide, ethyl iodide, allyl bromide, 3,3-dimethylallyl bro-
mide, and geranyl bromide in the presence of K2CO3 as the base,
followed by acid-base work-up and extraction with dichloro-
methane. The respective compounds were obtained in 73–94%
yields (Scheme 2).
to pro-apoptotic stimuli by means of the MTT colorimetric assay.
The lack of a reference drug in our assays is justified by the fact
that cell lines that have been selected in the current study display
various levels of resistance to different types of pro-apoptotic stim-
uli (as detailed in the references provided for these cell lines). We
estimate in fact that compounds 1 and 2 represent the reference
compounds in the current study.
Compounds were chemically synthesized following two
criteria: (a) esterification of the hydroxyl function of juglone 1
and plumbagin 2 with acids having different lengths of the carbon
skeleton (C2, C12, C14, C16, C18 either saturated or monounsatu-
rated). This structural modification may allow these highly lipophi-
lic derivatives to more easily permeate the cell membrane and to
release the active portion inside the cell after cleavage by
esterases;16,17 (b) decrease in polarity of 1 and 2 by etherification
of the OH function with 1–3, 5 and 10 carbon chains The general
chemical structure of the compounds under study is illustrated
below and detailed in Table 1.
O
R2
O
O
R2
R2
OR1
O
R1COCl (Ac2O)
E3N, Et2O
r.t. (120 °C)
30 min.
Among the 22 synthesized compounds, only four, other than
juglone and plumbagin, have been found in nature. Juglone methyl
ether 9 has been previously extracted from the exocarp of Juglans
mandshurica Maxim. and from the root of Plumbago zeylanica;19
OCOR1
O
OH
O
Scheme 1. (R2 = H, Me).
Table 1
In vitro growth inhibitory activity of the compounds under studya,b
R1
R2
MTT colorimetric assay (
SKMEL-28
lM)
Hs683
A549
MCF7
B16F10
U373
Mean SEM
1
2
3
4
5
6
7
8
H
H
Lauryl
Myristyl
Palmityl
Stearyl
Oleyl
COCH3
H
Me
H
H
H
H
H
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
7
4
7
3
13
3
7
5
6
1
8
17
5
9
4
21
32
96
2
1
4
1
9
29
36
>100
67
77
23
6
23
19
7
67
17
9
33
>100
35
5
29
36
>100
67
77
23
9
19
26
6
67
22
12
30
>100
36
5
24
30
86
30
61
8
39
24
21
16
>100
29
22
57
>100
57
4
15
33
>100
18
67
23
30
9
18
30
>100
65
91
8
27
53
19
61
7
10
5
4
44 10
72
15
17
18
21
8
5
3
6
4
4
9
CH3
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
CH2CH3
CH2CH@CH2
CH2CH@C(CH3)2
Geranyl
Lauryl
Myristyl
Palmityl
Stearyl
Oleyl
COCH3
CH3
CH2CH3
CH2CH@CH2
CH2CH@C(CH3)2
Geranyl
29
32
15
>100
16
16
48
>100
62
5
5
24
3
7
59
24
5
7
>100
3
2
>100
77
18
73
91
31
3
5
3
3
7
100
27 11
3
13
44
96
40
4
4
7
3
4
3
7
9
6
1
1
4
0
1
22
46
22
0,4
2
3
1
1
30
3
2
3
3
4
3
3
4
5
6
3
3
22
23
40
31
34
6
a
The 24 compounds studied belong to two chemical groups, that is, esters of juglone 1 (compounds 3–8) and plumbagin 2 (compounds 14–19), ethers of both (compounds
9–13 and 20–24).
b
The cell lines analyzed belong to three histological types. Human glioma model lines included the Hs683 oligodendroglioma (ATCC code HTB-138) and the U373
glioblastoma (ECACC code 08061901) cell lines. Melanoma models included the human SKMEL-28 (ATCC code HTB-72) and the mouse B16F10 (ATCC code CRL-6475) cell
lines. Human carcinoma models included the A549 (DSMZ code ACC107) and the MCF-7 breast (DSMZ code ACC115). The origin of the cell lines are detailed in the
Experimental section.
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S. Fiorito et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
O
O
it can be postulated that the presence of the methyl group allows
plumbagin and its derivatives to rearrange to 1,2-quinone methide
as the effective biologically active agent with protonation of the
oxygen atom in position 4, as depicted in Scheme 3, whereas
with juglone and its derivatives this would result in the inactive
1,4-semiquinone form.
Ethers are less polar than the parent phenolic products 1 and 2
and thus more capable to permeate cell membrane. This can partly
explain the differences in activities recorded between ethers and
juglone 1 and plumbagin 2. But it is also evident that such decrease
in polarity is not sufficient to retain the activity being geranyl
ethers virtually not active. Thus also the structure of the moiety
linked to the OH group in position may play a pivotal role. Ester
derivatives are by far less polar than compounds 9–12 and
20–23, and so in principle more able to permeate the membrane
and to reach higher concentrations inside cells. However once in
the endocellular environment they may not be hydrolysed by
O
R2
R2
R1X
K2CO3
acetone, 80 °C, 1h
OR1
O
OH
Scheme 2. (R2 = H, Me, X = Br, I).
Chemical stability of esters 3–8 and 14–19 was investigated by
incubation of each product in the cell culture medium used to per-
form pharmacological assays for 72 h. After this period in every
case the percentage of recovery of each ester was >95%.
In the current study, we evaluated the in vitro growth inhibitory
properties of several naturally occurring and semi-synthetic
juglone and plumbagin derivatives. Table 1 thus shows the data
of the synthesized compounds belonging to the groups of esters
and ethers of juglone 1 and plumbagin 2 in comparison to these
two parent compounds (used as reference ones) on six selected
cancer cell lines, namely Hs683 (human dendroglioma), A549
(human nonsmall lung carcinoma), MCF7 (human breast cancer),
SKMEL-28 (human melanoma), B16F10 (murine melanoma), and
U373 (human glioblastoma). In terms of structure activity relation-
ships, the growth inhibitory capacities of the synthesized chemi-
cals are tightly linked to the type of functionalization of the
naphthoquinone nucleus. The original hypothesis that addressed
the synthesis of esters of juglone 3–8 and plumbagin 14–19 (e.g.,
hydrolysis by endocellular lipases to get a higher concentration
of the parent naphthoquinones inside the cell)18 seems to be not
satisfactory in light of the recorded results. In fact we did not vir-
tually notice better effects for long chain fatty acid esters respect to
1 and 2, with the only exception of plumbagin palmitate 15 that
recorded slightly higher values in terms of mean IC50. For Hs683
and B16F10 cell lines, activities were comparable to that of plum-
bagin 2. Acetates 9 and 19 showed a potency comparable or even
equal to the parent product. Such differences in activities between
acetate and other fatty acid esters may reflect the reported higher
kinetics of acetate hydrolysis compared to other kind of esters by
cell carboxylesterases.23 In vitro growth inhibitory effects similar
to those observed by Onodera et al.16 and Kawamura et al.17 using
juglone and plumbagin laurate and palmitate have been observed
in our case. More interesting results have been obtained for ether
derivatives of both juglone 1 and plumbagin 2. The presence of a
small to medium sterically hindered apolar substituents provided
active products with potencies comparable or even better than
the parent naphthoquinones. Form data reported in Table 1 it is
in fact evident that long chains like the geranyl ones of 13 and
24 led to a huge decrease of the growth inhibitory activity. This
O
O
OR
O
OR
OH
Scheme 3.
CT
1 (17µM)
8 (8µM)
2 (5µM)
20 (5µM)
is particularly true for 13 having IC50 values >100 lM for 4/6 cell
lines analyzed. Among the shorter chains tested, the best results
have been obtained with the 3,3-dimethylallyloxy moiety like in
compounds 12 and 23. For both mean IC50 growth inhibitory con-
centrations were equal or less than the parent compounds 1 and 2.
Considering individual cell lines juglone 3,3-dimethylallyl ether 12
was more active than juglone 1 in 3/6 cell lines analyzed (i.e., A549,
SKMEL-28 melanoma, and U373 glioblastoma cell lines).
In contrast plumbagin ethers were all more active than plumba-
gin itself with the only exception of plumbagin ethyl ether 21
(Table 1). Comparing the growth inhibitory capacities of juglone
1 and plumbagin 2 ether derivatives, it is evident that in all cases
compounds 20–23 were more efficient than compounds 9–12.
Thus the presence of the methyl group in position 2 of the naph-
thoquinone nucleus may play a crucial role for the observed
growth inhibitory effects by plumbagin derivatives. To this concern
Figure 1. Quantitative videomicroscopy analysis of compounds 1, 2, 8, and 20 on
human U373 cells after 72 h of treatment. Analysis were carried out twice in
quadruplicate for each chemicals tested (CT = controls).
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S. Fiorito et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
lipases to a large extent to release in loco the more efficient pheno-
lic compounds 1 and 2 or either may not trigger any specific targets
sharing the same lipophilicity of inactive geranyl ethers.
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rather than a cytostatic effect induced numerous cell deaths.
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selected pro-apoptotic resistant cancer cells growth in vitro.
Juglone 1 and plumbagin 2 along with their ethers displayed the
best activity sharing a putative cytotoxic action. However studies
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exerted by other naphthoquinones are needed. Such a research
activity is now ongoing in our laboratories.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.12.
017.
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Please cite this article in press as: Fiorito, S.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.12.017