Unified synthesis and cytotoxic activity of 8-O -methylfusarubin and its analogues

Article abstract of DOI:10.1039/c9ob01221d

A simple and unified synthesis of four related pyranonaphthoquinone natural products, e.g. 8-O-methylfusarubin, 8-O-methylanhydrofusarubin, fusarubin and anhydrofusarubin, is reported. The key synthetic features include the precedented Diels-Alder cycload

Full text of DOI:10.1039/c9ob01221d

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DOI: 10.1039/C9OB01221D
Unified synthesis and cytotoxic activity of 8-O-methylfusarubin and its
analogues
Pongsit Vijitphan a, Vatcharin Rukachaisirikul a, Chatchai Muanprasat b,c, Panata Iawsipo d,e,
Jiraporn Panprasert d,e and Kwanruthai Tadpetch*a
aDepartment of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Prince of
Songkla University, Hat Yai, Songkhla 90112, Thailand
bDepartment of Physiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
cExcellent Center for Drug Discovery, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
dDepartment of Biochemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science,
Burapha University, Chonburi 20131, Thailand
eUnit of Bioactive Natural Compounds for Healthcare Products Development, Faculty of Science, Burapha
University, Chonburi 20131, Thailand
ABSTRACT
A simple and unified synthesis of four related pyranonaphthoquinone natural products e.g. 8-
O-methylfusarubin, 8-O-methylanhydrofusarubin, fusarubin and anhydrofusarubin is
reported. The key synthetic features include the precedented Diels-Alder cycloaddition to
assemble the naphthalene skeleton, selective formylation and acetonylation and
intramolecular acetalization to construct the pyran ring. Manipulation of the oxidation state of
the naphthoquinone core was utilized to construct the two analogues, fusarubin and
anhydrofusarubin. This work also highlights an unprecedented directing effect of the
hydroxymethylene group in the selective hypervalent iodine-mediated quinone oxidation. The
four synthetic compounds were evaluated for in vitro cytotoxic activity against six human
cancer cells. 8-O-Methylfusarubin is the most potent analogue and displays excellent
cytotoxic activity against MCF-7 breast cancer cells with an IC50 value of 1.01 M with no
cytotoxic effect to noncancerous Vero cells, which could potentially be a promising lead
compound for anti-breast cancer drug discovery.
* Corresponding author. Tel.: +66 74 288437; fax: +66 74 558841.
Email address: kwanruthai.t@psu.ac.th (K. Tadpetch).
1

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DOI: 10.1039/C9OB01221D
Introduction
Naphthoquinones are secondary metabolites isolated from various natural sources
such as plants, fungi, algae and bacteria.1 The core structure of naphthoquinones consists of a
naphthalene ring containing two carbonyl groups at 1,4 or 1,2 positions which can be named
as 1,4-napthoquinones and 1,2-naphthoquinones, respectively (Figure 1). Pyrano-
naphthoquinones are subgroups of naphthoquinones containing a pyran ring attached to
naphthoquinones. The basic skeleton of pyranonaphthoquinones is naphtho[2,3-c]pyran-5,10-
dione (Figure 1).2 This group of metabolites has been reported to exhibit diverse biological
activities particularly antimicrobial activity against a broad range of microbial and fungal
species e.g. Staphylococcus aureus, Escherichia coli and Candida rugosa.3 Furthermore,
some pyranonaphthoquinones were shown to exhibit antiplasmodial, antimalarial and
anticancer activities.4
Figure 1. The core structures of 1,4-, 1,2-naphthoquinones and pyranonaphthoquinones.
In 2010, our research group reported the isolation of new anthraquinone and
naphthoquinone derivatives along with known fusarubin5 (1) and anhydrofusarubin6 (2) from
sea fan-derived fungi, Fusarium spp. PSU-F14 and PSU-F135.7 Concurrently, our group also
isolated a known related pyranonaphthoquinone, 8-O-methylfusarubin8 (3) from a seagrass-
derived fungus Pestalotiopsis sp. PSU-ES180 (Figure 2).9 The isolated compounds 1, 2 and 3
were examined for selected biological activities and were found to exhibit excellent in vitro
cytotoxic activity against MCF-7 human breast cancer cells with IC50 values of 9.8, 1.06, 0.9
µM, respectively. Notably, the cytotoxic activities of compounds 2 and 3 were superior to a
standard drug doxorubicin (IC50 = 2.18 µM). In addition, compounds 1, 2 and 3 displayed
very low cytotoxic effect against noncancerous African green monkey kidney (Vero) cells
with IC50 values of 79, 49 and 58 µM, respectively. This finding has renewed the interest in
the anticancer activity of this class of naphthoquinones. Owing to promising cytotoxic
activity of these pyranonaphthoquinones and as part of our ongoing program for anticancer
drug discovery, we have been focusing on a synthetic course of compounds in this class.
2

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Herein, we report a unified synthetic approach to pyranonaphthoquinone natural products 1–3
DOI: 10.1039/C9OB01221D
as well as a related analogue, 8-O-methylanhydrofusarubin10 (4) and the cytotoxic activities
against six human cancer cell lines of the synthetic compounds.
Figure 2. Structures of fusarubin (1), anhydrofusarubin (2), 8-O-methylfusarubin (3) and 8-
O-methylanhydrofusarubin (4) and cytotoxic activities against MCF-7 cells of natural
compounds 1-3 compared with doxorubicin.
To our surprise, there has been no report on the synthesis of fusarubin and 8-O-
methylfusarubin whereas only one synthesis of anhydrofusarubin is precedented to date. In
2012, de Koning and co-workers disclosed the first total synthesis of 2 using Stobbe
condensation to construct the naphthalene core and Wacker oxidation to assemble the
isochromene ring as the key strategy.11 Their synthetic approach led to 2 in 11 steps with an
overall yield of 5%.
Results and Discussion
Structurally, fusarubin (1) and 8-O-methylfusarubin (3) only differ by the position of
the 1,4-quinone moiety of the naphthoquinone core. From this inherent structural similarity, it
was envisioned that compounds 1 and 3 could be synthesized by the same synthetic approach
via manipulation of the oxidation state of the naphthoquinone core. We would begin with the
syntheses of 3 and its dehydrated analogue 4, of which their retrosynthetic analysis is
depicted in Scheme 1. 8-O-methylanhydrofusarubin (4) would be derived from 3 via
dehydration. The dihydropyran hemiacetal moiety of 3 was envisaged to be constructed from
3

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acetonylnaphthoquinone 5 via intramolecular acetalization. The acetalization precursVoierw A5rticle Online
DOI: 10.1039/C9OB01221D
would then be obtained from acetonylation of the corresponding naphthoquinone prepared
from selective oxidation of naphthol derivative 6. The hydroxymethylene moiety of 6 would
be installed via Vilsmeier-Haack formylation/reduction of known pentaalkoxynaphthalene
intermediate 7, which in turn could be prepared from 5-bromovanillin following a protocol
reported by Green and co-workers.12
Scheme 1. Retrosynthetic analysis of 8-O-methylfusarubin (3) and 8-O-
methylanhydrofusarubin (4).
The Vilsmeier-Haack formylation precursor, pentaalkoxynaphthalene 7, was prepared
following a procedure reported by Green and co-workers with slight modification (Scheme
2). Starting with commercially available 5-bromovanillin (8), dibromobenzene 9 was
obtained in 4 steps in 83% yield.13 The key Diels-Alder cycloaddition between benzyne
intermediate generated from 9 and 2-methoxyfuran (10) in THF at –78 °C constructed
naphthols 11 and 12 in 62% combined yield as an inseparable mixture. Subsequent treatment
of a mixture of 11 and 12 with NaH and CH3I at 0 °C provided the desired naphthalene
intermediate 7 in good yield.14 Our modified synthesis led to improved yield of 7 in 42% over
6 steps. Our next task was to selectively install the formyl group at C-6 using formylation
reaction. Kozlowski et al. has previously reported the Vilsmeier-Haack formylation of a very
similar pentamethoxynaphthalene analogue of 7 which yielded the formylated product at C-
6.15As expected, subjection of 7 to Vilsmeier-Haack formylation using oxalyl chloride and
DMF resulted in the formation of naphthaldehyde 13 exclusively in 86% yield.16 The position
of the newly formed formyl group was confirmed by an nOe correlation between aldehyde
4

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proton ( 10.49) and C-5 methoxy proton ( 3.92) signals as well as HMBC correlations.
DOI: 10.1039/C9OB01221D
Subsequent NaBH4 reduction of 13 smoothly provided the targeted naphthyl alcohol 6 in
quantitative yield. The next challenge was to selectively oxidize the right-hand ring of 6 to
naphthoquinone. Electronically, the left-hand ring of 6 would be more likely to be oxidized
due to an extra electron-donating methoxy group. Precedents on regioselective oxidation of
this type of substrates mediated by cerium ammonium nitrate (CAN) have revealed the
exclusive oxidation of the left ring.18 Unsurprisingly, treatment of 6 with CAN in acetonitrile
at 0 °C gave only the undesired product naphthoquinone 14 in 50% yield, which prompted us
to revise this step of the synthesis.
Scheme 2. Synthesis of pentaalkoxynaphthalene 6
To overcome electronic bias in the oxidation step, we shifted our attention to using a
hydroquinone mono ether as an oxidation precursor in order to achieve the selective
oxidation of the right-hand ring of 6. It is well documented that oxidation of hydroquinone
mono ethers to 1,4-quinones is a very facile process.18b,19 In addition, the free hydroxyl group
of the hydroquinone mono ether would be a good directing group to induce the incoming
formyl group at the requisite ortho-like position.20Thus, to prove this, we subjected a mixture
of naphthols 11 and 12 to the aforementioned formylation conditions (Scheme 3a).
Gratifyingly, the desired naphthaldehyde 15 resulting from C-formylation was obtained in
42% yield along with O-formylation product, naphthyl formate 16 in 17% yield. The position
of the formyl group of 15 was confirmed by an nOe correlation between methoxy protons (
3.80) and an aromatic proton ( 7.02) on the right ring. Moreover, the structure of formate 16
was verified by subjection to hydrolysis using 10%KOH to give the naphthol regioisomer 12
in 70% yield (Scheme 3b). The structure of 12 was confirmed by nOe enhancement of the
5

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hydroxy proton ( 9.66) after irradiation of benzylic protons ( 5.12) of the Bn group. With
DOI: 10.1039/C9OB01221D
the requisite naphthaldehyde 15 in hand, we continued with NaBH4 reduction to obtain the
corresponding naphthyl alcohol 17. Unfortunately, compound 17 is very unstable and can
easily decompose in a few hours upon exposure to air. Due to the low stability of alcohol 17,
this compound was immediately taken to the next step without chromatographic purification.
To our delight, treatment of naphthol 17 with CAN in MeCN at 0 °C smoothly furnished the
desired naphthoquinone 18 in 94% over two steps.
Scheme 3. a) synthesis of naphthoquinone 18 b) basic hydrolysis of formate ester 16
Although we were successful in the selective oxidation to secure the appropriate
oxidation state of the naphthoquinone intermediate, the drawback of this route was the use of
a mixture of naphthols 11 and 12 as formylation precursors and only 11 was consumed to the
targeted product. The other precursor 12 underwent O-formylation to generate the naphthyl
formate byproduct, which lowered the total yield of this step. To avoid this limitation, we
decided to revisit the first pathway to convert pentaalkoxynaphthalene 6 to the desired
naphthoquinone 18 by screening of other oxidizing agents for quinone oxidation (Table 1).
Using silver(II) oxide in the presence of 6M nitric acid in dioxane at room temperature11,21
led to no reaction and the starting compound was recovered (entry 1). Changing the oxidizing
6

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agents to hypervalent iodines gave more satisfactory results. Treating 6 with
DOI: 10.1039/C9OB01221D
(bis(trifluoroacetoxy)iodo)benzene (PhI(OCOCF3)2) in a 9:1 mixture of MeCN and water at 0
°C for 30 minutes yielded the undesired naphthoquinone 14 as a major product in 51% yield
and the desired naphthoquinone 18 as a minor product in only 25% yield (entry 2). Increasing
the reaction temperature to room temperature slightly improved the yield of 18 to 32% while
the undesired 14 was still a major product (entry 3). The more electron-withdrawing ligands
of PhI(OCOCF3)2 might be attributed to oxidation of the more electron-rich left ring of
naphthalene 6.22 Therefore, the less reactive hypervalent iodine reagent with less electron-
withdrawing ability ligand, diacetoxyiodobenzene (PhI(OAc)2) was investigated. Subjection
of 6 to PhI(OAc)2 under the same solvent mixture at 0 °C for 30 minutes yielded the desired
naphthoquinone 18 as a major product in 58% yield along with 14 in 34% (entry 4).
Elevating the reaction temperature to room temperature further enhanced the yield of 18 to
67% (entry 5). Thus, we further increased the reaction temperature to 50 °C under the same
conditions for 1 min, which the reaction could be judged complete by the color change from
light yellow to dark orange, and found that the requisite naphthoquinone 18 was formed as a
sole product in 75% (entry 6). The excellent regioselectivity was attributed to the
hydroxymethylene moiety as a directing group in this PhI(OAc)2-mediated oxidation at
elevated temperature. To emphasize the importance of the hydroxymethylene directing group
in this selective oxidation, pentaalkoxynapthalene 7 absent of hydroxymethylene group was
subjected to the same conditions of entry 6 (Scheme 4). It was discovered that oxidation
occurred preferentially on the more electron-rich left ring of 7 to give 19 as a major product
in 65%, whereas naphthoquinone regioisomer 22 resulting from oxidation of the less
electron-rich right-hand ring was obtained as a minor product in only 16% yield. This control
experiment strongly suggested that the hydroxymethylene could be exploited as a directing
group in hypervalent iodine-mediated quinone oxidation.
Table 1. Screening of oxidation conditions to construct naphthoquinone 18 from
naphthalene 6
7

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DOI: 10.1039/C9OB01221D
yield (%)
solvent
temp
entry
1
reagents
time
(concentration)
14
18
AgO, 6M HNO3 1,4-dioxane (0.04M)
rt
0 °C
rt
overnight
30 min
30 min
30 min
30 min
1 min
no reaction
MeCN/H2O
PhI(OCOCF3)2
2
3
4
5
6
51
42
34
28
–
25
32
58
67
75
(9:1, 0.1M)
MeCN/H2O
(9:1, 0.1M)
MeCN/H2O
(9:1, 0.1M)
MeCN/H2O
(9:1, 0.1M)
MeCN/H2O
PhI(OCOCF3)2
PhI(OAc)2
0 °C
rt
PhI(OAc)2
PhI(OAc)2
50 °C
(9:1, 0.1M)
Scheme 4. PhI(OAc)2-mediated oxidation of pentaalkoxynaphthalene 7 at 50 °C.
Having established the optimized synthetic sequence to construct the key
naphthoquinone core 18, we proceeded to complete the syntheses of the natural products 3
and 4 (Scheme 5). Although acetonylation of 18 could be performed directly in the presence
of free alcohol functionality, the yield and purity of the corresponding product were quite
low. Hence, protection of the free alcohol was required. The protecting group of choice was
the tert-butyldimethylsilyl (TBS) group since it could be removed under acidic conditions
which would also induce the intramolecular hemiacetal formation. Therefore, alcohol 18 was
protected as silyl ether 21 using TBSCl and imidazole. Introduction of the acetonyl group
was accomplished using acetylmethylpyridinium chloride (22) in the presence of Et3N in
MeCN to give acetonylnaphthoquinone 23 in quantitative yield.23 Treatment of 23 with 1M
hydrochloric acid in MeCN at room temperature resulted in simultaneous removal of the TBS
protecting group and intramolecular acetalization to smoothly furnish pyranonaphthoquinone
24 in 74% yield. Alternatively, 24 could be synthesized in a remarkable 85% via a one-pot
fashion from 21 by simply quenching the acetonylation reaction with excess HCl and
prolonged stirring without isolation of 23. Subsequent hydrogenolytic deprotection of the
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Organic & Biomolecular Chemistry
benzyl group using catalytic Pd(OH)2 in ethyl acetate afforded the natural product 3 in V6ie0w%Article Online
DOI: 10.1039/C9OB01221D
yield. It should be noted that hydrogenolysis using typical Pd/C catalyst in alcoholic solvent
led to the formation of an unwanted pyran acetal product, which could not be hydrolyzed
back to the corresponding pyran hemiacetal. Finally, following a procedure reported by
Nguyen et al.,24 compound 3 was subjected to dehydration using catalytic TsOH in toluene at
105 °C to give 4 in 69% yield. The 1H and 13C NMR spectroscopic data of synthetic 3 are in
excellent agreement with those previously reported for the natural product.25 The specific
rotation of 3 was observed as []D26 = +7.33 (c 0.03, acetone), suggesting that synthetic 3 was
obtained as a scalemic mixture. Thus, no attempts were made to identify the absolute
configuration of synthetic 3. Although there is one literature precedent by Evidente et al. on
the determination of the absolute configuration of natural 3 isolated from a grass weed-
derived fungus Rutstroemia capillus-albis26, the specific rotation and absolute configuration
of 3 isolated from other natural sources particularly Fusarium species were not reported. For
1
1
compound 4, only the H NMR data were reported in the literature.9 The H NMR data of
synthetic 4 were identical to those of natural 4. We further confirmed the identity of synthetic
4 by 13C and 2D NMR as well as HRMS data.
Scheme 5. Completion of the synthesis of 8-O-methylfusarubin (3) and 8-O-
methylanhydrofusarubin (4).
As previously mentioned, the other two targets fusarubin (1) and anhydrofusarubin (2)
only differ from 3 and 4 by the position of the 1,4-quinone moiety of the naphthoquinone
core. Therefore, we envisioned to synthesize 1 and 2 by exploiting the same strategy and
utilizing the common intermediate from the syntheses of 3 and 4 via manipulation of the
9

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oxidation state of the naphthoquinone nucleus. The retrosynthetic analysis ofDO1I: a10n.1d03V92ie/wC9AiOsrtiBcl0e1O2n2l1inDe
shown in Scheme 6. Anhydrofusarubin (2) would again be obtained from dehydration of
fusarubin (1), which in turn would be assembled by intramolecular acetalization of 25.
Naphthoquinone 25 would be accessible from selective oxidation of naphthol 26. Naphthol
26 would then be derived from selective quinone reduction/protection of naphthoquinone
intermediate 23. The appropriate protecting group should be easily and globally removed
under acidic conditions which would be used in the acetalization step as well.
Scheme 6. Retrosynthetic analysis of fusarubin (1) and anhydrofusarubin (2).
We initially chose the TBS group as a protecting group of hydroquinone for the
purpose of global deprotection. Syntheses of 1 and 2 commenced with selective reduction of
the quinone moiety of 23 using Na2S2O4 in THF at room temperature to furnish the
corresponding hydroquinone,27 which was carried on to the next step without
chromatographic purification due to its facile autoxidation upon exposure to air (Scheme 7).
Treatment of the hydroquinone with TBSOTf in the presence of 2,6-lutidine in CH2Cl2
unexpectedly provided pyran TBS ether 27 in 94% over two steps. The next task was to
selectively oxidize the left-hand ring of 27. This involved hydrogenolytic removal of the
benzyl protecting group using Pd/C catalyst to deliver the corresponding unstable naphthol,
which was immediately taken to subsequent oxidation with PhI(OAc)2 under previously
described conditions to afford pyranonaphthoquinone 28 in moderate yield (57% over 2
steps). Compound 28 was then treated with various Brønsted acids e.g. HCl, TsOH and
AcOH or TBAF in order to mediate global deprotection and concomitant formation of the
pyran hemiacetal. Disappointingly, only decomposition of the starting material or formation
10

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Organic & Biomolecular Chemistry
of complex mixtures was observed. Thus, alternative hydroquinone protectingDOgIr: o10u.1p03V9iwe/wCa9AOsrtiBcl0e1O2n2l1inDe
sought.
Scheme 7. Attempted synthesis of fusarubin (1) via TBS protection.
To circumvent the problems encountered with the TBS protecting group, we decided
to switch to the ethoxymethyl (EOM) group which should also be easily removed under
acidic conditions.28 The revised synthesis is described in Scheme 8. Selective reduction of
naphthoquinone 23 using Na2S2O4 in diethyl ether provided the corresponding hydroquinone
in 87% yield. We discovered that using Et2O as a solvent in this step shortened the reaction
time and led to higher yield of the hydroquinone product.29 In addition, despite its facile air
oxidation, quick chromatographic purification of the resultant hydroquinone was required
because employing the crude hydroquinone directly in the EOM protection step gave the low
yield of 29 (22% over two steps). Therefore, after extensive experimentation, subjecting the
purified hydroquinone to EOM protection using NaH and EOMCl in DMF at 0 °C delivered
the requisite EOM ether 29 in 60% yield (53% over two steps).30 The benzyl group was then
removed under previously described conditions for hydrogenolysis in Scheme 7 to furnish
naphthol 30 in 82% yield. Subsequent oxidation of 30 with PhI(OAc)2 under the
aforementioned conditions provided the requisite para-naphthoquinone 31 in 50% yield
along with ortho-naphthoquinone byproduct in 15%. However, the ortho-quinone byproduct
is relatively unstable and decomposes at ambient temperature in a few days. With the
requisite naphthoquinone 31 readily available, final global deprotection of 31 using 1M HCl
in MeCN and concomitant pyran hemiacetal formation were performed to deliver fusarubin
(1) in 54% yield along with dehydrated derivative, anhydrofusarubin (2) in 22% yield.
Compound 1 could be further converted to 2 in 73% yield by treating with catalytic TsOH in
1
13
toluene at 105 °C. The H and C NMR spectroscopic data in CDCl3 solvent of synthetic 1
11

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and 2 are identical to those reported in the literature.11,31 However, due to very loDwOI:s1o0l.1u0b39i/lCit9yOB01221D
View Article Online
1
of 1 in CDCl3, we also reported herein the H and 13C NMR spectroscopic data of 1 in
26
DMSO-d6 solvent. The specific rotation of 1 was observed to be []D = +4.35 (c 0.036,
acetone), suggesting that synthetic 1 was also obtained as a scalemic mixture.
Scheme 8. Completion the synthesis of fusarubin (1) and anhydrofusarubin (2).
The four synthetic naphthoquinones 1-4 were evaluated for their in vitro cytotoxic
activity against the MCF-7 breast cancer cells as well as against noncancerous Vero cells by a
colorimetric method using the resazurin microplate assay (REMA, Table 2). Among the
compounds tested, 8-O-methylfusarubin (3) showed the most potent cytotoxic activity against
MCF-7 cells with an IC50 value of 1.01 M and no cytotoxic effect to Vero cells (IC50
=
47.49 M). This observation was consistent with the reported data for the natural product
(Figure 2). Synthetic 1 and 4 possessed comparably potent cytotoxic effect against MCF-7
cells with IC50 values of 3.19 M and 2.96 M, respectively. It is worth noting that
cytotoxicity of synthetic fusarubin (1) is greater compared to the reported data of natural 1. In
addition, contrary to the observed cytotoxicity of natural anhydrofusarubin (2) (IC50 = 0.9
M), we found that synthetic 2 displayed the least potent cytotoxic effect against MCF-7
cells with an IC50 value of 12.28 M. Nevertheless, these four synthetic analogues exhibited
more potent cytotoxic activity compared to standard drugs doxorubicin and tamoxifen.
12

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DOI: 10.1039/C9OB01221D
Table 2. Cytotoxic activity of synthetic compounds 1-4 against MCF-7 cancer and Vero cells
using the resazurin microplate assay.
cytotoxicity, IC50 (M)
compound
MCF-7 cells
Vero cells
19.36
77.54
47.49
20.54
–
3.19
12.28
1.01
2.96
15.25
17.23
–
1
2
3
4
doxorubicin
tamoxifen
ellipticine
–
3.97
To further verify the results of cytotoxic activity against MCF-7 cancer cells using
REMA, synthetic compounds 1-4 were subjected to in vitro cytotoxic activity evaluation
against MCF-7 cancer cells using MTT colorimetric assay (Table 3). We observed the similar
trend in potency of compounds 1-4 against MCF-7 cells compared to REMA i.e. compound 3
displayed the most potent cytotoxicity with an IC50 value of 2.85 M. Importantly,
compounds 1-4 were tested for MCF-7 cancer cell viability in 3D cancer spheroid models via
detection of live and dead cells using high-content imaging system (Figure 3). In this assay,
compound 3 still showed the highest potency in a long-term effect (day 3). These data
suggested that 8-O-methylfusarubin (3) is the most potent analogue of this series of
pyranonaphthoquinones against MCF-7 breast cancer cells and could be a very promising
lead compound for anti-breast cancer drug discovery.
Table 3. Cytotoxic activity of synthetic compounds 1-4 against MCF-7 cancer cells using the
MTT and 3D spheroid assays.
cytotoxicity, IC50 (M)
compound
3D cancer spheroid assay
MTT assay
day 1
18.70
57.09
4.44
25.49
–
day 2
12.50
39.67
1.83
16.42
–
day 3
13.14
31.46
2.29
5.79
–
11.31
20.88
2.85
1
2
3
8.63
4
doxorubicin
28.94
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DOI: 10.1039/C9OB01221D
Figure 3. High-content imaging of synthetic 1-4 on 3D MCF-7 breast cancer spheroids.
Additionally, synthetic analogues 1-4 were further assessed for cytotoxic activities by
MTT assay against five other human cancer cell lines including three cervical carcinoma
(C33A, HeLa and SiHa), one colorectal carcinoma (HCT116) and one hepatoma (HepG2)
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cells as well as noncancerous Vero cells (Table 4). The four synthetic compouDnOdI:s10s.1h0o39w/Ce9dOB01221D
antiproliferative effect against the five cancer cell lines tested with IC50 ranges of 4.73 –
>22.5 M albeit in lower extent compared to a standard drug doxorubicin. Notably,
compounds 1-4 exhibited significant and comparable cytotoxic effect against the HCT116
colorectal carninoma cells (IC50 = 4.73–6.08 M) suggesting that the naphthoquinone core
might play an important role in this inhibitory effect. Nevertheless, in this MTT assay,
synthetic compounds 1-4 displayed less pronounced selectivity on cancer cells over Vero
cells compared to REMA. This might be attributed to sensitivity of different cancer and Vero
cells to the compounds tested as well as the different assays used.
Table 4. Cytotoxic activity of synthetic 1-4 against five human cancer cell lines and Vero
cells.
cell
lines
cytotoxicity, IC50 (M)
doxorubicin
0.14±0.03
cisplatin
1
2
3
4
C33A
HeLa
SiHa
10.47±3.21
6.98±1.72
6.05±1.74
>22.5
7.3±1.08
13.15±0.35
14.6±1.14
6.87±0.33
4.73±2.10
7.44±0.77
14.12±1.86 13.4 ±5.03
0.16±0.01 12.73±6.41
0.185±0.02 15.33±4.28
14.75±3.36
4.87±0.33
4.87±0.53
19.8±0.54
5.42±1.43
HCT116 6.08±1.47
0.23±0.03
0.66±0.06
>1
>25
>25
HepG2
Vero
5.33±0.09
9±0.67
12.65±2.87 15.54±2.24
17.92±5.46 17.83±4.73 13.63±2.21
20.13±2.44
Conclusion
We herein reported
a
simple and unified synthesis of four related
pyranonaphthoquinone natural products 1-4. We relied on the precedented Diels-Alder
cycloaddition to assemble the naphthalene core. Selective installation of the formyl and
acetonyl groups was used to install the three carbons of the pyran rings, which were in turn
constructed by intramolecular acetalization. Our work also highlighted an unprecedented
directing effect of the hydroxymethylene group in the selective hypervalent iodine-mediated
quinone oxidation. Employing this strategy, 8-O-methylfusarubin (3) and 8-O-
methylanhydrofusarubin (4) have been synthesized from Green’s intermediate 7 in 5 and 6
steps in 41% and 28% overall yields, respectively. By manipulation of the oxidation state of
the naphthoquinone nuclei from the syntheses of 3 and 4, fusarubin (1) and anhydrofusarubin
(2) could be synthesized from 7 in 8 steps in 9% and 4% overall yields, respectively. The four
synthetic compounds were evaluated for cytotoxic activity against six human cancer cells
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using the resazurin microplate and MTT assays. 8-O-Methylfusarubin (3) is the DmOIo: 1s0t.1p03o9t/eCn9OtB01221D
analogue and displays excellent cytotoxic activity against MCF-7 breast cancer cells with an
IC50 value of 1.01 M with no cytotoxic effect to noncancerous Vero cells determined by
REMA. The four synthetic compounds also showed antiproliferative effect against other five
cancer cell lines tested with IC50 ranges of 4.73 – >22.5 M as determined by MTT assay.
Conflicts of interest
There are no conflicts of interest to declare.
Acknowledgements
This work was supported by The NSTDA Chair Professor grant (the Fourth Grant) of
the Crown Property Bureau and the National Science and Technology Development Agency
to Professor V. Rukachaisirikul. P. V. thanks the Faculty of Science Research Fund, Prince
of Songkla University for research assistantship (Contract No. 1-2559-02-004). The Graduate
School, Prince of Songkla University and the Center of Excellence for Innovation in
Chemistry (PERCH-CIC), Ministry of Higher Education, Science, Research and Innovation
are gratefully acknowledged for partial support.
Notes and References
1. (a) L. Ramos-Peralta, L. I. López-López, S. Y. Silva-Belmares, A. Zugasti-Cruz, R.
Rodríguez-Herrera and C. N. Aguilar-González, In The Battle Against Microbial Pathogens:
Basic Science, Technological Advances and Educational Programs, ed. A. Méndez-Vilas,
Formatex Research Center: Spain, vol. 1, 2015, December 2015, pp. 542–550.; (b) Y.
Kumagai, Y. Shinkai, T. Miura and A. K. Cho, Annu. Rev. Pharmacol. Toxicol., 2012, 52,
221–247.
2. M. A. Brimble, L. J. Duncalf and M. R. Nairn, Nat. Prod. Rep., 1999, 16, 267–281.
3. R. A. Baker, J. H. Tatum and S. Jr. Nemec, Mycopathologia, 1990, 111, 9–15.
4. (a) J. Sperry, P. Bachu and M. A. Brimble, Nat. Prod. Rep., 2008, 25, 376–400.; (b) B. J.
Naysmith, P. A. Hume, J. Sperry and M. A. Brimble, Nat. Prod.Rep., 2017, 34, 25–61.; (c) K.
P. Kumar, K. Javvaji, Y. Poornachandra, A. D. Allanki and S. Misra, J. Microbiol. Res.,
2017, 7, 23–30.
5. V. H. W. Ruelius and A. Gauhe, Justus Liebigs Ann Chem., 1950, 569, 38–59.
16

Page 17 of 18
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C9OB01221D
6. J. H. Tatum and R. A. Baker, Phytochemistry 1983, 22, 543–547.
7. K. Trisuwan, N. Khamthong, V. Rukachaisirikul, S. Phongpaichit, S. Preedanon and J.
Sakayaroj, J. Nat. Prod., 2010, 73, 1507–1511.
8. P. S. Steyn, P. S. Wessels and W. F. O. Marasas, Tetrahedron, 1979, 35, 1551–1555.
9. Unpublished results
10. J. H. Tatum, R. A. Baker and R. E. Berry, Phytochemistry, 1985, 24, 457–459.
11. A. Pillay, A. L. Rousseau, M. A. Fernandes and C. B. de Koning, Org.Biomol.Chem.,
2012, 10, 7809–7819.
12. R. G. F. Giles, I. R. Green and N. van Eeden, Synth. Commun., 2006, 36, 1695–1706.
13. See supplementary information for detailed experimental data.
14. A. Toy, T. A. Scahill and A. Yamashita, J. Org. Chem., 1989, 54, 3625–3634.
15. A. N. Lowell, M. W. Fennie and M. C. Kozlowski, J. Org. Chem., 2008, 73, 1911–1918.
16. A. Thiraporn, V. Rukachaisirikul, P. Iawsipo, T. Somwang and K. Tadpetch, Eur. J. Org.
Chem., 2017, 7133–7147.
17. Assignment of the methoxy groups at C-2 ( 4.05) and C-4 ( 4.00) was based on nOe
enhancements by irradiations of aromatic methine proton at H-3 ( 6.81). Assignment of the
methoxy group at C-8 ( 3.88) was based on nOe enhancement by irradiation of aromatic
methine proton at H-7 ( 7.13).
18. (a) S. Kotha, V. R. Aswar and A. Manchoju, Tetrahedron, 2016, 72, 2306–2315. (b) R.
G. F. Giles, I. R. Green and N. van Eeden, Synthesis, 2004, 10, 1601–1608.
19. (a) J.-h. Cui, W. Zhou and S.-s. Li, J. Chem. Res., 2012, 264–265. (b) W. R. Roush and R.
J. Neitz, J. Org. Chem., 2004, 69, 4906–4912. (c) C.-j. Sie, V. Patteti, Y.-R. Yang and K.-K.
T. Mong, Chem. Commun., 2018, 54, 1885–1888.
20. (a) G. Jones and S. P. Stanforth in Organic Reactions, ed. L. A. Paquette, John Wiley &
Sons, Inc., 1997, vol. 49, pp. 1–330. (b) Y. Tanoue, A. Terada and Y. Matsumoto, Bull.
Chem. Soc. Jpn., 1989, 62, 2736–2738. (c) J. Lin, W. Zhang, N. Jiang, Z. Niu, K. Bao, L.
Zhang, D. Liu, C. Pan and X. Yao, J. Nat. Prod., 2008, 71, 1938–1941.(d) D. Maes, S.
Vervisch, S. Debenedetti, C. Davio, S. Mangelinckx, N. Giubellina and N. De Kimpe,
Tetrahedron, 2005, 61, 2505–2511.
21. (a) C. B. de Koning, R. G. F. Giles, L. M. Engelhardt and A. H. White, J. Chem. Soc.,
Perkin Trans 1., 1988, 3209–3216. (b) C.-L. Lee, K. Nakagawa-Goto, D. Yu, Y.-N. Liu, K.
F. Bastow, S. L. Morris-Natschke, F.-R. Chang, Y.-C. Wu and K.-H. Lee, Bioorg. Med.
17

Organic & Biomolecular Chemistry
Page 18 of 18
View Article Online
Chem. Lett., 2008, 18, 4275–4277. (c) M. A. Brimble, N. P. S. Hassan, B. J. NayDsmOI:i1t0h.10a3n9d/C9JO.B01221D
Sperry, J. Org. Chem., 2014, 79, 7169–7178. (d) J. E. Y. Sumani, K. J. Ngwira, A. Lemmerer
and C. B. de Koning, Eur. J. Org. Chem., 2019, 1145–1153.
22. (a) V. V. Zhdankin, ARKIVOC, 2009, 1–62. (b) A. Yoshimura and V. V. Zhdankin,
Chem. Rev., 2016, 116, 3328–3435. (c) T. Dohi, and Y. Kita, Chem. Commun., 2009, 2073–
2085.
23. B. Kesteleyn, L. Van Puyvelde and N. De Kimpe, J. Org. Chem., 1999, 64, 438–440.
24. T. A. D. Thi, T. H. V. Thi, H. T. Phuong, T. H. Nguyen, C. P. The, C. V. Duc, Y.
Depetter, M. D’hooghe and T. V. Nguyen, Bioorg. Med. Chem. Lett., 2015, 25, 3355–3358.
25. L. Studt, P. Wiemann, K. Kleigewe, H.-U. Humpf and B. Tudzynski, Appl. Environ.
Microbiol., 2012, 78, 4468–4480.
26. M. Masi, S. Meyer, M. Górecki, G. Pescitelli, S. Clement, A. Cimmino and A. Evidente,
Molecules, 2018, 23, 1734/1–1734/12.
27. J. Padwal, W. Lewis and C. J. Moody, J. Org. Chem., 2011, 76, 8082–8087.
28. (a) X. Cai, K. Ng, H. Panesar, S.-J. Moon, M. Paredes, K. Ishida, C. Hertweck and T. G.
Minehan, Org. Lett., 2014, 16, 2962–2965. (b) N. V. S. Mudiganti, S. Claessens, P.
Habonimana, and N. De Kimpe, J. Org. Chem., 2008, 73, 3867–3874. (c) T. Taguchi, Y.
Suda, A. Hosoda and Y. Kobayashi, Tetrahedron Lett., 1988, 29, 5765–5766. (d) G. A.
Guerrero-Vásquez, N. Chinchilla, J. M. G. Molinillo and F. A. Macías, J. Nat. Prod., 2014,
77, 2029–2036.
29. Y. Suhara, M. Watanabe, S. Motoyoshi, K. Nakagawa, A. Wada, K. Takeda, K.
Takahashi, H. Tokiwa and T. Okano, J. Med. Chem., 2011, 54, 4918–4922.
30. C.-Y. Peng, J.-Y. Shen, Y.-T. Chen, P.-J. Wu, W.-Y. Hung, W.-P. Hu and P.-T. Chou, J.
Am. Chem. Soc., 2015, 137, 14349–14357.
31. N. S. Chowdhury, Md. H. Sohrab, Md. S. Rana, C. M. Hasan, S. Jamshidi and K. M.
Rahman, J. Nat. Prod. 2017, 80, 1173–1177.
18