Efficient, collective synthesis and nitric oxide inhibitory activity of rubrolides E, F, R, S and their derivatives

Article abstract of DOI:10.1016/j.tetlet.2016.11.096

An efficient first synthesis of biologically significant natural butenolides, rubrolides F (1f), R (1r), S (1s) & its 7″,8″-didehydro derivative (1sa), and 3″-bromo rubrolide (1fa) along with the synthesis of rubrolide E (1e) and its di-O-methyl derivative (1ea) is accomplished in a collective fashion from commercially available and inexpensive precursors in overall yields of 14–48.5%. Key features are Wittig-Horner reaction, SeO₂-induced tandem allylic hydroxylation/intramolecular cyclization and Knoevenagel condensation. Next, in their inhibitory activity towards nitric oxide (NO) production in lipopolysaccharide-induced RAW 264.7 macrophages as an indicator of anti-inflammatory activity, all compounds displayed good inhibitory activity in a concentration-dependent manner. None of the compound exhibited notable cytotoxicity at the highest concentration (10?μM) and IC₅₀values are found in the range from 8.53 to 17.85?μM.

Full text of DOI:10.1016/j.tetlet.2016.11.096

Accepted Manuscript
Efficient, collective synthesis and nitric oxide inhibitory activity of rubrolides
E, F, R, S and their derivatives
Kongara Damodar, Jin-Kyung Kim, Jong-Gab Jun
PII:
S0040-4039(16)31577-5
DOI:
http://dx.doi.org/10.1016/j.tetlet.2016.11.096
TETL 48382
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 September 2016
18 November 2016
22 November 2016
Please cite this article as: Damodar, K., Kim, J-K., Jun, J-G., Efficient, collective synthesis and nitric oxide inhibitory
activity of rubrolides E, F, R, S and their derivatives, Tetrahedron Letters (2016), doi: http://dx.doi.org/10.1016/
j.tetlet.2016.11.096
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Graphical Abstract
Efficient, collective synthesis and nitric oxide
inhibitory activity of rubrolides E, F, R, S
and their derivatives
Leave this area blank for abstract info.
Kongara Damodar, Jin-Kyung Kim, Jong-Gab Jun
- Unified synthetic strategy
- 14-48.5% overall yields
- Potent in vitro nitric oxide (NO) inhibition
- No cytotoxicity
- IC50 (µM): 1e: 10.53; 1ea: 13.01; 1f: 8.53;
1fa: 11.91; 1r: 12.51; 1s: 17.85; 1sa: 13.29

1
Tetrahedron Letters
Efficient, collective synthesis and nitric oxide inhibitory activity of rubrolides E,
F, R, S and their derivatives
Kongara Damodara, Jin-Kyung Kimb, Jong-Gab Juna,
a Department of Chemistry and Institute of Applied Chemistry, Hallym University, Chuncheon 24252, Korea.
b Department of Biomedical Science, College of Natural Science, Catholic University of Daegu, Gyeungsan-Si 38430, Korea.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
An efficient first synthesis of biologically significant natural butenolides, rubrolides F (1f),
R (1r), S (1s) & its 7",8"-didehydro derivative (1sa), and 3"-bromo rubrolide (1fa) along
with the synthesis of rubrolide E (1e) and its di-O-methyl derivative (1ea) is accomplished
in a collective fashion from commercially available and inexpensive precursors in overall
yields of 14-48.5%. Key features are Wittig-Horner reaction, SeO2-induced tandem allylic
hydroxylation/intramolecular cyclization and Knoevenagel condensation. Next, in their
inhibitory activity towards nitric oxide (NO) production in lipopolysaccharide-induced
RAW 264.7 macrophages as an indicator of anti-inflammatory activity, all compounds
displayed good inhibitory activity in a concentration-dependent manner. None of the
compound exhibited notable cytotoxicity at the highest concentration (10 µM) and IC50
values are found in the range from 8.53 to 17.85 μM.
Available online
Keywords:
Rubrolides
Wittig-Horner reaction
Knoevenagel condensation
Nitric oxide
Anti-inflammatory
2015 Elsevier Ltd. All rights reserved.
Butenolide, a five membered α,β-unsaturated γ-lactone
scaffold, is found in a plethora of natural products and
pharmaceutical agents.1 The structural discrepancy within the
butenolides is complemented by various biological activities.
Rubrolides are biologically active marine ascidian metabolites
belong to the family of butenolides with an alkylidene and
phenolic moieties appendages at C-5 and C-4 position,
respectively.
Rubrolides E (1e) and F (1f) were first isolated from a marine
tunicate Ritterella rubra along with rubrolides A-D, G, H and
possessed strong antibacterial as well as selective protein
phosphatases 1 and 2A inhibition activity.2 Recently, rubrolide R
(1r) and S (1s) have been isolated from the fermentation broth of
the marine-derived fungus Aspergillus terreus OUCMDZ-1925.3
Of note, rubrolide R (1r) was shown to display comparable or
superior antioxidant activity to those of trolox and ascorbic acid
with an IC50 value of 1.33 mM while rubrolide S (1s) exhibited
comparable or superior anti-influenza A (H1N1) virus activity to
that of ribavirin with an IC50 value of 87.1 mM.3 Halogenated
rubrolides such as 3"-bromorubrolide (1fa) (Fig. 1) were also
isolated from Synoicum globosum ascidian and showed
antibacterial activity.4 Other promising activities of rubrolides
and their analogues include inhibition of bacterial biofilm
formation,5 anticancer,6 human aldose reductase (ALR2)
inhibition,7 anti-tobacco mosaic virus (anti-TMV) activity8 and
Figure 1: Structures of rubrolides E (1e), F (1f), R (1r) and S (1s), 3"-bromo
rubrolide (1fa) and their derivatives (1ea and 1sa).
inhibition of photosynthesis.9
The uncomplicated molecular architecture in conjunction with
multifarious biological activities of rubrolides have attracted the
endeavours from synthetic community and, to date, syntheses of
rubrolides C, E, L and M have been achieved10 and some groups
have also made great efforts in their analogues preparation.11
Recently, Barbosa and co-workers disclosed a concise synthesis
of rubrolides B, I, K and O using Suzuki coupling and a late-
stage regioselective bromination reaction as key steps.12
———
 Corresponding author. Tel.: +82-33-248-2075; fax: +82-33-256-3421
e-mail: jgjun@hallym.ac.kr

2
ether afforded C-prenylated product 9 in 80% yield.15 The free
phenolic group of 9 was protected as a EOM ether by reacting
with EOM-Cl and K2CO3/TBAI system to afford compound 10.
Cyclization of
monohydrate (p-TsOH·H2O) gave 11 in 90% yield. Next, 2,3-
dichloro-5,6-dicyano-p-benzoquinone (DDQ)-promoted
9 using catalytic p-toluenesulfonic acid
oxidative cyclization of 9 in refluxing benzene resulted the
chromene aldehyde 12 in 81% yield.
To complete the synthesis of the butenolide natural
products, rubrolides, with 2, 3 and aldehydes 10-14, we chose a
two-step reaction sequence comprising the Knoevenagel
condensation and deprotection. Compound
2 underwent
Knoevenagel condensation with p-anisaldehyde (13) in the
presence of piperidine as a base in MeOH to give butenolide
1ea, which was subsequently subjected to demethylation with
boron tribromide (BBr3) to afford rubrolide E (1e) in 95% yield.
Next, Knoevenagel condensation of 3 with 13, 3-bromo-4-
hydroxybenzaldehyde (14) and aldehydes 10-12 afforded
butenolides 15-19 in 51-70% yields, respectively. To our delight,
deprotection of EOM-group of compounds 15-19 was smoothly
proceeded with Dowex® resin and furnished rubrolide F (1f), 3"-
bromo rubrolide (1fa), rubrolides R (1r), S (1s) and its derivative
(1sa), respectively. All the natural products (1e, 1f, 1fa, 1r and
1s) and their derivatives (1ea and 1sa) were settled from their
spectral (1H, 13C- NMR and MS) data.
Scheme1: General retrosynthetic analysis of rubrolides under the current
study.
In connection with our continued interest13 in the synthesis of
bioactive natural products and their analogues, herein, we
describe the first synthesis of rubrolides F (1f), R (1r), S (1s) &
its 7",8"-didehydro derivative (1sa), and 3"-bromo rubrolide
(1fa) along with the synthesis of rubrolide E (1e) and its di-O-
methyl derivative (1ea). We have envisioned that the target
compounds can be achieved by a unified synthetic sequence
comprising Wittig-Horner reaction, SeO2-induced tandem
oxidative cyclization and Knoevenagel condensation as pivotal
steps (Scheme 1).
Inhibition of nitric oxide (NO) production:
Inflammation is an essential host response to infection by
pathogens, damaged cells or irritants. This complex but highly
coordinated process results in the increased production of
various soluble mediators including chemokines, cytokines, free
radicals (such as nitric oxide (NO)), and eicosanoids
(prostaglandins) by resident cells (that is, tissue macrophages,
lymphocytes, fibroblasts, endothelial cells and mast cells) in the
injured or infected tissue.16 NO, produced from L-arginine by
nitric oxide synthase (endothelial-NOS, neuronal-NOS and
inducible-NOS), is one of the important one among the above
stated mediators. The role of NO in the pathogenesis of
inflammation tightly depends upon its concentration.17
Physiologically vital amounts of NO produced by inducible-
NOS (iNOS) in response to inflammatory stimuli help in
mounting an effective defense against pathogens, whereas,
overproduction of NO by iNOS could potentially result in tissue
damage and activation of proinflammatory mediators associated
with acute and chronic inflammations.18 Hence, more attention is
now being paid to the pharmacological interference with the NO
production cascade and can be considered as a promising
strategy for the development of new anti-inflammatory drugs.
To investigate the inhibitory effect of the synthesized
rubrolides (1e, 1f, 1r, 1s and 1fa) and their analogues (1ea and
1sa) against NO production, we measured the amount of nitric
oxide (NO) in lipopolysaccharide (LPS)-stimulated RAW 264.7
cells as an indicator of anti-inflammatory activity, following our
previous procedure.13a N-Monomethyl-L-arginine (L-NMMA)
was employed as a positive control.19 As shown in Table 1, all
the compounds had a concentration-dependent inhibitory effect
on NO production. At the highest concentration (10 µM),
rubrolide F (1f) had the strongest inhibitory effect that means
As shown in Scheme 2, our synthetic sequence commenced
with the Wittig-Horner olefination of commercially available 4-
methoxyacetophenone (5). Reaction of
5 with trimethyl
phosphonoacetate in the presence of sodium hydride (NaH) at 0
ºC, gave the corresponding E-isomer of α,β-unsaturated ester 4
in 83% yield. Ester 4 was subjected to selenium dioxide (SeO2)-
oxidation, wherein, the allylic methyl group underwent
hydroxylation and subsequent intramolecular cyclization
furnished rubrolides back bone, 4-(4-hydroxyphenyl)furan-
2(5H)-one (2). Treatment of 4 with 1.5 equiv. of SeO2 in 1,4-
dioxane under reflux condition for 3 h offered the compound 2 in
73% along with the corresponding over oxidised product,
aldehyde 6, in 13% as a E,Z-mixture. Varying the time and
equivalents of SeO2 resulted in the diminished yields of 2 due to
either remaining starting material or it’s over oxidation.
Previously, Kagabu and co-workers reported the similar strategy
with SeO2 in acetic acid and perchloric acid as a catalyst,
wherein, the reaction times are relatively longer and had tedious
work up procedure.14 Demethylation of compound 2 using BBr3
produced compound 7 in 97% yield. Compound 7 was protected
as 4'-ethoxymethyl (EOM) ether 3 (91%) using chloromethyl
ethyl ether (EOM-Cl), K2CO3 and catalytic tetrabutylammonium
iodide (TBAI).
With requisite key intermediates 2 and 3 in hand, we then
turned our attention to procure the corresponding aldehydes.
Dimethylallylation of OH group of 4-hydroxybenzaldehyde (8)
with tert-butyl (2-methylbut-3-en-2-yl) carbonate and catalytic
tetrakis(triphenylphosphine)palladium(0) [Pd(PPh3)4] followed
by microwave-promoted Claisen rearrangement of the resulting
low amount of NO release from the cells (45.19 ± 4.91; IC50
=
8.53 μM) which was in comparison with the positive control, L-

3
Scheme 2: Synthesis of rubrolides and derivatives. Reagents and conditions: (a) trimethyl phosphonoacetate, NaH, 0 ºC, 0.5 h, then 5, rt, 14 h, 83%. (b) SeO2,
1,4-dioxane, reflux, 3 h, 73% (2), 13% (6). (c) BBr3 (1.0 M in CH2Cl2), -78 0 ºC then rt, 24 h, 97%. (d) EOM-Cl, K2CO3, TBAI, 0 ºC then rt, 15 h, 91%. (e)
EOM-Cl, K2CO3, TBAI, 0 ºC then rt, 20 h, 69%. (f) p-TsOH·H2O, toluene, reflux, 2 h, 90%. (g) DDQ, benzene, reflux, 45 min, 81%. (h) piperidine, MeOH, 25-
30 ºC, 15 h, 80% (1ea), 69% (15), 53% (16), 51% (17), 70% (18), 68% (19). (i) BBr3 (1.0 M in CH2Cl2), -78 0 ºC then rt, 24 h, 95%. (j) Dowex® resin, MeOH,
35 ºC, 24 h, 95% (1f), 89% (1fa), 51% (1r), 95% (1s), 83% (1sa).
NMMA (44.72 ± 1.18; IC50 = 5.13 μM) followed by
rubrolide S derivative (1sa) (53.87 ± 3.17; IC50 = 13.29 μM) and
rubrolide E (1e) (54.47 ± 2.31; IC50 = 10.53 μM). Next, cell
viability assay was conducted to discern whether the inhibition
of NO production resulted from the cytotoxicity of the tested
compounds. All the compounds did not have significant
cytotoxicity at the highest concentration (10 µM) leading to the
effective inhibition of NO production (Table 1). No clear
structure-activity relationship was observed in this study,
however, our results specified further examples of the
importance of rubrolide compounds as significant NO
production inhibitors.
In conclusion, we have achieved an efficient first synthesis of
bioactive natural butenolides, rubrolides F (1f), R (1r), S (1s) &
its 7",8"-didehydro derivative (1sa), and 3"-bromo rubrolide
(1fa) along with synthesis of rubrolide E (1e) and its di-O-
methyl derivative (1ea) in a collective manner starting from
commercially available and inexpensive precursors and in
overall yields of 14-48.5%. This synthetic approach relies on the
application of Wittig-Horner reaction and SeO2-induced tandem
oxidative cyclization, which efficiently provides an access to the
rubrolide core skeleton. Additionally, synthesized rubrolides
screened for their inhibitory effect against NO production in
LPS-induced RAW 264.7 cells, in which, all compounds showed

4
significant inhibition in a concentration-dependent manner with
no cytotoxicity. IC50 values are found in the range from 8.53 to
17.85 µM. Moreover, this efficient and flexible synthetic route
offers opportunities to make analogues of rubrolides and may
facilitate their biological studies.
Table 1. NO production inhibitory activities of rubrolides E (1e), F (1f), R (1r), S (1s), 3"-bromo rubrolide (1fa) and their
derivatives (1ea and 1sa)
Compound
NO Productiona,b
Proliferationa
IC50 (µmol/L)
1 µmol/L
6.71 ± 1.61
10 µmol/L
6.71 ± 1.61
98.03 ± 2.50
1 µmol/L
10 µmol/L
100 ± 0.03
Medium
100 ± 0.03
LPS
98.03 ± 2.50
84.99 ± 2.77
87.15 ± 2.16
87.95 ± 0.33
86.92 ± 1.55
79.61 ± 4.11**
90.58 ± 5.67
89.03 ± 0.80
75.16 ± 5.12***
1e
1ea
54.47 ± 2.31***
66.25 ± 0.56***
45.19 ± 4.91***
57.8 ± 1.08***
61.58 ± 1.88***
68.31 ± 2.80***
53.87 ± 3.17***
44.72 ± 1.18***
103.83 ± 0.07
95.08 ± 0.03
100.55 ± 0.08
97.27 ± 0.04
98.91 ± 0.03
95.08 ± 0.03
97.81 ± 0.07
99.64 ± 0.02
108.74 ± 0.02
97.27 ± 0.05
103.28 ± 0.01
97.27 ± 0.05
97.81 ± 0.05
93.99 ± 0.04
92.35 ± 0.05
95.81 ± 0.06
10.53
13.01
8.53
1f
1fa
11.91
12.51
17.85
13.29
5.13
1r
1s
1sa
L-NMMA
a The results are reported as mean value ± SEM for n = 3. Statistical significance is based on the difference when compared with LPS-treated
groups (**P < 0.01 and ***P < 0.001).
b Inhibition is based on LPS.
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Supplementary Material
Supplementary
data
(experimental
procedures
and
characterization data and copies of H- and 13C-NMR spectra)
1
associated with this article can be found in the online version at
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5
Highlights
1. First synthesis of rubrolides F, R, S and their analogues in 14-36% overall yields.
2. Synthesis of rubrolide E has also been achieved with an overall yield of 46%.
3. Key steps: Wittig-Horner, SeO2-induced oxidative cyclization & Knoevenagel condensation.
4. Significant in vitro nitric oxide production inhibition without notable cytotoxicity.
5. IC50 values are found in the range from 8.53 to 17.85 μM.