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K. Damodar et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Fig. 1. Structures of portulacanones A–D (4, 8, 9 and 6) and their derivatives (1–3, 5 and 7).
diseases.11 Upon isolation these compounds were evaluated and
showed in vitro cytotoxic activities towards four human cancer cell
lines, including SGC-7901, NCI-H460, K-562 and SF-268.11 Intrigu-
ing bioactivity of POL, especially promising anti-inflammatory
activity,12 and no report on the synthesis of portulacanones A–D
led us to attempt the synthesis and study their NO inhibitory activ-
ity. As part of our ongoing interest13,14 in the synthesis of bioactive
natural products and their analogues as potent NO production
inhibitors, herein, we report the synthesis and in vitro study of
( )-portulacanones A–C (4, 8, and 9) portulacanone D (6) and their
derivatives (1–3, 5 and 7) (Fig. 1).
structural component of the outer wall of Gram-negative bacteria.
It is well known that treatment of RAW 264.7 macrophages with
LPS induces NO production. NG-Monomethyl-
L-arginine (L-NMMA)
has been reported to significantly suppress NO production.15 In
this study, RAW 264.7 macrophages were treated with LPS and
0.1, 1, 10, 25, 50 and 100 mM concentrations of compounds 1–9
and
measured. However, at 25, 50 and 100
pounds 1–9 exhibited almost same level activity (Fig. 2). Signifi-
cant NO suppression changes were observed at 1–10 M. Hence,
we discussed the activities at 1 and 10 M concentrations only.
L-NMMA as control, then NO production and cell viability were
lM concentrations, com-
l
l
The synthesis of homoisoflavonoids (1–9) commenced with the
Friedel-Crafts acylation of commercially available 1,3,5-
trimethoxybenzene (10) (Scheme 1). Treatment of 10 with acetic
anhydride in the presence of boron trifluoride diethyl etherate
(BF3ÁEt2O) provided compound 11 in 89% yield. Compound 11
underwent selective ortho-demethylation using AlCl3 to furnish
compound 12 in 87% yield. Another acetophenone 14 was accessed
from compound 13. Acetylation of compound 13 and subsequent
Fries rearrangement afforded 14 in an excellent yield of 93% over
two steps. Protection of salicylaldehyde (15) as its ethoxymethyl
(EOM) ether (16) using chloromethyl ethyl ether (EOM-Cl), K2CO3
and catalytic tetrabutylammonium iodide (TBAI) proceeded in
83% yield. Next, condensation of commercially available 2-
hydroxy-4-methoxyacetophenone (17), compounds 12 and 14
with N,N-dimethylformamide dimethyl acetal (DMF-DMA) fol-
lowed by acid treatment of the resulting enamino ketones gave
the corresponding 4H-chromen-4-ones 18a–18c in 88–91% yields,
respectively. Catalytic hydrogenation of 18a–18c delivered the cor-
responding chroman-4-ones 19a–19c in 83–87%, respectively.
Having the key fragments 19a–19c and 16 in hand, next, we set
out to explore the key Aldol condensation reaction. At first, we
tried the reactions between 19b and 15 using p-toluenesulfonic
acid (pTsOH) in benzene which was not successful. Next, conden-
sation of 19b and 16 using piperidine as a base in benzene as well
as in DMF at 30–100 °C resulted in low yield (<20%) of product
(20b) formation. Gratifyingly, we identified KOH in EtOH/H2O
(5/1) was effective for the condensation of 19b and 16. Subse-
quently, the reaction of 19a and 19c with 16 provided 20a (48%)
and 20c (25%), respectively. Deprotection of 20a–20c using 1 N
HCl gave the compounds 1, 3 and 7 in high yields. Catalytic hydro-
genation of 1, 3 and 7 afforded the homoisoflavonoids 2, 4 and 8 in
86–91% yields, respectively. Finally, selective ortho-demethylation
of 4, 8 and 3 using 1.0 M BCl3 (in CH2Cl2) smoothly furnished the
leftover three target compounds 5, 9 and 6, respectively. All the
final products (1–9) structures were settled from their spectral
data (1H and 13C NMR and MS) (see the Supplementary
information).
The magnitude of NO released from macrophages was determined
by measuring the concentration of nitrite, a stable oxidized pro-
duct of NO, in the culture supernatant using the Griess reagent.
All the compounds tested (1–9) had a concentration-dependent
inhibitory effect NO production by 264.7 macrophages (Table 1).
The percentage of NO inhibition ranged from 97.2% to 15.9% at
the highest (10
compounds i.e.,
(92.5%), 1 (91.4%) and 7 (83.0%) showed the strongest inhibitory
activities at 10 M (Table 1 and Fig. 2). At the lowest concentration
(1 M), compound 3 still significantly reduced the NO production
lM) concentrations. Of the 9 compounds (1–9), 4
3
(97.2%), followed by (portulacanone D)
6
l
l
(38.5%) by 264.7 macrophages. IC50 values of 1–9 were evaluated
by GraphPad Prism 4.0 software and showed 1.75, 14.17, 1.26,
14.17, 12.42, 2.09, 2.91, 13.43 and 12.16 mM respectively (Table 1).
The cytotoxicity of the compounds against RAW 264.7 macro-
phages was also tested by MTT assay to ascertain that the observed
NO inhibitory effect of 1–9 was not due to the cell death. None of
the tested compounds exerted detectable cytotoxicity at 10 lM
concentration for 24 h, which was leading to effective inhibition
of NO production (Table 2). Although, the compounds 1–9 had
good activity against NO production at 50
most likely due to cytotoxic effects of 1–9 on the macrophages
(Fig. 2 and Table 2). Percentages of cell viability at 50 M and
100 M respectively were only in the range of 10.7–58.7% and
lM and 100 lM, this is
l
l
10.9–44.0%. We further evaluated whether these inhibitory effects
are related to iNOS modulation using Western blot analysis. As
shown in Fig. 3, the results were in accordance with the findings
related to NO production (Table 1 and Fig. 2), the protein expres-
sion of iNOS induced by LPS in RAW 264.7 cells was markedly
inhibited by compounds 1, 3, 6 and 7 treatment. However, these
compounds did not affect the expression of b-actin, the housekeep-
ing gene. This indicates that the reduced expression of iNOS due to
these compounds exposure was responsible for the inhibition of
NO production. As we screened a limited number of compounds
i.e., only 9 compounds, thorough structure-activity relationship
(SAR) analysis is not possible. However, the following might be
worth noting: 1) double bond between C3 and C11 appears to be
crucial for the observed inhibition of NO as compounds 1, 3, 6
and 7 were the most efficacious. 2) Within the C3 and C11 double
bond containing compounds (1, 3, 6 and 7), compounds with two
NO inhibitory potential of the synthesized homoisoflavonoids
(1–9) was monitored in vitro by incubating RAW 264.7 macro-
phage cell lines with bacterial lipopolysaccharides (LPS), a major
Damodar, Kongara
