Organic Letters
Letter
this approach nevertheless provided 8 in higher overall yield
(15% over 4 steps as compared to those reported by Lin (10%
over 15 steps) and Fujimoto (4% over 9 steps)). Subsequent
conversion of the azide on 7 to acetamide using thioacetic acid
gave 8 in 64−70% yield.
from the NF-κB reporter assay (Figure 4c) indicated that LPS
significantly increased the NF-κB activity compared to control
J774A.1 macrophages, and this effect was further enhanced by
1e in a dose-dependent manner. It is noteworthy that 1e alone
did not alter the NF-κB activity in control cells. This suggests
that 1e increases LPS-induced TNF-α production in macro-
phages possibly through enhancing NF-κB activity.
Synthesis of the diacylglycerol fragment 4 started with the
benzylation of (S)-solketal (Scheme 3). Since the fatty acid in
the secondary position is labile to 1,2-acyl migration under
acidic/basic conditions,17 the benzyl moiety was chosen as the
protecting group of the primary alcohol. Subsequent removal
of the isopropylidene acetal gave compound 9 in 90% yield.
To attach different fatty acids on the two hydroxyl groups of
9, we attempted a regioselective esterification of the primary
alcohol using the DCC/DMAP system. However, the reaction
was only moderately selective for the primary ester. Attempts
to selectively esterify the primary alcohol using other reaction
conditions (Mukaiyama reagent, oxyma/EDC, B(OH)3, Zn-
(OTf)2, PPh3/DIAD, PPh3/I2, CDI, Fe(acac)3, DCC/2,6-
lutidine) resulted in either very low yields or no desired
product. Further optimization eventually showed that the
TBTU/base (base: TEA, DIEA,18 DBU, K2CO3, NMM, and
DABCO) system is highly selective for the primary alcohol
(>90%) with TBTU/NMM giving 10 with the best yield and
selectivity (yield: 73−85%; selectivity of 1°:2° = 99:1). A
second esterification with DCC/DMAP gave 11 (yield: 70−
91%), following which debenzylation using 10% Pd/C in
ethanol with 5% acetic acid provided the diacylglycerol 4 in
quantitative yield.
To investigate the mechanism of action of 1e on the
inhibition of IL-6 production, Western blots were performed.
The results showed that while LPS significantly increased
phosphorylation levels of ERK1/2, JNK1/2, and p38, treat-
ment of the same LPS-activated macrophages with 1e did not
alter the phosphorylation levels (Supporting Information,
Figure S2), implying that 1e-mediated modulation of IL-6
and TNF-α did not occur via the activation of mitogen-
activated protein kinases. Western blot results (Supporting
Information, Figure S3) also showed that treatment with 1e
did not change the expression levels of the autophagy markers
LC3, p62, and ATG5, indicating that 1e did not alter
autophagy induction either. Hence we decided to examine
the effects of 1e on the IL-6 mRNA expression in LPS-
activated macrophages using RT-qPCR. The results obtained
(Figure 4d) showed that treatment with LPS significantly
increased the IL-6 mRNA expression, but this expression was
reduced in the presence of 1e, suggesting a correlation between
1e treatment and IL-6 mRNA expression. Further experiments
are presently underway to elucidate the detailed mechanism of
action.
In summary, a shorter synthesis of the PGL-1 analogue 1 has
been developed. Immunomodulatory studies show that the
activity of 1 is dependent on the chain lengths of the three fatty
acid tails. Preliminary studies to elucidate the mechanism of
action suggest that the immunomodulatory activity of 1 could
proceed via NF-κB activation.
Treatment of 4 with PCl3 in the presence of imidazole and
TEA at 0 °C11 gave the H-phosphonate intermediate 12 which
was stable at room temperature, thus allowing easy handling
and installing of the same H-phosphonate intermediate on
different analogues of 8 (Scheme 4). Phosphorylation to
compound 13 was achieved under microwave conditions using
trichloroacetonitirile19 as an activator. Amberlyst-15 ion-
exchange resin was then used to remove all the TEAH+ ions
prior to hydrogenation with 10% Pd/C catalyst to yield 1 in
the acid form. A total of 19 PGL-1 analogues were synthesized
(Table 1).
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
To investigate the immunomodulatory properties of 1,
macrophages were treated with the respective compounds and
then activated with LPS. The levels of IL-6 and TNF-α
produced were quantified using ELISA. Results obtained
showed that most of the compounds reduced IL-6 production
(Figure 3a) and increased TNF-α production in LPS-activated
macrophages (Figure 3b), indicating that 1 exhibits immuno-
modulatory activities in macrophages. Among the PGL-1
derivatives, compounds 1b, 1e, and 1g exhibited stronger
inhibition of IL-6 production and larger induced TNF-α
production in LPS-activated macrophages and were thus
further evaluated for their cytotoxicities against J774A.1
macrophages (Supporting Information, Figure S1). The results
obtained showed that all three compounds reduced the
viability of J774A.1 macrophages in a dose-dependent manner,
with 1e being the least cytotoxic. Thus, compound 1e was used
in subsequent biological studies.
To investigate the dose response of 1e on IL-6 and TNF-α
in mouse macrophages, macrophages were treated with varying
concentrations of 1e and then activated with LPS. The levels of
IL-6 and TNF-α produced were quantified using ELISA.
Results showed that 1e dose-dependently reduced IL-6
production (Figure 4a) and increased TNF-α production in
LPS-activated macrophages (Figure 4b). In addition, results
Experimental procedures, characterization of the prod-
ucts, and cytotoxicity data of compounds 1b, 1e, and 1g
against J774A.1 macrophages (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
Shih-Hsiung Wu − Institute of Biological Chemistry, Academia
Kuo-Feng Hua − Department of Biotechnology and Animal
Science, National Ilan University, Yilan County 260, Taiwan;
Department of Pathology, Tri-Service General Hospital,
National Defense Medical Center, Taipei 11490, Taiwan;
Department of Medical Research, China Medical University
Hospital, China Medical University, Taichung 40402, Taiwan;
Yulin Lam − Department of Chemistry, National University of
Authors
Chin Heng Gan − Department of Chemistry, National
University of Singapore, Singapore 117543 Singapore;
D
Org. Lett. XXXX, XXX, XXX−XXX