Significant differences were considered for p values inferior to 0.05. *p<0.05,
**p<0.01, ***p<0.001 as compared to control.
test its Lp-PLA2 inhibitory activity. We were able to hydrolyze
efficiently 5 in 76% yield. The peptide coupling allowed 16
isolation in 80% yield. Both Woolford et al and Liu et al showed
that the thiouracile moiety and particularly the carbonyl oxygen
forms hydrogen bonding with Leu153, Phe274 and Gln352 while
the fluorophenyl moiety interacts with Leu107, Leu159, Ala355
and Phe357 in the active site of Lp-PLA2.25,26 Group 4 brings
lipophilic interactions with Phe125 along with π-π interactions
that improve the already strong affinity towards Lp-PLA2.
However, in the case of O-alkylated products, the carbonyl
oxygen may not be available to interact within the active site of
the enzyme.
To test this hypothesis, we decided to compare the ability of
14, 16, and esters 5, 6 to inhibit Lp-PLA2 (Figure 5) according to
the protocol described in the experimental section. 5 did not
inhibit Lp-PLA2 activity whereas 6 showed a dose-dependent
inhibitory effect. This underlines the importance of the
regioisomer since O-alkylated chains block the crucial interaction
within the enzyme recognition site. The same effect can be
observed with 14 and 16.
Our work summarizes different methods to synthesize 14 and
points out ambiguities in the literature during the alkylation of
the thiouracile intermediate. We have shown that, based on our
results, alkylation in DMF yield most likely the O-alkylated
analogs instead of the N1-alkylated isomers as claimed by the
literature. We showed that the method developed by GSK
(Mulholland’s method) is highly substrate-dependent and cannot
be used without triflated alcohols. 14, 6 as well as 16 and 5 were
tested for their ability to inhibit Lp-PLA2. In vitro experiments
demonstrated that the O-alkylated molecules did not modify Lp-
PLA2 activity compared to N1-alkylated compounds. In
conclusion, caution should be exerted when modifying
experimental conditions (temperature, solvent, etc.) that may lead
to the production of unexpected regioisomers. A more advanced
characterization analysis such as 2D NMR should be performed
to ascertain the exact structure of the desired compounds.
Scheme 5. Peptide coupling in the divergent synthesis of 14.
Acknowledgments
This work was supported by the ERDF (European Regional de-
velopment fund) number 33054 as well as a Philancia fund.
Florian Guibbal was supported by a CIFRE doctoral grant
n°2013/1522. The authors thank Dr. Fanny Lesage for her work
on statistical analysis. This work has benefited from the facilities
and expertise of the Small Molecule Mass Spectrometry platform
of ICSN and NMR service (Centre de Recherche de Gif-
www.icsn.cnrs-gif.fr).
References and notes
Scheme 6. Synthesis of 16. Reagents and conditions: (a) TFA, DCM, RT,
There are no conflicts to declare. The manuscript was written
through contributions of all authors. All authors have given
approval to the final version of the manuscript
76%; (b) COMU, DiPEA, DMF, 0°C then RT, 80%.
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Figure 5. Lp-PLA2 activity test of 14 compared to 3 synthesized analogs. A:
(6), B: (14), C: (5), D: (16). Data are expressed as means SEM of three
independent experiments performed in triplicate. Statistical evaluation was
performed by using one-way ANOVA and Tukey test (PRISM software).