M. Fantacuzzi et al. / Journal of Pharmaceutical and Biomedical Analysis 120 (2016) 419–424
423
was evaluated by measuring the l-citrulline level, equal to NO pro-
duction, and, for this reason, the enzymatic mixture was derivatized
with OPA/NAC, and the citrulline fluorescent derivative was quan-
tified with RP-HPLC. The introduction of 0.1% v/v of TFA as ion-pair
in the mobile phase improved the chromatographic peak shape.
the inhibition of iNOS and/or nNOS without the involvement of the
endothelial isoform, for a first biological screening 10 M concen-
tration of each compound in the presence of one NOS isoform was
used.
Fig. 2 shows the percentage of residual NOS activity in the
presence of each compound compared to the control (absence of
inhibitor) and blank (absence of NADPH, which starts the enzymatic
reaction). The percentage of activity of endothelial and neuronal
NOS are, for all acetamidine derivatives, superior to 95.4%, stat-
ing that these compounds do not affect these isoforms and can
be considered as selective iNOS inhibitors. In the case of iNOS,
on the contrary, all compounds show a decrease of NOS activ-
ity. In particular, in the series of 1400W derivatives, compounds
2–5, the percentage of activity raise with the methyl chain length
increasing between the indole ring and the amide; in fact, com-
pound 2, containing only a methylene shows 21.9% of activity
and compound 3, containing two methylene chain, possesses 34%
of activity. The chain of three or four methyl, compounds 3 and
4, generate a loss of activity (53.9 and 86.6% of activity, respec-
tively).
In the indole series, 6–13, the acetamidino group is directly
liked or through methyl chain in different position of the indole
ring. Compound 6, in which the acetaminidino function is linked
to C2 of the indole via a methylene linker, possesses 50.6% of
activity, and 10 M can be considered as the IC50 value. Com-
pounds 7–10, differing from each other only for the chain length
that connect the acetamidine to C3, possess percentage of activ-
ity depending on the chain length: longer is the chain, lesser is
the percentage of activity, until a length of three carbon atoms.
Compounds 8–10 show, respectively, 22.7%, 15.5%, and 20.5% of
activity. The best value is obtained with compound 9. Compound
11, in which the acetaminidino function is directly linked to C5,
possesses 81.4% of activity indicating that this modification did
not improve the inhibitory activity of this compound compared
to the lead. Little modification to the structure of 11, that is the
introduction of a methylene spacer between the acetamidine and
C5, compound 12, or the switch of the acetamidine to C6, com-
pound 13, decrease the percentage of activity to 18.2% and 13.6%,
respectively, making them good inhibitor for further investiga-
tions.
amino moiety of the lead 1 with an indole ring seems to be very
promising. In the series of quinoline derivatives, the IC50 values
were higher than 2 M.
3.4. Chemical stability
Compounds 2–18 were analyzed for the chemical stability in
aqueous buffer at pH 2.0 with hydrochloric acid solution to simu-
late a non-enzymatic gastric fluid, at pH 7.4 with phosphate buffer
to simulate a non-enzymatic intestinal fluid, and at pH 9.0 with
sodium hydroxide solution. Compounds were injected in the HPLC
system with UV detection at precise time (0, 1, 2, 4, 8, 24, 48, and
72 h) and all of them resulted stable.
4. Conclusion
In the present study, the synthesis and the biological evalu-
ation of novel acetamidine derivatives as Nitric Oxide Synthase
inhibitor were described. The calculation of NOS activity and selec-
tivity was realized monitoring the l-citrulline production during
the enzymatic assay by means of RP-HPLC fluorescence detection
of l-citrulline derivatized with OPA/NAC reagent before the chro-
matographic injection.
In this series of acetamidine emerged five compounds with
submicromolar inhibitor activity on iNOS that did not posses any
inhibitor activity on nNOS and eNOS at 10 M; thus they can be
considered selective iNOS inhibitors. In particular, compound 12,
containing the acetamidino group linked by a methyl chain to C5
of the indole ring, show a very good potency (IC50 of 53 nM) and
selectivity.
Funding
This study was supported by University “G. d’Annunzio” of Chieti
local grants.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
References
[1] R. Bruckdorfer, The basic about nitric oxide, Mol. Aspects Med. 26 (2005) 3–31.
[2] W.K. Alderton, C.E. Cooper, R.G. Knowles, Nitric oxide synthases: structure,
function and inhibition, Biochem. J. 357 (2001) 593–615.
[3] U. Forstermann, W.C. Sessa, Nitric oxide synthases: regulation and function,
Eur. Heart J. 33 (2012) 829–837.
[4] T.L. Poulos, H. Li, Structural basis for isoform-selective inhibition in nitric
oxide synthase, Acc. Chem. Res. 46 (2013) 390–398.
[5] P. Mukherjee, M.A. Cinelli, S. Kang, R.B. Silverman, Development of nitric
oxide synthase inhibitors for neurodegenerative and neuropathic pain, Chem.
Soc. Rev. 43 (2014) 6814–6838.
The quinoline derivatives, 14–15, which contain the acetami-
dine directly linked to C3 and C5, respectively, show 39.4 and 40.2%
value of percentage of activity. For the isoquinoline derivatives,
16–18, the percentage of activity is, instead, higher than 56.8% for
all compounds, containing the acetamidine in C3, C5, or C8.
[6] J.M. Hevel, M.A. Marletta, Nitric oxide synthase assay, Methods Enzymol. 233
(1994) 250–258.
3.3. iNOS IC50 calculation
[7] C. Maccallini, M. Di Matteo, A. Ammazzalorso, A. D’Angelo, B. De Filippis, S. Di
Silvestre, M. Fantacuzzi, L. Giampietro, A. Pandolfi, R. Amoroso,
Reversed-phase high-performance liquid chromatography method with
fluorescence detection to screen nitric oxide synthases inhibitors, J. Sep. Sci.
37 (2014) 1380–1385.
[8] C. Maccallini, A. Patruno, N. Besˇker, J.I. Alì, A. Ammazzalorso, B. De Filippis, S.
Franceschelli, L. Giampietro, M. Pesce, M. Reale, M.L. Tricca, N. Re, M. Felaco, R.
Amoroso, Synthesis, biological evaluation, and docking studies of
N-substituted acetamidines as selective inhibitors of inducible nitric oxide
synthase, J. Med. Chem. 52 (2009) 1481–1485.
[9] C. Maccallini, A. Patruno, F. Lannutti, A. Ammazzalorso, B. De Filippis, M.
Fantacuzzi, S. Franceschelli, L. Giampietro, S. Masella, M. Felaco, N. Re, R.
Amoroso, N-Substituted acetamidines and 2-methylimidazole derivatives as
selective inhibitors of neuronal nitric oxide synthase, Bioorg. Med. Chem. Lett.
20 (2010) 6495–6499.
was realized in the presence of five different concentrations of
the inhibitor (0.01, 0.1, 0.5, 1, and 10 M). Compounds 2–3, 8–10,
12–15, with a percentage of iNOS activity at 10 M under 50%,
were selected for the IC50 calculation. In Table 1 are shown the
IC50 obtained for the selected compounds.
Compounds 2–3, the 1400W derivatives, possess an IC50 value
of 0.888 and 1.838 M, respectively.
In the indole serie, the best IC50 value of 53 nM was obtained
by compound 12, in which the acetamidine moiety is liked to C5
by a methyl chain, followed by compound 9 (IC50 of 0.241 M) and
compound 13 (IC50 of 0.446 M). The substitution of the benzyl
[10] M. Fantacuzzi, C. Maccallini, F. Lannutti, A. Patruno, S. Masella, M. Pesce, L.
Speranza, A. Ammazzalorso, B. De Filippis, L. Giampietro, N. Re, R. Amoroso,