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F. Manna et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3629–3633
the access of substrates to the isoalloxazine nucleus
hampering their oxidation by the enzyme. Moreover,
the molecular modelling studies have highlighted that
computational approaches like the one we followed in
the past,11 are too simplistic to have importance in
assisting the drug design of new reversible AO inhibitors.
8. Parmar, S. S.; Pandey, B. R.; Dwivedi, C.; Harbison, R. D.
J. Pharm. Sci. 1974, 63, 1152.
9. Soni, N.; Pande, K.; Kalsi, R.; Kupta, T. K.; Parmar, S. S.;
Barthwal, J. P. Res. Commun. Chem. Pathol. Pharmacol. 1987,
56, 129.
10. Palaska, E.; Erol, D.; Demirdamar, R. Eur. J. Med. Chem.
1996, 31, 43.
11. Manna, F.; Chimenti, F.; Bolasco, A.; Bizzarri, B.; Befani,
O.; Pietrangeli, P.; Mondovı, B.; Turini, P. J. Enzyme Inhib.
1998, 13, 207.
In conclusion and in partial, even though fortuitous,
agreement with our preliminary hypotheses, the acetyl
group proved to contribute to the inhibitory activity
and selectivity towards MAOs of 1–6, likely taking part
in the interaction with the isoalloxazine nucleus. The
OH groups on the phenyl ring at C3, in a similar man-
ner, could participate in the formation of important
intermolecular hydrogen bonds, increasing in this way
the inhibitory activity towards MAOs of the most active
compounds.
12. (a) Wouters, J.; Ooms, F.; Jegham, S.; Koenig, J. J.;
George, P.; Durant, F. Eur. J. Med. Chem. 1997, 32, 721. (b)
Wouters, J.; Moureau, F.; Evrard, G.; Koenig, J.-J.; Jegham,
S.; George, P.; Durant, F. Bioorg. Med. Chem. 1999, 7, 1683.
13. Unpublished results. The pyrazoline ring and the phenyl
ring at C3, especially, were found to interact with the polar
pyridazine and pyrimidine portions of the isoalloxazine
nucleus, respectively, while the acetyl group at N1 was direc-
ted outwards, in direction of the catalytic site.12b
14. All chemicals were commercial reagents of analytical
grade and were used without purification. Bovine serum amine
oxidase (BSAO) was purified according to Turini et al.;15 its
specific activity was 0.3 U/mg. Swine kidney diamine oxidase
(SKDAO) was purified according to Mondovı et al.;16 its spe-
cific activity was 0.4 U/mg. Bovine brain mitochondria (MAO)
were isolated according to Basford.17 In all experiments the
AO activity of the beef brain mitochondria, SKDAO, and
BSAO were measured fluorimetrically according to Matsu-
moto et al.18 Briefly, the incubation mixtures contained the
following: 0.1 mL of 0.25 M potassium phosphate buffer (pH
7.5); 0.1 mL of peroxidase (25000 UI) solution (0.5 mg/mL);
0.1 mL of homovanillic acid solution (1 mg/mL); 0.1 mL of
sample (beef brain mitochondria, 6 mg/mL; SKDAO or BSAO
0.14 mg/mL), or water for the hydrogen peroxide assay;
0.1 mL of the appropriate substrate (tyramine for MAO, ben-
zylamine for BSAO and putrescine for SKDAO) under eva-
luation at four different final concentration ranging from 0.01
to 1 mM or hydrogen peroxide as standard, 22 nmol/mL, and
0.1 mL of water; 0.05 mL of each pyrazole derivative solution
to achieve the final concentration ranging from 0 to 10ꢀ8 M,
respectively. The solutions were incubated for 30 min at 38 ꢁC,
the reaction was then ended by addition of 2 mL of NaOH
0.1 M, and the fluorimetric assay was performed. Pyrazole
derivatives were dissolved in dimethyl-sulfoxide (DMSO),
added to the reaction mixture, pre-incubated 30 min before
adding the appropriate substrate, and then incubated for an
additional 30 min to determine enzyme activity. To study the
inhibition of pyrazole derivatives on both MAO A and B
activities separately, the mitochondrial fractions were pre-
incubated for 30 min at 38 ꢁC with the specific inhibitors
(l-deprenyl 1 mM to estimate the MAO A activity, and clor-
gyline 1 mM to assay the isoform B); the samples were then
processed as described above in the presence of their specific
substrates (serotonin for MAO A and benzylamine for the B).
The protein concentration was determined according to
Goa.19 Fluorimetric measurements were recorded with a Per-
kin–Elmer LS 50B Spectrofluorimeter. The results are repor-
ted in Table 2. Dixon plots were used to estimate the
inhibition constant (Ki) of the inhibitors. Data are the means
of three or more experiments each of them performed in
duplicate.
The information conveyed by the structure–activity
studies reported here are considered important for
ongoing projects of rational design and synthesis of new
potent reversible derivatives, structurally related to the
already synthesized compounds 1–6. A new method for
the synthesis of 1-acetyl-3-(2,4- and 2,6-dihydroxyphenyl)
- 5 - (disubstituted - phenyl)- 4,5- dihydro- (1H) -pyrazole
derivatives is under study with the dual-purpose to pre-
pare new derivatives, in homochiral form and with better
yields than those reported here. A full computational
approach will be applied as well, that will take into con-
sideration several reversible inhibitors, with the aim to
develop a SAR model of the inhibition of amine oxidases
activity by 1-acetyl-3,5-diphenyl-4,5-dihydro-(1H)-pyra-
zole derivatives, able to rationalize the influence of the
stereoisomers on the activity/selectivity.
Acknowledgements
The authors wish to thank the IBAF (Istituto di Bio-
tecnologie Applicate alla Farmacologia) of the CNR
(Consiglio Nazionale delle Ricerche) for computational
resources. This work was partly financed by MURST,
by a CNR project on Biotechnology e Molecular Biol-
ogy and by MIUR (cofin. 2000, ex 40%, prot.
MM03237197_002).
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