Synthesis and molecular modelling studies of prenylated pyrazolines as MAO-B inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.09.061

A series of N-substituted-3-[(2′-hydroxy-4′-prenyloxy)-phenyl]- 5-phenyl-4,5-dihydro-(1H)-pyrazolines were synthesized and tested on human monoamine oxidase-A and -B isoforms. Structure-activity relationships and molecular modelling showed that some subst

Full text of DOI:10.1016/j.bmcl.2010.09.061

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6479–6482
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and molecular modelling studies of prenylated pyrazolines as MAO-B
inhibitors
Rossella Fioravanti a, , Adriana Bolasco a, Fedele Manna a, Francesca Rossi a, Francisco Orallo b,
⇑
Matilde Yáñez b, Alberto Vitali c, Francesco Ortuso d, Stefano Alcaro d
a Dipartimento di Chimica e Tecnologie del Farmaco, Università di Roma ‘La Sapienza’ P.le Aldo Moro, 5, 00185 Rome, Italy
b Departamento de Farmacología and Instituto de Farmacia Industrial, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Universitario Sur,
E-15782 Santiago de Compostela (La Coruña), Spain
c Istituto di Chimica del Riconoscimento Molecolare, Sezione di Roma, Istituto di Biochimica e Biochimica Clinica, Università Cattolica del S. Cuore, Largo F. Vito1, 00168 Roma, Italy
d Dipartimento di Scienze Farmacobiologiche, Università di Catanzaro ‘Magna Græcia’, Complesso Ninì Barbieri, 88021 Catanzaro, Italy
a r t i c l e i n f o
a b s t r a c t
A series of N-substituted-3-[(20-hydroxy-40-prenyloxy)-phenyl]-5-phenyl-4,5-dihydro-(1H)-pyrazolines
were synthesized and tested on human monoamine oxidase-A and -B isoforms. Structure–activity rela-
tionships and molecular modelling showed that some substitutions, such as benzyloxy or chlorine atom,
improve the best interaction with active site of hMAO-B.
Article history:
Received 30 July 2010
Revised 9 September 2010
Accepted 10 September 2010
Available online 17 September 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Prenylated pyrazoline
4,5-Dihydro-(1H)-pyrazole
hMAO-B inhibitors
Docking
Monoamine oxidase (MAO, EC 1.4.3.4, amine–oxygen oxidore-
ductase) is a membrane-bound flavoenzyme responsible for the
oxidative deamination of xenobiotic amines1 and monoamine neu-
rotransmitters such as serotonin (5-HT), norepinephrine (NE), and
dopamine (DA).2
amines, including noradrenaline and serotonin which are patho-
logically decreased in depression. Inhibition of MAO-B leads to
an increase in the neurotransmitter, dopamine. Dopamine is in-
volved in reward reinforcement12 and low levels of this neuro-
transmitter occur in Parkinson’s disease.9
Mammals, including humans, contain two isozymes, MAO-A
and MAO-B, distinguished by substrate and inhibitor selectivity3,4
MAO-A preferentially catalyses the oxidation of serotonin and
norephinephrine and is inhibited by clorgyline, whereas MAO-B
catalyses the oxidation of phenyl ethylamine and benzyl amine
and is inhibited by (R)(ꢀ)-deprenyl. Tyramine, dopamine, and
tryptamine are substrates for both enzymes. Because of their role
in the metabolism of monoamine neurotransmitters, the MAO-A
and MAO-B are thought to be involved in psychiatric and neurolog-
ical disorders such as depression and Parkinson’s disease, respec-
tively.5 For this reason the identification of MAO inhibitors
(MAO-Is) is of great interest in drug discovery3,4 since inhibitors
of MAO are used as antidepressants and neuroprotectants.4,6 In
particular, MAO-A inhibitors are useful as antidepressants,7,8 and
MAO-B inhibitors in the treatment of Parkinson’s disease.9–11
Inhibition of MAO-A results in increased brain levels of biogenic
Bearing in mind what reported in the literature about heterocy-
clic monoamine oxidase inhibitors with pyrazole and pyrazoline
structures13 and pursuing our study in this field,14–16 here we re-
port the synthesis of new derivatives 3a–k (Fig. 1), the biological
data, and the molecular modelling study of the most active
compounds.
The newly compounds bearing, isoprenoid chain, on aromatic
group in C3, because as reported in literature the remarkable
O
CH3
X =
S
NH2
X
N
N
O
OH
R
3 a-k
⇑
Corresponding author. Address: Università degli Studi di Roma ‘La Sapienza’,
P.le A. Moro, 5, 00185 Rome, Italy. Tel.: +39 6 49693259; fax: +39 6 49913133.
Figure 1. Derivatives 3a–k: N-substituted-3(20-hydroxy-40-prenyloxy)phenyl-5-
phenyl-4,5-dihydro-(1H)-pyrazoles.
E-mail address: rossella.fioravanti@uniroma1.it (R. Fioravanti).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.061

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R. Fioravanti et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6479–6482
Table 2
properties of some natural compounds as prenylated flavonoids
are thought to reside in their enhanced interaction with biological
membranes and increased affinity for target proteins compared
with their non-prenylated analogues.17–20 Moreover, in the past,
we synthesized and tested some prenylated chalcones,21 which
showed a poor inhibitory activity on MAO-B; so this work would
enable us to compare the MAO inhibition activities of the chal-
cones to that of the pyrazoles.
The experimental IC50 values (racemic mixture) and theoretical interaction energies
of 3d, 3e, 3j and 3k with hMAO-B for all enantiomers.
Compound
hMAO-B interaction energy (kcal/mol)
Exp. pIC50
(R)
(S)
Average
3d
3e
3j
ꢀ13.00
ꢀ11.30
ꢀ12.30
ꢀ12.80
ꢀ13.20
ꢀ11.10
ꢀ13.20
ꢀ12.90
ꢀ13.10
ꢀ11.20
ꢀ12.75
ꢀ12.85
6.76
5.06
6.57
6.08
3k
The biological activities on novel and reference compounds are
reported in Table 1. As shown, the most of derivatives synthesized
showed an interesting inhibitory activity on hMAO-B isoform with
no efficacy towards hMAO-A, except derivative 3d, which shows
good inhibition for both isoforms. The more potent hMAO-B inhib-
itors are those with a benzyloxy group in para position at C5 (com-
pounds 3d and 3j) and those with chlorine substituent in the same
position (compounds 3e and 3k). The introduction of methyl and
methoxy groups (3b, 3c, 3g and 3h), in the same position, gave a
significant decrease or loss in the inhibition. Furthermore, we can
suppose that the different behaviour of the derivatives 3d, 3j and
3e, 3k is probably closely related to the substituents on N1 as well.
So, following our previously reported studies,17,19 and to
pointed out the importance of different substituents, we focused
by molecular modelling techniques our attention on the interac-
tion of both (R)- and (S)-stereoisomers of the most potent inhibi-
tors 3d, 3e, 3j and 3k within the hMAO binding clefts as reported
in Supplementary data. The interaction energy of complexation
has been considered to evaluate the binding modes proposed for
3d, 3e, 3j and 3k. Theoretical results have been able to reproduce
the experimental hMAO-B inhibition of all compounds but no rec-
ognition of the hMAO-A has been suggested. Actually all molecules,
including the 3d, highlighted unsatisfactory interaction to such an
isoform. So our analysis has been focused onto the hMAO-B only. In
this isoform tested compounds were ranked with interaction ener-
gies in the range from ꢀ11.10 to ꢀ13.20 kcal/mol with slight dif-
ferences between the enantiomers (Table 2).
the recognition of (R)-3d, (R)-3j, (S)-3j and (R)-3k (see Supplemen-
tary data). Taking into account the potency and the chemical
similarity we decided to, respectively, compare the most stable
complexes of 3d versus 3j and of 3e versus 3k (Fig. 2).
In both 3d and 3j cases the (S)-enantiomer showed a slightly
better interaction energy with respect to the (R)-one. Interestingly
the (S)-3d did not reported hydrogen bonds to the target, but it
highlighted two
p–p stacking contacts, respectively, involving
the pyrazoline ring and Tyr326, and the benzyloxy moiety and
Trp119. The quite chemically similar compound (S)-3j showed
remarkable difference with respect to (S)-3d: the thioamide group
established an hydrogen bond to the Ile199 backbone. This interac-
tion promoted two hydrogen bonds among the pyrazole ring and
the phenolic hydroxyl group to a water molecules bridging by
the same interaction to Thr201 side chain. Such a network of pro-
ductive interaction moved the (S)-3j far from the stacking involved
residues reported for the (S)-3d and could be considered as the
strongest recognition difference between these two analogues into
the hMAO-B. The comparison between 3e and 3k showed a differ-
ent scenario. First of all, the best interacting stereoisomer was
(R)- for 3e and (S)- for 3k. The 3e, the less potent among modelled
inhibitors, was not able to produce significant interactions to
hMAO-B, actually no hydrogen bond or stacking contacts were
reported. The driving force the hMAO-B 3e recognition can be
addressed to hydrophobic interaction only: actually the pyrazole
and the chloro-phenyl rings were located into a lipophilic cage
delimited by Ile199, Ile316 and Tyr326 and the phenolic ring was
surrounded by Leu171 and Ile198. The compound 3k, showed a
binding mode similar to 3j, two hydrogen bonds were highlighted,
All compounds reported similar binding modes into the
hMAO-B. In all cases the isoprenyloxy moiety was directed
towards the FAD cofactor and the C5 pyrazoline substituents
towards the outer side of the hMAO-B cleft. Solvent water mole-
cules, by means of hydrogen bonds, have contributed to stabilize
Table 1
Chemical structure and anti-MAO efficacy of derivatives 3a–k. IC50 values and MAO-B selectivity ratios [IC50 (MAO-A)]/[IC50 (MAO-B)] for the
inhibitory effects of test drugs (new compounds and reference inhibitors) on the enzymatic activity of human recombinant MAO isoforms expressed
in baculovirus infected BTI insect cells.
a
a
Compound
R
X
mp (°C)
hMAO-A (pIC50
)
hMAO-B (pIC50)
**
**
**
6.50
**
**
**
**
**
**
**
3a
3b
3c
3d
3e
3f
3g
3h
H
COCH3
COCH3
COCH3
COCH3
COCH3
CSNH2
CSNH2
CSNH2
CSNH2
CSNH2
274–275
248–249
237–239
160–163
175–177
205–207
235–236
202–205
175–176
186–189
CH3
OCH3
OCH2Ph
Cl
4.22
6.76
5.06
4.28
**
H
CH3
OCH3
OCH2Ph
Cl
**
3j
3k
6.57
6.08
4.21
7.70
5.12
*
**
Clorgylineb
Deprenylc
Iproniazidb
Moclobemideb
Isatind
8.35
4.17
5.18
3.44
4.25
5.10
All IC50 values (new compounds and references inhibitors) have been estimated in this work as described in Ref. 13 using p-tyramine (5 mM) as
*
common substrate for hMAO-A and (5 mM) for hMAO-B (1 mM). Results are mean S.E.M. from five experiments. Inactive at 1 mM (highest
**
concentration tested). Inactive at 100 lM (highest concentration tested).
a
pIC50 = ꢀlog IC50
See Ref. [22].
See Ref. [23].
See Ref. [24].
.
b
c
d

R. Fioravanti et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6479–6482
6481
Figure 2. Comparison among the most stable hMAO-B theoretical complexes of a) 3d (green carbons) and 3j (white carbons) and b) 3e (cyan carbons) and 3k (magenta
carbons). The enantiomers are reported in polytube. The enzyme is showed in transparent yellow cartoon, the FAD cofactor is displayed in spacefill rendering. Interacting
residues are reported in wireframe and coloured according to the respective complex model. Residues interacting to both displayed enantiomers are labelled in yellow.
Solvent molecules and hydrogen bonds are hidden for clarity.
R
O
OH
CH3
O
OH
HO
OH
CH3
Br
i
ii
O
O
iv
2a-e
O
iii
O
OH
N
O
OH
R
R
N
N
N
CH3
O
NH2
S
3a-e
3f-k
Scheme 1. Reagents and condition: (i) K2CO3, acetone, rt; (ii) benzaldehyde, Ba(OH)2ꢁ8H2O, EtOH 96%, 30 °C; (iii) NH2–NH2, CH3COOH, EtOH dry, 78 °C; (iv) NH2NHCSNH2,
EtOH dry; 78 °C.
respectively, between the phenol and the backbone of Phe168 and
between the thioamide group and the Pro102. The chloro-phenyl
substituent hydrophobically contacted Phe118, Leu164, Phe168,
Trp119 and Pro104.
Therefore, our results and in particular molecular modelling
studies interestingly pointed out that more substituents in the
molecules as isoprenyloxy, benzyloxy, thioamide, and phenolic hy-
droxyl, are important for interaction with active site of hMAO-B, so
our study opens new perspective for the design of a novel class of
hMAO-B inhibitors.
Derivatives 3a–k, were synthesized as reported in Scheme 1
(see Supplementary data). The newly synthesized derivatives
3a–k, were characterised by elemental analysis and the 1H NMR
spectral studies (see Supplementary data). All pyrazolines synthe-
sized were screened for inhibition on the two human isoforms of
human monoamine oxidase. (Chemical and physical data, anti-
MAO efficacy values are reported in Table 1).
The potential effects of the test drugs on MAO inhibition were
investigated by measuring their effects on the production of hydro-
gen peroxide H2O2 from p-tyramine, using the AmplexÒ Red MAO
assay kit (Molecular Probes, Inc., Eugene, Oregon, USA) and micro-
somal MAO isoforms prepared from insect cells (BTI-TN-5B1-4) in-
fected with recombinant baculovirus containing cDNA inserts for
human MAO-A or MAO-B (Sigma–Aldrich Química S.A., Alcoben-
das, Spain).
The production of H2O2 catalysed by MAO isoforms can be de-
tected using 10-acetyl-3,7-dihydroxyphenoxazine (AmplexÒ Red
reagent), a non-fluorescent and highly sensitive probe that reacts
with H2O2 in presence of horseradish peroxidase to produce a fluo-
rescent product, resorufin. In this study, MAO inhibition was eval-
uated using the above mentioned method, following the general
procedure previously described by us.25
As reported in our recent communications,14,15 molecular mod-
elling techniques have been applied in order to rationalise the rec-
ognition of most potent inhibitors with respect to human MAO-A
and MAO-B enzymes. The new, high resolution, Protein Data Bank26
(PDB) crystallographic structures 2Z5X27 and 2V6028 were consid-
ered as receptor model of hMAO-A and hMAO-B, respectively.
The compounds 3d, 3e, 3j and 3k were the selected ligands for
our computational study consisting in two main steps: (i) confor-
mational search of all enantiomers and (ii) flexible docking simula-
tion toward both hMAO isozyme. Details of the procedures
followed in both steps are reported in the Supplementary data.
Acknowledgments
This work was supported by grants from MURST (Italy), Minis-
terio de Sanidad y Consumo (Spain; FISS PI061537) and Consellería
de Innovación e Industria de la Xunta de Galicia (Spain; INCI-
TE07PXI203039ES, INCITE08E1R203054ES and 08CSA019203PR).

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R. Fioravanti et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6479–6482
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The financial support of FIRB RBNE034XSW is also gratefully
acknowledged. We also acknowledge Mr. Anton Gerada, a profes-
sional translator, Fellow of the Institute of Translation and
Interpreting of London and Member of AIIC (Association Internatio-
nale des Interprêtes de Conférences—Geneva) for revision of the
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Supplementary data
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