Towards development of selective and reversible pyrazoline based MAO-inhibitors: Synthesis, biological evaluation and docking studies

Article abstract of DOI:10.1016/j.bmcl.2009.11.015

Ten novel 3,5-diaryl pyrazolines were synthesized and investigated for their monoamine oxidase (MAO) inhibitory property. All the molecules were found to be reversible and selective inhibitor for either one of the isoform (MAO-A or MAO-B). Further insights in the theoretical evaluation of the possible interactions between the compounds and monoamine oxidases (MAO-A or MAO-B) have been developed through docking studies. The theoretical values are in congruence with their experimental values.

Full text of DOI:10.1016/j.bmcl.2009.11.015

Bioorganic & Medicinal Chemistry Letters 20 (2010) 132–136
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Towards development of selective and reversible pyrazoline based
MAO-inhibitors: Synthesis, biological evaluation and docking studies
Anasuya Sahoo a, Samiye Yabanoglu b, Barij N. Sinha a, Gulberk Ucar b, Arijit Basu a,
Venkatesan Jayaprakash a,
*
a Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi 835 215, India
b Department of Biochemistry, Faculty of Pharmacy, Hacettepe University, 06100 Sıhhıye, Ankara, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Ten novel 3,5-diaryl pyrazolines were synthesized and investigated for their monoamine oxidase (MAO)
inhibitory property. All the molecules were found to be reversible and selective inhibitor for either one of
the isoform (MAO-A or MAO-B). Further insights in the theoretical evaluation of the possible interactions
between the compounds and monoamine oxidases (MAO-A or MAO-B) have been developed through
docking studies. The theoretical values are in congruence with their experimental values.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 2 September 2009
Revised 3 November 2009
Accepted 6 November 2009
Available online 12 November 2009
Keywords:
Pyrazolines
MAO-inhibitors
Docking
Monoamine oxidase inhibitors (MAOIs) have shown therapeutic
value in variety diseases especially neurodegenerative diseases.1
Earlier, MAO-inhibitors introduced into clinical practice were
abandoned due to adverse side-effects, such as hepatotoxicity,
orthostatic hypertension and the so called ‘cheese effect’ character-
ized by hypertensive crises.2 These side-effects were hypothesized
to be related to nonselective and irreversible monoamine oxidase
inhibition. Ability of new generations of inhibitors to selectively in-
hibit two isoforms (MAO-A and MAO-B) led to a renewed interest
in the therapeutic potential of these compounds. These two forms
of MAO are characterized by their different sensitivities to inhibi-
tors and their different specificities to substrates.3 MAO-A prefera-
bly metabolizes serotonin, adrenaline, and noradrenaline,4
whereas b-phenylethylamine and benzylamine are predominantly
metabolized by MAO-B.5 Tyramine, dopamine, and some other
important amines are common substrates for both the isoen-
zymes.6 Nowadays, the therapeutic interest of MAOIs falls into
two major categories. MAO-A inhibitors have been used mostly
in the treatment of mental disorders, in particular depression
and anxiety,7–9 while MAO-B inhibitors could be used in the treat-
ment of Parkinson’s disease and Alzheimer’s disease.10,11 Efforts
have been oriented toward the discovery of reversible and selec-
tive inhibitors of MAO-A/MAO-B leading to a new generation of
compounds.
The classical period of the MAO-inhibitors started with hydra-
zine derivatives. Originally proposed as a tuberculostatic agent,
their prototype, iproniazid, was the first modern anti-depressant.
2-Pyrazolines can be considered as a cyclic hydrazine moiety. Pyr-
azolines were reported to have MAO inhibitory and anti-depres-
sant activity.12–30 Our group has earlier reported thirty two new
3,5-diaryl carbothioamide pyrazolines with MAO inhibitory activ-
ity.31,32 In an effort to develop selective inhibitors of MAO, ten no-
vel analogues were synthesized and screened MAO inhibitory
activity. Structure based docking was performed in order to gain
more insight towards their binding mode.
The compounds were synthesized as reported earlier.31,32 The
synthetic route has been outlined in Scheme 1. Hydroxy chalcones
1 and 2 were prepared through Claisen–Schmidt condensation of
2-hydroxy acetophenone with either 2-hydroxy benzaldehyde or
4-hydroxy benzaldehyde. Compounds 3 and 4 were synthesized
by the reaction of hydrazine hydrate with 1 and 2, respectively
in ethanol. Reaction of benzoyl chloride with compounds 3 and 4
in pyridine provided compounds 5 and 6, respectively. Reaction
of benzene sulfonyl chloride and p-toluene sulphonyl chloride with
compound 3 and 4 in tetrahydrofuran provided compounds 7–10.
Reaction of thiosemicarbazide with 1 and 2 in ethanol and 2 M
equivalent of sodium hydroxide provided compounds 11 and 12,
respectively. Compounds 11 and 12 upon treatment with methyl
iodide and then with hydroxylamine in methanol provided com-
pounds 13 and 14, respectively. Structures, physico-chemical and
spectral characterization of the synthesized compounds are given
in Supplementary data.
* Corresponding author. Tel.: +91 6512276247.
E-mail address: venkatesanj@bitmesra.ac.in (V. Jayaprakash).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.015

A. Sahoo et al. / Bioorg. Med. Chem. Lett. 20 (2010) 132–136
133
Scheme 1. Synthesis of compounds 3–14. Reagents and conditions: (a) NH2NH2.H2O (80%) excess, C2H5OH, reflux 3–6 h; (b) C6H5COCl, pyridine, reflux 3 h; (c) R2-C6H4SO2Cl,
THF, stirring, 0.5–1 h; (d) thiosemicarbazide, C2H5OH, reflux 8–10 h; (e) CH3I/NH2OH, stirring 6–12 h.
MAO was purified from the rat liver and total MAO activity was
measured spectrophotometrically according to the method of
Holt.33 Assay mixture contained a chromogenic solution consisted
of vanillinic acid, 4-aminoantipyrin and peroxidase type II in potas-
sium phosphate buffer (pH 7.6). Assay mixture was pre-incubated
with substrate p-tyramine before addition of enzyme. The reaction
was initiated by the addition of homogenate and increase in absor-
bance was monitored at 498 nm at 37 °C for 60 min. Molar absorp-
tion coefficient of 4654 Mꢀ1 cmꢀ1 was used to calculate the initial
shifts the selectivity towards MAO-A. Substitution with toluene
sulphonyl derivative (9 and 10) at 1N-position provided the most
active and most selective MAO-A inhibitor.
In order to gain more insight on the binding mode of the com-
pounds with MAO-A and MAO-B docking studies using AUTODOCK 4.0
(automated by separate UNIX script34) were employed. Marvin
Sketch (Chemaxon), Protein Preparation Wizard (Schrodinger
Inc.) and Open Babel were used for ligand and protein preparation.
Top scoring molecules from the largest cluster were considered for
interaction studies. The calculated inhibition constants (Ki) as well
as their experimental values for each enzyme–inhibitor complex
are shown in Table 1. Interestingly, the results obtained computa-
tionally are in good agreement with their experimental values. Ide-
ally, any docking program should be capable of sorting the
compounds according to their activity. Prediction of real binding
affinities is an important aspect of any scoring function and their
score (binding energy) should reflect their biological activity.
velocity of the reaction. Results were expressed as nmol hꢀ1 mgꢀ1
.
For selective measurement of MAO-A and MAO-B activities,
homogenates were incubated with the substrate p-tyramine fol-
lowing the inhibition of one of the MAO isoform with selective
inhibitors. After inhibition of homogenates with selective inhibi-
tors, total MAO activity was determined by method of Holt. Newly
synthesized compounds were dissolved in DMSO and used in the
concentration range of 1–1000 lM. Inhibitors were then incubated
with purified MAO at 37 °C for 0–60 min prior to adding to the as-
say mixture. Reversibility of the inhibition of MAO by these com-
pounds was assessed by dilution. Kinetic data for interaction of
the enzyme with these compounds were determined using Micro-
soft Excel package program. IC50 values were determined from
plots of residual activity percentage, calculated in relation to a
sample of the enzyme treated under the same conditions without
inhibitor, versus inhibitor concentration [I].
All the synthesized compounds were selective and reversible
inhibitor towards either MAO-A or MAO-B. Compounds 3 and 4
selectively inhibit MAO-B, with a selectivity index of 4.74 and
6.491, respectively. All the other analogues were selective towards
MAO-A, with outstanding selectivity index in the magnitude of
10ꢀ4–10ꢀ6 (5, 6 and 8–10, Fig. 1). Their structure–activity relation-
ship suggest that, any substitution at 1N-position of pyrazoline
Spearman’s rank order correlation coefficient q (1) was previously
employed35–37 to evaluate docking and scoring protocols.
n
P
2
6
½rðxiÞ ꢀ rðyiÞꢁ
n3 ꢀ n
i
q
¼ 1 ꢀ
ð1Þ
Where n is the number of pairs n = 10 in our case, r(xi) and r(yi)
are the rank of the activity and the interaction energy of the ith
sample in the testing set. In theory, the Spearman correlation coef-
ficient falls between ꢀ1 and +1, where +1 corresponds to a perfect
correlation, ꢀ1 corresponds to a perfect inverse correlation, and
zero corresponds to total disorder. An excellent rank order correla-
tion coefficient for MAO-A,
q = 0.964, and for MAO-B, q = 0.936
was obtained. Justifying the search algorithm and scoring function
employed in the current study (Figs. 1 and 2a and b).

134
A. Sahoo et al. / Bioorg. Med. Chem. Lett. 20 (2010) 132–136
500000
450000
400000
350000
300000
250000
200000
150000
S
E
L
E
C
T
I
V
I
T
Y
SI-experimental
SI-calculated
100000
50000
0
3
4
SI-calculated
SI-experimental
5
6
7
8
9
SI
10
13
14
code
Figure 1. Experimental and calculated selectivity indices (calculated as Ki (MAO-A)/Ki (MAO-B)) of the newly synthesized pyrazoline derivatives show a very close
correlation.
Table 1
Calculated and experimental Ki values corresponding to the inhibition of MAO isoforms by the newly synthesized pyrazoline derivatives
Code
aExperimental (Ki)
bCalculated (Ki)
M) MAO-B (lM)
Inhibition type
Reversibility
MAO inhibitory selectivity
MAO-A (
l
M)
MAO-B (
lM)
MAO-A (
l
3
4
5
6
7
8
9
10
13
14
SEL
MOC
3.7
3.9
0.80
0.78
0.60
1.94
2.65
0.83
3.82
0.39
0.16
0.28
0.34
17.09
8.97
ND
0.54
0.60
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Selective for MAO-B
Selective for MAO-B
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-B
Selective for MAO-A
99230
20330
1550
700.2
620
285900
96.8
210
349410
754150
7300
209.72
462.23
—
88.22
167.09
ND
5.77
0.91
0.68
0.36
0.60
15.66
10.6
105.66
0.005
1.35
1.08
ND
ND
SEL-Selegeline, MOC-Moclobemide.
a
Values were determined from the kinetic experiments in which p-tyramine (substrate) was used at 500
lM to measure MAO-A and 2.5 mM to measure MAO-B. Pargyline
or clorgyline were added at 0.50 M to determine the isoenzymes A and B. Newly synthesized compounds and the known inhibitors were pre-incubated with the
l
homoganates for 60 min at 37 °C. Each value represents the mean SEM of three independent experiments.
b
Values obtained through AUTODOCK program.
0
0
0
experimental rank
experimental rank
Figure 2. Plot of calculated versus experimental rank. (a) For MAO-A and (b) for MAO-B. Spearman’s rank order correlation coefficient
q
MAO-A, q = 0.964, and for MAO-B,
q
= 0.936 was obtained.
Colibus et al.38 meticulously depicted the structural comparison
of MAO-A from 210 to 216 (201–207 in MAO-B) which results in
of human MAO-A and MAO-B. According to their findings MAO-A
and MAO-B share a high sequence similarity of 72%, overall struc-
ture of human MAO-A is quite similar to that of MAO B (rms devi-
C
a
movements up to 6 Å.
Binding of these ligands to either MAO-A or MAO-B reveals the
importance substitution in 1N-position of pyrazolines. Their subtle
variation in this position can greatly influence both the selectivity
as well as their potency.
ation of 1.2 Å, 488 equiv C-
a atom). The only significant structural
difference observed is the conformation of the cavity-shaping loop

A. Sahoo et al. / Bioorg. Med. Chem. Lett. 20 (2010) 132–136
135
In complex of compounds 7–10 with MAO-A, hydrogen bonding
interaction was observed between the sulphonyl oxygen of the li-
gands and hydroxyl hydrogen of either TYR444 (compounds 7–8)
or TYR407 (compounds 9–10). Due these Hydrogen bonding inter-
actions ring B of compounds 7–8 and ring C of compounds 9–10
were positioned in the aromatic cage (FAD, TYR407 and TYR444,
Pocket1). A para methyl substitution of ring C of compounds 9–
10 considerably changes the orientation of the ligand in the active
site that results in significant increase in potency as well as selec-
tivity indices towards MAO-A compared with compounds 7–8. The
other two phenyl rings were well accommodated in pocket2
(ILE180, ILE335, LEU337, MET350, PHE352) and pocket3 (due to
cavity-shaping loop, GLY74, ARG206, ILE207, PHE208, GLU216,
TRP441). Interaction of compounds 4, 6, 8 and 10 with human
MAO-A (2BXR) was given in Figure 3a.
In complex of compounds 3–4 with MAO-B, the hydrogen bond-
ing interaction was found between 1N hydrogen of pyrazoline ring
and hydroxyl oxygen of TYR435 while 2N hydrogen with carbonyl
oxygen of ILE199. Due to this interaction the ring B and ring A of
compounds 3 and 4 were positioned in the pocket1 (aromatic cage)
and in narrow cavity (pocket 2 and 3 were reduced and merged to
narrow cavity in MAO-B due to different orientation of the cavity
forming loop, Figure 3b) respectively. Compounds 3–4 interacts
with MAO-B better than MAO-A due to the interaction of ring B
with catalytic pocket1 of MAO-B that is missing in case of MAO-
A (Fig. 3a and b, compound 4 represented in blue colour). Com-
pounds 5–10 with additional ring C experiences a significant steric
hindrance in the narrow cavity that accounts for its poor interac-
tion of these compounds with MAO-B. Interaction of compounds
4, 6, 8 and 10 with human MAO-B (2BYB) was given in Figure 3b.
Figure 3. (a) Interaction of compounds 4, 6, 8 and 10 with MAO-A (2BXR), (b) interaction of compounds 4, 6, 8 and 10 with MAO-A (2BYB). FAD in green colour, compound 4
in dark blue colour, compound 6 in red colour, compound 8 in light blue colour and compound 10 in yellow colour. TYR of aromatic cage are in tube representation coloured
by atom type and cavity-shaping loop residues of MAO-A and its equivalent in MAO-B are represented as ribbon.

136
A. Sahoo et al. / Bioorg. Med. Chem. Lett. 20 (2010) 132–136
14. Manna, F.; Chimenti, F.; Bolasco, A.; Bizzarri, B.; Befani, O.; Pietrangeli, P.;
From the above study we have successfully identified few com-
Mondovi, B.; Turini, P. J. Enzyme Inhib. 1998, 13, 207.
15. Manna, F.; Chimenti, F.; Bolasco, A.; Secci, D.; Bizzarri, B.; Befani, O.; Turini, P.;
Mondovi, B.; Alcaro, S.; Tafi, A. Bioorg. Med. Chem. Lett. 2002, 12, 3629.
16. Gökhan, N.; Yes_ilada, A.; Uçar, G.; Erol, K.; Bilgin, A. A. Arch. Pharm. 2003, 336,
362.
17. Chimenti, F.; Bolasco, A.; Manna, F.; Secci, D.; Chimenti, P.; Befani, O.; Turini, P.;
Giovannini, V.; Mondovi, B.; Cirilli, R.; La Torre, F. J. Med. Chem. 2004, 47, 2071.
18. Chimenti, F.; Maccioni, E.; Secci, D.; Bolasco, A.; Chimenti, P.; Granese, A.;
Befani, O.; Turini, P.; Alcaro, S.; Ortuso, F.; Cirilli, R.; La Torre, F.; Cardia, M. C.;
Distinto, S. J. Med. Chem. 2005, 48, 7113.
pounds which are reversible and selective inhibitor of either MAO-
A or MAO-B. A concise idea about their structure activity relation-
ship was also drawn and importance of different side chain substi-
tutions especially at 1N-position was understood. Presence any
substitution at 1N-position shifts the selectivity towards MAO-A.
There binding mode analysis with the help of molecular docking
provided imperative insights about their molecular recognition
process it also provided information which is helpful in designing
other selective and reversible MAO-inhibitors in future.
19. Gökhan Kelekçi, N.; Yabanog˘lu, S.; Küpeli, E.; Salgın Göks_en, U.; Özgen, Ö.;
Uçar, G.; Yes_ilada, E.; Kendi, E.; Yes_ilada, A.; Bilgin, A. A. Bioorg. Med. Chem.
2007, 15, 5775.
20. Soni, N.; Pande, K.; Kalsi, R.; Gupta, T. K.; Parmar, S. S.; Barthwal, J. P. Res.
Commun. Chem. Pathol. Pharmacol. 1987, 56, 129.
Acknowledgement
21. Bilgin, A. A.; Palaska, E.; Sunal, R. Arzneimittelforschung 1993, 43, 1041.
22. Palaska, E.; Erol, D.; Demirdamar, R. Eur. J. Med. Chem. 1996, 31, 43.
23. Palaska, E.; Aytemir, M.; Uzbay, I. T.; Erol, D. Eur. J. Med. Chem. 2001, 36, 539.
24. Rajendra Prasad, Y.; Lakshmana Rao, A.; Prasoona, L.; Murali, K.; Ravi Kumar, P.
Bioorg. Med. Chem. Lett. 2005, 15, 5030.
We are grateful to the Head, SAIF, CDRI, Lucknow for providing
1H NMR and FAB-MS Spectral data.
25. Manna, F.; Chimenti, F.; Bolasco, A.; Bizzarri, B.; Befani, O.; Pietrangeli, P.;
Mondovı‘, B.; Turini, P. J. Enzyme Inhib. 1998, 13, 207.
Supplementary data
26. Gökhan, N.; Yes_ilada, A.; Uçar, G.; Erol, K.; Bilgin, A. A. Arch. Pharm. Pharm.
Med. Chem. 2003, 336, 362.
27. Ucar, G.; Gokhan, N.; Yesilada, A.; Bilgin, A. A. Neurosci. Lett. 2005, 382, 327.
28. Chimenti, F.; Bolasco, A.; Manna, F.; Secci, D.; Chimenti, P.; Granese, A.; Befani,
O.; Turini, P.; Cirilli, R.; La Torre, F.; Alcaro, S.; Ortuso, F.; Langer, T. Curr. Med.
Chem. 2006, 13, 1411.
29. Yabanoglu, S.; Ucar, G.; Gokhan, N.; Salgin, U.; Yesilada, A.; Bilgin, A. A. J. Neural.
Transm. 2007, 114, 769.
30. Gokhan, N.; Yabanoglu, S.; Kupeli, E.; Salgın, U.; Ozgen, O.; Ucar, G.; Yesilada,
A.; Kendi, E.; Yesilada, E.; Bilgin, A. A. Bioorg. Med. Chem. 2007, 15, 5775.
31. Venkatesan, J.; Sinha, B. N.; Ucar, G.; Ercan, A. Bioorg. Med. Chem. Lett. 2008, 18,
6362.
32. Stirrett, K. L.; Ferreras, J. A.; Venkatesan, J.; Sinha, B. N.; Ren, T.; Quadri, L. E. N.
Bioorg. Med. Chem. Lett. 2008, 18, 2662.
33. Holt, A.; Sharman, D. F.; Baker, G. B.; Pelcic, M. M. Anal. Biochem. 1997, 244, 384.
34. Sharma, V.; Patnaik, K. K.; Jayprakash, V.; Basu, A.; Mishra, N. Bioinformation
2009, 4, 84.
35. Mishra, N.; Basu, A.; Jayaprakash, V.; Sharon, A.; Patnaik, K. K. Bioorg. Med.
Chem. Lett. 2009, 19, 5582.
36. Wang, R.; Lu, Y.; Wang, S. J. Med. Chem. 2003, 46, 2287.
37. Moitessier, N.; Therrien, E.; Hanessian, S. J. Med. Chem. 2006, 49, 5885.
38. Colibus, L. D.; Li, M.; Binda, C.; Lustig, A.; Edmondson, D. E.; Mattevi, A. Proc.
Natl. Acad. Sci. U.S.A. 2005, 102, 12684.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.11.015.
References and notes
1. Youdim, M. B. H.; Edmonson, D.; Tipton, K. F. Nat. Rev. Neurosci. 2006, 7, 295.
2. Oxidase Inhibitors; Gorrod, J. W., Gibson, G. G., Mitchard, M., Eds.Developments
of Drugs and Modern Medicines. Part I. Drug Design; Ellis Horwood Ltd:
Chichester, 1986; p 40.
3. Kalgutkar, A. S.; Castagnoli, N., Jr. Med. Res. Rev. 1995, 15, 325.
4. Johston, J. P. Biochem. Pharmacol. 1968, 17, 1285.
5. Knoll, J.; Magyar, K. Adv. Biochem. Psychophysiol. 1972, 5, 393.
6. O’Carroll, A.; Fowler, C. J.; Phillips, J. P.; Tobbia, I.; Tipton, K. F. N.-S. Arch.
Pharmacol. 1983, 322, 198.
7. Rudorfer, M. V.; Potter, W. Z. Drugs 1989, 37, 713.
8. Pacher, P.; Kohegyi, E.; Keckemeti, V.; Furst, S. Curr. Med. Chem. 2001, 8, 89.
9. Pacher, P.; Keckemeti, V. Curr. Med. Chem. 2004, 11, 925.
10. Wouters, J. Curr. Med. Chem. 1998, 5, 137.
11. Carreiras, M. C.; Marco, J. L. Curr. Pharm. Des. 2004, 10, 367.
12. Parmar, S. S.; Pandey, B. R.; Dwivedi, C.; Harbison, R. D. J. Pharm. Sci. 1974, 63,
1152.
13. Soni, N.; Pande, K.; Kalsi, R.; Gupta, T. K.; Parmar, S. S.; Barthwal, J. P. Res.
Commun. Chem. Pathol. 1987, 56, 129.