Structure-activity relationships in the inhibition of monoamine oxidase B by 1-methyl-3-phenylpyrroles

Article abstract of DOI:10.1016/j.bmc.2007.11.059

1-Methyl-3-phenyl-3-pyrrolines are structural analogues of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and like MPTP are selective substrates of monoamine oxidase B (MAO-B). As part of an ongoing investigation into the substrate properties of various 1-methyl-3-phenyl-3-pyrrolinyl derivatives, it is shown in the present study that their respective MAO-B catalyzed oxidation products act as reversible competitive inhibitors of the enzyme. The most potent inhibitor among the oxidation products considered was 1-methyl-3-(4-trifluoromethylphenyl)pyrrole with an enzyme-inhibitor dissociation constant (K_i value) of 1.30 μM. The least potent inhibitor was found to be 1-methyl-3-phenylpyrrole with a K_i value of 118 μM. The results of an SAR study established that the potency of MAO-B inhibition by the 1-methyl-3-phenylpyrrolyl derivatives examined here is dependent on the Taft steric parameter (E_s) and Swain-Lupton electronic constant (F) of the substituents attached to C-4 of the phenyl ring. Electron-withdrawing substituents with a large degree of steric bulkiness appear to enhance inhibition potency. Potency was also found to vary with the substituents at C-3, again with E_s and F being the principal substituent descriptors.

Full text of DOI:10.1016/j.bmc.2007.11.059

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2463–2472
Structure–activity relationships in the inhibition of
monoamine oxidase B by 1-methyl-3-phenylpyrroles
Modupe O. Ogunrombi,a Sarel F. Malan,a Gisella Terre’Blanche,a
Neal Castagnoli, Jr.,b Jacobus J. Bergha and Jacobus P. Petzera,*
aPharmaceutical Chemistry, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa
bDepartment of Chemistry, Virginia Tech, Blacksburg, VA 24061, USA
Received 20 July 2007; revised 13 November 2007; accepted 21 November 2007
Available online 28 November 2007
Abstract—1-Methyl-3-phenyl-3-pyrrolines are structural analogues of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP) and like MPTP are selective substrates of monoamine oxidase B (MAO-B). As part of an ongoing investigation into the
substrate properties of various 1-methyl-3-phenyl-3-pyrrolinyl derivatives, it is shown in the present study that their respective
MAO-B catalyzed oxidation products act as reversible competitive inhibitors of the enzyme. The most potent inhibitor among
the oxidation products considered was 1-methyl-3-(4-trifluoromethylphenyl)pyrrole with an enzyme-inhibitor dissociation constant
(Ki value) of 1.30 lM. The least potent inhibitor was found to be 1-methyl-3-phenylpyrrole with a Ki value of 118 lM. The results of
an SAR study established that the potency of MAO-B inhibition by the 1-methyl-3-phenylpyrrolyl derivatives examined here is
dependent on the Taft steric parameter (Es) and Swain–Lupton electronic constant (F) of the substituents attached to C-4 of the
phenyl ring. Electron-withdrawing substituents with a large degree of steric bulkiness appear to enhance inhibition potency. Potency
was also found to vary with the substituents at C-3, again with Es and F being the principal substituent descriptors.
ꢀ 2007 Elsevier Ltd. All rights reserved.
C6H5
1. Introduction
C6H5
The first step in the metabolic activation of the parkin-
sonian inducing proneurotoxin, 1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine [MPTP, (1)], is catalyzed by
the flavoenzyme, monoamine oxidase B (MAO-B)
(Scheme 1),1,2 to yield the ring a-carbon 2-electron oxi-
dation product, the corresponding 1-methyl-4-phenyl-
2,3-dihydropyridinium species MPDP+ (2H+). This met-
abolic intermediate, presumably via the corresponding
conjugate base 2, undergoes a second 2-electron oxida-
tion to generate the 1-methyl-4-phenylpyridinium
metabolite MPP+ (3+), the ultimate neurotoxin.3,4 Liter-
ature reports various structural analogues of MPTP that
have been found to act as good substrates of MAO-B5,6
as well as of MAO-A.7 Among these is 1-methyl-3-phe-
nyl-3-pyrroline (4) (Scheme 2) which, like MPTP, is a
cyclic tertiary allylamine exhibiting selectivity for the
MAO-B isoform.8 The MAO-B catalyzed ring a-carbon
MAO-B
N
N
CH3
CH3
2H+
1
-H+
C6H5
C6H5
-2e-, H+
?
N
N
CH3
3+
CH3
2
Scheme 1. The MAO-B catalyzed oxidation of MPTP (1) to yield the
corresponding dihydropyridinium product MPDP+ (2H+) which
spontaneously undergoes a second 2-electron oxidation to generate
MPP+ (3+).
Keywords: Monoamine oxidase B; Reversible inhibitors; Competitive
inhibition; 1-Methyl-3-phenylpyrrole; Structure–activity relationship.
*
Corresponding author. Tel.: +27 18 2992206; fax: +27 18 2994243;
e-mail: jacques.petzer@nwu.ac.za
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.11.059

2464
M. O. Ogunrombi et al. / Bioorg. Med. Chem. 16 (2008) 2463–2472
C6H5
MAO-B
C6H5
i
N
N
Ar
CO2H
Ar
6a-m
N
N
Ar
CH3
CH3
4
5H+
ii
C6H5
N
-H+
CH3
5a-m
N
Scheme 3. Synthetic pathway to 1-methyl-3-phenylpyrroles (5a–m).
Reagents and conditions: (i) DMF, POCl3, 80 ꢁC; (ii) NaOCH3,
pyridine, reflux.
CH3
5
Scheme 2. The MAO-B catalyzed oxidation of 1-methyl-3-phenyl-3-
pyrrolinyl derivatives (4) to yield the corresponding 1-methyl-3-
phenylpyrrolyl products (5). This oxidation most likely arises via
5H+, the short-lived conjugate acid of the pyrrolyl product 5.
solvent, the structures and purity of the compounds
1
were verified by mass spectrometry, H NMR, and 13C
NMR. For those compounds previously reported, the
physical data and melting points obtained were com-
pared to the corresponding literature values as cited in
Section 4.
2-electron oxidation of 4 yields 1-methyl-3-phenylpyr-
role (5) as the final product. This oxidation most likely
arises via 5H+, the short-lived conjugate acid of the
pyrrolyl product 5.9 As part of an ongoing investigation
into the substrate properties of various 1-methyl-3-phe-
nyl-3-pyrrolinyl derivatives, it is shown in the present
study that their respective MAO-B catalyzed oxidation
products act as reversible competitive inhibitors of the
enzyme. A literature survey reveals that reversible inhi-
bition of MAO-B by 1-methyl-3-phenylpyrrole (5a)
and its 4-chlorophenyl analogue (5b) has previously
been demonstrated.10 In an attempt to determine the ef-
fect that specific structural modifications of 1-methyl-3-
phenylpyrrole will have on MAO-B inhibition potency,
we have synthesized 13 1-methyl-3-phenylpyrrolyl deriv-
atives (5a–m) and determined their enzyme-inhibitor dis-
sociation constants (Ki values) for reversible interaction
with MAO-B. As part of the present SAR analysis, the
1-methyl-3-phenylpyrrolyl derivatives investigated dif-
fered only in the substituents on C-3 and C-4 of the phe-
nyl ring. Since inhibitors of MAO-B are currently in use
and being investigated for the treatment of neurodegen-
erative disorders,11,12 the results of this study may aid in
the identification and design of new reversible inhibitors.
2.2. Enzymology and inhibition studies
In the present study we have determined the enzyme-
inhibitor dissociation constants (Ki values) for reversible
interaction of MAO-B with members of a synthetic ser-
ies of 1-methyl-3-phenylpyrrolyl derivatives (5a–m).
MAO-B activity measurements were based on the ring
a-carbon oxidation of 1-methyl-4-(1-methylpyrrol-2-
yl)-1,2,3,6-tetrahydropyridine (MMTP) to yield the cor-
responding dihydropyridinium species (MMDP+).15 The
concentrations of MMDP+ produced by this enzymatic
reaction were measured spectrophotometrically since
MMDP+ absorbs light maximally at 420 nm. At this
wavelength neither the enzyme substrate nor the test
inhibitors absorb light. Because of these favorable chro-
mophoric characteristics and the in vitro chemical sta-
bility of MMDP+, this assay is frequently used to
evaluate the potencies of potential inhibitors of MAO-
B.16,17 The mitochondrial fraction obtained from ba-
boon liver tissue was employed as enzyme source since
it exhibits a high degree of MAO-B catalytic activity
and is devoid of MAO-A activity.15 Therefore, even
though MMTP is a MAO-A/B mixed substrate, its oxi-
dation by baboon liver mitochondria can be attributed
exclusively to the action of the MAO-B isoform. The
interaction of reversible inhibitors with MAO-B ob-
tained from baboon liver tissue appears to be similar
to the interaction with the human form of the enzyme
since inhibitors are approximately equipotent with both
enzyme sources.17
2. Results
2.1. Chemistry
The 1-methyl-3-phenylpyrrolyl derivatives (5a–m) exam-
ined in this study were prepared in relatively good yields
(26.6–72.7%) according to a previously reported proce-
dure (Scheme 3).13 The key starting materials were
the 2-aryl-3-(dimethylamino)allylidene(dimethyl)ammo-
nium perchlorates (6a–m) which were prepared in very
high yields from the appropriately substituted phenyla-
cetic acid derivatives and DMF.14 Cyclization of 6, to
yield the target pyrrolyl derivatives (5a–m), was
achieved by treatment with sodium methoxide in anhy-
drous pyridine. Following purification by column chro-
matography or by recrystallization from a suitable
The MAO-B inhibitory properties of 5a–m first were
investigated in order to determine whether the test inhib-
itors act as time-dependent inactivators or reversible
inhibitors of the enzyme. For this study 5e was selected
as a representative test inhibitor. When baboon liver
mitochondrial fractions were preincubated with 5e
(5.20 lM) for periods of 0, 15, 30, and 60 min, the rate
of MAO-B catalyzed oxidation of MMTP (90 lM) to
MMDP+ remained unchanged (results not shown).18

M. O. Ogunrombi et al. / Bioorg. Med. Chem. 16 (2008) 2463–2472
2465
Table 1. The Ki values for the inhibition of MAO-B by 1-methyl-3-
phenylpyrrolyl derivatives (5a–m)
From this result it can be concluded that 5e interacts
reversibly with the active site of MAO-B. The reversibil-
ity of enzyme inhibition by 5a–m was also apparent
from the Lineweaver–Burk plots constructed from the
kinetic data (see below).
Ar
N
All of the 1-methyl-3-phenylpyrrolyl derivatives (5a–m)
evaluated were found to be inhibitors of MAO-B. As
demonstrated with 1-methyl-3-(3-chlorophenyl)pyrrole
(5h) (Fig. 1), classical Lineweaver–Burk plots for com-
petitive inhibition were obtained for all of the test inhib-
itors. The enzyme-inhibitor dissociation constants (Ki
values) for the inhibition of MAO-B by 5a–m are pre-
sented in Table 1. The data lead to the conclusion that
substitution on the phenyl ring leads to inhibitors with
enhanced inhibition potencies since 5a was found to be
the weakest inhibitor. The most potent inhibitor of the
series was 1-methyl-3-(4-trifluoromethylphenyl)pyrrole
(5e) with a Ki value of 1.30 lM. The second most potent
inhibitor of the series was 1-methyl-3-(3-trifluoromethyl-
phenyl)pyrrole (5k) with a Ki value of 6.55 lM. Substi-
tution at C-4 or C-3 of the phenyl ring with the
electronegative CF3 functional group therefore appears
to be the best method of enhancing the binding affinity
of 1-methyl-3-phenylpyrrolyl derivatives for the active
site of MAO-B. Interestingly, no inhibition of beef liver
MAO-B was observed by 5e, even at a concentration of
200 lM (results not shown). Similarly, no inhibition of
beef liver MAO-B was observed by any of the other
pyrrolyl derivatives examined here. Further inspection
of Table 1 reveals that substitution with bromine at C-
4 (5c) and C-3 (5i) also results in pyrrolyl derivatives
with relatively high inhibition potencies. Since substitu-
tion at C-4 (5d) and C-3 (5j) of the phenyl ring with fluo-
rine resulted in relatively weaker inhibitors compared to
the bromine analogues 5c and 5i and chlorine analogues
CH3
Compound
Ar
Ki value (lM)a
Esb
0.00
Fb
5a
5b
5c
5d
5e
5f
C6H5
118
0.00
0.42
0.45
0.45
0.38
0.01
0.29
0.42
0.45
0.45
0.38
0.01
0.29
4-ClC6H4
4-BrC6H4
4-FC6H4
18.4
6.65
31.0
1.30
22.5
36.5
20.9
14.3
38.9
6.55
56.0
41.7
ꢀ0.97
ꢀ1.16
ꢀ0.46
ꢀ2.40
ꢀ1.24
ꢀ0.55
ꢀ0.97
ꢀ1.16
ꢀ0.46
ꢀ2.40
ꢀ1.24
ꢀ0.55
4-CF3C6H4
4-CH3C6H4
4-OCH3C6H4
3-ClC6H4
3-BrC6H4
3-FC6H4
5g
5h
5i
5j
5k
5l
3-CF3C6H4
3-CH3C6H4
3-OCH3C6H4
5m
The values of the selected physicochemical parameters used in the SAR
studies are also listed.
a The enzyme source used was baboon liver mitochondrial MAO-B.
b Values obtained from Ref. 19.
5b and 5h, it does not appear that potent inhibition is
linked exclusively to the presence of electronegative sub-
stituents in the phenyl ring.
2.3. Quantitative structure–activity relationships (QSAR)
In an attempt to quantify the relationship between
MAO-B inhibitory activity and the physicochemical
properties of the substituents, a Hansch-type SAR study
was carried out by multiple linear regression analysis.
Five parameters were used to describe each substituent.
The Taft steric parameter (Es)19 and the Van der Waals
volume (Vw)20 were used as descriptors of bulkiness
while the lipophilicities of the substituents were de-
scribed by the Hansch constant (p).19 The classical
Hammett (rm or rp) and Swain–Lupton (F) constants
served as electronic parameters.19 All the physicochemi-
cal values of the substituents were obtained from stan-
dard compilations.19,20 The analogues were divided
into two groups—those bearing substituents at C-4 of
the phenyl ring (5b–g) and those with substituents at
C-3 of the phenyl ring (5h–m). The unsubstituted 1-
methyl-3-phenylpyrrole (5a) was considered a member
of both groups. Results of the statistical analysis for
the two groups are shown in Tables 2 and 3,
respectively.
20
15
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
10
5
0
-40 -20
0
20 40 60 80
[I], μM
-0.01
0.00
0.01
1/[S], μM
0.02
0.03
0.04
-1
For analogues substituted at C-4 of the phenyl ring (Ta-
ble 2), the Taft steric parameter (Es) was the only substi-
tuent descriptor that showed a meaningful correlation
with the logarithm of the Ki values (expressed in micro-
molar). Regression analysis of logKi with Es exhibited a
relatively good correlation with a R2 value of 0.91. The
statistical F value for the correlation was found to be
51.7, which is higher than the Fmax value (25.32)21 for
Figure 1. Lineweaver–Burk plots of the oxidation of MMTP by
baboon liver MAO-B in the absence (filled circles) and presence of
various concentrations of 5h (open circles, 15 lM; filled triangles,
30 lM; and open triangles, 60 lM). The concentration of the baboon
liver mitochondrial isolate was 0.15 mg/mL and the rates are expressed
as nmol/min mg protein of MMDP+ formed. The inset is the replot of
the slopes versus the inhibitor concentration.

2466
M. O. Ogunrombi et al. / Bioorg. Med. Chem. 16 (2008) 2463–2472
Table 2. Correlations of the MAO-B inhibition constants (logKi) of
the 1-methyl-3-phenylpyrrolyl derivatives (5a–g) with steric, electronic,
and hydrophobic descriptors of the substituents at C-4 of the phenyl
ringa
(Fig. 2). The negative correlation between logKi and F
(ꢀ0.83 0.26) indicates that the potency (logKi) by
which 1-methyl-3-phenylpyrroles inhibit MAO-B may
be enhanced by substitution with electron-withdrawing
C-4 functional groups. Although not statistically signif-
icant, there also appears to be a moderate correlation
(R2 = 0.70) between inhibitor binding affinity (logKi)
and the lipophilicity (p) of the C-4 substituents. Since
more lipophilic substituents have increasingly positive
Hansch constant (p) values, the negative correlation
(ꢀ1.30 0.38) between p and the logKi values indicates
that enhancement of the lipophilicity of the C-4 substit-
uents may lead to better inhibition potency. The ob-
served linear correlations of logKi with both Es and p
are to be expected since, for the set of substituents exam-
ined here, there exists a moderate correlation (R2 = 0.68)
between these two substituent descriptors. No signifi-
cant correlations were observed for any other combina-
tion of two substituent descriptors.
Parameter Slope
y-Intercept R2 Fb
Significancec
rp
F
ꢀ1.76 0.64 1.40 0.17 0.60 7.54 0.041
ꢀ1.67 1.18 1.72 0.40 0.29 2.00 0.22
ꢀ1.17 1.05 1.01 0.31 0.20 1.23 0.32
ꢀ1.30 0.38 1.82 0.22 0.70 11.6 0.019
0.77 0.11 1.99 0.13 0.91 51.7 0.0008
0.71 0.07 2.16 0.09 0.98 79.7 0.0004
Vw
p
Es
Es + F
ꢀ0.83 0.26
0.032
a The logarithm of the Ki values expressed in micromolar was used in
the linear regression analysis.
b Higher F values indicate a better fit and a regression equation with an
F value higher than the critical F value may be judged as significant.
Critical F values may be calculated as described recently.21
c The significance is the fractional probability that the coefficient of the
added variable is zero.
For analogues substituted at C-3 of the phenyl ring
(Table 3), there appeared to be no single substituent
descriptor that showed a meaningful correlation with
Table 3. Correlations of the MAO-B inhibition constants (logKi) of
the 1-methyl-3-phenylpyrrolyl derivatives (5a and h–m) with steric,
electronic, and hydrophobic descriptors of the substituents at C-3 of
the phenyl ringa
Parameter Slope
y-Intercept R2 Fb
Significancec
2.4
rm
F
ꢀ1.69 0.49 1.86 0.14 0.71 12.0 0.018
ꢀ1.52 0.63 1.91 0.22 0.54 5.75 0.062
ꢀ0.47 0.29 1.91 0.30 0.34 2.52 0.17
ꢀ0.84 0.27 1.85 0.16 0.65 9.34 0.028
0.46 0.12 1.92 0.15 0.73 13.6 0.014
0.38 0.05 2.14 0.06 0.97 72.2 0.0013
2.0
Vw
H
OCH3
p
Es
1.6
CH3
Es + F
i
1.2
0.8
0.4
0.0
-0.4
F
Cl
K
ꢀ1.07 0.18
0.0039
a The logarithm of the Ki values expressed in micromolar were used in
the linear regression analysis.
b Higher F values indicate a better fit and a regression equation with an
F value higher than the critical F value may be judged as significant.
Critical F values may be calculated as described recently.21
c The significance is the fractional probability that the coefficient of the
added variable is zero.
Br
CF3
-2.5
-3.0
-2.0
-1.5
-1.0
-0.5
0.0
H
Es - 0.83
F
95% significance (a higher F value indicates a better fit).
All other single-parameter fits with the logKi values
exhibited poorer statistical correlations. Correlations
could be improved by the inclusion of an additional sub-
stituent parameter in the regression analysis. A two-
parameter fit with Es and the Swain–Lupton constant
(F) yielded a model with a R2 value of 0.98 and a statis-
tical F value of 79.7 (Fmax = 30.18). For this correlation,
the probabilities that Es and F are zero are 0.04% and
3.2%, respectively. Therefore, the best mathematical
description of binding affinity (logKi) of the C-4 substi-
tuted 1-methyl-3-phenylpyrroles (5a–g) to MAO-B is:
2.4
2.1
1.8
1.5
1.2
0.9
0.6
CH3
i
OCH3
K
F
Cl
Br
CF3
-3.0
-2.5
-2.0
-1.5
Es - 1.07
-1.0
-0.5
0.0
log Ki ¼ 0:71ðꢁ0:07ÞEs ꢀ 0:83ðꢁ0:26ÞF
þ 2:16ðꢁ0:09Þ
F
ðR2 ¼ 0:98 and F ¼ 79:7Þ
ð1Þ
Figure 2. Correlations of the logKi values for the inhibition of MAO-B
by 5a–m with the Taft steric parameter (Es) of the substituents at C-4
(top) and C-3 (bottom) of the phenyl ring. The Es values were adjusted
by the contribution of the Swain–Lupton F constant as indicated on
the x-axis titles. The linear regression lines are graphical representa-
tions of Eqs. 1 and 2 and the correlation coefficients are 0.98 and 0.97,
respectively.
Since bulky substituents have increasingly negative Taft
steric parameter (Es) values, the positive correlation ob-
served with Es (0.71 0.07) indicates that the MAO-B
inhibition potency (logKi) may be enhanced by substitu-
tion at C-4 of the phenyl ring with a bulky substituent

M. O. Ogunrombi et al. / Bioorg. Med. Chem. 16 (2008) 2463–2472
2467
the logKi values. A two-parameter fit with Es and the
Swain–Lupton constant (F), however, yielded a model
with a R2 value of 0.97 and a statistical F value of
72.2 (Fmax = 30.18). For this correlation, the probabili-
ties that Es and F are zero are 0.13% and 0.39%, respec-
tively. Therefore, the best mathematical description of
binding affinity (logKi) of the C-3 substituted
1-methyl-3-phenylpyrroles (5a and h–m) to MAO-B is:
examining the interactions of the inhibitors with the ac-
tive site of MAO-B.
The best-ranked docking solutions obtained for all com-
pounds examined (5a–m) indicate that the inhibitors tra-
verse both the entrance and substrate cavities of the
enzyme, with the pyrrolyl ring extending beyond the
boundary between the entrance and substrate cavities
while the phenyl ring binds within the substrate cavity.
This orientation is probably favored in order to prevent
unfavorable interactions between the N–CH3 and the
large polar regions of the substrate cavity and/or to
maximize hydrophobic interactions of the N–CH3 in
the entrance cavity.25 As shown by example with the
most potent inhibitor of the series, 5e (4-CF3), the phe-
nyl ring is located in the substrate cavity (Fig. 3) while
the pyrrolyl nucleus extends into the entrance cavity
where the N–CH3 is stabilized by the hydrophobic envi-
ronment defined by Phe-103, Trp-119, Leu-164, Leu-
167, Phe-168, Ile-316.25 The best-ranked docking solu-
tions of inhibitors 5b (4-Cl), 5c (4-Br), and 5f (4-CH3)
are virtually superimposed on the binding orientation
of 5e. Inhibitor 5a (H) and 5d (4-F) (Fig. 4) also adopt
log Ki ¼ 0:38ðꢁ0:05ÞEs ꢀ 1:07ðꢁ0:18ÞF
þ 2:14ðꢁ0:06Þ
ðR2 ¼ 0:97 and F ¼ 72:2Þ
ð2Þ
As observed with the analogues substituted at the C-4
position, the positive sign of the Es parameter coefficient
(0.38 0.05) and the negative sign of the F parameter
coefficient (ꢀ1.07 0.18) suggest that the potency (log-
Ki) of MAO-B inhibition may be enhanced by substitu-
tion with sterically bulky C-3 functional groups that are
electron-withdrawing (Fig. 2).
2.4. Modeling studies
In an attempt to better understand the outcomes of the
SAR study and to gain additional insight into the bind-
ing modes of the inhibitors, molecular docking of all the
1-methyl-3-phenylpyrroles (5a–m) within the active site
of human MAO-B was performed. Among the crystallo-
graphic structures of MAO-B deposited in the Brookha-
ven Protein Data Bank, the structure with trans,trans-
farnesol bound to the enzyme (2BK3.pdb)22 was selected
for the docking studies. The choice of this complex was
based on the high resolution of the crystallographic
structure and the observation that trans,trans-farnesol
spans both the entrance and substrate cavities of the en-
zyme active site. As a result, the side chain of Ile-199,
which acts as a ‘gate’ separating the two cavities, is ro-
tated out of its normal conformation to allow for the fu-
sion of the two cavities and the accommodation of
larger structures.23 In order to evaluate the accuracy
of the docking procedure, the co-crystallized ligand
was redocked within the active site using the LigandFit
application of the molecular docking software, Discov-
ery Studio 1.7.24 The ligand to be docked was first con-
structed within DS Visualizer Pro and then prepared for
the docking simulations using the Prepare Ligands
application of Discovery Studio. Following the docking
procedure, the docked ligand conformations were fur-
ther refined using in situ ligand minimization with the
Smart Minimizer algorithm. Even though trans,trans-
farnesol has a relatively high degree of flexibility, the
three best-ranked docking solutions obtained exhibited
relatively small RMSD from the co-crystallized ligand
Figure 3. Schematic representation of the most stable complex
between 5e and MAO-B. The inhibitor is displayed in blue, the flavin
in purple, and the residues of the enzymatic clefts in black.
˚
(1.26 0.18 A). Within the best-ranked docking solu-
tions, trans,trans-farnesol, however, also occupied re-
versed binding orientations which constituted
approximately 40% of the 10 best docking solutions.
Therefore, although the orientations of the best-ranked
docking solutions obtained with LigandFit closely
approximate that of the co-crystallized ligand, reversed
binding poses can be expected to be among the docking
solutions. Even with this limitation, LigandFit appears
to be an effective molecular docking protocol for
Figure 4. Schematic representation of the most stable complex
between 5d and MAO-B. The inhibitor is displayed in blue, the flavin
in purple, and the residues of the enzymatic clefts in black.

2468
M. O. Ogunrombi et al. / Bioorg. Med. Chem. 16 (2008) 2463–2472
MAO-B, even at a concentration of 200 lM which is
well above its Ki value (1.30 lM) for the inhibition of
baboon liver MAO-B. Beef liver MAO-B was also not
inhibited by any of the other pyrrolyl derivatives exam-
ined here. Other compounds (Scheme 4), 1,4-diphenyl-2-
butene (7),23 trans,trans-farnesol (8)26, and (E)-8-(3-
chlorostyryl)caffeine (9),27 have also been reported to in-
hibit baboon liver and recombinant human MAO-B
competitively while having no effect on beef MAO-B.
The crystal structures of human recombinant MAO-B
in complex with 7 and 8 have shown that these reversible
inhibitors exhibit a dual binding mode that involves tra-
versing both the entrance and substrate cavities of the
enzyme.23 The ‘gate’ separating the two cavities is the
side chain of Ile-199 which is rotated out of its normal
conformation to allow for the fusion of the two cavities
in order to accommodate these larger inhibitors.23 In
contrast to 7 and 8, the small molecule inhibitor isatin
(10) has been shown to inhibit competitively all known
MAO-B isoforms, including the beef isoform, with sim-
ilar potencies.22 Crystal structures of human recombi-
nant MAO-B in complex with isatin23 have shown that
isatin binds within the substrate cavity, leaving the en-
trance cavity unoccupied. In this instance the side chain
of Ile-199 exhibits the normal or ‘closed’ rotamer con-
formation. Ile-199 is conserved in all known MAO-B se-
quences with the exception of beef MAO-B where it is
replaced with Phe. The increased size of the Phe aro-
matic ring relative to Ile is suggested to prevent its occu-
pation of the ‘open’ rotamer conformation.22 The only
available space for the aromatic ring to occupy is in
the entrance cavity of the enzyme which prevents the
binding of inhibitors that must traverse both cavities.22
The observation that 5a–m does not inhibit beef liver
MAO-B suggests that binding of 1-methyl-3-phenylpyr-
roles to the active site of MAO-B is dependent upon the
rotation of Ile-199 out of its normal conformation. This
implies that 1-methyl-3-phenylpyrroles also exhibit a
Figure 5. Schematic representation of the most stable complex
between 5k and MAO-B. The inhibitor is displayed in blue, the flavin
in purple, and the residues of the enzymatic clefts in black.
similar binding modes, but the N–CH3 protrudes to a
lesser degree into the entrance cavity and hence is stabi-
lized to a lesser extent (than 5e) by the hydrophobic
environment of the entrance cavity.
The 1-methyl-3-phenylpyrroles substituted at C-3 of the
phenyl ring (5h–m) adopt similar binding orientations to
the C-4 substituted analogues (Fig. 5). The principal dif-
ference is that the C-3 substituents are directed toward
the small hydrophobic region defined by the apolar res-
idues, Tyr-60, Phe-343, and Tyr-398, while the C-4 sub-
stituents are directed in the general direction of the
aromatic cage defined by Tyr-398 and Tyr-435.25 This
binding mode presumably maximizes favorable hydro-
phobic contacts between the C-3 substituent and the en-
zyme while minimizing unfavorable interactions with
the polar environment of the substrate cavity. As ex-
pected, the reverse binding modes are also represented
among the 10 best-ranked docking solutions of the
inhibitors examined, with a frequency of approximately
30–40%. The best solutions were, however, always ori-
entated with the pyrrolyl ring directed toward the en-
trance cavity.
7
3. Discussion
OH
This investigation shows that substitution of 1-methyl-3-
phenylpyrroles on the phenyl ring has a considerable ef-
fect on the potency of MAO-B inhibition displayed by
these compounds. For example, substitution at C-4 with
a trifluoromethyl functional group (5e) increases the
inhibition potency by approximately 90-fold compared
to the unsubstituted 1-methyl-3-phenylpyrrole (5a).
The SAR analysis indicates that the inhibition activities
correlate with the Taft steric parameter and the Swain–
Lupton constant of the substituents at both C-4 and C-3
of the phenyl ring. Inhibitor binding to the enzyme is fa-
vored by an increase in both the steric bulk and the elec-
tronegativity of the para and meta substituents.
8
Cl
O
N
N
(E)
N
O
N
9
O
O
N
10
H
Interestingly 1-methyl-3-phenylpyrroles do not seem to
be inhibitors of beef liver MAO-B since the most potent
inhibitor examined here, 5e, does not inhibit beef liver
Scheme 4. The structures of the reversible MAO-B inhibitors 1,4-
diphenyl-2-butene (7), trans,trans-farnesol (8), (E)-8-(3-chlorosty-
ryl)caffeine (9), and isatin (10).

M. O. Ogunrombi et al. / Bioorg. Med. Chem. 16 (2008) 2463–2472
2469
dual binding mode that involves interactions with both
the entrance and substrate cavities.
In conclusion, even though none of the 1-methyl-3-phe-
nylpyrroles examined here were found to be exception-
ally potent inhibitors of MAO-B, this study reveals the
general structural features that are important for
MAO-B inhibition as well as the modifications that
can be made in order to enhance inhibition potency.
Features important for inhibition are coplanarity of
the aromatic rings and bulky electronegative substitu-
ents at the para and meta positions of the phenyl ring.
Structural modifications that may enhance inhibition
potency are the enhancement of the distance between
the pyrrolyl and phenyl rings (e.g., with an ethylene lin-
ker) and the inclusion of additional lipophilic substitu-
ents on the pyrrolyl ring (e.g., CH3 at position 2 and/
or 5). Both these modifications will have the effect of
promoting hydrophobic burial of the pyrrolyl ring in
the entrance cavity of MAO-B.
While the exact binding orientation of 1-methyl-3-phenyl-
pyrroles in the active site of MAO-B is unknown, molec-
ular docking studies support the argument that the
inhibitors occupy both cavities. The most stable protein-
inhibitor models generated indicate that the inhibitors
may bind to MAO-B with the pyrrolyl moiety protruding
into the entrance cavity while the phenyl ring is located
within the substrate cavity (Figs. 3–5). For this binding
mode to be possible, the side chain of Ile-199 must be ro-
tated into the ‘open’ conformation. The pyrrolyl N–CH3
appears to be stabilized by the hydrophobic environment
of the entrance cavity and as a result, the structural fea-
tures of the inhibitor that allow for a higher degree of pro-
jection of the N–CH3 into the entrance cavity, may
enhance inhibition potency. One such feature may be en-
hanced steric bulk of the phenyl substituents. In accor-
dance with this idea, the SAR analysis showed that the
inhibition activity correlated with the Taft steric parame-
ter of the substituents at both C-4 and C-3.
4. Experimental
Caution. MMTP is a structural analogue of the nigro-
striatal neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahy-
dropyridine (MPTP) and should be handled using
disposable gloves and protective eyewear. Procedures
for the safe handling of MPTP have been described
previously.30
The apparent contribution of the Swain–Lupton con-
stant (F) of the phenyl substituents toward the correla-
tions of the inhibition potency with Es and F (Eqs. 1
and 2) is not well understood. Electron-withdrawing
substituents at C-3 and C-4 enhance MAO-B inhibition
potency of 1-methyl-3-phenylpyrroles. This is also
apparent from the moderate correlations of 0.60 (Table
2) and 0.71 (Table 3) recorded between the logKi values
and the Hammett electronic parameters (rp or rm). A
possible explanation may be that electron-withdrawing
functionalities promote planarity between the phenyl
and pyrrolyl rings. The literature supports the idea that
planar, heterocyclic compounds frequently act as com-
petitive inhibitors of MAO-B.28,29 The shift to longer
wavelengths of maximal light absorption (kmax) upon
substitution of the phenyl ring by electron-withdrawing
functional groups (CF3, Br, and Cl) may support this
hypothesis. As shown in Table 4, the more potent inhib-
itors examined in this study (CF3, Br, and Cl) exhibited
kmax values of 276–286 nm while the relatively less po-
tent inhibitors (H, F, CH3, and OCH3) were found to
have kmax values of 264–272 nm.
4.1. Chemicals and instrumentation
All starting materials not described elsewhere were ob-
tained from Sigma–Aldrich and were used without puri-
fication. The oxalate salt of MMTP was prepared as
described previously.7 Petroleum ether used in this study
was of a distillation range of 40–60 ꢁC. Proton and car-
bon NMR spectra were recorded on a Varian Gemini
300 spectrometer. Proton (1H) spectra were recorded
in CDCl3 and d6-DMSO at a frequency of 300 MHz
and carbon (13C) spectra at 75 MHz. Chemical shifts
are reported in parts per million (d) downfield from
the signal of tetramethylsilane added to the deuterated
solvent. Spin multiplicities are given as s (singlet), d
(doublet), t (triplet), q (quartet), dd (doublet of dou-
blets) or m (multiplet) and the coupling constants (J)
are given in hertz (Hz). Direct insertion electron impact
ionization (EIMS), high resolution (HRMS), and fast
atom bombardment (FAB-MS) mass spectra were ob-
tained on a VG 7070E mass spectrometer. Melting
points (mp) were determined on a Gallenkamp melting
point apparatus and are uncorrected. UV–Vis spectra
were recorded on a Milton-Roy Spectronic 1201 spec-
trophotometer. Thin layer chromatography (TLC) was
carried out with neutral aluminium oxide 60 (Merck)
containing UV254 fluorescent indicator.
Table 4. The wavelengths of maximal light absorption (kmax) of
1-methyl-3-phenylpyrrolyl derivatives (5a–m)
Compound
kmax (nm)a
e (Mꢀ1
)
5a
5b
5c
5d
5e
5f
268
276
279
264
286
269
268
276
277
272
277
270
269
13,880
18,260
18,660
12,360
15,060
15,460
17,280
13,700
13,880
13,420
13,640
13,660
12,220
5g
5h
5i
4.2. Synthesis of 2-aryl-3-(dimethylamino)allylidene
(dimethyl)ammonium perchlorates (6a–m)
5j
5k
5l
Synthetic intermediates 6a–m were prepared in relatively
high yields from the corresponding phenylacetic acid,
DMF, and phosphoryl chloride according the method
described in the literature.14 The melting points of the
compounds reported previously were as follows: 6a mp
5m
The extinction coefficients (e) at these wavelengths are also listed.
a All UV/Vis spectral measurements were conducted in isopropanol.

2470
M. O. Ogunrombi et al. / Bioorg. Med. Chem. 16 (2008) 2463–2472
203–205 ꢁC (from ethanol), lit. mp 193–194 ꢁC14; 6b mp
149–151 ꢁC (from ethanol), lit. mp 142–144 ꢁC14; 6c mp
162–164 ꢁC (from ethanol), lit. mp 152–154 ꢁC14; 6f mp
165–166 ꢁC (from ethanol), lit. mp 150–152 ꢁC31; 6g mp
134–136 ꢁC (from ethanol), lit. mp 130–131 ꢁC14; 6h mp
186–188 ꢁC (from ethanol), lit. mp 180 ꢁC32; 6k mp 114–
116 ꢁC (from ethanol), lit. mp 141.5–143 ꢁC33; 6l mp
160–162 ꢁC (from ethanol), lit. mp 164–166 ꢁC.33 The
characterizations of compounds that are previously
unreported are summarized below.
(6a–m) according to a modification of the method de-
scribed in the literature.13 Sodium wire (143 mmol)
was reacted with methanol (48 mL) and the resulting
solution was added under an atmosphere of argon to a
solution of 6 (64.8 mmol) in 262 mL dry pyridine (dis-
˚
tilled over CaH2 and stored over 4 A molecular sieves).
The reaction was heated under reflux for 24 h. The pyr-
idine was removed via vacuum distillation to obtain a
yellow pasty residue to which 100 mL distilled water
was added. The resulting suspension was extracted with
ethylacetate (3 · 100 mL) and the combined organic
phases were dried over anhydrous magnesium sulfate
(10 g). After the solvent was removed under reduced
pressure an oily residue was obtained. The crude prod-
uct was dissolved in a minimal amount of ethylacetate
and purified on a short column (35 · 80 mm) by neutral
aluminium oxide chromatography (Fluka 507C) with
100% petroleum ether (5a–b, f, and k–l) or petroleum
ether/ethylacetate, 90:10 (5g and m), as mobile phase.
The fractions containing the product were in most cases
recrystallized from an appropriate solvent as cited be-
low. For the synthesis of 5c–e and 5h–j, the following
modifications were made: following the addition of the
100 mL distilled water to the yellow pasty residue ob-
tained from the vacuum distillation of the pyridine sol-
vent, a suspension was obtained which was stirred for
60 min at room temperature and filtered. A yellow solid
residue was obtained which was recrystallized from an
appropriate solvent as cited below. All reactions were
monitored using neutral aluminium oxide TLC (mobile
phase of 100% petroleum ether). The TLC plates were
visualized with UV light (254 nm) or by staining with io-
dine. The melting points of compounds reported previ-
ously were as follows: 5a mp 44–46 ꢁC (from
petroleum ether), lit. mp 46–47 ꢁC13; 5b mp 112–
114 ꢁC (from methanol), lit. mp 117.5–119.5 ꢁC13; 5c
mp 129–130 ꢁC (from methanol), lit. mp 132–133 ꢁC13;
5f mp 49–51 ꢁC (from methanol), lit. mp 55–56 ꢁC34;
5g mp 120–122 ꢁC (from methanol), lit. mp 126–
128 ꢁC13; 5j mp 73–75 ꢁC (from methanol), lit. mp 94–
96 ꢁC.13 The characterizations of compounds that were
previously unreported are summarized below.
2-(4-Fluorophenyl)-3-(dimethylamino)allylidene(dimethyl)
ammonium perchlorate (6d) was synthesized from 4-flu-
orophenylacetic acid in a yield of 67.8%: mp 144–
1
146 ꢁC (from ethanol); H NMR (d6-DMSO) d 2.44 (s,
6H), 3.24 (s, 6H), 7.23–7.37 (m, 4H), 7.69 (s, 2H); 13C
NMR (d6-DMSO) d 39.30, 48.53, 103.82, 115.25 (d),
128.73 (d), 134.12 (d), 160.47, 163.02, 163.73; FAB-MS
m/z 221 (M+).
2-(4-Trifluoromethylphenyl)-3-(dimethylamino)allylid-
ene(dimethyl)ammonium perchlorate (6e) was synthesized
from 4-(trifluoromethyl)phenylacetic acid in a yield of
1
72.9%: mp 151–154 ꢁC (from ethanol); H NMR (d6-
DMSO) d 2.42 (s, 6H), 3.26 (s, 6H), 7.56 (d, 2H,
J = 7.8 Hz), 7.75–7.79 (m, 4H); 13C NMR (d6-DMSO) d
48.59, 103.24, 122.16, 124.96 (q), 125.77, 128.99 (q),
132.95, 137.45, 162.83; FAB-MS m/z 271 (M+).
2-(3-Bromophenyl)-3-(dimethylamino)allylidene(dimethyl)
ammonium perchlorate (6i) was synthesized from 3-
bromophenylacetic acid in a yield of 80.4%: mp 177–
1
178 ꢁC (from ethanol); H NMR (d6-DMSO) d 2.46 (s, 6H),
3.24 (s, 6H), 7.31–7.41 (m, 2H), 7.57 (t, 1H, J = 1.6 Hz),
7.62–7.65 (m, 1H), 7.69 (s, 2H); 13C NMR (d6-DMSO) d
48.55, 103.32, 121.53, 130.21, 131.24, 131.57, 134.44, 135.15,
162.75; FAB-MS m/z 281, 283 (M+).
2-(3-Fluorophenyl)-3-(dimethylamino)allylidene(dimethyl)
ammonium perchlorate (6j) was synthesized from 3-fluor-
ophenylacetic acid in a yield of 78.1%: mp 158–161 ꢁC
1
(from ethanol); H NMR (d6-DMSO) d 2.46 (s, 6H),
3.24 (s, 6H), 7.14–7.22 (m, 2H), 7.24–7.31 (m, 1H),
7.43–7.51 (m, 1H), 7.70 (s, 2H); 13C NMR (d6-DMSO)
d 39.33, 48.54, 103.53, 115.65 (d), 118.97 (d), 128.56
(d), 130.25 (d), 134.98 (d), 159.90, 162.76, 163.17;
FAB-MS m/z 222 (MH+).
1-Methyl-3-(4-fluorophenyl)pyrrole (5d) was synthesized
from 6d in a yield of 31.5%: mp 98–100 ꢁC (from meth-
1
anol); H NMR (CDCl3) d 3.67 (s, 3H), 6.37 (dd, 1H,
J = 1.8, 2.7 Hz), 6.61 (t, 1H, J = 2.5 Hz), 6.83 (t, 1H,
J = 2.1 Hz), 7.00 (m, 2H), 7.42 (m, 2H); 13C NMR
(CDCl3) d 36.27, 106.26, 115.29 (d), 118.26, 122.78,
124.17, 126.32 (d), 132.13, 132.17; EIMS m/z 175
(MÅ+); HRMS calcd 175.0797, found 175.0811.
2-(3-Methoxyphenyl)-3-(dimethylamino)allylidene(dimethyl)
ammonium perchlorate (6m) was synthesized from 3-
methoxyphenylacetic acid in a yield of 75.8%: mp 148–
1
150 ꢁC (from ethanol); H NMR (d6-DMSO) d 2.47 (s,
6H), 3.23 (s, 6H), 3.77 (s, 3H), 6.85–6.87 (m, 2H),
6.97–7.01 (m, 1H), 7.31–7.36 (m, 1H), 7.67 (s, 2H); 13C
NMR (d6-DMSO) d 48.46, 55.16, 104.86, 114.47,
117.40, 124.48, 129.41, 133.79, 158.94, 162.73; FAB-
MS m/z 233 (M+).
1-Methyl-3-(4-trifluoromethylphenyl)pyrrole (5e) was
synthesized from 6e in a yield of 41.1%: mp 161–
1
162 ꢁC (from methanol); H NMR (CDCl3) d 3.68 (s,
3H), 6.45 (dd, 1H, J = 1.8, 2.8 Hz), 6.34 (t, 1H,
J = 2.5 Hz), 6.96 (t, 1H, J = 2.0 Hz), 7.00 (s, 4H); 13C
NMR (CDCl3) d 36.37, 106.53, 119.39, 123.19, 123.63,
124.75, 125.52 (q), 139.53; EIMS m/z 225 (MÅ+); HRMS
calcd 225.0765, found 225.0757.
4.3. Synthesis of 1-methyl-3-phenylpyrroles (5a–m)
The 1-methyl-3-phenylpyrrolyl derivatives (5a–m) were
synthesized from the corresponding 2-aryl-3-(dimethyl-
1-Methyl-3-(3-chlorophenyl)pyrrole (5h) was synthe-
sized from 6h in a yield of 33.6%: mp 77–80 ꢁC (from
amino)allylidene(dimethyl)ammonium
perchlorates

M. O. Ogunrombi et al. / Bioorg. Med. Chem. 16 (2008) 2463–2472
2471
methanol); 1H NMR (CDCl3) d 3.66 (s, 3H), 6.41 (t, 1H,
J = 2.3 Hz), 6.62 (t, 1H, J = 2.5 Hz), 6.90 (t, 1H,
J = 2.0 Hz), 7.09–7.13 (m, 1H), 7.20–7.26 (m, 1H),
7.34–7.38 (m, 1H), 7.48 (t, 1H, J = 1.9 Hz); 13C NMR
(CDCl3) d 36.30, 106.33, 118.93, 122.95, 123.66,
124.87, 125.05, 129.74, 134.39, 137.87; EIMS m/z 191
(MÅ+); HRMS calcd 191.0502, found 191.0490.
served as substrate for the inhibition studies. Incuba-
tions were carried out in sodium phosphate buffer
(100 mM, pH 7.4) and contained MMTP (30–120 lM),
the mitochondrial isolate (0.15 mg protein/mL), and
various concentrations of the test inhibitors. The final
volume of the incubations was 500 lL. The stock solu-
tions of the inhibitors were prepared in DMSO and were
added to the incubation mixtures to yield a final DMSO
concentration of 4% (v/v). DMSO concentrations higher
than 4% are reported to inhibit MAO-B.29 Following
incubation at 37 ꢁC for 15 min, the enzyme reactions
were terminated by the addition of 10 lL perchloric acid
(70%) and the samples were centrifuged at 16,000g for
10 min. The MAO-B catalyzed production of MMDP+
is reported to be linear for the first 15 min of incubation
under these conditions.15 The supernatant fractions
were removed and the concentrations of the MAO-B
generated product, MMDP+, were measured spectro-
1-Methyl-3-(3-bromophenyl)pyrrole (5i) was synthesized
from 6i in a yield of 36.9%: mp 82–84 ꢁC (from metha-
1
nol); H NMR (CDCl3) d 3.65 (s, 3H), 6.39 (dd, 1H,
J = 1.8, 2.7 Hz), 6.60 (t, 1H, J = 2.5 Hz), 6.87 (t, 1H,
J = 2.0 Hz), 7.15 (m, 1H), 7.22–7.26 (m, 1H), 7.36–7.40
(m, 1H), 7.61 (t, 1H, J = 1.9 Hz); 13C NMR (CDCl3) d
36.32, 106.34, 118.93, 122.75, 122.96, 123.40, 123.55,
127.80, 127.96, 130.03, 138.18; EIMS m/z 235, 237
(MÅ+); HRMS calcd 234.9997, found 235.0003.
1-Methyl-3-(3-trifluoromethylphenyl)pyrrole (5k) was
synthesized from 6k in a yield of 51.9%: mp 41–44 ꢁC;
1H NMR (CDCl3) d 3.68 (s, 3H), 6.45 (dd, 1H,
J = 1.8, 2.7 Hz), 6.64 (t, 1H, J = 2.4 Hz), 6.95 (t, 1H,
J = 2.0 Hz), 7.39 (m, 2H), 7.62–7.66 (m, 1H), 7.71 (m,
1H); 13C NMR (CDCl3) d 36.34, 106.37, 119.04,
121.49 (q), 121.69 (q), 122.61, 123.12, 123.69, 126.22,
127.99, 128.92, 136.79; EIMS m/z 225 (MÅ+); HRMS
calcd 225.0765, found 225.0764.
photometrically at
a
wavelength of 420 nm
(e = 25,000 Mꢀ1).15 The initial rates of oxidation at four
different substrate concentrations (30–120 lM) in the
absence and presence of three different concentrations
of the inhibitors were calculated and Lineweaver–Burk
plots were constructed. The slopes of the Lineweaver–
Burk plots were plotted versus the inhibitor concentra-
tion and the Ki value was determined from the x-axis
intercept (intercept = ꢀKi). Linear regression analysis
was performed using the SigmaPlot software package
(Systat Software Inc.). Each Ki value reported here is
representative of a single determination where the corre-
lation coefficient (R2 value) of the replot of the slopes
versus the inhibitor concentrations was at least 0.98.
1-Methyl-3-(3-methylphenyl)pyrrole (5l) was synthesized
1
from 6l in a yield of 72.7%: mp 39–42 ꢁC; H NMR
(CDCl3) d 2.38 (s, 3H), 3.68 (s, 3H), 6.45 (dd, 1H,
J = 1.8, 2.7 Hz), 6.62 (t, 1H, J = 2.5 Hz), 6.90 (t, 1H,
J = 2.0 Hz), 6.97–7.01 (m, 1H), 7.20–7.25 (m, 1H),
7.30–7.35 (m, 2H); 13C NMR (CDCl3) d 21.50, 36.24,
106.35, 118.51, 122.12, 122.58, 125.11, 125.79, 126.02,
128.43, 135.88, 137.96; EIMS m/z 171 (MÅ+); HRMS
calcd 171.1048, found 171.1049.
4.5. Time-dependent inhibition studies
In order to determine whether the test inhibitor 5e acts
as a time-dependent inactivator or reversible inhibitor
of MAO-B, baboon liver mitochondrial fractions
(0.3 mg of protein/mL) were preincubated with 5e
(5.20 lM) for periods of 0, 15, 30, and 60 min at
37 ꢁC.15 The solvent for this incubation was 100 mM so-
dium phosphate buffer (pH 7.4). The MAO-A/B mixed
substrate, MMTP, at final concentration of 90 lM,
was then incubated at 37 ꢁC for 15 min with 0.15 mg
protein/mL of the preincubated mitochondria. The final
volumes of these incubations were 500 lL and the final
concentration of the test inhibitor 2.60 lM. Following
termination of the reactions by the addition of 10 lL
of perchloric acid (70%), the concentrations of MMDP+
were measured as outlined above. These experiments
were carried out in triplicate.
1-Methyl-3-(3-methoxyphenyl)pyrrole (5m) was synthe-
1
sized from 6m in a yield of 62.0%: mp 67–70 ꢁC; H
NMR (CDCl3) d 3.67 (s, 3H), 3.84 (s, 3H), 6.45 (dd,
1H, J = 1.8, 2.7 Hz), 6.62 (t, 1H, J = 2.5 Hz), 6.71–6.75
(m, 1H), 6.91 (t, 1H, J = 2.0 Hz), 7.06 (dd, 1H,
J = 1.6, 2.5 Hz), 7.10–7.13 (m, 1H), 7.23–7.08 (m, 1H);
13C NMR (CDCl3) d 36.23, 55.12, 106.40, 110.59,
110.73, 117.66, 118.71, 122.63, 12.86, 129.46, 137.42,
158.89; EIMS m/z 187 (MÅ+); HRMS calcd 187.0997,
found 187.1004.
4.4. MAO-B inhibition studies
Mitochondria were isolated from baboon and beef liver
tissue as described by Salach and Weyler35 and stored at
ꢀ70 ꢁC in 300 lL aliquots. Following addition of an
equal volume of sodium phosphate buffer (100 mM,
pH 7.4) containing glycerol (50%, w/v) to the aliquots,
the protein concentration was determined by the method
of Bradford using bovine serum albumin as reference
standard.36 Since the mitochondrial fraction obtained
from baboon and beef liver tissue is reported to be de-
void of MAO-A activity,15 inactivation of this isoform
was unnecessary. The MAO-A and -B mixed substrate
MMTP (Km = 60.9 lM for baboon liver MAO-B)15
4.6. SAR studies
The values of the substituent descriptors rp, rm, F, p,
and Es were obtained from Hansch and Leo19 while
those for the Van der Waals volume (Vw) were obtained
from compilations by Van de Waterbeemb and Testa.20
Linear regression analysis of the logKi values as a func-
tion of the substituent descriptor values was carried out
with Statistica software package (StatSoft Inc.). In order
to estimate the significance of the regression equations,
the F statistic was employed. An F value higher than

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M. O. Ogunrombi et al. / Bioorg. Med. Chem. 16 (2008) 2463–2472
8. Wang, Y. X.; Mabic, S.; Castagnoli, N., Jr. Bioorg. Med.
Chem. 1998, 6, 143.
9. Williams, C. H.; Lawson, J. Biochem. J. 1998, 336, 63.
10. Williams, C. H.; Lawson, J. Neurobiology (Bp) 1999, 7,
225.
11. Youdim, M. B. H.; Bakhle, Y. S. Br. J. Pharmacol. 2006,
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the critical F value was judged to be significant. The crit-
ical F value (Fmax) for 95% significance for models con-
structed from seven logKi values (Tables 2 and 3) and
which contain one parameter (out of a possible five:
Vw, Es, p, rp/m, and F) was calculated to be 25.32, while
the Fmax value for models containing two parameters
was calculated to be 30.18.21
4.7. Molecular docking studies
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All the computational studies were carried out in the
Windows-based Discovery Studio 1.7 modeling and
simulation environment.24 The ligands to be docked
were constructed within DS Visualizer Pro and then
prepared for the docking simulations using the Pre-
pare Ligands application of Discovery Studio. The
crystallographic structure of trans,trans-farnesol in
complex with human MAO-B (2BK3.pdb)22 was re-
trieved from the Brookhaven Protein Data Bank
(www.rcsb.org/pdb) and the co-crystallized inhibitor
was manually deleted. Following typing of the recep-
tor model with the CHARMm forcefield, the binding
site was identified by the LigandFit flood-filling algo-
rithm. Automated docking was then carried out with
the LigandFit application of Discovery Studio. This
docking protocol employed total ligand flexibility
whereby the final ligand conformations were deter-
mined by the Monte Carlo conformation search meth-
od set to a variable number of trial runs. The docked
ligands were further refined using in situ ligand mini-
mization with the Smart Minimizer algorithm. All the
application modules within Discovery Studio were set
to their default values and 10 docking solutions were
allowed for each ligand.
16. Vlok, N.; Malan, S. F.; Castagnoli, N., Jr.; Bergh, J. J.;
Petzer, J. P. Bioorg. Med. Chem. 2006, 14, 3512.
17. Petzer, J. P.; Steyn, S.; Castagnoli, K. P.; Chen, J. F.;
Schwarzschild, M. A.; Van der Schyf, C. J.; Castagnoli,
N., Jr. Bioorg. Med. Chem. 2003, 11, 1299.
18. Khalil, A. A.; Steyn, S.; Castagnoli, N., Jr. Chem. Res.
Toxicol. 2000, 13, 31.
19. Hansch, C.; Leo, A. Exploring QSAR. Fundamentals and
Applications in Chemistry and Biology; American Chemical
Society: Washington, DC, 1995, pp 1–124.
20. Van de Waterbeemb, H.; Testa, B. In Advances in Drug
Research; Testa, B., Ed.; Academic Press: London, 1987;
pp 85–225.
21. Livingstone, D. J.; Salt, D. W. J. Med. Chem. 2005, 48,
661.
´
22. Hubalek, F.; Binda, C.; Khalil, A.; Li, M.; Mattevi, A.;
Castagnoli, N., Jr.; Edmondson, D. E. J. Biol. Chem.
2005, 280, 15761.
´
23. Binda, C.; Li, M.; Hubalek, F.; Restelli, N.; Edmondson,
D. E.; Mattevi, A. Proc. Natl. Acad. Sci. U.S.A. 2003, 100,
9750.
24. Accelrys Discovery Studio 1.7, Accelrys Software Inc.,
San Diego, CA, USA, http://www.accelrys.com.
25. Novaroli, L.; Daina, A.; Favre, E.; Bravo, J.; Carotti, A.;
Leonetti, F.; Catto, M.; Carrupt, P. A.; Reist, M. J. Med.
Chem. 2006, 49, 6264.
Acknowledgments
´
The NMR and MS spectra were recorded by Andre Jou-
26. Khalil, A. A.; Davies, B.; Castagnoli, N., Jr. Bioorg. Med.
Chem. 2006, 14, 3392.
bert, Johan Jordaan, and Louis Fourie of the SASOL
Centre for Chemistry, North-West University. This
work was supported by grants from the National Re-
search Foundation and the Medical Research Council,
South Africa.
27. Chen, J. F.; Xu, K.; Petzer, J. P.; Staal, R.; Xu, Y. H.;
Beilstein, M.; Sonsalla, P. K.; Castagnoli, K.; Castagnoli,
N., Jr.; Schwarzschild, M. A. J. Neurosci. 2001, 21, RC143.
28. Castagnoli, K.; Palmer, S.; Anderson, A.; Bueters, T.;
Castagnoli, N., Jr. Chem. Res. Toxicol. 1997, 10, 364.
29. Gnerre, C.; Catto, M.; Leonetti, F.; Weber, P.; Carrupt, P.
-A.; Altomare, C.; Carotti, A.; Testa, B. J. Med. Chem.
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