N-methyl-N-((1-methyl-5-(3-(1-(2-methylbenzyl)piperidin-4-yl)propoxy)-1H-indol-2-yl)methyl)prop-2-yn-1-amine, a new cholinesterase and monoamine oxidase dual inhibitor

Article abstract of DOI:10.1021/jm501501a

On the basis of N-((5-(3-(1-benzylpiperidin-4-yl)propoxy)-1-methyl-1H-indol-2-yl)methyl)-N-methylprop-2-yn-1-amine (II, ASS234) and QSAR predictions, in this work we have designed, synthesized, and evaluated a number of new indole derivatives from which we have identified N-methyl-N-((1-methyl-5-(3-(1-(2-methylbenzyl)piperidin-4-yl)propoxy)-1H-indol-2-yl)methyl)prop-2-yn-1-amine (2, MBA236) as a new cholinesterase and monoamine oxidase dual inhibitor.

Full text of DOI:10.1021/jm501501a

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Article
N-Methyl-N-((1-methyl-5-(3-(1-(2-methylbenzyl)piperidin-4-
yl)propoxy)-1H-indol-2-yl)methyl)prop-2-yn-1-amine, a New
Cholinesterase and Monoamine Oxidase Dual Inhibitor
Oscar M. Bautista-Aguilera, Abdelouahid Samadi, Mourad Chioua, Katarina Nikolic, Slavica
Filipic, Danica Agbaba, Elena Soriano, Laura de Andrés, María Isabel Rodríguez-Franco,
Stefano Alcaro, Rona R. Ramsay, Francesco Ortuso, Matilde Yañez, and José Marco-Contelles
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 24 Nov 2014
Downloaded from http://pubs.acs.org on December 7, 2014
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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NꢀMethylꢀNꢀ((1ꢀmethylꢀ5ꢀ(3ꢀ(1ꢀ(2ꢀmethylbenzyl)piperidinꢀ4ꢀ
yl)propoxy)ꢀ1Hꢀindolꢀ2ꢀyl)methyl)propꢀ2ꢀynꢀ1ꢀamine, a New
Cholinesterase and Monoamine Oxidase Dual Inhibitor
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¶
¶
¶
Oscar M. BautistaꢀAguilera, Abdelouahid, Samadi, Mourad Chioua, Katarina
∆
∆
∆
§
±
Nikolic, Slavica Filipic, Danica Agbaba, Elena Soriano, Lucía de Andrés, María
±
Ω
∫
Ω,*
Isabel RodríguezꢀFranco, Stefano Alcaro, Rona R. Ramsay, Francesco Ortuso,
‡
,*
¶,*
Matilde Yañez, and José MarcoꢀContelles
¶
Laboratorio de Química Médica (IQOG, CSIC), Juan de la Cierva 3, Eꢀ28006, Madrid, Spain
∆
Institute of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450,
11000 Belgrade, Serbia
§
SEPCO, (IQOG, CSIC), Juan de la Cierva, 3, 28006ꢀMadrid, Spain
±
Ω
Instituto de Química Médica (IQMꢀCSIC), C/ Juan de la Cierva, 3 – 28006ꢀMadrid, Spain
Dipartimento di Scienze della Salute, Università “Magna Græcia” di Catanzaro, Campus “S. Venuta”,
Viale Europa, 88100 Catanzaro, Italy
∫
Biomedical Sciences Research Complex, University of St Andrews; Biomolecular Sciences Building,
North Haugh, St Andrews KY16 9ST, UK
‡
Facultad de Farmacia, Departamento de Farmacología, Universidad de Santiago de Compostela,
Campus Vida, Santiago de Compostela (La Coruña), Spain
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ABSTRACT:
Based on Nꢀ((5ꢀ(3ꢀ(1ꢀbenzylpiperidinꢀ4ꢀyl)propoxy)ꢀ1ꢀmethylꢀ1Hꢀ
indolꢀ2ꢀyl)methyl)ꢀNꢀmethylpropꢀ2ꢀynꢀ1ꢀamine (II, ASS234), and QSAR predictions,
in this work we have designed, synthesized, and evaluated a number of new indole
derivatives from which we have identified NꢀmethylꢀNꢀ((1ꢀmethylꢀ5ꢀ(3ꢀ(1ꢀ(2ꢀ
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methylbenzyl)piperidinꢀ4ꢀyl)propoxy)ꢀ1Hꢀindolꢀ2ꢀyl)methyl)propꢀ2ꢀynꢀ1ꢀamine
MBA236) as a new cholinesterase and monoamine oxidase dual inhibitor.
(2,
Key Words: Drug design, QSAR, Synthesis, Biological evaluation, Multipotent
Molecules, MAO/ChE Inhibitors, PAMPAꢀBBB, Molecular modeling, ADMET,
Alzheimer’s disease.
INTRODUCTION
Alzheimer's disease (AD) is an ageꢀrelated neurodegenerative disorder that accounts for
1
approximatively 70% of adult dementia. Worldwide, it is estimated that 40 million
people suffer from AD, and the prevalence of AD is expected to rise significantly in the
2
next decades, as the average age of the population increases. AD is characterized by a
3
progressive memory loss, a decline in language skills and other cognitive impairments.
Although the etiology of AD is not completely known, common hallmarks, such as
4
5
6
amyloidꢀβ (Aβ) deposits, τꢀprotein aggregation, and oxidative stress, are thought to
7
play key roles in the pathophysiology of the disease. In addition, the selective loss of
cholinergic neurons in AD results in a deficit of acetylcholine (ACh) in specific brain
8
regions that mediate learning and memory. However, alterations in other
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neurotransmitter systems, specially serotoninergic, and dopaminergic, are also thought
9
to be responsible for the behavioural disturbances observed in AD patients.
Monoamine oxidase (MAO; EC 1.4.3.4), the enzyme that catalyses the oxidative
1
0
deamination of a variety of biogenic and xenobiotic amines, is also an important target
to be considered for the treatment of specific features of AD. MAO exists as two
distinct enzymatic isoforms, MAOꢀA and MAOꢀB, based on their substrate and
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inhibitor specificities. MAOꢀA preferentially deaminates serotonin, adrenaline and
noradrenaline and is selectively and irreversibly inhibited by clorgyline. In contrast,
MAOꢀB preferentially deaminates βꢀphenylethylamine and benzylamine and is
12
irreversibly inhibited by lꢀdeprenyl. Selective inhibitors for MAOꢀA are effective
antidepressants, whereas MAOꢀB inhibitors are useful in the treatment of Parkinson’s
13
14
disease (PD) and might also be valuable for the treatment of AD. In fact, MAOꢀB
inhibitors are also currently in clinical trials for the treatment of AD because an
increased level of MAOꢀB has been detected in the plaqueꢀassociated astrocytes of
15
brains from AD patients.
At present, there are three FDAꢀapproved drugs (donepezil, galantamine and
16,17
rivastigmine)
that improve AD symptoms by inhibiting acetylcholinesterase (AChE;
E.C.1.1.1.7), i.e. the enzyme responsible for the hydrolysis of ACh, and, thereby, raising
ACh content in the synapsis. Apart from the beneficial palliative properties of AChE
18,19
inhibitors in AD,
cholinergic drugs have shown little efficacy to prevent the
progression of the disease. Consequently, there is no efficient therapy to cure, stop or
even slow the progression of the disease; therefore, effective therapeutics are urgently
sought and needed.
The failure to find such a drug or treatment is possibly due to the multifactorial
nature of AD. Thus, a single drug that acts on a specific target to produce the desired
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clinical effects might not be suitable for the complex nature of AD. Accondingly, the
20ꢀ22
multiꢀtargetꢀdirected ligand (MTDL) approach
has been the subject of increasing
attention by many research groups, which have developed a variety of compounds
2
3ꢀ26
acting on very diverse targets.
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We have previously reported Nꢀ((5ꢀ(benzyloxy)ꢀ1Hꢀindolꢀ2ꢀyl)methyl)propꢀ2ꢀ
2
7
ynꢀ1ꢀamine (I, PF9601N) (Figure 1) as a potent and selective propargylamineꢀ
containing MAOꢀB inhibitor (MAOBI) possessing neuroprotective properties that,
unlike lꢀdeprenyl does not generate amphetamines when metabolised and possesses
2
8
antiꢀapoptotic properties. Next we used I as hit compound with therapeutic potential
for the treatment of neurodegeneratyive diseases, and started a project targeted to
finding an improved lead compound.
As a result of this effort, we found that Nꢀ((5ꢀ(3ꢀ(1ꢀbenzylpiperidinꢀ4ꢀ
yl)propoxy)ꢀ1ꢀmethylꢀ1Hꢀindolꢀ2ꢀyl)methyl)ꢀNꢀmethylpropꢀ2ꢀynꢀ1ꢀamine (II, ASS234,
Figure 1) is a multipotent drug able to inhibit human AChE (hAChE) and human
butyrylcholinesterase (hBuChE), showing inhibition of human MAOꢀA and human
29
MAOꢀB (hMAOꢀA, hMAOꢀB). The kinetic analysis demonstrated that compound II is
not only a reversible inhibitor of both hAChE and hBuChE with micromolar affinity,
3
0
but a highly potent irreversible inhibitor of hMAOꢀA, similarly to clorgyline. The
crystal structure of human MAOꢀB in complex with compound II highlighted the
covalent adduct formed with the flavin N5 atom which, based on the spectral changes,
30
occurs also with the MAOꢀA cofactor. Hybrid II inhibited Α
β
1ꢀ42 selfꢀaggregation
more efficiently than that of Α
β
1ꢀ40, limiting the formation of fibrillar and oligomeric
species, and completely blocked the aggregation mediated by AChE of both Αβ1ꢀ42
and Α 1ꢀ40. Interestingly, compound II significantly reduced Α 1ꢀ42ꢀmediated toxicity in
31
β
β
SHꢀSY5Y human neuroblastoma cells, and showed a significant ability to capture freeꢀ
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radical species in vitro, as well as a potent effect in preventing the Αβ1ꢀ42 induced
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1,32
depletion of catalase and SODꢀ1.
Finally, compound II is able to cross the blood
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brain barrier in vivo. Overall, these data indicate that hybrid II is the desired lead
compound in our search for neurodegenerative diseases therapy.
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N
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Me
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Me
II
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O
N
N
H
H
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6, 17
8, 19
I
Figure 1. Schematic structural and
functional modifications in compounds I
and II leading to the new target
molecules 1ꢀ19
.
Based on these results, we started a lead compound optimization program,
whose results are described now in this manuscript. From this study, NꢀmethylꢀNꢀ((1ꢀ
methylꢀ5ꢀ(3ꢀ(1ꢀ(2ꢀmethylbenzyl)piperidinꢀ4ꢀyl)propoxy)ꢀ1Hꢀindolꢀ2ꢀyl)methyl)propꢀ2ꢀ
ynꢀ1ꢀamine (2, MBA236) has emerged as a permeable, potent, irreversible, and quite
selective hMAOꢀA inhibitor (MAOAI) in the nanomolar range, showing also moderate
and almost equipotent hAChE vs hBuChE inhibition power.
RESULTS AND DISCUSSION
Design. In Figure 1 we show the modifications in compounds I and II which have
resulted into the new nineteen molecules that we have synthesized. Our selection was
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based on QSAR analysis (see below), and simple functional changes on compounds II
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Figure 2. Compounds 1ꢀ12 synthesized and
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MeO
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O
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HN
NBn
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Figure 3. Synthesized compounds 13ꢀ
9 designed from compound I.
1
.
As depicted, the Nꢀbenzyl motif in compound II has been eliminated (1), or
modified incorporating different substituents in the phenyl ring, such as oꢀMeC CH
2), pꢀCNC CH (3), and Nꢀ[(8ꢀOꢀmethylꢀ or 8ꢀOꢀ(N,N´ꢀdimethylcarbamoyl)]5ꢀ
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methylen]quinolone (4 and 5) (Figure 2). Taking into account the structure, inhibition
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power and selectivity of compound I for MAO B, the N(1)Me in compound II has
been
replaced
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leading
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the
N(1)Me
and
(
indole)C(2)CH
indole)C(2)CH
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N(Me)CH
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C≡CH in hybrid II has been replaced by a N1(H) and
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(
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N(H)CH C≡CH in 7 (Figure 2); compound 8 is like 7 (Figure 2), but
the acetylene has been substituted by a vinyl group. Compounds 9 and 10 (Figure 2) are
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the Nꢀbenzylꢀ and Nꢀphenylꢀ1,2,3ꢀtriazole derivatives, respectively, of compound II.
Similarly, compounds 11 and 12 (Figure 2) result from hybrid II by eliminating the
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C(2)CH
2 2 2 2
N(Me)CH OH, and CO Et, respectively, and in both
C≡CH side chain by CH
cases we have replaced the N(1)Me group by N(1)H.
27
Regarding compound I, related transformations have afforded compounds 13ꢀ
16 (Figure 3). Compounds 17ꢀ19 (Figure 3) are ladostigil/rasagilineꢀlike carbamates,
where we wanted to explore the known ability of compounds bearing this functional
3
6
motif to inhibit ChEs. Results from recent MAO/ChE literature, such as the
34
substitution of an acetylene by a triazole, or the use of a multifunctional chelator
37
derived from 8ꢀhydroxyquinoline, have been used to design molecules 5, 9, 10, 14,
and 15 (Figures 2 and 3).
QSAR analysis. The 3DꢀQSAR study has been carried out with main aim to explain
and predict the binding on the active sites of human MAOꢀA, MAOꢀB, AChE, and
27,31,35
BuChE enzymes based on the pharmacological activities
previously observed for
the leadꢀimproving program work with multipotent compound II (Supporting
Information), and the results are shown in Table 1.
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Table 1. Experimental and QSARꢀpredicted
activities of novel MAO/ChE inhibitors.
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Experimental
IC50 (hMAOꢀA)
6.3 ± 0.4 nM
10.3 ± 0.8 nM
257.6 ± 11.4 nM
310.6 ± 17.1 nM
630.1 ± 16.1 nM
Experimental
IC50 (hMAOꢀB)
183.6 ± 7.4 nM
164.7 ± 12.1 nM
196.3 ± 7.8 nM
273.1 ± 8.9 nM
7.9 ± 0.5 nM
Experimental
IC50 (hAChE)
2.8 ± 0.1 µM
8.4 ± 0.9 µM
**
Predicted
IC50 (MAOꢀA)
3.9 nM
CMPD
2
4
5
218.8 nM
306.2 nM
3.2 µM
1
1
4
3
23.7 µM
Predicted
IC50 (MAOꢀB)
100.7 nM
662.2 nM
38.8 µM
58.5 µM
73.1 µM
Predicted
IC50 (AChE)
849.2 nM
3.0 µM
CMPD
2
13
5
14
4
CMPD
2
5
14
4
4.0 µM
10.3 µM
11.1 µM
Predicted
***
**
13
Experimental
IC (hBuChE)
CMPD
IC (BuChE)
5
0
50
2
5
4
4.9 ± 0.2 µM
5.9 ± 0.4 µM
1.3 µM
4.4 µM
9.8 µM
14.8 µM
202.8 µM
**
**
**
1
1
4
3
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*
* Inactive at 100 µM (highest concentration tested).
*** 100 µM inhibits the corresponding MAO activity by
approximately 40ꢀ50%. At higher concentration the
compounds precipitate.
27,31.35
In the data set of previously characterised indole derivatives
the MAOꢀA
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pIC50 activity interval spanned 4 log units (3.07–9.10); for pIC50 (MAOꢀB) it was 5.6
log units (4.00–10.60), for pIC (AChE) 4.2 log units (4.00ꢀ8.17), and pIC (BuChE)
50
50
3.4 log units (4.00ꢀ7.44). The relatively wide pIC50 interval of the training set provide
broad applicability domain for the 3DꢀQSAR models created. The 3DꢀQSAR (MAO A)
2
model with two significant components (A= 2), R : 0.94, leaveꢀoneꢀout cross validation
2
2
Q : 0.66, Training Set parameters (R Observed vs. Predicted: 0.908 and RMSEE: 0.433), and
2
Test Set parameters (R Observed vs. Predicted: 0.809 and RMSEP: 0.617), was developed. The
2
3
DꢀQSAR (MAO B) model with two significant components (A=2), R : 0.97 and leaveꢀ
2
2
oneꢀout cross validation Q : 0.85, Training Set parameters (R Observed vs. Predicted: 0.957
2
and RMSEE: 0.303), and Test Set parameters (R Observed vs. Predicted: 0.787 and RMSEP:
0
.598), was formed. Similarly, the 3DꢀQSAR (AChE) model with three significant
2
2
components (A=3), R : 0.87 and leaveꢀoneꢀout cross validation Q : 0.61, Training Set
2
parameters (R Observed vs. Predicted: 0.873 and RMSEE: 0.431), and Test Set parameters
2
(
R
Observed vs. Predicted: 0.539 and RMSEP: 0.661), was developed. The 3DꢀQSAR (BuChE)
2
model with two significant components (A=2), R : 0.92 and leaveꢀoneꢀout cross
2
2
validation Q : 0.72, Training Set parameters (R Observed vs. Predicted: 0.922 and RMSEE:
2
0
.261), and Test Set parameters (R Observed vs. Predicted: 0.657 and RMSEP: 0.521), was
35
created.
The statistical parameters indicated that the 3DꢀQSAR (MAOꢀA, MAOꢀB,
AChE, BuChE) models could be used for prediction of MAOꢀA, MAOꢀB, AChE, and
BuChE inhibiting activities for novel analogs of compounds I and II. Reliability of the
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activities predictions is strongly related to the type and extent of structural changes of
the lead compounds.
3Dꢀpharmacophores are derived from 3Dꢀmaps of interaction energies between
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the examined molecule and four chemical probes: DRY (which represent hydrophobic
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interactions), O (sp carbonyl oxygen, representing Hꢀbond acceptor), N1 (neutral flat
NH, like in amide, Hꢀbond donor), and the TIP probe (molecular shape descriptor). The
3
Dꢀpharmacophores for MAOꢀA and MAOꢀB inhibiting activities of compound II
contain crucial favorable OꢀTIP/OꢀDRY/TIPꢀTIP/DRYꢀTIP interactions between the
propargylamine moiety and the benzyl group. Therefore, substitution of the benzyl
moiety of hybrid II with small groups, such as an oꢀMe in 2 (Figure 2), could enhance
the MAOꢀA and MAOꢀB inhibiting activity of the examined compounds by facilitating
the TIPꢀTIP/DRYꢀTIP favorable interactions. The 3DꢀQSAR study confirmed also
previous experimental findings that the propargylamine moiety provides an essential
35,38,39
positive influence on the MAOꢀA and MAOꢀB inhibiting activity.
The 3Dꢀpharmacophores for BuChE inhibiting activities of lead compound II
contain specific unfavorable TIPꢀTIP/DRYꢀTIP interactions between the indole and the
para position of the benzyl moieties. Therefore, substitution with bulky substituent at
para position of the benzyl moiety, such as a cyano group in 3 (Figure 2), could
decrease BuChE inhibiting activity of compound II derivatives (Table 1). The 3Dꢀ
pharmacophores for AChE inhibiting activities of compound II contain a crucial
favourable OꢀO interaction between the piperidine ring and ortho positions of the
benzyl group. Thus a small substituent at ortho position of the benzyl ring, such as the
methyl group in compound 2 (Figure 2) could enhance AChE inhibiting activity of lead-
compound II (Table 1).
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In addition to 2, compounds 4, 3, 13 and 14 have also been investigated by
QSAR analysis, and their predicted IC values are also shown in Table 1. The predicted
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IC50 values clearly suggest that: (a) compound IIꢀdonepezil type hybrids 2, 4, and 5, in
this order, should be the most potent MAOꢀAI; (b) the compound Iꢀlike derivative 14
should be a more potent inhibitor of MAOꢀA, AChE, and BuChE than 13; and (c) 2
should be the most potent inhibitor against all four enzymes.
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Next, the designed compound II analogues were synthesized and examined for
MAO/ChE inhibitory activity.
Chemistry. The synthesis of the new molecules (Figures 2 and 3) has been achieved by
standard or reported methods, in very efficient and short synthetic sequences, in good
overall yields (see Supporting Information). All new molecules gave satisfactory
analytical and spectroscopic data, in good agreement with their structures.
Table 2. IC values and MAOꢀB selectivity ratios [IC (MAOꢀA)]/[IC (MAOꢀB)] for
5
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50
the inhibitory effects of test compounds on the enzymatic activity of hMAO isoforms
expressed in baculovirus infected BTI insect cells. IC values for the inhibitory effects
5
0
of test compounds on the enzymatic activity of hAChE expressed in HEK 293 cells, and
hBuChE.
CMPD
2
hMAOꢀA
hMAOꢀB
Ratio
0.03
hAChE
hBuChE
Ratio
0.57
a
6.3 ± 0.4 nM
183.6 ± 7.4 nM
2.8 ± 0.1 µM
4.9 ± 0.2 µM
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b
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257.6 ± 11.4 nM
196.3 ± 7.8 nM
35.1 ± 2.8 µM
33.6 ± 1.5 µM
21.3 ± 1.9 µM
1.0 ± 0.02 µM
13.5 ± 1.1 µM
1.2 ± 0.1 µM
4.1 ± 0.7 nM
1.3
0.26
0.57
2.1
8.4 ± 0.9 µM
15.4 ± 0.9 µM
4.9 ± 0.3 µM
4.6 ± 0.3 µM
9.6 ± 0.08 µM
13.6 ± 1.4 µM
3.4 ± 0.2 µM
**
5.9 ± 0.4 µM
7.1 ± 0.5 µM
7.3 ± 0.8 µM
40.6 ± 2.2 µM
9.7 ± 0.7 µM
31.2 ± 3.1 µM
3.3 ± 0.2 µM
**
1.4
2.2
b
9.1 ± 0.7 µM
b
19.2 ± 1.3 µM
0.67
0.11
0.99
0.43
1.03
b
45.3 ± 1.6 µM
11
876.6 ± 25.2 nM
9.1 ± 0.3 µM
0.87
0.67
0.05
70
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a
II
I
58.2 ± 1.2 nM
a
287.3 ± 11.2 nM
a
0.0000
Clorgyline
4.7 ± 0.2 nM
65.8 ± 1.6 µM
**
**
7
1
a
lꢀDeprenyl
63.6 ± 1.3 µM
18.2 ± 0.9 nM
nd
3,494
**
**
c
Donepezil
nd
0.016 ± 0.001 µM
8.2 ± 0.2 µM
MAO: IC50 values were determined after 15 min preincubation of inhibitor with the enzyme. All IC50 values shown in this
Table are the mean ± SD from five experiments. Level of statistical significance: P < 0.01 or P < 0.05 versus the
corresponding IC50 values obtained against MAOꢀB, as determined by ANOVA/Dunnett´s. Ref. 23. ** Inactive at 100 µM
(highest concentration tested). ChE: All IC50 values shown in this Table are the mean ± SD from five experiments. nd: not
a
b
c
determined.
Biological evaluation. The biological evaluation, comprising the hMAOꢀA/ hMAOꢀB,
and hAChE/ hBuChE inhibition, has been carried out according to the protocols
40,41
described in the Supporting Information, following the usual methods.
In Table 1S
(
Supporting Information) we have shown the IC50 values for the inhibition of the
MAOs and ChEs with all the nineteeen new compounds, using compounds I and II, reꢀ
4
2
analyzed in this work as reference compounds. The established MAOI, clorgyline and
lꢀdeprenyl, and the ChEI, donepezil, are also reported for comparative purposes. Note
that only seven compounds, 2, 5ꢀ8 11, and 12, were active simultaneously in the four
enzymes (Table 2). As shown in Table 1S, compound 6 is a moderate hMAOꢀAI and
hBuChEI, in the high micromolar range, but shows very modest hMAOꢀB and hAChE
inhibition power. 12 is almost equipotent with 6 for the hMAOꢀA and hAChE
inhibition, but 2.6ꢀfold more, and 4.4ꢀfold less potent than 6, for the inhibition of
hMAOꢀB and hBuChE, respectively. 7 is a poor hMAOꢀAI and hMAOꢀBI, but a
moderate hAChEI and BuChEI, in the micromolar range. 8 is a quite poor hMAOꢀAI,
hMAOꢀBI and hBuChEI, but a modest hAChEI. Furthermore, 5, and 11 were nonꢀ
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selective, potent hMAOI in the low micromolar range, but moderate and equipotent
ChE inhibitors.
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Figure 4. Structure of compound 2, and its
IC50 values for the inhibition of MAO and
ChEs, compared with the the reference
compound II.
However, among all these products, as predicted by the QSAR analysis (see
above), compound 2 (Table 2) was the most potent hMAOꢀA, hMAOꢀB AChE, and
BuChE multipotent inhibitor found in this work. Compound 2 is a very potent, in the
nanomoloar range, and selective hMAOꢀAI (Ki = 7.5 ± 0.9 nM), showing a potent and
slightly selective hAChE inhibition profile (Ki = 1.3 ± 0.5 µM), in the micromolar
range. Compared with the reference compound II (Figure 4), the new compound 2 is
9.2ꢀfold and 6.5ꢀfold more potent hMAOꢀAI and hMAOꢀBI, respectively, 1.2ꢀfold more
potent hAChEI, and only 1.5ꢀfold less potent hBuChEI. Comparing with compound I
and the standard MAOIs, for the inhibition of MAOꢀA, compound 2 is 45ꢀfold more
potent than compound I, 1.3ꢀfold less than clorgyline, and 10095ꢀfold more potent than
lꢀdeprenyl. Conversely, for the inhibition of MAOꢀB, compound 2 is 45ꢀfold less, 358ꢀ
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fold more and 10ꢀfold less potent than compound I, clorgyline and lꢀdeprenyl,
respectively.
To sum up, compound 2 is a multipotent molecule of MAO and ChE, showing a
very attractive pharmacological profile, as a selective and very potent MAOꢀAI, with
high MAOꢀB and potent hAChE/ hBuChE inhibition power. It must be also concluded
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that all our present and past efforts to transform compound I into a multipotent
MAO/ChE compound, by transforming the benzyl group into the ChE donepezilꢀlike
pharmacophore have met with success, but at the cost of loosing significant MAOꢀB
inhibition capacity.
Taking into account these findings, we have performed further studies on the
inhibition mechanism of action of compound 2.
Figure 5. Recovery of hMAOꢀA activity after dilution
following incubation (30 min at room temperature) of
the 100xꢀenzyme concentration with 10ꢀfold IC50
concentration of compound 2 or clorgyline. The control
was carried out by preꢀincubating in the absence of
inhibitor and diluting in the same way. Results are
expressed as percentage of control. Represented data
are mean ± SD of five independent assays.
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Figure 6. Recovery of hMAOꢀB activity after dilution
following incubation (30 min at room temperature) of the
100xꢀenzyme
concentration
with
10ꢀfold
IC50
concentration of compound 2 or lꢀdeprenyl. The control
was carried out by preꢀincubating in the absence of
inhibitor and diluting in the same way. Results are
expressed as percentage of control. Represented data are
mean ± SD of five independent assays
.
43
Figures 5 and 6 show the results of the reversibility test on hMAO. Compound
2 binds as an irreversible inhibitor on both enzyme isoforms, maintaining near a 90%
inhibition after the dilution of the preꢀincubated mixture. The IC50 decreased from 14.6
±
1.2 nM in reversible conditions (no preicubation and substrate concentration 2xKm)
to 6.3 ± 0.4 nM assayed with saturating substrate after a 15 min incubation.
Figure 7. Recovery of hAChE (grey column) and
hBuChE (black column) activity after the dilution of
the incubation (30 min at room temperature) of the
100Xꢀenzyme concentration and 10ꢀfold IC50 of
compound 2. The control was carried out by preꢀ
incubating in the absence of inhibitor and diluting in
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the same way. Results are expressed as percentage of
control. Represented data are mean ± SD of five
independent assays.
Nevertheless, the recovery of compound 2 on ChEs was significantly greater
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(
around 20%), allowing us to classify it as a slow inhibitor (Figure 7). Kinetic assays
performed with this compound have shown ambiguous results, probably due to the low
degree of reversibility of inhibition achieved on hAChE and hBuChE. As a result of the
low reversibility, the pattern of the LineweaverꢀBurk representation appears to indicate
a mixed model for both enzymes (results not shown).
With the aim to investigate compound 2 recognition with respect to either MAOꢀ
A, MAOꢀB, AChE and BuChE enzymes, molecular modeling studies have been carried
out. The theoretical investigation has been focused on the ligand enzyme interaction and
target structural perturbation produced by compound 2 comparing these information
with respect to the same data coming from the related compound II (Supporting
Information). Only one proposed structure was found for compound 2 in hMAOꢀA
and two for hMAOꢀB, with a global miminum population equal to 96.11%. Compounds
II and 2 both adopted bent conformations in hMAOꢀA, but were linear in hMAO–B
(Figure S1, Supporting Information). Overall, the interactions between compounds II,
2
and the MAOs are driven by both steric hindrance and hydrophobic contribution with
an additional electrostatic term present in hMAOꢀA. Using the target RMSd matrix,
each MD trajectory was clustered into nine groups. The visual inspection of the ten
structures for each inhibitor with each enzyme clearly indicates the conformation
stabilizing effect of the oꢀmethyl group in hybrid 2 (Figure S4, Supporting
Information).
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In this work we have designed a new and multipotent MAOI. MAOI are well
known for showing diverse therapeutic potential as the result of combining selectivity
15
and (ir)reversible modes of binding. MAOꢀAI have been used mostly in the treatment
4
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of depression and anxiety, and MAOꢀBI for the treatment of PD. Recent studies have
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shown that MAOꢀA is involved in the regulation of serotonin for ventricular remodeling
46
via activation of the serotonin2A receptors. In the aged heart, the increased MAOꢀA
concentrations results in high levels of oxygen peroxide leading to cardiomyocyte
47
severe damage. A survey of the current literature on MAOIs shows also that one of the
major reasons for designing new and specific inhibitors is to function as
48
cardioprotectants for the ageꢀdependent increase of MAO A in the heart. Although
MAOꢀAIs enhance extracellular levels of dopamine produced from Lꢀdopa, irreversible,
and selective MAOꢀAI cannot be used in the PD therapy, because of cardiovascular
49
side effects. Another disadvantage of an irreversible MAOꢀAI is the hypertensive
50
“
cheese effect”, due to the predominance of MAOꢀA in the gut wall to metabolize
tyramine in fermented foods such as cheese and beer. Hypertensive crises may be
prevented by using reversible rather than irreversible MAOꢀAI, or tissueꢀspecific
51
inhibitors such as the brainꢀselective ladostigil.
Figure 8. Structures of ladostigil, and M30D.
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The cases of ladostigil and M30D (Figure 8) are of interest for this project
and future possible endeavours, because both are ChE, MAOꢀA and MAOꢀB
irreversible inhibitors with potential therapeutic applications as antidepressants,
4
4
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antiparkinson and antiꢀAlzheimer drugs. Ladostigil (Figure 8) was designed from
rasagiline, a MAOꢀBI, bearing a propargylamine motif, responsible for the
neuroprotective properties, and a carbamate group, affording ChE inhibitory activity.
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3
M30D (Figure 8) shows an inhibitory profile against ChE [hAChE IC = 0.52 ± 0.07
50
ꢀ
M; hBuChE IC50= 44.90 ± 6.10 ꢀM] and against MAO [ratMAOꢀA: IC50= 0.0077 ±
0.0007 ꢀM; ratMAOꢀB: IC = 7.90 ± 1.34 ꢀM] very similar the one observed for 2
5
0
(
Table 2), as well as other properties as potential drugs for treating AD.
In Vitro Blood–Brain Barrier Permeation Assay. Finally, in order to evaluate the
54
brain penetration we used the PAMPAꢀBBB method described by Di, and
subsequently optimised by RodríguezꢀFranco et al. for molecules with limited waterꢀ
55ꢀ57
solubility.
The in vitro permeability (P ) value of compound 2 through a lipid extract of
e
porcine brain was determined by using PBS: ethanol (70:30). In the same assay, 11
commercial drugs of known CNS penetration were also tested and their experimental
e
values were compared to reported values, giving a good lineal correlation, P (exp.) =
2
1
.4442 P
e
(bibl.) + 6.5263 (R = 0.95). From this equation and taking account the limits
5
4
established by Di for BBB permeation, we found that compounds with permeability
ꢀ
6
ꢀ1
e
values above 12.3 10 cm s could penetrate into the CNS. Derivative 2 showed P =
ꢀ
6
ꢀ1
14.4 ± 0.4 10 cm s and thus, it could cross BBB by passive diffusion (Supporting
Information)
.
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CONCLUSIONS
In this paper we have reported the design, synthesis and biological evaluation of
nineteen new donepezilꢀindolyl hybrids as multifunctional drugs able to bind MAO and
ChE enzymes, using the MAO B inhibitor I hit, and the multipotent II hybrid lead as
references for leadꢀoptimization in order the discover a new leadꢀcompound for deeper
in vivo preꢀclinical investigations targeted to neurological disorders. The QSARꢀ
designed compound II analogues have been synthesized and examined for MAO/ChE
inhibitory activity. As shown in Figure 4, the potent MAO/ChE inhibiting activity for
the novel ligand 2 confirmed the quality of the optimized 3Dꢀpharmacophores for use in
future work to design novel donepezilꢀindolyl hybrids. We have found significant
biological activity on the four enzymes studied in almost half of the synthesized
structures. The most promising compound of the series, 2, acted as an irreversible
hMAOꢀAI, potent in the nanomolar range, nine times more potent than the reference
compound II, and 29ꢀfold more selective for hMAOꢀA over hMAOꢀB. Inhibition of the
ChEs by hybrid 2 is in the micromolar range, slightly better than compound II for
hAChEs although slightly poorer for hBuChE. Although we are confronted with
unbalanced, highly preferred MAO vs ChE inhibition, the potency and desired balance
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between targets in MTDLꢀbased strategies is not known. Molecular modeling studies
reported similar binding modes of compounds II and 2 in all the evaluated biological
targets. The oꢀMe group in compound 2 improves the ligand recognition increasing the
ligandꢀenzyme hydrophobic interaction in hBuChE, and πꢀπ stacking in hMAOꢀA,
hMAOꢀB and hAChE. Thus, it is clear that simple modification of compound II, such
as the incorporation of an oꢀmethyl instead of a hydrogen in the phenyl ring of the Nꢀ
benzylpiperidine motif, produces significant qualitative and quantitative
pharmacological changes in the inhibition of MAO and ChE enzymes. Finally, the
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ADMET virtual analysis suggests that inhibitor 2 should present good drugꢀlike
characteristics similar to compound II, with a slightly better brain penetration ability, a
prediction experimentally confirmed by the in vitro blood–brain barrier permeation
assay.
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Consequently, we have fulfilled our initial expectations, that a new, easily
available, permeable multipotent MAO inhibitor has been discovered. The related
pharmacological properties with ladostigil, clearly support projected in vivo studies for
proof of concept targeted to probe the suitability of compounds 2 and II to treat
neurodegeneratives disorders such AD or PD.
ASSOCIATED CONTENT
Supporting Information
QSAR methods, ADMET analysis and descriptors, the synthesis of the target
1
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compounds, the H and C spectra, biological evaluation, molecular modeling, and in
vitro blood–brain barrier permeation assay of compound 2. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding authors
*
For JMC: Eꢀmail: iqoc21@iqog.csic.es; Tel.: +34ꢀ91ꢀ5622900; Fax: +34ꢀ91ꢀ5644853.
* For MY: Eꢀmail: matilde.yannez@uam.es; Tel:+34ꢀ881ꢀ815529; Fax:+34ꢀ881ꢀ545595
For FO: Eꢀmail: ortuso@unicz.it; Tel.:+39ꢀ09613694197; Fax: +39 0961391490.
Author Contributions
*
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OMBA carried out the synthesis of the target molecules. OMBA, MC, and AS carried
out the spectroscopic analyses. KT, SF and DA carried out the QSAR predictions. MY
undertook the enzyme inhibition and mechanistic studies. ES did the ADMETꢀanalysis.
FO and SA carried out the computational studies. LdA did the PAMPAꢀBBB
experiment, under MIRF supervision. RRR evaluated the data and contributed to
writing the manuscript. JMC conceived, designed and supervised the project.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
JMC thanks MINECO (Spain) for grant SAF2012ꢀ33304, and Universidad Camilo José
Cela (Madrid, Spain) for support (MULTIMOL 2013ꢀ20). Oscar Mauricio Bautistaꢀ
Aguilera thanks MINECO for a FPI fellowship. This work was supported by the COST
action CM1103. DA and KN acknowledge COST action CM1207 and the project
(Contract #172033) supported by the Ministry of Education and Science of the Republic
of Serbia. M.I.R.ꢀF. acknowledges the financial support from the Spanish Ministry of
Economy and Competitiveness (SAF2012ꢀ31035), CSIC (PIEꢀ201280E074), and
Fundación de Investigación Médica Mutua Madrileña Automovilística (AP103952012).
ABBREVIATIONS USED
ACh, acetylcholine; AChEI, acetylcholinesterase inhibitors; hAChE, human
acetylcholinesterase; AD, Alzheimer’s disease; ADMET, Absorption, Distribution,
Metabolism, Excretion, Toxicity; hBuChE, human butyrylcholinesterase; hERG, human
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Ether-à-go-goꢀRelated Gene; MAO, monoamine oxidaseꢀA/B; MAOI, monoamine
oxidase inhibitors; MTDL, MultiꢀTargetꢀDirected Ligand; PAMPAꢀBBB, Parallel
Artificial Membrane Permeation Assay/BloodꢀBrainꢀBarrier; QSAR, Quantitative
StructureꢀActivity Relationships.
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