Discovery of two new classes of potent monoamine oxidase-B inhibitors by tricky chemistry

Article abstract of DOI:10.1039/c4cc08798d

The discovery of potent and selective monoamine oxidase-B inhibitors for the management of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases is still a challenging endeavor. Herein, we report the discovery of two new classes of poten

Full text of DOI:10.1039/c4cc08798d

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Discovery of two new classes of potent
monoaminoxidaseꢀB inhibitors by tricky chemistry
Cite this: DOI: 10.1039/x0xx00000x
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F. Cagide,a T. Silvaa , J. Reisa, A. Gaspara, F. Borgesa*, L.R. Gomesb and J.N. Lowc
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
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emerged as a new scaffold for the development of IMAOꢀB (Fig.
1B).
The discovery of potent and selective monoamine oxidaseꢀB
inhibitors for the management of neurodegenerative
diseases such as Alzheimer’s disease and Parkinson’s
disease is still a challenging endeavor. Herein, we report
the discovery of two new classes of potent and selective
MAOꢀB inhibitors based on chromaneꢀ2,4ꢀdione and
chromoneꢀ3ꢀcarboxamide scaffolds.
Monoamine oxidase (MAO) is an enzyme present in mammals in
two isoforms: MAOꢀA and MAOꢀB. These isoforms have a crucial
role in the metabolism of neurotransmitters, representing strategic
and important drug targets in the therapy of neurodegenerative
diseases (MAOꢀB) and depression (MAOꢀA). Selective MAOꢀB
inhibitors (IMAOꢀB) as deprenyl and rasagiline are currently used in
the symptomatic treatment of Parkinson’s disease (PD), either alone
or in combination with levodopa. Currently, the side effects
associated with the use of deprenyl and rasagiline (Figure 1A) as
well as their potential application for Alzheimer’s disease (AD) are
the main factors driving the discovery of new IMAOꢀB with
increased potency and selectivity.1ꢀ3 Over the past years, the
privileged structure concept has emerged as a stimulus for the
acceleration of drug discovery and development processes, as
diversityꢀoriented synthesis is a fastꢀtrack practice. In this context,
the chromone (1,4ꢀbenzopyrone) scaffold has been selected by our
research group as the core for the design and development of new
IMAOꢀB.4 In this framework, a functionꢀoriented library based on
chromoneꢀ3ꢀcarboxylic acid, a promising IMAOꢀB,5 has been
synthesized to accomplish structureꢀactivityꢀrelationship (SAR)
studies. The effect of the incorporation of an amide bond, a longꢀ
established biological chemical connection, in the chromone core on
the activity was also assessed.6,7 Consequently, a set of chromone
carboxamide derivatives have been synthesized and their IMAO
Figure 1. (A) MAOꢀB inhibitors used in single or combined therapy;
(B) Discovery of IMAOꢀB based on chromone scaffold.
However, some queries arising from the previous studies still remain
open, namely those related to spectroscopic data and the putative
presence of tautomeric forms in solution.8 To provide further insight,
NMR experiments at variable temperatures were performed along
with theoretical studies using NMR spectral predictors, but no
spectral changes were observed, even at low temperatures. Thus,
additional efforts had to be carried to address and clarify the
remaining issues. Accordingly, the chromoneꢀ3ꢀcarboxamide
derivatives were synthesized by two different amidation reactions
using the same starting materials (chromoneꢀ3ꢀcarboxylic acid and
the appropriate amine). The selected reactions involve different
methods of activating the carboxylic acid function and have in
common the subsequent addition of an arylamine: a) the use of
phosphonium salt (benzotriazolꢀ1ꢀyloxy)tripyrrolidinophosphonium
hexafluorophosphate (PyBOP) as coupling agent and b) the in situ
generation of an acyl chloride with phosphorus (V) oxychloride
activity evaluated. In addition,
a combined theoretical and
experimental study has been carried out to explain the superior
activity of chromone 3ꢀcarboxamide versus chromone 2ꢀ
carboxamide as IMAOꢀB.7,8 As a result, chromoneꢀ3ꢀcarboxamide
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(POCl3) in DMF. From the array of coupling reagents described so
far, PyBOP was selected since it can minimize some of the wellꢀ
known drawbacks of this type of reactions, such as laborious
purification steps due to the presence of byꢀproducts, namely those
9ꢀ11
from dicyclohexylcarbodiimide type coupling reagents.
In
addition, PyBOP operates in mild and slightly basic conditions and
presents less toxicity constraints as compared to other phosphonium
coupling agents, such as BOP. Accordingly, our first attempt to
obtain chromoneꢀ3ꢀcarboxamides was performed using a oneꢀpot
condensation procedure and PyBOP as coupling reagent. Briefly, to
a solution of the chromoneꢀ3ꢀcarboxylic acid in dimethylformamide
(DMF) in the presence of N,Nꢀdiisopropylethylamine (DIPEA),
PyBOP was added at 0ºC. This step led to the activation of the
chromoneꢀ3ꢀcarboxylic acid and in situ formation of an activated
ester intermediate. Then, the amine with the appropriate aromatic
substitution pattern was added to the mixture. In the PyBOPꢀassisted
experiments, the structural elucidation (NMR and EM/IE) data of the
(3)
Figure 2. X Ray structure of 3ꢀ((phenylamino)methylene)chromaneꢀ
2,4ꢀdione (2) and Nꢀphenylꢀ4ꢀoxoꢀ4Hꢀchromeneꢀ3ꢀcarboxamide (3).
Details for data acquisition, structural solving and refinement are
given in files deposited in the Cambridge Structural Database:
CCDC 895638 for 2 and CCDC 932188 for 3.
main reaction products was consistent with the previous reported
6ꢀ8
results.
(see Supplementary Information). In parallel, and as
mentioned before, a different methodology using acidic conditions
(POCl3 in DMF) was performed. Briefly, the synthetic strategy
involved the in situ generation of the chromoneꢀ3ꢀacyl chloride, and
subsequent addition of aniline, or its derivatives. This procedure led
to the formation of chromone type compounds that were fully
characterized by NMR and EM/IE (see Supplementary Information).
The spectroscopic data were divergent from the aforementioned.
From the data gathered, it was concluded that two types of chemical
entities have been synthesized from chromoneꢀ3ꢀcarboxylic acid
(Scheme 1): one based on chromaneꢀ2,4ꢀdione and the other on the
chromoneꢀ3ꢀcarboxamide core.
After the identification of chromaneꢀ2,4ꢀdione and chromoneꢀ3ꢀ
carboxamide, and their derivatives, other studies were implemented
to acquire evidence to explain their formation from the same starting
material. As the reactivity of chromoneꢀ3ꢀcarboxylic acid (1)
towards primary and secondary amines has been recently reported,12,
13
the first step was to check if the arylamine could promote a
nucleophilic attack at the C2 position of the starting material. To test
this hypothesis, chromoneꢀ3ꢀcarboxylic acid (1) and aniline were
mixed in dichloromethane, in the presence of DIPEA but in the
absence of PyBOP, and stirred for the same reactional period of time
(Scheme 2).
POCl3
DMF
O
O
PyBOP
O
O
H
N
O
COOH
O
O
O
HN
R
(1)
(2) R=H
(3) R=H
(4) R= OH
(6) R=Cl
(5) R= OH
(7) R=Cl
R
(8) R=CH3
(9) R=CH3
Scheme 1. Synthesis of chromaneꢀ2,4ꢀdione (2, 4, 6 and 8) and
chromoneꢀ3ꢀ carboxamide (3, 5, 7 and 9) based derivatives from
chromone ꢀ3ꢀcarboxylic acid (1).
Scheme
2.
Formation
of
(Z)ꢀ1ꢀ(2ꢀhydroxyphenyl)ꢀ3ꢀ
A complete characterization of compounds 2 and 3 (Scheme 1) (phenylamino)propꢀ2ꢀenꢀ1ꢀone from chromoneꢀ3ꢀcarboxylic acid
(1).
obtained in PyBOP and POCl3/DMF reactional conditions,
respectively, was made by single crystal XꢀRay diffractometry (Fig.
2). Details for hydrogen bonding geometries as well as the Upon completion a new compound was detected by TLC analytical
discussion of molecular geometries are given in Supplementary control and it was identified as (Z)ꢀ1ꢀ(2ꢀhydroxyphenyl)ꢀ3ꢀ
(phenylamino)propꢀ2ꢀenꢀ1ꢀone (Scheme 2). Its formation is in
Information. In both cases the XꢀRay structural analysis corroborate
the spectroscopic data.
12, 13
accordance with previously published data.
Hence, it can be
concluded that in these conditions the amidation of chromoneꢀ3ꢀ
carboxylic acid (1) does not take place. The formation of chromaneꢀ
2,4ꢀdione nucleus must then be directly connected to the PyBOP
activation process and the presence of aniline, or its derivatives.
From the experiments performed with chromoneꢀ3ꢀcarboxylic acid
(1) in the presence of PyBOP and DIPEA, and in the absence of the
aromatic amines, only the benzotriazolyl ester derivative (compound
B, Scheme 3) resultant from the activation of –COOH function was
detected. Up to now, it seems that a nucleophilic attack by the amine
at the C2 position occurs after in situ formation of the ester
intermediate, causing ringꢀopening and ring closing processes that at
the end lead to the formation of chromaneꢀ2,4ꢀdione derivatives.
Moreover, two main compounds were identified, either in the
presence or absence of the nonꢀnucleophilic base DIPEA : (Z)ꢀ1ꢀ(2ꢀ
hydroxyphenyl)ꢀ3ꢀ(phenylamino)propꢀ2ꢀenꢀ1ꢀone and the chromaneꢀ
(2)
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2,4ꢀdione based derivatives (Schemes 1 and 3). Chromoneꢀ3ꢀ each chromaneꢀ2,4ꢀdione/ chromaneꢀ3ꢀcarboxamide pair (see Table
carboxamide based compounds were only detected in vestigial 1) bearing the same substitution pattern, the chromoneꢀ3ꢀ
amounts. As the same process does not occur in the same conditions carboxamide counterparts generally display a higher activity towards
with chromoneꢀ2ꢀcarboxylic acid, one must conclude that the C2 hMAOꢀB, except for compounds with the 4’ꢀhydroxyaryl substituent
position of chromoneꢀ3ꢀcarboxylic acid (1) is more activated (Scheme 1, compounds 4 and 5).
towards nitrogen nucleophiles. It is worth noting that when
chromoneꢀ2ꢀcarboxylic acid was used as starting material only Table 1. MAO inhibitory activities of chromone carboxamides and
chromoneꢀ2ꢀcarboxamides were obtained in both methods. 13, 14
reference inhibitors.
Compound
IC50 (nM)a
S.I.a
hMAOꢀA
hMAOꢀB
b
b
b
267.9 ± 7.8
> 37
> 140
>202
2
3
4
5
70.7 ± 1.7
49.5 ± 1.6
1382.4 ±
2.5
b
> 7.2
b
b
b
b
64.9 ± 1.4
2.9 ± 1.2
42.3 ± 0.3
8.0 ± 3.2
> 154
> 3448
> 236
6
7
8
9
> 1250
(R)ꢀ(−)ꢀ
Deprenyl
Clorgyline
68734 ± 4.2 19.44 ± 0.7
6.27 ± 0.3
3536
63410 ± 1.1 0.000099
a All IC50 values are the mean ± SD from three experiments. SI:
hMAOꢀB selectivity index = IC50(hMAOꢀA)/IC50(hMAOꢀB);
b Inactive at 10 µM (highest concentration tested).
These results point out that the presence of the amide spacer between
the heterocyclic and the exocyclic ring is of utmost importance for
MAOꢀB inhibitory activity. The presence of a pꢀsubstituent in the
exocyclic ring (Cl or CH3) favors the activity and selectivity, with
the exception for the 4’ꢀhydroxyaryl chromone (compound 5). In
fact, compounds 7 and 9 are potent and selective IMAOꢀB, being
more potent than the standard inhibitor deprenyl 6.7ꢀ and 2.4ꢀfold,
respectively. The differences in IMAOꢀB activity found for 2 and 3
sparked a closer look into the geometrical differences between them,
as determined by single crystal XꢀRay diffractometry, which are
schematically depicted in Fig. 3.
Scheme 3. Mechanistic proposal for the formation of chromaneꢀ2,4ꢀ
dione and its derivatives from chromoneꢀ3ꢀcarboxylic acid (1).
After the chemical findings, the next step involved MAOꢀB
inhibition studies of chromaneꢀ2,4ꢀdione derivatives (Scheme 1,
compounds 2, 4, 6 and 8) and chromoneꢀ3ꢀcarboxamide derivatives
6ꢀ8
(Scheme 1, compounds 3, 5, 7 and 9). Our previous studies point
out that, in general, chromoneꢀ3ꢀcarboxamide derivatives have
superior inhibitory activity towards MAOꢀB than the chromoneꢀ2ꢀ
carboxamide counterparts. The inhibitory activity of the compounds
on human MAO (hMAOꢀA and hMAOꢀB isoforms) was evaluated
by measuring their effect on the production of hydrogen peroxide
(H2O2) from pꢀtyramine, which was detected using 10ꢀacetylꢀ3,7ꢀ
dihydroxyphenoxazine (Amplex Red reagent), a nonꢀfluorescent and
highly sensitive probe that reacts with H2O2 in the presence of
horseradish peroxidase to produce a fluorescent product, resorufin
(see Supplementary Information). The data of the hMAO in vitro
studies, inhibitory potencies and selectivity indexes (SI) for the
compounds under study and reference compounds is shown in Table
1. The results show that regardless of the functionalization of the
dihydropyranꢀ2,4ꢀdione and pyrone ring the majority of the
compounds display potent and selective hMAOꢀB inhibitory activity
(micromolar to nanomolar range). The presence of C3ꢀsubstituents
in the scaffold seems to be important for MAOꢀB inhibition. For
(2)
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DOI: 10.1039J/Co4uCrCn0a8l7N98aDme
†
Electronic supplementary information (ESI) available: Experimental
chemical and biological details, characterization data and Xꢀray data
collection, structure solution and refinement.
The authors thank the Foundation for Science and Technology (FCT) of
Portugal
(PEstꢀC/QUI/UI0081/2013).
F.
Cagide
(SFRH/BPD/74491/2010), T. Silva (SFRH/BD/79671/2011), J. Reis
(SFRH/BD/96033/2013) and A. Gaspar (SFRH/BPD/93331/2013) grants
are supported by FCT. Thanks are also due to the staff at the National
Crystallographic Service, University of Southampton for the data
collection, help and advice. 16
(3)
Figure 3. Schematic diagram of the geometrical differences of 3ꢀ
(phenylamino)methylene)chromaneꢀ2,4ꢀdione (2) and Nꢀphenylꢀ4ꢀ
oxoꢀ4Hꢀchromeneꢀ3ꢀcarboxamide (3).
1
2
J. Reis, I. Encarnação, A. Gaspar, A. Morales, N. Milhazes
and F. Borges, Curr. Top. Med. Chem., 2012, 12, 2116ꢀ2130.
A.M. Helguera, G. PerezꢀMachado, M.N. Cordeiro and F.
Borges, Mini Rev. Med. Chem., 2012, 12, 907ꢀ919.
T. Thomas, Neurobiol. Aging, 2000, 21, 343ꢀ348.
A. Gaspar, M.J. Matos, J. Garrido, E. Uriarte and F. Borges,
Chem. Rev., 2014, 114, 4960ꢀ4992.
S. Alcaro, A. Gaspar, F. Ortuso, N. Milhazes, F. Orallo, E.
Apart from the slight differences found in molecular conformation
with respect to planarity, see supplementary information for values ,
both compounds 3ꢀ(phenylamino)methylene)chromaneꢀ2,4ꢀdione (2)
and Nꢀphenylꢀ4ꢀoxoꢀ4Hꢀchromeneꢀ3ꢀcarboxamide (3) present a
framework consisting of 5 similiar rings: A and B of the benzopyran
3
4
moiety, the E benzyl ring and two extra pseudoꢀcyclic rings: a sixꢀ
5
6
7
8
9
15
membered S(6) ring C and a fiveꢀmember S(5) ring
identified as
Uriarte, M. Yáñez and F. Borges, Bioorg. Med. Chem. Lett.
2010, 20, 2709ꢀ2712.
,
D, both resulting from the formation of intramolecular hydrogen
interactions. Moreover, in Nꢀphenylꢀ4ꢀoxoꢀ4Hꢀchromeneꢀ3ꢀ
carboxamide (3) an additional sixꢀmembered S(6) ring 15, identified
as F was projected. The geometric molecular differences may help in
the understanding of the enzymeꢀinhibitor interactions taking place
within the substrate pocket and they seem to be fundamentally
related with: (i) the relative positions of the rings; compound 2
presents a framework of three fused “anthracene like” 6 member
rings while compound 3 displays a skeleton of three fused
“phenantrene like” 6 member rings: (ii) the relative positions of the
hydrogen bonding donors and acceptors; the 4ꢀketo oxygen atom of
the pyran type ring is identically positioned in both molecules but
the nitrogen donors and the other carbonyl acceptors are placed
differently; iii) the position of the phenyl substituent differs as a
consequence of the assumed configurations around the enamine and
amide spacers between the aromatic residues.
A. Gaspar, J. Reis, A. Fonseca, N. Milhazes, D. Viña, E.
Uriarte and F. Borges, Bioorg Med Chem Lett, 2011, 21, 707ꢀ
709.
A. Gaspar, T. Silva, M. Yáñez, D. Vina, F. Orallo, F. Ortuso,
E. Uriarte, S. Alcaro and F. Borges, J. Med. Chem., 2011, 54
,
5165ꢀ5173.
A. Gaspar, F. Teixeira, E. Uriarte, N. Milhazes, A. Melo,
M.N. Cordeiro, F. Ortuso, S. Alcaro and F. Borges,
ChemMedChem, 2011, 6, 628ꢀ632.
C.A.G.N. Montalbetti and V. Falque, Tetrahedron, 2005, 61
,
10827ꢀ10852.
10 E. Valeur and M. Bradley, Chem Soc Rev, 2009, 38, 606ꢀ631.
11 M.M. Joullie and K.M. Lassen, Arkivoc, 2010, viii, 189ꢀ250.
12 M.A. Ibrahim, Tetrahedron, 2009. 65, 7687ꢀ7690.
13 M.A. Ibrahim, Arkivoc, 2008, xvii, 192ꢀ204.
14 F. Cagide, J. Reis, A. Gaspar and F. Borges, Tetrahedron
Lett., 2011, 52, 6446ꢀ6449.
15 A. Gaspar, F. Cagide, E. Quezada, J. Reis, E. Uriarte and F.
Borges, Magn. Reson. Chem., 2013, 51, 251ꢀ254.
16 S.J. Coles and P.A. Gale, Chem. Sci., 2012, 3, 683ꢀ689
Conclusions
In summary, chromaneꢀ2,4ꢀdione and chromoneꢀ3ꢀcarboxamide
derivatives were obtained from chromoneꢀ3ꢀcarboxylic acid by a
process that is dependent on the nature of the activating reagents and
not reliant on the type of aniline used in the study. Although
chromaneꢀ2,4ꢀdione and chromaneꢀ3ꢀcarboxamide derivatives are
potent
and
selective
IMAOꢀB,
chromoneꢀ3ꢀcarboxamide
counterparts generally display higher activity The geometrical
differences found between the two chemical frameworks can justify
the biological data as they can hinder or assist the ligandꢀenzyme
interactions with the enzyme pocket.
Notes and references
CIQUP/Departamento de Química
a
e Bioquímica, Faculdade de
Ciências, Universidade do Porto, 4169ꢀ007Porto, Portugal.
b
FPꢀENASꢀFaculdade de Ciências de Saúde, Escola Superior de Saúde
da UFP, Universidade Fernando Pessoa, Pꢀ4200ꢀ150 Porto, Portugal
c
Department of Chemistry, University of Aberdeen, Meston Walk, Old
Aberdeen, AB24 3UE, Scotland
¥ These authors contributed equally to this work
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