Indazole-and indole-5-carboxamides: Selective and reversible monoamine oxidase B inhibitors with subnanomolar potency

Article abstract of DOI:10.1021/jm500729a

Indazole-and indole-carboxamides were discovered as highly potent, selective, competitive, and reversible inhibitors of monoamine oxidase B (MAO-B). The compounds are easily accessible by standard synthetic procedures with high overall yields. The most potent derivatives were N-(3,4- dichlorophenyl)-1-methyl-1H-indazole-5-carboxamide (38a, PSB-1491, IC ₅₀ human MAO-B 0.386 nM, >25000-fold selective versus MAO-A) and N-(3,4-dichlorophenyl)-1H-indole-5-carboxamide (53, PSB-1410, IC₅₀ human MAO-B 0.227 nM, >5700-fold selective versus MAO-A). Replacement of the carboxamide linker with a methanimine spacer leading to (E)-N-(3,4- dichlorophenyl)-1-(1H-indazol-5-yl)methanimine (58) represents a further novel class of highly potent and selective MAO-B inhibitors (IC₅₀ human MAO-B 0.612 nM, >16000-fold selective versus MAO-A). In N-(3,4- difluorophenyl-1H-indazole-5-carboxamide (30, PSB-1434, IC₅₀ human MAO-B 1.59 nM, selectivity versus MAO-A >6000-fold), high potency and selectivity are optimally combined with superior physicochemical properties. Computational docking studies provided insights into the inhibitors' interaction with the enzyme binding site and a rationale for their high potency despite their small molecular size.

Full text of DOI:10.1021/jm500729a

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Article
Indazole- and Indole-5-carboxamides: Selective and Reversible
Monoamine Oxidase B Inhibitors with Subnanomolar Potency
Nikolay Tzvetkov Tzvetkov, Sonja Hinz, Petra Küppers, Marcus Gastreich, and Christa E Müller
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm500729a • Publication Date (Web): 23 Jun 2014
Downloaded from http://pubs.acs.org on July 8, 2014
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Indazoleꢀ and Indoleꢀ5ꢀcarboxamides: Selective and Reversible
Monoamine Oxidase B Inhibitors with Subnanomolar Potency
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Nikolay T. Tzvetkov, * Sonja Hinz, Petra Küppers, Marcus Gastreich, and Christa E. Müller *
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PharmaCenter Bonn, University of Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, Bonn,
Germany
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BioSolveIT GmbH, An der Ziegelei 79, 53757 St. Augustin, Germany
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To whom correspondence should be addressed: Nikolay Tzvetkov Tzvetkov and Christa E. Müller,
PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, An der Immenburg 4, Dꢀ
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3121 Bonn, Germany. Phone: +49ꢀ179ꢀ5284358 (N.T.T.); +49ꢀ228ꢀ73ꢀ2301 (C.E.M.). Fax: +49ꢀ228ꢀ
3ꢀ2567. Eꢀmail: ntzvetkov@uniꢀbonn.de (N.T.T.); christa.mueller@uniꢀbonn.de (C.E.M.).
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ABSTRACT
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Indazoleꢀ and indoleꢀcarboxamides were discovered as highly potent, selective, competitive and
reversible inhibitors of monoamine oxidase B (MAOꢀB). The compounds are easily accessible by
standard synthetic procedures with high overall yields. The most potent derivatives were Nꢀ(3,4ꢀ
dichlorophenyl)ꢀ1ꢀmethylꢀ1Hꢀindazoleꢀ5ꢀcarboxamide (38a, PSBꢀ1491, IC human MAOꢀB 0.386 nM,
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>25000ꢀfold selective versus MAOꢀA) and Nꢀ(3,4ꢀdichlorophenyl)ꢀ1Hꢀindoleꢀ5ꢀcarboxamide (53, PSBꢀ
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410, IC50 human MAOꢀB 0.227 nM, >5700ꢀfold selective versus MAOꢀA). Replacement of the
carboxamide linker with a methanimine spacer leading to (E)ꢀNꢀ(3,4ꢀdichlorophenyl)ꢀ1ꢀ(1Hꢀindazolꢀ5ꢀ
yl)methanimine (58) represents a further novel class of highly potent and selective MAOꢀB inhibitors
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IC human MAOꢀB 0.612 nM, >16000ꢀfold selective versus MAOꢀA). In Nꢀ(3,4ꢀdifluorophenylꢀ1Hꢀ
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indazoleꢀ5ꢀcarboxamide (30, PSBꢀ1434, IC50 human MAOꢀB 1.59 nM, selectivity versus MAOꢀA
6000ꢀfold) high potency and selectivity are optimally combined with superior physicochemical
>
properties. Computational docking studies provided insights into the inhibitors’ interaction with the
enzyme binding site and a rationale for their high potency despite their small molecular size.
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KEYWORDS: Alzheimer’s disease, carboxamides, docking studies, indazoles, indoles, reversible
inhibitors, molecular modeling, monoamine oxidase A and B, neuroprotection, neurodegenerative
diseases, Parkinson’s disease, physicochemical properties, species differences, structureꢀactivity
relationships, synthesis
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INTRODUCTION
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Monoamine oxidases (МАО, EC 1.4.3.4) are flavin adenine dinucleotide (FAD) containing enzymes
localized on the mitochondrial outer membrane, which catalyze the oxidative deamination of biogenic
amines and monoamine neurotransmitters. Two isoforms of MAO are present in most mammalian
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tissues, MAOꢀA and MAOꢀB, distinguished by their substrate and inhibitor selectivity. Epinephrine
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(adrenaline), norepinephrine (noradrenaline), dopamine (DA), tyramine and tryptamine are substrates
for both isoforms. MAOꢀA displays higher affinity for the substrates norepinephrine and serotonin
(5ꢀHT) than MAOꢀB and is inhibited by low concentrations of clorgyline, whereas MAOꢀB exhibits
higher affinity towards phenylethylamine (PEA) and benzylamine, and is potently inhibited by of
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selegiline. The reaction catalyzed by MAO results in the production of hydrogen peroxide (H O ) and
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other reactive oxygen species (ROS), which may contribute to oxidative stress and cell damage. The
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protein sequences of MAOꢀA and MAOꢀB are ~70% identical. Both MAO isoforms show regional
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differences in enzyme activity and distribution in the human brain. Highest MAO activity is observed
in the basal ganglia (striatum) and hypothalamus, whereas the cerebellum and neocortex show low
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levels of MAO activity. Serotonergic neurons and astrocytes contain predominantly MAOꢀB, while the
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MAOꢀA isoform is mainly located in catecholaminergic neurons. The activity and the expression levels
of MAOꢀB in the human brain, but not those of MAOꢀA, increase with aging and may be associated
with the loss of dopaminergic neurons in the substantia nigra, where MAOꢀB is the main form in glial
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cells. Increased MAOꢀB activity in the substantia nigra is thus observed in patients with Parkinson’s
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diseases (PD). The relationship between oxidative stress and progressive neuronal impairment
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indicates that inhibition of MAOꢀB activity may have neuroprotective effects.
Selective irreversible and reversible MAOꢀA inhibitors are used in the treatment of depression and
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anxiety disorders. Selective MAOꢀB inhibitors are currently applied for the therapy of PD, mostly in
combination with the dopamine prodrug levodopa, to reduce the metabolic degradation of dopamine and
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increase its halfꢀlife. Due to their potential neuroprotective effects, MAOꢀB inhibitors may be useful
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for the treatment of other neurodegenerative diseases as well including Alzheimer´s disease (AD).
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treatment of PD and AD.
Examples for this new strategy include dualꢀacting acetylcholinesterase
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(AChE) and MAO inhibitors for AD,
dualꢀacting A2A adenosine receptor (A2A AR) antagonists and
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MAOꢀB inhibitors for PD,
and tripleꢀtarget drugs acting as A and A AR antagonist in addition to
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inhibition of MAOꢀB for the treatment of PD and AD.
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Several selective MAOꢀB inhibitors, including the irreversible inhibitors selegiline (1) and rasagiline
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2) as well as the reversible inhibitor safinamide (3) , are currently in clinical use for the treatment of
PD (for structures, see Figure 1). Selegiline (1) is a potent and selective “suicide type” irreversible
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MAOꢀB inhibitor (human MAOꢀB, IC50 = 6.79 nM; human MAOꢀA, IC50 = 1700 nM)
used in
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combination with levodopa therapy. One major drawback of 1 are sideꢀeffects evoked by its
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amphetamine metabolites.
Figure 1. Structures of irreversible (Irr) and reversible (Rev) MAOꢀB inhibitors in clinical use.
The irreversible MAOꢀB inhibitor rasagiline (2, human brain MAOꢀB, IC = 14.0 nM; human brain
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MAOꢀA, IC50 = 710 nM) is not metabolized to amphetamine derivatives and is used for both,
monotherapy in early PD and as adjunctive therapy in lateꢀstage patients with PD experiencing motor
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fluctuations.
However, irreversible MAOꢀB inhibitors may show safety issues and pharmacological
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Therefore, selective and reversible inhibition of MAO is believed to be advantageous. For example, the
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reversible MAOꢀA inhibitor moclobemide is successfully used to treat depression and anxiety.
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Reversible MAOꢀB inhibitors are currently being developed. Safinamide (3) is such a compound,
which is being evaluated in advanced clinical trials as an addꢀon therapy to dopamine agonists or to
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levodopa in PD patients with motor fluctuations. Besides MAOꢀB inhibition (human brain MAOꢀB,
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IC50 = 9.0 nM; human brain MAOꢀA, IC50 = 45 ꢀM) it shows additional mechanisms of action
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including inhibition of DA reuptake, inhibition of excessive glutamate release , and sodium channel
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inhibition.
A wide range of MAOꢀB inhibitors with common structural features has been developed to date (for
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examples, see Figure 2).
compounds such as the 2,5ꢀdisubstituted indole 4, 5ꢀnitroindazole (5), 7ꢀ[(3ꢀchlorobenzyloxy)]ꢀ4ꢀ
(methylamino)methyl]coumarin (6), isatine (7) and its C5ꢀsubstituted analogues (E)ꢀ5ꢀstyrylisatin (8)
and 5ꢀ(4ꢀphenylbutyl)isatin (9). The 2ꢀpropargylamineꢀsubstituted compound 4 is an irreversible
These include small monoꢀ or disubstituted heterobicyclic
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MAOꢀB inhibitor that is similarly potent as selegiline (1). MAOꢀB inhibitor 4 was found to exhibit
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neuroprotective effects in several in vivo models of PD.
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Figure 2. Structures and potencies of previously described MAOꢀB inhibitors.
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Compound 5 was described as the most potent MAOꢀB inhibitor within the class of nitroindazoles.
Recently, aminocoumarin derivative 6 has been identified as a promising clinical candidate with high
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MAOꢀB inhibitory activity and suitable pharmacokinetic properties. Another small molecule, isatin
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(
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more potent, reversible MAOꢀB inhibitors.
Due to the promising pharmacological properties combined with only minor side effects of selective,
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reversible MAOꢀB inhibitors, we aimed at developing such inhibitors with improved properties. In the
present study, novel classes of most potent irreversible MAOꢀB inhibitors were discovered, namely
indazoleꢀ5ꢀcarboxamides (designated class I), indoleꢀ5ꢀcarboxamides (class II) and (indazolꢀ5ꢀ
yl)methanimine derivatives (class III). The new compounds were evaluated at rat and human MAO A
and B, and optimized in order to improve their MAOꢀB affinity and selectivity, as well as their
physicochemical and drugꢀlike properties. Computational studies were performed to understand their
binding modes and to explain their exceedingly high affinities.
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RESULTS AND DISCUSSION
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Compound Design. Our design of novel classes of MAOꢀB inhibitors was based on the general formula
I containing a heterobicyclic ring system linked to a phenyl moiety as displayed in Figure 3 (L = linker,
R = H or halogen, and Het = heterocycle). The pharmacophore in I labeled in red incorporates the key
structural features found in all previously described MAOꢀB inhibitors 4–9 (cf. Figure 2). For
simplification, we divided I into two scaffold fragments representing the common structural motifs
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(building blocks) used for the construction of these MAOꢀB inhibitors.
Figure 3. Rational design strategy to obtain MAOꢀB inhibitor 10, and further structural optimization to
obtain class I MAOꢀB inhibitor 15.
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We prepared and evaluated only 6 compounds (10-15) in our rational design strategy to obtain a
compound with the desired high, subnanomolar potency and MAOꢀB selectivity. In order to design the
initial compound 10, we studied the core structural characteristics of the known MAOꢀB inhibitors
considering their potential for further scaffold variation, novelty and accessibility (see Figure 2).
Structureꢀactivity relationship (SAR) examination for MAOꢀB potency and selectivity showed that
electronꢀrich spacers such as a double bond, an ether, or a methylamine group were well tolerated by
MAOꢀB, while C3ꢀ to C4ꢀalkyl linkers led to less potent and less selective compounds. Since linkers
like C1ꢀ to C4ꢀalk(en)yl (8 and 9) or ethers (3, 4 and 6) are already wellꢀestablished in a number of
structures with MAOꢀB activity, we decided to replace these by an amide group in 10. Furthermore, C5ꢀ
substitution in several heterobicyclic motifs has been observed to be beneficial for potent, reversible
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MAOꢀB inhibitors. Except for the coumarine derivative 6, which is substituted at position 7, the
MAOꢀB inhibitors 4, 5, 8, and 9 are C5ꢀsubstituted (see Figure 2). Based on previous studies targeted
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towards dual A2A adenosine receptor antagonists / MAOꢀB inhibitors, we conjugated a 5ꢀchloroꢀ
substituted morpholinopyridine motif (blue box) at the C5 position of an indazole moiety (gray box) to
obtain compound 10. The 5ꢀchloroꢀ6ꢀmorpholinopyridine was selected as a bioisosteric replacement of
halogenꢀsubstituted phenyl residues due to its polarity and favorable physicochemical properties.
Similar to the benzyloxy group in the potent MAOꢀB inhibitors safinamide (3) and compound 4, the
bulky moiety in 10 may extend through the substrate cavity into the entrance cavity of the bipartite
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active site of human MAOꢀB. In fact, the first compound 10 designed through a rational design
strategy provided a moderate inhibitory activity at human MAOꢀB in the high nanomolar range (IC50
220 nM) being 45ꢀfold selective for MAOꢀB over MAOꢀA. These results encouraged further
exploration within the series of indazoleꢀcontaining compounds related to 10. In the next step, we
performed a systematic modification of 10 by introducing a hydrophilic 1ꢀmethylꢀpyridone moiety
(modification A) or a more lipophilic 2ꢀchloroꢀ6ꢀfluorophenyl ring (modification B), respectively, to
obtain compounds 11 and 12 (Figure 3). While 11 showed no activity at all, compound 12 exhibited
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lower potency than the parent 10 with a micromolar IC50 value of 3.30 ꢀM at human MAOꢀB.
Compound 12 was subsequently modified with 2,3ꢀ and 3,4ꢀdichloro substituents at the phenyl ring to
obtain 13 and 14, respectively. The chlorine substituent at the aryl C3 position was kept constant. Both
compounds showed progressive improvement of MAOꢀB inhibition from micromolar (13, human
MAOꢀB, IC 2.59 ꢀM) to low nanomolar potency (14, human MAOꢀB, IC 12.4 nM). The choice of
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5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
50
the 3,4ꢀdichlorophenylꢀsubstitution pattern appeared to be essential for obtaining very high MAOꢀB
inhibitory activity. Finally, we obtained a further dramatic increase in MAOꢀB inhibitory potency by
inverting the amide linker leading to compound 15. Thus, we developed indazoleꢀ5ꢀcarboxamides as a
new structural class of extraordinarily potent MAOꢀB inhibitors (designated class I). Nꢀ(3,4ꢀ
dichlorophenyl)ꢀ1Hꢀindazoleꢀ5ꢀcarboxamide (15) was evaluated at rat and human MAOꢀA and B: it was
identified as a highly potent inhibitor (20ꢀfold more active than 14) with remarkable selectivity for the
MAOꢀBꢀisoform (human MAOꢀB, IC 0.586 nM; human, MAOꢀA, IC >10000 nM) surpassing the
5
0
50
activity of all recently developed MAOꢀBꢀselective inhibitors. The indazolꢀ5ꢀcarboxamide scaffold
subsequently served as a starting point for the design, synthesis and biological evaluation of a number of
compounds, many of them with MAOꢀB inhibitory activity in the subnanomolar range and significantly
improved physicochemical properties. We also prepared two additional classes of MAOꢀB inhibitors
differing from 15 either by replacement of the 5ꢀsubstituted indazole by a 5ꢀsubstituted indole
(designated class II), or by exchange of the amide connection by a methanimine linker (class III).
3
6
Chemistry. All new compounds of the present study were prepared by amide coupling reactions,
3
7
38
Nꢀalkylation and iminoalkylation reactions using a variety of reagents and conditions as illustrated in
Schemes 1ꢀ5. Amide coupling was performed by reaction of the differently substituted carboxylic acids
1
8-20, 21a-c, 22, 39, and 50-52 with substituted amines 17, 23a-k, 25, or aminopyridine 24,
respectively. With the exception of carboxylic acids 18 and 19, all building blocks used for the
syntheses of the final products were commercially available. Amide coupling was conducted in the
presence of Hünig’s base (N,Nꢀdiisopropylethylamine, DIPEA), using the condensation reagent Oꢀ
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Journal of Medicinal Chemistry
(
benzotriazolꢀ1ꢀyl)ꢀN,N,N',N'ꢀtetramethyluronium tetrafluoroborate (TBTU) in acetonitrile or,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
alternatively, by applying 1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide hydrochloride (EDCꢀHCl) as
3
4
a coupling reagent in methanol. In general, amide coupling reactions were performed at room
temperature for several hours. The conversion of the staring materials was monitored by thinꢀlayer
chromatography (TLC) or by high performance liquid chromatography (HPLC) coupled to an ultraviolet
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(UV) detector and to electrospray ionisation mass spectrometry (ESIꢀMS) analysis. Products 10-14 and
36,39
1
6 were obtained as shown in Scheme 1. Compound 14 had previously been described,
but in the
present study we used different reaction conditions to obtain 14 in higher yields. The carboxylic acids
8 and 19 were prepared by twoꢀ or threeꢀstep reaction procedures according to published procedures
for details see Supporting Information). A methylene group in 16 was introduced in order to study an
1
(
extension of the spacer between the 3,4ꢀdichloroꢀphenyl ring and the indazole moiety.
a
Scheme 1. Synthesis of indazolꢀ5ꢀylꢀamides 10-14 and 16
a
39
Reagents and conditions: (i) for 10-14: carboxylic acid 18, 19 or 21a-c (1.0 equiv.), 5ꢀaminoꢀ1Hꢀ
indazole (17, 1.0 equiv.), TBTU/DIPEA (1.2 equiv.), acetonitrile, RT, 3ꢀ72 h, yield 23–73%; (ii) for 16:
2ꢀ(3,4ꢀdichlorophenyl)acetic acid (20, 1.0 equiv.), 5ꢀaminoꢀ1Hꢀindazole (17, 1.0 equiv.), EDCꢀHCl (1.1
equiv.), methanol, RT, 3ꢀ4 h, yield 56% (16).
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The preparation of indazoleꢀ5ꢀcarboxamides 15, 26-37 and 38a-b is depicted in Scheme 2. The
compounds were prepared by amide coupling reaction of commercially available 1Hꢀindazoleꢀ5ꢀ
carboxylic acid (22) with differently substituted anilines and related amines (23a-k, 24, 25). In order to
investigate the impact of the substitution pattern of the phenyl ring on biological activity, we introduced
a broad variety of substituents mainly in the 3ꢀ and 4ꢀposition including halogen atoms, methoxy and
hydroxyl functions. To introduce structural diversity in position N1 or N2 of the indazole moiety, 15
was alkylated with methyl iodide yielding a 3:1 mixture of N1ꢀ/N2ꢀmethylated products 38a and 38b
(Scheme 2). The reaction occurred in the presence of potassium carbonate in N,Nꢀdimethylformamide
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
7
(
DMF) at room temperature and the reaction time was optimized by monitoring of the conversion. The
mixture of 38a and 38b was separated by column chromatography. The structure determination of 38a
and 38b was carried out by heteronuclear multiple bond correlation (HMBC) NMR in combination with
1
13
H and C NMR spectroscopy.
a
Scheme 2. Synthesis of indazoleꢀ5ꢀcarboxamides 15, 26-37 and 38a-b
a
Reagents and conditions: (i) for 15, 32 and 34: 1Hꢀindazoleꢀ5ꢀcarboxylic acid (22, 1.0 equiv.),
differently substituted anilines 23a,h,j (1.0 equiv.), TBTU/DIPEA (1.2 equiv.), acetonitrile, RT, 3ꢀ72 h,
yield 11–30%; (ii) for 15, 26-31, 33, and 35-37: 1Hꢀindazoleꢀ5ꢀcarboxylic acid (22, 1.0 equiv.),
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Journal of Medicinal Chemistry
differently substituted anilines 23a-g,i,k, 24 and 25 (1.0 equiv.), EDCꢀHCl (1.1 equiv.), methanol, RT,
ꢀ4 h, yield 18ꢀ67%; (iii) Nꢀ(3,4ꢀdichlorophenyl)ꢀ1Hꢀindazoleꢀ5ꢀcarboxamide (15, 1.0 equiv.), MeI
(1.3–2.0 equiv), K CO (1.3 equiv), DMF, RT, 16–20 h, yield 47% (38a) and 9% (38b).
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
2
3
In order to increase the compounds’ polarity and waterꢀsolubility, we introduced a bioisosteric
replacement for the 3,4ꢀdichloroꢀphenyl residue, namely a 3,4ꢀdichloropyridine moiety (compound 36).
An extension of the spacer between the indazole core and the substituted phenyl ring was introduced in
37 by coupling of 22 with the corresponding 3,4ꢀdichlorobenzylamine (25).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Indazoleꢀ6ꢀcarboxamides 40-49, which represent 6ꢀsubstituted analogues or isomers of the indazoleꢀ5ꢀ
carboxamides (15, 27-35) described above were obtained by coupling of 1Hꢀindazoleꢀ6ꢀcarboxylic acid
(39) with anilines 23a,c-k as shown in Scheme 3. This set of compounds was prepared in order to
investigate the role of the indazole nitrogen atoms for MAOꢀB inhibition, in particular, the supposed
role (and position) of the NH as a hydrogen bond donor in protein binding.
a
Scheme 3. Synthesis of indazoleꢀ6ꢀcarboxamides 40-49
a
Reagents and conditions: (i) 1Hꢀindazoleꢀ6ꢀcarboxylic acid (39, 1.0 equiv.), differently substituted
anilines 23a,c-k (1.0 equiv.), EDCꢀHCl (1.1 equiv.), methanol, RT, 3ꢀ4 h, yield 22–71%.
Replacement of the indazole ring of 15 by an indole, an imidazo[1,2ꢀa]pyridine, or a
[1,2,4]triazoloꢀ
[
4,3ꢀa]pyridine moiety led to compounds 53, 55 and 56 (Scheme 4). These were synthesized in order to
create diversity of the heterobicyclic core via variation of the electronic properties of the ring system
while keeping the 3,4ꢀdichlorophenyl substituent of lead structure 15 constant. Furthermore, alkylation
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Page 14 of 81
of 53 was performed by Nꢀalkylation with methyl iodide under mild reaction conditions yielding 54
Scheme 4).
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
Scheme 4. Synthesis of indoleꢀ5ꢀcarboxamides 53 and 54 and Nꢀ(3,4ꢀdichlorophenyl)carboxamide
a
derivatives 55 and 56
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Reagents and conditions: (i) carboxylic acids 50-52 (1.0 equiv.), 3,4ꢀdichloroaniline (23a, 1.0 equiv.),
EDCꢀHCl (1.1 equiv.), methanol, RT, 3ꢀ4 h, yield 17–36%; (ii) Nꢀ(3,4ꢀdichlorophenyl)ꢀ1Hꢀindoleꢀ5ꢀ
carboxamide (53, 1.0 equiv.), MeI (1.3–2.0 equiv), K CO (1.3 equiv), DMF, RT, 16–20 h, yield 68%.
2
3
Finally, the amide group was replaced by an imine linker. For the preparation of 58 and 59, aldehydes
7a,b were reacted with 3,4ꢀdichloroaniline 23a in the presence of a catalytic amount of acetic acid in
5
4
0
ethanol under reflux (Scheme 5). The chemical stability of product 59 was investigated by LC/ESIꢀMS
and compared to that of amide 54. For this purpose we analyzed stock solutions of the compounds in
DMSO (10 mM), which had been prepared for biological testing and kept for 70 days at room
temperature (for details see Supporting Information, Figures S3, S4 and Table S2). We observed that the
imine 59 was relatively stable and showed only moderate degradation under these conditions (15%
hydrolysis), while the amide 54 was found to be completely stable. Imines 58 and 59 will offer further
opportunities for modification to obtain more stable compounds: e.g. in analogy to the preparation of
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Journal of Medicinal Chemistry
4
0
safinamide (3), the imine double bond may be reduced to an aminoalkyl group. Molecular mechanics
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
calculations showed that the formation of the Eꢀisomer was favoured over the Zꢀdiastereomer (for
1
13
details, see Supporting Information, Figure S5). NMR analyses ( H and C) confirmed the sole
formation of the Eꢀisomer. However, the possibility of an E/Zꢀisomerization of 58, e.g. after exposing it
4
1
to daylight in dilute solution, cannot be excluded.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Scheme 5. Synthesis of 3,4ꢀdichlorophenylꢀsubstituted (1Hꢀindazolꢀ5ꢀyl)methanimines 58 and 59
Cl
O
i
H
N
Cl
Cl
Cl
R
R
N
N
N
N
H N
2
57a R = H
58 R = H
59 R = Me
23a
57b R = Me
a
Reagents and conditions: (i) 1Hꢀindazoleꢀ or 1ꢀmethylꢀ1Hꢀindazoleꢀ5ꢀcarboxadehyde (57a,b, 1.0
equiv.), 3,4ꢀdichloroaniline (23a, 1.0 equiv.), acetic acid (0.2 equiv.), ethanol, reflux, 1ꢀ24 h, yields:
9% (58) and 90% (59).
8
Our synthetic strategy to obtain novel classes of MAOꢀB inhibitors provides several advantages. These
include easy accessibility of the final products by only one or two steps using commercially available
starting materials. The procedures allow the possibility for broad structural variation in the last step, and
the preparation of compound libraries for fast analysis of SARs. Moreover, the procedures will enable
simple scaleꢀup from milligram amounts to large, multigram or even kilogram quantities.
The new compounds were isolated by column chromatography and subsequently reꢀcrystallized to
1
13
obtain pure products. All final products were fully characterized by NMR spectroscopy ( H and C) and
mass spectrometry (LC/ESIꢀMS) and their structures were confirmed. The purity of all compounds was
determined by HPLCꢀUV to be at least 95% (see Experimental Section and Supporting Information).
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Monoamine Oxidase Inhibition Studies. The compounds were investigated for inhibition of human
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
and rat MAOꢀA and MAOꢀB using recombinant enzymes (human) expressed in baculovirusꢀinfected
4
2
insect cells, and mitochondriaꢀenriched rat liver fractions as sources for the rat MAO isoforms,
respectively. Enzyme inhibition assays of reference and test compounds at human MAOꢀA and MAOꢀB
were performed by a fluorescenceꢀbased assay with the substrate pꢀtyramine measuring the production
of hydrogen peroxide formed as a byꢀproduct of the enzymatic deamination reaction (also see
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
43
Experimental Section and Supporting Information). The inhibitors clorgyline for MAOꢀA and
selegiline for MAOꢀB were used to block the respective isoenzyme in rat liver fractions for determining
selective inhibition of the other isoenzyme. The selective inhibitors were also used as positive controls
in human recombinant MAOꢀA and MAOꢀB assays, respectively. Determined inhibitory potencies (IC50
values) for reference inhibitors and all new synthesized compounds are collected in Table 1.
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Journal of Medicinal Chemistry
Table 1. Inhibitory Potencies of Compounds at Monoamine Oxidases
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
IC ± SEM (nM)
50
MAO-A
h= human, r = rat)
MAO-B
Selectivityb
Compd.
(
(h = human, r = rat)
index (SI)
Standard inhibitors
18
Selegiline (1)
1700 (human brain)
6.59 ± 1.09 (h)
9.97 ± 1.33 (r)
>250
18
9
44 (rat brain)
18
6
.79 (human brain)
18
3
.63 (rat brain)
1
8
18
Rasagiline (2)
Safinamide (3)
710 (human brain)
14.0 (human brain)
>50
18
18
4
12 (rat brain)
4.43 (rat brain)
44
5000
5.18 ± 0.04 (h)
8.0 ± 4.9 (r)
.0 (human brain)
8 (rat brain)
4
5000 (human brain)
27
1
5
80000 (rat brain)
44
9
27
9
5-Carboxamidoindazole derivatives 10-16
R
10
>10000 (h)
>10000 (r)
220 ± 23 (h)
1990 ± 110 (r)
>9
1
1
1
2
>10000 (h)
>10000 (h)
>10000 (r)
c
–
–
>
10000 (r)
>10000 (h)
10000 (r)
3300 ± 400 (h)
>10000 (r)
>
>
10000 (h)
2590 ± 410 (h)
>10000 (r)
–
1
3
>10000 (r)
>
>
10000 (h)
10000 (r)
12.4 ± 0.2 (h)
138 ± 18 (r)
806
>11
1
1
4
6
>
>
10000 (h)
10000 (r)
882 ± 61 (h)
>10000 (r)
Indazole-5-carboxamides 15, 26-38 (class I inhibitors)
R
15
≥10000 (h)
>10000 (r)
0.586 ± 0.087 (h)
1.43 ± 0.11 (r)
17065
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2
2
6
7
>10000 (h)
117 ± 13 (h)
08 ± 34 (r)
>86
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7
>
10000 (r)
<10000 (h)
10000 (r)
2.75 ± 0.40 (h)
2.73 ± 0.21 (r)
3636
>
28
>10000 (h)
>10000 (r)
0.679 ± 0.044 (h)
2.36 ± 0.17 (r)
14727
14970
6289
2
3
9
0
<10000 (h)
0.668 ± 0.053 (h)
2.61 ± 0.68 (r)
>
10000 (r)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
>10000 (h)
>10000 (r)
1.59 ± 0.16 (h)
8.89 ± 0.05 (r)
(
PSBꢀ
1434)
31
>10000 (h)
>10000 (r)
185 ± 21 (h)
1400 ± 56 (r)
>54
2924
>81
3
3
2
3
>10000 (h)
3.42 ± 0.28 (h)
21.9 ± 3.4 (r)
>
10000 (r)
>10000 (h)
123 ± 16 (h)
941 ± 51 (r)
<
10000 (r)
34
8790 ± 200 (h)
>10000 (r)
4.36 ± 0.08 (h)
43.1 ± 4.2 (r)
2016
80
3
3
5
6
3000 ± 122 (h)
37.5 ± 7.3 (h)
385 ± 9 (r)
>
10000 (r)
<10000 (h)
10000 (r)
>10000 (h)
5.42 ± 0.20 (h)
26.6 ± 1.5 (r)
1845
<
3
7
388 ± 52 (h)
837 ± 28 (r)
>25
>
10000 (r)
38a
see structure above
see structure above
>10000 (h)
0.386 ± 0.052 (h)
1.32 ± 0.09 (r)
25906
>
10000 (r)
(PSBꢀ
1491)
3
8b
420 ± 24 (h)
1.44 ± 0.41 (h)
22.5 ± 0.6 (r)
292
1
740 ± 60 (r)
Indazole-6-carboxamides 40-49
R
4
4
0
1
3310 ± 288 (h)
67.1 ± 9.1 (h)
466 ± 39 (r)
>49
>29
<
10000 (r)
>10000 (h)
335 ± 11 (h)
423 ± 13 (r)
>
10000 (r)
4
2
>10000 (h)
10000 (r)
234 ± 38 (h)
1070 ± 44 (r)
>42
>31
>7
–
>
43
>10000 (h)
>10000 (r)
316 ± 29 (h)
2670 ± 152 (r)
4
4
4
5
>10000 (h)
1300 ± 176 (h)
<10000 (r)
>
10000 (r)
>10000 (h)
>10000 (h)
>10000 (r)
>
10000 (r)
46
>10000 (h)
1280 ± 141 (h)
<10000 (r)
8
>
10000 (r)
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Journal of Medicinal Chemistry
4
7
>10000 (h)
<10000 (h)
3280 ± 174 (r)
–
4
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
<
10000 (r)
48
>10000 (h)
2760 ± 973 (h)
>10000 (r)
>
10000 (r)
4
9
>10000 (h)
6220 ± 825 (h)
>10000 (r)
>
10000 (r)
Indole-5-carboxamides 53 and 54 (class II inhibitors)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
R
c
5
3
H
1300 ± 68 (h)
790 ± 121 (r)
0.227 ± 0.039 (h)
5727
4425
6
1.01 ± 0.16 (r)
(PSBꢀ
1410)
5
4
Me
>10000 (h)
2.26 ± 0.16 (h)
1.11 ± 0.11 (r)
>
10000 (r)
Imidazopyridine 55 and triazolopyridine 56
R
5
5
5
6
see structure above
>10000 (h)
>10000 (r)
141 ± 12 (h)
1140 ± 108 (r)
71
>4
see structure above
>10000 (h)
2270 ± 246 (h)
>10000 (r)
>
10000 (r)
(Indazol-5-yl)methanimines 58 and 59 (class III inhibitors)
R
58
H
<10000 (h)
>10000 (r)
0.612 ± 0.065 (h)
3.69 ± 0.17 (r)
16340
9709
5
9
Me
<10000 (h)
1.03 ± 0.09 (h)
4.55 ± 0.31 (r)
>
10000 (r)
a
b
c
n = 3, unless otherwise noted. SI = IC (human MAOꢀA)/IC (human MAOꢀB). n = 4.
50
50
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Page 20 of 81
Structure-Activity Relationships at Monoamine Oxidase B. The biological evaluation of 37 novel
compounds at human and rat MAOꢀA and ꢀB enzymes resulted in the identification of highly potent,
specific MAOꢀB inhibitors from three related chemical series: indazoleꢀ5ꢀcarboxamides (class I,
compounds 15, 26-38a and 38b); indoleꢀ5ꢀcarboxamides (class II, 53 and 54); and (indazolꢀ5ꢀ
yl)methanimines (class III, 58 and 59). Most of these compounds contain a disubstituted phenyl moiety
attached to the heterobicyclic core structure by an amide or an imine linker. Among the tested
compounds, 29 showed high inhibition of MAOꢀB ranging from submicromolar to subnanomolar
potency. The majority of potent compounds showed high selectivity for MAOꢀB without noticeable
inhibitory activity at MAOꢀA.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5ꢀChloroꢀNꢀ(1Hꢀindazolꢀ6ꢀyl)ꢀ6ꢀmorpholinonicotinamide (10) showed a moderate inhibitory activity at
human MAOꢀB (IC 220 nM), and was significantly weaker at the rat enzyme (IC 1990 nM, 9ꢀfold).
5
0
50
Considering the hydrophobic character and the bipartite structure of the active site of the human MAOꢀ
35,44,45
B enzyme and the binding mode of known inhibitors,
the bulky and polar 6ꢀmorpholinoꢀ
substituted pyridine moiety of 10 might be too large, e.g. as compared to the reference inhibitors 1-3.
Therefore, we focused on reducing the molecular mass and also introduced more lipophilic residues into
the target structures. In fact, the introduction of the smaller, but more polar Nꢀmethylpyridone residue
(
compound 11) abolished MAO inhibition completely. Better results were obtained by introducing a
phenyl ring substituted with electronꢀwithdrawing groups (mainly F and Cl). Introduction of a 2ꢀchloroꢀ
ꢀfluoroꢀ or a 2,3ꢀdichloroꢀsubstituted phenyl ring resulted in compounds 12 and 13 which were weaker
6
than 10 indicating that orthoꢀphenyl substituents were not well tolerated. To evaluate the importance of
meta,paraꢀsubstitution at the phenyl ring, we evaluated the dichloroꢀsubstituted isomer 14. Compared to
the lead structure 10, 3,4ꢀdichlorophenyl derivative 14 showed a remarkable, 18ꢀfold improvement in
MAOꢀB inhibition (human, IC = 12.4 nM; rat, IC = 138 nM ). Thus, 14 was the first highly potent
50
50
and selective MAOꢀB inhibitor within the series of Nꢀ(indazoꢀ5ꢀyl)benzamide derivatives. The structure
of 14 indicated that a lipophilic 3,4ꢀdisubstituted phenyl ring and a carboxamide linker might be
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Journal of Medicinal Chemistry
favorable when connected to the indazole C5ꢀposition. In order to further study the role of the linker, we
introduced a methylene group between the aromatic ring and the amide carbonyl group producing
(indazolꢀ5ꢀyl)acetamide derivative 16. The elongation of the linker led to a 71ꢀfold decrease in potency
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(human, IC50 882 nM) in 16 compared to the corresponding benzamide derivative 14. At the rat
orthologue 16 was virtually inactive. Because of this result, we decided to keep the carboxamide linker
for connecting the two ring systems. However in the next series of compounds we inverted the
carboxamide function by connecting indazoleꢀ5ꢀcarboxylic acid with aniline derivatives (compounds 15
and 26ꢀ38). This modification of the linker provided a new class of extraordinarily potent and selective
MAOꢀB inhibitors some of which displayed subnanomolar potency. Indazoleꢀ5ꢀcarboxamide derivative
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
5 exhibited 9ꢀfold higher inhibitory activity at human MAOꢀB than the standard inhibitor safinamide
(3); it was 21ꢀfold more potent than its isomer 14. Compound 15 exhibited a slight preference for human
versus rat MAOꢀB (human MAOꢀB, IC 0.586 nM; rat MAOꢀB, IC 1.43 nM) and did not inhibit rat
5
0
50
and human MAOꢀA at a high concentration of 10000 nM. We therefore considered Nꢀ(3,4ꢀ
dichlorophenyl)ꢀ1Hꢀindazoleꢀ5ꢀcarboxamide (15) as a new lead structure for further structural variations
to explore the SARs of this new class of MAOꢀB inhibitors. The corresponding derivative without
substituents at the phenyl ring (compound 26) led to decreased inhibition (human MAOꢀB, IC 117
50
nM; rat MAOꢀB, IC 708 nM). 3,5ꢀDichlorophenylꢀsubstitution (compound 27) resulted in a potent
5
0
compound which was only 2ꢀ5ꢀfold less potent than the 3,4ꢀdichlorophenylꢀsubstituted analogue 15
(human MAOꢀB, IC 2.75 nM; rat MAOꢀB, IC 2.73 nM). It should be noted that 27 was the only
5
0
50
compound of the present series that showed no species differences. Thus, metaꢀ and paraꢀsubstituents at
the phenyl ring appeared to be crucial for high inhibitory activity at MAOꢀB. Following this
observation, we subsequently introduced different substituents in the 3ꢀ and 4ꢀposition of the phenyl
ring. 3ꢀChloroꢀ4ꢀfluoroꢀ or 4ꢀchloroꢀ3ꢀfluoroꢀsubstitution (compounds 28 and 29) led to remarkably
potent inhibitors of human MAOꢀB (28, IC 0.679 nM; 29, IC 0.668 nM), comparable to the potency
50
50
of the parent compound, the dichlorophenyl derivative 15. The 3,4ꢀdifluoroꢀphenyl substituent provided
MAOꢀB inhibitor 30, which was similarly potent (IC , human MAOꢀB 1.59 nM); it was approximately
5
0
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Page 22 of 81
6
ꢀfold less potent at the rat enzyme (IC = 8.89 nM). In contrast, the introduction of 3,4ꢀdimethoxyꢀ
50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
substitution at the phenyl ring (compound 31, IC human MAOꢀB 185 nM) was not as well tolerated by
5
0
MAOꢀB leading to a >300ꢀfold decrease in potency compared to the dichlorophenyl derivative 15.
Consequently, we continued by replacing only one of the 3,4ꢀchloro substituents in 15 with a methoxy
or hydroxyl group (compounds 32-35). While two methoxy substituents had been unfavourable with
regard to MAOꢀB inhibition, the exchange of only one chloro substituent of 15 for a methoxy group
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(compounds 32 and 34) was tolerated and resulted in an only slight reduction in inhibitory potency.
Both, the 3ꢀchloroꢀ4ꢀmethoxyphenyl (32) and the 4ꢀchloroꢀ3ꢀmethoxyphenyl derivatives (34) were
almost equipotent at human MAOꢀB (32, IC human MAOꢀB 3.42 nM; 34, human MAOꢀB IC 4.36
5
0
50
nM). At rat MAOꢀB, both compounds were less potent (32, rat MAOꢀB, IC 21.9 nM; 34, rat MAOꢀB,
50
IC 43.1 nM). In contrast, a hydroxyl instead of a methoxy residue in the same position of the phenyl
5
0
ring resulted in a significant decrease in affinity (33, human MAOꢀB, IC 123 nM; 35, human MAOꢀB,
5
0
IC 37.5 nM). In contrast to the methoxyꢀsubstituted analogues, we observed a preference for the 3ꢀOH
50
(35) versus the 4ꢀOH substitution (33) with regard to MAOꢀB affinity. Our SARs thus revealed that
electronꢀdonating groups such as OMe and OH resulted in lower MAOꢀB inhibitory activity than
electronꢀwithdrawing substituents (e.g., Cl and F) at one or both, paraꢀ and metaꢀposition of the phenyl
ring in lead structure 15. In order to enhance waterꢀsolubility of this relatively lipophilic series of MAOꢀ
B inhibitors, the benzene ring of 15 was bioisosterically replaced by a 5,6ꢀdichloropyridine residue
(compound 36). The resulting indazoleꢀ5ꢀcarboxamide derivative 36 exhibited high MAOꢀB inhibition
(human MAOꢀB, IC 5.42 nM; rat MAOꢀB, IC 26.6 nM) even though somewhat weaker than that of
50 50
lead structure 15. Introduction of a 3,4ꢀdichloroꢀsubstituted benzyl moiety to probe a linker extension
(compound 37) led to a considerable decrease in MAOꢀB inhibitory potency (human MAOꢀB, IC 388
5
0
nM; rat MAOꢀB, IC50 837 nM), in analogy to the effect observed for the indazolꢀ5ꢀylꢀacetamide
homolog 16. Because of the high MAOꢀB inhibitory potency and selectivity of compound 15, we used
the 3,4ꢀdichlorophenyl and the carboxamide linker as fixed motifs for further modification of the C5ꢀ
substituted indazole unit. As a next step we introduced a methyl substituent on one of the indazole
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Journal of Medicinal Chemistry
nitrogen atoms resulting in isomers 38a and 38b. Methylation at the indazole N1 position (38a) slightly
increased potency at MAOꢀB, providing the best inhibitor of the present series (human MAOꢀB, IC50
0.386 nM; rat MAOꢀB, IC 1.43 nM). Compound 38b, an N2ꢀmethylated regioisomer of 38a, was less
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
50
potent than its N1ꢀsubstituted analogue and than the unmethylated lead structure 15, but it still showed
high MAOꢀB inhibitory potency (human MAOꢀB, IC 1.44 nM,). Its affinity for the human enzyme
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
50
was 15ꢀfold higher than that for the rat enzyme (rat MAOꢀB, IC50 22.5 nM), being one of the
compounds that showed major species differences. Moreover, 38b was also identified as the only
example in all series having notable inhibitory activity at MAOꢀA (human MAOꢀA, IC 420 nM; rat
5
0
MAOꢀA, IC 1740 nM). Thus, the N2ꢀmethyl substitution of the indazole moiety was found to be
5
0
beneficial for MAOꢀA inhibition, although 38b was still >290ꢀfold selective for human MAOꢀB over
human MAOꢀA. Our results indicate that the indazole NH is not required as a hydrogen bond donor, and
the binding pocket of MAOꢀB is able to accommodate substituents like methyl groups at indazole N1 or
N2. The high potency of compounds 15, and 27-30 shows that 3,4ꢀdisubstituted phenylꢀ5ꢀ
amidoindazoles represent a new class of highly potent and selective MAOꢀB inhibitors, which may be
suitable for further development as diagnostic or therapeutic drugs.
Further systematic SAR analysis led us to investigate a small set of C6ꢀconnected indazoleꢀ
carboxamides (40-49) with the same substitution pattern as the corresponding isomeric indazoleꢀ5ꢀ
carboxamides (compounds 15, 27-35). Compared to the indazoleꢀ5ꢀcarboxamides, the corresponding
indazoleꢀ6ꢀcarboxamides displayed greatly reduced MAOꢀB inhibition. Only 3,4ꢀ or 3,5ꢀ
halogenophenylꢀsubstituted indazoleꢀ6ꢀcarboxamide derivatives (i.e., 40-44) possessed some, although
only moderate, inhibitory activity at human MAOꢀB, whereas methoxyꢀ or hydroxylꢀsubstituted
compounds (45-49) were inactive or only very weakly active. The most potent compound in the
indazoleꢀ6ꢀcarboxamide series was the 3,4ꢀdichlorophenylꢀsubstituted derivative 40 (human MAOꢀB,
IC 67.1 nM; rat MAOꢀB, IC 466 nM). However, 40 was still 115ꢀfold (human MAOꢀB) and 326ꢀ
50
50
fold (rat MAOꢀB) less potent than the related indazoleꢀ5ꢀcarboxamide analogue 15. The 3,5ꢀ
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dichlorophenyl derivative 41 had a moderate inhibitory effect (human MAOꢀB, IC 335 nM; rat MAOꢀ
5
0
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
B, IC50 423 nM). Similar potencies were observed for the corresponding 4ꢀfluoroꢀ and 3ꢀfluoroꢀ
substituted analogues 42 and 43, which were moderately active only at the human, but not at the rat
MAOꢀB (42, human MAOꢀB, IC 234 nM; 43: human MAOꢀB, IC 316 nM). Within the class of
5
0
50
indazoleꢀ6ꢀcarboxamides, in which the position of the indazole NH is altered in comparison with the
indazoleꢀ5ꢀcarboxamide series, the MAOꢀB potency decreased in the same rank order as observed for
the more potent indazoleꢀ5ꢀcarboxamide isomers (15 and 27-35): 3,4ꢀdiꢀCl ≈ 3ꢀF, 4ꢀCl ≈ 4ꢀF, 3ꢀCl >
3,5ꢀdiꢀCl > 3ꢀCl, 4ꢀOMe ≈ 3ꢀOMe, 4ꢀCl > 3ꢀCl, 4ꢀOH ≈ 3ꢀOH, 4ꢀCl. One exception was the 3,4ꢀ
difluoroꢀsubstituted compound 44, which was less potent than expected (compare to 30).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In order to further explore the heterobicyclic part in the so far most potent series of indazoleꢀ5ꢀ
carboxamides, the indazole residue was replaced by different heterobicycles resulting in compounds 53-
5
6, while the 3,4ꢀdichloroꢀsubstituted phenyl ring of 15 was kept constant. Replacement of the indazole
by an indole residue (compound 53) resulted in an extremely high MAOꢀB inhibitory potency (human
MAOꢀB, IC 0.227 nM; rat MAOꢀB, IC 1.01 nM) showing only weak inhibition of MAOꢀA (human
5
0
50
MAOꢀA, IC 1300 nM; rat MAOꢀA, IC 6790 nM). Thus, the indole derivative 53 was 3ꢀfold more
50
50
potent than the indazoleꢀ5ꢀcarboxamide analogue 15 at human MAOꢀB; it represents the most potent
MAOꢀB inhibitor of all compounds investigated. The activity of its Nꢀmethylated derivative 54 at
human MAOꢀB was 10ꢀfold lower (IC50 2.26 nM). Based on their high bioactivity, which was
comparable or even superior to the indazoleꢀ5ꢀcarboxamide series, the indoleꢀ5ꢀcarboxamides 53 and 54
may as well be considered as suitable candidates for further drug development (class II MAOꢀB
inhibitors).
Next, we continued with the modification of the indazole residue by replacing it by an
imidazo[1,2ꢀa]pyridine or a
[1,2,4]triazolo[4,3ꢀa]pyridine moiety (compounds 55 and 56). In
comparison to the indazoleꢀ5ꢀcarboxamides and the indoleꢀ5ꢀcarboxamides, compounds 55 and 56
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Journal of Medicinal Chemistry
showed a large decrease in inhibitory activity at MAOꢀB. The 3,4ꢀdichloroꢀphenylꢀsubstituted
imidazo[1,2ꢀa]pyridineꢀ6ꢀcarboxamide (55) containing a bridge nitrogen atom had only moderate
potency at the human (IC50 141 nM) and rat MAOꢀB (IC50 1140 nM), while the
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
[1,2,4]triazolo[4,3ꢀa]pyridine motif of 56 led to an almost complete loss of activity. This indicates that
the position of the nitrogen atom (N1) present in the indazole and indole moieties is essential for MAOꢀ
B binding and has a strong impact on potency. A second nitrogen atom is well tolerated only in the
neighboring position (e.g., N2ꢀposition of the indazole). Introduction of a third nitrogen atom
(compound 56) resulted in a virtually complete loss of MAOꢀB inhibitory activity (cf. docking studies
below).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Finally, the carboxamide linker in the 3,4ꢀdichlorophenylꢀsubstituted indazole derivate 15 was replaced
by an imine function producing Schiff bases 58 and 59. Both compounds showed similarly high
MAOꢀB inhibitory potencies as the corresponding indoleꢀ5ꢀcarboxamides (53 and 54). The
unsubstituted indazole derivative 58 (human MAOꢀB, IC 0.612 nM; rat MAOꢀB, IC 3.69 nM) was
5
0
50
slightly more potent at MAOꢀB than its N1ꢀmethylꢀsubstituted derivative 59 (human MAOꢀB, IC 1.03
5
0
nM; rat MAOꢀB, IC 4.55 nM).
50
From all 37 tested compounds, 29 were found to be potent inhibitors of MAOꢀB. In the series of
indazoleꢀ5ꢀcarboxamide derivatives (15, 27-38a, 38b), all compounds were found to be active at
MAOꢀB. The highest inhibitory potencies were achieved with the compounds containing a 3,4ꢀ
dihalogenophenyl residue (15, 28 and 29) and an Nꢀunsubstituted or an N1ꢀmethylated indazole core
(38a) inhibiting MAOꢀB in the subnanomolar range. These compounds are significantly more potent
than the current irreversible (selegiline, rasagiline) and reversible (safinamide) standard MAOꢀB
inhibitors (>13ꢀfold). In the classes of indoleꢀ5ꢀcarboxamides (class II, 53 and 54) and (indazolꢀ5ꢀ
yl)methanimines (class III, 58 and 59) selective MAOꢀB inhibitors with similarly high potencies as
those of the best indazoleꢀ5ꢀcarboxamides (class I derivatives) were discovered. The most potent
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MAOꢀB inhibitor of the whole series was indoleꢀ5ꢀcarboxamide 53 (class II inhibitor), which contains a
,4ꢀdichlorophenyl residue. It inhibits human MAOꢀB with subnanomolar potency and was found to be
23ꢀfold more potent than the standard inhibitor safinamide (3). The optimized structural features were a
ꢀindazole or a 5ꢀindole moiety connected to a lipophilic, substituted phenyl ring through a polar linker
1
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1
1
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3
5
with a length of two atoms.
0
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0
Species Selectivity. Significant species differences and moderate correlations between human and rat
1
4,28,46
IC values were previously described for other classes of MAOꢀB inhibitors.
Therefore, all new
5
0
compounds were tested at human as well as rat MAOꢀA and MAOꢀB in order to assess potential species
differences. Since most compounds were inactive at MAOꢀA, species differences at MAOꢀA could only
be noticed for very few compounds. However, for many compounds major species differences in the
inhibitory potencies at MAOꢀB could be observed. With the exception of 27 and 54, which were
virtually equipotent at rat and human MAOꢀB, the potency of the compounds at human MAOꢀB was
generally higher (5.2ꢀfold on average, with compound 38b showing the highest deviation of
approximately 1.2 log units between IC values at human and rat MAOꢀB) than at the rat orthologue.
5
0
The plot of all determined pIC values at rat MAOꢀB versus pIC values at human MAOꢀB gave a
50
50
2
linear regression with a slope = 1.00, yꢀintercept = ꢀ0.67 and R = 0.89, thus displaying a clear
correlation between rat and human MAOꢀB enzyme inhibitory activity (for details, see Figure S8,
Supporting Information). Nonetheless it appears to be indispensable to determine MAOꢀB inhibition not
only at the human enzyme, but also at the species used for preclinical studies.
Mechanism of Monoamine Oxidase B Inhibition. The goal of the present study was to prepare novel
reversible MAOꢀB inhibitors. Reversible inhibition is expected to have considerable advantages
compared to irreversible inhibition, as discussed above. To investigate whether the indazoleꢀ5ꢀ
carboxamides (class I compounds) are reversible or irreversible inhibitors of human MAOꢀB, we
performed reactivation experiments with the representative human MAOꢀB inhibitor 15. For this
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Journal of Medicinal Chemistry
purpose, the inhibition of reactivated human MAOꢀB by 15 was evaluated at a concentration that
represents its IC value versus pꢀtyramine as a substrate. The enzyme activity of 15 was measured for
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9
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0
2
2 min in the presence of low concentration of the substrate pꢀtyramine (10 ꢀM preꢀincubation)
followed by a large increase in substrate concentration (to 1.0 mM). The irreversible inhibitor selegiline
0
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0
1 and the reversible inhibitor safinamide 3 were tested as reference inhibitors (Figure 4).
Figure 4. Reactivation of MAOꢀB: recombinant human MAOꢀB enzyme was treated under assay
conditions with inhibitor 15 at a concentration that presents its IC value (1.0 nM) in the presence of
8
0
the substrate pꢀtyramine. The irreversible MAOꢀB inhibitor selegiline (1) and the reversible inhibitor
safinamide (3) were applied at concentrations of 30 nM and 50 nM, respectively. After a preꢀincubation
period of 22 min, the substrate concentration was increased from 10 ꢀM to 1.0 mM, and the
fluorescence was measured over a period of 5 h.
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Journal of Medicinal Chemistry
Page 28 of 81
In case of a reversible inhibition, after preꢀincubation followed by an increase in the substrate
concentration, the MAOꢀB inhibitor will be replaced by the competing excess of pꢀtyramine and a
subsequent enzyme reactivation can be observed. In the experiment with the inhibitor 15, an elevated
fluorescence can be detected after increasing the pꢀtyramine concentration indicating, that 15 acts as a
reversible MAOꢀB inhibitor. The measurements with the reversible reference inhibitor safinamide 3
clearly showed the expected reversible mode of interaction with MAOꢀB, which is proven by an
elevation of the fluorescence after increasing the substrate concentration, similar as that observed for 15.
In contrast, in the experiment with the irreversible inhibitor selegiline (1) the residual activity was not
significantly enhanced after increasing the substrate concentration. These experiments clearly indicate
that the indazolꢀ5ꢀcarboxamides are reversible inhibitors of MAOꢀB. This was expected since they do
not contain any reactive moieties.
1
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9
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1
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0
To further examine the interaction mode of the indazolꢀ5ꢀcarboxamides with the binding site of
MAOꢀB, the type of enzyme inhibition was determined by MichaelisꢀMenten kinetic experiments. For
this purpose, the initial rates of the MAOꢀBꢀcatalyzed oxidation of pꢀtyramine at six different substrate
concentrations in the absence and in the presence of three different concentrations of the selected
representative inhibitor 15 were measured. The results are depicted in Figure 5. MichaelisꢀMenten
kinetic parameters K and V
of human MAOꢀB inhibition were determined in the presence and
max
m
absence of inhibitor 15. The maximal velocity (Vmax) remained almost constant at different
concentrations of 15, whereas the Michaelis constant (K ) rose with increasing inhibitor concentrations.
m
The LineweaverꢀBurk plots for different concentrations of 15 were linear and intersected at the yꢀaxis
with the plot for the uninhibited enzyme. In addition, a Dixon plot was calculated (see Figure S9,
Supporting Information). The obtained results indicate that the indazolꢀ5ꢀcarboxamides are competitive
MAOꢀB inhibitors.
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Journal of Medicinal Chemistry
1
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Figure 5. Mode of MAOꢀB inhibition: saturation curves (top) and LineweaverꢀBurk plot (bottom) of the
inhibition of recombinant human MAOꢀB enzyme by different concentrations of 15 (0, 0.1, 0.5, and 1.0
nM) in the presence of pꢀtyramine (0.05, 0.1, 0.25, 0.5, 1.0, and 1.5 mM) as a substrate. In the
LineweaverꢀBurk plot the reciprocal MAOꢀB inhibitory activity was plotted against the reciprocal
substrate concentration (double reciprocal plot, n = 2).
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