Discovery of New Chemical Entities for Old Targets: Insights on the Lead Optimization of Chromone-Based Monoamine Oxidase B (MAO-B) Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.6b00527

The discovery of new chemical entities endowed with potent, selective, and reversible monoamine oxidase B inhibitory activity is a clinically relevant subject. Therefore, a small library of chromone derivatives was synthesized and screened toward human monoamine oxidase isoforms (hMAO-A and hMAO-B). The structure-activity relationships studies strengthen the importance of the amide spacer and the direct linkage of carbonyl group to the γ-pyrone ring, along with the presence of meta and para substituents in the exocyclic ring. The most potent MAO-B inhibitors were N-(3′-chlorophenyl)-4-oxo-4H-chromene-3-carboxamide (20) (IC₅₀ = 403 pM) and N-(3′,4′-dimethylphenyl)-4-oxo-4H-chromene-3-carboxamide (27) (IC₅₀ = 669 pM), acting as competitive and noncompetitive reversible inhibitors, respectively. Computational docking studies provided insights into enzyme-inhibitor interactions and a rationale for the observed selectivity and potency. Compound 27 stands out due to its favorable toxicological profile and physicochemical properties, which pointed toward blood-brain barrier permeability, thus being a valid candidate for subsequent animal studies.

Full text of DOI:10.1021/acs.jmedchem.6b00527

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Article
Discovery of new chemical entities for old targets: insights on the lead
optimization of chromone-based monoamine oxidase B (MAO-B) inhibitors
Joana Reis, Fernando Cagide, Daniel Chavarria, Tiago B. Silva, Carlos Fernandes, Alexandra Gaspar,
Eugenio Uriarte, Fernando Remião, Stefano Alcaro, Francesco Ortuso, and Fernanda M. Borges
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00527 • Publication Date (Web): 31 May 2016
Downloaded from http://pubs.acs.org on May 31, 2016
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Discovery of new chemical entities for old targets: insights on the lead optimization
of chromoneꢀbased monoamine oxidase B (MAOꢀB) inhibitors
[a]
¥
[a]
¥
[a]
[a]
[a]
Joana Reis , Fernando Cagide , Daniel Chavarria , Tiago Silva , Carlos Fernandes ,
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Alexandra Gaspar , Eugenio Uriarte , Fernando Remião , Stefano Alcaro , Francesco
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Ortuso , Fernanda Borges*
[a]
CIQUP/Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto,
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169ꢀ007, Porto, Portugal.
[b]
Department of Organic Chemistry, Faculty of Pharmacy, University of Santiago de
Compostela, 15782 Santiago de Compostela, Spain
[c]
UCIBIOꢀREQUIMTE, Laboratory of Toxicology, Department of Biological Sciences, Faculty
of Pharmacy, University of Porto, 4050ꢀ313, Porto, Portugal
[d]
Department of “Scienze della Vita”, University “Magna Græcia” of Catanzaro, 88100
Catanzaro, Italy
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ABSTRACT
The discovery of new chemical entities endowed with potent, selective and reversible
monoamine oxidaseꢀB inhibitory activity is a clinically relevant subject. Therefore, a
small library of chromone derivatives was synthesized and screened towards human
monoamine oxidase isoforms (hMAOꢀA and hMAOꢀB). The structureꢀactivity
relationships studies strengthen the importance of the amide spacer and the direct
linkage of carbonyl group to the γꢀpyrone ring, along with the presence of meta and
para substituents in the exocyclic ring. The most potent MAOꢀB inhibitors were Nꢀ(3’ꢀ
chlorophenyl)ꢀ4ꢀoxoꢀ4Hꢀchromeneꢀ3ꢀcarboxamide (20) (IC50 = 403 pM) and Nꢀ(3’,4’ꢀ
dimethylphenyl)ꢀ4ꢀoxoꢀ4Hꢀchromeneꢀ3ꢀcarboxamide (27) (IC50 = 669 pM), acting as
competitive and nonꢀcompetitive reversible inhibitors, respectively. Computational
docking studies provided insights into enzyme−inhibitor interactions and a rationale for
the observed selectivity and potency. Compound 27 stands out due to its favorable
toxicological profile and physicochemical properties, which pointed toward blood brain
barrier permeability, thus being a valid candidate for subsequent animal studies.
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Keywords: MAOꢀB inhibitors, New Chemical Entities, Chromone
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INTRODUCTION
Parkinson’s disease (PD) is one of the most prevalent neurodegenerative disorders,
affecting up to 10 million people worldwide. Its current pharmacological therapy is
mainly focused on improving the motor symptomatology and the patients’ quality of
life by slowing down the progression of symptoms and reducing morbidity. In spite of
all the research performed in the field, there are only a few classes of drugs approved
for the treatment of the motor related symptoms of PD, namely those acting primarily
on the dopaminergic system, such as Lꢀdopa, dopamine agonists, monoamine oxidaseꢀB
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, 2
(
MAOꢀB) and catecholꢀOꢀmethyltransferase inhibitors.
Monoamine oxidases (MAOs) are intracellular flavineꢀcontaining enzymes that play a
major role in the in vivo inactivation of biogenic amines, both in peripheral and central
neuronal tissues. MAOꢀA and MAOꢀB are two isoforms of MAOs expressed in
mammals, which can be distinguished based on their substrate preference and their
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ꢀ5
interaction with specific inhibitors. The primary substrates of MAOs in the human
brain are neurotransmitters and, albeit dopamine can be metabolised by both isoforms, it
has a higher affinity for MAOꢀB. The expression of MAOꢀB in neuronal tissues
enhances 4ꢀfold with aging, resulting in an increased level of dopamine metabolism and
higher production of hydrogen peroxide, which are thought to play a relevant role in the
aetiology of neurodegenerative diseases. Accordingly, the use of selective MAOꢀB
inhibitors (IMAOꢀB), alone or combined with dopamine or its precursors, are important
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therapeutic approaches for neurodegenerative disorders, such as PD.
Currently, there are two IMAOꢀB in the market: selegiline (Rꢀ(−)ꢀdeprenyl) and
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rasagiline which are selective but irreversible (Figure 1). More recently, safinamide has
been developed as a multiꢀtarget drug based on the enhancement of the dopaminergic
function (through reversible inhibition of MAOꢀB and of dopamine reuptake) and
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inhibition of the excessive release of glutamate in patients with early or midꢀ to lateꢀ
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stage PD (Figure 1).
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Figure 1. Structures of known MAOꢀB inhibitors.
However, due the lack of significant disease modifying properties, a pressing need for
new chemical entities endowed with selective and reversible IMAOꢀB still exists.
Chromones, a class of heterocyclic compounds widely distributed in nature, have
attracted attention in the field of drug discovery and development due to their
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interesting biological activities.
Several lines of evidence reported by our research
group have shown that chromone (4Hꢀbenzopyranꢀ4ꢀone) is a privileged structure for
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the rational discovery and development of new IMAOꢀB.
In this context, it was
shown that the structural modification of the chromone scaffold with the insertion of a
phenylcarboxamide moitey at position C3 of the γꢀpyrone ring yielded selective IMAOꢀ
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B.
Focusing on the lead optimization of chromoneꢀ3ꢀphenylcarboxamide as IMAOꢀ
B and guided by the rational design strategy shown in Figure 2, the work herein reports
the synthesis of a novel chromone library and the establishment of new structureꢀ
activity relationships (SAR). The evaluation of drugꢀlike properties, kinetics and
mechanism of enzymatic inhibition and cytotoxicity studies of the best chromoneꢀbased
IMAOꢀB were also performed. Finally, in order to rationalize our results, docking
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experiments were performed using models built on the basis of the crystal structures of
human MAOꢀA and MAOꢀB.
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Figure 2. Structure of the lead compound described in previous work
and rational design strategy
followed for lead optimization.
RESULTS AND DISCUSSION
Chemistry. A lead optimization process of chromoneꢀ3ꢀphenylcarboxamide was
performed to improve its IMAOꢀB potency and selectivity. The rational design strategy
described in Figure 2 was mainly focused on the study of: (i) the effect of different
substituents (type, number and position) located on the aromatic exocyclic ring C and
(ii) the importance of the amide linker located between the γꢀpyrone nucleus (ring B)
and the exocyclic aromatic ring (ring C) by the insertion of different spacers (amide,
inverse amide, ester, thioester, amine and vinyl). As a result, a library of new chromoneꢀ
based compounds was obtained (Schemes 1ꢀ3). The first chromone series (Scheme 1,
compounds 2ꢀ31) incorporated carboxamide, thioester or ester spacers directly attached
to Cꢀ3 and an aromatic exocyclic ring with different electron donating or withdrawing
substituents, variable in type, number and position. The chromoneꢀbased compounds
were synthesized in moderate yields through a reaction that encompassed the in situ
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generation of an acyl chloride intermediate, using phosphoryl chloride (POCl ) in N,Nꢀ
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dimethylformamide (DMF), and the subsequent addition of the appropriate arylamine,
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ꢀmethylphenol or 4ꢀmethylbenzenethiol. Compound 10 was obtained from compound
12 by cleavage of the tertꢀbutoxycarbonyl protecting group with trifuoroacetic acid
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(TFA).
Scheme 1. General strategy followed for the synthesis of chromoneꢀ3ꢀphenylcarboxamide derivatives (2ꢀ
31) from chromoneꢀ3ꢀcarboxylic acid (1). Reagents and conditions: (a) POCl , DMF, appropriate
arylamine (a ), Nꢀmethylaniline (a ), 4ꢀmethylphenol (a ) or 4ꢀmethylbenzenethiol (a ), r.t., 1ꢀ5h; (b)
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TFA, r.t., 2h.
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Scheme 2. Synthetic strategy followed for the synthesis of chromone derivative 34. Reagents and
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conditions: (a) 5ꢀ(2ꢀhydroxyethyl)ꢀ3,4ꢀdimethylthiazolium iodide, DBU, r.t., 5h; (b) POCl
DIPEA, 4ꢀmethylbenzoic acid, r.t., 2h.
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, DMF,
Compound 34, bearing an inverted carboxamide linker at Cꢀ3, was obtained by a twoꢀ
step synthetic strategy. Initially, an intramolecular cyclization between aldehyde and
nitrile of compound 32 was performed in the presence of 5ꢀ(2ꢀhydroxyethyl)ꢀ3,4ꢀ
dimethylthiazolium iodide and 1,8ꢀdiazabicyclo[5.4.0]undecꢀ7ꢀene (DBU), leading to
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the chromone amine 33 (Scheme 2). Subsequently, chromone 34 was synthesized by
an in situ amidation reaction using 4ꢀmethylbenzoic acid, POCl and DMF. In turn,
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compound 36 (Scheme 3) was obtained by a reductive amination, in the presence of
sodium triacetoxyborohydride, of the imine intermediate formed from the reaction of 3ꢀ
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formylchromone (35) with 4ꢀmethylaniline.
Scheme 3. Synthetic strategy followed for the synthesis of chromone derivatives (36ꢀ41) from 3ꢀ
formylchromone (35). Reagents and conditions: (a) 4ꢀmethylaniline, Na(AcO) BH, r.t., 15 min; (b)
3
malonic acid, pyridine, r.t., 1.5 h;(c) PyBOP, appropriate arylamine, DMF, DIPEA, r.t., 6h.
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The synthetic procedure followed to obtain the chromone derivatives bearing a vinyl
spacer linked to the γꢀpyrone ring involved two steps (Scheme 3). Firstly, the 3ꢀ(4ꢀoxoꢀ
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Hꢀchromenꢀ3ꢀyl)acrylic acid (compound 37) was obtained from the reaction between
ꢀformylchromone (35) and malonic acid through a KnoevenagelꢀDoebner
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condensation. The corresponding chromone carboxamides (compounds 38ꢀ41) were
then synthesized by a oneꢀpot condensation reaction. Briefly, this amidation was
performed using compound 37 and aniline or its ringꢀsubstituted derivatives as starting
materials, using as coupling agent (benzotriazolꢀ1ꢀyloxy)ꢀtripyrrolidinophosphonium
2
0
hexafluorophosphate (PyBOP) in basic medium (N,Nꢀdiisopropylethylamine, DIPEA).
Monoamine oxidase inhibition studies. The evaluation of hMAO inhibitory activity of
the chromones under study was performed by a fluorescenceꢀbased assay measuring
2 2
their effect on the production of hydrogen peroxide (H O ) from pꢀtyramine (a common
substrate for both hMAOꢀA and hMAOꢀB), using the Amplex Red MAO assay kit and
microsomal MAO isoforms prepared from insect cells (BTIꢀTNꢀ5B1ꢀ4) infected with
14
recombinant baculovirus containing cDNA inserts for hMAOꢀA or hMAOꢀB. The
production of H O catalyzed by MAO isoforms was detected using 10ꢀacetylꢀ3,7ꢀ
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dihydroxyphenoxazine, a nonꢀfluorescent and highly sensitive probe that reacts with
in the presence of horseradish peroxidase to produce a fluorescent product
2 2
H O
14
(
resorufin). The chromone derivatives under study and standard inhibitors did not
react directly with the Amplex Red reagent, which indicated that they did not interfere
with the measurements.
The results of the in vitro evaluation of inhibitory potency (IC50 values) and selectivity
expressed as SI ([IC50 (MAOꢀA)]/[IC50 (MAOꢀB)]) towards hMAOꢀA and hMAOꢀB of
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the chromones under study and standard inhibitors (clorgyline for MAOꢀA and (R)ꢀ(−)ꢀ
deprenyl, rasagiline, safinamide for MAOꢀB, Figure 1) are shown in Tables 1 and 2.
Structure−activity relationship studies. Following the previous results obtained for
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chromoneꢀ3ꢀphenylcarboxamides (compounds 2ꢀ4) , and in order to establish SAR f
the MAO inhibitory activity of the newly developed chromone library (Figure 2) was
evaluated and the results are depicted in Tables 1 and 2.
Table 1. MAO inhibitory activities of chromoneꢀ3ꢀ phenylcarboxamides 6ꢀ28 and standard inhibitors.
IC50 (nM)
Compound
R
SI
hMAOꢀB
hMAOꢀA
15
b
2
70.70 ± 1.70
2.90 ± 1.20
8.00 ± 3.20
1382 ± 25
*
*
*
*
>
141
15
b
b
3
>
>
3448
1250
15
4
15
b
5
>
7.2
b
b
6
7
8
9
1.15 ± 0.16
11.43 ± 1.19
7.30 ± 1.08
***
*
*
>
>
8696
b
>
875
*
1370
ꢀ
***
*
b
1
0
1
4574 ± 410
7396 ± 84
>
2.2
1.3
b
1
*
>
1
2
3
*
*
*
ꢀ
b
1
24.00 ± 1.10
>417
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b
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15
26.45 ± 2.11
3.31 ± 0.25
*
*
>378
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>
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b
16
427 ± 30
1.03 ± 0.15
736 ± 12
*
*
*
*
>23
b
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>9709
b
1
8
9
>
13
1
239 ± 21
>
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c
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21
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23
24
0.40 ± 0.023
1.33 ± 0.010
1.73 ± 1.01
2.65 ± 0.31
***
**
*
>
250000
b
>
>
7519
5780
b
*
b
*
>
3773
***
ꢀ
2
5
6
***
***
ꢀ
b
2
579 ± 30
*
>17
c
27
0.67 ± 0.13
**
>
149254
b
28
31.61 ± 1.32
*
>316
a
Rꢀ(
ꢀ
)ꢀDeprenyl
ꢀ
ꢀ
ꢀ
ꢀ
16.73 ± 1.48
49.66 ± 2.26
23.07 ± 2.07
63410 ± 120
68730 ± 421
52974 ± 742
**
4108
1067
Rasagiline
Safinamide
Clorgyline
c
4335
4.46 ± 0.32
0.000070
* Inactive at 10 µM; ** Inactive at 100 µM (highest concentration tested); *** Not soluble in DMSO;
b
Values obtained under the assumption that the corresponding IC50 against MAOꢀA or MAOꢀB is the highest concentration tested (10 µM).
Values obtained under the assumption that the corresponding IC50 against MAOꢀA or MAOꢀB is the highest concentration tested (100 µM).
c
SI: hMAOꢀB selectivity index = IC50 (hMAOꢀA) /IC50 (hMAOꢀB)
.
Effect of ring C substituents on MAO inhibitory activity. The introduction of halogens at
the para position of the exocyclic ring yielded different results: iodine (compound 7)
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and fluorine (compound 8) caused a decrease on activity when compared with the
chlorine (compound 3, IC50 = 2.90 nM), while bromine (compound 6) led to a more
potent compound (IC50 = 1.15 nM). Properties like atomic radius and/or
electronegativity may thus play a significant role on the ligandꢀenzyme interaction. The
evaluation of the effect of the introduction of a pꢀthiol substituent in compound 9 was
not performed due to its poor solubility. The introduction of a pꢀamine substituent
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(compound 10) was unfavorable, leading to a ~500ꢀfold activity decrease when
compared to the halogenated or methyl substituted derivatives (compounds 3, 4, 6ꢀ8).
This result is in accordance with recently published data, which showed that the
introduction of a strong electron donating group (pꢀOH, compound 5) decreased MAOꢀ
15
B inhibitory capacity (IC50 = 1382 nM). Additionally, the presence of bulky para
substituents led to a dramatic decrease (compound 11) or complete loss (compound 12)
of MAOꢀB inhibitory activity. These observations also provided valuable data
concerning the volume restrictions of the substituents on the exocyclic ring, since only
small substituents seemed to favor an appropriate accommodation of the ligand on the
enzyme’s active site. The presence of the pꢀethynyl (compound 13, IC = 24.00 nM)
5
0
and pꢀvinyl (compound 14, IC50 = 26.45 nM) substituents yielded compounds with a
remarkable inhibitory potency, although a superior activity was obtained for the
saturated pꢀethyl derivative (compound 15, IC50 = 3.31 nM). The data showed that the
presence of an unsaturated system in the mentioned position is not crucial for the
IMAOꢀB activity of chromoneꢀ3ꢀphenylcarboxamide based ligands. Overall, SAR
studies indicated that the type of substituents on the exocyclic aromatic ring of
chromoneꢀ3ꢀphenylcarboxamide (Figure 1) exerted a noteworthy influence on MAOꢀB
inhibitory activity.
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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
Subsequently, the effect of the most interesting substituents (Cl, Br, OH and CH3)
located at the ortho and meta positions of the exocyclic ring was evaluated. With the
exception of the bromine group (compound 17), the insertion of oꢀsubstituents
3
(compounds 16 (Cl), 18 (OH) and 19 (CH )) resulted in a noticeably decrease on MAOꢀ
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
B inhibitory activity, when compared to the corresponding para derivatives (compounds
3ꢀ5). However, when these substituents were located at the meta position (compounds
2
0 (Cl), 22 (OH) and 23 (CH )) a significant enhancement of IMAOꢀB potency was
3
observed, with the exception of the mꢀbromine derivative (21), for which no potency
change was observed. The type and position of substituents had a noteworthy effect on
MAOꢀB inhibitory activity. In particular, the presence of a mꢀchlorine substituent at the
aromatic exocyclic ring remarkably enhanced IMAOꢀB potency, with chromone 20
(
IC50 = 0.40 nM and a SI >250000) emerging as the most potent and selective
derivative.
Additional studies were performed with chromone carboxamides in which the exocyclic
ring was derivatized with two or three chlorine or methyl substituents. However, the
low solubility of the corresponding derivatives (compounds 24 and 25) precluded their
biological evaluation. Thus, the biological screening was only performed with the
dimethyl (compounds 26 and 27) and trimethyl (compound 28) derivatives. Compound
27 (IC50 = 0.67 nM and a SI >149254) displayed a notorious increase of MAOꢀB
potency and selectivity when compared with compound 4 (IC50 = 8.00 nM and a SI
>
1250), due to the introduction of a mꢀmethyl substituent. However, when the same
substituents were located at ortho and meta positions (compound 26), a significant
decrease of potency and selectivity (IC50 = 579 nM and a SI of 17) was observed. For
compound 28, with a third 5’ꢀmethyl group, a decrease on IMAOꢀB activity was also
observed (IC50 = 31.61 nM).
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SAR studies pointed that chromoneꢀ3ꢀphenylcarboxamides with a mꢀchlorine
substituent (20) or a 3’,4’ꢀdimethyl substituent (compound 27) presented IMAOꢀB IC50
values within the picomolar range. Both compounds 20 (IC50 = 0.40 nM, SI >250000)
and 27 (IC50 = 0.67 pM, SI > 149254) showed respectively a 42ꢀ and 25ꢀfold increase in
inhibitory potency when compared to (R)ꢀ(−)ꢀdeprenyl, a 123ꢀ and 74ꢀfold increase
compared to rasagiline and, finally, a 57ꢀ and 34ꢀfold increase compared to safinamide.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Effect of the spacer between the chromone and exocyclic aromatic ring. The insertion
of a methyl group in the amide spacer completely abolished MAO inhibitory activity
(compound 2 vs 29). Similarly, the replacement of the amide nitrogen with oxygen
(
compound 4 vs 30) caused a marked decrease of IMAOꢀB activity from 8.00 nM
compound 4) to 2038 nM (compound 30). A comparable outcome was observed when
(
the nitrogen was replaced by sulphur (compound 4 vs 31), with 67ꢀfold decrease on
IMAOꢀB activity observed for compound 31 (IC50 = 538 nM). This outcome suggests
the inexorable significance of hydrogen bonding with the residues in the enzyme’s
active pocket. The importance of the carbonyl moiety was then subsequently explored.
First, an exchange on the carbonyl and nitrogen group position (compound 4 vs 34) was
performed and the IMAO activity evaluated: although the compound was still active, a
12ꢀfold decrease on IMAOꢀB activity (IC50 = 102 nM) was observed. As the results
obtained so far indicated the importance of the spatial position of the amide group for
the observed IMAO activity, a subsequent structural modification was performed in
which the carbonyl group was removed (compound 36). Biological screening of 36
showed that the presence of the amine yielded an active IMAOꢀB, a 71ꢀfold decrease on
inhibitory potency was observed (IC50 = 568 nM), suggesting that the carbonyl moiety
is also a very important structural requirement to establish the appropriate interactions
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2
2
2
2
2
2
2
2
2
3
3
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3
3
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3
3
3
4
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4
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4
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5
5
5
6
with the target residues. To further characterize the importance of the location of the
carboxamide spacer, a vinylic spacer between the pyrone ring and the carboxamide
function of the chromoneꢀ3ꢀphenylcarboxamide was introduced (compound 38), leading
to complete loss of MAO inhibitory activity. The same effect was observed when pꢀ
chlorine (compound 39) or pꢀmethyl (compound 40) substituents were placed in the
exocyclic aromatic ring and with the introduction of a 3´,4´ꢀdimethyl substituent
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
(
compound 41). The existence of a vinylic spacer may lead to steric constrains in the
MAOꢀB pocket and, consequently, an unfavorable effect on activity. Moreover, the data
reinforces the hypothesis established in our previous work, in which the carboxamide
function linked directly to the pyrone ring ensured a key role in the ligandꢀenzyme
11
complex. The vinylic spacer present in compounds 38ꢀ41 may displace the
carboxamide function from the appropriate binding site, leading to complete loss of
MAO inhibitory activity.
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Table 2. MAO inhibitory activities of chromone derivatives (29ꢀ31, 34, 36 and 38ꢀ41).
hMAOꢀB
hMAOꢀA
Compound
29
R
SI
(
IC50 in nM)
(IC50 in nM)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
*
*
ꢀ
3
3
3
0
1
4
2038 ± 135
538 ± 46
102 ± 9
568 ± 38
*
79455 ± 2974
39
2.0
1100 ± 78
b
*
>98
36
94297 ± 7399
*
166.0
ꢀ
O
O
NH
38
O
3
9
0
*
*
*
*
ꢀ
ꢀ
4
41
*
*
ꢀ
*
Inactive at 10 µM (highest concentration tested);
b
Values obtained under the assumption that the corresponding IC50 against MAOꢀA or MAOꢀB is the highest concentration
tested (10 µM).
SI: hMAOꢀB selectivity index = IC50 (hMAOꢀA) /IC50 (hMAOꢀB)
.
Evaluation of drugꢀlike properties. The 10 most potent and selective IMAOꢀB
compounds 3, 4, 6, 7, 15, 17, 20, 21, 22 and 27) were selected and their drugꢀlike
(
properties calculated (Table 3). The selection criteria was an IC50 towards human MAOꢀ
B < 12 nM and selectivity higher than 1000ꢀfold for MAOꢀB over MAOꢀA.
Remarkably, all the selected compounds were more potent hMAOꢀB inhibitors than the
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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
standard inhibitors. The calculated physicochemical parameters include: molecular
weight (MW), number of heavy atoms (N), partition coefficient (clog P), topological
2
polar surface area (tPSA in Å ), number of hydrogen acceptors (HBA), number of
hydrogen donors (HBD) and number of rotatable bonds (nrotb) (Table 3). In addition,
the following multiparameter scores were also calculated: ligandꢀlipophilicity
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
2
1
22
efficiency (LLE), blood (plasma)ꢀbrain partitioning (logBB) and absorption,
23
distribution, metabolism, excretion and toxicity (ADMET) efficiency index (AEI)
Table 3). The same parameters were calculated for the MAOꢀB standard inhibitors
(
selegiline, safinamide and rasagiline (Table 3).
Overall, the selected chromones were in line with general drugꢀlikeness requirements,
24
namely concerning the wellꢀknown ruleꢀofꢀ5. Additionally, they exhibited clogP and
tPSA values within the optimal range for oral bioavailability and bloodꢀbrain barrier
2
21,22,24,25
(
BBB) permeability (log P 2−4, MW < 400 and tPSA 50−90 Å ).
With the
2
1
exception of compounds 7 and 15, the multiparameter score LLE obtained for all
21
derivatives was within the optimal range for suitable drug candidates (5 < LLE < 7).
Chromones 20, 22 and 27 (LLE ≥ 6) may be suitable candidates for in vivo studies.
Another key measurement is the prediction of BBB permeability properties, as
determined by the logBB, which is the ratio of the steadyꢀstate concentrations of the
drug in the brain and in the blood. Compounds with logBB below −1 are poorly
22
distributed to the brain and are unlikely to function as effective CNS drugs. All
chromone derivatives depicted on Table 3 displayed logBB > −1, pointing towards
potential BBB permeability. Incorporating the contribution of drugꢀtransporters, the
AEI score links lipophilic efficiency and ADMET properties. It has been suggested that
23
AEI values over 7 identify compounds with improved ADMET properties. In line with
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the AEI score calculated for the new chiral IMAOꢀB safinamide, all the selected
23
chromones displayed AEI > 7.
Overall, the selected chromones presented drugꢀlike properties similar to those of the
standard inhibitor safinamide. Moreover, rasagiline and (R)ꢀ(−)ꢀdeprenyl have outlier
behaviour for a significant number of parameters, including N, tPSA, log BB and AEI
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(see Table 3).
Table 3. Drugꢀlike properties of selected chromones and MAOꢀB standard inhibitors
Log
a
a
a
2 a
a
a
a
Compound
MW
pIC50
N
cLogP
tPSA (Å )
HBA
HBD
nrotb
LLE
AEI
BB
ꢀ0.236
ꢀ0.271
ꢀ0.214
ꢀ0.173
ꢀ0.199
ꢀ0.222
ꢀ0.239
ꢀ0.219
ꢀ0.714
ꢀ0.213
0.280
ꢀ0.371
0.492
≥ ꢀ1
3
4
6
7
299.71
279.30
344.16
391.16
293.32
344.16
299.71
344.16
281.27
293.32
171.24
302.35
187.29
<450
8.54
8.10
8.94
7.94
8.48
8.99
9.39
8.88
8.76
9.17
7.30
7.64
7.77
___
21
21
21
21
22
21
21
21
21
22
13
22
14
___
3.31
3.08
3.45
3.72
3.55
3.40
3.29
3.42
2.13
3.46
2.10
2.91
2.64
<5
59.31
59.31
59.31
59.31
59.31
59.31
59.31
59.31
79.54
59.31
12.03
64.36
3.24
4
4
1
1
2
2
5.23
5.02
5.49
4.22
4.93
5.59
6.10
5.46
6.63
5.71
5.20
4.73
5.13
>5
8.81
8.46
9.25
7.12
8.31
9.42
10.29
9.20
8.34
9.64
43.26
7.34
158.32
___
4
1
2
4
1
2
15
17
20
21
4
1
3
4
1
2
4
1
2
4
1
2
22
27
5
2
2
4
1
2
Rasagiline
Safinamide
R)ꢀ(−)ꢀDeprenyl
1
1
2
4
3
7
(
1
0
4
+
21ꢀ23, 26
CNS drugs
<60ꢀ70
<7
<3
<8
MW: molecular weight; N: number of heavy atoms; clog P: calculated logarithm of the octanolꢀwater partition coefficient; HBA:
number of hydrogen acceptors; HBD: number of hydrogen donors; nrotb: number of rotatable bonds; LLE: ligandꢀlipophilicity
efficiency; Log BB: logarithm of the ratio of the concentration of a drug in the brain and in the blood; AEI: ADMET efficiency
a
index. Properties calculated using Cheminformatics software [http://www.molinspiration.com].
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1
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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
Evaluation of enzymeꢀinhibition mechanism. To examine the inhibition mechanism
of the most promising hMAOꢀB inhibitors (compounds 20 and 27), kinetic experiments
were performed. For this purpose, the initial rates of the MAOꢀBꢀcatalyzed oxidation of
pꢀtyramine at five different substrate concentrations, in the absence or presence of the
selected chromone inhibitors at different concentrations, were measured. The results are
depicted in Figure 4. Graphical analyses of the reciprocal Lineweaver−Burk plots
allowed the determination of Michaelis−Menten reaction kinetic parameters (Michaelis
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
constant, K
m
and maximum velocity, Vmax). Concerning compound 20, it was found that
increased. The Lineweaver−Burk plots for
the Vmax remained unchanged while K
m
different concentrations of 20 (Figure 4A) were linear and intersected at the yꢀaxis,
indicating that this compound acted as a competitive IMAOꢀB. As for compound 27, the
m
K remained almost constant at different concentrations of the inhibitor whereas Vmax
decreased. The LineweaverꢀBurk plots obtained for different concentrations of
compound 27 (Figure 4B) displayed a series of converging lines on the same point of
the xꢀaxis (1/[S]), profiling a nonꢀcompetitive inhibition mechanism.
2
500
2000
500
000
00
B
2500
000
500
A
2
1
1
1000
500
1
100
1
00
No Inhibitor
0.8 nM
5
No Inhibitor
0.5 nM
80
-0.5
0.5
1.0
1.5
80
-500
[I] (nM)
-0.5
0.0
0.5
1.0
1.5
[
I] (nM)
60
40
1 nM
1 nM
6
0
40
20
20
-0.01
0.01
0.02
0.03
-1)
0.04
-0.01
0.01
0.02
0.03
0.04
-20
-20
1/[p-tyramine] (µM-1)
1/[p-tyramine] (µM
Figure 4 ꢀ Kinetic studies on the mechanism of hMAOꢀB inhibition by chromones 20 (Figure 4A) and 27
Figure 4B). The effect of the inhibitors on the enzyme was determined from the double reciprocal plot of
(
1/rate (1/V) versus 1/substrate concentration in presence of varying concentrations of the inhibitors. The
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Ki values were calculated by the intersection of the curves obtained by plotting 1/V versus the inhibitor
concentration for each substrate concentration (Dixon plots insets on the top left).
From the Dixon plots, obtained from the replots of the slopes of the Lineweaver–Burk
plots vs inhibitor concentrations (upper left corner), the hMAOꢀB inhibition binding
affinities, determined as inhibition constants (Ki), were calculated. Chromones 20
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
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(Figure 4A) and 27 (Figure 4B) displayed Ki values of 0.79 and 0.67 nM, respectively.
The estimated Ki values correlated well with the inhibition mechanism suggested by the
kinetic experiments: compound 20 (IC50 = 0.40 nM) displayed IC50 and Ki values
slightly different but within the picomolar range, and compound 27 (IC50 = 0.67 nM), as
is a nonꢀcompetitive inhibitor, showed a Ki equal to its IC50. Compound 27 can bind to
the allosteric site distorting the 3D tertiary structure of the enzyme.
The reversibility of MAOꢀB inhibition by chromones 20 and 27 was then assessed by
timeꢀdependent inhibition studies. The behavior of standard irreversible ((R)ꢀ(−)ꢀ
deprenyl and rasagiline) and reversible (safinamide) inhibitors was also evaluated under
the same experimental conditions. hMAOꢀB activity (% of control) was measured along
a 60 min incubation with the enzyme inhibitors (Figure 5).
A
B
8
0
0
7
0
(
R)-(-)-Deprenyl
Chromone 20
Chromone 27
6
Rasagiline
Safinamide
60
40
50
20
40
0
0
20
40
60
0
20
40
60
t (min)
t (min)
Figure 5. Timeꢀdependent inhibition of recombinant human MAOꢀB by standard compounds (R)ꢀ(−)ꢀ
deprenyl (50 nM), safinamide (40 nM) and rasagiline (200 nM) (Figure 4A) and test compounds 20 (0.7
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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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
nM), and compound 27 (2 nM) (Figure 4B). The remaining activity was expressed as % of activity. Data
are the mean ± S.D. of three different experiments.
The analysis of timeꢀdependent enzyme inhibition studies performed with the
irreversible inhibitors (Figure 5A) showed that the enzyme residual activity decayed
continuously after the first 15 minutes of incubation, which is consistent with
irreversible enzymatic inhibition. In case of the reversible inhibitor safinamide (Figure
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
5A) an enhancement on enzymatic activity was observed along the analysis time. The
same behavior was observed for chromones 20 and 27 (Figure 5B), which points
towards reversible MAOꢀB inhibition.
Modeling studies. The hMAOs binding modes of the most potent inhibitors, molecular
modeling studies were carried out. Docking experiments were performed for
compounds 20 and 27 with respect to hMAOꢀA and hMAOꢀB theoretical receptor
models (see Experimental section). Both targets were recognized by the chromone
based inhibitors by means of two opposite binding modes: a) with the chromone
scaffold directed to the flavin adenine dinucleotide (FAD) cofactor (BMA) or b)
towards the entrance gorge (BMB). Excluding 27 into the hMAOꢀA, whose BMB was
predicted as best pose all best ranked complexes reported a BMA ligand
accommodation. The graphical inspection of all sampled poses revealed BMA and
BMB of 20 and 27 in both hMAOs (Table S1, Experimental section). All available
docking score functions were in agreement to the experimentally observed hMAOꢀB
selectivity. However, no correlation was observed comparing compounds’ 20 and 27
different affinities. Actually, even if docking simulation suggested for both compounds
a similar affinity with respect to the same target, 27 was predicted as more active than
20. With the aim to progress in our analysis, ligand free targets (LF), the best poses
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(TC) of 20 and 27 in both targets and the top ranked configuration opposite (SC) to the
corresponding TC were submitted to explicit water solvent molecular dynamics
simulation (Table S1, Figure S1, Experimental section). Firstly, the number of solvent
water molecules available into the LF active sites and displaced by ligands TC and SC
recognition was evaluated taking into account the molecular dynamics (MD) starting
structures (Table 4).
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Table 4. Number of hMAOs active sites water molecules displaced by compounds 20 and 27 recognition
in MD starting structures.
Target
Compound Recognition Displaced Waters
TC
SC
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Such a preliminary analysis, and according to experimental selectivity data, addressed a
desolvation penalty to hMAOꢀA recognition. Actually, both inhibitor binding modes
always required the displacement of a larger number of water molecules in hMAOꢀA
with respect to hMAOꢀB. Then, the perturbations of hMAOs conformational properties
were investigated by comparing MD generated trajectories of the targetsꢀinhibitors
complexes with respect to the corresponding target ligand free (see Experimental
section). The resulting RMSd matrix (Figure S2, Supplementary information) attained
for both inhibitors clearly showed wider hMAOꢀA than hMAOꢀB distortions. Taking
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into account that previous observation not appeared strictly dependent from the binding
mode, an involvement of ligand steric hindrance in the isoform selectivity could be
suggested. In fact, hMAOꢀA and hMAOꢀB active sites show similar primary structure
but the former exhibits a monopartite substrate cavity whereas the second one has a
larger and bipartite binding cleft. The differences in terms of shape and volume could
reduce the hMAOꢀB perturbation induced by our ligands recognition. With the aim to
understand the role of the chromone inhibitors exocyclic aromatic nucleous substituents
and their contribution to the target binding, a graphical inspection of MD trajectories
was performed (Figure 6). This approach disclosed the favorable effect of the solvent in
target recognition. In all trajectories, compounds 20 and 27 established stable water
bridged hydrogen bonds to the hMAOs. In the case of compound 20 the water bridges
involved also chlorine atom located on the exocyclic aromatic substituent. Such an atom
also revealed target direct halogen bonds. Moreover, the MD frames graphical
inspection highlighted a key role of stacking in complexes stabilization. In hMAOꢀA,
Tyr407 established a πꢀπ interaction with both TC and SC ligand orientation. The same
contribution was also observed for hMAOꢀA Phe208 and, with lower frequency, for
Tyr444. In hMAOꢀB, solvent molecules improved the ligands recognition, more
remarkably than in hMAOꢀA: water bridges between chromone moiety and Tyr435 or
Phe102 or between amide sp2 oxygen and Thr201 were observed in 20 TC and 27 SC,
respectively. Notably, the compound 20 SC binding mode in hMAOꢀB showed a wide
interaction network involving isoform exclusive residues: the ligand established
hydrogen bond to Cys172 and stacking contacts to Tyr326 and Phe208 that in hMAOꢀA
are replaced by Asn181, Ile335 and Ile199 respectively. Moreover during the MD
simulation a water bridge was underlined between compound 20 chlorine atom and
Tyr188 side chain.
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Figure 6. Last MD trajectory frames of 20 and 27 interacting to hMAOꢀA and hMAOꢀB. Ligands are
depicted in polytube, FAD cofactor in spacefill, ligand interacting residues in wireframe and water
molecules in ball and sticks notation. Chemical structures are CPK colour compliant. Cyan, purple or
yellow dotted lines represent stacking contacts, halogen or hydrogen bonds respectively.
Compound 27 reported similar interactions with respect to 20: stacking contacts were
observed with hMAOꢀB Tyr326 and Ile199. Probably due to its steric hindrance, larger
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than 20, and due to the lack of the halogen atom, its hMAOꢀB recognition was less
stable and its location within the active site was less deep preventing the hydrogen bond
to Cys172.
Overall, the acquired data pointed out that chromoneꢀ3ꢀphenylcarboxamides should not
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be considered as panꢀassay interference compounds. In fact, they are not MAO
promiscuous ligands, as they behaved as selective MAOꢀB inhibitors with absent or
negligible MAOꢀA activity. Furthermore, as shown for the most promising chromones,
they did not behave as covalent inhibitors, since MAOꢀB inhibition was a reversible
process. Furthermore, a good correlation between biological data and molecular docking
experiences was attained.
Evaluation of the cytotoxicity. The cytotoxicity of the most promising compounds 20
and 27 was evaluated by the determination of the cellular viability in human
neuroblastoma cells (SHꢀSY5Y cell line), after 24h incubation period and at two
different concentrations (1 µM and 10 µM). Cellular viability was estimated using the
methylthiazolyldiphenylꢀtetrazolium bromide (MTT) reduction and the neutral red (NR)
uptake assays. Metabolically active cells reduce MTT to formazan, which can be
spectrophotometrically quantified, providing a direct measure of mitochondrial
28
function. The NR uptake assay is based on the ability of viable cells to incorporate and
bind the supravital dye neutral red in the lysosomes, thus providing a quantitative
estimation of the number of viable cells in culture. Control experiments were performed
for both viability endpoints by adding vehicle (serum free medium with 0.1% DMSO)
instead of the compound solution. The results obtained are shown in Figure 7. In the
MTT reduction assay, compound 20 caused a significant decrease on cellular viability,
when compared to control groups (101.6 ± 11.7 %), for both concentrations (43.70 ±
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5.98 % at 10 µM and 64.31 ± 5.96 % at 1 µM, p < 0.001) (Figure 7A). For compound
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7 no significant decrease on cellular viability was detected at 1 µM and only a slight
decrease on the cellular viability was observed at 10 µM (74.55 ± 7.08 %, p < 0.01)
Figure 7A). However, no significant decrease of the cellular viability for both
(
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compounds´ concentrations was observed when considering the NR uptake endpoint
(Figure 7B).
B
A
1
50
150
100
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CTRL
uM
10 uM
CTRL
1
1
1
µM
100
0 µM
**
*
**
*
**
5
0
0
0
2
0
27
20
27
Figure 7. Cell viability of human neuroblastoma SHꢀSY5Y cells after a 24h exposure to chromones 20
and 27 at 1 and 10 µM as determined in the MTT reduction (A) and the NR uptake (B) assays. Untreated
cells were used as control. ** p < 0.01 and *** p < 0.001.
Since the main effects were observed in the MTT reduction assay, the preliminary
toxicological screening under the current experimental conditions points towards
potential effects of 20 on mitochondrial function even at low concentrations (1 µM),
which may be indicative of a potential drugꢀinduced toxicity. Compound 27 showed
only a mild mitochondrial effect at the highest concentration, suggesting a wider safety
window.
Overall, the pharmacological and toxicological data acquired so far sustain the project
progression wherein additional preclinical studies will be performed.
CONCLUSIONS
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We successfully reported the optimization of the Nꢀphenylchromoneꢀ3ꢀcarboxamide
scaffold for the development of selective and reversible MAOꢀB inhibitors with
subnanomolar potency. Structural optimization and SAR studies strengthen the
importance of the amide spacer and the direct linkage of the carbonyl group to the γꢀ
pyrone ring. While oꢀsubsituents in the exocyclic ring generally led to a decrease on
inhibitory potency, the presence of substituents in meta and para positions significantly
enhanced MAOꢀB inhibition. The most potent and selective derivatives were Nꢀ(3’ꢀ
chlorophenyl)ꢀ4ꢀoxoꢀ4Hꢀchromeneꢀ3ꢀcarboxamide (20) (MAOꢀB IC50 = 0.40 nM), a
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competitive
IMAOꢀB
and
Nꢀ(3’,4’ꢀdimethylphenyl)ꢀ4ꢀoxoꢀ4Hꢀchromeneꢀ3ꢀ
carboxamide (27) (MAOꢀB IC
5
0 = 0.67 nM), a nonꢀcompetitive IMAOꢀB. Both acted as
reversible MAOꢀB inhibitors with remarkable potency within the subnanomolar range
and showed a drugꢀlike profile similar to that of standard inhibitor safinamide.
Computational docking studies provided insights into the inhibitors’ interaction with the
enzyme binding site and a rationale for their high potency. Compound 27 can be
highlighted due to its wide safety range, as determined by preliminary cytotoxicity
screenings in human neuroblastoma SHꢀSY5Y cells. Due to its appropriate
physicochemical profile, which points towards BBB permeability, compound 27 is a
valid candidate for subsequent animal studies and for the development of promising
drug candidates for the therapy of neurodegenerative diseases.
EXPERIMENTAL SECTION
Chemistry. Reagents and general conditions. Chromoneꢀ3ꢀcarboxylic acid, 3ꢀ
formylchromone, benzotriazolꢀ1ꢀyloxy)tripyrrolidinophosphonium hexafluorophosphate
(PyBOP), pyridine, malonic acid, potassium carbonate, sodium sulphate, N,Nꢀ
diisopropylethylamine (DIPEA), dimethylformamide (DMF), 1,8ꢀdiazabicycloundecꢀ7ꢀ
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ene (DBU), phosphorus oxychloride (POCl ), aniline and its derivatives were purchased
3
from SigmaꢀAldrich Química S.A. (Sintra, Portugal). All other reagents and solvents
were pro analysis grade and were acquired from Carlo Erba Reagents and Scharlab. and
were used without additional purification.
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Thinꢀlayer chromatography (TLC) was carried out on precoated silica gel 60 F254
(Merck) with layer thickness of 0.2 mm. For analytical control the following systems
were used: ethyl acetate/petroleum ether, ethyl acetate/methanol, chloroform/methanol,
diethyl acetate in several proportions. The spots were visualized under UV detection
(254 and 366 nm) and iodine vapor. Flash chromatography was performed using silica
gel 60 0.2ꢀ0.5 or 0.040ꢀ0.063 mm (Carlo Erba Reagents). Following the workup, the
organic phases were dried over Na SO . Solutions were decolorized with activated
2
4
charcoal, when necessary. The recrystallization solvents were ethyl acetate,
dichloromethane or ethyl ether/nꢀhexane. Solvents were evaporated in a Buchi
Rotavapor.
The purity of the final products (>97% purity) was verified by highꢀperformance liquid
chromatography (HPLC) equipped with a UV detector. Chromatograms were obtained
in an HPLC/DAD system, a Jasco instrument (pumps model 880ꢀPU and solvent mixing
model 880ꢀ30, Tokyo, Japan), equipped with a commercially prepacked Nucleosil RPꢀ
18 analytical column (250 mm x 4.6 mm, 5 µm, MachereyꢀNagel, Duren, Germany),
and UV detection (Jasco model 875ꢀUV) at the maximum wavelength of 254 nm. The
mobile phase consisted of a methanol/water or acetonitrile/water (gradient mode, room
temperature) at a flow rate of 1 mL/min. The chromatographic data was processed in a
Compaq computer, fitted with CSW 1.7 software (DataApex, Czech Republic).
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Apparatus. NMR data were acquired, at room temperature, on a Brüker AMX 400
1
13
spectrometer operating at 400.15 MHz for H and 100.62 MHz for C. Chemical shifts
were expressed in δ (ppm) values relative to tetramethylsilane (TMS) as internal
reference; coupling constants (J) were given in Hz. Assignments were also made from
DEPT (Distortionless Enhancement by Polarization Transfer) (underlined values).
Electron impact mass spectra (EIꢀMS) were carried out on a VG AutoSpec instrument;
the data were reported as m/z (% of relative intensity of the most important fragments).
Microwaveꢀassisted synthesis was performed in a Biotage Initiator Microwave
Synthesizer. Melting point data were obtained in Stuart Scientic Melting Point SMP1.
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A) Synthesis of chromone carboxamides (2ꢀ9, 11ꢀ29), ester (30) and thioester (31).
To a solution of chromoneꢀ3ꢀcarboxylic acid (1, 2.6 mmol) in DMF (4 mL) POCl (2.6
3
mmol) was added. The mixture was stirred at room temperature for 30 min for the in
situ formation of the acyl chloride. Then, an aromatic amine with the desired aromatic
pattern (for compounds 2ꢀ9, 11ꢀ29), 4ꢀmethylphenol (for compound 30), 4ꢀ
methylbenzenethiol (for compound 31) were added to the reaction. After 1ꢀ5 hours, the
mixture was diluted with dichloromethane (20 mL), washed with H O (2 x 10 mL) and
2
3
with saturated NaHCO solution (2 x 10 mL). The organic phase was dried, filtered and
concentrated under reduced pressure. The crude product was purified by flash
chromatography and/or crystallization.
N
ꢀ(4’ꢀBromophenyl)ꢀ4ꢀoxoꢀ4
H
ꢀchromeneꢀ3ꢀcarboxamide (6). The compound was
1
obtained in 45% yield and recrystallized from DMF: mp 278−280 °C. H NMR (CDCl )
3
δ = 7.50 – 7.45 (2H, m, H3’, H5’), 7.55 (1H, ddd, J = 8.1, 7.2, 1.0 Hz, H6), 7.61 (1H,
dd, J = 8.5, 0.6 Hz, H8), 7.68 – 7.63 (2H, m, H2’, H6’), 7.81 (1H, ddd, J = 8.7, 7.2, 1.7
13
Hz, H7), 8.34 (1H, dd, J = 8.0, 1.4 Hz, H5), 9.07 (1H, s, H2), 11.47 (1H, s, NH).
C
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NMR (CDCl ): 115.8 (C3), 117.1 (C4’), 118.6 (C8), 122.1 (C2’, C6’), 124.0 (C4a),
3
126.3 (C6), 126.6 (C5), 132.0 (C3’, C5’), 135.0 (C7), 137.1 (C1’), 156.2 (C8a), 160.8
+
+
(
CONH), 162.9 (C2), 177.4 (C4). MS/EI m/z (%): 345 (M +2, 31), 343 (M , 29), 173
(100), 121 (23).
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N
ꢀ(4’ꢀIodophenyl)ꢀ4ꢀoxoꢀ4
H
ꢀchromeneꢀ3ꢀcarboxamide (7). The compound was
1
obtained in 16% yield and recrystallized from DMF: mp 277−280 °C. H NMR (CDCl )
3
δ = 7.60 – 7.53 (3H, m, H6, H3’, H5’), 7.63 (1H, d, J = 8.5 Hz, H8), 7.72 – 7.66 (2H, m,
H2’, H6’), 7.83 (1H, ddd, J = 8.6, 7.2, 1.6 Hz, H7), 8.36 (1H, dd, J = 8.0, 1.5 Hz, H5),
1
3
9.09 (1H, s, H2), 11.48 (1H, s, NH). C NMR (CDCl
3
): δ = 87.9 (C4’), 116.0 (C3),
118.7 (C8), 122.5 (C2’, C6’), 124.2 (C6), 126.4 (C5), 126.8 (C4a), 135.2 (C7), 137.2
(C1’), 138.1 (C3’, C5’), 156.3 (C8a), 161.0 (CONH), 163.1 (C2), 177.6 (C4). MS/EI
+
m/z (%): 390 (M , 93), 173 (100), 121 (25).
N
ꢀ(4’ꢀFluorophenyl)ꢀ4ꢀoxoꢀ4
H
ꢀchromeneꢀ3ꢀcarboxamide (8). The compound was
1
obtained in 23% yield and recrystallized from DMF: mp 254−257°C. H NMR (CDCl )
3
δ = 7.11 – 7.02 (2H, m, H3’, H5’), 7.55 (1H, ddd, J = 8.1, 7.3, 1.0 Hz, H6), 7.61 (1H,
dd, J = 8.5, 0.5 Hz, H8), 7.75 – 7.67 (2H, m, H2’, H6’), 7.80 (1H, ddd, J = 8.7, 7.2, 1.7
13
Hz, H7), 8.34 (1H, dd, J = 8.0, 1.4 Hz, H5), 9.08 (1H, s, H2), 11.40 (1H, s, NH).
C
NMR (CDCl ): δ = 115.7 (d, JFC = 22.5 Hz, C3’, C5’), 115.9 (C3), 118.6 (C8), 122.2 (d,
3
JFC = 7.8.5 Hz, C2’, C6’), 124.1 (C4a), 126.3 (C6), 126.6 (C5), 134.1 (d, JFC = 2.8 Hz,
C1’), 135.0 (C7), 156.2(C8a), 159.5 (d, JFC = 244.7 Hz, C4’), 160.7 (CONH), 162.9
+
+
(C2), 177.5 (C4). MS/EI m/z (%): 384 (M +1, 31), 383 (M , 94), 174 (43), 173 (100),
121 (81).
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