Alkynyl-coumarinyl ethers as MAO-B inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.01.046

In this study, alkynyl-coumarinyl ethers were developed as inhibitors of human monoamine oxidase B (MAO-B). A series of 31 new, ether-connected coumarin derivatives was synthesized via hydroxycoumarins, whose phenolic group at position 6, 7 or 8 was converted by means of the Mitsunobu reaction. The majority of the final products were produced from primary alcohols with a terminal alkyne group. The inhibitors were optimized with respect to the structure of the alkynyloxy chain and its position at the fused benzene ring as well as the residue at position 3 of the pyran-2H-one part. A hex-5-ynyloxy chain at position 7 was found to be particular advantageous. Among the 7-hex-5-ynyloxy-coumarins, the 3-methoxycarbonyl derivative 36 was characterized as a dual-acting inhibitor with IC₅₀ values of less than 10 nM towards MAO-A and MAO-B, and the 3-(4-methoxy)phenyl derivative 44 was shown to combine strong anti-MAO-B potency (IC₅₀ = 3.0 nM) and selectivity for MAO-B over MAO-A (selectivity >3400-fold).

Full text of DOI:10.1016/j.bmc.2014.01.046

Bioorganic & Medicinal Chemistry 22 (2014) 1916–1928
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Alkynyl–coumarinyl ethers as MAO-B inhibitors
⇑
⇑
Matthias D. Mertens, Sonja Hinz, Christa E. Müller , Michael Gütschow
Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, alkynyl–coumarinyl ethers were developed as inhibitors of human monoamine oxidase B
(MAO-B). A series of 31 new, ether-connected coumarin derivatives was synthesized via hydroxycouma-
rins, whose phenolic group at position 6, 7 or 8 was converted by means of the Mitsunobu reaction. The
majority of the final products were produced from primary alcohols with a terminal alkyne group.
The inhibitors were optimized with respect to the structure of the alkynyloxy chain and its position at
the fused benzene ring as well as the residue at position 3 of the pyran-2H-one part. A hex-5-ynyloxy
chain at position 7 was found to be particular advantageous. Among the 7-hex-5-ynyloxy-coumarins,
the 3-methoxycarbonyl derivative 36 was characterized as a dual-acting inhibitor with IC50 values of
less than 10 nM towards MAO-A and MAO-B, and the 3-(4-methoxy)phenyl derivative 44 was
shown to combine strong anti-MAO-B potency (IC50 = 3.0 nM) and selectivity for MAO-B over MAO-A
(selectivity >3400-fold).
Received 31 October 2013
Revised 20 January 2014
Accepted 23 January 2014
Available online 8 February 2014
Keywords:
Alkynes
Coumarins
Distribution coefficients
Mitsunobu reaction
Monoamine oxidase
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Both isoforms of MAO are important targets for drug discov-
ery and development. Tranylcypromine represents an irreversible
The degradation of certain neurotransmitters as well as dietary
and xenobiotic amines is catalyzed by monoamine oxidase (MAO,
EC 1.4.3.4). MAO is a flavin-dependent enzyme found in the outer
mitochondrial membrane of neuronal, glial and all other mamma-
lian cells. Two isoforms of monoamine oxidase are known, MAO-A
and MAO-B. Both isoforms share about 70% of their amino acid
identity, but they differ in their tissue distribution, substrate and
inhibitor preferences.1 While MAO-A is ubiquitously present in hu-
man tissues, MAO-B predominantly resides in the brain, in partic-
ular in the basal ganglia. MAO-B preferentially catalyzes the
deamination of 2-phenylethylamine, and MAO-A favorably oxi-
dizes serotonin; while dopamine is a substrate for both isoforms.2
During aging, the expression of MAO-B increases, and MAO-B is be-
lieved to be involved in aging-related neurodegenerative diseases.
The increased expression is connected with an enhanced dopamine
metabolism. Hydrogen peroxide is produced during the MAO-cat-
alyzed deamination reaction, resulting in oxidative damage and
apoptotic signaling events.3 X-ray crystal structures of MAO-A
and MAO-B from different species and cocrystallized with various
ligands are available and provided a closer insight into the active
site of the two enzymes.1
inhibitor for both isoforms. Its lack of selectivity and the unde-
sired ‘cheese effect’ have led to a limited use, restricted to the
treatment of therapy-resistant depression.4 Fluorinated ana-
logues of this drug showed better selectivity and expectedly
fewer side effects.5,6 Moclobemide, a reversible MAO-A inhibitor,
is currently used in the clinic against depression and anxiety.
MAO-B inhibitors are frequently administered in the therapy of
neurodegenerative disorders such as Alzheimer’s and Parkinsons
diseases. Two irreversible MAO-B inhibitors, that is, rasagiline
and selegiline (1, Fig. 1) are successfully applied in the treatment
of Parkinson’s disease.7 Another promising MAO-B inhibitor, safi-
namide (2), has been evaluated in phase III clinical trials with a
positive outcome.8 This reversible inhibitor additionally blocks
dopamine reuptake.9 Several dual-acting MAO inhibitors have
been developed which address a second target. Inhibition of ace-
tylcholinesterase, for example, leads to a prolonged action of
acetylcholine in the synaptic cleft and proved to be beneficial
in Alzheimer’s disease.10–14 For the treatment of Parkinson’s
disease, xanthine and benzothiazinone derivatives have been de-
scribed combining MAO-B inhibition and adenosine A2A receptor
antagonism.15,16
The simple molecular scaffold of the coumarin (benzopyran-2-
one) has attracted much attention in drug research because of
the variety of biological activities of its representatives. Coumarins
are known to act as inhibitors or modulators on various target
⇑
Corresponding authors. Tel.: +49 228 732301; fax: +49 228 732567 (C.E.M.);
tel.: +49 228 732317; fax: +49 228 732567 (M.G.).
E-mail addresses: christa.mueller@uni-bonn.de (C.E. Müller), guetschow@
uni-bonn.de (M. Gütschow).
http://dx.doi.org/10.1016/j.bmc.2014.01.046
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

M. D. Mertens et al. / Bioorg. Med. Chem. 22 (2014) 1916–1928
Me
1917
2. Results and discussion
Me
N
NH2
N
H
A small library of substituted hydroxycoumarins (13–22) was
prepared as outlined in Scheme 1 and Table 1. Different established
methods were applied to convert salicylaldehydes or resorcinol to
coumarins. Starting from commercially available salicylaldehydes
9–11, Knoevenagel condensation with either dimethyl malonate
or ethyl 3-(4-methoxyphenyl)-3-oxopropanoate as reactive meth-
ylene compounds and a catalytic amount of piperidine afforded
coumarins 13–17 in good yields. Perkin condensation with N-ace-
tylglycine or 4-methoxyphenylacetic acid in acetic anhydride gave
the corresponding acetoxy precursors, whose deprotection under
basic conditions furnished the hydroxycoumarins 18–20. Umbellif-
erone (21) was available through a one-pot Wittig protocol. Pech-
mann condensation of ethyl acetoacetate and resorcinol (12) in the
presence of concentrated sulfuric acid resulted in 4-methylumbel-
liferone (22) in good yield.
F
O
O
Me
1
2 (Safinamide)
(Selegiline)
Me
H
N
Me
Me
O
Et
O2S
Cl
O
O
O
O
O
3
(Esuprone)
4
(NW-1772)
Me
O
Me
O
NH2
N
H
F
F
O
O
O
O
5
6
Me
With these substituted hydroxycoumarins in hand, several final
products (24–52) were synthesized (Scheme 2 and Table 2). Four
different primary alcohols with a terminal alkyne group (to pro-
duce 24–29, 33–44) and five other alcohols (to produce 45–52)
were subjected to the Mitsunobu reaction using PPh3 and diethyl
azodicarboxylate (DEAD) or diisopropyl azodicarboxylate (DIAD)
in tetrahydrofuran (THF). Most of the reactions proceeded
smoothly within two hours at room temperature, and recrystallisa-
tion furnished the coumarinyl ethers in high purity and moderate
to good yields. Similarly, 55 was prepared via Mitsunobu reaction
with the non-coumarin phenol 23. Cleavage of the methyl ester of
26 with lithium hydroxide gave the free carboxylic acid 30, which
was coupled to 4-fluoroaniline or Boc-piperazine with N-[(dimeth-
ylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-
methylmethanaminium hexafluorophosphate N-oxide (HATU) in
the presence of diisopropylethylamine (DIPEA), affording the
amide-type coumarins 31 and 32 (Scheme 2).
The synthesis of two aza-analogues 53 and 54, in which a meth-
ylene unit of the alkynyloxy substituent of 36 and 42 is replaced by
NH, is depicted in Scheme 3. The route started again with a Mitsun-
obu reaction of hydroxycoumarins (13 and 21) and 2-bromoethanol,
followed by alkylation of 2-nitro-N-(prop-2-ynyl)benzenesulfon-
amide in the presence of potassium carbonate. Thiophenol-medi-
ated nosyl deprotection furnished the free amines (53 and 54) in
good yields.
Me
SO2
O
Me
N
H
O
O
O
O
7
8
Figure 1. Structures of known MAO inhibitors including those with coumarin
scaffold.
structures; for example, derivatives have been developed that
possess anticoagulant or antiproliferative activities.17,18 Certain
coumarins act as inhibitors of aromatase,19 17b-hydroxysteroid
dehydrogenase 1,20 or acetylcholinesterase.21,22 Furthermore, cou-
marins have been described as GABAA receptor modulators,23 as
cannabinoid receptor ligands,24,25 or as ligands at other G pro-
tein-coupled receptors.26 Thus, the coumarin scaffold can be re-
garded as a privileged structure in medicinal chemistry.25 The
facile synthetic accessibility and the versatile possibilities to mod-
ify the coumarin core have contributed to the wide utilization of
this heterocycle. Noteworthy, coumarins or coumarin-like struc-
tures have been utilized for the design of activity-based probes
for MAO enzymes,27–30 and differently substituted coumarins have
been synthesized or were isolated from natural sources and evalu-
ated for their potential as MAO inhibitors, showing selective inhi-
bition of either MAO-A or MAO-B isoforms from the micromolar to
the picomolar range (Fig. 1).31,32 Depending on their substitution
pattern, the selectivity towards the two isoforms can be modu-
lated. For example, coumarins with a sulfonic ester moiety at-
tached to the 7-position exhibit a preferred affinity for MAO-A,
as can be seen in esuprone (3).33 For MAO-B, halo-substituted ben-
zyloxy residues in position 7 of the coumarin core, as in 4 and 5, are
well accepted.34,35 Such a substitution pattern is, however, not
essential, as the carbohydrazide 6 revealed high potency and
MAO-B selectivity.36 Aryl substituents at position 3, either directly
connected to the coumarin core (7)37 or via amide linker (8)38
caused strong activity against MAO-B.
Coumarins 24–55 were investigated for inhibition of MAO-A
and MAO-B using human recombinant enzymes. The Amplex Red
monoamine oxidase assay was performed. The inhibitory potencies
of the coumarins are listed in Table 2. Except for the inhibitor 24,
all compounds, if active at all, displayed a higher affinity for
MAO-B than for MAO-A. First, the impact of the chain length on
biological activity was examined on the basis of compounds with
CHO
methodsA-E
method F
HO
R2
O
OH
R3
O
6
7
9-11
HO
The present study was performed to develop alkynyl–coumari-
nyl ethers as MAO-B inhibitors. Thus, coumarins bearing an alky-
8
nyloxy substituent with
a terminal triple bond have been
13 22
-
synthesized and evaluated for their inhibitory profile towards both
human MAO isoforms. We attempted to optimize the inhibitors
with respect to the structure of the alkynyloxy chain and its posi-
tion at the fused benzene ring as well as the residue at position 3 of
the pyran-2H-one part. Among the 31 new coumarin derivatives,
several exhibited strong affinity to the MAO enzymes. Compound
44 was identified to be a highly potent and particularly selective
inhibitor of MAO-B.
OH
HO
12
Scheme 1. Synthesis of hydroxycoumarins 13–22 (for structures see Table 1).
Reagents and conditions: method A, dimethyl malonate, piperidine, MeOH; method
B, ethyl 3-(4-methoxyphenyl)-3-oxopronoate, piperidine, MeOH; method C, (i)
N-acetylglycine, NaOAc, Ac2O; (ii) K2CO3, MeOH; method D, (i) 4-methoxyphenyl-
acetic acid, pyridine, Ac2O; (ii) K2CO3, MeOH; method E, PPh3, ethyl bromoacetate,
EtOH, KOH; method F, ethyl acetoacetate, H2SO4.

1918
M. D. Mertens et al. / Bioorg. Med. Chem. 22 (2014) 1916–1928
Table 1
compounds were included in the study for comparative evaluation.
Synthesized hydroxycoumarins 13–22
Their design was based on known coumarin-type MAO inhibitors,
for example, coumarins with halo-substituted arylalkoxy
chains,10,34–36,38 as realized in 45 and 47. The preparation of 50
was prompted by the geranyl structure of auraptene, a coumarin
derivative from Dictamnus albus and the related natural product
geiparvarin from Geijera parviflora, both processing anti-MAO
activities.39,40 The coumarin core was decorated with a hexyloxy
chain (in 52) in order to verify the impact of the triple bond by
comparison with the hex-5-ynyloxy derivative 36. Compound 53
represents an aza-analogous structure of 36 with improved water
solubility. Among this subseries of six 7-substituted coumarin
3-methyl esters, inhibitors 36, 45, 47 and 52 exhibited IC50 values
of less than 2 nM. Compounds 45, 47, 50 and 52 showed remark-
able MAO-B selectivity. Thus, to achieve potent and selective
coumarin-type MAO-B inhibitors, the 7-alkynyloxy substituent is
not necessary.
In addition to the synthetic work and the enzyme inhibition as-
says, the lipophilicity of representative coumarins was examined.
The log D7.4 values (Table 2) were experimentally determined by
an HPLC-based procedure.41 As expected, a replacement of the
CH2 unit in 36 (log D7.4 = 2.5) by NH decreased the lipophilicity
of the aza analogue 53 (log D7.4 = 0.5), which was only moderately
active. The aforementioned potent compounds, 36, 45, 47 and 52,
turned out to be relatively lipophilic with log D7.4 values between
2.5 and 4.2. An undesired high log D7.4 value resulted from the ex-
change of the C„CH terminus (36, log D7.4 = 2.5) by CH2CH3 (52,
logD7.4 = 4.2). Although both 6-carbon chains led to highly potent
MAO-B inhibitors, the 7-hex-5-ynyloxy substitution pattern of 36
was maintained for further structural optimization.
Compd
Hydroxy subst.
R2
R3
Method
13
14
15
16
17
18
19
20
21
22
7-
6-
8-
7-
6-
7-
6-
7-
7-
7-
H
H
H
H
H
H
H
H
H
Me
CO2Me
CO2Me
CO2Me
COC6H4-4-OMe
COC6H4-4-OMe
NHCOMe
NHCOMe
C6H4-4-OMe
H
A
A
A
B
B
C
C
D
E
F
H
R2
R2
O
R3
O
6
R3
a
HO
R1O
7
8
O
O
24-29, 33-52
13-22
R3 = CO2Me
R3
26
30
31
b
R3 = CO2H
O
O
O
c
F
O
R3 =
N
H
O
32 R3 =
N
NBoc
To produce molecular diversity at the pyran-2H-one part, com-
pounds 38, 40, 42, 43 have been synthesized, considering struc-
tures of known MAO inhibiting coumarins.14,35,42 Introduction of
the bulky aroyl residue in 38 resulted in a reduction of MAO-B
inhibitory activity but an enhanced selectivity. The acetylamino
derivative 40 was neither potent nor selective. On the other hand,
removal of the methoxycarbonyl residue improved the selectivity
of the corresponding inhibitors 42 and 43. These trends for 7-hex-
5-ynyloxy compounds were confirmed for the corresponding
7-pent-4-ynyloxy analogues (38 and 29 vs 36 and 26; 40 and
33 vs 36 and 26, 42 and 34 vs 36 and 26, 43 and 35 vs 36 and
26). Comparison of pairs of 6-carbon chain and 5-carbon chain
homologues revealed a more advantageous profile of the former
ones (38 vs 29, 40 vs 33, 42 vs 34, 43 vs 35). A final coumarin
modification in the course of this study was carried out by exci-
sion of the exocyclic carbonyl group in 38, leading to 44. Other-
wise, 44 can be regarded to result from a replacement of the
CO2 unit in 36 by a phenylene spacer. In recent years, 3-aryl sub-
stitution in coumarins was established to be suitable for MAO-B
inhibition. In particular, the 4-methoxyphenyl moiety at 3-posi-
tion was combined with small substituents at positions 6 and 8
to achieve potent and MAO-B selective coumarin inhibi-
tors.31,37,42–45 Compound 44 inhibited MAO-B with an IC50 value
of 3.0 nM and showed a more than 3400-fold selectivity over
MAO-A. Thus, in future studies it would be interesting to
decorate the 3-(4-methoxyphenyl)coumarin with other elongated
substituents at 7-position (as present in 45–52). However, the
lipophilicity of 44 (logD7.4 = 4.3) was increased compared to 36.
The hex-5-ynyloxy chain was also placed at the corresponding
position of an appropriate non-coumarin scaffold46 demonstrating
the feasibility of obtaining selective MAO-B inhibitors that way.
The resulting dihydroquinolin-2(1H)-one 55 (logD7.4 = 1.6) had
an IC50 value of 53 nM.
a
HO
N
H
O
O
N
H
O
23
55
Scheme 2. Synthesis of alkynyloxy-substituted coumarins 24–52 and 55 (for
structures see Table 2). Reagents and conditions: (a) R1OH, DEAD or DIAD, PPh3,
THF; (b) LiOH, THF/H2O; (c) 4-fluoroaniline or 1-Boc-piperazine, HATU, DIPEA, DMF.
a fixed pyran-2H-one part (R2 = H, R3= CO2Me). The 7-propargyl-
oxy-substituted compound 24 inhibited both isoforms to the same
extent (IC50 values of ꢀ2
lM). The elongation of the 7-alkynyloxy
chain by methylene units led to a stepwise improvement of the
inhibitory potency (24 vs 25 vs 26 vs 36). This correlation was par-
ticularly impressive for our main target enzyme, MAO-B, where the
exchange of the 3-carbon chain (24) by a 6-carbon chain (36) in-
creased the affinity by three orders of magnitude. The coumarin es-
ter 36 already represented the most potent dual-acting inhibitor of
this study, exhibiting IC50 values of 1.4 and 9.6 nM for MAO-B and
MAO-A, respectively.
Using the example of the 5-carbon chain, its linkage to the ben-
zene ring was analyzed. Whereas the attachment at the 8-position
(28) resulted in a loss of activity, the 7- and 6-substituted isomers
were both slightly selective for MAO-B, but the former was 17-fold
more active at MAO-B (26 vs 27). Thus, in the following inhibitor
design, we focused on an ether-attachment to position 7 of the
coumarin. Further isomeric pairs of 6- and 7-substituted couma-
rins, provided in a later stage of this study, revealed a trend in that
6-isomers showed a better MAO-B selectivity, but 7-isomers were
more potent (36 vs 37; 38 vs 39; 40 vs 41; 47 vs 48; 50 vs 51).
Furthermore, the hex-5-ynyloxy chain of 36 was replaced by
other ether-connected moieties, such as substituted benzyl and
phenethyl, geranyl, hexyl and propargylaminoethyl. The resulting
Currently used MAO-B inhibitors display an irreversible mode
of action. However, reversible inhibition may have advantages
including better controllability. Two selected potent MAO-B

M. D. Mertens et al. / Bioorg. Med. Chem. 22 (2014) 1916–1928
1919
Table 2
MAO-A and MAO-B inhibitory activities of compounds 24–55
R2
O
6
7
R3
O
R1O
8
24 54
-
Compd
Alkoxy subst.
R1
R2
R3
MAO-A IC50 SEMa (nM)
MAO-B IC50 SEMa (nM)
MAO-B selectivityb
24
25
26
27
28
29c
30
31
32
33
34
35
36c
37
38c
39
40
41c
42
43
44c
45c
46
47c
48
49
50
51
52c
53c
54
7-
7-
7-
6-
8-
7-
7-
7-
7-
7-
7-
7-
7-
6-
7-
6-
7-
6-
7-
7-
7-
7-
6-
7-
6-
7-
7-
6-
7-
7-
7-
CH2C„CH
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
COC6H4-4-OMe
CO2H
CONHC6H4-4-F
1570 80
818 36
953 78
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
930 82
3520 270
553 19
9.64 0.84
>10,000
1790 110
258 27
82.4 1.1
1440 10
>10,000
1
3
12
>7
—
>19
—
—
—
21
38
30
7
>81
>150
—
(CH2)2C„CH
(CH2)3C„CH
(CH2)3C„CH
(CH2)3C„CH
(CH2)3C„CH
(CH2)3C„CH
(CH2)3C„CH
(CH2)3C„CH
(CH2)3C„CH
(CH2)3C„CH
(CH2)3C„CH
(CH2)4C„CH
(CH2)4C„CH
(CH2)4C„CH
(CH2)4C„CH
(CH2)4C„CH
(CH2)4C„CH
(CH2)4C„CH
(CH2)4C„CH
(CH2)4C„CH
CH2C6H4-3-F
CH2C6H4-3-Cl
(CH2)2C6H4-4-Cl
(CH2)2C6H4-4-Cl
CH2C6H4-3-Cl
Geranyl
535 60
>10,000
>10,000
ꢁ10,000
CON(C2H4)2N-Boc
NHCOMe
H
43.6 1.9
91.8 9.1
18.2 2.0
1.41 0.15
123 12
H
CO2Me
CO2Me
COC6H4-4-OMe
COC6H4-4-OMe
NHCOMe
NHCOMe
H
>10,000
>10,000
67.1 7.2
>10,000
66.8 2.5
>10,000
367 21
59.9 2.3
>10,000
77.2 7.7
>10,000
74.8 2.4
>10,000
62.7 6.7
ꢁ10,000
>10,000
66.1 1.7
2680 160
ꢁ10,000
41.7 2.5
24.7 2.0
2.62 0.40
0.770 0.074
2.96 0.10
0.588 0.054
31.5 1.9
1.41 0.02
2
>400
140
78
>3400
130
>320
53
>83
5
130
>31
75
H
C6H4-4-OMe
CO2Me
CO2Me
CO2Me
CO2Me
NHCOMe
CO2Me
CO2Me
CO2Me
CO2Me
H
120
3
13.5 1.2
78.9 1.9
325 53
0.875 0.047
377 78
Geranyl
Hexyl
(CH2)2NHCH2C„CH
(CH2)2NHCH2C„CH
7
12
852 27
55c
>10,000
52.6 1.5
>190
O
N
H
O
a
b
c
IC50 value are means from three experiments.
IC50 (MAO-A) divided by IC50 (MAO-B).
LogD7.4 values were determined as follows, 3.1 (29), 2.5 (36), 3.5 (38), 2.4 (41), 4.3 (44), 3.2 (45), 3.9 (47), 4.2 (52), 0.5 (53), 1.6 (55).
inhibitors of the present series were investigated for reversibility
of the inhibitory effect. Thus, MAO-B was preincubated with inhib-
itor, subsequently the substrate p-tyramine (10 lM) was added,
MAO-B, and the 6-isomers to be more selective for MAO-B over
MAO-A. It was found that a hex-5-ynyloxy chain at position 7
was suitable to receive strong MAO-B inhibition. For example,
the dual-acting compound 36 exhibited high affinity towards both
MAO isoforms (IC50 = 1.4 and 9.6 nM for MAO-B and MAO-A),
whereas 44 was the most promising candidate of this series with
respect to both potency and selectivity (MAO-B IC50 = 3.0 nM,
selectivity >3400-fold). Experimentally determined logD7.4 values
indicated that the introduction of the triple bond (as present in
36) significantly reduced the lipophilicity (in comparison to the
saturated analogue 52). When appropriately placed at various
bicyclic scaffolds, the hex-5-ynyloxy chain is considered to be a
valuable substructure for the development of MAO-B inhibitors.
and after 22 min the substrate concentration was increased to
1 mM (Fig. 2). Inhibitors 36 and 44 were studied at concentrations
roughly correlating to their IC80 values. For comparison, the revers-
ible inhibitor safinamide (2), and the irreversible inhibitor selegi-
line (1) were investigated under the same conditions. The large
increase in substrate concentration leads to a displacement of
reversible inhibitors, while no significant change is observed for
irreversible inhibitors. In fact, 36 and 44 behaved like the revers-
ible inhibitor safinamide. Therefore it can be concluded that the
triple bond in the new inhibitors 24–44 and 55 does not lead to
irreversible enzyme inhibition in contrast to the propargyl group
present in the irreversible inhibitor selegiline.
4. Experimental section
3. Conclusion
4.1. General methods and materials
In this study, a stepwise approach to structurally optimized
coumarins as MAO-B inhibitors has been undertaken. A compari-
son of isomeric coumarin pairs with the same ether-connected
chain showed that the 7-isomers tend to be more potent at
Thin-layer chromatography was carried out on Merck alumi-
num sheets, silica gel 60 F254. Detection was performed with UV
light at 254 nm. Preparative column chromatography was
performed on Merck silica gel 60 (70–230 mesh). Melting points

1920
M. D. Mertens et al. / Bioorg. Med. Chem. 22 (2014) 1916–1928
CH3), 7.15 (dd, 1H, 3J = 8.8 Hz, 4J = 2.9 Hz, 7-H), 7.19 (d, 1H,
R3
O
R3
O
a
4J = 2.9 Hz, 5-H), 7.27 (d, 1H, 8-H, 3J = 8.8 Hz), 8.66 (s, 1H, 4-H),
9.86 (br s, 1H, OH); 13C NMR (125 MHz, DMSO-d6) d 53.2, 114.7,
118.0, 118.4, 119.1, 123.6, 148.8, 149.7, 154.9, 157.1, 164.2. Anal.
Calcd for C11H8O5: C, 60.00; H, 3.66. Found: C, 59.87; H, 3.71.
LC–MS (ESI) (90% H2O to 100% MeOH in 10 min, then 100% MeOH
to 20 min, DAD 220–400 nm), 99.9% purity, m/z = 221.20 ([M+H]+).
Br
HO
O
O
O
13 R3 = CO2Me
21
56
R3 = CO2Me
R3 = H
57
R3 = H
NO2
b
c
R3
SO2
N
4.2.2. Methyl 8-hydroxy-2-oxo-2H-chromene-3-carboxylate (15)
2,3-Dihydroxy-benzaldehyde (2.76 g, 20 mmol) was dissolved
in MeOH (30 mL). Dimethylmalonate (2.90 g, 2.51 mL, 22 mmol)
and piperidine (0.17 g, 0.2 mL, 2.0 mmol) were added. The solution
was refluxed for 2 h. The reaction mixture was cooled to 0 °C. The
product crystallized from the reaction mixture was filtered off,
washed with MeOH (20 mL) and dried to give a light yellow solid
(2.71 g, 62%): mp 203–204 °C; 1H NMR (500 MHz, DMSO-d6) d
3.82 (s, 3H, CH3), 7.17–7.21 (m, 2H, arom.H), 7.30–7.32 (m, 1H, ar-
om.H), 8.69 (s, 1H, 4-H), 10.33 (s, 1H, OH); 13C NMR (125 MHz,
DMSO-d6) d 52.5, 117.4, 118.8, 120.3, 120.8, 125.0, 143.4, 144.6,
149.5, 156.0, 163.4. HRMS-ESI m/z [M+Na]+ calcd for C11H8O5Na:
243.0264, found: 243.0269. LC–MS (ESI) (90% H2O to 100% MeOH
in 10 min, then 100% MeOH to 20 min, DAD 220–400 nm), 100%
purity, m/z = 221.20 ([M+H]+).
O
O
O
O
R3 = CO2Me
58
59
R3 = H
R3
O
H
N
O
53 R3 = CO2Me
R3 = H
54
Scheme 3. Synthesis of propargylaminoethoxy-substituted coumarins 53 and 54.
Reagents and conditions: (a) 2-bromoethanol, DEAD, PPh3, THF; (b) 2-nitro-N-
(prop-2-ynyl)benzenesulfonamide, K2CO3, DMF; (c) thiophenol, K2CO3, DMF.
1.8×106
1.5×106
1.3×106
1.0×106
without
inhibitor
4.2.3. 7-Hydroxy-3-(4-methoxybenzoyl)-2H-chromen-2-one
(16)
36 (8 nM)
44 (10 nM)
7.5×105
2,4-Dihydroxybenzaldehyde (2.76 g, 20.0 mmol), ethyl
3-(4-methoxyphenyl)-3-oxopropanoate (4.44 g, 20.0 mmol) and
piperidine (0.43 g, 0.5 mL, 5.0 mmol) were refluxed in MeOH (50 mL)
for 4 h. A yellow solid precipitated and was filtered off, washed
with MeOH (10 mL) to give a yellow powder (3.92 g, 66%): mp
190–191 °C; 1H NMR (500 MHz, DMSO-d6) d 3.84 (s, 3H, CH3), 6.71
(d, 1H, 4J = 2.2 Hz, 8-H), 6.79 (dd, 2H, 4J = 2.2 Hz, 3J = 8.5 Hz, 6-H),
2 (50 nM)
1 (30 nM)
5.0×105
2.5×105
0
0
50 100 150 200 250 300 350
Time (min)
3
7.03 (d, 2H, J = 8.9 Hz, 20,60-H), 7.62 (d, 1H, 3J = 8.5 Hz, 5-H), 7.83
Figure 2. Human MAO-B was preincubated for 1 h with several inhibitors each at
the concentration that approximately represents its IC80 value. The enzyme reaction
was started by the addition of p-tyramine. After 22 min, the substrate concentration
3
(d, 2H, J = 9.2 Hz, 30,50-H), 8.22 (s, 1H, 4-H); 13C NMR (125 MHz,
DMSO-d6, 30 °C) d 55.7, 102.3, 110.3, 114.0, 114.6, 120.8, 129.7,
131.4, 132.0, 146.0, 156.9, 158.7, 163.6, 164.7, 190.5. HRMS-ESI
m/z [M+Na]+ calcd for C17H12O5Na: 319.0577, found: 319.0580.
LC–MS (ESI) (90% H2O to 100% MeOH in 10 min, then 100% MeOH
to 20 min, DAD 220–450 nm), 99.8% purity, m/z = 297.38 ([M+H]+).
was increased from 10
period of 5 h.
lM to 1 mM, and fluorescence was measured over a time
were determined by a Boëtius melting point apparatus from VEB
Wägetechnik Rapido PHMK and are uncorrected. 1H NMR
(500 MHz) and 13C NMR (125 MHz) were recorded on a Bruker
Avance DRX 500. Chemical shifts d are given in ppm referring to
the signal center using the solvent peaks for reference: DMSO-d6
2.49/39.7 ppm. Elemental analyses were performed with a Vario
EL apparatus. LC-DAD chromatograms and ESI-MS spectra were re-
corded on an Agilent 1100 HPLC system with Applied Biosystems
API-2000 mass spectrometer. HRMS-ESI spectra were recorded
on a micrOTOF-Q spectrometer. Solvents and reagents were ob-
tained from Acros (Geel, Belgium), Fluka (Taufkirchen, Germany),
Alfa Aesar (Karlsruhe, Germany), Activate Scientific (Prien, Ger-
many) or Sigma–Aldrich (Steinheim, Germany). Hydroxycouma-
rins 13 and 20 were prepared as described elsewhere.47
Compound 23 was purchased from Activate Scientific (Prien,
Germany).
4.2.4. 6-Hydroxy-3-(4-methoxybenzoyl)-2H-chromen-2-one
(17)
2,5-Dihydroxybenzaldehyde (0.69 g, 5.0 mmol), ethyl 3-(4-
methoxyphenyl)-3-oxopropanoate (1.33 g, 6.0 mmol) and piperi-
dine (0.17 g, 0.2 mL, 2.0 mmol) were refluxed in MeOH (20 mL)
for 2 h. A yellow precipitate was filtered off and washed with
MeOH (10 mL) to give the product as yellow powder (1.34 g,
91%): mp 239–241 °C; 1H NMR (500 MHz, DMSO-d6) d 3.84 (s,
3H, CH3), 6.71 (d, 1H, 4J = 2.2 Hz, 8-H), 6.79 (dd, 2H, 4J = 2.2 Hz,
3
3J= 8.5 Hz, 6-H), 7.03 (d, 2H, J = 8.9 Hz, 20,60-H), 7.62 (d, 1H,
3
3J = 8.5 Hz, 5-H), 7.83 (d, 2H, J = 9.2 Hz, 30,50-H), 8.22 (s, 1H,
4-H); 13C NMR (125 MHz, DMSO-d6) d 55.8, 113.5, 114.2, 117.3,
118.9, 121.5, 127.1, 129.0, 132.2, 144.2, 147.5, 154.2, 158.5,
164.0, 190.3. HRMS-ESI m/z [M+Na]+ calcd for C17H12O5Na:
319.0577, found: 319.0581. LC–MS (ESI) (90% H2O to 100% MeOH
in 10 min, then 100% MeOH to 20 min, DAD 220–400 nm), 100%
purity, m/z = 297.25 ([M+H]+).
4.2. Preparation of hydroxycoumarins 14–19, 21, 22
4.2.1. Methyl 6-hydroxy-2-oxo-2H-chromene-3-carboxylate (14)
2,5-Dihydroxy-benzaldehyde (2.76 g, 20 mmol) was dissolved
in absolute MeOH (30 mL). Dimethyl malonate (2.90 g, 2.51 mL,
22 mmol) and piperidine (0.17 g, 0.2 mL, 2.0 mmol) were added
and the mixture was refluxed for 2 h. The reaction mixture was
cooled to 0 °C. The product was filtered off and washed with cold
EtOH (10 mL) and dried to yield a light brown solid (2.62 g, 60%):
mp 206–208 °C; 1H NMR (500 MHz, DMSO-d6) d 3.81 (s, 3H,
4.2.5. N-(7-Hydroxy-2-oxo-2H-chromen-3-yl)acetamide (18)
2,4-Dihydroxy-benzaldehyde (2.76 g, 20.0 mmol), N-acetylgly-
cine (2.34 g, 20.0 mmol) and anhydrous sodium acetate (4.92 g,
60.0 mmol) were refluxed in Ac2O (100 mL) for 4 h. The reaction
mixture was evaporated in vacuo and the residue was recrystallized
from EtOH (100 mL). The product was filtered off and washed with
cold EtOH (10 mL) to yield 3-acetamido-2-oxo-2H-chromen-7-yl

M. D. Mertens et al. / Bioorg. Med. Chem. 22 (2014) 1916–1928
1921
acetate as a light brown solid (1.77 g, 34%): mp 233–234 °C; 1H
NMR (500 MHz, DMSO-d6) d 2.16 (s, 3H, CH3), 2.29 (s, 3H, CH3),
7.12 (dd, 1H, 4J = 2.2 Hz, 3J = 8.6 Hz, 6-H), 7.25 (d, 1H, 4J = 2.3 Hz,
8-H), 7.72 (d, 1H, 3J = 8.5 Hz, 5-H), 8.60 (s, 1H, 4-H), 9.71 (br s, 1H,
NH); 13C NMR (125 MHz, DMSO-d6) d 21.0, 24.1, 109.8, 117.6,
119.1, 123.3, 124.2, 128.6, 150.0, 151.1, 157.4, 169.0, 170.3. LC–
MS (ESI) (90% H2O to 100% MeOH in 10 min, then 100% MeOH to
20 min, DAD 220–400 nm), 98.1% purity, m/z = 262.23 ([M+H]+).
3-Acetamide-2-oxo-2H-chromen-7-yl acetate (1.31 g, 5.0 mmol),
K2CO3 (1.38 g, 20.0 mmol) were refluxed in MeOH (25 mL) for
1.5 h The reaction mixture was evaporated and solved in water
(25 mL). After acidification with 2 N HCl to pH ꢀ3, the resulting pre-
cipitate was filtered off to yield a light brown solid (0.42 g, 38%): mp
303–304 °C (lit. mp 305 °C);48 1H NMR (500 MHz, DMSO-d6) d 2.16
(s, 3H, CH3), 2.29 (s, 3H, CH3), 7.12 (dd, 1H, 4J = 2.2 Hz, 3J = 8.6 Hz, 6-
H), 7.25 (d, 1H, 4J = 2.3 Hz, 8-H), 7.72 (d, 1H, 3J = 8.5 Hz, 5-H), 8.60 (s,
1H, 4-H), 9.71 (br s, 1H, NH); 13C NMR (125 MHz, DMSO-d6) d 21.0,
24.1, 109.8, 117.6, 119.1, 123.3, 124.2, 128.6, 150.0, 151.1, 157.4,
169.0, 170.3. LC–MS (ESI) (90% H2O to 100% MeOH in 10 min, then
100% MeOH to 20 min, DAD 200–400 nm), 99.1% purity, m/
z = 220.09 ([M+H]+).
(d, 1H, 3J = 8.5 Hz, 5-H), 7.91 (d, 1H, 3J = 9.2 Hz, 4-H), 10.52 (br s,
1H, OH); 13C NMR (125 MHz, DMSO-d6) d 102.3, 111.4, 111.5,
113.3, 129.8, 144.6, 155.7, 160.6, 161.4. LC–MS (ESI) (90% H2O to
100% MeOH in 10 min, then 100% MeOH to 20 min, DAD 220–
400 nm), 98.1% purity, m/z = 163.30 ([M+H]+).
4.2.8. 7-Hydroxy-4-methyl-2H-chromen-2-one (22)
Resorcinol (2.20 g, 20.0 mmol) and ethyl acetoacetate (2.60 g,
2.53 mL, 20.0 mmol) were stirred for 10 min at 90 °C. Concentrated
H2SO4 (0.5 mL) was added to the mixture and stirring was contin-
ued for 1 h. The mixture was then poured onto icewater (50 mL)
and extracted with EtOAc (3 ꢂ 50 mL). The organic phase was dried
over Na2SO4, filtrated and evaporated in vacuo. The residue was
recrystallized from H2O/EtOH (1:1, 30 mL) to give light brown
crystals (2.09 g, 11.9 mmol, 59%): mp 180–182 °C; 1H NMR
(500 MHz, DMSO-d6) d 2.34 (d, 3H, 4J = 1.3 Hz, CH3), 6.10 (d, 1H,
4J = 1.3 Hz, 3-H), 6.68 (d, 1H, 3J = 2.2 Hz, 8-H), 6.78 (dd, 1H,
4J = 2.5 Hz, 3J = 8.7 Hz, 6-H) 7.56 (d, 1H, 3J = 8.5 Hz, 5-H), 10.46 (br
s, 1H, OH); 13C NMR (125 MHz, DMSO-d6) d 18.2, 102.3, 110.4,
112.2, 113.0, 126.7, 153.6, 155.0, 160.4, 161.3. Anal. Calcd for
C
10H8O3 ꢂ 0.15 H2O: C, 67.15; H, 4.68. Found: C, 67.10, H, 4.85.
LC–MS (ESI) (90% H2O to 100% MeOH in 10 min, then 100% MeOH
4.2.6. N-(6-Hydroxy-2-oxo-2H-chromen-3-yl)acetamide (19)
2,5-Dihydroxy-benzaldehyde (2.76 g, 20 mmol), N-acetylgly-
cine (2.34 g, 20 mmol) and anhydrous sodium acetate (4.92 g,
60 mmol) were refluxed in Ac2O (40 mL) overnight. The reaction
mixture was allowed to cool to room temperature. The product
was filtered off, washed with MeOH (30 mL) and dried to yield
3-acetamido-2-oxo-2H-chromen-6-yl acetate as a light grey solid
(1.23 g, 24%): mp 222–223 °C; 1H NMR (500 MHz, DMSO-d6) d
2.16 (s, 3H, CH3), 2.29 (s, 3H, CH3), 7.12 (dd, 1H, 4J = 2.2 Hz,
3J = 8.6 Hz, 6-H), 7.25 (d, 1H, 4J = 2.3 Hz, 8-H), 7.72 (d, 1H,
3J = 8.5 Hz, 5-H), 8.60 (s, 1H, 4-H), 9.71 (br s, 1H, NH); 13C NMR
(125 MHz, DMSO-d6) d 21.0, 24.1, 109.8, 117.6, 119.1, 123.3,
124.2, 128.6, 150.0, 151.1, 157.4, 169.0, 170.3. LC–MS (ESI) (90%
H2O to 100% MeOH in 10 min, then 100% MeOH to 20 min, DAD
220–400 nm), 97.5% purity, m/z = 262.15 ([M+H]+). 3-Acetamide-
2-oxo-2H-chromen-6-yl acetate (1.04 g, 4.0 mmol), K2CO3 (1.10 g,
8.0 mmol) were refluxed in MeOH (20 mL) for 1.5 h. The reaction
mixture was evaporated and dissolved in water (50 mL). After acid-
ification with 2 N HCl to pH ꢀ3, the resulting precipitate was fil-
tered off and recrystallized from MeOH (80 mL) to yield a light
brown solid (0.22 g, 25%): mp 217-218 °C; 1H NMR (500 MHz,
DMSO-d6) d 2.15 (s, 3H, CH3), 6.90 (dd, 1H, 4J = 2.9 Hz, 3J = 8.8 Hz,
7-H), 6.94 (d, 1H, 4J = 2.9 Hz, 5-H), 7.20 (d, 1H, 3J = 8.9 Hz, 8-H),
9.63-9.64 (2s overlapping, 2H, NH and OH); 13C NMR (125 MHz,
DMSO-d6) d 24.1, 112.0, 116.8, 117.6, 120.3, 123.7, 124.7, 143.2,
154.3, 157.8, 170.3. HRMS-ESI m/z [M+Na]+ calcd for C11H19NO4Na:
242.0424, found: 242.0420. LC–MS (ESI) (90% H2O to 100% MeOH
in 10 min, then 100% MeOH to 20 min, DAD 220–400 nm), 100%
purity, m/z = 220.14 ([M+H]+).
to 20 min, DAD 220–400 nm), 96.7% purity, m/z = 177.30 ([M+H]+).
4.2.9. Preparation of alkynyloxy-substituted coumarins 24–52
and 55–57
4.2.9.1. General procedure for the Mitsunobu reaction to obtain
24–52 and 55–57.
A mixture of the appropriate hydroxy-
coumarin (13–22; 2.0 mmol), triphenyl phosphine (1.04 g,
4.0 mmol), the corresponding alcohol (3.0 mmol) and THF (30 ml)
was cooled to 0 °C. DEAD (0.52 g, 0.49 mL, 3.0 mmol) or DIAD
(0.61 g, 0.59 mL, 3.0 mmol) was added dropwise and the solution
was stirred overnight at room temperature. The volume was re-
duced in vacuo the residue was diluted with EtOAc (100 mL),
washed with 1 N NaOH (3 ꢂ 100 mL) and brine (80 mL), dried over
Na2SO4 and evaporated to dryness. The products were obtained
either after recrystallization or column chromatography.
4.2.9.2. Methyl 2-oxo-7-(prop-2-ynyloxy)-2H-chromene-3-car-
boxylate (24).
Methyl 7-hydroxy-2-oxo-2H-chromene-3-car-
boxylate (13, 440 mg) was reacted with propagyl alcohol (168 mg)
and DIAD. The residue was recrystallized from MeOH (90 mL) to
give the product as light green solid (279 mg, 54%): mp 188–
189 °C; 1H NMR (500 MHz, DMSO-d6) d 3.65 (t, 1H, 4J = 2.5 Hz,
CH), 3.80 (s, 3H, CH3), 4.97 (d, 2H, 4J = 2.3 Hz, CH2–O), 7.04 (dd,
1H, 4J = 2.2 Hz, 3J = 8.7 Hz, 6-H), 7.07 (d, 1H, 4J = 2.5 Hz, 8-H), 7.85
(d, H, 3J = 8.8 Hz, 5-H), 8.75 (s, 1H, 4-H); 13C NMR (125 MHz,
DMSO-d6) d 52.4, 56.6, 78.4, 79.3, 101.5, 112.1, 113.7, 113.8,
131.8, 149.4, 156.2, 156.8, 162.7, 163.4. Anal. Calcd for C14H10O5:
C, 65.12; H, 3.90. Found: C, 64.80; H, 4.09. LC–MS (ESI) (90% H2O
to 100% MeOH in 10 min, then 100% MeOH to 20 min, DAD 220–
400 nm), 99.2% purity, m/z = 259.34 ([M+H]+).
4.2.7. 7-Hydroxy-2H-chromen-2-one (21)
Ethyl bromoacetate (1.67 g, 1.11 mL, 10 mmol) and PPh3
(3.15 g, 12 mmol) were solved in dry EtOH (40 mL) and refluxed
for 4 h. The mixture was cooled to room temperature, KOH
(0.90 g, 16 mmol) and 2,4-dihydroxybenzaldehyde (1.31 g,
9.5 mmol) were added and the mixture was refluxed for 1.5 h.
After evaporation in vacuo, the residue was suspended in water
(50 mL) and acidified to pH 2. The aqueous phase was extracted
with EtOAc (3 ꢂ 50 mL). The organic phase was dried, filtered
and purified by column chromatography using CH2Cl2/MeOH
(29:1) as eluent. The product was obtained as a white solid
(0.42 g, 27%): mp 227–228 °C (lit. mp 226.5–228 °C);49 1H NMR
(500 MHz, DMSO-d6) d 6.18 (d, 1H, 3J = 9.5 Hz, 3-H), 6.69 (d, 1H,
4J = 2.2 Hz, 8-H), 6.77 (dd, 1H, 4J = 2.2 Hz, 3J = 8.5 Hz, 6-H), 7.50
4.2.9.3. Methyl 2-oxo-7-(but-3-ynyloxy)-2H-chromene-3-car-
boxylate (25).
Methyl 7-hydroxy-2-oxo-2H-chromene-3-car-
boxylate (13, 440 mg) was reacted with 3-butyn-1-ol (210 mg) and
DEAD. The residue was recrystallized from MeOH (75 mL) to give
the product as colorless needles (323 mg, 60%): mp 167–168 °C;
1H NMR (500 MHz, DMSO-d6) d 1.92 (app quint, 2H, 3J = 6.3 Hz,
CH2), 2.33 (dt, 2H, 4J = 2.5 Hz, 3J = 7.0 Hz, CH2–CCH), 2.88 (t, 1H,
4J = 2.5 Hz, CH), 3.77 (s, 3H, CH3), 4.18 (t, 2H, 3J = 6.3 Hz, CH2–O),
7.00 (dd, 1H, 4J = 2.5 Hz, 3J = 8.7 Hz, 6-H), 7.03 (d, 1H, 4J = 2.6 Hz,
8-H), 7.82 (d, H, 3J = 8.5 Hz, 5-H), 8.73 (s, 1H, 4-H); 13C NMR
(125 MHz, DMSO-d6) d 18.8, 52.3, 66.9, 72.7, 81.2, 101.0, 111.7,
113.3, 113.7, 131.9, 149.5, 156.3, 157.1, 163.5, 163.8. Anal. Calcd
for C15H12O5: C, 66.17; H, 4.44. Found: C, 65.93; H, 4.64. LC–MS

1922
M. D. Mertens et al. / Bioorg. Med. Chem. 22 (2014) 1916–1928
(ESI) (90% H2O to 100% MeOH in 10 min, then 100% MeOH to
H, 5.01. Found: C, 72.56; H, 5.11. LC–MS (ESI) (90% H2O to 100%
MeOH in 10 min, then 100% MeOH to 20 min, DAD 220–400 nm),
98.9% purity, m/z = 363.37 ([M+H]+).
20 min, DAD 220–400 nm), 96.3% purity, m/z = 273.21 ([M+H]+).
4.2.9.4. Methyl 2-oxo-7-(pent-4-ynyloxy)-2H-chromene-3-car-
boxylate (26).
Methyl 7-hydroxy-2-oxo-2H-chromene-3-car-
4.2.9.8. N-(2-Oxo-7-(pent-4-ynyloxy)-2H-chromen-3-yl)acetam-
boxylate (13, 440 mg) was reacted with 4-pentyn-1-ol (252 mg)
and DIAD. The residue was recrystallized from MeOH (50 mL) to
give the product as colorless needles (403 mg, 70%): mp 130–
132 °C; 1H NMR (500 MHz, DMSO-d6) d 1.92 (app quint, 2H,
3J = 6.3 Hz, CH2), 2.33 (dt, 2H, 4J = 2.5 Hz, 3J = 7.0 Hz, CH2–CCH),
2.88 (t, 1H, 4J = 2.5 Hz, CH), 3.77 (s, 3H, CH3), 4.18 (t, 2H,
3J = 6.3 Hz, CH2–O), 6.99–7.02 (m, 2H, 6-H, 8-H), 7.82 (d, H,
3J = 8.6 Hz, 5-H), 8.73 (s, 1H, 4-H); 13C NMR (125 MHz, DMSO-d6,
25 °C) d 14.5, 27.5, 52.3, 67.3, 71.9, 83.6, 100.9, 111.6, 113.2,
113.7, 131.9, 149.6, 156.4, 157.1, 163.5, 164.2. Anal. Calcd for
ide (33).
N-(7-Hydroxy-2-oxo-2H-chromen-3-yl)acetamide
(18, 438 mg) was reacted with 4-pentyn-1-ol (252 mg) and DEAD.
The residue was recrystallized from MeOH (70 mL) to give the
product as pale brown needles (294 mg, 52%): mp 172–173 °C;
1H NMR (500 MHz, DMSO-d6) d 1.90 (app quint, 2H, 3J = 6.6 Hz,
CH2), 2.13 (s, 3H, CH3), 2.33 (dt, 2H, 4J = 2.5 Hz, 3J = 5.8 Hz, CH2–
CCH), 2.80 (t, 1H, 4J = 3.1 Hz, CH), 4.11 (t, 2H, 3J = 6.0 Hz, CH2–O),
6.94 (dd, 2H, 4J = 2.2 Hz, 3J = 8.7 Hz, 6-H), 6.98 (d, H, 4J = 2.6 Hz,
8-H), 7.59 (d, 1H, 3J = 8.9 Hz, 5-H), 8.52 (s, 1H, 4-H), 9.57 (s, 1H,
NH); 13C NMR (125 MHz, DMSO-d6) d 14.6, 23.9, 27.7, 66.8, 71.8,
83.7, 101.2, 112.9, 113.2, 122.0, 125.1, 128.9, 151.4, 157.9, 160.1,
170.0. Anal. Calcd for C16H15NO4: C, 67.36; H, 5.30; N, 4.91. Found:
C, 67.11; H, 5.41; N, 4.98. LC–MS (ESI) (90% H2O to 100% MeOH in
10 min, then 100% MeOH to 20 min, DAD 220–400 nm), 99.3% pur-
ity, m/z = 286.19 ([M+H]+).
C16H14O5: C, 67.13; H, 4.93. Found: C, 67.10; H, 5.01. LC–MS (ESI)
(90% H2O to 100% MeOH in 10 min, then 100% MeOH to 20 min,
DAD 220–400 nm), 99.1% purity, m/z = 287.19 ([M+H]+).
4.2.9.5.
acid (27).
2-Oxo-6-(pent-4-ynyloxy)-2H-chromene-3-carboxylic
Methyl 6-hydroxy-2-oxo-2H-chromene-3-carbox-
ylate (14, 2.0 mmol) weas reacted with 4-pentyn-1-ol (252 mg)
and DEAD. The residue was recrystallized from MeOH (35 mL) to
give the product as green solid (471 mg, 82%): mp 133–134 °C;
1H NMR (500 MHz, DMSO-d6) d 1.92 (app quint, 2H, 3J = 6.6 Hz,
2-CH2), 2.33 (dt, 2H, 3J = 7.3 Hz, 4J = 2.6 Hz, 3-CH2), 2.80 (t, 1H,
4J = 2.5 Hz, CH), 4.08 (t, 2H, 3J = 6.3 Hz, CH2–O), 7.33 (dd, 1H,
3J = 9.0 Hz, 4J = 2.9 Hz, 7-H), 7.36 (d, H, 3J = 8.9 Hz, 8-H), 7.48 (d,
1H, 4J = 2.9 Hz, 5-H), 8.71 (s, 1H, 4-H); 13C NMR (125 MHz,
DMSO-d6) d 14.6, 27.7, 52.6, 66.9, 71.8, 83.7, 112.8, 117.4, 117.8,
118.4, 122.9, 148.9, 149.2, 155.1, 156.2, 163.3. Anal. Calcd for
4.2.9.9. 7-(Pent-4-ynyloxy)-2H-chromen-2-one (34). Umbel-
liferone (21, 324 mg) was reacted with 4-pentyn-1-ol (252 mg)
and DEAD. The residue was recrystallized from MeOH (20 mL) to
give the product as colorless needles (387 mg, 85%): mp 97–98 °C;
1H NMR (500 MHz, DMSO-d6) d 1.92 (app quint, 2H, 3J = 6.3 Hz,
CH2), 2.33 (dt, 2H, 4J = 2.6 Hz, 3J = 7.1 Hz, CH2–CCH), 2.80 (t, 1H,
4J = 2.5 Hz, CH), 4.18 (t, 2H, 3J = 6.3 Hz, CH2–O), 6.27
(d, 1H, 3J = 6.3 Hz, 3-H), 6.94 (dd, 1H, 4J = 2.5 Hz, 3J = 8.5 Hz, 6.97
(d, H, 4J = 2.6 Hz, 5-H), 7.61 (d, 1H, 3J = 8.5 Hz, 6-H), 7.97 (d, 1H,
3J = 9.2 Hz, 4-H); 13C NMR (125 MHz, DMSO-d6) d 14.6, 27.6, 66.9,
71.8, 83.6, 101.4, 112.5, 112.6, 112.8, 129.7, 144.4, 155.5, 160.4,
161.8. Anal. Calcd for C14H12O3: C, 73.67; H, 5.30. Found: C, 73.62;
H, 5.35. LC–MS (ESI) (90% H2O to 100% MeOH in 10 min, then
100% MeOH to 20 min, DAD 220–400 nm), 99.5% purity,
m/z = 229.36 ([M+H]+).
C15H12O5: C, 67.13; H, 4.93. Found: C, 66.73; H, 5.01. LC–MS (ESI)
(90% H2O to 100% MeOH in 10 min, then 100% MeOH to 20 min,
DAD 220–400 nm), 99.5% purity, m/z = 287.16 ([M+H]+).
4.2.9.6. Methyl 2-oxo-8-(pent-4-ynyloxy)-2H-chromene-3-car-
boxylate (28).
Methyl 8-hydroxy-2-oxo-2H-chromene-3-car-
boxylate (15, 440 mg) was reacted with 4-pentyn-1-ol (252 mg)
and DEAD. The residue was purified by column chromatography
using petroleum ether/EtOAc (3:2) as eluent. The product was ob-
tained as pale green solid (411 mg, 72%): mp 98–99 °C; 1H NMR
(500 MHz, DMSO-d6,) d 1.93 (app quint, 2H, 3J = 7.0 Hz, 2-CH2),
2.37 (dt, 2H, 3J = 7.1 Hz, 4J = 2.5 Hz, 3-CH2), 2.80 (t, 1H, 4J = 2.6 Hz,
CH), 3.83 (s, 3H, CH3), 4.19 (t, 2H, 3J = 6.0 Hz, CH2–O), 7.31 (app.
d, 1H, 3J = 8.2 Hz, 3J = 7.9 Hz, 6-H), 7.42–7.46 (m, 2H, 5-H, 7-H),
8.74 (s, 1H, 4-H); 13C NMR (125 MHz, DMSO-d6) d 14.6, 27.8,
52.6, 67.6, 71.9, 83.6, 117.7, 117.8, 118.6, 121.6, 124.9, 144.3,
145.6, 149.3, 155.8, 163.2. HRMS-ESI m/z [M+Na]+ calcd for C16H15-
O5Na: 309.0733, found: 309.0736. LC–MS (ESI) (90% H2O to 100%
MeOH in 10 min, then 100% MeOH to 20 min, DAD 220–400 nm),
98.6% purity, m/z = 287.17 ([M+H]+).
4.2.9.10. 4-Methyl-7-(pent-4-ynyloxy)-2H-chromen-2-one
(35).
7-Hydroxy-4-methyl-2H-chromen-2-one (22, 352 mg)
was reacted with 4-pentyn-1-ol (252 mg) and DEAD. The residue
was recrystallized from MeOH (15 mL) to give the product as col-
orless needles (200 mg, 0.83 mmol, 41%): mp 102–103 °C; 1H
NMR (500 MHz, DMSO-d6) d 1.91 (app quint, 2H, 3J = 6.0 Hz, CH2),
2.33 (dt, 2H, 4J = 2.9 Hz, 3J = 7.1 Hz, CH2–CCH), 2.38 (d, 3H,
4J = 1.3 Hz, CH3), 2.80 (t, 1H, 4J = 2.9 Hz, CH), 4.14 (t, 2H,
3J = 6.4 Hz, CH2–O), 6.19 (d, 1H, 4J = 1.3 Hz, 3-H), 6.94–6.97 (m,
2H, 6-H, 8-H), 7.65–7.67 (d, H, 3J = 8.6 Hz, 5-H); 13C NMR
(125 MHz, DMSO-d6) d 14.6, 18.2, 27.6, 66.9, 71.8, 83.6, 101.4,
111.3, 112.5, 113.3, 126.6, 153.5, 154.9, 160.2, 161.7. Anal. Calcd
for C15H14O3: C, 74.36; H, 5.82. Found: C, 74.32; H, 5.87. LC–MS
(ESI) (90% H2O to 100% MeOH in 10 min, then 100% MeOH to
20 min, DAD 220–400 nm), 99.0% purity, m/z = 243.46 ([M+H]+).
4.2.9.7. 3-(4-Methoxybenzoyl)-7-(pent-4-ynyloxy)-2H-chromen-
2-one (29).
7-Hydroxy-3-(4-methoxybenzoyl)-2H-chromen-2-
4.2.9.11. Methyl 2-oxo-7-(hex-5-ynyloxy)-2H-chromene-3-car-
one (16, 594 mg) was reacted with 4-pentyn-1-ol (252 mg) and
DEAD. The residue was recrystallized from MeOH (120 mL) to give
the product as yellow crystals (550 mg, 1.52 mmol, 76%): mp
151–152 °C; 1H NMR (500 MHz, DMSO-d6) d 1.93 (app quint, 2H,
3J = 6.3 Hz, CH2), 2.35 (dt, 2H, 4J = 2.8 Hz, 3J = 7.1 Hz, CH2–CCH), 2.81
(t, 1H, 4J = 2.9 Hz, CH), 3.85 (s, 3H, CH3), 4.19 (t, 2H, 3J = 6.3 Hz,
CH2–O), 7.02 (dd, 1H, 4J = 2.5 Hz, 3J = 8.8 Hz, 6-H), 7.05 (d, 2H,
3J = 8.9 Hz, 20,60-H), 7.07 (d, 1H, 4J = 2.5 Hz, 8-H), 7.76 (d, 1H,
boxylate (36).
Methyl 7-hydroxy-2-oxo-2H-chromene-3-car-
boxylate (13, 440 mg) was reacted with 5-hexyn-1-ol (294 mg)
and DIAD. The residue was recrystallized from MeOH (50 mL) to
give the product as colorless needles (374 mg, 62%): mp
104–105 °C; 1H NMR (500 MHz, DMSO-d6) d 1.60 (app quint, 2H,
3J = 6.9 Hz, CH2), 1.83 (quint, 2H, 3J = 6.3 Hz, CH2), 2.23 (dt, 2H,
4J = 2.9 Hz, 3J = 7.1 Hz, CH2CCH), 2.76 (t, 1H, 4J = 2.5 Hz, CH), 3.80
(s, 3H, CH3), 4.13 (t, 2H, 3J = 6.3 Hz, CH2–O), 6.98–7.01 (m, 2H,
6-H, 8-H), 7.81 (d, H, 3J = 8.5 Hz, 5-H), 8.72 (s, 1H, 4-H); 13C NMR
(125 MHz, DMSO-d6) d 17.5, 24.6, 27.6, 52.3, 68.3, 71.6, 84.3,
100.8, 111.5, 113.1, 113.8, 131.8, 149.6, 156.4, 157.2, 163.5,
3
3J = 8.8 Hz, 5-H), 7.87 (d, 2H, J = 8.9 Hz, 30,50-H), 8.28 (s, 1H, 4-H);
13C NMR (125 MHz, DMSO-d6) d 14.6, 27.6, 55.8, 67.2, 71.9, 83.6,
101.2, 112.0, 113.5, 114.1, 123.1, 129.3, 131.1, 132.1, 145.3, 156.4,
156.4, 158.5, 163.2, 163.8, 190.3. Anal. Calcd for C22H18O5: C, 72.92;
164.4. Anal. Calcd for
C17H16O5: C, 67.99; H, 5.37. Found:

M. D. Mertens et al. / Bioorg. Med. Chem. 22 (2014) 1916–1928
1923
C, 67.90; H, 5.65. LC–MS (ESI) (90% H2O to 100% MeOH in 10 min,
then 100% MeOH to 20 min, DAD 220–400 nm), 99.7% purity, m/
z = 301.17 ([M+H]+).
3J = 6.3 Hz, CH2–O), 6.93 (dd, 2H, 4J = 2.6 Hz, 3J = 8.7 Hz, 6-H), 6.96
(d, H, 4J = 2.6 Hz, 8-H), 7.58 (d, 1H, 3J = 8.5 Hz, 5-H), 8.52 (s, 1H,
4-H), 9.56 (s, 1H, NH); 13C NMR (125 MHz, DMSO-d6) d 17.5,
23.9, 24.7, 27.7, 67.8, 71.5, 84.4, 101.1, 112.7, 113.2, 121.9, 125.2,
128.9, 151.4, 157.9, 160.2, 170.0. Anal. Calcd for C17H17NO4: C,
68.21; H, 5.72; N, 4.68. Found: C, 68.34; H, 5.78; N, 4.78. LC–MS
(ESI) (90% H2O to 100% MeOH in 10 min, then 100% MeOH to
20 min, DAD 220–400 nm), 98.1% purity, m/z = 300.24 ([M+H]+).
4.2.9.12. Methyl 6-(hex-5-ynyloxy)-2-oxo-2H-chromene-3-car-
boxylate (37).
Methyl 6-hydroxy-2-oxo-2H-chromene-3-car-
boxylate (14, 440 mg) was reacted with 5-hexyn-1-ol (294 mg)
and DEAD. The residue was recrystallized from MeOH (30 mL) to
give the product as pale green needles (442 mg, 74%): mp 124–
125 °C; 1H NMR (500 MHz, DMSO-d6) d 1.60 (app quint, 2H,
3J = 7.3 Hz, CH2), 1.82 (m, 2H, CH2), 2.23 (dt, 2H, 4J = 2.5 Hz,
3J = 7.1 Hz, CH2CCH), 2.76 (t, 1H, 4J = 2.6 Hz, CH), 3.82 (s, 3H,
CH3), 4.03 (t, 2H, 3J = 6.3 Hz, CH2–O), 7.32 (dd, 1H, 4J = 2.8 Hz,
3J = 8.8 Hz, 7-H), 7.36 (d, H, 3J = 9.2 Hz, 8-H), 7.46 (d, H,
4J = 2.9 Hz, 5-H), 8.69 (s, 1H, 4-H); 13C NMR (125 MHz, DMSO-d6)
d 17.5, 24.7, 27.8, 52.5, 67.9, 71.5, 84.4, 112.8, 117.4, 117.7, 118.3,
122.8, 148.9, 149.1, 155.2, 156.2, 163.3. Anal. Calcd for C16H14O5:
C, 67.99; H, 5.37. Found: C, 67.52; H, 5.56. LC–MS (ESI) (90% H2O
to 100% MeOH in 10 min, then 100% MeOH to 20 min, DAD 220–
400 nm), 97.5% purity, m/z = 301.22 ([M+H]+).
4.2.9.16. N-(2-Oxo-6-(hex-5-ynyloxy)-2H-chromen-3-yl)acetam-
ide (41).
N-(6-Hydroxy-2-oxo-2H-chromen-3-yl)acetamide
(19, 438 mg) was reacted with 5-hexyn-1-ol (294 mg) and DIAD.
The residue was recrystallized from MeOH (40 mL) to give the
product as white solid (468 mg, 78%): mp 158–160 °C; 1H NMR
(500 MHz, DMSO-d6) d 1.60 (app quint, 2H, CH2), 1.81 (m, 2H,
CH2), 2.13 (s, 3H, CH3), 2.23 (dt, 2H, 4J = 2.5 Hz, 3J = 7.1 Hz, CH2–
CCH), 2.75 (t, 1H, 4J = 2.5 Hz, CH), 4.02 (t, 2H, 3J = 6.3 Hz, CH2–O),
7.05 (dd, 2H, 4J = 2.8 Hz, 3J = 9.0 Hz, 7-H), 7.27 (d, H, 4J = 2.9 Hz,
5-H), 7.28 (d, 1H, 3J = 9.1 Hz, 8-H), 8.57 (s, 1H, 4-H), 9.67 (s, 1H,
NH); 13C NMR (125 MHz, DMSO-d6) d 17.5, 24.1, 24.7, 27.8, 67.7,
71.5, 84.4, 111.0, 116.9, 117.6, 120.3, 123.6, 124.9, 144.0, 155.5,
157.7, 170.3. Anal. Calcd. For C17H17NO4: C, 68.21; H, 5.72; N,
4.68. Found: C, 68.06; H, 5.66; N, 4.79. LC–MS (ESI) (90% H2O to
100% MeOH in 10 min, then 100% MeOH to 20 min, DAD 220–
400 nm), 99.4% purity, m/z = 300.19 ([M+H]+).
4.2.9.13.
men-2-one
3-(4-Methoxybenzoyl)-7-(hex-5-ynyloxy)-2H-chro-
(38). 7-Hydroxy-3-(4-methoxybenzoyl)-2H-
chromen-2-one (16, 592 mg) was reacted with 5-hexyn-1-ol
(294 mg) and DEAD. The residue was recrystallized from MeOH
(100 mL) to give the product as yellow crystals (475 mg, 63%):
mp 145–146 °C; 1H NMR (500 MHz, DMSO-d6) d 1.62 (app quint,
2H, CH2), 1.84 (m, 2H, CH2), 2.25 (dt, 2H, 4J = 2.9 Hz, 3J = 7.1 Hz,
CH2–CCH), 2.77 (t, 1H, 4J = 2.9 Hz, CH), 3.85 (s, 3H, CH3), 4.15 (t,
2H, 3J = 6.3 Hz, CH2–O), 7.00 (dd, 1H, 4J = 2.6 Hz, 3J = 8.7 Hz, 6-H),
4.2.9.17. 7-(Hex-5-ynyloxy)-2H-chromen-2-one (42).
Umbel-
liferone (21, 324 mg) was reacted with 5-hexyn-1-ol (294 mg) and
DEAD. The residue was recrystallized from EtOAc/n-hexane
(10/30 mL) to give the product as colorless solid (349 mg, 71%):
mp 82–83 °C; 1H NMR (500 MHz, DMSO-d6) d 1.60 (app quint,
2H, CH2), 1.82 (m, 2H, CH2), 2.23 (dt, 2H, 4J = 2.5 Hz, 3J = 7.1 Hz,
CH2–CCH), 2.76 (t, 1H, 4J = 2.5 Hz, CH), 4.08 (t, 2H, 3J = 6.4 Hz,
CH2–O), 6.26 (d, 1H, 3J = 9.5 Hz, 3-H), 6.93 (dd, 1H, 4J = 2.6 Hz,
3J = 8.7 Hz), 6.97 (d, H, 4J = 2.2 Hz, 5-H), 7.60 (d, 1H, 3J = 8.6 Hz,
6-H), 7.96 (d, 1H, 3J = 9.5 Hz, 4-H); 13C NMR (125 MHz, DMSO-d6)
d 17.5, 24.7, 27.7, 67.9, 71.5, 84.4, 101.3, 112.4, 112.5, 112.8,
129.6, 144.4, 155.6, 160.4, 162.0. Anal. Calcd for C15H14O3: C,
74.36; H, 5.82. Found: C, 74.36; H, 6.04. LC–MS (ESI) (90% H2O
to 100% MeOH in 10 min, then 100% MeOH to 20 min, DAD
220–400 nm), 100% purity, m/z = 243.31 ([M+H]+).
3
7.04 (d, 2H, J = 8.9 Hz, 20,60-H), 7.06 (d, 1H, 4J = 2.2 Hz, 8-H), 7.74
3
(d, 1H, 3J = 8.9 Hz, 5-H), 7.86 (d, 2H, J = 8.9 Hz, 30,50-H), 8.28 (s,
1H, 4-H); 13C NMR (125 MHz, DMSO-d6) d 17.5, 24.6, 27.6, 55.8,
68.2, 71.5, 84.4, 101.2, 111.9, 113.5, 114.1, 123.0, 129.3, 131.0,
132.1, 145.4, 156.4, 158.5, 163.3, 163.8, 190.3. Anal. Calcd for
C23H20O5: C, 73.39; H, 5.36. Found: C, 73.24; H, 5.56. LC–MS (ESI)
(90% H2O to 100% MeOH in 10 min, then 100% MeOH to 20 min,
DAD 220–400 nm), 98.9% purity, m/z = 377.30 ([M+H]+).
4.2.9.14.
men-2-one
3-(4-Methoxybenzoyl)-6-(hex-5-ynyloxy)-2H-chro-
(39). 6-Hydroxy-3-(4-methoxybenzoyl)-2H-
chromen-2-one (17, 592 mg) was reacted with 5-hexyn-1-ol
(294 mg) and DIAD. The residue was recrystallized from MeOH
(30 mL) to give the product as yellow crystals (348 mg, 46%): mp
117–118 °C; 1H NMR (500 MHz, DMSO-d6) d 1.61 (app quint, 2H,
3J = 7.2 Hz, CH2), 1.82 (m, 2H, CH2), 2.23 (dt, 2H, 4J = 2.9 Hz,
3J = 7.1 Hz, CH2–CCH), 2.76 (t, 1H, 4J = 2.5 Hz, CH), 3.86 (s, 3H,
CH3), 4.04 (t, 2H, 3J = 6.4 Hz, CH2–O), 7.05 (d, 2H, 3J = 9.2 Hz, ar-
om.H), 7.30 (dd, 1H, 4J = 2.9 Hz, 3J = 9.0 Hz, 7-H), 7.38 (d, 2H,
4J = 2.8 Hz, 5-H), 7.41 (d, 1H, 3J = 9.1 Hz, 8-H), 7.90 (d, 1H,
3J = 9.2 Hz, arom.H), 8.24 (s, 1H, 4-H); 13C NMR (125 MHz, DMSO-
d6) d 17.6, 24.7, 27.8, 55.8, 68.9, 71.5, 84.4, 112.4, 114.2, 117.5,
118.8, 121.5, 127.2, 128.9, 132.2, 144.2, 148.5, 155.3, 158.3,
164.0, 190.2. Anal. Calcd for C22H20O5 ꢂ 0.5 MeOH: C, 71.93; H,
5.65. Found: C, 71.85; H, 5.65. LC–MS (ESI) (90% H2O to 100% MeOH
in 10 min, then 100% MeOH to 20 min, DAD 220–400 nm), 95.9%
purity, m/z = 377.23 ([M+H]+).
4.2.9.18. 4-Methyl-7-(hex-5-ynyloxy)-2H-chromen-2-one
(43).
7-Hydroxy-4-methyl-2H-chromen-2-one (22, 352 mg)
was reacted with 5-hexyn-1-ol (294 mg) and DEAD. The residue
was recrystallized from MeOH (15 mL) to give the product as col-
orless needles (350 mg, 68%): mp 75–76 °C; 1H NMR (500 MHz,
DMSO-d6) d 1.60 (app quint, 2H, CH2), 1.82 (m, 2H, CH2), 2.23
(dt, 2H, 4J = 2.9 Hz, 3J = 7.1 Hz, CH2–CCH), 2.38 (d, 3H, 4J = 1.3 Hz,
CH3), 2.76 (t, 1H, 4J = 2.5 Hz, CH), 4.10 (t, 2H, 3J = 6.3 Hz, CH2–O),
6.18 (d, 1H, 4J = 1.3 Hz, 3-H), 6.93–6.95 (m, 2H, 6-H, 8-H), 7.65–
7.67 (d, H, 3J = 7.3 Hz, 5-H); 13C NMR (125 MHz, DMSO-d6) d 17.5,
18.2, 24.7, 27.7, 67.9, 71.5, 84.4, 101.3, 111.2, 112.6, 113.2, 126.6,
153.5, 154.9, 160.3, 161.9. Anal. Calcd for C16H16O3: C, 74.98, H,
6.29. Found: C, 74.94; H, 6.51. LC–MS (ESI) (90% H2O to 100% MeOH
in 10 min, then 100% MeOH to 20 min, DAD 220–400 nm), 98.0%
purity, m/z = 257.39 ([M+H]+).
4.2.9.15. N-(2-Oxo-7-(hex-5-ynyloxy)-2H-chromen-3-yl)acetam-
4.2.9.19. 7-(Hex-5-ynyloxy)-3-(4-methoxyphenyl)-2H-chromen-
ide (40).
N-(7-Hydroxy-2-oxo-2H-chromen-3-yl)acetamide
2-one (44).
7-Hydroxy-3-(4-methoxyphenyl)-2H-chromen-
(18, 438 mg) was reacted with 5-hexyn-1-ol (294 mg) and DIAD.
The residue was recrystallized from MeOH (20 mL) to give the
product as pale brown needles (346 mg, 58%): mp 146–147 °C;
1H NMR (500 MHz, DMSO-d6) d 1.60 (app quint, 2H, CH2), 1.81
(m, 2H, CH2), 2.13 (s, 3H, CH3), 2.23 (dt, 2H, 4J = 2.9 Hz,
3J = 7.1 Hz, CH2–CCH), 2.75 (t, 1H, 4J = 2.5 Hz, CH), 4.06 (t, 2H,
2-one (20, 536 mg) was reacted with 5-hexyn-1-ol (294 mg) and
DIAD. The residue was recrystallized from MeOH (30 mL) to give
to product a pale green platelets (552 mg, 79%): mp 127–128 °C;
1H NMR (500 MHz, DMSO-d6) d 1.58–1.64 (m, 2H, CH2), 1.83 (m,
2H, CH2), 2.24 (dt, 2H, 4J = 2.6 Hz, 3J = 6.9 Hz, CH2), 2.76 (t, 1H,
4J = 2.5 Hz, CH), 3.79 (s, 3H, CH3), 4.10 (t, 2H, 3J = 6.3 Hz, OCH2),

1924
M. D. Mertens et al. / Bioorg. Med. Chem. 22 (2014) 1916–1928
6.96 (dd, H, 4J = 2.5 Hz, 3J = 8.5 Hz, 6-H), 6.99–7.00 (m, 3H, arom.H),
7.64–7.67 (m, 3H, arom.H), 8.11 (s, 1H, 4-H); 13C NMR (125 MHz,
DMSO-d6) d 17.5, 24.7, 27.7, 55.3, 67.9, 71.5, 84.4, 100.8, 113.0,
113.3, 113.8, 122.9, 127.3, 129.6, 129.7, 139.5, 154.7, 159.4,
160.2, 161.6. Anal. Calcd for C22H20O4: C, 75.84; H, 5.79. Found:
C, 76.07; H, 5.83. LC–MS (ESI) (90% H2O to 100% MeOH in
10 min, then 100% MeOH to 20 min, DAD 220–400 nm), 98.5% pur-
ity, m/z = 349.13 ([M+H]+).
122.9, 128.4, 131.0, 131.2, 137.3, 148.9, 149.2, 154.9, 156.2,
163.3. Anal. Calcd for C19H15ClO5: C, 63.61; H, 4.21. Found: C,
63.27; H, 4.46. LC–MS (ESI) (90% H2O to 100% MeOH in 10 min,
then 100% MeOH to 20 min, DAD 220–400 nm), 100% purity,
m/z = 359.12 ([M+H]+).
4.2.9.24. N-(7-(3-Chlorobenzyloxy)-2-oxo-2H-chromen-3-yl)acet-
amide (49).
N-(7-Hydroxy-2-oxo-2H-chromen-3-yl)acetamide
(18, 441 mg, 2 mmol) was reacted with 3-chloro benzylalcohol
(427 mg) and DIAD. The residue was recrystallized from MeOH/H2O
(100 mL, 9/1) to give the product as pale brown needles (136 mg,
20%): mp 218–219 °C; 1H NMR (500 MHz, DMSO-d6) d 2.13 (s, 3H,
CH3), 5.21 (s, 2H, CH2), 7.02 (dd, 2H, 4J = 2.5 Hz,3J = 8.7 Hz, 6-H),
7.07 (d, H, 4J = 2.5 Hz, 8-H), 7.39–7.44 (m, 3H, arom.H), 7.54 (s, 1H, ar-
om.H), 7.62 (d, 1H, 3J = 8.5 Hz, 5-H), 8.53 (s, 1H, 4-H), 9.58 (s, 1H, NH);
13C NMR (125 MHz, DMSO-d6) d 24.0, 69.0, 101.7, 113.2, 113.5, 122.2,
124.9, 126.5, 127.6, 128.1, 129.0, 130.6, 133.3, 139.1, 151.3, 157.8,
159.6, 170.0. Anal. Calcd for C18H14ClNO4 ꢂ 0.6 H2O: C, 60.97; H,
4.32; N, 3.95. Found: C, 60.98; H, 4.16; N, 3.91. LC–MS (ESI) (90%
H2O to 100% MeOH in 10 min, then 100% MeOH to 20 min, DAD
220–400 nm), 95.7% purity, m/z = 344.18 ([M+H]+).
4.2.9.20. Methyl 7-(3-fluorobenzyloxy)-2-oxo-2H-chromene-3-
carboxylate (45).
Methyl 7-hydroxy-2-oxo-2H-chromene-3-
carboxylate (13, 440 mg) was reacted with (3-fluorophenyl)methanol
(252 mg, 2.0 mmol) and DEAD. The residue was recrystallized from
MeOH (80 mL) to give the product as white needles (456 mg, 70%):
mp 188–189 °C; 1H NMR (500 MHz, DMSO-d6) d 3.78 (s, 3H, CH3),
5.26 (s, 2H, CH2), 7.06–7.09 (m, 2H), 7.14–7.18 (m, H), 7.29–7.30
(m, 1H), 7.41–7.46 (m, 1H), 7.83 (d, 1H, 3J = 8.5 Hz, 5-H), 8.71 (s,
1H, 4-H); 13C NMR (125 MHz, DMSO-d6) d 52.3, 69.4, 101.4, 111.9,
2
2
113.4, 113.9, 114.6 (d, JC,F = 21.8 Hz), 115.1 (d, JC,F = 21.8 Hz),
3
3
123.9, 130.7 (d, JC,F = 8.2 Hz), 131.9, 139.0 (d, JC,F = 7.4 Hz), 149.5,
156.3, 157.0, 162.3 (d, JC,F = 243 Hz), 163.5, 163.7. Anal. Calcd for
1
C18H13FO5: C, 65.85; H, 3.99. Found: C, 65.53; H, 4.04. LC–MS (ESI)
(90% H2O to 100% MeOH in 10 min, then 100% MeOH to 20 min,
4.2.9.25. (E)-Methyl 7-(3,7-dimethylocta-2,6-dienyloxy)-2-oxo-
DAD 220–400 nm), 97.7% purity, m/z = 329.36 ([M+H]+).
2H-chromene-3-carboxylate (50).
Methyl 7-hydroxy-2-oxo-
2H-chromene-3-carboxylate (13, 440 mg) was reacted with
geraniol (463 mg) and DEAD. The residue was purified by column
chromatography using petroleum ether/EtOAc (3:1) as eluent.
The product was obtained colorless powder (232 mg, 33%): mp
81–82 °C; 1H NMR (500 MHz, DMSO-d6) d 1.55 (d, 3H, 4J = 0.6 Hz,
CH3), 1.59 (d, 3H, 4J = 1.0 Hz, CH3), 1.72 (d, 3H, 4J = 1.3 Hz, CH3),
2.05 (m, 4H, 2 ꢂ CH2) 3.80 (s, 3H, OCH3), 4.69 (d, 2H, 3J = 6.6 Hz,
OCH2), 5.04 (m, 1H, CH), 5.44 (m, 1H, CH), 6.98 (dd, 1H,
4J = 2.5 Hz, 3J = 8.5 Hz, 6-H), 7.00 (d, 1H, 4J = 2.6 Hz, 8-Hv), 7.82
(d, H, 3J = 8.5 Hz, 5-H), 8.72 (s, 1H, 4-H); 13C NMR (125 MHz,
DMSO-d6) d 16.5, 17.7, 25.5, 25.9, 52.3, 65.6, 101.0, 111.5, 113.0,
114.0, 118.8, 123.8, 131.2, 131.7, 141.8, 149.6, 156.4, 157.1,
163.5, 164.2. Anal. Calcd for C14H14O6: C, 70.77; H, 6.79. Found:
C, 70.45; H, 6.88. LC–MS (ESI) (90% H2O to 100% MeOH in
10 min, then 100% MeOH to 20 min, DAD 220–400 nm), 97.9% pur-
ity, m/z = 357.40 ([M+H]+).
4.2.9.21. Methyl 6-(3-chlorobenzyloxy)-2-oxo-2H-chromene-3-
carboxylate (46).
Methyl 6-hydroxy-2-oxo-2H-chromene-3-
carboxylate (14, 440 mg) was reacted with (3-chlorophenyl)methanol
(426 mg) and DIAD. The residue was recrystallized from MeOH
(90 mL) to give the product as light green needles (350 mg, 51%):
mp 167–168 °C; 1H NMR (500 MHz, DMSO-d6) d 3.82 (s, 3H,
CH3), 5.16 (s, 2H, CH2), 7.37–7.43 (m, 5H, arom.H), 7.53 (br s, 1H,
arom.H), 7.55 (d, 1H, 4J = 2.6 Hz, 5-H), 8.69 (s, 1H, 4-H); 13C NMR
(125 MHz, DMSO-d6) d 52.5, 69.1, 113.4, 117.5, 117.9, 118.3,
123.0, 126.5, 127.6, 128.1, 130.5, 133.3, 139.2, 148.7, 149.4,
154.6, 156.1, 163.3. Anal. Calcd for C18H13ClO5: C, 62.71; H, 3.80.
Found: C, 62.43; H, 3.82. LC–MS (ESI) (90% H2O to 100% MeOH in
10 min, then 100% MeOH to 20 min, DAD 220–400 nm), 100%
purity, m/z = 345.27 ([M+H]+).
4.2.9.22. Methyl 7-(4-chlorophenethoxy)-2-oxo-2H-chromene-
3-carboxylate (47).
Methyl 7-hydroxy-2-oxo-2H-chromene-
4.2.9.26. (E)-Methyl 6-(3,7-dimethylocta-2,6-dienyloxy)-2-oxo-
3-carboxylate (13, 440 mg) was reacted with 2-(4-chlorophenyl)ethanol
(470 mg) and DIAD. The residue was recrystallized from MeOH
(35 mL) to give the product as white needles (496 mg, 69%): mp
2H-chromene-3-carboxylate (51).
Methyl 6-hydroxy-2-oxo-
2H-chromene-3-carboxylate (14, 440 mg) was reacted with
geraniol (463 mg) and DEAD. The residue was purified by column
chromatography using petroleum ether/EtOAc (3:1) as eluent.
The product was obtained yellow syrup (388 mg, 54%): 1H NMR
(500 MHz, DMSO-d6) d 1.55 (s, 3H, CH3), 1.60 (d, 3H, 4J = 1.0 Hz,
CH3), 1.71 (d, 3H, 4J = 1.3 Hz, CH3), 2.03–2.08 (m, 4H, 2 ꢂ CH2)
3.82 (s, 3H, OCH3), 4.58 (d, 2H, 3J = 6.6 Hz, OCH2), 5.03–5.06 (m,
1H, CH), 5.42–5.45 (m, 1H, CH), 7.32 (dd, 1H, 4J = 2.9 Hz,
3J = 9.2 Hz, 7-H), 7.36 (d, 1H, 3J = 8.8 Hz, 8-H), 7.46 (d, H,
4J = 2.9 Hz, 5-H), 8.69 (s, 1H, 4-H); 13C NMR (125 MHz, DMSO-d6)
d 16.5, 17.7, 25.5, 25.9, 52.5, 65.2, 113.2, 117.3, 117.7, 118.3,
119.3, 123.0, 123.8, 131.1, 141.1, 148.9, 149.1, 155.0, 156.2,
163.3. HRMS-ESI m/z [M+Na]+ calcd for C21H24O5Na: 379.1516,
found: 379.1522. LC–MS (ESI) (90% H2O to 100% MeOH in
10 min, then 100% MeOH to 20 min, DAD 220–400 nm), 97.4% pur-
ity, m/z = 356.99 ([M+H]+).
143–145 °C; 1H NMR (500 MHz, DMSO-d6)
d 3.06 (t, 2H,
3J = 6.6 Hz, CH2), 3.80 (s, 3H, CH3), 4.34 (t, 2H, 3J = 7.0 Hz, O-CH2),
6.97 (dd, 1H, 4J = 2.5 Hz, 3J = 8.8 Hz, 6-H), 7.02 (d, 1H, 4J = 2.6 Hz,
8-H), 7.36 (m, 4H, arom.H), 7.80 (d, 1H, 3J = 8.9 Hz, 5-H), 8.72
(s, 1H, 4-H); 13C NMR (125 MHz, DMSO-d6) d 33.9, 52.3, 68.9,
101.0, 111.6, 113.2, 113.7, 128.4, 131.0, 131.2, 131.8, 137.1,
149.5, 156.3, 157.1, 163.5, 164.0. Anal. Calcd for C19H15ClO5:
C, 63.61; H, 4.21. Found: C, 63.26; H, 4.37. LC–MS (ESI) (90% H2O
to 100% MeOH in 10 min, then 100% MeOH to 20 min, DAD
220–400 nm), 98.8% purity, m/z = 359.13 ([M+H]+).
4.2.9.23. Methyl 6-(4-chlorophenethoxy)-2-oxo-2H-chromene-
3-carboxylate (48).
Methyl 6-hydroxy-2-oxo-2H-chromene-
3-carboxylate (14, 440 mg) was reacted with 2-(4-chlorophenyl)ethanol
(470 mg) and DIAD. The residue was recrystallized from MeOH
(30 mL) to give the product as light green needles (249 mg, 35%):
mp 142–143 °C; 1H NMR (500 MHz, DMSO-d6) d 3.05 (t, 2H,
3J = 7.0 Hz, CH2), 3.82 (s, 3H, CH3), 4.22 (t, 2H, 3J = 7.0 Hz, O–CH2),
7.30 (dd, 1H, 4J = 2.9 Hz, 3J = 8.8 Hz, 6-H), 7.36 (m, 5H, arom.H),
7.48 (d, 1H, 4J = 2.9 Hz, 5-H), 8.68 (s, 1H, 4-H); 13C NMR
(125 MHz, DMSO-d6) d 34.1, 52.5, 68.7, 112.9, 117.4, 117.8, 118.3,
4.2.9.27. Methyl 2-oxo-7-(hexyloxy)-2H-chromene-3-carboxyl-
ate (52).
Methyl 7-hydroxy-2-oxo-2H-chromene-3-carboxyl-
ate (13, 440 mg) was reacted with hexan-1-ol (252 mg) and
DIAD. The residue was recrystallized from MeOH (20 mL) to give
the product as colorless needles (434 mg, 71%): mp 104–105 °C;
1H NMR (500 MHz, DMSO-d6) d 0.86 (t, 3H, 3J = 7.0 Hz, CH3),

M. D. Mertens et al. / Bioorg. Med. Chem. 22 (2014) 1916–1928
1925
1.28–1.31 (m, 4H, CH2), 139–1.42 (m, 2H, CH2), 1.72 (q, 2H,
3J = 6.6 Hz, CH2), 3.80 (s, 3H, CH3), 4.09 (t, 2H, 3J = 6.6 Hz, CH2–O),
6.96–6.98 (m, 2H, 6-H, 8-H), 7.80 (d, H, 3J = 8.9 Hz, 5-H), 8.71 (s,
1H, 4-H); 13C NMR (125 MHz, DMSO-d6) d 14.0, 22.1, 25.1, 28.4,
31.0, 52.3, 68.8, 100.8, 111.4, 113.0, 113.7, 131.8, 149.6, 156.3,
157.2, 163.5, 164.4. Anal. Calcd for (C17H20O5) C, 67.09; H, 6.62.
Found C, 67.00; H, 6.56. LC–MS (ESI) (90% H2O to 100% MeOH in
10 min, then 100% MeOH to 20 min, DAD 220–400 nm), 98.7% pur-
ity, m/z = 305.21 ([M+H]+).
The product precipitated and was filtered off, washed with H2O
(10 mL) to give a light green solid (252 mg, 93%): mp 196–
198 °C; 1H NMR (500 MHz, DMSO-d6) d 1.92 (app quint, 2H,
3J = 6.6 Hz, 2-CH2), 2.33 (dt, 2H, 3J = 7.1 Hz, 4J = 2.9 Hz, 3-CH2),
2.80 (t, 1H, 4J = 2.9 Hz, CH), 4.18 (t, 2H, 3J = 6.3 Hz, CH2–O), 6.99
(dd, 1H, 3J = 8.7 Hz, 4J = 2.5 Hz, 6-H), 7.02 (d, H, 4J = 2.6 Hz, 8-H),
7.81 (d, 1H, 3J = 8.5 Hz, 5-H), 8.70 (s, 1H, 4-H), 12.94 (br s, 1H,
OH); 13C NMR (125 MHz, DMSO-d6) d 14.5, 27.6, 67.3, 71.9, 83.6,
100.9, 111.8, 113.7, 114.0, 131.7, 149.1, 157.0, 157.4, 164.0,
164.2. Anal. Calcd for C15H12O5: C, 66.17; H, 4.44. Found: C,
65.86; H, 4.47. LC–MS (ESI) (90% H2O to 100% MeOH in 10 min,
then 100% MeOH to 20 min, DAD 220–400 nm), 99.0% purity, m/
z = 273.21 ([M+H]+).
4.2.9.28.
(55).
7-(Hex-5-ynyloxy)-3,4-dihydroquinolin-2(1H)-one
7-Hydroxy-3,4-dihydroquinolin-2(1H)-one (23,
326 mg) was reacted with 5-hexyn-1-ol (294 mg) and DIAD. The
residue was purified by column chromatography with petroleum
ether/EtOAc (1:1) to obtain a white solid (182 mg, 37%): mp 84–
86 °C; 1H NMR (500 MHz, DMSO-d6) d 1.54–1.60 (m, 2H, CH2),
1.72–1.79 (m, 2H, CH2), 2.21 (dt, 2H, 4J = 2.5 Hz, 3J = 7.3 Hz, CH2),
2.38–2.41 (m, 2H, CH2), 2.75 (t, 1H, 4J = 7.3 Hz, CH), 2.77.278 (m,
2H, CH2), 3,89 (t, 2H, 3J = 6.3 Hz, CH2), 6.42 (d, 1H, 4J = 2.5 Hz, 8-
H), 6.46 (dd, 1H, 4J = 2.6 Hz, 3J = 8.3 Hz, 6-H), 7.02 (d, 1H,
3J = 8.2 Hz, 8-H), 9.92 (s, 1H, NH); 13C NMR (125 MHz, DMSO-d6)
d 17.6, 24.1, 24.8, 27.9, 30.9, 67.1, 71.5, 84.4, 101.9, 107.7, 115.7,
128.5, 139.3, 158.0, 170.4. HRMS-ESI m/z [M+Na]+ calcd for C15H17-
NO2Na: 266.1151, found: 266.1159. LC–MS (ESI) (90% H2O to 100%
MeOH in 10 min, then 100% MeOH to 20 min, DAD 220–400 nm),
95.7% purity, m/z = 244.32 ([M+H]+).
4.2.10.2.
chromene-3-carboxamide (31).
N-(4-Fluorophenyl)-2-oxo-7-(pent-4-ynyloxy)-2H-
2-Oxo-7-(pent-4-ynyloxy)-
2H-chromene-3-carboxilic acid (30, 272 mg, 1.0 mmol),
4-Fluoro-aniline (111 mg, 1.0 mmol) and DIPEA (0.26 g, 0.36 mL,
2.0 mmol) were dissolved in DMF (10 mL). HATU (456 mg,
1.2 mmol) was added and the solution was stirred at room temper-
ature. After 1 h a precipitate occurred and the solution was stirred
for an additional hour. After evaporation in vacuo the residue was
suspended in EtOAc (100 mL). The organic phase was washed with
10% citric acid solution, saturated NaHCO3 and brine. After drying
(Na2SO4) the solvent was removed under reduced pressure. The
residue was recrystallized from MeOH (30 mL) to give the product
as a light green solid (135 mg, 37%): mp 189–191 °C; 1H NMR
(500 MHz, DMSO-d6) d 1.93 (app quint, 2H, 3J = 6.3 Hz, 2-CH2),
2.33 (dt, 2H, 3J = 7.1 Hz, 4J = 2.5 Hz, 3-CH2), 2.81 (t, 1H, 4J = 2.8 Hz,
CH), 4.21 (t, 2H, 3J = 6.0 Hz, CH2–O), 7.07 (dd, 1H, 3J = 8.7 Hz,
4J = 2.2 Hz, 6-H), 7.15 (d, H, 4J = 2.6 Hz, 8-H), 7.19–7.22 (m, 2H, ar-
om.H), 7.73–7.76 (m, 2H, arom.H), 7.92 (d, 1H, 3J = 8.5 Hz, 5-H),
8.89 (s, 1H, 4-H), 10.62 (s, 1H, NH); 13C NMR (125 MHz, DMSO-
d6) d 14.5, 27.5, 67.4, 71.9, 83.6, 101.0, 112.4, 114.2, 115.6, 115.6,
115.8, 121.9, 122.0, 131.9, 134.6, 148.2, 156.4, 157.7, 159.6,
160.2, 161.1, 164.0. HRMS-ESI m/z [M+Na]+ calcd for C21H16FNO4-
Na: 388.0956, found: 388.0952. LC–MS (ESI) (90% H2O to 100%
MeOH in 10 min, then 100% MeOH to 20 min, DAD 220–400 nm),
99.7% purity, m/z = 366.19 ([M+H]+).
4.2.9.29. Methyl 7-(2-bromoethoxy)-2-oxo-2H-chromene-3-car-
boxylate (56).
Methyl 7-hydroxy-2-oxo-2H-chromene-3-car-
boxylate (13, 440 mg) was reacted with 2-bromoethanol (0.38 g,
0.21 mL) and DEAD. The residue was recrystallized from MeOH
(40 mL) and obtained as white needles (535 mg, 82%): mp 169–
170 °C; 1H NMR (500 MHz, DMSO-d6) d 3.80 (s, 3H, CH3), 3.84 (t,
2H, 3J = 5.4 Hz, CH2), 4.48 (t, 2H, 3J = 5.4 Hz, CH2), 7.03 (dd, 1H,
4J = 2.5 Hz, 3J = 8.5 Hz, 6-H), 7.05 (d, 1H, 4J = 2.5 Hz, 8-H), 7.84 (d,
H, 3J = 8.5 Hz, 5-H), 8.74 (s, 1H, 4-H); 13C NMR (125 MHz, DMSO-
d6, 30 °C) d 31.0, 52.3, 68.8, 101.1, 111.9, 113.5, 113.7, 131.9,
149.5, 156.3, 157.0, 163.5, 163.5. Anal. Calcd for C13H11BrO5: C,
47.73; H, 3.39. Found: C, 48.10; H, 3.48. LC–MS (ESI) (90% H2O to
100% MeOH in 10 min, then 100% MeOH to 20 min, DAD 220–
400 nm), 99.5% purity, m/z = 329.14 ([M+H]+).
4.2.10.3. tert-Butyl 4-(2-oxo-7-(pent-4-ynyloxy)-2H-chromene-
3-carbonyl)piperazine-1-carboxylate (32).
4-ynyloxy)-2H-chromene-3-carboxylic acid
2-Oxo-7-(pent-
(30, 136 mg,
4.2.9.30. 7-(2-Bromoethoxy)-2H-chromen-2-one (57).
Umbel-
liferone (21, 324 mg) was reacted with 2-bromoethanol (0.38 g,
0.218 mL) and DEAD. The residue was purified by column chromatogra-
phy (petroleum ether/EtOAc 3:1) followed by recrystallisation from
MeOH (20 mL) to obtain colorless needles (330 mg, 62%): mp 130–
132 °C (lit. mp 130.5 °C);50 1H NMR (500 MHz, DMSO-d6) d 3.82 (t, 2H,
3J = 5.7 Hz, CH2), 4.43 (t, 2H, 3J = 5.4 Hz, CH2), 6.29 (d, 1H, 3J = 9.5 Hz,
3-H), 6.97 (dd, 1H, 4J = 2.6 Hz, 3J = 8.5 Hz, 6-H), 7.01 (d, 1H, 4J = 2.6 Hz,
8-H), 7.84 (d, H, 3J = 8.5 Hz, 5-H), 7.98 (d, 1H, 3J = 9.5 Hz, 4-H); 13C
NMR (125 MHz, DMSO-d6) d 31.1, 68.5, 101.6, 112.9, 112.9, 129.7,
144.4, 155.5, 160.3, 161.1. Anal. Calcd for C11H9BrO3: C, 49.10; H, 3.37.
Found: C, 49.08; H, 3.45. LC–MS (ESI) (90% H2O to 100% MeOH in
10 min, then 100% MeOH to 20 min, DAD 220–400 nm), 100% purity,
m/z = 268.98 ([M+H]+).
0.5 mmol) was dissolved in DMF (10 mL) and cooled to 0 °C.
1-Boc-piperazine (95 mg, 0.6 mmol) and DIPEA (0.13 g, 0.18 mL,
1.0 mmol) were added. HATU (228 mg, 0.6 mmol) was added to
the mixture and the solution was stirred for 3 h at room tempera-
ture. After evaporation, the residue was dissolved in EtOAc
(50 mL). The organic phase was washed with 10% citric acid (3 ꢂ
50 mL), saturated NaHCO3 (3 ꢂ 50 mL) and brine (50 mL). The or-
ganic extract was dried over Na2SO4, filtrated and purified by col-
umn chromatography with petroleum ether/EtOAc 1:1 to obtain a
white solid (179 mg, 81%): mp 117–119 °C; 1H NMR (500 MHz,
DMSO-d6) d 1.40 (s, 9H, (CH3)3), 1.92 (app. quint, 2H, 3J = 6.6 Hz,
2-CH2), 2.34 (dt, 2H, 3J = 7.3 Hz, 4J = 2.5 Hz, 3-CH2), 2.81 (t, 1H,
4J = 2.6 Hz, 5-CH), 3.31–3.37 (m, 6H, CH2), 3.56 (br s, 2H, CH2),
4.17 (t, 2H,, 3J = 6.0 Hz, 1-CH2), 7.00 (d, 1H, 3J = 8.7 Hz, 4J = 2.5 Hz,
6-H), 7.04 (dd, 1H, 4J = 2.5 Hz, 8-H), 7.68 (d, H, 3J = 8.5 Hz, 5-H),
8.12 (s, 1H, 4-H); 13C NMR (125 MHz, DMSO-d6) d 14.5, 27.6,
28.2, 41.4, 46.3, 67.1, 71.9, 79.4, 83.6, 101.3, 112.1, 113.3, 120.9,
130.3, 143.1, 154.0, 155.7, 158.0, 162.5, 163.6. Anal. Calcd for
4.2.10. Preparation of compounds 30–32
4.2.10.1. 2-Oxo-7-(pent-4-ynyloxy)-2H-chromene-3-carboxylic
acid (30).
Methyl 2-oxo-7-(pent-4-ynyloxy)-2H-chromene-
3-carboxylate (26, 286 mg, 1.0 mmol) was dissolved in THF
(12 mL) and water (18 mL). Lithium hydroxide monohydrate
(252 mg, 6.0 mmol) was added and the solution was stirred at
room temperature for 2 h. The solution was filtrated and the fil-
trate was cooled on an ice bath before 10% HCl (10 mL) was added.
C24H28N2O6: C, 65.44; H, 6.41; N, 6.36. Found: C, 65.05; H, 6.44;
N, 6.17. LC–MS (ESI) (90% H2O to 100% MeOH in 10 min, then
100% MeOH to 20 min, DAD 220–400 nm), 97.7% purity,
m/z = 441.50 ([M+H]+).

1926
M. D. Mertens et al. / Bioorg. Med. Chem. 22 (2014) 1916–1928
4.2.11. Preparation of compounds 58, 59, 53, 54
4.2.11.1. Methyl 7-(2-(-2-nitro-N-(prop-2-ynyl)phenylsulfonam-
ido)ethoxy)-2-oxo-2H-chromene-3-carboxylate (58).
155.4, 160.3, 161.1. HRMS-ESI m/z [M+H]+ calcd for C20H17N2O7S:
429.0751, found: 429.0749. LC–MS (ESI) (90% H2O to 100% MeOH
in 10 min, then 100% MeOH to 20 min, DAD 220–400 nm), 100%
purity, m/z = 429.16 ([M+H]+), 446.18 ([M+NH4]+).
Prop-
argylamine (1.65 g, 1.92 mL, 30 mmol) was dissolved in CH2Cl2
(50 mL). Triethylamine (6.06 g, 8.35 mL, 60 mmol) was added. A
solution of 2-nitrobenzenesulfonyl chloride (6.65 g, 30 mmol) in
CH2Cl2 (50 mL) was added dropwise over 30 min. After stirring
for 4 h at room temperature, the solution was washed with 1 N
HCl solution (100 mL), saturated NaHCO3 (100 mL) and brine
(100 mL). The organic phase was dried over Na2SO4, filtrated and
evaporated in vacuo. The residue was purified by column chroma-
tography using petroleum ether/EtOAc (3:1) as eluent. 2-Nitro-N-
(prop-2-ynyl)benzenesulfonamide was obtained as a pale green
solid (4.80 g, 67%): mp 95–96 °C; 1H NMR (500 MHz, DMSO-d6) d
3.03 (t, 1H, 4J = 2.5 Hz, CH), 3.84 (d, 2H, 4J = 2.5 Hz, CH2), 7.84–
7.87 (m, 2H, arom.H), 7.95–7.98 (m, 1H, arom.H), 8.02–8.06 (m,
1H, arom.H), 8.51 (br s, 1H, NH); 13C NMR (125 MHz, DMSO-d6)
d 32.2, 75.0, 79.2, 124.5, 130.1, 132.7, 133.1, 134.3, 147.8. Anal.
Calcd for C9H8N2O7S: C, 45.00; H, 3.36; N, 11.66. Found: C, 44.90;
H, 3.50; N, 11.59. LC–MS (ESI) (90% H2O to 100% MeOH in
10 min, then 100% MeOH to 20 min, DAD 220–400 nm), 99.0% pur-
ity, m/z = 258.38 ([M+NH4]+). 2-Nitro-N-(prop-2-ynyl)benzenesul-
fonamide (240 mg, 1.0 mmol), K2CO3 (276 mg, 2.0 mmol) and
4.2.11.3. Methyl 2-oxo-7-(2-(prop-2-ynylamino)ethoxy)-2H-
chromene-3-carboxylate (53).
Methyl 7-(2-(-2-nitro-N-
(prop-2-ynyl)phenylsulfonamido)ethoxy)-2-oxo-2H-chromene-3-
carboxylate (58, 260 mg, 0.53 mmol), K2CO3 (221 mg, 1.59 mmol)
and thiophenol (0.12 g, 0.10 mL, 1.06 mmol) were stirred for 3 h
in DMF (10 mL). After evaporation in vacuo, the residue was dis-
solved in H2O (50 mL) and the aqueous phase was extracted with
EtOAc (3 ꢂ 50 mL). The combined organic layers were washed with
brine (50 mL), dried over Na2SO4, filtrated and evaporated. The res-
idue was purified by column chromatography using EtOAc + 1%
Et3N as eluent. The product was obtained as white solid (109 mg,
68%). The product was finally lyophilized with HCl to obtain the
salt: mp 233–234 °C; 1H NMR (500 MHz, DMSO-d6) d 3.42 (t, 2H,
3J = 5.1 Hz, NCH2), 3.71 (t, 1H, 4J = 2.6 Hz, CH), 3.81 (s, 3H, CH3),
3.98 (d, 2H, 4J = 2.6 Hz, CH2), 4.43 (t, 2H, 3J = 5.4 Hz, CCH2), 7.06
(dd, 1H, 4J = 2.5 Hz, 3J = 8.7 Hz, 6-H), 7.09 (d, 1H, 4J = 2.2 Hz, 8-H),
7.88 (d, 1H, 3J = 8.8 Hz, 5-H), 8.76 (s, 1H, 4-H), 9.61 (br s, 2H,
NH2); 13C NMR (125 MHz, DMSO-d6) d 36.1, 45.0, 52.4, 64.4, 75.2,
79.7, 101.2, 112.1, 113.7, 131.9, 149.5, 156.3, 156.9, 163.2, 163.4.
Anal. Calcd for C16H16ClNO5: C, 56.90; H, 4.77; N, 4.15. Found: C,
56.71; H, 4.82; N, 4.06. LC–MS (ESI) (90% H2O to 100% MeOH in
10 min, then 100% MeOH to 20 min, DAD 220–400 nm), 100% pur-
ity, m/z = 302.17 ([M+H]+).
methyl
7-(2-bromoethoxy)-2-oxo-2H-chromene-3-carboxylate
(56, 325 mg, 1.0 mmol) were suspended in DMF (10 mL). The reac-
tion mixture was stirred for 2 d at room temperature. After evapo-
ration in vacuo, the residue was dissolved in H2O (50 mL) and the
aqueous phase was extracted with EtOAc (3 ꢂ 50 mL). The com-
bined organic layers were dried over Na2SO4 and evaporated. The
residue was purified by column chromatography using petroleum
ether/EtOAc (1:1). The product was obtained as a white solid
(300 mg, 62%): mp 169–172 °C; 1H NMR (500 MHz, DMSO-d6) d
3.23 (t, 1H, 4J = 2.2 Hz, CH), 3.77 (t, 2H, 3J = 5.4 Hz, CH2), 3.80 (s,
3H, OCH3), 4.31–4.33 (m, 4H, CH2, CH2), 6.87 (dd, 1H, 4J = 2.2 Hz,
3J = 8.7 Hz, 6-H), 6.95 (d, 1H, 4J = 2.2 Hz, 8-H), 7.80 (d, 1H,
3J = 8.8 Hz, 5-H), 7.81–7.84 (m, 1H, arom.H), 7.87–7.90 (m, 1H, ar-
om.H), 7.96–7.98 (m, 1H, arom.H), 8.09–8.11 (m, 1H, arom.H), 8.72
(s, 1H, 4-H); 13C NMR (125 MHz, DMSO-d6) d 37.5, 46.0, 52.3, 66.3,
76.6, 77.6, 101.0, 111.8, 113.5, 113.6, 124.4, 130.4, 131.3, 131.8,
132.6, 135.0, 147.8, 149.5, 156.3, 157.0, 163.5. HRMS-ESI m/z
[M+Na]+ calcd for C22H18N2O9SNa: 509.0625, found: 509.0630.
LC–MS (ESI) (90% H2O to 100% MeOH in 10 min, then 100% MeOH
to 20 min, DAD 220–400 nm), 100% purity, m/z = 487.26 ([M+H]+),
504.44 ([M+NH4]+).
4.2.11.4. 7-(2-(Prop-2-ynylamino)ethoxy)-2H-chromen-2-one
(54).
2-Nitro-N-(2-(2-oxo-2H-chromen-7-yloxy)ethyl)-N-
(prop-2-ynyl)benzenesulfonamide (59, 300 mg, 0.7 mmol), K2CO3
(290 mg, 2.1 mmol) and thiophenol (0.15 g, 0.16 mL, 1.4 mmol)
were stirred for 6 h in DMF (10 mL). After evaporation in vacuo,
the residue was solved in H2O (50 mL) and the aqueous phase
was extracted with EtOAc (3 ꢂ 50 mL). The organic layers were
washed with brine (50 mL), dried over Na2SO4, filtrated and evap-
orated. The residue was purified by column chromatography using
EtOAc + 1% Et3N as eluent. The product was obtained as white solid
(92 mg, 54%): mp 103–104 °C; 1H NMR (500 MHz, DMSO-d6) d 2.25
(br s, 1H, NH), 2.94 (t, 2H, 3J = 5.4 Hz, CH2), 3.05 (t, 1H, 4J = 2.5 Hz,
CH), 3.37 (d, 2H, 4J = 2.5 Hz, CH2), 4.13 (t, 2H, 3J = 5.4 Hz, CH2), 4.33
(d, 2H, 4J = 2.6 Hz, CH2CCH), 6.26 (d, 1H, 3J = 9.8 Hz, 3-H), 6.94 (dd,
1H, 4J = 2.5 Hz, 3J = 8.7 Hz, 6-H), 6.97 (d, 2H, 4J = 2.2 Hz, 8-H), 7.61
(d, 1H, 3J = 8.5 Hz, 5-H), 7.97 (d, 1H, 3J = 9.5 Hz, 4-H); 13C NMR
(125 MHz, DMSO-d6) d 37.5, 46.7, 68.2, 73.8, 83.1, 101.4, 112.5,
112.6, 112.8, 129.6, 144.4, 155.5, 160.4, 161.9. Anal. Calcd for
4.2.11.2.
2-Nitro-N-(2-(2-oxo-2H-chromen-7-yloxy)ethyl)-N-
2-Nitro-N-(prop-2-
(prop-2-ynyl)benzenesulfonamide (59).
ynyl)benzenesulfonamide (240 mg, 1.0 mmol, prepared as noted
in the aforementioned procedure), K2CO3 (553 mg, 4.0 mmol) and
7-(2-bromoethoxy)-2H-chromen-2-one (57, 267 mg, 1.0 mmol)
were suspended in DMF (10 mL). The mixture was stirred for 4 d
at room temperature. After evaporation in vacuo, the residue was
dissolved in H2O (50 mL) and the aqueous phase was extracted
with EtOAc (3 ꢂ 50 mL). The organic layer was dried over Na2SO4,
filtrated and evaporated. The residue was purified by column chro-
matography using petroleum ether/EtOAc (1:1). The product was
obtained as a white solid (320 mg, 75%): mp 104–105 °C; 1H
NMR (500 MHz, DMSO-d6) d 3.23 (t, 1H, 4J = 2.6 Hz, CH), 3.76 (t,
2H, 3J = 5.4 Hz, CH2), 4.28 (t, 2H, 3J = 5.4 Hz, CH2), 4.33 (d, 2H,
4J = 2.6 Hz, CH2CCH), 6.28 (d, 1H, 3J = 9.5 Hz, 3-H), 6.91 (d, 1H,
4J = 2.2 Hz, 8-H), 7.06 (d, 2H, 3J = 8.5 Hz, 5-H), 7.81–7.84 (m, 1H, ar-
om.H), 7.87–7.90 (m, 1H, arom.H), 7.96 (d, 1H, 3J = 9.5 Hz, 4-H),
7.96–7.98 (m, 1H, arom.H), 8.09–8.11 (m, 1H, arom.H); 13C NMR
(125 MHz, DMSO-d6) d 37.5, 46.0, 66.0, 76.6, 77.6, 101.4, 112.7,
112.8, 124.4, 129.6, 130.4, 131.4, 132.5, 134.9, 144.4, 147.8,
C14H13NO3: C, 69.13; H, 5.39; N, 5.76. Found C, 68.89; H, 5.31; N,
5.58. LC–MS (ESI) (90% H2O to 100% MeOH in 10 min, then 100%
MeOH to 20 min, DAD 220–400 nm), 99.6% purity, m/z = 244.34
([M+H]+).
4.3. Procedure for logD7.4 estimation
LogD7.4 estimation was performed by an HPLC method as
described.41 HPLC grade acetonitrile was from VWR international
(Leuven, Belgium). Atenolol was from Fagron (Barsbüttel, Ger-
many), metoprolol tartrate from Synopharm (Barsbüttel, Ger-
many), diltiazem hydrochloride from Hexal (Munich, Germany),
labetalol hydrochloride from the EDQM (Strasbourg, France) and
triphenylene from Alfa Aesar (Karlsruhe, Germany). Analytical
HPLC was performed on a Jasco HPLC 2000 instrument with a Po-
laris C18-A column (2 mm I.D., 50 mm length, 3 lm particle size)
from Varian Inc. (Lake Forest, CA, USA). A linear mobile phase gra-
dient was used with 10 mM ammonium acetate (adjusted to pH

M. D. Mertens et al. / Bioorg. Med. Chem. 22 (2014) 1916–1928
1927
5. Yoshida, S.; Rosen, T. C.; Meyer, O. G.; Sloan, M. J.; Ye, S.; Haufe, G.; Kirk, K. L.
Bioorg. Med. Chem. 2004, 12, 2645–2652.
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and acetonitrile as mobile phase B. The gradient table was as
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5.5 min/0% B. A flow rate of 0.8 mL/min and a column temperature
of 40 °C were applied. UV detection wavelength was 254 nm. Solu-
tions of test compounds in DMSO (0.5 mg/mL) were injected and
measurements were performed in duplicate.
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4.4. MAO inhibition assays
Recombinant human MAO-A and MAO-B enzymes expressed
in baculovirus-infected insect cells were purchased from Sigma
Aldrich (M7441, M7316). The assays were carried out at room
temperature in 96-well plates in a final volume of 200
compound (2 L) dissolved in DMSO was pipetted into the 96-
well plates. Enzyme solution (90 L) in sodium phosphate buf-
lL. Test
l
l
fer (50 mM, pH 7.4) was subsequently added, and the mixture
was preincubated for 30 min at room temperature. To each
well, 0.3
lg of recombinant human MAO-A, or 0.9
lg MAO-B,
L of freshly prepared
17. Frederick, R.; Robert, S.; Charlier, C.; de Ruyck, J.; Wouters, J.; Pirotte, B.;
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54, 3839.
respectively, was added, followed by 90
Amplex Red reagent (Invitrogen A12214), prepared according
to the manufacturer’s recommendation as follows: for each
l
19. Stefanachi, A.; Favia, A. D.; Nicolotti, O.; Leonetti, F.; Pisani, L.; Catto, M.;
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plate, 1 mg of Amplex Red, dissolved in 200
100 L of reconstituted horseradish peroxidase (HRP 200 U/mL,
Sigma Aldrich P6782) were added to 9700 of sodium
phosphate buffer (50 mM, pH 7.4). To each well, 20 L of an
aqueous solution (final concentration 150 M) of p-tyramine
(Alfa Aesar A12220) was added to start the enzymatic reaction,
which was kept in the dark. The production of hydrogen per-
oxide produced resorufin which was monitored and quantified
by a microplate fluorescence reader (PHERAstar BMG Labtech,
excitation 544 nm, emission 590 nm) over 45 min. Data were
analyzed using GRAPH PAD PRISM Version 4 (San Diego, CA,
USA).
lL of DMSO and
l
lL
l
l
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4.5. MAO-B reactivation experiment
Time-dependent activity measurements were performed using
human MAO-B under assay conditions. Inhibitors 36 and 44 as well
as two reference compounds were examined at concentrations
approximately corresponding to their IC80 value. The enzyme/
inhibitor mixture was preincubated for 1 h. The enzyme reaction
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was started by the addition of 10 lM p-tyramine. After 22 min,
the substrate concentration was increased to a final concentration
of 1 mM of p-tyramine. The reactivation of the enzyme was moni-
tored by fluorescence measurements over a period of 5 h.
34. Pisani, L.; Muncipinto, G.; Misciosci, T. F.; Nicolotti, O.; Leonetti, F.; Catto, M.;
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Acknowledgments
36. Secci, D.; Carradori, S.; Bolasco, A.; Chimenti, P.; Yáñez, M.; Ortuso, F.; Alcaro, S.
Eur. J. Med. Chem. 2011, 46, 4846.
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1935.
We thank Marion Schneider for performing LC/MS analyses and
Sabine Terhart-Krabbe and Annette Reiner for recording NMR spec-
tra. Technical assistance in performing MAO inhibition assays by
Anika Püsche and Angelika Fischer is gratefully acknowledged.
39. Jeong, S. H.; Han, X. H.; Hong, S. S.; Hwang, J. S.; Hwang, J. H.; Lee, D.; Lee, M. K.;
Ro, J. S.; Hwang, B. Y. Arch. Pharm. Res. 2006, 29, 1119.
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41. Blättermann, S.; Peters, L.; Ottersbach, P. A.; Bock, A.; Konya, V.; Weaver, C. D.;
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.01.046.
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