6-Substituted 2-oxo-2H-1-benzopyran-3-carboxylic acid derivatives in a new approach of the treatment of cancer cell invasion and metastasis

Article abstract of DOI:10.1016/j.ejmech.2008.01.024

Novel 6-substituted 2-oxo-2H-1-benzopyran-3-carboxylic acid derivatives were synthesized and their potency in reducing the invasive behaviour of HT 1080 fibrosarcoma cells was evaluated. Structure-activity relationships were deduced from biological results and will be used in further design of new active compounds. In particular, the acetoxymethyl substituent found at the 6-position of previously described active compounds can be replaced by an acetamidomethyl substituent without loss of potency; while the presence of an aryl ester function at the 3-position was preferred to a thioester or an amide function to induce marked biological activity. This work confirms the interest of aryl esters of 6-substituted coumarin-3-carboxylic acids as potential new anti-cancer agents.

Full text of DOI:10.1016/j.ejmech.2008.01.024

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 2735e2750
http://www.elsevier.com/locate/ejmech
Original article
6-Substituted 2-oxo-2H-1-benzopyran-3-carboxylic acid
derivatives in a new approach of the treatment of
cancer cell invasion and metastasis
a
Isabelle Kempen , Marc Hemmer , Stephane Counerotte , Lionel Pochet ,
a
a
b
´
a
c
c
c
`
Pascal de Tullio , Jean-Michel Foidart , Silvia Blacher , Agnes Noel ,
¨
Francis Frankenne c, Bernard Pirotte a,
*
a Drug Research Center, Laboratoire de Chimie Pharmaceutique, Universite´ de Lie`ge, 1 Avenue de l’Hoˆpital,
tour 4 (þ5), Sart-Tilman, B-4000 Lie`ge, Belgium
b De´partement de Pharmacie, Universite´ de Namur, FUNDP, 61 rue de Bruxelles, B-5000 Namur, Belgium
c Laboratoire de Biologie des Tumeurs et du De´veloppement, Universite´ de Lie`ge, 1 Avenue de l’Hoˆpital, tour 3 (þ4),
Sart-Tilman, B-4000 Lie`ge, Belgium
Received 22 November 2007; received in revised form 14 January 2008; accepted 17 January 2008
Available online 31 January 2008
Abstract
Novel 6-substituted 2-oxo-2H-1-benzopyran-3-carboxylic acid derivatives were synthesized and their potency in reducing the invasive be-
haviour of HT 1080 fibrosarcoma cells was evaluated. Structureeactivity relationships were deduced from biological results and will be
used in further design of new active compounds. In particular, the acetoxymethyl substituent found at the 6-position of previously described
active compounds can be replaced by an acetamidomethyl substituent without loss of potency; while the presence of an aryl ester function
at the 3-position was preferred to a thioester or an amide function to induce marked biological activity.
This work confirms the interest of aryl esters of 6-substituted coumarin-3-carboxylic acids as potential new anti-cancer agents.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Coumarin derivatives; Cancer; Cell invasion; Anti-cancer drug
1. Introduction
now focussed on the search of compounds interfering with
the cancer cell invasion process and expressing anti-angiogenic
properties [1]. Examples of drugs, among which monoclonal
antibodies and small molecules, are currently under clinical in-
vestigation and some of them have recently been approved for
use in man in the treatment of several cancers associated or not
with conventional chemotherapies or radiation therapies [2e9].
These drugs comprise vascular endothelial growth factor
(VEGF) and epidermal growth factor (EGF) inhibitors (mono-
clonal antibodies) as well as VEGF and EGF receptor tyrosine
kinase inhibitors (small-molecule inhibitors), but also matrix
metalloproteinase (MMP), urokinase (uPA), cycloogygenase-
2 (COX-2) or methionine aminopeptidase inhibitors [1].
We recently reported that two synthetic 6-substituted
coumarin-3-carboxylic acid derivatives, 3-chlorophenyl
One of the most difficult problems arising during cancer
therapy is the occurrence of cancer cell invasion responsible
for the spread of tumour cells throughout the body. Such an
event leading to metastases is an important cause of the bad
prognosis of numerous cancers. Although anti-cancer chemo-
therapy mainly consists in the use of cytotoxic agents interfer-
ing with cell division, another research field has recently
emerged with the design and identification of agents able to in-
hibit or limit the metastatic process. Indeed, many efforts are
* Corresponding author. Tel.: þ32 4 366 43 65; fax: þ32 4 366 43 62.
E-mail address: b.pirotte@ulg.ac.be (B. Pirotte).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.01.024

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I. Kempen et al. / European Journal of Medicinal Chemistry 43 (2008) 2735e2750
6-acetoxymethyl-2-oxo-2H-1-benzopyran-3-carboxylate (1)
and 3-bromophenyl 6-acetoxymethyl-2-oxo-2H-1-benzo-
pyran-3-carboxylate (2) (Fig. 1), expressed a marked potency
in inhibiting cancer cell invasion in vitro and tumour growth
in vivo [10]. Simple natural and synthetic coumarins such as es-
culetin (3), osthole (4) and the synthetic 6-nitro-substituted
coumarin (5) (Fig. 1) are also known for a long time to exert
anti-cancer properties, but, for most of them, by an unknown
mechanism [11e14]. Recently reported coumarin derivatives
such as G8935 (6) (Fig. 1), structurally related to 1 and 2 and
identified as mitogen activated protein (MAP) kinase/extracel-
lular signal-regulated kinase (ERK) kinase inhibitors (MEK1
inhibitors), raise the possibility that the coumarin-3-carboxyl-
ates 1 and 2 may exert their anti-cancer activity through inhibi-
tion of MEK1 [15]. Another recent work showed that
coumarin-3-carboxamides (7, Fig. 1) tightly related to the
chemical structure of 1 and 2 selectively inhibited the tumour
cells that have a high level of epidermal growth factor receptor
(EGRF or erbB1) and erbB2 (HER2) receptor expression [16].
Interestingly, the chemical structure of the active coumarins
1 and 2 may be compared to that of linomide (roquinimex) (8,
Fig. 2), a quinolinone isostere of coumarins known to be an
immunomodulator, but also expressing anti-angiogenic activ-
ity in vitro and in vivo [17e19]. Moreover, the preferred con-
formation adopted by linomide, as found in the crystal [20]
(80; Fig. 2), can be superimposed to the preferential conforma-
tion adopted, in the solid state and probably in solution, by
aryl esters of coumarin-3-carboxylic acid derivatives (90;
Fig. 2) in accordance to recent published crystallographic
and molecular modelling studies [21,22]. In contrast to aryl es-
ters of 6-substituted coumarin-3-carboxylic acids, the corre-
sponding aryl amides 10 (Fig. 2) are not able to adopt such
conformation because a strong intra-molecular hydrogen
bound is established between the hydrogen atom of the exocy-
clic NH group and the lactonic carbonyl group [21].
Using coumarins 1 and 2 as lead compounds, the present
work aimed at describing new examples of coumarin-3-car-
boxylic acid derivatives in order to better define structuree
activity relationships that could explain their anti-invasive
activity. Particular attention was paid to the replacement of the
oxygen atom included in the exocyclic ester function in the 3-
position by a sulphur atom (thioesters) or by a nitrogen atom
(amides) with or without a methyl group. The importance of
the nature and the size of the substituent in the 6-position was
also examined. Among them, the replacement of the meta-
bolically labile ester function (acetoxymethyl moiety) in the
6-position by a more stable amide function (acetamidomethyl
moiety) was investigated. In these series, the presence of a halo-
gen atom in the meta position of the phenyl ring in the 3-position
(preferably a chlorine or a bromine atom) was maintained like in
the structure of the lead compounds 1 and 2.
2. Chemistry
Scheme 1 illustrates the synthetic pathway giving access to
the coumarinic derivatives characterised by the introduction of
a thioester function or an amide function in the 3-position.
O
O
O
HO
H3C
X
O
O
HO
O
O
O
1: X = Cl
2: X = Br
3
O2N
HO
H3C
O
O
O
O
O
4
5
O
O
N
H
Y
X
H3C
N
O
F
O
O
O
O
CH3
6
7
Fig. 1. Chemical structure of coumarins known to exert anti-cancer activity.

I. Kempen et al. / European Journal of Medicinal Chemistry 43 (2008) 2735e2750
2737
H3C
OH
N
O
OH
O
O
O
N
CH3
N
N
CH3
CH3
8'
8
O
O
O
Y
X
X
O
O
Y
O
O
O
9'
9
O
O
X
N
H
Y
O
10
Fig. 2. Structure of the anti-angiogenic drug linomide (8) in two different conformations. The ‘cis’ conformation of linomide (80) can be superimposed to the low
energy ‘cis’ conformation of the coumarinic aryl esters (90) but not to the stable conformation of the coumarinic aryl amides (10) characterised by the existence of
a strong intra-molecular hydrogen bond.
Compound 12 obtained from 11 by acetylation was con-
verted into the corresponding acid chloride 13 as previously
described [21,23]. The acyl chloride intermediate 13 then re-
acted with appropriate thiophenols and anilines to provide
the thioesters 14, the secondary amides 15 and the tertiary am-
ides 16 (Scheme 1).
acid were obtained according to Scheme 4. Compounds 25
previously described [23] reacted with hexamethylenetetra-
mine to give the hexaminium salts 26. The ‘Delepine’ reaction
conditions of hydrolysis were then used to convert the hexami-
nium salts into the corresponding primary amines 27. In the
last step, acetylation of the amine function of 27 provided
the corresponding acetamides 28.
Starting from 11, acylation, with the appropriate anhydride,
of the exocyclic OH function located on the hydroxymethyl
group in the 6-position led to the acid esters 17 (Scheme 2).
The latter intermediates were converted into the acyl chlorides
18 and then into the 3-bromophenyl esters 19 after reaction of
18 with 3-bromophenol in the presence of a base (Scheme 2).
Examples of coumarin derivatives with a nitro group in the
6-position were obtained according to Scheme 3. The com-
mercially available 5-nitrosalicylaldehyde 20 was converted
into ethyl 6-nitrocoumarin-3-carboxylate 21 after reaction
with ethyl malonate in the ‘Knoevenaegel’ reaction conditions.
The ethyl ester 21 was hydrolyzed to provide the correspond-
ing carboxylic acid 22. The latter was converted into the acyl
chloride 23 which then reacted with the appropriate meta-
halophenol to provide the aryl esters 24.
3. Results and discussion
The new synthetic coumarins were examined in a ‘Boyden
chamber’ invasion assay using HT 1080 fibrosarcoma cells in
order to determine their potency in reducing the invasive be-
haviour of tumour cells [10]. The assay consists in measuring
the ability of cells treated or not with the coumarinic deriva-
tives tested at different concentrations to pass through type
IV collagen-coated Transwell cell culture inserts (chemoinva-
sion assay). Cell invasion in the absence of coumarin deriva-
tive was considered as 100%. Results were compared to
those observed with a reference compound, GI 129471 (29,
Fig. 3), a well-known broad-spectrum hydroxamate-type
MMP inhibitor, used at 1 mM concentration, previously re-
ported to be active in this model [10], and also compared to
Finally, 6-aminomethyl- (27) and 6-acetamidomethyl-
substituted (28) halophenyl esters of coumarin-3-carboxylic

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I. Kempen et al. / European Journal of Medicinal Chemistry 43 (2008) 2735e2750
O
O
O
O
O
i
OH
HO
OH
H3C
O
O
O
12
11
ii
O
O
O
O
O
iii
H3C
S
X
Cl
H3C
O
O
O
O
O
14
14a :X = H
14b: X = Cl
14c: X = Br
13
iv
v
O
O
O
O
O
H3C
N
O
H3C
O
N
H
X
X
CH3
O
O
O
15
15a: X = Cl
16 16a: X = 2-Cl
16b: X = 3-Cl
16c: X = 4-Cl
Scheme 1. Reagents: (i) Ac2O; (ii) SOCl2; (iii) ArSH, pyridine, dioxane; (iv) ArNH2, pyridine, dioxane; and (v) ArNHCH3, pyridine, dioxane.
those obtained with the active coumarin derivatives 1 and 2 at
three different concentrations: 0.1, 1 and 10 mM. The activity
at 1 mM of another synthetic MMP inhibitor, Ro 28-2653 (30,
Fig. 3), an MMP-2/MMP-9/MT-MMP-selective inhibitor, was
also examined in the same chemoinvasion assay.
Table 1 indicates that GI 129471 at 1 mM concentration was
able to reduce by 62% the invasion of HT 1080 cells through
type IV collagen. The other MMP inhibitor, Ro 28-2653, was
found to be equally active to GI 129471 at 1 mM. As previ-
ously reported, the bromo-substituted coumarin 2 giving
78% inhibition of cell invasion at 1 mM concentration was
found to be more potent than the reference compound GI
129471, whereas the corresponding chloro-substituted ana-
logue 1 was also active (51% inhibition of cell invasion at
1 mM) but less potent than the two formers [10].
compound 1 by a NH group, was also found to be less active
than 1 giving 33% inhibition of cell invasion at 1 mM (versus
51% for 1) and 57% inhibition at 10 mM (versus 94% for 1).
Such an effect could be explained by the fact that amides
such as 15a may adopt a stable conformation in which a strong
hydrogen bond interaction is established between the NH
group and the lactonic carbonyl oxygen. By contrast, a greater
conformational freedom is given to esters such as 1 and 2
which may adopt the putative favourable ‘cis’ conformation
of the two carbonyl groups (see structure 90, Fig. 2).
Surprisingly, compound 16b, the N-methyl derivative of
15a, was not very potent in the cell invasion assay, although
the presence of a methyl group on the nitrogen atom warranted
the absence of intra-molecular H-bond interaction with the
lactonic carbonyl group.
Replacement of the oxygen atom of the exocyclic ester
function in the 3-position by a sulphur atom (thioesters
14aec) resulted in a significant reduction of the ability of
the drugs at inhibiting HT 1080 cell invasion through type
IV collagen (compare the results obtained with 14b versus 1
and with 14c versus 2 at 1 and 10 mM). The absence of a
halogen atom on the phenyl ring (see 14a) appeared to be un-
favourable for the anti-invasive activity compared to halo-
substituted compounds.
Table 1 also reports examples of analogues of the lead com-
pound 2 for which the size and the branching of the alkyl chain
on the acyloxymethyl group in the 6-position were modified.
An increase in the size of the chain conducted to a progressive
reduction of drug efficacy in inhibiting cell invasion. Interest-
ingly, the branching of the alkyl chain with 19f (isopropyl) and
19g (tert-butyl) led to a less pronounced decrease of their bi-
ological activity compared to compound 2. Marked differences
can be seen especially at 0.1 mM between the linear (19ae19e)
and the branched (19fe19g) 6-acyloxymethylcoumarin
derivatives.
The secondary amide 15a, resulting from the replacement
of the oxygen atom of the exocyclic ester function of

I. Kempen et al. / European Journal of Medicinal Chemistry 43 (2008) 2735e2750
2739
O
O
O
O
O
i
HO
OH
R
OH
O
O
O
17
11
ii
O
O
O
O
O
iii
R
Br
R
Cl
O
O
O
O
O
O
17a, 18a, 19a:R = -CH2CH3
19
18
17b, 18b, 19b:R = -(CH2)2CH3
17c, 18c, 19c:R = -(CH2)3CH3
17d, 18d, 19d:R = -(CH2)4CH3
17e, 18e, 19e:R = -(CH2)5CH3
17f, 18f, 19f:R = -CH(CH3)2
17g, 18g, 19g:R = -C(CH3)3
Scheme 2. Reagents: (i) (RCO)2O; (ii) SOCl2; and (iii) 3-bromophenol, pyridine, dioxane.
The replacement of the acetyloxymethyl substituent in the
6-position of the coumarin ring of compound 1 or compound
2 by another substituent such as a nitro, an aminomethyl and
an acetamidomethyl moiety was also examined. The
biological results obtained in the ‘Boyden chamber’ assay
with the compounds are reported in Table 2. The efficacy of
the new coumarin derivatives in this test was compared to
that of previously reported coumarins bearing a hydrogen
O
O
O2N
i
O2N
H
OC2H5
OH
O
O
O
21
20
ii
O
O
iii
O2N
O2N
Cl
OH
O
O
O
22
23
iv
O
O
O2N
O
X
24a: X = H
24b: X = F
24c: X = Cl
24d: X = Br
24e: X = I
O
24
Scheme 3. Reagents: (i) diethyl malonate, piperidine, HOAc, EtOH; (ii) NaOH, H2O; (iii) SOCl2; and (iv) 3-halophenol, pyridine, dioxane.

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I. Kempen et al. / European Journal of Medicinal Chemistry 43 (2008) 2735e2750
-
O
O
O
O
.Cl
N
N
+
Cl
X
X
N
O
i
O
N
O
O
25
26
ii
O
O
O
O
O
iii
H2N
.HCl
X
H3C
N
H
X
O
O
O
O
27
28
25a, 26a, 27a, 28a: X = H
25b, 26b, 27b, 28b: X = F
25c, 26c, 27c, 28c: X = Cl
25d, 26d, 27d, 28d: X = Br
25e,26e,27e,28e: X = I
Scheme 4. Reagents: (i) hexamethylenetetramine, CHCl3; (ii) HCl, EtOH; and (iii) Ac2O, TEA, DMF.
atom (31), a methyl (32), a chloromethyl (25c) and an acetox-
ymethyl group (1 and 2) in the 6-position [10].
When the amino group is acetylated, the resulting com-
pounds (28aee) are the amide isosteres of compounds 1 and
2. The iodophenyl ester 28e was found to be as potent as the
reference compound 2 at a 1 mM concentration. The chloro-
substituted amide compound 28c expressed a better activity
at 1 mM than its corresponding ester isostere 1 in the chemo-
invasion assay, while the bromo-substituted amide 28d was
found to be slightly less potent than its ester analogue 2 at
0.1 and 1 mM. It may be concluded that the ester function sen-
sitive to metabolic hydrolysis can be advantageously replaced
by a more stable amide function without dramatic decrease of
biological activity.
Compared to 2, the replacement of the acetoxymethyl moi-
ety in the 6-position by a nitro group gave rise to a less potent
bromophenyl ester 24d when tested at 1 mM in the chemoinva-
sion assay, but the latter was the most potent compound among
the 6-nitro-substituted coumarins (24aee) indicating that the
presence of a bromine atom on the phenyl ring was the best
choice of halogen atom in this series of coumarinic
compounds.
In the case of the 6-aminomethyl-substituted coumarins
(27aee), the bromophenyl ester 27d was less potent than the
corresponding bromophenyl reference compound 2. Interest-
ingly, the best choice of halogen atom on the phenyl ring
was the most bulky and lipophilic iodine atom giving rise to
the very potent compound 27e. Thus, except in the case of
an iodine atom, another halogen atom on the phenyl ring
didn’t improve the activity.
4. Conclusion
The present work aimed at describing new examples of
coumarin-3-carboxylic acid derivatives structurally related to
lead coumarins 1 and 2 expressing anti-invasive activity in
O
HN
NH
O
OH
N
O
NHOH
S
O
O
O
N
N
CH3
NO2
GI 129471 (29)
Ro28-2653 (30)
Fig. 3. Chemical structure of two synthetic MMP inhibitors.

I. Kempen et al. / European Journal of Medicinal Chemistry 43 (2008) 2735e2750
2741
Table 1
Effect of the new coumarin derivatives 14aec, 15a, 16aec and 19aeg at 0.1, 1 and 10 mM on HT 1080 fibrosarcoma cell invasion (‘Boyden chamber’ chemo-
invasion assay) compared to the coumarins 1 and 2 and the MMP inhibitors GI 129471 and Ro 28-2653
O
O
R
X
O
Y
O
O
Compound no.
X
Y
R
0.1 mMa
1 mMa
10 mMa
14a
14b
14c
S
S
S
H
eCH3
eCH3
eCH3
89.45 ꢀ 5.78
76.54 ꢀ 5.54
79.58 ꢀ 7.84
83.45 ꢀ 6.45
65.84 ꢀ 3.75
70.86 ꢀ 6.14
73.84 ꢀ 5.45
61.86 ꢀ 4.48
67.84 ꢀ 5.58
3-Cl
3-Br
15a
16a
16b
16c
NH
3-Cl
2-Cl
3-Cl
4-Cl
eCH3
eCH3
eCH3
eCH3
79.55 ꢀ 2.04
66.85 ꢀ 6.14
62.78 ꢀ 5.16
82.56 ꢀ 6.86
98.56 ꢀ 5.12
42.75 ꢀ 3.58
NCH3
NCH3
NCH3
n.d.b
n.d.
n.d.
n.d.
n.d.
n.d.
19a
19b
19c
19d
19e
19f
O
O
O
O
O
O
O
3-Br
3-Br
3-Br
3-Br
3-Br
3-Br
3-Br
eCH2CH3
84.36 ꢀ 2.36
88.55 ꢀ 2.88
92.36 ꢀ 4.42
94.25 ꢀ 2.96
89.36 ꢀ 3.25
54.36 ꢀ 4.23
65.24 ꢀ 5.63
40.23 ꢀ 2.35
50.47 ꢀ 2.89
63.25 ꢀ 1.61
59.32 ꢀ 3.54
55.56 ꢀ 2.89
37.80 ꢀ 1.64
48.21 ꢀ 8.32
21.32 ꢀ 1.96
24.67 ꢀ 2.23
32.26 ꢀ 0.42
33.94 ꢀ 4.01
35.96 ꢀ 6.85
21.07 ꢀ 0.45
23.64 ꢀ 3.46
e(CH2)2CH3
e(CH2)3CH3
e(CH2)4CH3
e(CH2)5CH3
eCH(CH3)2
eC(CH3)3
19g
1c
2c
O
O
3-Cl
3-Br
eCH3
eCH3
95.20 ꢀ 10.54
49.11 ꢀ 7.64
6.17 ꢀ 3.72
39.47 ꢀ 2.04
21.81 ꢀ 7.14
11.77 ꢀ 1.24
GI 129471
Ro 28-2653
a
n.d.
n.d.
38.45 ꢀ 2.36
41.60 ꢀ 4.35
n.d.
n.d.
Results are expressed as the number of migrated cells in % of control (100%) corresponding to the assay in the absence of tested compound (mean ꢀ s.d.,
n ꢁ 3).
b
c
n.d.: Not determined.
Published results (Ref. [10]).
vitro and anti-tumoral effect in vivo. Among the different
chemical modifications examined, the replacement of the ester
function in the 6-position of 1 and 2 by an amide function was
allowed without loss of potency in the ‘Boyden chamber’ che-
moinvasion assay with HT 1080 fibrosarcoma cells. By con-
trast, an aryl ester function in the 3-position was strongly
recommended since its replacement by a thioester or an amide
function was responsible for a significant decrease of the anti-
invasive activity.
(80 MHz) or a Bruker Avance (500 MHz) instrument using
d6-DMSO as the solvent with TMS as an internal standard;
chemical shifts are reported in d values (ppm) relative to
that of internal TMS. The abbreviations s ¼ singlet, d ¼
doublet, t ¼ triplet, q ¼ quadruplet, m ¼ multiplet, and
b ¼ broad are used throughout. Elemental analysis (C, H,
N, and S) was realized on a Carlo-Erba EA 1108-elemental
analyser and was within ꢀ0.4% of the theoretical values.
All reactions were routinely checked by TLC on silica gel
The increase of the size of the alkyl chain located on
the acyloxymethyl moiety in the 6-position reduced the
anti-invasive activity, although a short branched chain
such as an isopropyl or a tert-butyl chain was allowed with-
out markedly reducing the activity in the chemoinvasion
assay.
Merck 60 F254.
GI129471 was synthesized by Roche Diagnostics according
to the PCT Patent Applications WO 90/05719. Ro 28-2653
was obtained according to a synthetic route described in the
literature [24].
6-Hydroxymethyl-2-oxo-2H-1-benzopyran-3-carboxylic
acid (11), 6-acetoxymethyl-2-oxo-2H-1-benzopyran-3-car-
boxylic acid (12), phenyl 6-chloromethyl-2-oxo-2H-1-benzo-
pyran-3-carboxylate (25a), 3-fluorophenyl 6-chloromethyl-2-
oxo-2H-1-benzopyran-3-carboxylate (25b), 3-chlorophenyl
6-chloromethyl-2-oxo-2H-1-benzopyran-3-carboxylate (25c),
5. Experimental protocols
5.1. Chemistry
Melting points were determined with a Buchi-Tottoli capil-
¨
3-bromophenyl
6-chloromethyl-2-oxo-2H-1-benzopyran-3-
lary apparatus and are uncorrected. IR spectra were recorded
as KBr pellets on a PerkineElmer1000FTIR spectrophotometer.
The 1H NMR spectra were recorded on a Bruker AW-80
carboxylate (25d) and 3-iodophenyl 6-chloromethyl-2-oxo-
2H-1-benzopyran-3-carboxylate (25e) were obtained accord-
ing to previously described procedures [21,23].

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I. Kempen et al. / European Journal of Medicinal Chemistry 43 (2008) 2735e2750
Table 2
Effect of the new coumarin derivatives 24aee, 27aee and 28aee on HT 1080 fibrosarcoma cell invasion (‘Boyden chamber’ chemoinvasion assay) compared to
coumarins 1, 2, 31, 32 and 25c and the MMP inhibitor GI 129471
O
X
Y
O
O
O
Compound no.
X
Y
0.1 mMa
1 mMa
10 mMa
24a
24b
24c
24d
24e
27a
27b
27c
27d
27e
28a
28b
28c
28d
28e
eNO2
eNO2
eH
eF
n.d.b
n.d.
76.68 ꢀ 5.64
53.56 ꢀ 2.04
65.78 ꢀ 5.16
35.22 ꢀ 4.13
63.58 ꢀ 5.12
85.53 ꢀ 6.54
45.25 ꢀ 7.14
86.56 ꢀ 4.13
79.25 ꢀ 7.15
25.34 ꢀ 7.15
63.45 ꢀ 5.54
65.58 ꢀ 1.24
36.58 ꢀ 0.86
36.94 ꢀ 5.34
23.47 ꢀ 6.32
n.d.
n.d.
eNO2
eNO2
eNO2
eCl
eBr
eI
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
eCH2NH2
eCH2NH2
eCH2NH2
eCH2NH2
eCH2NH2
eCH2NHCOCH3
eCH2NHCOCH3
eCH2NHCOCH3
eCH2NHCOCH3
eCH2NHCOCH3
eH
eF
eCl
eBr
eI
n.d.
n.d.
n.d.
n.d.
88.26 ꢀ 5.12
n.d.
n.d.
63.58 ꢀ 6.32
n.d.
n.d.
eH
eF
n.d.
n.d.
n.d.
n.d.
eCl
eBr
eI
34.38 ꢀ 3.44
n.d.
27.14 ꢀ 4.50
n.d.
31c
eH
eCH3
eCH2Cl
eCl
eCl
eCl
52.40 ꢀ 11.13
81.50 ꢀ 5.12
98.35 ꢀ 5.47
45.50 ꢀ 9.97
52.49 ꢀ 1.44
79.76 ꢀ 8.71
35.65 ꢀ 6.94
27.62 ꢀ 1.50
67.00 ꢀ 7.73
32c
25cc
1
eCH2OCOCH3
eCH2OCOCH3
eCl
eBr
95.20 ꢀ 10.54
39.47 ꢀ 2.04
n.d.
49.11 ꢀ 7.64
21.81 ꢀ 7.14
38.45 ꢀ 2.36
6.17 ꢀ 3.72
11.77 ꢀ 1.24
n.d.
2
GI 129471
a
Results are expressed as the number of migrated cells in % of control (100%) corresponding to the assay in the absence of tested compound (mean ꢀ s.d.,
n ꢁ 3).
b
c
n.d.: Not determined.
Published results (Ref. [10]).
5.2. General procedure for the preparation of the
6-acyloxymethyl-2-oxo-2H-1-benzopyran-3-carboxylic
acids (17aeg)
7.50 (d, 1H, 8-H), 7.75 (s, 1H, 5-H), 7.77 (d, 1H, 7-H), 8.94
(s, 1H, 4-H). Anal. C14H12O6 (C, H).
5.2.2. 6-Butyryloxymethyl-2-oxo-2H-1-
benzopyran-3-carboxylic acid (17b)
Three grams of 6-hydroxymethyl-2-oxo-2H-1-benzopyran-
3-carboxylic acid (11) were refluxed with the appropriate an-
hydride for 3 h. After cooling, 200 mL of distilled water were
added and the suspension was stirred overnight. The resulting
precipitate was collected by filtration, washed with distilled
water and dried. The crude product was then crystallized in
the appropriate solvent (yields: 60e80%).
The title compound was obtained as described above after
the reaction of 11 with butyric anhydride (30 mL). The crude
product was crystallized in CH3CN:water: mp 146e147.5 ꢂC;
IR 3046 (CeH arom), 1740 (C]O lactone and ester), 1692
(C]O carboxylic acid), 1626, 1581, 1415, 1372 cmꢃ1; H
1
NMR 0.96 (t, 3H, CH3), 1.68 (m, 2H, CH2CH2CH3), 2.37 (t,
2H, CH2CH2CH3), 5.19 (s, 2H, CH2OCO(CH2)2CH3), 7.49
(d, 1H, 8-H), 7.71 (d, 1H, 7-H), 7.75 (s, 1H, 5-H), 8.94 (s,
1H, 4-H). Anal. C15H14O6 (C, H).
5.2.1. 6-Propionoxymethyl-2-oxo-2H-1-
benzopyran-3-carboxylic acid (17a)
The title compound was obtained as described above after
the reaction of 11 with propionic anhydride (30 mL). The
crude product was crystallized in CHCl3:petroleum ether
40e60 ꢂC: mp 174e176.5 ꢂC; IR 3055 (CeH arom), 1754
(C]O ester), 1741 (C]O lactone), 1689 (C]O carboxylic
5.2.3. 6-Valeryloxymethyl-2-oxo-2H-1-
benzopyran-3-carboxylic acid (17c)
The title compound was obtained as described above after
the reaction of 11 with valeric anhydride (30 mL). The crude
product was crystallized in CHCl3:diisopropylether: mp
108.5e109 ꢂC; IR 3055 (CeH arom), 1741 (C]O lactone
and ester), 1689 (C]O carboxylic acid), 1624, 1578, 1409,
1
acid), 1622, 1577, 1414, 1378 cmꢃ1; H NMR 1.18 (t, 3H,
CH3), 2.42 (q, 2H, CH2CH3), 5.21 (s, 2H, CH2OCOC2H5),

I. Kempen et al. / European Journal of Medicinal Chemistry 43 (2008) 2735e2750
2743
1375 cmꢃ1
;
1H NMR 0.88 (t, 3H, CH3), 1.42 (m, 2H,
5.2.8. Ethyl 6-nitro-2-oxo-2H-1-benzopyran-3-
carboxylate (21)
CH2CH3), 1.55 (m, 2H, CH2CH2CH3), 2.35 (t, 2H,
CH2CH2CH2CH3), 5.20 (s, 2H, CH2OCO(CH2)3CH3), 7.50
(d, 1H, 8-H), 7.78 (d, 1H, 7-H), 7.97 (s, 1H, 5-H), 8.96 (s,
1H, 4-H). Anal. C16H16O6 (C, H).
A mixture of 5-nitro-salicylaldehyde (20) (1 g, 5.98 mmol),
diethyl malonate (1.05 g, 6.59 mmol), piperidine (0.076 mL)
and acetic acid (0.03 mL) in ethanol (4 mL) was heated under
reflux for 18 h. The resulting precipitate was collected by fil-
tration and washed with ethanol. The crude product was sus-
pended in methanol, collected by filtration, washed with
methanol and dried (yield: 64%): mp 195.5e196.5; IR 3078,
3056 (CeH arom), 1779 (C]O ester), 1757 (C]O lactone),
5.2.4. 6-Hexanoyloxymethyl-2-oxo-2H-1-benzopyran-3-
carboxylic acid (17d)
The title compound was obtained as described above after
the reaction of 11 with hexanoic anhydride (20 mL). The
crude product was crystallized in CH3CN:water: mp 103e
105.5 ꢂC; IR 3052 (CeH arom), 1740 (C]O lactone & ester),
1
1692, 1619, 1570, 1528, 1348, 1256 cmꢃ1; H NMR 1.33 (t,
3H, CH3), 4.33 (q, 2H, CH2), 7.66 (d, 1H, 8-H), 8.51 (d,
1H, 7-H), 8.93e8.94 (m, 2H, 4-H, 5-H). Anal. C12H9NO6
(C, H, N).
1697 (C]O carboxylic acid), 1625, 1579, 1414, 1375 cmꢃ1
;
1H NMR 0.85 (t, 3H, CH3), 1.24 (m, 4H, CH2CH2CH3),
1.56 (m, 2H, CH2(CH2)2CH3), 2.35 (t, 2H, CH2(CH2)3CH3),
5.20 (s, 2H, CH2OCO(CH2)4CH3), 7.54 (d, 1H, 8-H), 7.78
(d, 1H, 7-H), 7.97 (s, 1H, 5-H), 9.02 (s, 1H, 4-H). Anal.
C17H18O6 (C, H).
5.2.9. 6-Nitro-2-oxo-2H-1-benzopyran-3-carboxylic acid (22)
The suspension of ethyl 6-nitro-2-oxo-2H-1-benzopyran-3-
carboxylate (21) (5 g, 18.9 mmol) in 10% aqueous NaOH
(150 mL) was heated under reflux for 30 min. After cooling,
pH was adjusted to 1 by 12 N HCl, while stirring over an
ice bath. The resulting precipitate was collected by filtration,
washed with distilled water and crystallized in CH3CN (yield:
91%): mp 229.5e230 ꢂC; IR 3116, 3076 (CeH arom), 1723
(C]O lactone and carboxylic acid), 1617, 1536, 1523,
5.2.5. 6-Heptanoyloxymethyl-2-oxo-2H-1-benzopyran-3-
carboxylic acid (17e)
The title compound was obtained as described above after
the reaction of 11 with heptanoic anhydride (35 mL). The
crude product was crystallized in CHCl3:petroleum ether
40e60 ꢂC: mp 102e102.5 ꢂC; IR 3055 (CeH arom), 1743
(C]O lactone and ester), 1690 (C]O carboxylic acid),
1
1354, 1233, 1208 cmꢃ1; H NMR 7.65 (d, 1H, 8-H), 8.50
(d, 1H, 7-H), 8.90 (s, 1H, 4-H), 8.91 (s, 1H, 5-H), 13.51 (s,
1H, COOH). Anal. C10H5NO6 (C, H, N).
1624, 1578, 1410, 1375 cmꢃ1; H NMR 0.88 (t, 3H, CH3),
1
5.3. General procedure for the preparation of the
thioesters (14), the secondary (15) and tertiary (16)
amides of 6-acetoxymethyl-2-oxo-2H-1-benzopyran-3-
carboxylic acid, the esters of 6-acyloxymethyl-2-oxo-2H-
1-benzopyran-3-carboxylic acid (19) and 6-nitro-2-oxo-
2H-1-benzopyran-3-carboxylic acid (24)
1.24 (m, 6H, (CH2)3CH3), 1.55 (m, 2H, CH2(CH2)3CH3),
2.38 (t, 2H, CH2(CH2)4CH3), 5.20 (s, 2H, CH2OCO
(CH2)5CH3), 7.46 (d, 1H, 8-H), 7.78 (d, 1H, 7-H), 7.97
(s, 1H, 5-H), 9.04 (s, 1H, 4-H). Anal. C18H20O6 (C, H).
5.2.6. 6-Dimethylacetoxymethyl-2-oxo-2H-1-benzopyran-3-
carboxylic acid (17f)
The appropriate carboxylic acid (1 g) was refluxed in thi-
onyl chloride (10 mL) for 3 h. The resulting solution was con-
centrated to dryness under reduced pressure and the residue
was dispersed in dry toluene (10 mL). The solvent was elimi-
nated under reduced pressure. Dispersion in dry toluene and
solvent elimination was repeated twice. The residue was dis-
persed in anhydrous dioxane (3 mL) and added with stirring
to a solution of the appropriate phenol, thiophenol or aniline
and anhydrous pyridine in anhydrous dioxane. The progress
of the reaction was followed by TLC. Yields are usually com-
prised between 50% and 80%.
The title compound was obtained as described above after
the reaction of 11 with isobutyric anhydride (30 mL). The
crude product was crystallized in CHCl3:petroleum ether
40e60 ꢂC: mp 134e135 ꢂC; IR 3049 (CeH arom), 1753
(C]O ester), 1726 (C]O lactone), 1684 (C]O carboxylic
1
acid), 1620, 1576, 1416, 1386 cmꢃ1; H NMR 1.20 (d, 6H,
(CH3)2), 2.63 (q, 1H, CH ), 5.20 (s, 2H, CH2), 7.49 (d, 1H,
8-H), 7.74 (s, 1H, 5-H), 7.76 (d, 1H, 7-H), 8.94 (s, 1H, 4-
H). Anal. C15H14O6 (C, H).
5.2.7. 6-Trimethylacetoxymethyl-2-oxo-2H-1-benzopyran-3-
carboxylic acid (17g)
5.3.1. S-Phenyl 6-acetoxymethyl-2-oxo-2H-1-benzopyran-3-
carbothioate (14a)
The title compound was obtained as described above after
the reaction of 11 with pivaloic anhydride (30 mL). The crude
product was crystallized in CHCl3:petroleum ether 40e60 ꢂC:
mp 136e137.5 ꢂC; IR 3044 (CeH arom), 2969 (CeH aliph),
1743 (C]O lactone and ester), 1674 (C]O carboxylic acid),
The title compound was obtained as described in the gen-
eral procedure after the reaction of the acyl chloride of 6-ace-
toxymethyl-2-oxo-2H-1-benzopyran-3-carboxylic acid (13)
(w3.8 mmol) with thiophenol (0.49 g, 4.5 mmol) and pyridine
(1 mL, 12.5 mmol). After stirring overnight at room tempera-
ture, the solvents were eliminated by distillation under reduced
pressure. The residue was dissolved in warm ethyl acetate and
the solution was treated with charcoal. After reducing the
1
1620, 1575, 1417, 1157 cmꢃ1; H NMR 1.24 (s, 9H, (CH3)3),
5.20 (s, 2H, CH2), 7.50 (d, 1H, 8-H), 7.74 (s, 1H, 5-H), 7.76
(d, 1H, 7-H), 8.95 (s, 1H, 4-H). Anal. C16H16O6 (C, H).

2744
I. Kempen et al. / European Journal of Medicinal Chemistry 43 (2008) 2735e2750
volume of the filtrate, precipitation was obtained by addition
of petroleum ether 40e60 ꢂC. The crude product was crystal-
lized twice in ethyl acetate: mp 140.5e142 ꢂC; IR 3047 (CeH
arom), 1729 (C]O lactone and ester), 1662 (C]O thioester),
dioxane. The crude product was dissolved in CHCl3 and
treated with charcoal, then cyclohexane was added and the re-
sulting precipitate was collected by filtration and dried: mp
187e190 ꢂC; IR 3263, 3124 (CeH arom), 1742 (C]O lac-
tone and ester), 1699 (C]O amide), 1617, 1591, 1575,
1
1568, 1182 cmꢃ1; H NMR 2.09 (s, 3H, CH3), 5.16 (s, 2H,
1
CH2), 7.54e7.53 (m, 6H, 8-H, C6H5), 7.79 (d, 1H, 7-H),
8.01 (s, 1H, 5-H), 8.81 (s, 1H, 4-H). Anal. C19H14O5S (C,
H, S).
1541, 1427, 1240 cmꢃ1; H NMR 2.10 (s, 3H, CH3), 5.17
(s, 2H, CH2), 7.21 (d, 1H, 8-H), 7.42 (t, 1H, 50-H), 7.56e
7.59 (m, 2H, 8-H, 40-H), 7.77 (d, 1H, 7-H), 7.99 (d, 2H, 20-
H, 60-H), 8.89 (s, 1H, 4-H), 10.75 (s,1H, NH ). Anal.
C19H14O5NCl (C, H, N).
5.3.2. S-3-Chlorophenyl 6-acetoxymethyl-2-oxo-
2H-1-benzopyran-3-carbothioate (14b)
The title compound was obtained as described in the gen-
eral procedure after the reaction of the acyl chloride of 6-ace-
toxymethyl-2-oxo-2H-1-benzopyran-3-carboxylic acid (13)
(w3.8 mmol) with 3-chlorothiophenol (0.65 g, 4.5 mmol)
and pyridine (1 mL, 12.5 mmol). After stirring overnight at
room temperature, the solvents were eliminated by distillation
under reduced pressure. The residue was dissolved in warm
ethyl acetate and the solution was treated with charcoal. After
reducing the volume of the filtrate, precipitation was obtained
by addition of petroleum ether 40e60 ꢂC. The crude product
was crystallized twice in ethyl acetate: mp 135.5e137 ꢂC;
IR 3066 (CeH arom), 1738 (C]O ester), 1728 (C]O lac-
5.3.5. N-methyl-N-(2-chlorophenyl)-6-acetoxymethyl-2-oxo-
2H-1-benzopyran-3-carboxamide (16a)
The title compound was obtained as described in the gen-
eral procedure after the reaction of the acyl chloride of 6-ace-
toxymethyl-2-oxo-2H-1-benzopyran-3-carboxylic acid (13)
(w3.8 mmol) with 2-chloro-N-methylaniline (0.7 g, 5 mmol)
and pyridine (1 mL, 12.5 mmol). After 6 h at room tempera-
ture, water was added and the mixture was cooled on an ice
bath, then the resulting precipitate was collected by filtration
and dried: mp 119e121 ꢂC; IR 3061, 2938 (CeH arom),
1732 (C]O lactone and ester), 1648 (C]O amide), 1623,
1
1580, 1486, 1233 cmꢃ1; H NMR 2.09 (s, 3H, CH3), 3.24
tone), 1656 (C]O thioester), 1569, 1181 cmꢃ1
;
1H NMR
(s, 3H, NCH3), 5.09 (s, 2H, CH2), 7.28e7.33 (m, 2H, 40-H,
8-H), 7.46e7.49 (m, 3H, 30-H, 50-H, 7-H), 7.59 (d, 1H, 60-
H), 7.73 (s, 1H, 5-H), 8.23 (s, 1H, 4-H). Anal. C20H16O5NCl
(C, H, N).
2.08 (s, 3H, CH3), 5.16 (s, 2H, CH2), 7.49 (d, 1H, 40-H),
7.53e7.55 (m, 2H, 8-H, 50-H), 7.59e7.61 (m, 20-H, 60-H),
7.80 (d, 1H, 7-H), 8.02 (s,1H, 5-H), 8.83 (s, 1H, 4-H). Anal.
C19H13O5SCl (C, H, S).
5.3.6. N-Methyl-N-(3-chlorophenyl)-6-acetoxymethyl-2-oxo-
2H-1-benzopyran-3-carboxamide (16b)
5.3.3. S-3-Bromophenyl 6-acetoxymethyl-2-oxo-
2H-1-benzopyran-3-carbothioate (14c)
The title compound was obtained as described in the gen-
eral procedure after the reaction of the acyl chloride of 6-ace-
toxymethyl-2-oxo-2H-1-benzopyran-3-carboxylic acid (13)
(w3.8 mmol) with 3-chloro-N-methylaniline (0.7 g, 5 mmol)
and pyridine (1 mL, 12.5 mmol). After 8 h at room tempera-
ture, the resulting precipitate was collected by filtration and
washed with dioxane. The crude product was crystallized
twice in methanol: mp 106e108 ꢂC; IR 3062, 2963 (CeH
arom), 1729 (C]O lactone and ester), 1648 (C]O amide),
The title compound was obtained as described in the gen-
eral procedure after the reaction of the acyl chloride of 6-ace-
toxymethyl-2-oxo-2H-1-benzopyran-3-carboxylic acid (13)
(w3.8 mmol) with 3-bromothiophenol (0.85 g, 4.5 mmol)
and pyridine (1 mL, 12.5 mmol). After stirring overnight at
room temperature, the solvents were eliminated by distillation
under reduced pressure. The residue was dissolved in warm
ethyl acetate and the solution was treated with charcoal. After
reducing the volume of the filtrate, precipitation was obtained
by addition of petroleum ether 40e60 ꢂC. The crude product
was crystallized twice in ethyl acetate: mp 129.5e132.5 ꢂC;
IR 3062 (CeH arom), 1736 (C]O ester), 1725 (C]O lac-
1
1623, 1591, 1580, 1481, 1230 cmꢃ1; H NMR 2.07 (s, 3H,
CH3), 3.33 (s, 3H, NCH3), 5.12 (s, 2H, CH2), 7.25 (m, 1H,
40-H),7.30 (m, 2H, 50-H, 8-H), 7.37 (m, 1H, 7-H), 7.47 (s,
1H, 5-H), 7.62 (d, 1H, 60-H), 7.73 (s, 1H, 20-H), 8.26 (s, 1H,
4-H). Anal. C20H16O5NCl (C, H, N).
tone), 1657 (C]O thioester), 1568, 1181 cmꢃ1 1H NMR
;
2.09 (s, 3H, CH3), 5.16 (s, 2H, CH2), 7.48 (d, 1H, 60-H),
7.52e7.56 (m, 2H, 8-H, 50-H), 7.72e7.74 (m, 20-H, 40-H),
7.80 (d, 1H, 7-H), 8.02 (s,1H, 5-H), 8.83 (s, 1H, 4-H). Anal.
C19H13O5SBr (C, H, S).
5.3.7. N-Methyl-N-(4-chlorophenyl)-6-acetoxymethyl-2-oxo-
2H-1-benzopyran-3-carboxamide (16c)
The title compound was obtained as described in the gen-
eral procedure after the reaction of the acyl chloride of 6-ace-
toxymethyl-2-oxo-2H-1-benzopyran-3-carboxylic acid (13)
(w3.8 mmol) with 4-chloro-N-methylaniline (0.7 g, 5 mmol)
and pyridine (1 mL, 12.5 mmol). After 6 h at room tempera-
ture, water was added and the mixture was cooled on an ice
bath, then the resulting precipitate was collected by filtration
and dried. The crude product was crystallized in methanol:
mp 117e119 ꢂC; IR 3054, 2951 (CeH arom), 1744 (C]O es-
ter), 1731 (C]O lactone), 1649 (C]O amide), 1622, 1580,
5.3.4. N-3-Chlorophenyl 6-acetoxymethyl-2-oxo-
2H-1-benzopyran-3-carboxamide (15a)
The title compound was obtained as described in the gen-
eral procedure after the reaction of the acyl chloride of 6-ace-
toxymethyl-2-oxo-2H-1-benzopyran-3-carboxylic acid (13)
(w3.8 mmol) with 3-chloroaniline (0.63 g, 5 mmol) and pyri-
dine (1 mL, 12.5 mmol). After 6 h at room temperature, the re-
sulting precipitate was collected by filtration and washed with

I. Kempen et al. / European Journal of Medicinal Chemistry 43 (2008) 2735e2750
2745
1
1494, 1224 cmꢃ1; H NMR 2.06 (s, 3H, CH3), 3.33 (s, 3H,
temperature, the solvent was removed by evaporation under
reduced pressure. The residue was dissolved in CHCl3 and
the organic layer was washed three times with 0.1 N HCl,
then dried over MgSO4, filtered, and evaporated to dryness un-
der reduced pressure. The residue was then dissolved in warm
methanol, treated with charcoal and crystallized by cooling on
ice bath. The resulting precipitate was collected by filtration
and dried: mp 133e134.5 ꢂC; IR 3066 (CeH arom), 1736
(C]O esters), 1690 (C]O lactone), 1628, 1579, 1471,
NCH3), 5.11 (s, 2H, CH2), 7.32e7.37 (m, 5H, 30-H, 50-H, 5-
H, 7-H, 8-H), 7.62 (d, 1H, 60-H), 7.71 (s, 1H, 20-H), 8.24 (s,
1H, 4-H). Anal. C20H16O5NCl (C, H, N).
5.3.8. 3-Bromophenyl 6-propionoxymethyl-2-oxo-
2H-1-benzopyran-3-carboxylate (19a)
The title compound was obtained as described in the gen-
eral procedure after the reaction of the acyl chloride of 6-pro-
1
pionoxymethyl-2-oxo-2H-1-benzopyran-3-carboxylic
acid
1367, 1247 cmꢃ1; H NMR 0.87 (t, 3H, CH3), 1.30 (m, 2H,
(18a) (w3.5 mmol) with 3-bromophenol (0.71 g, 4 mmol)
and pyridine (0.4 mL, 5 mmol). After 12 h at room tempera-
ture, the solvent was removed by evaporation under reduced
pressure. The residue was dissolved in CHCl3 and the organic
layer was washed three times with 0.1 N HCl, then dried over
MgSO4, filtered, and evaporated to dryness under reduced
pressure. The residue was then dissolved in warm methanol,
treated with charcoal and crystallized by cooling on an ice
bath. The resulting precipitate was collected by filtration and
dried: mp 124e124.5 ꢂC; IR 3064 (CeH arom), 1775 and
1737 (C]O esters), 1690 (C]O lactone), 1624, 1577,
CH2CH3), 1.54 (m, 2H, CH2CH2CH3), 2.38 (t, 2H,
CH2CH2CH2CH3), 5.18 (s, 2H, CH2OCO(CH2)3CH3), 7.33
(d, 1H, 60-H), 7.47 (t, 1H, 50-H), 7.50 (d, 1H, 8-H), 7.55 (d,
1H, 40-H), 7.56 (s, 1H, 20-H), 7.78 (d, 1H, 7-H), 7.97 (s, 1H,
5-H), 9.09 (s, 1H, 4-H). Anal. C22H19BrO6 (C, H).
5.3.11. 3-Bromophenyl 6-hexanoyloxymethyl-2-oxo-
2H-1-benzopyran-3-carboxylate (19d)
The title compound was obtained as described in the gen-
eral procedure after the reaction of the acyl chloride of 6-hex-
anoyloxymethyl-2-oxo-2H-1-benzopyran-3-carboxylic acid
(18d) (w3 mmol) with 3-bromophenol (0.6 g, 3.5 mmol) and
pyridine (0.4 mL, 5 mmol). After 12 h at room temperature,
the solvent was removed by evaporation under reduced pres-
sure. The residue was dissolved in CHCl3 and the organic
layer was washed three times with 0.1 N HCl, then dried
over MgSO4, filtered, and evaporated to dryness under reduced
pressure. The residue was then dissolved in warm methanol,
treated with charcoal and crystallized by cooling on an ice
bath. The resulting precipitate was collected by filtration and
dried: mp 122.5e126 ꢂC; IR 3067 (CeH arom), 1732
(C]O esters), 1692 (C]O lactone), 1628, 1580, 1471,
1
1470, 1377, 1246 cmꢃ1; H NMR 1.07 (t, 3H, CH3), 2.41
(q, 2H, CH2CH3), 5.19 (s, 2H, CH2OCOC2H5), 7.33 (d, 1H,
60-H), 7.46 (t, 1H, 50-H), 7.50 (d, 1H, 8-H), 7.55 (d, 1H, 40-
H), 7.60 (s, 1H, 20-H), 7.88 (d, 1H, 7-H), 7.97 (s, 1H, 5-H),
9.09 (s, 1H, 4-H). Anal. C20H15BrO6 (C, H).
5.3.9. 3-Bromophenyl 6-butyryloxymethyl-2-oxo-
2H-1-benzopyran-3-carboxylate (19b)
The title compound was obtained as described in the gen-
eral procedure after the reaction of the acyl chloride of 6-bu-
tyryloxymethyl-2-oxo-2H-1-benzopyran-3-carboxylic
acid
1
(18b) (w3.3 mmol) with 3-bromophenol (0.7 g, 4 mmol) and
pyridine (0.4 mL, 5 mmol). After 12 h at room temperature,
the solvent was removed by evaporation under reduced pres-
sure. The residue was dissolved in CHCl3 and the organic
layer was washed three times with 0.1 N HCl, then dried
over MgSO4, filtered, and evaporated to dryness under reduced
pressure. The residue was then dissolved in warm methanol,
treated with charcoal and crystallized by cooling on an ice
bath. The resulting precipitate was collected by filtration and
dried: mp 140.5 ꢂC; IR 3067 (CeH arom), 1736 (C]O es-
ters), 1691 (C]O lactone), 1628, 1579, 1471, 1367,
1367, 1247 cmꢃ1; H NMR 0.85 (t, 3H, CH3), 1.26 (m, 4H,
CH2CH2CH3), 1.56 (m, 2H, CH2(CH2)2CH3), 2.38 (t, 2H,
CH2(CH2)3CH3), 5.18 (s, 2H, CH2OCO(CH2)4CH3), 7.32 (d,
1H, 60-H), 7.51 (t, 1H, 50-H), 7.54 (d, 1H, 8-H), 7.56 (d, 1H,
40-H), 7.60 (s, 1H, 20-H), 7.78 (d, 1H, 7-H), 7.97 (s, 1H, 5-
H), 9.09 (s, 1H, 4-H). Anal. C23H21BrO6 (C, H).
5.3.12. 3-Bromophenyl 6-heptanoyloxymethyl-2-oxo-2H-1-
benzopyran-3-carboxylate (19e)
The title compound was obtained as described in the gen-
eral procedure after the reaction of the acyl chloride of 6-hep-
tanoyloxymethyl-2-oxo-2H-1-benzopyran-3-carboxylic acid
(18e) (w2.9 mmol) with 3-bromophenol (0.6 g, 3.5 mmol)
and pyridine (0.4 mL, 5 mmol). After 12 h at room tempera-
ture, the solvent was removed by evaporation under reduced
pressure. The residue was dissolved in CHCl3 and the organic
layer was washed three times with 0.1 N HCl, then dried over
MgSO4, filtered, and evaporated to dryness under reduced
pressure. The residue was then dissolved in warm methanol,
treated with charcoal and crystallized by cooling on an ice
bath. The resulting precipitate was collected by filtration and
dried: mp 121e122.5 ꢂC; IR 3067 (CeH arom), 1734
(C]O esters), 1689 (C]O lactone), 1628, 1580, 1471,
1247 cmꢃ1 1H NMR 0.89 (t, 3H, CH3), 1.58 (m, 2H,
;
CH2CH3), 2.37 (t, 2H, CH2CH2CH3), 5.19 (s, 2H, CH2OC-
O(CH2)2CH3), 7.33 (d, 1H, 60-H), 7.47 (t, 1H, 50-H), 7.50
(d, 1H, 8-H), 7.55 (d, 1H, 40-H), 7.60 (s, 1H, 20-H), 7.78 (d,
1H, 7-H), 7.97 (s, 1H, 5-H), 9.09 (s, 1H, 4-H). Anal.
C21H17BrO6 (C, H).
5.3.10. 3-Bromophenyl 6-valeryloxymethyl-2-oxo-
2H-1-benzopyran-3-carboxylate (19c)
The title compound was obtained as described in the gen-
eral procedure after the reaction of the acyl chloride of 6-
valeryloxymethyl-2-oxo-2H-1-benzopyran-3-carboxylic acid
(18c) (w3.1 mmol) with 3-bromophenol (0.65 g, 3.7 mmol)
and pyridine (0.4 mL, 5 mmol). After 12 h at room
1
1367, 1247 cmꢃ1; H NMR 0.84 (t, 3H, CH3), 1.24 (m, 6H,
(CH2)3CH3), 1.55 (m, 2H, CH2(CH2)3CH3), 2.38 (t, 2H,

2746
I. Kempen et al. / European Journal of Medicinal Chemistry 43 (2008) 2735e2750
CH2(CH2)4CH3), 5.18 (s, 2H, CH2OCO(CH2)5CH3), 7.33 (d,
1H, 60-H), 7.47 (t, 1H, 50-H), 7.50 (d, 1H, 8-H), 7.56 (d, 1H,
40-H), 7.60 (s, 1H, 20-H), 7.78 (d, 1H, 7-H), 7.97 (s, 1H, 5-
H), 9.09 (s, 1H, 4-H). Anal. C24H23BrO6 (C, H).
at 30 ꢂC. The crude product was stirred in methanol and col-
lected by filtration. It was then dissolved in warm DMF,
treated with charcoal and filtered. The filtrate was kept at
4 ꢂC for 2 h. The resulting precipitate was collected, washed
with DMF and methanol and dried for one night at 30 ꢂC.
The crude product was suspended in methanol and collected
by filtration (precipitate 1). Methanol was added to DMF fil-
trate and the resulting precipitate was collected by filtration,
washed with methanol and pooled with precipitate 1: mp
236.5e237.5 ꢂC; IR 3070 (CeH arom), 1775 (C]O ester),
5.3.13. 3-Bromophenyl 6-dimethylacetoxymethyl-2-oxo-
2H-1-benzopyran-3-carboxylate (19f)
The title compound was obtained as described in the gen-
eral procedure after the reaction of the acyl chloride of 6-di-
methylacetoxymethyl-2-oxo-2H-1-benzopyran-3-carboxylic
acid (18f) (w3.3 mmol) with 3-bromophenol (0.69 g, 4 mmol)
and pyridine (0.4 mL, 5 mmol). After 12 h at room tempera-
ture, the solvent was removed by evaporation under reduced
pressure. The residue was dissolved in CHCl3 and the organic
layer was washed three times with 0.1 N HCl, then dried over
MgSO4, filtered, and evaporated to dryness under reduced
pressure. The residue was then dissolved in warm methanol,
treated with charcoal and crystallized by cooling on an ice
bath. The resulting precipitate was collected by filtration and
dried: mp 152e153 ꢂC; IR 3067 (CeH arom), 1735 (C]O es-
1722 (C]O lactone), 1618, 1573, 1539, 1345, 1256 cmꢃ1
;
1H NMR 7.28e7.36 (m, 3H, 20-H, 40-H, 60-H), 7.49e7.52
(m, 2H, 30-H, 50-H), 7.71 (d, 1H, 8-H), 8.56 (d, 1H, 7-H),
8.99 (s, 1H, 5-H), 9.25 (s, 1H, 4-H). Anal. C16H9NO6 (C,
H, N).
5.3.16. 3-Fluorophenyl 6-nitro-2-oxo-2H-1-benzopyran-3-
carboxylate (24b)
The title compound was obtained as described in the gen-
eral procedure after the reaction of the acyl chloride of 6-ni-
tro-2-oxo-2H-1-benzopyran-3-carboxylic acid (23) (w4 mmol)
with 3-fluorophenol (1 g, 9 mmol) and pyridine (1 mL,
12.5 mmol). After 8 h at room temperature, the resulting precip-
itate was collected by filtration, washed with dioxane and
kept overnight at 30 ꢂC. The crude product was stirred in
methanol and collected by filtration. It was then dissolved
in warm DMF, treated with charcoal and filtered. The filtrate
was kept at 4 ꢂC for 2 h. The resulting precipitate was col-
lected by filtration, washed with DMF and methanol and
dried for one night at 30 ꢂC. The crude product was sus-
pended in methanol and collected by filtration (precipitate
1). Methanol was added to DMF filtrate and the resulting
precipitate was collected by filtration, washed with methanol
and pooled with precipitate 1: mp 226e226.5 ꢂC; IR 3086
(CeH arom), 1779 (C]O ester), 1721 (C]O lactone),
ters and lactone), 1628, 1579, 1472, 1368, 1247 cmꢃ1 1H
;
NMR 1.12 (d, 6H, CH3), 2.63 (q, 1H, CH ), 5.18 (s, 2H,
CH2OCOCH(CH3)2), 7.33 (d, 1H, 60-H), 7.46 (t, 1H, 50-H),
7.50 (d, 1H, 8-H), 7.54 (d, 1H, 40-H), 7.60 (s, 1H, 20-H),
7.78 (d, 1H, 7-H), 7.97 (s, 1H, 5-H), 9.10 (s, 1H, 4-H).
Anal. C21H17BrO6 (C, H).
5.3.14. 3-Bromophenyl 6-trimethylacetoxymethyl-2-oxo-
2H-1-benzopyran-3-carboxylate (19g)
The title compound was obtained as described in the gen-
eral procedure after the reaction of the acyl chloride of 6-tri-
methylacetoxymethyl-2-oxo-2H-1-benzopyran-3-carboxylic
acid (18g) (w3.1 mmol) with 3-bromophenol (0.6 g,
3.5 mmol) and pyridine (0.4 mL, 5 mmol). After 12 h at
room temperature, the solvent was removed by evaporation
under reduced pressure. The residue was dissolved in CHCl3
and the organic layer was washed three times with 0.1 N
HCl, then dried over MgSO4, filtered, and evaporated to dry-
ness under reduced pressure. The residue was then dissolved
in warm methanol, treated with charcoal and crystallized by
cooling on an ice bath. The resulting precipitate was collected
by filtration and dried: mp 158e161 ꢂC; IR 3067 (CeH arom),
1732 (C]O esters), 1686 (C]O lactone), 1628, 1580, 1471,
1621, 1572, 1532, 1346, 1248 cmꢃ1 1H NMR 7.16e7.28
;
(m, 3H, 20-H, 40-H, 60-H), 7.55 (t, 1H, 50-H), 7.72 (d, 1H,
8-H), 8.56 (d, 1H, 7-H), 8.99 (s, 1H, 5-H), 9.28 (s, 1H, 4-
H). Anal. C16H8NO6F (C, H, N).
5.3.17. 3-Chlorophenyl 6-nitro-2-oxo-2H-1-benzopyran-3-
carboxylate (24c)
The title compound was obtained as described in the general
procedure after the reaction of the acyl chloride of 6-nitro-2-
oxo-2H-1-benzopyran-3-carboxylic acid (23) (w4 mmol)
with 3-chlorophenol (1.15 g, 9 mmol) and pyridine (1 mL,
12.5 mmol). After 8 h at room temperature, the resulting precip-
itate was collected by filtration, washed with dioxane and left
overnight at 30 ꢂC. The crude product was stirred in metha-
nol and collected by filtration. It was then dissolved in warm
acetone (w150 mL), treated with charcoal and filtered. The
filtrate was kept at 4 ꢂC for 2 h and the resulting precipitate
was collected by filtration, washed with cold acetone and
dried: mp 245.5e246 ꢂC; IR 3075 (CeH arom), 1770
(C]O ester), 1723 (C]O lactone), 1620, 1572, 1541,
1
1367, 1247 cmꢃ1; H NMR 1.22 (s, 9H, CH3), 5.18 (s, 2H,
CH2OCOC(CH3)3), 7.33 (d, 1H, 60-H), 7.47 (t, 1H, 50-H),
7.51 (d, 1H, 8-H), 7.55 (d, 1H, 40-H), 7.60 (s, 1H, 20-H),
7.78 (d, 1H, 7-H), 7.97 (s, 1H, 5-H), 9.12 (s, 1H, 4-H).
Anal. C22H19BrO6 (C, H).
5.3.15. Phenyl 6-nitro-2-oxo-2H-1-benzopyran-3-
carboxylate (24a)
The title compound was obtained as described in the
general procedure after the reaction of the acyl chloride of 6-ni-
tro-2-oxo-2H-1-benzopyran-3-carboxylic acid (23) (w4 mmol)
with phenol (0.85 g, 9 mmol) and pyridine (1 mL, 12.5 mmol).
After 8 h at room temperature, the resulting precipitate was
collected by filtration, washed with dioxane and left overnight
1347, 1248 cmꢃ1
;
1H NMR 7.31 (d, 1H, 60-H), 7.45
(d, 1H, 40-H), 7.48 (s, 1H, 20-H), 7.55 (t, 1H, 50-H), 7.72

I. Kempen et al. / European Journal of Medicinal Chemistry 43 (2008) 2735e2750
2747
(d, 1H, 8-H), 8.57 (d, 1H, 7-H), 8.99 (s, 1H, 5-H), 9.27 (s,
1H, 4-H). Anal. C16H8NO6Cl (C, H, N).
(26a) (2.2 g, 4.8 mmol) and 12 N HCl (3.8 mL) in ethanol
(16 mL) were heated under reflux for 90 min. After cooling,
the resulting precipitate was collected by filtration, washed
with ethanol and discarded. The filtrate was evaporated to dry-
ness under reduced pressure and the residue was suspended in
a 1% aqueous solution of NaHCO3. The resulting precipitate
was collected by filtration and washed with water: mp
>260 ꢂC; IR 3067 (CeH arom), 2885 (NþeH), 1775 (C]O
ester), 1728 (C]O lactone), 1624, 1576, 1463, 1371, 1256,
5.3.18. 3-Bromophenyl 6-nitro-2-oxo-2H-1-benzopyran-3-
carboxylate (24d)
The title compound was obtained as described in the gen-
eral procedure after the reaction of the acyl chloride of 6-ni-
tro-2-oxo-2H-1-benzopyran-3-carboxylic acid (23) (w4 mmol)
with 3-bromophenol (1.56 g, 9 mmol) and pyridine (1 mL,
12.5 mmol). After 4 h at room temperature, the resulting pre-
cipitate was collected by filtration, washed with dioxane and
kept overnight at 30 ꢂC. The crude product was stirred in
methanol and collected by filtration. It was then dissolved
in warm DMF, treated with charcoal and filtered. The filtrate
was kept at 4 ꢂC for 2 h. The resulting precipitate was col-
lected by filtration, washed with cold DMF and dried for
one night at 30 ꢂC. The crude product was dissolved in
warm dioxane and treated with charcoal. The filtrate was
cooled and the resulting precipitate was collected by filtra-
tion, washed with dioxane and water: mp 268e270 ꢂC; IR
3070 (CeH arom), 1772 (C]O ester), 1719 (C]O lactone),
1203 cmꢃ1; H NMR 3.39 (b, 3H, NH3þ), 4.13 (s, 2H, CH2),
1
7.20e7.26 (m, 3H, 20-H, 40-H, 60-H), 7.50e7.57 (m, 3H, 50-
H, 60-H, 8-H), 7.92 (d, 1H, 7-H), 7.99 (d, 1H, 5-H), 9.01 (s,
1H, 4-H). Anal. C17H13NO4$HCl (C, H, N).
5.3.21. 3-Fluorophenyl 6-aminomethyl-2-oxo-
2H-1-benzopyran-3-carboxylate chloride salt (27b)
3-Fluorophenyl
6-hexamethylenetetrammoniomethyl-2-
oxo-2H-1-benzopyran-3-carboxylate chloride salt (26b) was
obtained as described for 26a after the reaction of hexamethy-
lenetetramine (1.15 g, 8.2 mmol) with 3-fluorophenyl 6-chlor-
omethyl-2-oxo-2H-1-benzopyran-3-carboxylate (25b) (1.2 g,
3.6 mmol). The resulting precipitate was collected by filtration
and washed with cold CHCl3 (yield: 65%): mp 182 ꢂC. 3-
fluorophenyl 6-hexamethylenetetrammoniomethyl-2-oxo-2H-
1-benzopyran-3-carboxylate chloride salt (26b) (1.6 g,
3.4 mmol) and 12 N HCl (2.7 mL) in ethanol (11 mL) were
heated under reflux for 90 min. After cooling, the resulting pre-
cipitate was collected by filtration and washed with ethanol.
The crude product was suspended in water (elimination of
NH4Cl) and the resulting precipitate was collected by filtration
and washed with water: mp >260 ꢂC; IR 2963; 2876 (NþeH),
1779 (C]O esters), 1722 (C]O lactone), 1626, 1580, 1485,
1
1618, 1572, 1537, 1346, 1248 cmꢃ1; H NMR 7.34 (d, 1H,
60-H), 7.48 (t, 1H, 50-H), 7.57 (d, 1H, 40-H), 7.61 (s, 1H,
20-H), 7.71 (d, 1H, 8-H), 8.57 (d, 1H, 7-H), 8.98 (d, 1H, 5-H),
9.26 (s, 1H, 4-H). Anal. C16H8NO6Br (C, H, N).
5.3.19. 3-Iodophenyl 6-nitro-2-oxo-2H-1-benzopyran-3-
carboxylate (24e)
The title compound was obtained as described in the gen-
eral procedure after the reaction of the acyl chloride of 6-ni-
tro-2-oxo-2H-1-benzopyran-3-carboxylic acid (23) (w4 mmol)
with 3-iodophenol (1.98 g, 9 mmol) and pyridine (1 mL,
12.5 mmol). After 8 h at room temperature, the resulting pre-
cipitate was collected by filtration, washed with dioxane and
kept overnight at 30 ꢂC. The crude product was stirred in
methanol and collected by filtration. It was then dissolved
in warm DMF, treated with charcoal and filtered. The filtrate
was kept at 4 ꢂC for 2 h. The resulting precipitate was col-
lected by filtration, washed with DMF and methanol and
dried for one night at 30 ꢂC: mp >260 ꢂC; IR 3068 (CeH
arom), 1770 (C]O ester), 1720 (C]O lactone), 1616,
1
1376, 1248 cmꢃ1; H NMR 3.37 (b, 3H, NH3þ), 4.12 (s, 2H,
CH2), 7.19e7.28 (m, 3H, 20-H, 40-H, 60-H), 7.52e7.59 (m,
2H, 50-H, 8-H), 7.94 (d, 1H, 7-H), 8.04 (d, 1H, 5-H), 9.03 (s,
1H, 4-H). Anal. C17H12NO4F$HCl (C, H, N).
5.3.22. 3-Chlorophenyl 6-aminomethyl-2-
oxo-2H-1-benzopyran-3-carboxylate chloride salt (27c)
3-Chlorophenyl
6-hexamethylenetetrammoniomethyl-2-
oxo-2H-1-benzopyran-3-carboxylate chloride salt (26c) was
obtained as described for 26a after the reaction of hexameth-
ylenetetramine (1.8 g, 12.8 mmol) with 3-chlorophenyl 6-
1
1570, 1532, 1344, 1247 cmꢃ1; H NMR 7.32 (d, 1H, 60-H),
7.45 (t, 1H, 50-H), 7.58 (d, 1H, 40-H), 7.60 (s, 1H, 20-H),
7.71 (d, 1H, 8-H), 8.57 (d, 1H, 7-H), 8.98 (d, 1H, 5-H),
9.28 (s, 1H, 4-H). Anal. C16H8NO6I (C, H, N).
chloromethyl-2-oxo-2H-1-benzopyran-3-carboxylate
(25c)
(2 g, 5.7 mmol). The resulting precipitate was collected by fil-
tration and washed with cold CHCl3: mp 210 ꢂC. The title
compound was obtained as described for 27b after the reac-
tion of 3-chlorophenyl 6-hexamethylenetetrammoniomethyl-
2-oxo-2H-1-benzopyran-3-carboxylate chloride salt (30c)
(2 g, 4 mmol) with 12 N HCl (3.2 mL) in ethanol (13.3 mL)
(yield: 40%): mp >260 ꢂC; IR 2987, 2882 (NþeH), 1777
(C]O esters), 1720 (C]O lactone), 1626, 1579, 1488,
5.3.20. Phenyl 6-aminomethyl-2-oxo-2H-1-benzopyran-3-
carboxylate chloride salt (27a)
Hexamethylenetetramine (1.55 g, 11 mmol) was added to
a solution of phenyl 6-chloromethyl-2-oxo-2H-1-benzopyran-
3-carboxylate (25a) (1.5 g, 3.2 mmol) in CHCl3 (45 mL).
The mixture was heated under reflux for 18 h. The resulting
precipitate of phenyl 6-hexamethylenetetrammoniomethyl-2-
oxo-2H-1-benzopyran-3-carboxylate chloride salt (26a) was
collected by filtration and washed with warm CHCl3 (yield:
92%): mp 169e171 ꢂC. Phenyl 6-hexamethylenetetrammonio-
methyl-2-oxo-2H-1-benzopyran-3-carboxylate chloride salt
1
1382, 1246 cmꢃ1; H NMR 3.33 (b, 3H, NH3þ), 4.12 (s, 2H,
CH2), 7.30 (d, 1H, 60-H), 7.43 (d, 1H, 40-H), 7.48 (s, 1H, 20-
H), 7.52e7.59 (m, 2H, 50-H, 8-H), 7.95 (d, 1H, 7-H), 8.04
(d, 1H, 5-H), 9.03 (s, 1H, 4-H). Anal. C17H12NO4Cl$HCl
(C, H, N).

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I. Kempen et al. / European Journal of Medicinal Chemistry 43 (2008) 2735e2750
5.3.23. 3-Bromophenyl 6-aminomethyl-2-oxo-
5.3.26. 3-Fluorophenyl 6-acetamidomethyl-2-oxo-
2H-1-benzopyran-3-carboxylate chloride salt (27d)
2H-1-benzopyran-3-carboxylate (28b)
3-Bromophenyl
6-hexamethylenetetrammoniomethyl-2-
3-Fluorophenyl 6-aminomethyl-2-oxo-2H-1-benzopyran-3-
carboxylate chloride salt (27b) (0.1 g, 0.3 mmol) and TEA
(0.055 ml, 0.43 mmol) were suspended in acetic anhydride
(4 mL, 42.4 mmol). After 1 h, distilled water (50 mL) was
added and the suspension was stirred at room temperature
for 1 h. The resulting white precipitate was collected by filtra-
tion and washed with water: mp 249e252 ꢂC; IR 3070 (CeH
arom), 1775 (C]O ester), 1759 (C]O lactone), 1712 (C]O
amide), 1649, 1621, 1574, 1486, 1377 cmꢃ1; 1H NMR 1.90 (s,
3H, CH3), 4.33 (s, 2H, CH2), 7.16e7.27 (m, 3H, 20-H, 40-H, 60-
H), 7.46 (d, 1H, 8-H), 7.52e7.55 (m, 1H, 50-H), 7.68 (d, 1H, 7-
H), 7.82 (s, 1H, 5-H), 8.45 (s, 1H, NH ), 9.09 (s, 1H, 4-H).
Anal. C19H14NO5F (C, H, N).
oxo-2H-1-benzopyran-3-carboxylate chloride salt (26d) was
obtained as described for 26a after the reaction of hexamethy-
lenetetramine (2.9 g, 20.7 mmol) with 3-bromophenyl 6-chlor-
omethyl-2-oxo-2H-1-benzopyran-3-carboxylate (25d) (4 g,
10 mmol). The resulting precipitate was collected by filtration
and washed with cold CHCl3: mp 174e178 ꢂC. The title com-
pound was obtained as described for 27b after the reaction of
3-bromophenyl 6-hexamethylenetetrammoniomethyl-2-oxo-
2H-1-benzopyran-3-carboxylate chloride salt (26d) (3 g,
5.6 mmol) with 12 N HCl (4.5 mL) in ethanol (18.7 mL)
(yield: 65%): mp >260 ꢂC; IR 2986, 2885 (NþeH), 1776
(C]O esters), 1720 (C]O lactone), 1627, 1580, 1469,
1381, 1246 cmꢃ1; H NMR 3.36 (b, 3H, NH3þ), 4.13 (s, 2H,
1
CH2), 7.34 (d, 1H, 60-H), 7.47 (t, 1H, 50-H), 7.55e7.61 (m,
3H, 20-H, 40-H, 8-H), 7.94 (d, 1H, 7-H), 8.03 (d, 1H, 5-H),
9.05 (s, 1H, 4-H). Anal. C17H12NO4Br$HCl (C, H, N).
5.3.27. 3-Chlorophenyl 6-acetamidomethyl-2-oxo-
2H-1-benzopyran-3-carboxylate (28c)
3-Chlorophenyl 6-aminomethyl-2-oxo-2H-1-benzopyran-3-
carboxylate chloride salt (27c) (0.25 g, 0.68 mmol) and TEA
(0.103 g, 10 mmol) were suspended in acetic anhydride
(10 mL, 0.106 mol). After 1 h, distilled water (50 mL) was
added and the suspension was stirred at room temperature
for 1 h. The resulting white precipitate was collected by filtra-
tion and washed with water: mp 241e242 ꢂC; IR 3067 (CeH
arom), 1777 (C]O ester), 1760 (C]O lactone), 1714 (C]O
amide), 1647, 1621, 1572, 1472, 1376 cmꢃ1; 1H NMR 1.90 (s,
3H, CH3), 4.33 (s, 2H, CH2), 7.29 (d, 1H, 60-H), 7.42 (d, 1H,
8-H), 7.45e7.48 (m, 2H, 20-H, 40-H), 7.53 (t, 1H, 50-H), 7.68
(d, 1H, 7-H), 7.82 (s, 1H, 5-H), 8.46 (s, 1H, NH ), 9.10 (s, 1H,
4-H). Anal. C19H14NO5Cl (C, H, N).
5.3.24. 3-Iodophenyl 6-aminomethyl-2-oxo-
2H-1-benzopyran-3-carboxylate chloride salt (27e)
3-Iodophenyl 6-hexamethylenetetrammoniomethyl-2-oxo-
2H-1-benzopyran-3-carboxylate chloride salt (26e) was
obtained as described for 26a after the reaction of hexameth-
ylenetetramine (1 g, 7 mmol) with 3-iodophenyl 6-chloro-
methyl-2-oxo-2H-1-benzopyran-3-carboxylate (25e) (1.2 g,
2.7 mmol). The resulting precipitate was collected by filtration
and washed with cold CHCl3 (yield: 70%): mp 171e175 ꢂC.
The title compound was obtained as described for 27b after
the reaction of 3-iodophenyl 6-hexamethylenetetrammonio-
methyl-2-oxo-2H-1-benzopyran-3-carboxylate chloride salt
(26e) (1.1 g, 1.9 mmol) with 12 N HCl (1.5 mL) in ethanol
(6.3 mL): mp >260 ꢂC; IR 2987, 2890 (NþeH), 1778
(C]O esters), 1722 (C]O lactone), 1627, 1581, 1466,
5.3.28. 3-Bromophenyl 6-acetamidomethyl-2-oxo-
2H-1-benzopyran-3-carboxylate (28d)
3-Bromophenyl 6-aminomethyl-2-oxo-2H-1-benzopyran-
3-carboxylate chloride salt (27d) (0.5 g, 1.2 mmol) and
TEA (0.18 g, 1.8 mmol) were suspended in acetic anhydride
(15 mL, 159 mmol) and heated under reflux for 1 h. After
cooling, distilled water (50 mL) was added and the suspen-
sion was stirred at room temperature for 1 h. The resulting
brown pasty precipitate was collected by filtration and
washed with water. The crude product was suspended in
ethyl acetate and the resulting precipitate was collected by
filtration, washed with ethyl acetate and dried: mp 233.5e
235 ꢂC; IR 3064 (CeH arom), 1776 (C]O ester), 1761
(C]O lactone), 1715 (C]O amide), 1646, 1620, 1572,
1380, 1245 cmꢃ1; H NMR 3.38 (b, 3H, NH3þ), 4.13 (s, 2H,
1
CH2), 7.28e7.34 (m, 2H, 50-H, 60-H), 7.58 (d, 1H, 8-H),
7.70e7.74 (m, 2H, 20-H, 40-H), 7.93 (d, 1H, 7-H), 8.03 (d,
1H, 5-H), 9.02 (s, 1H, 4-H). Anal. C17H12NO4I$HCl (C, H, N).
5.3.25. Phenyl 6-acetamidomethyl-2-oxo-2H-1-benzopyran-
3-carboxylate (28a)
Phenyl 6-aminomethyl-2-oxo-2H-1-benzopyran-3-carbox-
ylate chloride salt (27a) (0.1 g, 0.3 mmol) and TEA
(0.06 ml, 0.45 mmol) were suspended in acetic anhydride
(4 mL, 42.4 mmol). After 1 h, distilled water (50 mL) was
added and the suspension was stirred at room temperature
for 1 h. The resulting white precipitate was collected by filtra-
tion and washed with water. The crude product was then sus-
pended in acetone for 15 min, then collected by filtration: mp
>260 ꢂC; IR 3070 (CeH arom), 1774 (C]O ester), 1757
(C]O lactone), 1710 (C]O amide), 1651, 1621, 1574,
1470, 1376 cmꢃ1 1H NMR 1.90 (s, 3H, CH3), 4.33 (s,
;
2H, CH2), 7.33 (d, 1H, 60-H), 7.45e7.48 (m, 2H, 50-H, 8-
H), 7.55 (d, 1H, 40-H), 7.60 (s, 1H, 20-H), 7.67 (d, 1H, 7-
H), 7.82 (s, 1H, 5-H), 8.45 (s, 1H, NH ), 9.10 (s, 1H,
4-H). Anal. C19H14NO5Br (C, H, N).
1
1491, 1380 cmꢃ1; H NMR 1.91 (s, 3H, CH3), 4.32 (s, 2H,
5.3.29. 3-Iodophenyl 6-acetamidomethyl-2-oxo-
CH2), 7.29e7.35 (m, 3H, 20-H, 40-H, 60-H), 7.48 (d, 1H, 8-
H), 7.50e7.51 (m, 2H, 30-H, 50-H), 7.67 (d, 1H, 7-H), 7.80
(s, 1H, 5-H), 8.45 (s, 1H, NH ), 9.12 (s, 1H, 4-H). Anal.
C19H15NO5 (C, H, N).
2H-1-benzopyran-3-carboxylate (28e)
3-Iodophenyl 6-aminomethyl-2-oxo-2H-1-benzopyran-3-
carboxylate chloride salt (27e) (0.25 g, 0.55 mmol) and TEA
(0.08 g, 0.8 mmol) were suspended in acetic anhydride

I. Kempen et al. / European Journal of Medicinal Chemistry 43 (2008) 2735e2750
2749
(10 mL, 106 mmol). After 30 min, the mixture was gently
heated for another 30 min. Then, distilled water (50 mL)
was added and the suspension was stirred at room temperature
for 1 h. The resulting white precipitate was collected by filtra-
tion and washed with water: mp 223e224 ꢂC; IR 3056 (CeH
arom), 1763 (C]O ester and lactone), 1718 (C]O amide),
with 20% FCS (acting as chemoattractant), 1% BSA and
6 mL of an inhibitor solution (10ꢃ5, 10ꢃ4 and 10ꢃ3 M in
DMEM supplemented with 10% dimethylsulphoxyde). Filters
were recovered with 100 mL of cell suspension (6 ꢄ 104 cells),
200 mL DMEM supplemented with 0.1% BSA and 3 mL of in-
hibitor solution. Chambers were subsequently incubated for
48 h (in order to maintain a chemotactic gradient), media
from both lower and upper wells were renewed after 24 h in
a humid atmosphere at 37 ꢂC. After incubation, cells at the
bottom of filters were fixed in 4% paraformaldehyde for
15 min at 4 ꢂC, permeabilised for 10 min in absolute methanol
at ꢃ20 ꢂC and stained with Giemsa (4% in sterile water)
(Fluka 48900) for 15 min. The exceeding dye was removed
by washing with water and cells on the upper surface of filters
were eliminated by scrapping with a cotton swab. Cells having
reached the lower surface of the filters were visually counted
using a light microscope (Vanox AH3, Olympus, Hamburg,
Germany). Each essay was performed in triplicate (n ¼ 3). Re-
sults were expressed as percentage (ꢀs.d.) of the migration of
control cells.
1
1650, 1622, 1573, 1466, 1376 cmꢃ1; H NMR 1.90 (s, 3H,
CH3), 4.32 (s, 2H, CH2), 7.28e7.35 (m, 2H, 50-H, 60-H),
7.46 (d, 1H, 8-H), 7.66e7.73 (m, 3H, 20-H, 40-H, 7-H), 7.81
(s, 1H, 5-H), 8.46 (s, 1H, NH ), 9.08 (s, 1H, 4-H). Anal.
C19H14NO5I (C, H, N).
5.4. Biological assays
5.4.1. Chemoinvasion assay with HT 1080 fibrosarcoma cells
5.4.1.1. Cell culture. Human fibrosarcoma HT 1080 cells were
maintained in Dulbecco’s Modified Eagle’s Medium (DMEM)
supplemented with 10% (v/v) fetal calf serum (FCS), penicil-
linestreptomycin (100 UI/mLe100 mg/mL), 2 mM glutamine
and 10 mM HEPES buffer at 37 ꢂC in a humid atmosphere
(5% CO2 and 95% O2).
Acknowledgements
5.4.1.2. Coating of culture wells with type IV collagen. Twenty
four-well plates (Falcon, BectoneDickinson, USA) and
6.5 mm polycarbonate filters (8 mm pore size) of Transwell
cell culture chamber inserts (Costar, Netherlands) were coated
with type IV collagen purified from human placenta diluted at
200 mg/mL in phosphate-buffered saline (PBS): 3 mL, then
5 mL, then 10 mL of this solution were added successively to
the centre of filters. Collagen solution was then diluted to
100 mg/mL and two more additions were done. After each ad-
dition, coats were left to air-dry in a laminar flow hood under
UV light. Thus, the final amount of collagen was about
ꢀ24 mg. Before use, coated plates and inserts were washed
twice with sterile water and incubated for at least 1 h in se-
rum-free DMEM at 37 ꢂC.
This study was supported by grants from the National Fund
for Scientific Research (F.N.R.S., Belgium) from which P. de
Tullio is Research Associate. The technical assistance of Y.
Abrassart and F. Olivier is gratefully acknowledged.
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