Further characterization of a putative serine protease contributing to the γ-secretase cleavage of β-amyloid precursor protein

Article abstract of DOI:10.1016/j.bmc.2012.11.045

The 3-alkoxy-7-amino-4-chloro-isocoumarins JLK-6 and JLK-2 have been shown to markedly reduce the production of Amyloid β-peptide (Aβ) by Amyloid-β Precursor Protein (APP) expressing HEK293 cells by affecting the γ-secretase cleavage of APP, with no effect on the cleavage of the Notch receptor. This suggested that these compounds do not directly inhibit the presenilin-dependent γ-secretase complex but more likely interfere with an upstream target involved in γ-secretase-associated pathway. The mechanism of action of these compounds is unknown and there are high fundamental and therapeutical interests to unravel their target. Isocoumarin compounds were previously shown to behave as potent mechanism-based irreversible inhibitors of serine proteases, suggesting that the JLK-directed target could belong to such enzyme family. To get further insight into structure-activity relationships and to develop more potent isocoumarin derivatives, we have synthesized and evaluated a series of isocoumarin analogues with modifications at positions 3, 4 and 7. In particular, the 7-amino group was substituted with either acyl, urethane, alkyl or aryl groups, which could represent additional interaction sites. Altogether, the results highlighted the essential integrity of the 3-alkoxy-7-amino-4-chloro-isocoumarin scaffold for Aβ-lowering activity and supported the involvement of a serine protease, or may be more generally, a serine hydrolase. The newly reported 7-N-alkyl series produced the most active compounds with an IC₅₀ between 10 and 30 μM. Finally, we also explored peptide boronates, a series of reversible serine protease inhibitors, previously shown to also lower cellular Aβ production. The presented data suggested they could act on the same target or interfere with the same pathway as isocoumarins derivatives.

Full text of DOI:10.1016/j.bmc.2012.11.045

Bioorganic & Medicinal Chemistry 21 (2013) 1018–1029
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Further characterization of a putative serine protease contributing to
the c-secretase cleavage of b-amyloid precursor protein
Marine Peuchmaur a, ,k, Marie-Agnès Lacour a,à,k, Jean Sévalle b,§, Vincent Lisowski a, Youness Touati-Jallabe a,
Fabien Rodier a,–, Jean Martinez a, Frédéric Checler b, Jean-François Hernandez a,
⇑
a Institut des Biomolécules Max Mousseron, UMR5247 CNRS, Universités Montpellier 1 and 2, Faculté de Pharmacie, 15 avenue Charles Flahault, 34093 Montpellier Cedex 5, France
b Institut de Pharmacologie Moléculaire et Cellulaire, UMR7275 CNRS, Université de Nice-Sophia-Antipolis, Labelled Team ‘Fondation pour la Recherche Médicale’, LABEX Distalz,
660 route des Lucioles, Sophia-Antipolis, 06560 Valbonne, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The 3-alkoxy-7-amino-4-chloro-isocoumarins JLK-6 and JLK-2 have been shown to markedly reduce the
Received 24 August 2012
Revised 5 November 2012
Accepted 15 November 2012
Available online 5 December 2012
production of Amyloid b-peptide (Ab) by Amyloid-b Precursor Protein (APP) expressing HEK293 cells by
affecting the
gested that these compounds do not directly inhibit the presenilin-dependent
more likely interfere with an upstream target involved in -secretase-associated pathway. The mecha-
c-secretase cleavage of APP, with no effect on the cleavage of the Notch receptor. This sug-
c
-secretase complex but
c
nism of action of these compounds is unknown and there are high fundamental and therapeutical inter-
ests to unravel their target. Isocoumarin compounds were previously shown to behave as potent
mechanism-based irreversible inhibitors of serine proteases, suggesting that the JLK-directed target could
belong to such enzyme family. To get further insight into structure–activity relationships and to develop
more potent isocoumarin derivatives, we have synthesized and evaluated a series of isocoumarin ana-
logues with modifications at positions 3, 4 and 7. In particular, the 7-amino group was substituted with
either acyl, urethane, alkyl or aryl groups, which could represent additional interaction sites. Altogether,
the results highlighted the essential integrity of the 3-alkoxy-7-amino-4-chloro-isocoumarin scaffold for
Ab-lowering activity and supported the involvement of a serine protease, or may be more generally, a ser-
ine hydrolase. The newly reported 7-N-alkyl series produced the most active compounds with an IC50
Keywords:
Isocoumarin
Amyloid b peptide
Alzheimer’s disease
c
-Secretase
Serine proteases
between 10 and 30 lM. Finally, we also explored peptide boronates, a series of reversible serine protease
inhibitors, previously shown to also lower cellular Ab production. The presented data suggested they
could act on the same target or interfere with the same pathway as isocoumarins derivatives.
Ó 2012 Elsevier Ltd. All rights reserved.
Abbreviations: Ab, Amyloid b-peptide; AICD, APP intracellular domain; APP,
Amyloid-b Precursor Protein; BACE, beta-site APP cleaving enzyme; DCM, dichlo-
romethane; DMSO, dimethylsulfoxide; ESI, electrospray ionization; EtOAc, ethyl
1. Introduction
The Amyloid b-peptide (Ab) is very likely a key element in the
pathogenesis of Alzheimer’s disease.1,2 Ab is proteolytically pro-
duced from Amyloid-b Precursor Protein (APP) by the sequential
contribution of two aspartyl-proteases. BACE-1 is responsible of
the b-secretase cleavage of APP, releasing C99 that begins at the
acetate; FBS, fetal bovine serum; GSAP,
c-secretase activating protein; GSI, c-
secretase inhibitor; GSM, -secretase modulator; HEK, Human Embryonic Kidney;
c
Hex, hexane; HRMS, high resolution mass spectrometry; LC/MS, liquid chromatog-
raphy coupled to mass spectrometry; LDH, lactate dehydrogenase; NICD, Notch
intracellular domain; PBS, phosphate buffered saline; PS, presenilin; RIPA, radio-
immunoprecipitation assay; SDS–PAGE, sodium dodecylsulfate–polyacrylamide gel
electrophoresis; TFA, trifluoroacetic acid; THF, tetrahydrofurane.
N-terminal end of Ab.3–6 The
c-secretase cleavage of C99, which
⇑
Corresponding author. Tel.: +33 04 11 75 96 03; fax: +33 04 11 75 96 41.
occurs in the transmembrane domain of APP, yields Ab-40/42
and is mainly associated to a complex of at least four different pro-
teins including a presenilin (PS) as the catalytic subunit.7–9 Addi-
tional cleavage of APP by the so-called
7–10 residues downstream to the -site and close to the cytoplas-
E-mail address: jean-francois.hernandez@univ-montp1.fr (J.-F. Hernandez).
Present address: Département de Pharmacochimie Moléculaire, UMR5063 CNRS,
Université Joseph Fourier, Pôle Chimie, Bât. E, BP 53, 38041 Grenoble Cedex 9, France.
e-secretase occurs on C99,
à
Present address: Institut Charles Gerhardt, UMR5253 CNRS, Universités Mont-
c
pellier 1 and 2, ENSCM, 8 rue de l’Ecole Normale, 34296 Montpellier Cedex 5, France.
§
Present address: Tanz Centre for Research in Neurodegenerative Disease,
mic membrane boundary and produces a fragment called APP
IntraCellular Domain (AICD).10,11 It has been proposed that both
University of Toronto, 6 Queens Park Cr West, Toronto, Ontario, Canada M5S 3H2.
–
Present address: Institut des Sciences Moléculaires de Marseille, UMR7313 CNRS,
c
- and e-secretases depend on presenilin, but that they are differ-
Aix-Marseille Université, Campus Saint Jérôme, Avenue Escadrille Normandie-
Niémen, 13394 Marseille Cedex 20, France.
entially regulated.12 Accumulation of Ab and in particular of the
most aggregation-prone Ab-42 species first leads to the formation
k
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.11.045

M. Peuchmaur et al. / Bioorg. Med. Chem. 21 (2013) 1018–1029
1019
of soluble oligomers that likely account for the Ab-related toxic po-
tential.13,14 b- and
-Secretases early appeared as potentially rele-
PS is the likely target of these compounds,29 the Ab region of APP
was also labelled by one of these probes based on tarenflurbil.30
Imatinib (GleevecÒ), a tyrosine kinase inhibitor, was also found
to potently reduce Ab production without blocking NICD genera-
tion.31 Photoactivatable and biotinylated probes of Imatinib tar-
c
vant targets of therapeutic strategies aimed at slowing-down or
blocking the Ab-dependent neurodegenerative processes. Consid-
erable efforts have been made over the last years to develop Ab-
reducing drugs, but with very disappointing results so far.15–17
Although BACE-1 was initially considered as an ideal target, none
of the inhibitors able to potently block BACE-1 in vitro or in animal
models have yet proved beneficial in clinical trials. The main rea-
sons are the existence of other important substrates like neuregu-
lin-1,18 which is involved in myelination, a wide substrate-binding
domain and the necessity to cross the blood–brain barrier. More
recently, Rajapaksha et al. suggested that BACE-1 could be involved
in axonal guidance and that BACE-1 inhibitors could potentially
display deleterious effects on sensory neurons.19 Targeting PS-
geted GSAP (
partner of PS.32 Imatinib inhibits the binding of APP to GSAP, which
does not interact with Notch, decreasing -cleavage of APP and
-cleavage, thereby enhancing the production of
c-secretase activating protein), a newly discovered
c
increasing its
AICD.
e
Another important class of Notch-sparing GSI is a series of iso-
coumarins that were shown to markedly reduce recovery of both
Ab-40 and Ab-42 in a cell-based assay without affecting NICD gen-
eration.33 The most efficient isocoumarins have been called JLK-6
and JLK-2 (Fig. 1). These compounds also do not alter Notch signal-
ling in vivo and do not interfere with the processing of two other
dependent
c-secretase complex is even more complicated. This
generic term indeed comprises at least six distinct sub-complexes
with specific protein composition involved in the intra-membrane
cleavage of more than 60 proteins identified to date.20 Among
other vital functions related to their catalytic properties, PS-depen-
PS-dependent
CD44, nor with
c
a
-secretase substrates, namely cadherins and
- and b-secretases and GSK3b kinase.34 JLK-2 also
does not inhibit purified 20S proteasome,34 while it was shown to
interfere with the ubiquitin–proteasome pathway in a cell assay
according to an unknown mechanism.35 Finally, they are devoid
of non-specific effects on the secretory machinery and do not dis-
dent
c-secretase is involved in the essential maturation of the
Notch receptor leading to the production of NICD (Notch IntraCel-
lular Domain).21 NICD is involved in cell differentiation and embry-
onic development, the blockade of which leads to severe
abnormalities.17 This is probably one of the main reasons of prema-
ture interruption of two large phase III clinical trials of semagace-
play cell toxicity at 100
anism of action remains elusive, but they are believed to act
upstream of the PS-dependent -secretase as they do not physi-
l
M on various cell lines.34,36,37 Their mech-
c
cally interact with PS.36,38 Therefore, they act differently from
other claimed Notch-sparing GSI like begacestat or avagacestat,
which interact with PS. JLK-6 and JLK-2 belong to the family of
3-alkoxy-7-amino-4-chloro-isocoumarins, which were originally
described as mechanism-based irreversible inhibitors of serine
proteases by James C. Powers.39,40 They were first reported as sui-
cide inhibitors of elastases and chymotrypsin.39,41 JLK-6 was also
found to inhibit some other enzymes such as rhomboids,42,43
which are intra-membrane cleaving proteases of the serine cata-
lytic type, the mitochondrial pro-apoptotic serine protease
HtrA2/Omi [Sévalle, J. et al., unpublished] and cholesterol ester-
ase,44 which is another kind of serine hydrolase. All these data sup-
port the hypothesis that JLK isocoumarins target a putative serine
protease, or more generally a serine hydrolase acting upstream the
stat, a non-Notch-sparing
c-secretase inhibitor (GSI), which not
only worsened the cognition state of the patients, but also pro-
duced detrimental neoplastic effects related to Notch signalling
interruption.17 Notch-sparing GSI have been developed.17,22 How-
ever, recent studies challenged the claimed selectivity of some of
these compounds like begacestat or avagacestat, the latter being
currently tested in phase II clinical trial.23,24 Several classes of com-
pounds were found to selectively modulate the
-secretase modulators, GSM), sparing Notch proteolysis.17,22
subset of non-steroidal anti-inflammatory drugs were found to
modulate the -cleavage of APP, decreasing Ab-40 and Ab-42 pro-
c-cleavage of APP
(c
A
c
duction with increased generation of the poorly amyloidogenic Ab-
38 fragment.25 These compounds also modulate the scissile bond
position of Notch receptor but do not prevent NICD production.26
Two large phase 3 clinical studies with one of these compounds,
that is, tarenflurbil, were also completely negative,27 due to low
potency and brain penetration. However, they still represent the
most promising Notch-sparing and Ab-40/42 lowering approach.28
Several modulator-based chemical probes carrying photoaffinity
and biotin moieties were developed to identify their molecular tar-
get. Probably because the utilized agents belonged to different
structural classes, these studies raised conflicting results. Whereas
c
-cleavage of APP. It is therefore important to identify this (these)
putative cellular target(s) in order to better understand how JLK
isocoumarins affect -secretase activity. This might help to devel-
c
op new therapeutic strategies aimed at lowering Ab content. The
advantage of these isocoumarins is that they can form highly stable
and even irreversible covalent complexes with their serine
protease targets, according to a mechanism proposed by James C.
Powers and shown in Figure 1S (Supplementary data, S1).45
Successful isolation of serine proteases was already achieved by
O
O
O
H2N
H2N
O
O
O
O
Cl
Cl
JLK-6
JLK-2
A
Figure 1. Upper panel, structures of JLK-6 and JLK-2. Lower panel, SDS–PAGE analysis of the concentration-dependent effect of JLK-6 on Ab production by Swedish-mutated
APP-transfected HEK293 cells.

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M. Peuchmaur et al. / Bioorg. Med. Chem. 21 (2013) 1018–1029
Table 1
Structure of compounds 1–12 and their effect on Ab production by transfected HEK293 cells
O
R1
7
O
3
R2
O
4
X
Compound
R1
R2
X
Acta
Compound
R1
R2
X
Acta
1
2
3
4
5
6
NO2
NH2
NO2
NH2
NO2
NH2
Ethyl
Ethyl
Butyl
Butyl
Isobutyl
Isobutyl
Cl
Cl
Cl
Cl
Cl
Cl
ꢀ
+
7
8
9
10
11
12
NO2
NH2
NO2
NH2
NO2
NH2
Methyl
Methyl
Methoxyethyl
Methoxyethyl
2-Bromoethyl
2-Bromoethyl
H
H
H
H
H
H
+/ꢀ
ꢀ
ꢀ
+
T
ꢀ
ꢀ
T
+
ꢀ
a
Inhibitory activity of Ab production by HEK293 cells overexpressing swedish mutant bAPP. Compounds 1–12 have been tested at 60
lM concentration. + indicated an
inhibition >50% at the maximal concentration tested; ꢀ indicated inactive; T, significant cell toxicity. Toxic compounds were inactive at 10
lM.
several series of 3-alkoxy-isocoumarins modified at the 3, 4 and
7 positions. Several crystal structures of elastase–inhibitor com-
plexes have been resolved. Various binding modes such as interac-
tion of the 3-alkoxy or the 7-substituent in S1 subsite or a
positioning of the 3-alkoxy group in the S3 subsite or away from
the enzyme have been reported, indicating the impossibility to
establish a canonical binding mode for these isocoumarin deriva-
tives.40,41,49 Therefore, it is important to vary the 3-alkoxy group
and to explore the substitution of the 7-amino group. The latter
modification also adds potential interaction sites. Finally, the pres-
ence of the mechanistically important chlorine at position 4 was
evaluated.
R2OH
H+
O2N
COOH
COOH
O2N
COOH
COOR2
PCl5
(X = Cl) (X = H)
(CF3CO)2O
O
X
O
H2
Pd/C
H2N
O2N
O
O
OR2
OR2
X
Scheme 1. Synthesis of 3-alkoxy-isocoumarins 1–12.
2.1.1. Synthesis
The 3-alkoxy-4-chloro/H-7-nitro/amino-isocoumarins 1–12
(Table 1) were obtained according to published procedures
(Scheme 1).50,51,35
Briefly, 4-nitro-homophthalic acid was mono esterified by
treatment with the corresponding alcohol as solvent in the pres-
ence of catalytic amounts of sulfuric acid. The resulting alkyl 2-car-
boxy-4-nitrophenylacetate derivatives were cyclized by treatment
with PCl5 or trifluoroacetic anhydride to yield the 3-alkoxy-4-
the introduction of a biotin label to isocoumarin derivatives and
therefore, such strategy could also be envisioned for JLK-6
target.46,47 However, the low potencies of JLK-6/2 in cell assay
(IC50 of about 30–60 lM) and their low selectivity require prior
development of more potent analogues.
We have synthesized several series of JLK-6-derived isocouma-
rins and evaluated their biological activity to further characterize
the structural requirements driving the interaction with their tar-
get. Furthermore, we explored a series of peptide–boronate com-
pounds, another class of serine protease inhibitors, which were
found to possess similar Ab-lowering activity in cell culture.48
chloro-7-nitroisocoumarins 1, 3,
5 or the 3-alkoxy-7-nitro-
isocoumarins 7, 9, 11, respectively. Catalytic reduction of the nitro
group by hydrogenolysis gave 7-amino analogues 2, 4, 6, 8, 10, 12
(Table 1). Compounds 8 and 10 are the chlorine lacking analogues
of JLK-6 and JLK-2, respectively.
The acyl derivatives of JLK-6 13–17 (Table 2) were obtained in
good to moderate yields (35–85%) by reaction of JLK-6 with the
corresponding acyl chlorides or anhydrides (see Section 4)
(Scheme 2). The urethane derivatives of JLK-6 18–24 (Table 2) were
similarly obtained in good to moderate yields (30–85%) by reaction
of JLK-6 with the corresponding halogenoformates or anhydrides.
2. Results and discussion
2.1. 3-Alkoxy-isocoumarin derivatives
To get further insight into the structural requirements of the JLK
target for developing more potent compounds, we synthesized
Table 2
Structure of compounds 13–24 and their effect on Ab production by transfected HEK293 cells
Compound
R
Acta
Compound
R
Acta
13
14
15
16
17
18
Acetyl
Glutaryl
Pentafluoropropanoyl
Octanoyl
Pivaloyl
ꢀ
19
20
21
22
23
24
Adamantyloxycarbonyl
Fluorenylmethyloxycarbonyl
Ethyloxycarbonyl
t-Butyloxycarbonyl
Trichloroethoxycarbonyl
Allyloxycarbonyl
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+/ꢀ
ꢀ
Benzyloxycarbonyl
a
Inhibitory activity of Ab production by HEK293 cells overexpressing swedish mutant bAPP. Compounds 13–24 have been tested at 60
inhibition >50% at the maximal concentration tested; ꢀ indicated inactive.
l
M concentration. + indicated an

M. Peuchmaur et al. / Bioorg. Med. Chem. 21 (2013) 1018–1029
1021
O
O
O
O
H
N
H
N
R'-CHO, NaBH3CN
DCM (R = R'-CH2-)
7
7
H2N
H2N
R
R-Cl or R2O
O
O
R
O
O
Me
Me
Me
Me
NaHCO3
THF
R-B(OH)2, Cu(OAc)2
DCM (R = Ar)
O
O
O
O
Cl
Cl
Cl
Cl
Scheme 2. Synthesis of N-acylated (13–17) and urethane (18–24) derivatives of
Scheme 3. Synthesis of 7-N-alkylated (25–42) or arylated (43, 44) analogues of JLK-
JLK-6.
6.
The synthesis of the N-alkylated/arylated derivatives of JLK-6
required careful attention according to the instability of the
3-alkoxy-isocoumarin in alcoholic, nucleophilic or acidic medium
(Table 3, Scheme 3).
nucleophilic components including thiols and amines present in
small molecules or proteins.45,51,54 In a cell assay, the effect of sub-
stituents and/or structural modifications on cell entrance could be
a questionable parameter in the interpretation of activity data.
However, most introduced substituents were hydrophobic and
structural modifications were limited. Then, these molecules re-
mained sufficiently small. For these reasons, we assumed that
the efficiency of the modified compounds to reach their target
was not significantly influenced by structural variations.
The N-alkylated derivatives 25–42 were prepared by reductive
amination of JLK-6 with various alkyl- or aryl-aldehydes. Their syn-
thesis was first attempted following two classical protocols using
NaB(OAc)3H/dichloroethane in the presence of acetic acid or
NaBH3CN/MeOH. The isocoumarin was not enough stable in the
presence of acetic acid or in methanol, leading to complex mix-
tures. Conditions using NaBH3CN were varied and dichlorometh-
ane was finally chosen as the most convenient solvent.
Compounds were obtained in low to moderate yields (10–50%)
but in good purity. N-arylation was performed by Suzuki coupling
of arylboronic acids in anhydrous conditions in the presence of
copper acetate.52
2.1.2.1. Modifications of JLK-6 at positions
(Table 1). All 3-alkoxy-4-chloro-7-nitro-isocoumarins (1, 3,
5) were found non toxic but inactive in reducing Ab production
at a 60 M concentration, in accordance with previous results ob-
3
and
4
l
tained with the 7-nitro analogues of JLK-6 and JLK-2.33 Although
the nitro derivatives are usually more reactive inhibitors of serine
proteases than the amino analogues, they decompose rapidly in
aqueous medium (half-lives of less than 20 min in buffers) and
the acylated species formed by reaction with the catalytic serine
hydrolyses spontaneously with rapid reactivation of the enzyme
and concomitant release of henceforth inactive opened isocouma-
rin-derived product.45 In the test conditions, the inhibition by
these compounds would therefore be too transitory (until total
2.1.2. Biological evaluation
All compounds were evaluated in a cell-based assay and the Ab
production by APPswe overexpressing HEK293 cells53 was as-
sessed after 8 h incubation. In this context, it is important to high-
light the limited stability and lifetime of the isocoumarin
derivatives in aqueous medium and in the presence of various
Table 3
Structure of compounds 25–44 and their effect on Ab production by transfected HEK293 cells
Compound
R
Acta
Compound
R
Acta
25
T
35
ꢀ
26
+
36b
+
27
+/ꢀ
37
38
ꢀ
ꢀ
28b
+
29
+
39
+
30
31
32
33
T
T
T
T
40
41
42
43
ꢀ
ꢀ
ꢀ
T
34
ꢀ
44
ꢀ
a
Inhibitory activity of Ab production by HEK293 cells overexpressing Swedish mutant bAPP. Compounds 25, 26, 29, 31, 34, 35, 39, 41–44 have been tested at 60
l
M. All
others, compounds 27, 28, 30, 32, 33, 36–38, 40 have been tested at 10 and 30
lM concentration. + indicated an inhibition >50% at the maximal concentration tested; ꢀ
indicated inactive; T, significant cell toxicity.
b
IC50 between 10 and 30 lM.

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M. Peuchmaur et al. / Bioorg. Med. Chem. 21 (2013) 1018–1029
found inactive at 60 lM concentration. This might be due to unfa-
A
vourable interactions of the substituents with the target. Another
reason might be their inability to form irreversible complexes as
it was reported for most N-acyl-JLK-6 elastase inhibitors of the
above mentioned series,49 indicating that acylation of the 7-amino
group decreases the ability of the inhibitor to alkylate serine prote-
ases by lowering the strong electron-releasing properties of the
free amino group. As a result, these acylated compounds might
be slow ‘enzyme alkylators’.55 With this view, compounds 13–16
could form reversible acyl enzyme complexes, and then undergo
rapid hydrolysis with concomitant enzyme reactivation. A similar
negative result was previously obtained with JLK-8, the 7-N-phen-
ylalanyl derivative of JLK-6.33 The observed activity of 17 with a
pivaloyl, nevertheless less efficient than JLK-6, could be the result
of a highly stable complex between 17 and its target as stated
above and as it has also been observed for a few N-acyl-JLK-6 elas-
tase inhibitors.49
Compounds
DMSO
7
8
9
11 12 10
DMSO
Figure 2. Effect of compounds 7–12 (60
over-expressing HEK293 cells (DMSO and JLK-2/6 60
l
M) on the production of Ab by bAPPswe
lM as controls).
destruction of the inhibitor) to observe any Ab-lowering effect in
the time of the assay. The 7-amino analogue 2 with an ethoxy
group at position 3 behaved like JLK-6 (3-methoxy group) whereas
the two other 3-alkoxy-7-amino-4-chloro-isocoumarins (4, 6 with
a butoxy and an isobutoxy group, respectively) were toxic at
60 lM and inactive at 10 lM concentration (not shown). The activ-
ity of 2 and JLK-6 in this cell assay would have two reasons. First,
only the 3-alkoxy-7-amino-4-chloro analogues could form irre-
versible covalent complexes with their putative serine protease
target, the activity of which being recovered only after a new bio-
synthesis. Second, these analogues are more stable to hydrolysis
than the nitro derivatives in buffers (about 3.5 h in Hepes buf-
fer45,55). The toxicity of compounds 4 and 6 could be explained
by the targeting of other protease(s), the function of which being
essential for cell survival. Therefore, it appeared difficult to further
explore the structural requirements at position 3.
The results obtained with compounds 7–12 lacking the 4-chlo-
rine were quite contradictory to those obtained in the 4-chloro ser-
ies since the 7-amino derivatives 8, 10, 12 were inactive, while the
7-nitro analogues 7, 9, 11 showed Ab-lowering activity (Fig. 2). As a
matter of fact, the inactivity of compounds 8, 10 and 12 was in
good agreement with a previous study in which compound 8
was shown to inactivate serine proteases quite slowly or not at
all, indicating the significance of the chlorine atom. In the same
study the nitro analogue 7 was found much more active and the
enzymes inactivated by 7 were shown to undergo only transient
inactivation and to rapidly regain full activity.45 However the ob-
served Ab-lowering activity of the nitro analogues 7, 9, 11 could
be explained by a higher stability of the resulting acyl enzyme
complex formed with their target, as it is known that this stability
both depends on the structure of the 3-substituent and on the in-
volved enzyme.41,45 Overall, 7, 9, 11 were found less active than
JLK-6 and no modification could be easily considered in this series
to increase their potency.
None of the compounds 18–24 (Table 2) of the carbamate series
showed Ab-lowering activity at the same 60 lM concentration.
Again, these negative results could result from unfavourable bind-
ing caused by the substituents. It can also be hypothesized that the
more electro-attracting oxycarbonyl group, compared to an acyl
group, could result in a further decrease of the 7-nitrogen lone pair
mobility toward the phenyl ring, disfavouring the irreversible
alkylation of an enzyme active site (Fig. 1S). It remains that the
negative result obtained with compound 19 with a 7-Adoc substi-
tuent is contradictory to that reported for the equivalent analogue
of JLK-2 (JLK-733), which showed Ab-lowering activity in the cell
assay. Again, such results could be due to the unpredictable stabil-
ity of a particular complex, even for close compounds, when no
irreversible link between the inhibitor and the putative enzyme
is expected.
In these N-acyl and carbamate series, it was difficult to establish
structure–activity relationships, which could help in choosing
further modifications of the isocoumarin derivatives and the for-
mation of a sufficiently stable complex appeared highly unpredict-
able. Therefore, we developed a last series of JLK-6 analogues with
an alkylated (25–42) or arylated (43, 44) 7-amino group, in which
the 7-nitrogen lone-pair was supposedly more prone to electron
delocalization (Table 3). All substituents were hydrophobic and
classified as linear (25–27) or branched (28–33) alkyl chains, aryl-
alkyl (34–42) and aryl groups (43–44). Several compounds (30–33,
43) showed significant cytotoxicity (measured by an in vitro lac-
tate dehydrogenase (LDH) release assay) at the maximal concen-
2.1.2.2. Substitution of the 7-amino group of JLK-6.
The 7-
tration tested (30 or 60 lM, see note of Table 3). It could be
amino group was the most evident position to substitute with var-
ious groups to add possible interaction sites and therefore increase
the affinity for the target. This was shown for several N-acyl, ure-
thane and ureido derivatives of 7-amino-4-chloro-3-methoxy-iso-
coumarin (JLK-6), which were better elastase inhibitors compared
to the unsubstituted compound.45,49,55
noticed that compounds 30–33 shared a branched and bulky alkyl
chain. On the contrary, with the exception of compound 25
with an ethyl substituent, all other compounds with a linear (26,
27) or a small branched (28, 29) alkyl chain showed significant
Ab-lowering activity in the cell assay (Table 3). 7-Arylalkyl
derivatives were devoid of toxicity at the tested concentrations
but most of them did not show any significant activity, with the
exception of compounds 36 and 39 with a 4-nitrophenylmethyl
A series of acylated derivatives of JLK-6 was prepared (com-
pounds 13–17, Table 2). With exception of compound 17, all were
Compound 28
Compound 36
DMSO 10 µM 30 µM
DMSO 10 µM 30 µM
Figure 3. Inhibition of Ab production of bAPPswe expressing HEK293 cells by compounds 28 and 36. ns, Non significant.

M. Peuchmaur et al. / Bioorg. Med. Chem. 21 (2013) 1018–1029
1023
and 1-naphthalen-1-ylmethyl substituent, respectively. A limited
dose–response study was performed for the two most active com-
pounds, 28 and 36, which were shown to possess similar and
slightly higher potency compared to JLK-6, respectively (Fig. 3).
The significant Ab-lowering activity of several N-alkylated JLK-6
analogues and the cytotoxicity of some others were consistent
with an expected higher ability of these compounds to form an
irreversible complex with cellular serine proteases compared to
acylated and urethane analogues. Therefore, they represented the
most interesting series of JLK-6 analogues to further develop to
try to increase affinity.
was found less potent. This might be due to a lower cell penetra-
tion of the compound related to a lower hydrophobicity compared
to the pinanediol ester. An IC50 ranging between 10 and 30 lM was
measured for 47, indicating a lower potency than that reported in
Christie’s study for the free boronic acid. This discrepancy could
come from different cell type or assay conditions. A striking result
was that none of the pyrazine-containing compounds was found
active at 60 lM concentration. This included Bortezomib (com-
pound 46), a well-known proteasome inhibitor approved for the
treatment of multiple myeloma.60 In Christie’s study, Bortezomib
was found active but with a fivefold reduction in potency com-
pared to the phenyl-containing counterpart (the two compounds
have similar Ki values on proteasome). According to the low activ-
ity of 47 and 45 in our assay, a similar fivefold lower activity of
Bortezomib 46 (compared to 45) or its pinanediol ester 48 (com-
pared to 47) might not allow to observe any activity for the pyraz-
2.2. Peptide boronates
It was shown that small peptide-boronates efficiently block Ab
formation by APP-transfected IMR-32 neuronal cells through inhi-
bition of
c
-secretase cleavage.48 Known as proteasome inhibitors,
inyl-containing compounds at an only 60 lM concentration. At
it was first suggested that the multicatalytic complex proteasome
could be directly or indirectly responsible for Ab production. How-
ever, such activity has not been confirmed so far. Another hypoth-
esis is that these compounds act through an unknown target.
Peptide–boronates have also been described as potent and revers-
ible serine–protease inhibitors.56,57 For instance, it was recently re-
ported that Bortezomib belonging to this family efficiently inhibits
cathepsin G, chymase and, in particular a mitochondrial protease
involved in neuronal cell survival, HtrA2/Omi58 as do isocoumarins
[Ref. 45 and Sévalle J. et al. unpublished results]. That peptide–bor-
onates and isocoumarins share a common target could therefore be
envisioned.
To further characterize these compounds, we synthesized a ser-
ies of 8 peptide boronates (45–52) differing by the stereochemistry
of the amino-boronate residue, the nature of the N-terminal substi-
tuent (phenyl or pyrazine) and the boronate state (free boronic
acid or pinanediol ester) (Table 4). The peptide boronates were pre-
pared by the general method of Kettner and Shenvi57 and Matteson
et al.56 (Supplementary data, S2, Scheme 1S). All compounds were
least, these results indicated that the peptide boronates target in-
volved in Ab production was distinct from the proteasome and
would possess a high specificity at the level of the P3 substituent
(phenyl vs pyrazinyl). It is noteworthy that isocoumarin JLK-6 does
not inhibit proteasome.34 A second striking result was that the (S)
isomers of 45 and 47 (compounds 49 and 51, respectively) also
inhibited Ab production, although with lower potency compared
to the (R) isomers (IC50 of compound 51 between 30 and 60
Again, the boronic acid 49 was found less active than the pinanedi-
ol ester 51. The (S) stereochemistry corresponds to a -amino acid.
lM).
D
This observation was somewhat unexpected regarding ‘normal’
interaction of a ‘L’ P1 residue with a protease subsite. However,
as this residue is the C-terminal one, it is not impossible that the
differently oriented boronic moiety can still be accomodated by a
protease for covalent linking with the catalytic serine residue, al-
beit with a lower efficiency. Similar results were reported for close
inhibitors showing only three to fivefold differences in IC50 be-
tween the two isomers toward chymotrypsin and proteasome,
respectively.57,59
tested on bAPPswe overexpressing HEK293 cells at a 60
l
M con-
None of the peptide boronates was able to inhibit Ab production
from recombinant C100 substrate present in membrane prepara-
tions (Fig. 3S).61 This result indicates that like isocoumarins, the
peptide–boronates did not directly interact with the presenilin-
centration (Fig. 2S). As already noticed,57,59 the pinanediol esters
showed similar behaviours as their free boronic counterparts, con-
sistent with ester hydrolysis occurring in the culture medium lead-
ing to free boronic acids. The most active compound was the
pinanediol ester 47 with a benzoyl N-terminal residue and a (R)
stereochemistry. The corresponding boronic acid 45, which is the
most active compound in Christie’s study (IC50 of about 9 nM),48
dependent c-secretase complex.
Altogether, although there is still no direct proof, the similar
behaviour of isocoumarins and peptide–boronates observed
in our assays suggest they could share an identical target.
Table 4
Structure of compounds 45–52 and their effect on Ab production by transfected HEK293 cells
X
O
H
N
OR
X
N
B
H
O
OR
Compound
X
Stereo-chemistry
R
Acta
Compound
X
Stereo-chemistry
R
Acta
45
46
47b
48
CH
N
CH
N
R
R
R
R
H
H
+
49
50
51
52
CH
N
CH
N
S
S
S
S
H
H
+
ꢀ
ꢀ
Pinane-diol ester
Pinane-diol ester
+
Pinane-diol ester
Pinane-diol ester
+
ꢀ
ꢀ
a
Inhibitory activity of Ab production by HEK293 cells overexpressing swedish mutant bAPP. Compounds 45–52 have been tested at 60
inhibition >50% at the maximal concentration tested; ꢀ indicated inactive.
lM concentration. + indicated an
b
IC50 between 10 and 30 lM.

1024
M. Peuchmaur et al. / Bioorg. Med. Chem. 21 (2013) 1018–1029
Peptide–boronates are reversible serine protease inhibitors. They
might be modified with a reactive (photoreactive for instance)
moiety and used as probes to fish the target.
(1%/min) of 0.1% TFA/acetonitrile in 0.1% aqueous TFA over
30 min (flow rate: 28 mL/min). Mass spectrometry: samples were
prepared in acetonitrile/water (50/50 v/v) mixture. The LC/MS sys-
tem consisted of a Waters Alliance 2690 HPLC, coupled to a
Waters-Micromass ZQ spectrometer (electrospray ionization
mode, ESI+). All analyses were carried out using a RP C18 mono-
lithic Onyx Phenomenex column (25 ꢁ 4.6 mm) by means of a lin-
ear gradient (0–100%) of eluent B in A over 3 min (flow rate: 3 mL/
min). Eluent A: 0.1% aqueous formic acid; eluent B: acetonitrile/
0.1% formic acid. Positive ion electrospray mass spectra were ac-
3. Conclusions
According to the expected limited stability of the isocoumarins
in culture medium, an irreversible inactivation of the target should
occur in order to observe the inhibition of cellular Ab production
after 8-hours incubation. Supporting this irreversible inactivation
was the observation that 1 h treatment of APPswe overexpressing
quired at a solvent flow rate of 100–500 lL/min. Nitrogen was used
as both the nebulizing and drying gas. The data were obtained in a
scan mode in 0.1 s intervals; 10 scans were summed up to get the
final spectrum. HRMS were recorded on Q-TOF spectrophotometer
using ESI positive mode.
HEK293 cells with 60 lM JLK-6, followed by 7 h incubation in JLK-
6-free medium, resulted in Ab-lowering at a level close to that ob-
served after a 8 h standard treatment (Sévalle et al., unpublished).
These results suggested that the isocoumarins performed most of
their effect at the beginning of the assay and that this effect is
durable, supporting an irreversible target blockade. In the case of
peptide–boronates, which are reversible inhibitors, their activity
could account for a higher stability, which warrants their presence
in the medium for all duration of the assay. The most interesting
results were obtained with analogues of the 7-N-alkyl-isocoumarin
series, which showed Ab-lowering activity comparable to that of
JLK-6. These results together with the inactivity of the acyl and
urethane derivatives of JLK-6, except for compound 17, strongly
suggested the significance of the nitrogen electron pair mobility,
an essential feature for the irreversible inhibition of serine prote-
ases. These results therefore reinforced the idea that a serine pro-
tease, or maybe more generally a serine hydrolase, contributes to a
Compounds 1–6 were synthesized as previously described.50,55
Their physico-chemical characteristics are reported in the Supple-
mentary data.
4.1.1. General procedure for the preparation of 4H analogues of
JLK6 (7–12) from alkyl 2-carboxy-4-nitrophenylacetate
To a solution of alkyl 2-carboxy-4-nitrophenylacetate in anhy-
drous DCM was added 1.1 equiv of trifluoroacetic acid. After stir-
ring for 1 night at room temperature, the mixture was washed
with water and saturated aqueous NaHCO3. The organic phase
was then dried on Na2SO4, filtered and evaporated under vacuum.
The 3-alkoxy-7-nitro-isocoumarin was precipitated in propan-2-ol.
A solution of the preceding compound in THF was treated with
hydrogen in the presence of a catalytic amount of 10% Pd/C. After
5 h stirring at room temperature, the mixture was filtered through
celite and the celite was washed with THF. The filtrate was evapo-
rated to yield the 3-alkoxy-7-amino-isocoumarins.
metabolic pathway leading to APP c-secretase cleavage.
Although a few N-alkylated analogues showed slightly higher
potency than JLK-6, this was not sufficient to design efficient
molecular probes based on these compounds. We nevertheless
synthesized two biotinylated probes of JLK-6, one with a bioti-
nyl–acyl moiety46 and the other with a biotinyl–aminoalkyl moiety
(Supplementary Data, S4; Scheme 2S). The first one was inactive in
the cell assay as expected and it was also the case for the second
one, indicating that there are still efforts to elaborate isocouma-
rin-derived efficient probes. The development of isocoumarins
substituted at unexplored positions is now considered.
4.1.1.1. 3-Methoxy-7-nitro-isocoumarin [3-methoxy-7-nitro-1H-
2-benzopyran-1-one] (7)39,45
.
Pale yellow solid. Yield, 68% from
500 mg (2.1 mmol) of methyl 2-carboxy-4-nitrobenzeneacetate.50
HPLC tR = 2.24 min. 1H NMR (300 MHz, DMSO-d6):
8.67 (d,
d
J = 2.3 Hz, 1H), 8.47 (dd, J = 8.8, 2.3 Hz, 1H), 7.71 (d, J = 8.8 Hz, 1H),
6.23 (s, 1H), 4.01 (s, 3H). 13C NMR (75 MHz, DMSO-d6): d 163.2,
160.2, 146.8, 145.3, 130.3, 127.5, 125.9, 117.7, 80.1, 58.2. m/z (ES+)
222.1 (M+H+). HRMS, calcd for C10H8NO5 (M+H+) 222.0402, found
222.0408.
4. Experimental
4.1. Chemistry
4.1.1.2. 7-Amino-3-methoxy-isocoumarin [7-amino-3-methoxy-
1H-2-benzopyran-1-one] (8)39,45
.
Pale yellow solid. Yield, 50%
from 7 after HPLC purification. HPLC tR = 1.09 min. 1H NMR
(300 MHz, DMSO-d6): 7.38 (d, J = 2.2 Hz, 1H), 7.32 (d,
Reagents and solvents were obtained from Aldrich, Acros
Organics or Alfa Aesar and used without further purification.
1H and 13C NMR spectra were recorded on a 300 MHz Bruker
instrument using CDCl3 or DMSO-d6 solutions. Splitting patterns
of 1H spectra are designated as follows: s, singlet; d, doublet; t,
triplet; q, quartet; m, multiplet; br, broad. Chemical shifts are re-
ported in ppm relative to solvant signal (CDCl3: d = 7.26 ppm
(1H); 77.16 (13C); DMSO-d6: d = 2.50 ppm (1H); 39.52 (13C)).
Thin-layer chromatographies were performed on aluminium
backed sheets of silica gel F254 (0.2 mm, Fluka), and were visualised
under 254 nm light and by spraying with a 2% EtOH solution of
ninhydrin followed by heating, or by charring with an aqueous
solution of ammonium sulfate and sulfuric acid [200 g (NH4)2SO4
and 40 mL concentrated sulfuric acid in 1 L of water]. Column chro-
matography separations were performed using Merck silica gel 60
d
J = 8.4 Hz, 1H), 7.19 (dd, J = 8.4, 2.2 Hz, 1H), 5.83 (s, 1H), 3.83 (s,
3H). 13C NMR (75 MHz, DMSO-d6): d 160.5, 157.6, 143.0, 131.1,
126.1, 125.0, 117.7, 113.3, 78.4, 56.2. m/z (ES+) 192.1 (M+H+).
HRMS, calcd for C10H10NO3 (M+H+) 192.0661, found 192.0677.
4.1.1.3. 3-(2-Methoxyethoxy)-7-nitro-isocoumarin [3-(2-methoxy
ethoxy)-7-nitro-1H-2-benzopyran-1-one] (9).
Yellow solid.
Yield, 28% from 300 mg (1.06 mmol) of 2-methoxyethyl 2-car-
boxy-4-nitrobenzeneacetate.35 HPLC tR = 2.30 min. 1H NMR
(300 MHz, DMSO-d6): d 8.73 (d, J = 2.2 Hz, 1H), 8.51 (dd, J = 8.8,
2.2 Hz, 1H), 7.73 (d, J = 8.8 Hz, 1H), 6.28 (s, 1H), 4.40 (t, J = 4.3 Hz,
2H), 3.75 (t, J = 4.3 Hz, 2H), 3.37 (s, 3H). 13C NMR (75 MHz,
DMSO-d6): d 161.9, 160.0, 146.5, 145.1, 130.1, 127.3, 125.7,
117.5, 80.7, 70.2, 70.1, 59.1. m/z (ES+) 266.1 (M+H+).
of particle size 40–63 lm. Reverse phase HPLC analysis was per-
formed on a Chromolith SpeedRod C18 column (0.46 ꢁ 5 cm) by
means of a linear gradient (0–100%) of acetonitrile in 0.1% aqueous
TFA over 5 min (flow rate: 3 mL/min). Preparative reverse phase
HPLC purification was performed on a Waters Delta Pak C18 col-
4.1.1.4. 7-Amino-3-(2-methoxyethoxy)-isocoumarin [7-amino-
3-(2-methoxyethoxy)-1H-2-benzopyran-1-one] (10).
Yellow
solid. Yield, 70% from 9 after HPLC purification. HPLC tR = 1.24 min.
1H NMR (300 MHz, DMSO-d6): d 7.36 (d, J = 1.8 Hz, 1H), 7.32 (d,
umn (40 ꢁ 100 mm, 15
lm, 100 Å) by means of a linear gradient

M. Peuchmaur et al. / Bioorg. Med. Chem. 21 (2013) 1018–1029
1025
J = 8.4 Hz, 1H), 7.18 (dd, J = 8.4, 1.8 Hz, 1H), 5.88 (s, 1H), 4.22 (t, J = 4.3,
2H), 3.70 (t, J = 4.3 Hz, 2H), 3.35 (s, 3H). 13C NMR (75 MHz, DMSO-d6):
d 161.5, 157.1, 145.4, 134.1, 126.9, 125.3, 118.7, 113.1, 80.5, 70.6,
69.2, 59.1. m/z (ES+) 236.1 (M+H+).
CF3). m/z (ES+) 372.0 and 374.0 (M+H+). HRMS, calcd for
13H8ClF5NO4 (M+H+) 372.0062, found 372.0066.
C
4.1.2.4.
[N-(4-chloro-3-methoxy-1-oxo-1H-2-benzopyran-7-yl)-octana-
mide] (16). Obtained from octanoyl chloride. Pale yellowsolid.
7-(Octanoylamino)-4-chloro-3-methoxyisocoumarin
4.1.1.5. 3-(2-Bromoethoxy)-7-nitro-isocoumarin [3-(2-bromo-
ethoxy)-7-nitro-1H-2-benzopyran-1-one] (11)44,62. Yellow solid.
Yield, 45% from 200 mg (0.6 mmol) of 2-bromoethyl 2-carboxy-4-
nitrobenzeneacetate.35,62 HPLC tR = 2.71 min. 1H NMR (300 MHz,
DMSO-d6): d 8.60 (d, J = 2.4 Hz, 1H), 8.44 (dd, J = 8.8, 2.4 Hz, 1H),
7.66 (d, J = 8.8 Hz, 1H), 6.26 (s, 1H), 4.54 (t, J = 5.3 Hz, 2H), 3.84 (t,
J = 5.3 Hz, 2H). 13C NMR (75 MHz, DMSO-d6): d 160.8, 159.4,
145.8, 144.8, 129.7, 126.9, 125.2, 117.3, 80.7, 69.9, 30.7. m/z (ES+)
314.0 and 316.0 (M+H+).
Yield, 35%. mp 148 °C. HPLC tR = 3.46 min. 1H NMR (300 MHz,
DMSO-d6): d 8.50 (d, J = 2.1 Hz, 1H), 8.05 (dd, J = 8.7, 2.2 Hz, 1H),
7.60 (d, J = 8.7 Hz, 1H), 3.95 (s, 3H), 2.14 (m, 2H), 1.44 (m, 2H), 1.21
(m, 8H), 0.82 (m, 3H). 13C NMR (75 MHz, DMSO-d6): d 171.8, 158.7,
152.6, 137.9, 132.1, 127.2, 122.5, 118.1, 117.4, 89.6, 57.4, 36.5,
31.2, 28.6, 28.5, 25.0, 22.1, 13.9. m/z (ES+) 352.0 and 354.0 (M+H+).
HRMS, calcd for C18H23ClNO4 (M+H+) 352.1316, found 352.1328.
4.1.2.5. 7-(Pivaloylamino)-4-chloro-3-methoxyisocoumarin [N-
(4-chloro-3-methoxy-1-oxo-1H-2-benzopyran-7-yl)-pivala-
4.1.1.6. 7-Amino-3-(2-bromoethoxy)-isocoumarin [7-amino-3-(2-
bromoethoxy)-1H-2-benzopyran-1-one] (12)44,62
.
Yellow solid.
mide] (17).
Obtained from pivaloyl chloride. Pale yellow solid.
Yield, 63% from 11 after HPLC purification. HPLC tR = 1.55 min. 1H
NMR (300 MHz, DMSO-d6): d 7.28–7.36 (m, 2H), 7.17 (dd, J = 8.4,
2.1 Hz, 1H), 5.94 (s, 1H), 4.45 (t, J = 5.5, 2H), 3.87 (t, J = 5.5 Hz, 2H).
13C NMR (75 MHz, DMSO-d6): d 162.5, 155.7, 145.7, 129.9, 126.5,
124.6, 118.4, 112.2, 80.8, 69.3, 30.8. m/z (ES+) 284.0 and 286.0 (M+H+).
Yield, 65%. mp 187 °C. HPLC tR = 2.80 min. 1H NMR (300 MHz, DMSO-
d6): d 9.60 (s, 1H), 8.50 (d, J = 2.0 Hz, 1H), 8.15 (dd, J = 8.7, 2.0 Hz, 1H),
7.60 (d, J = 8.7 Hz, 1H), 4.05 (s, 3H), 1.25 (s, 9H). 13C NMR (75 MHz,
DMSO-d6): d 177.4, 159.2, 153.1, 138.4, 137.1, 132.6, 128.7, 122.6,
119.8, 117.6, 89.8, 57.7, 27.5. m/z (ES+) 310.0 and 312.0 (M+H+).
HRMS, calcd for C15H17ClNO4 (M+H+) 310.0846, found 310.0832.
4.1.2. General procedure for the preparation of N-acyl analogues
of JLK-6 (13–17)
4.1.3. General procedure for the preparation of carbamoyl
analogues of JLK6 (18–24)
Amides 13–17 were obtained by acylation of JLK-6 with the
appropriate acid chlorides or anhydrides. To a solution of JLK-6
(100 mg, 0.44 mmol) in DCM (10 mL) placed at 0 °C were added
dropwise 5 equiv of acid chloride or anhydride in DCM solution
(5 mL). After 2 h stirring at room temperature, the solution was di-
luted in DCM. The organic phase was washed with a 5% aqueous
NaHCO3 solution and brine, and dried over Na2SO4, filtered and
evaporated to dryness. The final pure isocoumarin was crystallized
from petroleum ether.
Carbamates 18–24 were obtained by acylation of JLK-6 with the
appropriate halogenoformates or anhydrides. In a typical experi-
ment, 50 mg (0.22 mmol) of JLK-6 was suspended in dry THF
(1 mL). After cooling at 0 °C, NaHCO3 (41 mg, 0.49 mmol) was
added, followed by R-CO2X (X = Cl, F) or (R-CO)2O (1.1 equiv,
0.24 mmol). After warming to room temperature, the reaction mix-
ture was stirred until disappearance of the starting product (3–
48 h). The reaction was then quenched with water and extracted
with AcOEt. Organic phases were finally dried on Na2SO4, filtered
and evaporated under vacuum.
4.1.2.1. 7-(Acetylamino)-4-chloro-3-methoxyisocoumarin [N-
(4-chloro-3-methoxy-1-oxo-1H-2-benzopyran-7-yl)-acetamide]
(13)49,50
.
Obtained from acetic anhydride. Yellow solid. Yield,
4.1.3.1. 7-[((Benzyloxy)carbonyl)amino]-4-chloro-3-methoxyiso-
85%. mp 220 °C (Lit. >210 °C). HPLC tR = 2.24 min. 1H NMR (300 MHz,
DMSO-d6): d 8.50 (d, J = 1.8 Hz, 1H), 8.00 (dd, J = 8.7, 2.1 Hz, 1H), 7.6
(d, J = 8.7 Hz, 1H), 4.00 (s, 3H), 2.05 (s, 3H). 13C NMR (75 MHz,
DMSO-d6): d 169.1, 159.1, 153.0, 138.2, 132.4, 127.5, 122.8, 117.7,
117.2, 57.7, 30.8, 17.5. m/z (ES+) 268.0 and 270.0 (M+H+). HRMS, calcd
for C12H10ClNO4 (M+H+) 268.0377, found 268.0374.
coumarin [phenylmethyl-(4-chloro-3-methoxy-1-oxo-1H-2-benz-
opyran-7-yl)-carbamate]
(18)49
.
Obtained
from
benzyl
chloroformate (42 mg, 35
lL). Yellow solid. Yield, 51%. HPLC
tR = 3.24 min. 1H NMR (300 MHz, DMSO-d6): d 10.18 (s, 1H), 8.28
(s, 1H), 7.87 (dd, J = 8.7, 1.2 Hz, 1H), 7.59 (d, J = 8.7 Hz, 1H), 7.31–
7.42 (m, 5H), 5.15 (s, 2H), 3.97 (s, 3H). 13C NMR (75 MHz, DMSO-
d6): d 159.1, 158.9, 153.8, 138.2, 136.7, 132.0, 128.9, 128.6, 128.5,
126.9, 123.1, 118.0, 117.4, 92.9, 66.5, 57.8. m/z (ES+) 360.1 and
362.1 (M+H+). HRMS, calcd for C18H15ClNO5 (M+H+) 360.0639
and 362.0609, found 360.0645 and 362.0650.
4.1.2.2. 7-(4-Carboxybutyramido)-4-chloro-3-methoxyisocoum-
arin [5-[(4-chloro-3-methoxy-1-oxo-1H-2-benzopyran-7-yl)-
amino]-5-oxo-pentanoic acid] (14)49
. Obtained from glutaric
anhydride. Yellow solid. Yield, 40%. mp 194 °C (Lit. 194 °C).
HPLC tR = 2.25 min. 1H NMR (300 MHz, DMSO-d6): d 8.50 (d,
J = 1.8 Hz, 1H), 8.00 (dd, J = 8.7, 2.0 Hz, 1H), 7.60 (d, J = 8.7 Hz,
1H), 3.95 (s, 3H), 2.72 (m, 2H), 2.41 (m, 1H), 2.27 (m, 1H), 1.82
(m, 2H). 13C NMR (75 MHz, DMSO-d6): d 174.6, 174.6, 159.2,
153.1, 138.2, 132.5, 127.7, 122.9, 118.6, 117.8, 89.9, 57.8, 35.8,
33.2, 20.8. m/z (ES+) 340.0 and 342.0 (M+H+). HRMS, calcd for
4.1.3.2. 7-[((Adamantyloxy)carbonyl)amino]-4-chloro-3-methoxy-
isocoumarin [adamantyl-(4-chloro-3-methoxy-1-oxo-1H-2-benz-
opyran-7-yl)-carbamate] (19).
Obtained from adamantyl
fluoroformate (48 mg). Yellow solid. Yield, 76%. HPLC tR = 3.75 min.
1H NMR (300 MHz, DMSO-d6): d 9.81 (s, 1H), 8.29 (s, 1H), 7.84 (d,
J = 9.0 Hz, 1H), 7.54 (d, J = 8.7 Hz, 1H), 3.96 (s, 3H), 1.52–2.10 (m,
15H). 13C NMR (75 MHz, DMSO-d6): d 159.1, 152.8, 152.6, 138.6,
131.7, 126.9, 122.9, 117.9, 117.3, 90.0, 79.9, 57.8, 45.7, 41.6, 40.1,
36.3, 36.0, 35.5, 31.1, 30.8, 30.5. m/z (ES+) 404.2 and 406.2
(M+H+). HRMS, calcd for C21H23ClNO5 (M+H+) 404.1265 and
406.1235, found 404.1268 and 406.1257.
C
15H15ClNO6 (M+H+) 340.0588, found 340.0596.
4.1.2.3. 7-(Pentafluoropropionylamino)-4-chloro-3-methoxyiso-
coumarin [N-(4-chloro-3-methoxy-1-oxo-1H-2-benzopyran-7-
yl)-2,2,3,3-pentafluoro-propionamide] (15). Obtained from
pentafluoropropionic anhydride. Orange powder. Yield, 47%. mp
184 °C. HPLC tR = 3.06 min. 1H NMR (300 MHz, DMSO-d6): d 8.45
(d, J = 1.8 Hz, 1H), 8.15 (dd, J = 8.7, 2.0 Hz, 1H), 7.65 (d, J = 8.8 Hz,
1H), 4.05 (s, 3H). 13C NMR (75 MHz, DMSO-d6): d 158.8, 155.9,
153.9, 135.1, 134.8, 132.8, 129.4, 123.1, 121.3, 117.7, 106.5, 89.3,
57.7; 19F NMR (not decoupled, DMSO-d6): 122 (s, CF2), 82 (s,
4.1.3.3. 7-[((9-Fluorenylmethoxy)carbonyl)amino]-4-chloro-3-meth-
oxyisocoumarin [9-fluorenylmethyl-(4-chloro-3-methoxy-1-oxo-1H-
2-benzopyran-7-yl)-carbamate]
(20)49
.
Obtained
from
9-fluorenylmethyl chloroformate (62 mg), the solid was washed
with MeCN. Pale yellow solid. Yield, 30%. HPLC tR = 3.67 min. 1H

1026
M. Peuchmaur et al. / Bioorg. Med. Chem. 21 (2013) 1018–1029
NMR (300 MHz, DMSO-d6): d 10.09 (s, 1H), 8.27 (s, 1H), 7.87 (m,
3H), 7.71 (d, J = 6.9 Hz, 1H), 7.56 (d, J = 8.1 Hz, 1H), 7.29–7.41 (m,
4H), 4.50 (d, J = 5.4 Hz, 2H), 4.29 (t, J = 5.7 Hz, 1H), 3.96 (s, 3H).
13C NMR (75 MHz, DMSO-d6): d 159.0, 153.8, 152.9, 144.1, 141.2,
138.1, 132.1, 128.1, 127.5, 127.0, 125.5, 123.0, 120.6, 118.0,
117.6, 89.9, 66.2, 57.8, 47.0. m/z (ES+) 448.2 and 450.2 (M+H+).
HRMS, calcd for C25H19ClNO5 (M+H+) 448.0952 and 450.0922,
found 448.0958 and 450.0801.
vacuo. When possible, the product was precipitated by addition
of H2O/CH3CN (2:1). The yellow precipitate was filtered, washed
with H2O/CH3CN (1:1) and dried. As an alternative, the product
was purified by preparative reverse phase HPLC.
4.1.4.1. 4-Chloro-7-(ethylamino)-3-methoxy-isocoumarin [4-
chloro-7-(ethylamino)-3-methoxy-1H-2-benzopyran-1-one]
(25). Obtained from acetaldehyde (23 mg) and purified by re-
verse phase HPLC. Yellow solid. Yield, 9%. Rf (Hex/EtOAc, 4:1)
0.36. HPLC tR = 2.15 min. 1H NMR (300 MHz, DMSO-d6): d 7.51 (d,
J = 8.7 Hz, 1H), 7.27 (dd, J = 8.7, 2.5 Hz, 1H), 7.20 (d, J = 2.4 Hz,
1H), 4.00 (s, 3H), 3.15 (q, J = 7.1 Hz, 2H), 1.23 (t, J = 7.1 Hz, 3H).
13C NMR (75 MHz, DMSO-d6): d 160.2, 151.5, 148.6, 126.5, 123.9,
123.3, 119.4, 109.2, 92.0, 58.6, 38.4, 14.8. m/z (ES+) 254.1 and
256.1 (M+H+). HRMS, calcd for C12H13ClNO3 (M+H+) 254.0584
and 256.0554, found 254.0597 and 256.0580.
4.1.3.4. 7-[((Ethyloxy)carbonyl)amino]-4-chloro-3-methoxyiso-
coumarin [ethyl-(4-chloro-3-methoxy-1-oxo-1H-2-benzopyran-
7-yl)-carbamate] (21)49
.
Obtained from ethyl chloroformate
(26 mg, 23
l
L). Yellow solid. Yield, 54%. HPLC tR = 2.77 min. 1H
NMR (300 MHz, DMSO-d6): d 10.02 (s, 1H), 8.27 (s, 1H), 7.87 (dd,
J = 8.7, 1.2 Hz, 1H), 7.58 (d, J = 8.7 Hz, 1H), 4.12 (q, J = 6.9 Hz, 2H),
3.97 (s, 3H), 1.23 (t, J = 6.9 Hz, 3H). 13C NMR (75 MHz, DMSO-d6): d
159.1, 153.9, 152.8, 138.4, 131.9, 126.9, 123.0, 118.0, 117.4, 89.9,
61.0, 57.8, 14.9. m/z (ES+) 298.1 and 300.1 (M+H+). HRMS, calcd for
C13H13ClNO5 (M+H+) 298.0482 and 300.0453, found 298.0490 and
300.0463.
4.1.4.2. 4-Chloro-7-(hexylamino)-3-methoxy-isocoumarin [4-
chloro-7-(hexylamino)-3-methoxy-1H-2-benzopyran-1-one]
(26).
Obtained from hexanal (53 mg). Yellow solid. Yield, 9%.
HPLC tR = 3.43 min. 1H NMR (300 MHz, DMSO-d6): d 7.46 (d,
J = 8.5 Hz, 1H), 7.20 (dd, J = 8.5, 1.6 Hz, 1H), 7.13 (d, J = 1.6 Hz,
1H), 3.96 (s, 3H), 3.06 (t, J = 6.7 Hz, 2H), 1.50–1.62 (m, 2H), 1.19–
1.44 (m, 6H), 0.87 (t, J = 5.3 Hz, 3H). 13C NMR (75 MHz, DMSO-
d6): d 160.2, 151.4, 149.1, 126.0, 123.9, 123.0, 119.5, 108.7, 92.0,
58.6, 43.6, 32.0, 29.2, 27.2, 23.0, 14.8. m/z (ES+) 310.1 and 312.1
(M+H+). HRMS, calcd for C16H21ClNO3 (M+H+) 310.1210 and
312.1180, found 310.1181 and 312.1205.
4.1.3.5. 7-[((tButyloxy)carbonyl)amino]-4-chloro-3-methoxyiso-
coumarin [tbutyl-(4-chloro-3-methoxy-1-oxo-1H-2-benzopyran-
7-yl)-carbamate] (22).
Obtained from di-tertbutyldicarbonate
(52 mg). Yellow–orange solid. Yield, 67%. HPLC tR = 3.12 min. 1H
NMR (300 MHz, DMSO-d6): d 9.77 (s, 1H), 8.30 (s, 1H), 7.84 (dd,
J = 8.7, 0.6 Hz, 1H), 7.55 (d, J = 8.7 Hz, 1H), 3.96 (s, 3H), 1.45 (s,
9H). 13C NMR (75 MHz, DMSO-d6): d 159.1, 153.1, 152.8, 138.7,
131.7, 126.8, 122.9, 117.9, 117.2, 90.0, 80.2, 57.8, 28.5. m/z (ES+)
326.1 and 328.1 (M+H+). HRMS, calcd for C15H17ClNO5 (M+H+)
326.0795 and 328.0766, found 326.0789 and 328.0782.
4.1.4.3. 4-Chloro-3-methoxy-7-(nonylamino)-isocoumarin [4-
chloro-3-methoxy-7-(nonylamino)-1H-2-benzopyran-1-one]
(27).
Obtained from nonanal (76 mg) and purified by reverse
4.1.3.6. 7-[((Trichloroethoxy)carbonyl)amino]-4-chloro-3-methoxy-
isocoumarin [trichloroethyl-(4-chloro-3-methoxy-1-oxo-1H-2-
benzopyran-7-yl)-carbamate] (23). Obtained from trichloroethyl
phase HPLC. Yellow solid. Yield, 13%. HPLC tR = 4.70 min. 1H NMR
(300 MHz, DMSO-d6): d 7.41 (d, J = 8.7 Hz, 1H), 7.18 (dd, J = 8.7,
2.4 Hz, 1H), 7.09 (d, J = 2.4 Hz, 1H), 3.92 (s, 3H), 3.01 (m, 2H),
1.51 (m, 2H), 1.21 (m, 12H), 0.81 (m, 3H). 13C NMR (75 MHz,
DMSO-d6): d 159.7, 155.0, 148.5, 125.6, 123.4, 122.5, 119.0,
108.3, 91.7, 58.1, 43.2, 31.7, 29.4, 29.3, 29.1, 28.7, 27.0, 22.5,
14.3. m/z (ES+) 352.1 and 354.1 (M+H+). HRMS, calcd for
chloroformate (153 lL). Yellow solid. Yield, 85%. mp 203 °C. HPLC
tR = 3.30 min. 1H NMR (300 MHz, DMSO-d6): d 8.35 (d, J = 1.8 Hz,
1H), 7.95 (dd, J = 8.8, 2.1 Hz, 1H), 7.65 (d, J = 8.8 Hz, 1H), 5.00 (s,
2H), 4.05 (s, 3H). 13C NMR (75 MHz, DMSO-d6): d 159.0, 153.1,
152.2, 137.4, 132.6, 128.9, 125.3, 123.1, 118.0, 96.1, 74.0, 57.7,
29.4. m/z (ES+) 400.0, 402.0 and 404.0 (M+H+).
C
19H27ClNO3 (M+H+) 352.1679 and 354.1650, found 352.1689
and 354.1671.
4.1.3.7. 7-[((Allyloxy)carbonyl)amino]-4-chloro-3-methoxyiso-
coumarin [allyl-(4-chloro-3-methoxy-1-oxo-1H-2-benzopyran-
4.1.4.4. 4-Chloro-7-(isobutylamino)-3-methoxy-isocoumarin [4-
chloro-7-(isobutylamino)-3-methoxy-1H-2-benzopyran-1-one]
(28). Obtained from isobutyraldehyde (19 mg) and purified by
reverse phase HPLC. Yellow solid. Yield, 11%. HPLC tR = 2.97 min.
1H NMR (300 MHz, DMSO-d6): d 7.41 (d, J = 8.7 Hz, 1H), 7.20 (dd,
J = 8.7, 2.4 Hz, 1H), 7.10 (d, J = 2.1 Hz, 1H), 3.92 (s, 3H), 2.86 (d,
J = 6.6 Hz, 2H), 1.83 (sept., J = 6.3 Hz, 1H), 0.91 (d, J = 6.6 Hz, 6H).
13C NMR (75 MHz, DMSO-d6): d 159.7, 150.9, 148.8, 125.5, 123.9,
122.4, 119.0, 108.3, 91.6, 58.2, 27.7, 20.8. m/z (ES+) 282.1 and
284.1 (M+H+). HRMS, calcd for C14H17ClNO3 (M+H+) 282.0897
and 284.0867, found 282.0897 and 284.0878.
7-yl)-carbamate] (24).
Obtained from allyl chloroformate
(29 mg, 25
l
L). Yellow solid. Yield, 60%. HPLC tR = 2.89 min. 1H
NMR (300 MHz, DMSO-d6): d 10.14 (s, 1H), 8.27 (s, 1H), 7.88 (dd,
J = 8.7, 0.6 Hz, 1H), 7.59 (d, J = 8.7 Hz, 1H), 5.95 (m, 1H), 5.35 (d,
J = 17.7 Hz, 1H), 5.21 (d, J = 10.2 Hz, 1H), 4.60 (d, J = 4.8 Hz, 2H),
3.97 (s, 3H). 13C NMR (75 MHz, DMSO-d6): d 159.1, 153.6, 152.9,
138.2, 133.5, 132.1, 126.9, 123.1, 118.3, 118.0, 117.4, 89.9, 65.4,
57.8. m/z (ES+) 310.1 and 312.1 (M+H+). HRMS, calcd for
C
14H13ClNO5 (M+H+) 310.0482 and 312.0453, found 310.0496
and 312.0516.
4.1.4.5. 4-Chloro-3-methoxy-7-[(3-methyl-butyl)amino]-iso-
4.1.4. General procedure for the preparation of N-alkyl
analogues of JLK6 (25–42)
coumarin [4-chloro-3-methoxy-7-[(3-methyl-butyl)amino]-1H-
2-benzopyran-1-one] (29).
Obtained from isovaleraldehyde
The N-alkyl analogues of JLK-6 (25–42) were obtained by reduc-
tive amination of JLK-6 with the appropriate aldehydes. In a typical
experiment, 100 mg (0.44 mmol) of JLK-6 and the aldehyde
(1.2 equiv, 0.53 mmol) were solubilized in dry DCM (3 mL) and
the reaction mixture was stirred at room temperature for 15 min.
Then, NaBH3CN (2 equiv, 55 mg, 0.88 mmol) was added and stir-
ring was continued for 16 h. After addition of a saturated aqueous
NaHCO3 solution, the mixture was extracted with DCM, and the
organic layer was dried over MgSO4, filtered and concentrated in
(46 mg) and purified by reverse phase HPLC. Yellow solid. Yield,
20%. mp 52 °C. Rf (Hex/EtOAc, 4:1) 0.63. HPLC tR = 3.21 min. 1H
NMR (300 MHz, DMSO-d6): d 7.51 (d, J = 8.7 Hz, 1H), 7.22–7.32
(m, 2H), 4.01 (s, 3H), 3.14 (t, J = 7.2 Hz, 2H), 1.68–1.84 (m, 1H),
1.52 (dd, J = 7.2, 7.2 Hz, 2H), 0.96 (d, J = 6.6 Hz, 6H). 13C NMR
(75 MHz, DMSO-d6): d 160.1, 151.6, 148.2, 126.8, 123.9, 123.5,
119.4, 109.7, 91.9, 58.6, 42.3, 38.0, 26.2, 23.3 (2C). m/z (ES+)
296.1 and 298.1 (M+H+). HRMS, calcd for C15H19ClNO3 (M+H+)
296.1053, found 296.1046.

M. Peuchmaur et al. / Bioorg. Med. Chem. 21 (2013) 1018–1029
1027
4.1.4.6. 4-Chloro-7-[(2-ethyl-hexyl)amino]-3-methoxy-isocouma-
rin [4-chloro-7-[(2-ethyl-hexyl)amino]-3-methoxy-1H-2-benzopy-
ran-1-one] (30). Obtained from 2-ethylhexanal (68 mg) and purified
by flash column chromatography on silica gel (eluent hexane to hex-
ane/EtOAc (90:10). Yellow solid. Yield, 7%. HPLC tR = 4.81 min. 1H
NMR (300 MHz, DMSO-d6): d 7.41 (d, J = 8.7 Hz, 1H), 7.19 (dd, J = 8.7,
2.4 Hz, 1H), 7.11 (d, J = 2.4 Hz, 1H), 3.92 (s, 3H), 3.26 (s, 1H), 2.94 (m,
2H), 1.51 (m, 1H), 1.25–1.41 (m, 8H), 0.83 (m, 6H). 13C NMR
(75 MHz, DMSO-d6): d 159.7, 150.8, 148.8, 125.4, 123.4, 122.5, 119.0,
108.1, 90.0, 58.2, 46.4, 38.4, 31.0, 28.8, 24.2, 23.0, 14.4, 11.1. m/z
(ES+) 338.2 and 340.2 (M+H+). HRMS, calcd for C18H25ClNO3 (M+H+)
338.1523 and 340.1493, found 338.1515 and 340.1508.
solid with H2O/MeCN (50:50) and cold diethylether. Yellow solid.
Yield, 52%. mp 141–142 °C. Rf (Hex/EtOAc, 4:1) 0.35. HPLC
tR = 3.12 min. 1H NMR (300 MHz, DMSO-d6): d 7.47 (d, J = 8.9 Hz,
1H), 7.33 (d, J = 8.4 Hz, 2H), 7.28 (dd, J = 8.9, 2.1 Hz, 1H), 7.20 (d,
J = 2.1 Hz, 1H), 6.93 (d, J = 8.4 Hz, 2H), 4.32 (d, J = 5.3 Hz, 2H),
3.99 (s, 3H), 3.76 (s, 3H). 13C NMR (75 MHz, DMSO-d6): d 160.1,
159.2, 151.5, 148.7, 131.8, 129.3 (2C), 126.5, 123.8, 123.3, 119.3,
114.7 (2C), 109.5, 91.9, 58.6, 55.9, 46.6. m/z (ES+) 346.1 and
348.1 (M+H+). HRMS, calcd for C18H17ClNO4 (M+H+) 346.0846
and 348.0817, found 346.0850 and 348.0851.
4.1.4.12. 4-Chloro-3-methoxy-7-[(4-nitrophenylmethyl)amino]-
isocoumarin
[4-chloro-3-methoxy-7-[(4-nitrophenylmethyl)
4.1.4.7. 4-Chloro-7-[(2,2-dimethyl-propyl)amino]-3-methoxy-iso-
coumarin [4-chloro-7-[(2,2-dimethyl-propyl)amino]-3-methoxy-
1H-2-benzopyran-1-one] (31). Obtained from 2,2-dimethylpropanal
(46 mg) and purified by preparative HPLC. Yellow solid. Yield, 34%. mp
141 °C. Rf (Hex/EtOAc, 4:1) 0.74. HPLC tR = 3.44 min. 1H NMR
(300 MHz, DMSO-d6): d 7.48 (d, J = 8.7 Hz, 1H), 7.36 (d, J = 8.7 Hz,
1H), 7.27 (s, 1H), 4.00 (s, 3H), 2.94 (s, 2H), 1.01 (s, 9H). 13C NMR
(75 MHz, DMSO-d6): d 160.2, 151.3, 149.8, 125.8, 123.8, 122.9, 119.4,
108.8, 92.1, 58.6, 55.4, 33.1, 28.4 (3C). m/z (ES+) 296.1 and 298.1
(M+H+). HRMS, calcd for C15H19ClNO3 (M+H+) 296.1053 and
298.1024, found 296.1048 and 298.1037.
amino]-1H-2-benzopyran-1-one] (36). Obtained from 4-nitro-
benzaldehyde (80 mg) and purified by washing the solid with
H2O/MeCN (50:50) and cold diethylether. Yellow solid. Yield,
30%. Rf (Hex/EtOAc, 4:1) 0.12. HPLC tR = 3.05 min. 1H NMR
(300 MHz, DMSO-d6): d 8.16 (d, J = 8.1 Hz, 2H), 7.58 (d, J = 8.1 Hz,
2H), 7.41 (d, J = 8.7 Hz, 1H), 7.21 (d, J = 8.7 Hz, 1H), 7.08 (s, 1H),
4.50 (d, J = 5.4 Hz, 2H), 3.90 (s, 3H). 13C NMR (75 MHz, DMSO-
d6): d 159.5, 159.0, 148.3, 147.7, 147.0, 128.5, 126.5, 124.0, 123.5,
122.9, 118.9, 109.2, 91.3, 58.1, 46.1. m/z (ES+) 361.0 and 363.0
(M+H+). HRMS, calcd for C17H14ClN2O5 (M+H+) 361.0591 and
363.0562, found 361.0604 and 363.0592.
4.1.4.8. 4-Chloro-7-[(2,2-dimethylbutyl)amino)]-3-methoxy-iso-
coumarin [4-chloro-7-[(2,2-dimethylbutyl)amino]-3-methoxy-
1H-2-benzopyran-1-one] (32). Obtained from 2,2-dimethylbutanal
(53 mg) and purified by reverse phase HPLC. Yellow solid. Yield, 16%.
4.1.4.13. 4-Chloro-3-methoxy-7-[(2-nitrophenylmethyl)amino]-
isocoumarin [4-chloro-3-methoxy-7-[(2-nitrophenylmethyl)
amino]-1H-2-benzopyran-1-one] (37).
Obtained from 2-nitro-
benzaldehyde (80 mg) and purified by washing the solid with H2O/
MeCN (50:50) and cold diethylether. Yellow solid. Yield, 29%. Rf
(Hex/EtOAc, 4:1) 0.2. HPLC tR = 3.05 min. 1H NMR (300 MHz,
DMSO-d6): d 8.04 (d, J = 8.1 Hz, 1H), 7.65 (m, 1H), 7.54 (m, 1H),
7.42 (d, J = 8.7 Hz, 1H), 7.20 (dd, J = 8.7, 2.1, 1H), 7.10 (d,
J = 1.8 Hz, 1H), 6.94 (m, 1H), 4.67 (d, J = 6.0 Hz, 2H), 3.91 (s, 3H).
13C NMR (75 MHz, DMSO-d6): d 159.5, 148.7, 147.8, 134.9, 134.2,
132.3, 129.6, 128.8, 126.6, 125.4, 123.5, 122.6, 118.9, 109.1, 93.1,
58.1, 44.1. m/z (ES+) 361.0 and 363.0 (M+H+). HRMS, calcd for
HPLC tR = 3.84 min. 1H NMR (300 MHz, DMSO-d6):
d 7.42 (d,
J = 8.7 Hz, 1H), 7.15 (dd, J = 8.7, 2.4 Hz, 1H), 7.10 (d, J = 2.4 Hz, 1H),
3.92 (s, 3H), 3.04 (m, 2H), 1.46 (m, 2H), 0.92 (s, 9H). 13C NMR
(75 MHz, DMSO-d6): d 159.7, 150.9, 148.4, 125.8, 123.4, 122.7,
119.0, 108.4, 91.6, 58.1, 42.4, 39.7, 30.0, 29.7. m/z (ES+) 310.1 and
312.2 (M+H+). HRMS, calcd for C16H21ClNO3 (M+H+) 310.1210 and
312.1180, found 310.1214 and 312.1186.
4.1.4.9. 4-Chloro-7-(cyclohexylmethylamino)-3-methoxy-isocoum-
arin [4-chloro-7-(cyclohexylmethylamino)-3-methoxy-1H-2-benz-
opyran-1-one] (33). Obtained from cyclohexylcarbaldehyde
(60 mg) and purified by flash column chromatography on silica
gel (Hex/EtOAc, 99:1). Yellow solid. Yield, 7%. Rf (Hex/EtOAc, 9:1)
0.73. HPLC tR = 4.67 min. 1H NMR (300 MHz, DMSO-d6): d 7.40 (d,
J = 8.7 Hz, 1H), 7.18 (dd, J = 8.7, 2.4 Hz, 1H), 7.09 (d, J = 2.4 Hz,
1H), 3.92 (s, 3H), 2.89 (d, J = 7.5 Hz, 2H), 1.19–1.65 (m, 11H). 13C
NMR (75 MHz, DMSO-d6): d 159.8, 153.3, 148.8, 123.5, 123.4,
122.5, 119.0, 108.2, 92.4, 58.2, 42.1, 37.1, 31.1, 28.0, 25.9. m/z
(ES+) 322.1 and 324.1 (M+H+). HRMS, calcd for C17H21ClNO3
(M+H+) 322.1210 and 324.1180, found 322.1196 and 324.1154.
C
17H14ClN2O5 (M+H+) 361.0591 and 363.0562, found 361.0603
and 363.0597.
4.1.4.14. 7-[(4-Benzyloxyphenylmethyl)amino]-4-chloro-3-meth-
oxy-isocoumarin 7-[(4-benzyloxyphenylmethyl)amino]-[4-chloro-
3-methoxy-1H-2-benzopyran-1-one]
(38). Obtained
from
4-benzyloxybenzaldehyde (113 mg) and purified by washing the
solid with H2O/MeCN (50:50) and cold diethylether. Yellow solid.
Yield, 13%. HPLC tR = 3.48 min. 1H NMR (300 MHz, DMSO-d6): d
6.79–7.37 (m, 12H), 5.02 (s, 2H), 4.23 (m, 1H), 3.90 (s, 3H), 3.25
(s, 2H). 13C NMR (75 MHz, DMSO-d6): d 159.6, 157.7, 151.0,
148.2, 137.6, 131.6, 128.9, 128.8, 128.2, 128.0, 126.0, 123.3,
122.8, 118.9, 115.1, 109.0, 92.3, 69.6, 58.1, 46.1. m/z (ES+) 422.2
and 424.2 (M+H+). HRMS, calcd for C24H21ClNO4 (M+H+)
422.1159 and 424.1130, found 422.1170 and 424.1152.
4.1.4.10. 4-Chloro-3-methoxy-7-(phenylmethylamino)-isocouma-
rin [4-chloro-3-methoxy-7-(phenylmethylamino)-1H-2-benzopy-
ran-1-one] (34). Obtained from benzaldehyde (56 mg) and purified
by washing the solid with H2O/MeCN (50:50). Yellow solid. Yield,
10%. mp 122 °C. Rf (Hex/EtOAc, 4:1) 0.52. HPLC tR = 3.15 min. 1H
NMR (300 MHz, DMSO-d6): d 7.49 (d, J = 8.7 Hz, 1H), 7.24–7.44 (m,
6H), 7.21 (d, J = 2.4 Hz, 1H), 4.41 (s, 2H), 3.99 (s, 3H). 13C NMR
(75 MHz, DMSO-d6): d 160.0, 151.6, 148.5, 140.0, 129.3 (2C), 128.1
(2C), 127.8, 126.7, 123.9, 123.5, 119.4, 109.7, 92.0, 58.6, 47.2. m/z
(ES+) 316.1 and 318.1 (M+H+). HRMS, calcd for C17H15ClNO3 (M+H+)
316.0740 and 318.0711, found 316.0740 and 318.0699.
4.1.4.15. 4-Chloro-3-methoxy-7-[(1-naphthalenylmethyl) amino]-
isocoumarin [4-chloro-3-methoxy-7-[(1-naphthalenylmethyl)
amino]-1H-2-benzopyran-1-one] (39). Obtained from 1-naph-
thaldehyde (83 mg) and purified by washing the solid with cold
MeCN. Yellow solid. Yield, 34%. mp 162 °C. Rf (Hex/EtOAc, 4:1)
0.38. HPLC tR = 3.55 min. 1H NMR (300 MHz, DMSO-d6): d 8.19 (d,
J = 7.7 Hz, 1H), 8.01 (dd, J = 8.7, 2.0 Hz, 1H), 7.89 (d, J = 7.7 Hz,
1H), 7.44–7.65 (m, 4H), 7.35 (dd, J = 8.7, 2.0, 1H), 7.27 (d,
J = 2.0 Hz, 1H), 6.95–7.02 (m, 1H), 4.84 (d, J = 5.2 Hz, 2H), 3.99 (s,
3H). 13C NMR (75 MHz, DMSO-d6): d 160.1, 151.5, 148.9, 134.9,
134.4, 132.0, 129.4, 128.5, 127.1, 126.7, 126.6, 126.3, 125.9,
124.5, 123.9, 123.2, 119.4, 109.3, 92.0, 58.6, 45.4. m/z (ES+) 366.0
4.1.4.11. 4-Chloro-3-methoxy-7-[(4-methoxyphenylmethyl)amino]-
isocoumarin [4-chloro-3-methoxy-7-[(4-methoxyphenylmeth-
yl)amino]-1H-2-benzopyran-1-one]
(35). Obtained
from
4-methoxybenzaldehyde (72 mg) and purified by washing the

1028
M. Peuchmaur et al. / Bioorg. Med. Chem. 21 (2013) 1018–1029
and 368.0 (M+H+). HRMS, calcd for C21H17ClNO3 (M+H+) 366.0897
and 366.0867, found 366.0898 and 368.0899.
4.1.5.2. 4-Chloro-3-methoxy-7-[(3-nitrophenyl)amino]-isocouma-
rin [4-chloro-3-methoxy-7-[(3-nitrophenyl)amino]-1H-2-benzo-
pyran-1-one] (44). Obtained from 3-nitrophenylboronic acid
(148 mg) and purified by washing the solid with MeCN and cold dieth-
ylether. Brown solid. Yield, 16%. Rf (Hex/EtOAc, 4:1) 0.32. HPLC
tR = 3.19 min. 1H NMR (300 MHz, C3D6O): d 8.34 (s, 1H), 7.99 (m, 2H),
7.80 (m, 3H), 7.62 (d, J = 6.0 Hz, 2H), 4.13 (s, 3H). 13C NMR (75 MHz,
C3D6O): d 161.9, 159.5, 144.8, 131.6, 131.2, 127.3, 124.3, 123.3, 119.1,
116.3, 115.4, 110.9, 109.9, 102.9, 92.4, 58.3. m/z (ES+) 347.0 and
349.0 (M+H+). HRMS, calcd for C16H12ClN2O5 (M+H+) 347.0435 and
349.0405, found 347.0420 and 349.0413.
4.1.4.16. 4-Chloro-3-methoxy-7-[(2-naphthalenylmethyl) amino]-
isocoumarin [4-chloro-3-methoxy-7-[(2-naphthalenylmethyl)
amino]-1H-2-benzopyran-1-one] (40). Obtained from 2-naph-
thaldehyde 83 mg) and purified by flash column chromatography
on silica gel (Hex/EtOAc, 9:1). Yellow solid. Yield, 19%. Rf (Hex/
EtOAc, 9:1) 0.81. HPLC tR = 4.47 min. 1H NMR (300 MHz, DMSO-
d6): d 7.79–7.87 (m, 4H), 7.44–7.51 (m, 3H), 7.41 (d, J = 8.4 Hz,
1H), 7.26 (dd, J = 8.7, 2.4 Hz, 1H), 7.16 (d, J = 2.4 Hz, 1H), 4.50 (s,
2H), 3.89 (s, 3H), 3.29 (sl, 1H). 13C NMR (75 MHz, DMSO-d6): d
159.6, 151.0, 148.2, 137.2, 133.4, 132.6, 128.5, 128.0, 127.9,
126.6, 126.1 (2C), 126.0, 125.6, 123.4, 122.9, 118.9, 109.1, 91.4,
58.1, 46.9. m/z (ES+) 366.1 and 368.1 (M+H+). HRMS, calcd for
4.1.6. Peptide boronates (45–52)
The peptide boronates were prepared by the general method of
Kettner and Shenvi57 and Matteson and Sadhu.56 Their physico-
chemical properties are reported in the Supplementary data.
C
21H17ClNO3 (M+H+) 366.0897 and 366.0867, found 366.0890
and 368.0887.
4.2. Biology
4.1.4.17. 4-Chloro-3-methoxy-7-[(3-phenylpropyl)amino]-iso-
coumarin [4-chloro-3-methoxy-7-[(3-phenylpropyl)amino]-
1H-2-benzopyran-1-one] (41). Obtained from 3-phenylpropanal
(71 mg) and purified by HPLC. Yellow solid. Yield, 20%. mp 98 °C. Rf
(Hex/EtOAc, 4:1) 0.60. HPLC tR = 3.37 min. 1H NMR (300 MHz,
DMSO-d6): d 7.46 (d, J = 8.7 Hz, 1H), 7.20–7.32 (m, 6H), 7.14 (d,
J = 2.4 Hz, 1H), 3.96 (s, 3H), 3.09 (t, J = 6.9 Hz, 2H), 2.70 (t,
J = 7.5 Hz, 2H), 1.88 (dt, J = 7.5, 6.9 Hz, 2H). 13C NMR (75 MHz,
DMSO-d6): d 160.2, 151.5, 148.8, 142.6, 129.2 (4C), 126.7, 126.3,
123.9, 123.1, 119.5, 109.0, 92.0, 58.6, 43.2, 33.5, 31.0. m/z (ES+)
344.1 and 346.1 (M+H+). HRMS, calcd for C19H19ClNO3 (M+H+)
344.1053 and 346.1024, found 344.1071 and 346.1020.
4.2.1. Effect of compounds on Ab production by stably
transfected HEK293 cells overexpressing bAPPswe (Swedish
mutant K670N/M671L)
HEK cells have been established as described.53 Cellular in vitro
Ab production was measured by following the expression of Ab-40
and Ab-42 by means of FCA3340 and FCA3542 antibodies as previ-
ously described.63 These antibodies, developed by us are particu-
larly useful for immunoprecipitation of Ab and Western blot
analysis. Briefly, the cells were incubated for 7 h in secretion med-
ium (OptiMem supplemented with 1% FBS) in the presence of var-
ious inhibitor concentrations. Media were collected, completed
with one tenth of 10ꢁ RIPA buffer and incubated with a 200-fold
dilution of antibodies overnight at 4 °C under agitation. Protein
A-Agarose was then added to the media and stirred three more
hours at 4 °C. After a quick centrifugation step to remove superna-
tants, agarose beads were boiled 5 min in Tris–Tricine loading
buffer. Recovered proteins were resolved on 16.5% Tris–Tricine
poly-acrylamide gels, transferred onto nitrocellulose membranes,
which were heated in boiling 1ꢁ PBS for 5 min and blocked in 5%
skimmed milk in 1ꢁ PBS containing 0.05% Tween 20. Membranes
were then incubated overnight with 6E10 monoclonal antibody
and blots were visualized by enhanced chemiluminescence (ECL,
Amersham). Images were acquired using Fuji’s Luminescence Im-
age Analyser LAS-3000 (Raytest, Courbevoie, France) and quanti-
fied using Aida analyser software.
4.1.4.18.
mino]-isocoumarin [4-chloro-3-methoxy-7-[(3-phenyl-2-pro-
pen-1-yl)amino]-1H-2-benzopyran-1-one] (42). Obtained
4-Chloro-3-methoxy-7-[(3-phenyl-2-propen-1-yl)a-
from 3-phenyl-2-propenal (70 mg) and purified by washing the so-
lid with H2O/MeCN (40:60). Yellow solid. Yield, 41%. mp 142 °C. Rf
(Hex/EtOAc, 4:1) 0.32. HPLC tR = 3.34 min. 1H NMR (300 MHz,
DMSO-d6): d 7.51 (d, J = 8.7 Hz, 1H), 7.41–7.46 (m, 2H), 7.22–7.39
(m, 5H), 6.65 (d, J = 16.0 Hz, 1H), 6.40 (dt, J = 16.0, 5.3 Hz, 1H),
3.85–4.08 (m, 5H). 13C NMR (75 MHz, DMSO-d6): d 160.1, 151.5,
148.7, 137.5, 131.3, 129.5 (2H), 128.3, 127.9, 127.0 (2C), 126.5,
123.9, 123.2, 119.4, 109.5, 92.0, 58.6, 45.5. m/z (ES+) 342.1 and
344.1 (M+H+). HRMS, calcd for C19H17ClNO3 (M+H+) 342.0897
and 344.0867, found 342.0899 and 344.0873.
4.1.5. General procedure for the preparation of N-aryl analogues
of JLK6 (43 and 44)
4.2.2. In vitro
The production of the recombinant
Flag), the preparation of the ‘solubilized’ membranes fraction and
the in vitro -secretase assay were described in details in a previ-
ous study.61
In brief, ‘solubilized membranes’ (5
cells were first diluted in sodium citrate buffer and then incubated
with 10 L of reaction buffer containing recombinant C100-Flag
(50 g/mL). The 20 L resulting reaction mixtures were incubated
c
-secretase assay on membrane preparations
c-secretase substrate (C100-
To a solution of JLK-6 (100 mg, 0.44 mmol) in dry DCM contain-
ing 4 Å molecular sieves, were added the arylboronic acid
(2 equiv), Cu(OAc)2 (1.5 equiv) and Et3N (2 equiv). After 3 days stir-
ring at room temperature, the suspension was filtered and the fil-
trate was evaporated to dryness. The residue was taken up in H2O/
MeCN (50:50) to precipitate the desired product.52
c
l
L) obtained from HEK293
l
l
l
4.1.5.1. 4-Chloro-3-methoxy-7-(phenylamino)-isocoumarin [4-
chloro-3-methoxy-7-(phenylamino)-1H-2-benzopyran-1-one]
over constant agitation for 16 h at 37 °C (or 4 °C for negative con-
trols). Samples were then supplemented with 2ꢁ Tris–Tricine load-
ing buffer, boiled for 5 min and subjected to western blot as
described above.
(43).
Obtained from phenylboronic acid (108 mg) and puri-
fied by washing the solid with H2O/MeCN (50:50). Brown solid.
Yield, 64%. Rf (Hex/EtOAc, 4:1) 0.46. HPLC tR = 3.23 min. 1H NMR
(300 MHz, DMSO-d6): d 7.74 (m, 1H), 7.62 (m, 2H), 7.36 (m, 2H),
7.18 (d, J = 7.5 Hz, 2H), 6.99 (m, 1H), 4.02 (s, 3H). 13C NMR
(75 MHz, DMSO-d6): d 159.8, 152.4, 143.8, 142.8, 130.3 (2C),
129.4, 125.9, 124.2, 122.2, 119.2, 119.0 (2C), 113.6, 91.2,
58.4. m/z (ES+) 302.0 and 304.0 (M+H+). HRMS, calcd for
4.2.3. Cytotoxicity
Cell toxicity of the different compounds was analyzed using an
in vitro lactate deshydrogenase (LDH) release assay (CytoTox-One,
Promega).
Briefly, cells were grown on 24-wells plates and treated with
the compounds at the maximal concentrations as described above.
C
16H13ClNO3 (M+H+) 302.0584 and 304.0554, found 302.0611
and 304.0611.
After treatment, 20 lL of media were collected and analyzed for

M. Peuchmaur et al. / Bioorg. Med. Chem. 21 (2013) 1018–1029
1029
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analyses.
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Supplementary data
Supplementary data (S1, the hypothesized mechanism of inhi-
bition of serine proteases by isocoumarins (Fig. 1S); S2, character-
ization data for compounds 1–6; S3, synthetic pathway
(Scheme 1S) and characterization data and biological evaluation
of peptide-boronates 45–52 (Figs. 2S and 3S); S4, structure of bio-
tinylated probes of JLK-6 (Scheme 2S)) associated with this article
can be found, in the online version, at http://dx.doi.org/10.1016/
j.bmc.2012.11.045.
36. Esler, W. P.; Das, C.; Campbell, W. A.; Kimberly, W. T.; Kornilova, A. Y.; Diehl, T.
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37. Alves da Costa, C.; Ayral, E.; Hernandez, J.-F.; St George-Hyslop, P.; Checler, F. J.
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