Design, synthesis and biological evaluation of small-azo-dyes as potent Vesicular Glutamate Transporters inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.03.056

Vesicular Glutamate Transporters (VGLUTs) allow the loading of presynapic glutamate vesicles and thus play a critical role in glutamatergic synaptic transmission. VGLUTs have proved to be involved in several major neuropathologies and directly correlated to clinical dementia in Alzheimer and Parkinson's disease. Accordingly VGLUT represent a key biological target or biomarker for neuropathology treatment or diagnostic. Yet, despite the pivotal role of VGLUTs, their pharmacology appears quite limited. Known competitive inhibitors are restricted to some dyes as Trypan Blue (TB) and glutamate mimics. This lack of pharmacological tools has heavily hampered VGLUT investigations. Here we report a rapid access to small molecules that combine benefits of TB and dicarboxylic quinolines (DCQs). Their ability to block vesicular glutamate uptake was evaluated. Several compounds displayed low micromolar inhibitory potency when size related compounds are thirty to forty times less potent (i.e. DCQ). We then confirmed the VGLUT selectivity by measuring the effect of the series on vesicular monoamine transport and on metabotropic glutamate receptor activity. These inhibitors are synthesized in only two steps and count among the best pharmacological tools for VGLUTs studies.

Full text of DOI:10.1016/j.ejmech.2014.03.056

European Journal of Medicinal Chemistry 78 (2014) 236e247
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Design, synthesis and biological evaluation of small-azo-dyes as
potent Vesicular Glutamate Transporters inhibitors
,
c
,
,
c
,
,c,d
Franck-Cyril Favre-Besse a, Odile Poirel b d, Tiphaine Bersot b d, Elodie Kim-Grellier b
,
Stephanie Daumas b d, Salah El Mestikawy b
e, Francine C. Acher a
,
,
c
,
,
c
,
d,
,
*
,
Nicolas Pietrancosta a
*
a Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR8601 CNRS, Université Paris Descartes, Sorbonne Paris Cité, 45 rue des
Saints-Pères, 75270 Paris 06, France
b Institut National de la Santé et de la Recherche Médicale (INSERM), U952, Université Pierre et Marie Curie, 9 quai Saint Bernard, 75005 Paris, France
c Centre National de la Recherche Scientifique (CNRS) UMR 7224, 9 quai Saint Bernard, 75005 Paris, France
d Université Pierre et Marie Curie (UPMC) Paris 06, Pathophysiology of Central Nervous System Disorders, 9 quai Saint Bernard, 75005 Paris, France
e Douglas Hospital Research Center, Department of Psychiatry, McGill University, 6875 boulevard Lasalle Verdun, QC, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Vesicular Glutamate Transporters (VGLUTs) allow the loading of presynapic glutamate vesicles and thus
play a critical role in glutamatergic synaptic transmission. VGLUTs have proved to be involved in several
major neuropathologies and directly correlated to clinical dementia in Alzheimer and Parkinson’s dis-
ease. Accordingly VGLUT represent a key biological target or biomarker for neuropathology treatment or
diagnostic. Yet, despite the pivotal role of VGLUTs, their pharmacology appears quite limited. Known
competitive inhibitors are restricted to some dyes as Trypan Blue (TB) and glutamate mimics. This lack of
pharmacological tools has heavily hampered VGLUT investigations. Here we report a rapid access to
small molecules that combine benefits of TB and dicarboxylic quinolines (DCQs). Their ability to block
vesicular glutamate uptake was evaluated. Several compounds displayed low micromolar inhibitory
potency when size related compounds are thirty to forty times less potent (i.e. DCQ). We then confirmed
the VGLUT selectivity by measuring the effect of the series on vesicular monoamine transport and on
metabotropic glutamate receptor activity. These inhibitors are synthesized in only two steps and count
among the best pharmacological tools for VGLUTs studies.
Received 12 December 2013
Received in revised form
4 March 2014
Accepted 17 March 2014
Available online 18 March 2014
Keywords:
Neurotransmitter
Glutamate
Vesicular uptake
VGLUT
Inhibitors
Azo dye
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
into synaptic vesicles by VGLUT1-3 for additional cycles of release
[4,5].
Glutamate (Glu) is the major excitatory neurotransmitter in the
Central Nervous System (CNS) and is involved in most, if not all,
physiological functions as well as neuropathologies of the CNS [1].
To secure synaptic transmission, the concentration of Glu in the
synaptic vesicles has to be finely regulated during the neuro-
transmitter cycle [2]. Glu released from synaptic vesicles by
exocytosis binds to glutamate receptors to trigger biological re-
sponses. Released glutamate is rapidly removed from the synaptic
cleft by plasma membrane transporters named EAAT1-5 (Excit-
atory Amino Acid Transporters) [3]. Reuptaken Glu is accumulated
In this study, we focus our attention on one step of this gluta-
mate cycle: the vesicular uploading and accumulation by VGLUTs.
Despite their key role in excitatory transmission, pharmacology of
VGLUTs appears very poor. Indeed, only few chemical compounds
efficiently target these transporters [3]. Accordingly development
of pharmacological tools appears crucial to better understand the
functional implication of VGLUT1-3 in normal and pathological
conditions and to use these transporters as biomarkers of human
“synaptopathologies” for diagnosis or therapeutic purposes. In
spite of their recent characterization, therapeutic implication has
been demonstrated in Parkinson’s disease model, Alzheimer dis-
ease or epilepsy [6]. Presently, three types of VGLUT inhibitors have
been identified [3] : dyes [5,7e10] with Ki in the 20 nMe10
range, substituted quinolines (DCQ) [11,12] with Ki in the 40e
300 M range and glutamate analogs with IC50 > 230 M. Dyes
display higher affinities, but a lower selectivity than quinolines
mM
* Corresponding authors.
E-mail addresses: francine.acher@parisdescartes.fr (F.C. Acher), Nicolas.
pietrancosta@parisdescartes.fr (N. Pietrancosta).
m
m
http://dx.doi.org/10.1016/j.ejmech.2014.03.056
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

F.-C. Favre-Besse et al. / European Journal of Medicinal Chemistry 78 (2014) 236e247
237
[13]. The third type includes the glutamate-like inhibitors known to
interact with glutamate receptors but these compounds show very
weak affinity on VGLUT [3]. We have previously shown that po-
tency of Rose Bengal (RB) may not be increased by chemical
modulation [10]. On the other hand Trypan Blue (TB) or Evans Blue
(EB) are well known universal cellular dyes thus lacking selectivity
and chemical variations of these dyes are not easily accessible. DCQ
derivatives remain a potential structure in terms of pharmacolog-
ical purpose but Carrigan et al. have shown their limit considering
their moderate affinities. Moreover these derivatives have been
synthesized in very low yields (<5% for best compounds) [12,14]. In
this work, we propose to increase the potency and improve the
synthesis of these derivatives by replacing DCQ by the di-sulfonic
naphthyl moiety present in TB inhibitor as a bioisostere. DCQs
(Fig. 1) can be replaced by the newly synthesized analogs in high
yields and in less synthetic steps. Comparison between the dyes’
structures and DCQs, allowed the design of a hybrid azo-linked-
biaryl-disulfonic- naphthyl shown in Fig. 1 and discussed below.
Synthesis of such a series and VGLUT uptake evaluation are
described herein. Compounds 7c, 7e, 4i and 4p are the most potent
-withdrawing groups. Since in the quinoline series, the type of
bond between the phenyl and quinoline moiety has little effect on
inhibitory activity [12], we selected a diazo-link for its synthetic
simplicity. In order to increase solubility, we selected p-carboxylic
acid substituted analogs and corresponding p-halogeno analogs to
increase hydrophobicity. The major advantage of the diazo-
naphtalene bioisostere is its synthetic route allowing easy access
to various derivatives such as compounds 4 and 7 (Scheme 1). They
may be obtained in only two steps performed in one pot: diazo-
tation of aniline followed by electrophilic aromatic substitution of a
naphthyl derivative. Due to the relative instability of diazonium
salt, we prepared this intermediate in situ and used it directly,
without purification. Nevertheless kinetics of diazonium formation
needs to be controlled when used directly.
As previously mentioned, the first step consists in the formation
of a diazonium salt 2 or 6 in acidic aqueous solution. The mecha-
nism of the reaction has been already described by several groups
since 1958 [16,17] and different mechanisms postulated. To clarify
the kinetics, we monitored the diazonium/aniline ratio obtained in
various conditions by 1H NMR. As anticipated, electronic properties
of aniline substituents highly impact the diazotation kinetics.
Indeed, deactivation of the aromatic ring by electron-withdrawing
or sigma-attracting substituents (i.e. nitro or fluorine) led to a
mixture of diazonium compounds and starting material
(Supplementary Fig. 1 and Table 1). Moreover when aniline was
unsubstituted or substituted by an electron-donating group, the
reaction was complete. Thus, two major factors appear to directly
influence reaction completion: the aniline nucleophilicity and its
solubility in acidic media. Comparison between pKa and reaction
completion clearly indicates that diazonium formation is limited by
aniline nucleophilic attack on nitrosonium ion (assuming that
amine pKa can be correlated to its nucleophilicity). But this only
factor may not explain the difference between fluorine or chlorine
substituted anilines diazotation. We also observed that aniline
solubility is critical as illustrated when using poorly soluble 2,4-
dinitro aniline. No reaction was observed in water while 70% of
conversion was estimated when reaction was run in ethanol as
solvent. This particular reactivity is even more spectacular when
working on the mono-diazotation of benzidine derivatives 5. As for
aniline, benzidine holding withdrawing substituents (2-F or 2-Cl)
reacts partially with sodium nitrite and leads to only mono-
diazotized derivative. In contrast, as already observed with ani-
line, benzidine bearing electro-donating or sigma-donor sub-
stituents (eMe, eOMe) are more reactive and when treated with
even less than 1 equivalent of sodium nitrite a mixture of di-
diazonium salt, starting material and mono-diazotized analog is
obtained. Taking into account these observations, diazotation re-
action of electron-poor aromatic anilines has been performed in
water/ethanol solvent using 1.3 equivalents of sodium nitrite.
Concerning compound 7, a preliminary study has been limited to
easily accessible diazotized benzidine (electro-withdrawing
substituted aniline).
derivatives with IC50’s of 1.6, 1.8, 2.1 and 2.1 mM respectively. These
new VGLUT inhibitors provide a valuable contribution to the
pharmacology of these transporters that is amazingly limited.
2. Results and discussion
2.1. Chemistry
Analogs of the mono-azo dyes 4 where generated by modula-
tion of the phenyl ring substituents while keeping the same di-
sulfonic naphthalene entity as deduced from observed variations
of EB (IC50 ¼ 87 nM), TB (IC50 ¼ 50 nM) and Chicago Sky Blue (CSB)
(IC50 ¼ 3
mM) potencies. Indeed EB and TB display similar VGLUT
inhibitory activity whereas replacing the methyl group of EB by a
methoxy group in CSB decreases 34-fold the inhibitory activity
[9,15]. SAR of DCQs also revealed the critical role of aromatic sub-
stitution [11,12]. We also decided to investigate the phenyl ring
substituent effect in
4 by introducing electro-donating or
The second step of the synthesis of 4 and 7, consists in an aro-
matic electrophilic substitution of a poly-functionalized naphthyl
moiety (Compound 3). Again at this step, literature appears quiet
unreliable. When some authors described an attack specifically on
the ortho position of a phenol [18e20], others confirmed the
regioselective addition in the para position of the hydroxyl sub-
stituent [21]. Based on this literature, aromatic substitution should
also occur in ortho/para position of the hydroxyl substituent in
alkaline conditions and in ortho position of the amino group in
acidic ones (Supplementary Fig. 1). To avoid side product formation
and a fastidious purification step when using compound 2 bearing
both amino and hydroxyl substituent, we decided to clarify the
regioselectivity of this step and thus studied first diazonium
HO3S
Fig. 1. Strategy used for compounds 4 and 7 design: we combined quinoline key
features (i.e. hydrophobic substituent (circled in green), aromatic bicyclic negative
charges (circled in pink)) and easily accessible naphthyl blue dyes counterpart (i.e.
Compound 3). (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)

238
F.-C. Favre-Besse et al. / European Journal of Medicinal Chemistry 78 (2014) 236e247
When diazonium 2k was added to compound 10 in the same
conditions, only single ortho substitution (product 11) occurred and
no addition in para position of the hydroxyl was detected [24]. As
previously described no addition occurred in C5 and C6 positions
(presence of doublets in the naphtyl moiety correlating with the
ketone C3 in HMBC experiment). Cosy correlation between H6 and
H8 and HSQC experiment permitted the identification of H8 and C8.
Finally, a correlation between C8 and a singulet (H1) in HMBC
experiment confirmed the addition on C3.
H3OS
Surprisingly, when compound 12 was treated in these condi-
tions, only the di-substituted compound 13 was obtained. At this
step no mono-addition was observed with mass spectrometry and
the single pic of di-coupled compounds ([MꢁH]- ¼ 766 in ESI-) was
detected. Moreover, the doublet between H5 and H6 of naphtyl
moiety confirmed that no addition occurred on the corresponding
carbons. The di-addition could be either at the C3 and C1 positions
or C3 and C8 or C8 and C1. Nevertheless, the NOESY experiment
highlighted a correlation between a proton of the aryl substituent
(HeC600) and a proton presents on the naphthyl moiety. This ruled
out an addition on the C1 and C8 position. To discriminate between
an addition on C1eC8 or C3eC8, an HMBC experiments were used.
Indeed, the common correlations of H6 (129.3 ppm, 120.4 ppm) and
the only naphthyl singulet (H1 or H8) confirmed its position on C1
(also visible by NOESY). All these data validated the addition on
both C8 and C3 positions (See Supplementary data).
This last example led us to question about the amine influence
on regioselectivity in basic conditions that has never been previ-
ously described. Compound 14 was also engaged in the same re-
action conditions and as awaited, no reaction (even at ortho to
amino position) was observed (Scheme 2).
Altogether, these results allow concluding about the necessity of
a hydroxyl substituent for regioselective substitution in basic con-
ditions and warned us about the possible reactivity of ortho amino
position even in basic condition when the naphthyl moiety is both
hydroxyl and amino substituted. We proceeded to the synthesis of
compounds 4 and 7 by coupling various diazonium 2 to compound
3. Compounds were purified by classical precipitation at pH 2 in a
water/ethanol mixture. Alternative preparative HPLC purification
was used when no precipitate had formed or when the purity was
not sufficient; this second purification highly impacted global
yields as shown in Table 1.
Interestingly, when coupling 4-carboxy or 4-bromo-tetra-
fluorophenyl diazonium 2 to naphthyl moiety 3, an aromatic sub-
stitution occurred and one ortho-fluorine substituent was replaced
by a hydroxyl group in 4f and 4n. This substitution has only been
observed in few cases mostly without further explanation and only
with phase transfer agent in organic solvent. Marriot et al. postu-
lated an assistance of a sodium ion that could help the hydroxyl
group attack where Feldman et al. postulated an enhance reactivity
of hydroxyl ion in organic solvent [25,26]. It should be also noted
that no substitution occurred when stirring tetrafluoro-aniline
alone at pH 9 (data not shown). Regarding the regioselectivity, as
previously observed for compound 13, (Scheme 2), some di-
substitutions affording compounds 16, were observed but only
when coupling highly reactive diazonium 2 (i.e. ortho substituted
by electro-withdrawing group (R1 ¼ -F, compound 2d, R1 ¼ eCF3,
compound 2e) with compound 3. (Supplementary Scheme 1) As
observed with compound 13, the second addition occurred at ortho
position of the aniline substituent and not at C1.
Scheme 1. Synthesis of compounds 4 and 7. a) NaNO2, HCl/H2O, b) Compound 3, NaOH
10%, pH ¼ 9.
addition in simpler structures (compounds 8, 10, 12, 14, Scheme 2)
[22,23].
A representative diazonium salt (compound 2k) was added to a
range of naphthyls (8, 10, 12, 14) at pH 9.0. Mono or di-additions
was confirmed firstly by Mass spectrometry. Regioselectivity of
additions were elucidated by NMR spectroscopic experiments
(NOESY, HMQC.) (See Supplementary data). As expected, com-
pound 8 gave only one substituted isomer 9 diazotized in C1 po-
sition (i.e. no other para or ortho position was accessible) in a
quantitative yield after 1 h of stirring at 0e5 ꢀC. Regioselectivity
was confirmed by 1H NMR and 13C NMR: the doublet between H5
and H6 of naphthyl moiety confirmed that no addition occurred on
the corresponding carbons. Moreover the chemical shift of C3
2.2. Pharmacology
All synthesized compounds 4 and 7 were then evaluated for
their ability to inhibit 3H-glutamate uptake into isolated vesicles
from rat brain. As previously described, inhibition was measured by
characteristic of a ketone function (d (C3) >170 ppm) established
that the coupling occurred in C4 position.

F.-C. Favre-Besse et al. / European Journal of Medicinal Chemistry 78 (2014) 236e247
239
Table 1
20
m
M, were next determined. CCCP (vATPase inhibitor) and TB
Yields of compounds 4 and 7 over the two steps.
(competitive inhibitor (IC50 ¼ 0.1
mM)) were used as negative
controls (100% of inhibition). No Inhibitor was added for positive
control (i.e. tritiated glutamate is typically accumulated in vesicles:
0% of inhibition), allowing the complete inhibitory calibration.
A first series of monoazo-dyes 4, 9, 11 and 13 was tested and
revealed that most of 4 were able to reduce vesicular glutamate
Entry
R1
R2
R3
R4
R5
Yields
4aa
CH3
H
Cl
F
CF3
F
OCH3
CH3
CH3
H
Cl
F
CF3
F
OCH3
CH3
CH3
H
Cl
F
CF3
F
H
H
H
H
H
F
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
F
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
I
Br
I
I
I
H
H
H
H
H
F
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
F
H
H
H
H
H
OH
H
CH3
H
H
H
H
H
OH
H
CH3
H
H
H
H
37%
80%
84%
12%
13%
52%
85%
74%
18%
24%
6%
4ba
4ca
4da,b
4ea,b
4fa
uptake at 20 mM (Table 2) whereas 9, 11 and 13 were inactive. These
4ga
results demonstrate that the di-sulfonic naphthalene moiety
bearing an amino and a hydroxyl group builds up a core that dis-
plays increased potency as compared to the di-carboxylic quino-
lines DCQs (Fig. 1) [28]. Interestingly when best analogs such as
DCQ1 described by Carrigan et al. [12] inhibit VGLUT with an IC50
4ha
4ia
4ja
4ka
4la,b
4ma,b
4na
4oa
4pa
7aa
3%
2%
around 40
promising at 20
mM, many of our easily accessible compounds appear
Br
Br
74%
20%
24%
2%
25%
40%
2%
mM (4i, 4p, 4m) (Tables 2and 3).
The first observation allows concluding that p-carboxylic acid
compounds appear mostly less active than corresponding p-halo-
geno substituted analogues. Only 4e, the 20-CF3, 40-COOH analog is
Br
c
7ba
7ca
c
c
c
c
c
found among the most potent inhibitors, with an IC50 of 6.8 mM. The
7da,b
7ea,b
7fa,b
weaker activity of these compounds may be correlated to the
negative charge of carboxylate function and not to its electro-
withdrawing character. Indeed, compounds 4i and 4m bearing
respectively ortho-methyl, para-iodo and ortho-trifluoromethyl,
para-iodo substituent showed similar activity at 20
83%) despite the opposite character of their ortho substituent.
H
F
42%
6%
a
Purified by precipitation.
Purified by HPLC.
Substituted phenyl as described in Scheme 1.
b
c
mM (76 and
Again, the ortho-methoxy para-bromo 4o and ortho -fluoro; para-
iodo 4l similarly blocked glutamate uptake (42 and 46% at 20 mM).
quantification of tritiated glutamate uptake by purified vesicles
[7,27]. Compounds were firstly examined for their capacity to block
The same result is observed when comparing activity of 4g (ortho-
methoxy; para-carboxylic acid) with 4d (ortho-fluoro; para-car-
boxylic acid) both moderately active (respectively 25% and 18% of
glutamate uptake at 20
mM level (Tables 2and 3). IC50’s of de-
rivatives 4 and 7 that displayed more than 60% of inhibition at
inhibition at 20 mM). A hydrophobic counterpart in para position
appears more beneficial as observed for halogenated derivatives.
Similarly to EB and CSB, replacement of a methyl group (4i,
IC50 ¼ 2.1
mM) by a methoxy (4o, IC50 > 20 mM) leads to a marked
decrease of activity. The relatively moderate activity of methoxy
derivatives appears to be independent of substituent electronic
properties. As a matter of fact, electro-withdrawing substituted
compounds like mono-fluorinated (R1 ¼ F) 4l or chlorinated
(R1 ¼ Cl) 4k as well as electro-donating substituted derivatives
(R1 ¼ OMe) 4o inhibit glutamate uptake with similar potency at
20
mM (46, 45% and 42% inhibition respectively). A plausible
explanation for the reduced inhibitory activity could be the limited
volume available around R1 position to interact with VGLUTs. The
bulk (Van der walls) of substituent seems to affect directly inhibi-
tory properties and to be optimal with a methyl group. Compounds
4i and 4p are the most active derivatives (IC50 ¼ 2.1
An increase of the substituent size diminishes the potency of
compounds e.g. 4o compared to 4i (42% of inhibition at 20
mM for both).
mM
compared to 83% for 4i). On the other hand reducing the volume
decreases also inhibitory efficacy of compounds (e.g. 83% and 46%
inhibition for 4m and 4l). Moreover, compound 4n appears one of
the less active compounds of this series probably because of the
presence of a polar substituent (hydroxyl) in the hydrophobic
environment.
Additionally the azo-hydrazo tautomer equilibrium may also
influence the inhibitory capacity of 4 but according to spectroscopic
data all compounds 4 are found only in the hydrazo form (d
13C
(C4) ¼ 180 ppm) [23] and no azo form have been detected in NMR.
As described previously for DCQs, a hydrophobic substituent at
para-phenyl position of compounds is favorable for inhibition; we
then decided to increase the size of compounds at this position by
adding one functionalized phenyl ring.
The series of compounds 7 (Scheme 1) may be viewed as
biphenyl analogs of 4 but also as TB derivatives shortened by one
naphthyl. As for previous series, we focused our effort on the biaryl
Scheme 2. Regioselective addition of diazonium 2k to compounds 8, 10, 12, and
14.aReagents and conditions: NaOH 10%, pH ¼ 9, 0e2 ꢀC.

240
F.-C. Favre-Besse et al. / European Journal of Medicinal Chemistry 78 (2014) 236e247
Table 2
even if the parent drug TB has been characterized as a competitive
VGLUT inhibitory activity of compounds 4.a
inhibitor [3] other dyes such as Rose Bengal can modulate the
transport of monoamine, probably by indirect interaction (potential
or proton gradients are crucial and could be targeted by non-
competitive drugs) [10]. Moreover, purified vesicles used for up-
take experiments are extracted from a brain region where both
VMAT2 and VGLUTs highly co-localize. [29] Compounds 4 and 7
show a good selectivity because no effect on dopamine uptake has
been detected at 200 mM (data not shown). These results confirm
that these analogs did not interact with ATPase or with other
proteins implicated in proton uptake. On the other hand, we also
verified that glutamate competitive transporter inhibitors did not
interact with glutamate receptors such as mGlu1-8 receptors. We
evaluated the activity of the best analogs 4i and 7c on all receptor
subtypes in order to cover the wide range of glutamate affinity for
Entry
R1
R2
R3
R4
R5
% Inhibition
(at 20 mM)
IC50 (mM)
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
CH3
H
Cl
F
CF3
F
OCH3
CH3
CH3
H
Cl
F
H
H
H
H
H
F
H
H
H
H
H
H
H
F
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
I
Br
I
I
I
H
H
H
H
H
F
H
H
H
H
H
H
H
F
H
H
H
H
H
OH
H
CH3
H
H
H
H
H
OH
H
15
15
55
18
76
18
25
5
83
33
45
46
83
39
42
77
/
/
/
/
mGluRs (1 mM to 1
mM). As expected, no significant agonist or
antagonist effect has been characterized at 200
mM in cell based
6.8 ꢂ 1.8
assays showing a particular non cytotoxicity of these azo dyes as
previously described [30,31].
/
/
/
2.1 ꢂ 1.6
3. Conclusion
/
/
/
In conclusion an SAR study in a TB derived series allows the
characterization of key features required for potent VGLUT uptake
inhibition. A comparison between DCQ and naphthyl analogs
confirms the importance of an amino group in this scaffold,
whereas the positions of negative charges are not crucial as
underlined by similar activity of TB and EB. More precisely, this
study has disclosed strategic features for VGLUT inhibitor design
such as volume limitation for biaryl functionalization or the detri-
mental effect of a negative charge. In the present study, we have
synthesized a mini chemical library of small VGLUTs ligands with
improved synthetic methodology, yields and biological potencies.
Once the synthetic experimental conditions were optimized, access
to various structures was straightforward and allowed a rapid
characterization of key features for VGLUT uptake inhibition. The
preliminary observations made by Carrigan et al. [12] have been
confirmed and completed. Among these characteristics, bi-aryl
derivatives and 8-hydrolylated substituent are needed for
improved potencies. Our results should be considered in the
context of the very limited pharmacology of VGLUTs [3,32]. More-
over, the affinity of glutamate for VGLUTs is very low (Km ¼ 1e
3 mM) whereas the best structural analog interact with VGLUT with
an IC50 of 0.2 mM [3]. DCQs, which have been designed for the same
CF3
F
OCH3
CH3
5.8 ꢂ 1.2
Br
Br
Br
/
/
H
H
H
H
CH3
2.1 ꢂ 1.3
a
All experiment have been assayed at least in triplicate.
substituents and kept an azo-link between the bi-aryl and naphthyl
moieties (Fig. 1). In contrast to what was observed for smaller
structures, it appears that the steric bulk is not the only factor
influencing inhibitory properties of compounds (Table 3).
Indeed, direct comparison between uptake activities and van
der Walls volumes does not explain the activity variation. Results
with the optimal substituent size characterized previously, provide
a compound with moderate inhibitory activity (7a, R1 ¼ CH3); and
compounds 7c (R1 ¼ Cl) or 7d (R1 ¼ F), bearing respectively larger
and smaller groups, are significantly more potent (IC50 ¼ 1.6 and
4.8 mM). In contrast to their analogs 4, biphenyl substituted naph-
thyl 7 activities seems to be correlated to the electronic character of
biphenyl substituent. Indeed, electro-withdrawing substituted 7c,
7d, 7e, 7f showed significant inhibition where electro-donating
analogs 7a, 7b appears quite weaker. These results question about
the strict equivalent position of this series compared to the previ-
ous and confirm the relative flexibility of the binding site of VGLUT
due to its physiological transport function and mechanism. In this
group of compounds, a compromise between steric bulk and
electronic properties appears crucial for activity. Compared to the
previous one, chlorinated analog 7c is more potent than the
methylated (7a) or the unsubstituted (7b) which are less active
than the poly-fluorinated compound 7f.
purpose as 4 and 7, displayed IC50’s of 40 and 95
mM [12]. Our
compounds, in particular 7c, 7e, 4i and 4p, are active in the
micromolar range, and are thus one of the best compromise be-
tween activity and selectivity for pharmacological VGLUT inhibi-
tion. They pave the way as pharmacological tools to the regulation
of glutamate neurotransmission, a crucial player in a large number
of CNS pathologies.
In the two series of inhibitors 4 and 7, CF3 substituted analogs
display particular biological properties and are all found to be
potent (Tables 2 and 3). Indeed, among the “half” TB analogs para-
substituted with a carboxylic acid, only 4e shows significant
inhibitory activity all other analogs being much less active. As for
other para-halogenated compounds 4m is most potent with an IC50
4. Experimental section
4.1. General
All chemicals and solvents were purchased from commercial
suppliers (Acros, Aldrich, Alpha Aesar, Apollo Scientific) and used
as received. Most solvents were purchased from Carlo Erba-SDS.
value of 5.8 mM. The best analog remains the bi-aryl substituted
ꢀ
compound 7e. Another explanation could be the conformation
adopted by compounds particularly between azo and phenyl/
biphenyl parts but preliminary studies didn’t allow to conclude
(Supplementary Table 2).
Best compounds have also been evaluated for their ability to
interfere with Vesicular Monoamine Transporter uptake. Indeed,
Solvents for reactions were dried on 4 A molecular sieves (Carlo
Erba e SDS) or were distillated. All reactions were monitored by
thin layer chromatography (TLC) on precoated silica on aluminum
(Merck 60 F254, aluminum sheets) and revealed by UV. Silica gel
chromatography was performed with Silica gel 60 MerckÒ, 40e
63 mm.

F.-C. Favre-Besse et al. / European Journal of Medicinal Chemistry 78 (2014) 236e247
241
Table 3
50% A e 50% B from 40 min to 44 min, linear decrease from 50 to
20% B between 44 and 46 min, 80% A between 46 and 56 min. Flow:
VGLUT inhibitory activity of compounds 7.a
8 mL minꢁ1
.
l1 ¼ 254 nm, l2 ¼ 550 nm. Gradient P2 is using solvent
A: ammonium acetate buffer 0.05 M, solvent B: acetonitrile, 80% A
for 4 min, linear increase from 20 to 50% B between 4 and 32 min,
50% A e 50% B from 32 min to 47 min, linear decrease from 50 to
20% B between 47 and 50 min, 80% A between 50 and 55 min. Flow:
8 mL minꢁ1
.
l1 ¼ 254 nm, l2 ¼ 550 nm. Gradient A is using solvent
A: water-formic acid 1&, solvent B: acetonitrile 90% A for 5 min,
linear increase from 10 to 60% B between 5 and 22 min, 40% A e 60%
B from 22 min to 30 min, linear decrease from 60 to 10% B between
30 and 32 min, 90% A between 32 and 36 min. Flow: 0.75 mL minꢁ1
.
l
1
¼
254 nm,
l
2
¼
550 nm. Prior to use: 5-Amino-4-
Entry
R1
R2
R4
R5
% Inhibition
IC50
(m
M)
hydroxynaphthalene-2,7-disulfonic acid monosodium salt was
(at 20 mM)
recrystallized three times at pH ¼ 3 in a mixture of EtOH/H2O ꢁ1/1.
7a
7b
7c
7d
7e
7f
CH3
H
Cl
F
CF3
F
H
H
H
H
H
F
H
H
H
H
H
F
H
H
H
H
H
F
52
25
88
74
98
96
/
/
4.2.1. General procedure A
1.6 ꢂ 1.1
4.8 ꢂ 1.6
1.8 ꢂ 1.5
3.3 ꢂ 1.4
Aniline derivative (1 eq) was mixed with iced water and/or
absolute EtOH (2 mL by mmol of reagent) and stirred at 0 ꢀC then
hydrochloric acid 37% (4 eq) was added dropwise rapidly. Sodium
nitrite (1.1 eq or 1.3 eq aniline substituent depending) was added
solid or in solution (H2O/EtOH 1 M) and the reaction mixture was
stirred at 0e2 ꢀC for 30 min. The solvent was removed carefully
under reduced pressure without heating and can be used without
further purification.
a
All experiment have been assayed at least in triplicate.
Reversed phase TLC were performed using RP-18 F254 plates
(MerckÒ), and reversed-phase chromatography with silica gel
100C18-reversed phase (FlukaÒ). Compounds were purified on a
Gilson analytical instrument with a 321 pump, eluted peaks were
detectedbya UVevis 156detectorand retentiontimesarereportedin
minutes. System was equipped with a MachereyeNagel NucleodurÒ
4.2.2. General procedure B
5-Amino-4-hydroxynaphtalene-2,7-disulfonic acid sodium salt
(0.9 eq) was dissolved in iced water (5 mL by mmol of reagent) then
pH was adjusted to 9 by adding 30% NaOH solution. The diazonium
salt was added to the naphthalene moiety and pH maintained at 9
by addition of 10% NaOH solution. After complete addition of the
diazonium salt to the 5-amino-4-hydroxynaphthalene-2,7-
disulfonic acid monosodium salt, stirring was maintained at 0 ꢀC
for 2 h. Degraded diazonium was removed after extraction with
EtOAc and water soluble diazo compounds were recrystallized
three times from EtOH/(H2O; pH ¼ 2) (1/1). All organic impurities
were then extracted by washing with small portions of Et2O. The
precipitated compounds were dried under vacuum.
100-10C18 (250 mm ꢃ 32 mm, 10
m
m). 1H (250.13 or 400.13 or
500.16MHz),13C (62.9or100.62or125.78 MHz)and19F(376.49MHz)
NMR spectra were respectively recorded on an ARX 250 or an Avance
II 400 Bruker or an Avance II 500 Bruker spectrometer.
Chemical shifts (d) are given in d (ppm) with reference to re-
sidual 1H or 13C of deuterated solvents, respectively CD3OD 3.31,
49.0; D2O 4.80; CDCl3 7.24, 77.00; (CD3)2CO 2.05, 29.84 and 206.26
or (CD3)2SO 2.50, 39.52). Mass spectra (MS) were recorded with an
LCQ-advantage (ThermoFinnigan) mass spectrometer with positive
(ESIþ) or negative (ESIꢁ) electrospray ionization (ionization tension
4.5 kV, injection temperature 240 ꢀC). HPLC-MS analyses were
performed on a Thermo Finnigan LCQ Advantage Instrument as
described above, equipped for HPLC with a MachereyeNagel
4.2.3. General procedure C
Benzidine derivative (1 eq) was mixed with iced water and/or
absolute EtOH (2 mL by mmol of reagent) and stirred at 0 ꢀC then
hydrochloric acid 37% (8 eq) was added dropwise rapidly. Sodium
nitrite (2.2 eq or 2.6 eq aniline substituent depending) was added in
solid or in solution (H2O/EtOH 1 M) below 2 ꢀC and reaction
mixture was stirred at 0e2 ꢀC for 30 min. Naphtalenesulfonique
compound (1.8 eq) was dissolved in iced water (10 mL by mmol of
reagent) then pH was adjusted to 9 by adding NaOH 30% solution.
The diazonium salt was coupled in alkaline media (pH 9) below 2 ꢀC
by adding NaOH 10% solution. After complete addition of the dia-
zonium salt to the 5-amino-4-hydroxynaphthalene-2,7-disulfonic
acid monosodium salt, stirring was maintained at 0 ꢀC for 2 h.
Degraded diazonium was removed after extraction with EtOAc and
water soluble diazo compounds were recrystallized three times
from EtOH/(H2O; pH ¼ 2) (1/1). All organic impurities were then
extracted by washing with small portion of Et2O. The precipitated
compounds were dried under vacuum.
NucleosilÒ 100-5 C18 (125 mm ꢃ 4 mm, 4
mm). Products were
eluted as the same manner as described above.
HPLC analyses were carried out on two devices: a Gilson
analytical instrument with a 321 pump, column temperature of
NucleosilÒ columns was controlled with an Igloo-CIL Peltier effect
thermostat or column oven Shimadzu CTO-10A, eluted peaks were
detected by a UVevis 156 detector and retention times are reported
in minutes. A Gilson analytical instrument with a 306/307 pump,
column temperature of NucleosilÒ columns was controlled with an
Igloo-CIL Peltier effect thermostat or column oven Shimadzu CTO-
10A, eluted peaks were detected by a UVevis 155 detector and
retention times are reported in minutes. Purity of the tested com-
pounds was established by analytical HPLC-MS, by HPLC or by
elementary analysis and was at least 95%. UV spectra were per-
formed Saphire spectrofluorimeter in pure water. Modeling figure
has been made using discovery Studio 2.5 software.
4.2. General procedure
The following gradients were used preparative (Gradient P1e
P2) and analytical (Gradient A) HPLC: Gradient P1 is using solvent
A: ammonium acetate buffer 0.05 M, solvent B: acetonitrile, 80% A
for 4 min, linear increase from 20 to 50% B between 4 and 40 min,
Atom numbering

242
F.-C. Favre-Besse et al. / European Journal of Medicinal Chemistry 78 (2014) 236e247
4.2.3.1. 4-Amino-3,5-dimethylbenzoic
acid,
1h.
EtOH. Compound was obtained as a white powder in 100% yield
3,5-dimethyl-4-nitrobenzoic acid (976 mg, 5 mmol) was suspended
in water (20 mL) under vigorously stirring for 30 min, until mixture
was homogenized); Na2CO3 (300 mg, 2.8 mmol) and zinc dust (3 g,
30 mmol) were added to the solution then the mixture was heated
fat 30 ꢀC and HCl conc. (10 mL) was added dropwise over 20 min.
Thereafter, the solution was filtered, and the filter cake was washed
twice with water (2 ꢃ 7 mL). To remove unreacted 3,5-dimethyl-4-
nitrobenzoic acid, the filtrate was washed twice with ethyl acetate
(2 ꢃ 40 mL). The aqueous layer was adjusted to pH 9.35 by addition
of saturated Na2CO3 solution (35 mL), and the precipitated ZnOH
was filtered off. Once more, the filter cake was washed twice with
5% NaHCO3 (2 ꢃ 10 mL). To remove by products, the filtrate was
washed twice with EtOAc (2 ꢃ 80 mL). Subsequently, the aqueous
layer was adjusted to pH 3.5 by addition of 1 N HCl (w120 mL).
Finally, the solution was extracted five times with ethyl acetate
(5 ꢃ 80 mL). The combined organic layers were dried with MgSO4
and evaporated dryness. Compound was obtained as a clear brown
(460 mg). 1H NMR (500 MHz, MeOD):
d
9.61 (d, J ¼ 2.9 Hz, 1H, H3 );
0
0
0
8.96 (dd, J ¼ 9.4 Hz, J ¼ 2.9 Hz, 1H, H5 ); 7.97 (d, J ¼ 9.4 Hz, 1H, H6 ).
4.2.3.8. (E)-5-ammonio-3-(2-(4-carboxy-2-methylphenyl)hydra-
zono)-4-oxo-3,4-dihydronaphthalene-2,7-disulfonate, 4a (LSP9-
2129). Following the general procedure B, corresponding diazo-
nium salt (1.4 mmol) was added to 5-Amino-4-hydroxy-
naphthalene-2,7-disulfonic acid (445 mg, 1.3 mmol). Compound
was obtained as a dark blue powder in 37% yield (300 mg). 1H NMR
0
(500 MHz, DMSO):
d
15.57 (s, 1H, NH); 8.04 (d, J ¼ 8.4 Hz, 1H, H6 );
0
0
7.85 (s, 1H, H3 ); 7.83 (d, J ¼ 8.4 Hz, 1H, H5 ); 7.33 (s, 1H, H1); 7.09 (s,
1H, H6); 6.87 (s, 1H, H8); 2.42 (s, 3H, H7 ). 13C NMR (126 MHz,
0
0
DMSO):
d
181.9 (C4); 166.3 (C7’); 153.7 (C7); 153.5 (C2); 144.8 (C4 );
0
0
0
142.9 (C5); 135.9 (C9); 130.8 (C3 ); 130. 3 (C3); 129.1 (C2 ); 128.3 (C5 );
0
0
124.4 (C1); 123.6 (C1 ); 113.2 (C6 ); 114.3 (C8); 113.1 (C6); 111.5 (C10);
0
17.05
(C8 ).
Elemental
analysis
found
(calcd.)
(þNaCl; þNaþ; þ3H3Oþ): C 33.99 (34.05); H 3.80 (3.65); N 6.79
(6.62); S 10.15 (10.10). HPLC (Gradient A) tR ¼ 8.20 min. MS (ESI)ꢁ:
m/z 480.1 [M ꢁ H]ꢁ. UV vis lmax ¼ 543 nm.
solid in 76% yield (629 mg). 1H NMR (500 MHz, CD3CN):
d
8.94 (brs,
1H, OH); 7.55 (s, 2H, H30, H50); 4.52 (brs, 2H, NH2); 2.15 (s, 6H, H70)
13C NMR (126 MHz, CD3CN):
d
168.5 (C80); 149.9 (C10); 131.0 (C30,
C50); 122.4(C40); 121.4 (C20,C60); 17.9 (C70). MS (ESI)þ: m/z 166
[M þ H]þ.
4.2.3.9. (E)-5-Ammonio-3-(2-(4-carboxyphenyl)hydrazono)-4-oxo-
3,4-dihydronaphthalene-2,7-disulfonate,
4b
(LSP9-2177).
Following the general procedure B, corresponding diazonium salt
(1.9 mmol) was added to 5-Amino-4-hydroxynaphthalene-2,7-
disulfonic acid (581 mg, 1.7 mmol). Compound was obtained as a
dark blue powder in 10% yield (92 mg). 1H NMR (500 MHz, DMSO):
4.2.3.2. 4-carboxy-2-Methylbenzenediazonium
chloride,
2a.
Following the general procedure A, 4-Amino-3-methylbenzoic acid
(453 mg, 3 mmol), HCl 37% (1 mL, 12 mmol), NaNO2 in solution
(227 mg, 2.3 mL), solvent H2O/EtOH. Compound was obtained as a
white powder in 100% yield (595 mg). 1H NMR (500 MHz, MeOD):
0
0
d
15.24 (s, 1H, NH); 7.94 (d, J ¼ 8.8 Hz, 2H, H3 eH5 ); 7.72 (d,
0
0
J ¼ 8.8 Hz, 2H, H2 eH6 ); 7.30 (s, 1H, H1); 7.08 (s, 1H, H6); 6.86 (s, 1H,
d
8.69 (d, J ¼ 8.5 Hz, 1H, H60); 8.39 (d, J ¼ 1.2 Hz, 1H, H30); 8.31 (dd,
H8). 13C NMR (126 MHz, DMSO):
d 181.9 (C4); 166.8 (C7’); 153.9 (C7);
J ¼ 8.5 Hz, J ¼ 1.2 Hz, 1H, H50); 2.87 (s, 3H, H70).
153.4 (C2); 146.4 (C4 ); 142.8 (C5); 136.2 (C9); 130.7 (C3 eC5 ); 130. 3
0
0
0
0
0
0
(C3); 125.6 (C1 ); 123.7 (C1); 115.7 (C2 eC6 ); 113.7 (C8); 112.5 (C6);
111.5 (C10). Elemental analysis found (calcd.) (þNaþ; þ3H3Oþ): C
37.81 (37.36); H 3.41 (3.87); N 7.46 (7.86); S 11.02 (11.74). HPLC
(Gradient A) tR ¼ 5.65 min. MS (ESI)ꢁ: m/z 465.9 [M ꢁ H]ꢁ. UV vis
lmax ¼ 534 nm.
4.2.3.3. 4-carboxybenzenediazonium chloride 2b. Following the
general procedure A, 4-Aminobenzoic acid (685 mg, 5 mmol), HCl
37% (1.67 mL, 20 mmol), NaNO2 in solid (380 mg, 5.5 mmol), solvent
H2O. Compound was obtained as a white powder in 100% yield
0
(925 mg). 1H NMR (500 MHz, MeOD):
d
8.87 (d, J ¼ 8.8 Hz, 2H, H2 e
0
0
0
H6 ); 8.39 (d, J ¼ 8.8 Hz, 2H, H3 eH5 )
4.2.3.10. (E)-5-Ammonio-3-(2-(4-carboxy-2-chlorophenyl)hydra-
zono)-4-oxo-3,4-dihydronaphthalene-2,7-disulfonate, 4c (LSP9-
2192). Following the general procedure B, corresponding diazo-
4.2.3.4. 4-carboxy-2-fluorobenzenediazonium
chloride,
2d.
Following the general procedure A, 4-Amino-3-fluorobenzoic acid
(620 mg, 4 mmol), HCl 37% (1.33 mL, 16 mmol), NaNO2 in solid
(303 mg, 4.4 mmol), solvent H2O. Compound was obtained as a
white powder in 95% yield (770 mg). 1H NMR (500 MHz, MeOD):
nium
salt
(1.6
mmol)
was
added
to
5-Amino-4-
hydroxynaphthalene-2,7-disulfonic acid (479 mg, 1.4 mmol).
Compound was obtained as a dark blue powder in 84% yield
(630 mg). 1H NMR (500 MHz, DMSO):
d
15.24 (s, 1H, NH); 8.11 (d,
0
0
d
8.74 (dd, J ¼ 8.5 Hz, J ¼ 5.6 Hz, 1H, H6’); 8.32 (d, J ¼ 9.8 Hz, 1H, H3’);
J ¼ 8.6 Hz, 1H, H6 ); 8.0 (d, J ¼ 1.6 Hz, 1H, H3 ); 7.93 (dd, J ¼ 8.6 Hz,
0
0
8.25 (d, J ¼ 8.5 Hz, 1H, H5 ).
J ¼ 1.6 Hz, 1H, H5 ); 7.36 (s, 1H, H1); 7.12 (s, 1H, H6); 6.88 (s, 1H, H8).
13C NMR (126 MHz, DMSO):
d
182.2 (C4); 165.9 (C7 ); 154.2 (C7);
0
0
0
4.2.3.5. Iodobenzenediazonium chloride, 2j. Following the general
procedure A, 4-iodoaniline (1095 mg, 5 mmol), HCl 37% (1.67 mL,
20 mmol), NaNO2 in solide (380 mg, 5.5 mmol), solvent H2O.
Compound was obtained as a white powder in 100% yield
0
153.8 (C2); 142.9 (C5); 142.8 (C4 ); 136.0 (C9); 131.7 (C3); 130.4 (C3 );
0
0
0
0
129.4 (C5 ); 126.2 (C1 ); 125.2 (C1); 119.1 (C2 ); 116.1 (C6 ); 114.2 (C8);
113.2 (C6); 111.0 (C10). Elemental analysis found (calcd.)
(þNaþ; þ3H3Oþ): C 37.81 (37.36); H 3.41 (3.87); N 7.46 (7.86); S
11.02 (11.74). HPLC (Gradient A) tR ¼ 8.36 min. MS (ESI)ꢁ: m/z 500.0
[M ꢁ H]ꢁ. UV vis lmax ¼ 536 nm.
(1330 mg). 1H NMR (500 MHz, MeOD):
d
8.81 (d, J ¼ 8.8 Hz, 2H, H2 ,
0
0
0
H6 ); 8.34 (d, J ¼ 8.8 Hz, 2H, H3 , H5 )
4.2.3.6. 4-bromo-2-fluorobenzenediazonium
chloride,
2l.
4.2.3.11. (E)-5-Ammonio-3-(2-(4-carboxy-2-fluorophenyl)hydra-
zono)-4-oxo-3,4-dihydronaphthalene-2,7-disulfonate, 4d (LSP9-
2135). Following the general procedure B, corresponding diazo-
Following the general procedure A, 4-Bromo-2-fluoroaniline
(760 mg, 4 mmol), HCl 37% (1.33 mL, 16 mmol), NaNO2 in solu-
tion (3 mL, 3 mmol), solvent H20eEtOH. Compound was obtained
as a white powder in 100% yield (810 mg). 1H NMR (500 MHz,
0
nium
salt
(1.4
mmol)
was
added
to
5-Amino-4-
hydroxynaphthalene-2,7-disulfonic acid (445 mg, 1.3 mmol). The
crude product was purified by preparative HPLC gradient P2.
Compound LSP9-2135 was the first eluted and obtained as a violet
MeOD):
d
8.62 (dd, J ¼ 9 Hz, J ¼ 6.3 Hz, 1H, H6 ); 8.35 (dd, J ¼ 9 Hz,
0
0
J ¼ 1.8 Hz, 1H, H5 ); 8.08 (d, J ¼ 9 Hz, 1H, H3 ).
powder in 12% yield (80 mg). 1H NMR (500 MHz, DMSO):
d 15.38 (s,
0
4.2.3.7. 2,4-Dinitrobenzenediazonium chloride, 2q. Following the
general procedure A, 2,4-Dinitroaniline (366 mg, 2 mmol), HCl 37%
(0.7 mL, 8 mmol), NaNO2 in solution (2.6 mL, 2.6 mmol), Solvent
1H, NH); 8.02 (t, J ¼ 8.3 Hz, 1H, H6 ); 7.80 (dd, J1 ¼8.3 Hz, J2 ¼ 1.5 Hz,
0
0
1H, H5 ); 7.75 (dd, J1 ¼ 11.8 Hz, J2 ¼ 1.5 Hz, 1H, H3 ); 7.33 (s, 1H, H1);
7.10 (d, J ¼ 1.5 Hz, 1H, H6); 6.87 (d, J ¼ 1.5 Hz, 1H, H8). 13C NMR

F.-C. Favre-Besse et al. / European Journal of Medicinal Chemistry 78 (2014) 236e247
243
(126 MHz, DMSO):
d
0
182.3 (C4); 166.7 (C7 ); 153.9 (C7); 153.8 (C2);
(300 mg). 1H NMR (500 MHz, DMSO):
1H, H1); 7.07 (s,1H, H6); 6.87 (s,1H, H8); 2.62 (s, 6H, eCH3). 13C NMR
0
0 0
d 7.68 (s, 2H, H3 , H5 ); 7.34 (s,
0
0
149.2 (C2 ); 148.1 (C4 ); 142.5 (C5); 136.2 (C9); 131.4 (C3); 126.7 (C5 );
0
0
124.8 (C1); 116.4 (C6 ); 116.2 (C3 ); 114.2 (C8); 113.1(C6); 111.4 (C10).
(126 MHz, DMSO): d 181.2 (C4); 167.0 (C7’); 153.4 (C7); 153.2 (C2);
Elemental analysis found (calcd.) (þ2Naþ; þ1Clꢁ; 3H3Oþ): C 33.12
142.5 (C4 ); 142.0 (C5); 136.2 (C9); 130.5 (C3 eC5 ); 130.4 (C3); 128.4
0
0
0
0 0 0
(C2 eC6 ); 125.9 (C1); 123.1 (C1 ); 113.4 (C8); 112.1 (C6); 112.0 (C10);
(32.78);
H
3.17 (3.24);
N
6.26 (6.75). HPLC (Gradient A)
tR ¼ 8.77 min. MS (ESI)ꢁ: m/z 484.1 [M ꢁ H]ꢁ. UV vis lmax ¼ 532 nm.
20.2
(C8 eC9 )
Elemental
analysis
found
(calcd.)
0
0
(þNaCl; þNaþ; þ4H3Oþ): C 35.19 (35.00); H 4.30 (4.33); N 6.39
(6.44); S 9.71 (9.84). HPLC (Gradient A) tR ¼ 8.34 min. MS (ESI)ꢁ: m/
z 494.1 [M ꢁ H]ꢁ. UV vis lmax ¼ 530 nm.
4.2.3.12. (E)-5-Ammonio-3-(2-(4-carboxy-2-(trifluoromethyl)
phenyl)hydrazono)-4-oxo-3,4-dihydronaphthalene-2,7-disulfonate,
4e (LSP9-3041). Following the general procedure B, corresponding
diazonium salt (1.4 mmol) was added to 5-Amino-4-
hydroxynaphthalene-2,7-disulfonic acid (445 mg, 1.3 mmol). The
crude product was purified by preparative HPLC gradient P2.
Compound was the first eluted and obtained as a violet powder in
4.2.3.16. (E)-5-Ammonio-3-(2-(4-iodo-2-methylphenyl)hydrazono)-
4-oxo-3,4-dihydronaphthalene-2,7-disulfonate,
4i
(LSP9-2114).
Following the general procedure B, corresponding diazonium salt
(5 mmol) was added to 5-Amino-4-hydroxynaphthalene-2,7-
disulfonic acid monosodium salt (1591 mg, 4.7 mmol). Compound
was obtained as a violet powder in 18% yield (506 mg). 1H NMR
0
13% yield (98 mg). 1H NMR (500 MHz, DMSO):
d 15.82 (s, 1H, NH);
0
0
0
8.16 (m, 3H, H3 , H5 , H6 ); 7.32 (s,1H, H1); 7.10 (s,1H, H6); 6.86 (s,1H,
H8). 13C NMR (126 MHz, DMSO): 182.0 (C4); 172.3 (C7 ); 154.2 (C7);
(500 MHz, DMSO):
d
15.62 (s, 1H, NH); 7.77 (d, J ¼ 8.9 Hz, 1H, H5 );
0
0
0
0
0
0
153.7 (C2); 142.6 (C4 ); 141.1 (C5 ); 136.1 (C1 ); 134.1 (C5); 131.1 (C9);
7.65 (s, 1H, H3 ); 7.62 (d, J ¼ 8.9 Hz, 1H, H6 ); 7.30 (s, 1H, H1); 7.06 (s,
126.7 (C3 ); 125.4 (C3); 124.5 (C6 ); 117.0 (C7 ); 116.5 (C1); 114.1 (C8);
1H, H6); 6.87 (s, 1H, H8); 2.38 (s, 3H, H7 ). 13C NMR (126 MHz,
0
0
0
0
0
0
113.5 (C2 ); 112.9 (C6); 111.0 (C10); Elemental analysis found (calcd.)
DMSO):
d
0
181.4 (C4); 153.5 (C7); 153.2 (C2); 142.7 (C5); 140.7 (C2 );
(þ2NaClþ; þH2O): C 32.35 (32.30); H 2.00 (1.96); N 6.27 (6.28); S
9.60 (9.58). HPLC (Gradient A) tR ¼ 11.80 min. MS (ESI)ꢁ: m/z 534.1
[M ꢁ H]ꢁ. UV vis lmax ¼ 529 nm.
138.5 (C6 ); 136.9 (C9); 135.6 (C3 ); 130.0 (C3); 126.9 (C1 ); 123.0 (C1);
0
0
0
0
0
117.4 (C5 ); 113.4 (C8); 112.1 (C6); 111.7 (C10); 88.0 (C4 ); 16.0 (C8 ).
Elemental analysis found (calcd.) (þ2Naþ; þH3Oþ): C 32.55 (32.73);
H 2.57 (2.61); N 6.70 (6.56); S 10.22 (9.99). HPLC (Gradient A)
4.2.3.13. (E)-5-Ammonio-3-(2-(4-carboxy-2,3,5-trifluoro-6-
hydroxyphenyl)hydrazono)-4-oxo-3,4-dihydronaphthalene-2,7-
disulfonate, 4f (LSP9-3031). Following the general procedure B,
corresponding diazonium salt (1.4 mmol) was added to 5-Amino-4-
hydroxynaphthalene-2,7-disulfonic acid (410 mg, 1.2 mmol).
Compound was obtained as a violet powder in 52% yield (335 mg).
tR
¼
11.78 min. MS (ESI)ꢁ: m/z 562.0 [M
ꢁ
H]ꢁ. UV vis
lmax ¼ 543 nm.
4.2.3.17. (E)-5-Ammonio-3-(2-(4-bromophenyl)hydrazono)-4-oxo-
3,4-dihydronaphthalene-2,7-disulfonate, 4j (LSP9-2120).
Following the general procedure B, corresponding diazonium salt
(1.4 mmol) was added to 5-Amino-4-hydroxynaphthalene-2,7-
disulfonic acid monosodium salt (445 mg, 1.3 mmol). Compound
was obtained as a violet powder in 24% yield (545 mg). 1H NMR
0
1H NMR (500 MHz, DMSO):
d 15.48 (s, 1H, NH); 10.60 (s, 1H, OH);
7.3 (s, 1H, H1); 7.16 (d, J ¼ 1.2 Hz, 1H, H6); 6.9 (d, J ¼ 1.2 Hz, 1H, H8).
13C NMR (126 MHz, DMSO):
d
182.6 (C4); 161. 6 (C7 ); 154.6 (C7);
0
0
0
0
154.2 (C2); 145.4 (C5 ); 140.8 (C5); 136.4 (C2 ); 135.8 (C9); 133.8 (C6 );
(500 MHz, DMSO):
0
d
15.34 (s, 1H, NH); 7.64 (d, J ¼ 8.8 Hz, 2H, H2 ,
0
0
0
130.2 (C3); 125.1 (C1); 121.4 (C1 ); 114.9 (C8); 113.9 (C6); 110.9 (C10);
H6 ); 7.56 (d, J ¼ 8.8 Hz, 2H, H3 , H5 ); 7.26 (s, 1H, H1); 7.06 (d,
106.6 (C4 ). 19F NMR (376 MHz, DMSO):
d
ꢁ139.22 (dd, J ¼ 8.7 Hz,
J ¼ 1.5 Hz, 1H, H8); 6.84 (d, J ¼ 1.5 Hz, 1H, H6). 13C NMR (126 MHz,
0
0
0
J ¼ 6.4 Hz, 1F, F5 ); ꢁ151.52 (dd, J ¼ 23.6 Hz, J ¼ 6.4 Hz, 1F,
DMSO):
0
d
181.7 (C4); 153.8 (C7); 153.6 (C2); 142.6 (C5); 118.7 (C2 e
0
0
0
0
0
F2 ); ꢁ159.85 (dd, J ¼ 23.6 Hz, J ¼ 8.7 Hz, 1F, F3 ). Elemental analysis
found (calcd.) (þNaþ; þ2 Clꢁ; þ2H3Oþ; þH2O): C 29.69 (29.66); H
2.36 (2.34); N 5.97 (6.10). HPLC (Gradient A) tR ¼ 8.39 min. MS
(ESI)ꢁ: m/z 535.9 [M ꢁ H]ꢁ. UV vis lmax ¼ 539 nm.
C6 ); 137.1 (C9); 132.6 (C3 eC5 ); 130.9 (C3); 124.7 (C1 ); 123.3 (C1);
0
113.9 (C8); 112.6 (C6); 111.9 (C10); 88.9 (C4 ). Elemental analysis
found (calcd.) (þ2 Naþ): C 34.42 (33.93); H 2.96 (2.49); N 7.55
(7.42); S 10.79 (11.32). HPLC (Gradient A) tR ¼ 10.08 min. MS (ESI)-:
m/z 499.9 [MꢁH]-. UV vis lmax ¼ 536 nm.
4.2.3.14. (E)-5-Ammonio-3-(2-(4-carboxy-2-methoxyphenyl)hydra-
zono)-4-oxo-3,4-dihydronaphthalene-2,7-disulfonate, 4g (LSP9-
2137). Following the general procedure B, corresponding diazo-
4.2.3.18. (E)-5-Ammonio-3-(2-(2-chloro-4-iodophenyl)hydrazono)-
4-oxo-3,4-dihydronaphthalene-2,7-disulfonate,
4k
(LSP9-2179).
nium
salt
(1.4
mmol)
was
added
to
5-Amino-4-
Following the general procedure B, corresponding diazonium salt
(1.4 mmol) was added to 5-Amino-4-hydroxynaphthalene-2,7-
disulfonic acid (445 mg, 1.3 mmol). Compound was obtained as a
dark blue powder in 6% yield (37 mg). 1H NMR (500 MHz, DMSO):
0
hydroxynaphthalene-2,7-disulfonic acid (445 mg, 1.3 mmol).
Compound was obtained as a dark blue powder in 69% yield
(480 mg). 1H NMR (500 MHz, DMSO):
d
15.30 (s, 1H, NH); 7.98 (d,
0
0
J ¼ 8.2 Hz, 1H, H6 ); 7.63 (dd, J ¼ 8.2 Hz, J ¼ 1.8 Hz, 1H, H5 ); 7.59 (d,
d
15.47 (s,1H, NH); 7.90 (d, J ¼ 1.7 Hz,1H, H3 ); 7.83 (d, J ¼ 8.7 Hz,1H,
0
0
0
J ¼ 1.8 Hz, 1H, H3 ); 7.29 (s, 1H, H1); 7.07 (s, 1H, H6); 6.85 (s, 1H, H8);
H6 ); 7.76 (dd, J ¼ 8.7 Hz, J ¼ 1.7 Hz, 1H, H5 ); 7.33 (s, 1H, H1); 7.10 (d,
4.04 (s, 3H, H8 ). 13C NMR (126 MHz, DMSO):
d
181.8 (C4); 169.9
J ¼ 1.2 Hz, 1H, H6); 6.88 (d, J ¼ 1.2 Hz, 1H, H8). 13C NMR (126 MHz,
0
0
0
0
(C7 ); 153.8 (C7); 153.4 (C2); 146.80 (C2 ); 142.8 (C5); 136.2 (C9);
DMSO):
d
181.9 (C4); 153.9 (C7); 153.5 (C2); 142.5 (C5); 139.3 (C1 );
0
0
0
0
0
0
0
135.5 (C4 ); 130.7 (C3); 126.0 (C1 ); 123.8 (C1); 123.1 (C5 ); 114.8 (C6 );
137.1 (C3 ); 136.8 (C9); 136.1 (C5 ); 130.8 (C3); 124.3 (C1); 120.4 (C2 );
0
0
0
0
113.7 (C8); 112.5 (C6); 111.8 (C10); 111.5 (C3 ); 56.2 (C8 ). Elemental
analysis found (calcd.) (þNaCl; þNaþ; þ3H3Oþ): C 33.99 (34.05); H
3.80 (3.65); N 6.79 (6.62); S 10.15 (10.10). HPLC (Gradient A)
tR ¼ 8.80 min. MS (ESI)ꢁ: m/z 495.9 [M ꢁ H]ꢁ. UV vis lmax ¼ 544 nm.
118.6 (C6 ); 113.9 (C8); 112.8 (C6); 111.2 (C10); 87.0 (C4 ). Elemental
analysis found (calc) (þNaþ; þ4H2O): C 28.20 (28.35); H 2.64
(2.68);
tR
lmax ¼ 527 nm.
N 5.08 (6.20); S 9.96 (9.46). HPLC (Gradient A)
¼
12.12 min. MS (ESI)ꢁ: m/z 581.8 [M
ꢁ
H]ꢁ. UV vis
4.2.3.15. (E)-5-Ammonio-3-(2-(4-carboxy-2,6-dimethylphenyl)
hydrazono)-4-oxo-3,4-dihydronaphthalene-2,7-disulfonate,
4h
4.2.3.19. (E)-5-Ammonio-3-(2-(2-fluoro-2-iodophenyl)hydrazono)-
4-oxo-3,4-dihydronaphthalene-2,7-disulfonate 4l (LSP9-2126).
Following the general procedure B, corresponding diazonium salt
(2.5 mmol) was added to 5-Amino-4-hydroxynaphthalene-2,7-
disulfonic acid (445 mg, 1.3 mmol). The crude product was
(LSP9-2139). Following the general procedure B, corresponding
diazonium salt (1.4 mmol) was added to 5-Amino-4-
hydroxynaphthalene-2,7-disulfonic acid (445 mg, 1.3 mmol).
Compound was obtained as a dark blue powder in 74% yield

244
F.-C. Favre-Besse et al. / European Journal of Medicinal Chemistry 78 (2014) 236e247
0
purified by preparative HPLC gradient P2. Compound was the first
(dd, J ¼ 8.6 Hz, J ¼ 1.7 Hz, 1H, H5 ); 7.06 (s, 1H, H8); 6.87 (s, 1H, H6);
eluted and obtained as a violet powder in 3% yield (25 mg). 1H NMR
4.00 (s, 3H, H7 ). 13C NMR (126 MHz, DMSO):
d
181.4 (C4); 153.5 (C7);
0
0
0
(500 MHz, DMSO):
d
15.36 (s, 1H, NH); 7.76 (d, J ¼ 8.5 Hz, 1H, H6’);
153.1 (C2); 148.1 (C2 ); 142.8 (C5); 136.3 (C9); 131.1 (C1 ); 129.9 (C3);
0
0
0
0
7.75 (dd, J ¼ 10.3 Hz, J ¼ 1.7 Hz, 1H, H3’); 7.64 (dd, J ¼ 8.5 Hz,
124.1 (C5 ); 123.0 (C1); 116.9 (C6 ); 116.3 (C4 ); 114.6 (C3 ); 113.5 (C8);
0
0
J ¼ 1.7 Hz, 1H, H5 ); 7.29 (s, 1H, H1); 7.08 (d, J ¼ 1.3 Hz, 1H, H6); 6.84
112.1 (C6); 111.8 (C10); 56.6 (C7 ). Elemental analysis found (calcd.)
(d, J ¼ 1.3 Hz, 1H, H8). 13C NMR (126 MHz, DMSO):
d
180.8 (C4);
(þNaCl; þ3H3Oþ): C 30.48 (30.56); H 3.31 (3.29); N 6.27 (6.12); S
9.57 (9.41). HPLC (Gradient A) tR ¼ 10.53 min. MS (ESI)ꢁ: m/z 530.1
[M ꢁ H]ꢁ. UV vis lmax ¼ 543 nm.
0
0
0
156.8 (C7); 154.6 (C2); 150.7 (C3 ); 146.3 (C5 ); 145.4 (C5); 140.5 (C6 );
0
0
134.6 (C9); 134.1 (C1 ); 130.5 (C3); 124.7 (C1); 123.5 (C2 ); 117.2 (C8);
0
115.1 (C6); 111.2 (C10); 89.3 (C4 ). Elemental analysis found (calc)
(þNaCl; þ3H3Oþ): C 28.61 (28.93); H 2.58 (2.66); N 6.09 (6.10).
HPLC (Gradient A) tR ¼ 10.61 min. MS (ESI)ꢁ: m/z 565.9 [M ꢁ H]ꢁ.
UV vis lmax ¼ 536 nm.
4.2.3.23. (E)-5-Ammonio-3-(2-(4-bromo-2,6-dimethylphenyl)hydra-
zono)-4-oxo-3,4-dihydronaphthalene-2,7-disulfonate, 4p (LSP9-
2125). Following the general procedure B, corresponding diazo-
nium salt (1.4 mmol) was added to 5-Amino-4-hydroxy-
naphthalene-2,7-disulfonic acid monosodium salt (445 mg,
1.3 mmol). Compound was obtained as a purple powder in 24%
Compound 16l was the second eluted in the purification of 4l
and obtained as a blue powder in 5% yield (42 mg). 1H NMR
(500 MHz, DMSO):
d
10.53 (s, 1H, NH); 10.30 (s, 1H, NH); 7.90 (dd,
00
yield (180 mg). 1H NMR (500 MHz, DMSO):
d 16.31 (s, 1H, NH); 7.33
J ¼ 10 Hz, J ¼ 1.6 Hz, 1H, H3 ); 7.85 (dd, J ¼ 10.4 Hz, J ¼ 1.4 Hz, 1H,
0
00
0
H3 ); 7.81 (t, J ¼ 8.4 Hz, 1H, H6 ); 7.80 (t, J ¼ 8.3 Hz, 1H, H6 ); 7.73 (dd,
0
0
(s, 2H, H3 , H5 ); 7.31 (s,1H, H1); 7.04 (s,1H, H6); 6.87 (s, 1H, H8); 2.55
00
(s, 6H, H7 ). 13C NMR (126 MHz, DMSO):
d 181.0 (C4); 153.5 (C7);
J ¼ 8.6 Hz, J ¼ 1.4 Hz, 1H, H5 ); 7.70 (dd, J ¼ 8.6 Hz, J ¼ 1.4 Hz, 1H,
0
H5 ); 7.47 (s, 1H, H6); 7.41 (s, 1H, H8). 13C NMR (126 MHz, DMSO):
0
0
0
0
153.0 (C2); 142.7 (C5); 138.0 (C1 ); 136.9 (C9); 131.6 (C3 eC5 ); 129.8
(C3); 122.3 (C1); 116.7 (C2 eC6 ); 113.1 (C8); 112.1 (C6); 111.6 (C10);
106.8 (C4 ); 19.5 (C7 ). Elemental analysis found (calcd.)
(þNaCl; þ2H3Oþ): C 33.49 (33.42); H 3.47 (3.26); N 6.16 (6.48); S
9.76 (9.88). HPLC (Gradient A) tR ¼ 11.96 min. MS (ESI)ꢁ: m/z 530.0
[M ꢁ H]ꢁ. UV vis lmax ¼ 523 nm.
d
180.7; 169.2; 154.7; 146.4; 145.3; 140.6; 140.3; 134.5; 134.0;
0
0
130.5; 129.0; 125.4; 124.5; 123.4; 122.5; 120.0; 119.1; 117.1; 115.0;
109.7; 96.2 HPLC (Gradient A) tR ¼ 14.11 min. MS (ESI)ꢁ: m/z 813.8
[M ꢁ H]ꢁ. UV vis lmax ¼ 600 nm.
0
0
4.2.3.20. (E)-5-Ammonio-3-(2-(4-iodo-2-(trifluoromethyl)phenyl)
hydrazono)-4-oxo-3,4-dihydronaphthalene-2,7-disulfonate,
4m
4.2.3.24. 5d LSP9-3024 and 5b -LSP9-3088. NaOH (20 g, 0.5 mol)
was dissolved in 60 mL of water. 4-Bromo-2-fluoroaniline (19 g,
0.1 mol), sodium formate (6.8 g, 0.1 mol), cetyl trimethy-
lammonium bromide (CTAB) (4 g, 0.01 mol) and palladium on
activated charcoal (2 g) were added to the solution. The reaction
mixture was stirred under reflux for 4 h. Afterwards, another
batch of sodium acetate (6.8 g, 0.1 mol), was added to the so-
lution. The reaction mixture was heated under reflux for another
20 h. After cooling down to room temperature, the reaction
mixture was filtered and the residual solid washed with EtOAc
(500 mL). This layer was filtered to remove salts. Solvents were
removed under reduced pressure. The crude product was puri-
fied by column chromatography on silica gel (Cyclohexane/
EtOAc: 70/30). The first eluted product was 3,30-difluor-
obiphenyl-4,40-diamine (5d), followed by 2-fluorobenzidine and
benzidine 5b).
(LSP9-2119). Following the general procedure B, corresponding
diazonium salt (3.7 mmol) was added to 5-Amino-4-
hydroxynaphthalene-2,7-disulfonic acid (1130 mg, 3.3 mmol). The
crude product was purified by preparative HPLC gradient P2.
Compound was the first eluted and obtained as a dark blue powder
in 2% yield (40 mg). 1H NMR (500 MHz, DMSO):
d
11.05 (brs, 1H,
NH); 8.00 (m, 3H, H3’, H5’, H6’); 7.43 (s, 1H, H1); 7.18 (s, 1H, H6); 7.00
(s, 1H, H8). 13C NMR (126 MHz, DMSO):
d
172.3 (C4); 150.3 (C7);
0
0
0
150.2 (C2); 145.2 (C5 ); 143.8 (C1 ); 142.1 (C5); 136.2 (C9); 134.1 (C3 );
0
0
0
125.8 (C3); 125.1 (C6 eC7 ); 120.3 (C1); 117.2 (C8); 115.2 (C2 ); 113.6
0
(C6); 110.1 (C10); 91.5 (C4 ). HPLC (Gradient A) tR ¼ 12.38 min. MS
(ESI)ꢁ: m/z 615.9 [M ꢁ H]ꢁ. UV vis lmax ¼ 521 nm.
4.2.3.21. (E)-5-Ammonio-3-(2-(4-bromo-2,3,5-trifluoro-6-
hydroxyphenyl)hydrazono)-4-oxo-3,4-dihydronaphthalene-2,7-
disulfonate, 4n (LSP9-3032). Following the general procedure B,
corresponding diazonium salt (1.4 mmol) was added to 5-Amino-4-
hydroxynaphthalene-2,7-disulfonic acid (410 mg, 1.2 mmol) in
10 mL of H2O. The crude product was purified by preparative HPLC
using gradient P2. Compound was obtained as a dark blue solid in
3,30-Difluoro-[1,10-biphenyl]-4,40-diamine 5d was obtained as
an orange powder in 24% yield (2.62 g) 1H NMR (500 MHz, CD3CN):
0
d
7.20 (dd, J ¼ 13 Hz, J ¼ 2 Hz, 2H, H2, H2 ); 7.14 (dd, J ¼ 8.2 Hz,
0
0
J ¼ 2 Hz, 2H, H6, H6 ); 6.82 (t, J ¼ 8.8 Hz, 2H, H5, H5 ); 4.19 (brs, 4H,
2 ꢃ NH2). 13C NMR (126 MHz, CD3CN):
d
152.7 (d, J ¼ 236.8 Hz, C3,
74% yield (510 mg). 1H NMR (500 MHz, DMSO):
d 15.55 (s, 1H, NH);
0
0
0
10.72 (s, 1H, OH); 7.28 (s, 1H, H1); 7.14 (s, 1H, H6); 6.9 (s, 1H, H8). 13
C
C3 ); 135.5 (d, J ¼ 13.4 Hz, C4, C4 ); 130.9 (d, J ¼ 6.3 Hz, C1, C1 ); 123.1
0
0
(d, J ¼ 2.4 Hz, C6, C6 ); 117.9 (d, J ¼ 4.4 Hz, C5, C5 ); 113.6 (d,
NMR (126 MHz, DMSO):
d 182.4 (C4); 154.5 (C7); 154.1 (C2); 145.5
0
J ¼ 19.5 Hz, C2, C2 ).
0
0
0
0
(C5 ); 140.8 (C5); 140.1 (C3 ); 136.5 (C2 ); 135.9 (C9); 134.4 (C6 ); 129.8
Benzidine 5b was obtained as an orange powder in 10% yield
0
(C3); 124.6 (C1); 119.0 (C1 ); 114.7 (C8); 113.7 (C6); 111.0 (C10); 92.54
19
(1.12 g) 1H NMR (500 MHz, CD3CN):
d
7.31 (d, J ¼ 8.2 Hz, 4H, H2, H6,
0
0
(C4 ). F NMR (376 MHz, DMSO): ꢁ133.90 (s, 1F, F5 ); ꢁ145.20 (d,
0
0
0
0
H2 , H6 ); 6.62 (d, J ¼ 8.2 Hz, 4H, H3, H5, H3 , H5 ); 4.09 (brs, 4H,
0
0
J ¼ 28 Hz, 1F, F2 ); ꢁ157.93 (d, J ¼ 28 Hz, 1F, F3 ). Elemental analysis
found (calcd) (þNaþ; þ2 Clꢁ; þ2H3Oþ; þH2O): C 26.57 (26.57); H
2.20 (2.09); N 5.46 (5.81). HPLC (Gradient A) tR ¼ 12.30 min. MS
(ESI)ꢁ: m/z 569.9 [M ꢁ H]ꢁ. UV vis lmax ¼ 539 nm.
2 ꢃ NH2).
4.2.3.25. 3,30-Dimethyl-[1,10-biphenyl]-4,40-bis(diazonium),
6a.
Following the general procedure A, tolidine (637 mg, 3 mmol), HCl
37% (1 mL, 12 mmol), NaNO2 in solid (166 mg, 2.4 mmol), solvent
H2O/EtOH. The crude product was concentrated in vacuo then
extracted by adding 200 mL of HCL 1 M solution to pH ¼ 2 and
EtOAc. Compound was obtained as an orange powder in 41% yield
0
4.2.3.22. (E)-5-Ammonio-3-(2-(4-bromo-2-methoxyphenyl)hydra-
zono)-4-oxo-3,4-dihydronaphthalene-2,7-disulfonate, 4o (LSP9-
2122). Following the general procedure B, corresponding diazo-
nium salt (2.4 mmol) was added to 5-Amino-4-hydroxy-
naphthalene-2,7-disulfonic acid monosodium salt (716 mg,
2.1 mmol). Compound was obtained as a violet powder in 20% yield
(300 mg). 1H NMR (500 MHz, MeOD):
d
8.59 (d, J ¼ 8.8 Hz, 1H, H6 );
0
0
8.15 (d, J ¼ 1.2 Hz, 1H, H3 ); 8.07 (dd, J ¼ 8.8 Hz, J ¼ 1.2 Hz, 1H, H5 );
(245 mg). 1H NMR (500 MHz, DMSO):
d
15.35 (s, 1H, NH); 7.85 (d,
7.84 (d, J ¼ 1.8 Hz, 1H, H3 ); 7.79 (dd, J ¼ 8.2 Hz, J ¼ 1.8 Hz, 1H, H5 );
00
00
0
0
00
00
J ¼ 8.6 Hz, 1H, H6 ); 7.33 (d, J ¼ 1.7 Hz, 1H, H3 ); 7.29 (s, 1H, H1); 7.24
7.37 (d, J ¼ 8.2 Hz, 1H, H6 ); 2.85 (s, 3H, H7’); 2.45 (s, 3H, H7 ).

F.-C. Favre-Besse et al. / European Journal of Medicinal Chemistry 78 (2014) 236e247
245
4.2.3.26. 40-Ammonio-3,30-dichloro-[1,10-biphenyl]-4-diazonium, 6c.
Following the general procedure A, 3,30-Dichlorobenzidine
(489 mg, 1.5 mmol), HCl 37% (0.5 mL, 6 mmol), NaNO2 in solid
(62 mg, 0.9 mmol), solvent H2O. The crude product was concen-
trated in vacuo then extracted by adding 100 mL of HCL 1 M so-
lution to pH ¼ 2 and EtOAc. The aqueous layer was evaporated.
Compound was obtained as a brown-orange powder in 45% yield
0
7.47 (dd, J ¼ 8.2 Hz, J ¼ 1.9 Hz, 1H, H5 ); 7.32 (s, 1H, H1); 7.08 (s, 1H,
00
H6); 6.91 (d, J ¼ 8.2 Hz, 1H, H6 ); 6.88 (s, 1H, H8). 13C NMR (126 MHz,
00
DMSO):
d 181.5; 153.8; 153.3; 142.7; 137.5; 136.20; 135.9; 130.4;
126.7; 125.8; 125.5; 123.5; 120.4; 117.5; 113.8; 113.7; 112.4; 11.5.
Elemental analysis found (calcd) (þNaCl; þH3Oþ; þ2H2O): C 40.18
(40.48); H 3.72 (3.69); N 8.15 (8.21); S 8.68 (9.40). HPLC (Gradient
A): tR ¼ 12.71 min. MS (ESI)ꢁ: m/z 583.01[MꢁH]ꢁ. UV vis
lmax ¼ 551 nm.
(140 mg). 1H NMR (500 MHz, MeOD):
d
8.55 (d, J ¼ 8.2 Hz, 1H, H6 );
0
0
8.26 (s, 1H, H3 ); 8.05 (d, J ¼ 8.2 Hz, 1H, H5 ); 7.93 (d, J ¼ 1.3 Hz, 1H,
H3 ); 7.70 (dd, J ¼ 8.1 Hz, J ¼ 1.3 Hz, 1H, H5 ); 7.08 (d, J ¼ 8.1 Hz, 1H,
4.2.3.31. (E)-5-ammonio-3-(2-(40-ammonio-3,30-difluoro-[1,10-
biphenyl]-4-yl)hydrazono)-4-oxo-3,4-dihydronaphthalene-2,7-
disulfonate, 7d (LSP9-3073). Following the general procedure C,
corresponding diazonium salt (4 mmol) was mixed 5-Amino-4-
00
00
0
H6 ).
4.2.3.27. 40-Ammonio-3,30-difluoro-[1,10-biphenyl]-4-diazonium, 6d.
Following the general procedure A, LSP9-3024 (880 mg, 4 mmol),
HCl 37% (1.33 mL, 16 mmol), NaNO2 in solution (3.6 mL, 3.6 mmol),
solvent EtOH. The crude product was concentrated in vacuo then
extracted by adding 100 mL of HCL 1 M solution to pH ¼ 2 and
EtOAc. The aqueous layer was evaporated. Compound was obtained
as an orange powder in 50% yield (545 mg). 1H NMR (500 MHz,
0
hydroxynaphthalene-2,7-disulfonic
acid
monosodium
salt
(445 mg, 0.7 mmol). The crude product was purified by preparative
HPLC gradient P2. Compound was obtained as a dark blue powder
in 41% yield (159 mg). 1H NMR (500 MHz, DMSO):
d 15.63 (s, 1H,
00
NH); 8.02 (t, J ¼ 8.6 Hz, 1H, H6 ); 7.63 (dd, J ¼ 13.1 Hz, J ¼ 1.4 Hz, 1H,
00
00
H3 ); 7.58 (dd, J ¼ 8.6 Hz, J ¼ 1.4 Hz, 1H, H5 ); 7.50 (dd, J ¼ 12.7 Hz,
0
0
MeOD):
d
8.55 (dd, J ¼ 9.1 Hz, J ¼ 6.5 Hz 1H, H5 ); 8.15 (dd,
J ¼ 1.7 Hz, 1H, H3 ); 7.37 (dd, J ¼ 8.6 Hz, J ¼ 1.7 Hz, 1H, H5 ); 7.31 (s,
0
0
J ¼ 11.9 Hz, J ¼ 1.7 Hz, 1H, H3 ); 8.04 (dd, J ¼ 9.1 Hz, J ¼ 1.7 Hz, 1H,
1H, H1); 7.08 (s, 1H, H6); 6.90 (t, J ¼ 8.6 Hz, 1H, H6 ); 6.87 (s, 1H, H8).
H6 ); 7.72 (m, 2H, H3 eH5 ); 7.11 (t, J ¼ 8.7 Hz, 1H, H6 ).
13C NMR (126 MHz, DMSO):
d
181.6 (C4); 153.7 (C7); 153.3 (C2);
0
00
00
00
00
0
00
152.2 (C2 ); 150.3 (C2 ); 142.6 (C5); 139.2 (C4 ); 136.2 (C9); 136.0
4.2.3.28. (E)-5-Ammonio-3-(2-(40-ammonio-3,30-dimethyl-[1,10-
biphenyl]-4-yl)hydrazono)-4-oxo-3,4-dihydronaphthalene-2,7-
disulfonate, 7a (LSP9-2187). Following the general procedure C,
corresponding diazonium salt (0.8 mmol) was added to 5-Amino-
4-hydroxynaphthalene-2,7-disulfonic acid monosodium salt
(205 mg, 0.6 mmol). Compound was obtained as a red powder in
(C4 ); 130.3 (C3); 129.0 (C1 ); 123.3 (C1); 122.5 (C5 ); 122.4 (C5 );
0
0
00
0
00
0
0
00
117.4 (C6 ); 117.2 (C6 ); 113.6 (C8); 113.0 (C3 ); 112.5 (C1 ); 112.3 (C6);
00
112.2 (C3 ); 111.6 (C10). Elemental analysis found (calcd)
(þNH4Cl þ H3OþClꢁ þ 3H2O): C 37.19 (37.08); H 4.12 (4.10); N 9.85
(9.83); S 8.65 (9.00). HPLC (Gradient A): tR ¼ 10.63 min. MS (ESI)ꢁ:
m/z 549.2 [MꢁH]ꢁ. UV vis lmax ¼ 548 nm.
25% yield (82 mg). 1H NMR (500 MHz, DMSO):
d 15.82 (s, 1H, NH);
4.2.3.32. (E)-5-Ammonio-3-(2-(40-ammonio-3,30-bis(tri-
fluoromethyl)-[1,10-biphenyl]-4-yl)hydrazono)-4-oxo-3,4-
dihydronaphthalene-2,7-disulfonate, 7e (LSP9-3070). Following the
general procedure C, corresponding diazonium salt (2 mmol) was
mixed with 5-Amino-4-hydroxynaphthalene-2,7-disulfonic acid
monosodium salt (223 mg, 0.7 mmol). The crude product was pu-
rified twice by preparative HPLC using gradient P2 then gradient P1.
Compound was obtained as a dark blue powder in 2% yield (11 mg).
0
00
00
0
0
8.02 (d, J ¼ 8.4 Hz, 1H, H6 ); 7.60 (s, 1H, H3 ); 7.55 (m, 3H, H3 , H6 ,
00
0
H5 ); 7.32 (s, 1H, H1); 7.31 (d, J ¼ 8.2 Hz, 1H, H5 ); 7.06 (s, 1H, H6);
6.88 (s, 1H, H8); 2.47 (s, 3H, H7 ); 2.36 (s, 3H, H7 ). 13C NMR
00
0
(126 MHz, DMSO): 181.2; 153.3; 153.1; 142.7; 140.2; 136.3; 134.7;
d
133.4; 129.8; 128.9; 128.5; 125.3; 125.0; 124.6; 122.7; 122.7; 122.6;
122.6; 115.9; 113.4; 112.0; 111.9. Elemental analysis found (calcd)
(þNaCl; þH3Oþ; þ2H2O): C 42.00 (41.98); H 4.29 (4.29); N 7.75
(8.10); S 8.68 (9.27). HPLC (Gradient A): tR ¼ 9.95 min. MS (ESI)ꢁ: m/
z 541.1 [M ꢁ H]ꢁ. UV vis lmax ¼ 545 nm.
1H NMR (500 MHz, DMSO):
d 15.69 (s, 1H, NH); 8.10 (m, 6H, H3 e
0
0
00
00
00
H5 eH6 eH3 eH5 eH6 ); 7.35 (s, 1H, H1); 7.12 (s, 1H, H6); 6.89 (s, 1H,
4.2.3.29. (E)-5-Ammonio-3-(2-(40-ammonio-[1,10-biphenyl]-4-yl)
H8). 13C NMR (126 MHz, DMSO):
d
182.0; 172.0; 171.4; 162.8; 154.2;
hydrazono)-4-oxo-3,4-dihydronaphthalene-2,7-disulfonate,
(LSP9-2142). Following the general procedure C, corresponding
diazonium salt (1.5 mmol) was mixed with 5-Amino-4-
7b
153.8; 150.2; 145.7; 142.6; 139.9; 136.2; 136.1; 133.6; 132.3; 131.2;
126.0; 125.3; 124.6; 119.0; 118.1; 114.1; 113.4; 113.0; 111.1. Elemental
analysis found (calcd) (þ0.5 (NH4)2SO4 þ NH4Cl þ 4H2O): C 32.93
(32.98); H 3.92 (3.92); N 10.21 (10.42); S 9.15 (9.17). HPLC (Gradient
A): tR ¼ 11.042 min. MS (ESI)ꢁ: m/z 649.6 [MꢁH]ꢁ. UV vis
lmax ¼ 566 nm.
hydroxynaphthalene-2,7-disulfonic
(308 mg, 0.9 mmol). Compound was obtained as a dark blue
powder in 40% yield (186 mg). 1H NMR (500 MHz, DMSO):
15.24
(s, 1H, NH); 7.70 (m, 6H, H2 eH3 eH5 eH6 eH3 eH5 ); 7.36 (d,
acid
monosodium
salt
d
00
00
00
00
0
0
J ¼ 8.6 Hz, 2H, H2 eH6 ); 7.30 (s, 1H, H1); 7.06 (s, 1H, H6); 6.88 (s, 1H,
4.2.3.33. (E)-5-Ammonio-3-(2-(40-ammonio-2,20,3,30,5,50,6,60-octa-
fl u o r o - [ 1,1 0- b i p h e n y l ] - 4 - y l ) h y d r a z o n o ) - 4 - o x o - 3 , 4 -
dihydronaphthalene-2,7-disulfonate, 7f (LSP9-3071). Following the
general procedure C, corresponding diazonium salt (2 mmol) was
mixed 5-Amino-4-hydroxynaphthalene-2,7-disulfonic acid mono-
sodium salt (223 mg, 0.7 mmol). The crude product was purified by
preparative HPLC gradient P2. Compound was obtained as a dark
0
0
H8). 13C NMR (126 MHz, DMSO):
d
181.3; 153.5; 153.0; 142.6;
142.30; 135.1; 134.6; 129.1; 127.3; 127.3; 122.8; 116.8; 113.4; 112.0;
111.9. Elemental analysis found (calcd) (þNaþ; þ3H3Oþ): C 44.18
(44.44); H 4.31 (4.58); N 9.06 (9.42); S 10.09 (10.79). HPLC
(Gradient A): tR ¼ 8.44 min. MS (ESI)ꢁ: m/z 513.1 [MꢁH]ꢁ. UV vis
lmax ¼ 537 nm.
blue powder in 2% yield (9 mg). 1H NMR (500 MHz, DMSO):
(s,1H, NH); 7.29 (s,1H, H1); 7.11 (s,1H, H6); 6.87 (s,1H, H8). 13C N MR
(126 MHz, DMSO): 183.6; 155.6; 155.2; 154.2; 152.3; 152.3; 144.6;
d 16.12
4.2.3.30. (E)-5-Ammonio-3-(2-(40-ammonio-3,30-dichloro-[1,10-
biphenyl]-4-yl)hydrazono)-4-oxo-3,4-dihydronaphthalene-2,7-
disulfonate, 7c (LSP9-3075). Following the general procedure C,
corresponding diazonium salt (3 mmol) was mixed with 5-Amino-
4-hydroxynaphthalene-2,7-disulfonic acid monosodium salt
(285 mg, 0.9 mmol). Compound was obtained as a dark blue
d
138.2; 138.0; 138.0; 132.3; 131.0; 131.0; 125.3; 124.5; 124.4; 119.3;
115.6; 115.0; 114.8; 114.3; 114.2; 113.6. HPLC (Gradient A):
tR ¼ 11.709 min. MS (ESI)ꢁ: m/z 680.6 [MꢁH þ Naþ]ꢁ. UV vis
lmax ¼ 568 nm.
powder in 42% yield (220 mg). 1H NMR (500 MHz, DMSO):
d
15.69
0
0
(s, 1H, NH); 8.10 (d, J ¼ 8.7 Hz, 1H, H6 ); 7.76 (d, J ¼ 1.3 Hz, 1H, H3 );
4.2.3.34. (Z)-4-(2-(2-Chloro-4-iodophenyl)hydrazono)-3-oxo-3,4-
dihydronaphthalene-2,7-disulfonate, 9. Following the general
0
00
7.69 (dd, J ¼ 8.7 Hz, J ¼ 1.3 Hz, 1H, H5 ); 7.64 (d, J ¼ 1.9 Hz, 1H, H3 );

246
F.-C. Favre-Besse et al. / European Journal of Medicinal Chemistry 78 (2014) 236e247
procedure A, diazonium of 2-Chloro-4-iodoaniline (508 mg,
2 mmol), was formed and added to 2-Naphtol-3,6-disulfonic acid
(488 mg, 1.4 mmol) following Procedure B. Compound was ob-
tained as an orange-red powder in 75% yield (594 mg). 1H NMR
by inhibition by CCCP or Trypan Blue were subtracted from those in
the presence of ATP to calculate ATP dependent uptake activity. The
amount of radioactive Glu trapped on GF/C glass-fiber filter paper
was indistinguishable from that trapped on the Millipore HAWP
filter (data not shown). Since the rate of filtration with GF/C glass-
fiber filter paper is higher this type of filter was used in all exper-
iments described here. Similar uptake protocols were used with
other neurotransmitters. Statistical and IC50 calculation have been
performed using BioDataFit 1.02 software of Chang Bioscience.
(500 MHz, DMSO):
d
16.40 (s, 1H, NH); 8.42 (d, J ¼ 8.2 Hz, 1H, H5);
0
0
8.31 (s, 1H, H1); 8.04 (s, 1H, H3 ); 7.98 (d, J ¼ 8.4 Hz, 1H, H5 ); 7.96 (s,
1H, H8); 7.86 (d, J ¼ 8.4 Hz, 1H, H6 ); 7.83 (d, J ¼ 8.4 Hz, 1H, H6) 13C
0
NMR (126 MHz, DMSO):
d
172.9 (C4); 147.1 (C7); 141.1 (C2); 139.6
0
0
0
(C1 ); 139.5 (C1); 137.5 (C3 , C6 ); 132.8 (C9); 131.1 (C4); 127.5 (C6);
0
0
0
126.6 (C8); 125.92 (C10); 123.8 (C2 ); 121.5 (C5); 118.7 (C5 ); 91.2 (C4 ).
HPLC (Gradient A): tR ¼ 9.87 min. MS (ESI)þ: m/z 567.1 [MþH]þ. UV
vis lmax ¼ 493 nm.
Acknowledgment
The authors thank Isabelle Brabet for mGluR assays under the
supervision of Cyril Goudet and Jean-Philippe Pin at the Functional
Genomic Institute (Montpellier, France), Drs R. Azerad and M. Maurs
(UMR8601) for helpful advice in chromatography. This work was
supported by grants from the Agence Nationale pour la Recherche
(Grant ANR-06-NEUR0-048-02 and ANR-10-MALZ-0105).
4.2.3.35. (E)-3-(2-(2-Chloro-4-iodophenyl)hydrazono)-4-oxo-3,4-
dihydronaphthalene-2,7-disulfonate, 11. Following the general pro-
cedure A, 2-Chloro-4-iodoaniline (508 mg, 2 mmol), HCl 37%
(0.7 mL, 8 mmol), NaNO2 in solid (180 mg, 2.6 mmol), solvent H2O.
1-naphtol-3,6-disulfonic acid (455 mg, 1.4 mmol). Compound was
obtained as a red powder in 40% yield (321 mg). 1H NMR (500 MHz,
DMSO):
d
16.12 (s, 1H, NH); 8.26 (d, J ¼ 8.2 Hz, 1H, H5); 8.00 (d,
Appendix A. Supplementary data
0
0
J ¼ 1.4 Hz, 1H, H3 ); 7.89 (d, J ¼ 8.7 Hz, 1H, H6 ); 7.88 (dd, J ¼ 8.7 Hz,
0
J ¼ 1.4 Hz, 1H, H5 ); 7.87 (d, J ¼ 1.6 Hz, 1H, H8); 7.75 (dd, J ¼ 8.2 Hz,
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.03.056.
J ¼ 1.6 Hz, 1H, H6); 7.59 (s, 1H, H1). 13C NMR (126 MHz, DMSO):
0
0
d
176.9 (C4); 152.9 (C7); 143.5 (C2); 139.2 (C2 ); 137.4 (C6 ); 137.2
0
(C3 ); 135.4 (C10); 130.3 (C3); 129.8 (C9); 126.9 (C5); 125.5 (C8); 125.1
0
0
0
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A): tR ¼ 10.53 min. MS (ESI)þ: m/z 567.1 [MþH]þ. UV vis
lmax ¼ 500 nm.
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